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https://openalex.org/W4287693601	https://zenodo.org/records/6367371/files/2011.02838.pdf	English	null	Real-time parameter inference in reduced-order flame models with heteroscedastic Bayesian neural network ensembles	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	2,938	Real-time parameter inference in reduced-order ﬂame models with heteroscedastic Bayesian neural network ensembles Real-time parameter inference in reduced-order ﬂame models with heteroscedastic Bayesian neural network ensembles Matthew P. Juniper Department of Engineering University of Cambridge Cambridge, UK mpj1001@cam.ac.uk Ushnish Sengupta ∗ Department of Engineering University of Cambridge Cambridge, UK us271@cam.ac.uk Matthew P. Juniper Department of Engineering University of Cambridge Cambridge, UK mpj1001@cam.ac.uk Matthew P. Juniper Department of Engineering University of Cambridge Cambridge, UK mpj1001@cam.ac.uk Ushnish Sengupta ∗ Department of Engineering University of Cambridge Cambridge, UK us271@cam.ac.uk Maximilian L. Croci ∗ Department of Engineering University of Cambridge Cambridge, UK mlc70@cam.ac.uk Maximilian L. Croci ∗ Department of Engineering University of Cambridge Cambridge, UK mlc70@cam.ac.uk v:2011.02838v1 [cs.LG] 11 Oct 2020 arXiv:2011.02838v1 [cs.LG] 11 Oct 2020 arXiv:2011.02838v1 [cs.LG] 11 Oct Abstract The estimation of model parameters with uncertainties from observed data is an ubiquitous inverse problem in science and engineering. In this paper, we suggest an inexpensive and easy to implement parameter estimation technique that uses a heteroscedastic Bayesian Neural Network trained using anchored ensembling. The heteroscedastic aleatoric error of the network models the irreducible uncertainty due to parameter degeneracies in our inverse problem, while the epistemic uncertainty of the Bayesian model captures uncertainties which may arise from an input observation’s out-of-distribution nature. We use this tool to perform real-time parameter inference in a 6 parameter G-equation model of a ducted, premixed ﬂame from observations of acoustically excited ﬂames. We train our networks on a library of 2.1 million simulated ﬂame videos. Results on the test dataset of simulated ﬂames show that the network recovers ﬂame model parameters, with the correlation coefﬁcient between predicted and true parameters ranging from 0.97 to 0.99, and well-calibrated uncertainty estimates. The trained neural networks are then used to infer model parameters from real videos of a premixed Bunsen ﬂame captured using a high-speed camera in our lab. Re-simulation using inferred parameters shows excellent agreement between the real and simulated ﬂames. Compared to Ensemble Kalman Filter-based tools that have been proposed for this problem in the combustion literature, our neural network ensemble achieves better data-efﬁciency and our sub-millisecond inference times represent a savings on computational costs by several orders of magnitude. This allows us to calibrate our reduced order ﬂame model in real-time and predict the thermoacoustic instability behaviour of the ﬂame more accurately. ∗The ﬁrst two authors contributed equally to this work. Machine Learning and the Physical Sciences workshop at the 34th Conference on Neural Information Processing Systems (NeurIPS 2020), Vancouver, Canada. 1 Introduction Complex, nonlinear physical models of engineering systems give rise to challenging inverse problems which require the estimation of unknown model parameters from observations, along with uncertainty quantiﬁcation. With the growing popularity of digital twins [1], there is also a need to do bring down the computational cost of parameter inference such that these models may be updated in real-time using the latest sensor observations. Traditional ﬁltering-based techniques require the system to be simulated in parallel, which becomes infeasible if the underlying model is computationally expensive to simulate. Amortized inference using neural networks [2] circumvents this problem by having an expensive ofﬂine training phase, where a surrogate of the approximate posterior p(θ\|z) is learnt from a library of simulator-generated observations zi corresponding to input parameters θi, typically using normalizing ﬂow-based methods [3] [4]. The surrogate can then be rapidly evaluated online to perform parameter inference on new observed data. Machine Learning and the Physical Sciences workshop at the 34th Conference on Neural Information Processing Systems (NeurIPS 2020), Vancouver, Canada. The physical model that motivated this paper is a reduced order kinematic model for ﬂames that captures the response of a ducted premixed ﬂame to acoustic perturbations, known as the G-equation[5]. An accurate description of this response is necessary because the interplay between acoustic waves and the heat release rate of the ﬂame can lead to undesirable thermoacoustic instabilities, which have presented an obstinate challenge to designers of high-energy density combustors [6]. Previous studies [7][8] have reported that calibrating the parameters of the G-equation model parameters using the Ensemble Kalman Filter can make it quantitatively accurate and reproduce the experimentally observed behaviour of acoustically forced ﬂames. To achieve a converged estimate of the parameters, however, the ensemble Kalman ﬁlter needs a long sequence of ﬂame observations, the assimilation of which requires that multiple instances of the G-equation solver be run for hours. The Ensemble Kalman Filter can also get trapped in local optima if the ensemble of simulations is initialized poorly. Our current study evaluates the feasibility of performing the same task accurately with a drastically lower compute budget and fewer observations, using an amortized neural network-based inference technique. 2.1 Experimental observations of ducted premixed Bunsen ﬂame Figure 1: The experimental setup. Figure 1: The experimental setup. Figure 1: The experimental setup. Figure 1: The experimental setup. Figure 1 shows a schematic of the experimental setup. It has a premixed, laminar Bunsen ﬂame inside a cylindrical enclosure, with an optical access window for a high-speed camera. A loudspeaker, driven by an ampliﬁed sinusoidal signal, is mounted upstream of the ﬂame for acoustic forcing. Experiments were performed at different fuel compositions (methane: ethene ratios), ﬂow rates, excitation frequencies (250 - 450 Hz) and excitation amplitudes. The ﬂame dynamics are recorded with a Phantom v4.2 CMOS camera at a resolution of 1200x800 pixels and a frame rate fs = 2500 fps. Thresholding is applied to the grayscale frames to detect a thick band around the ﬂame front. For each row of pixels in the image, the mean x coordinate of the pixels within the band, weighted by their intensities, is used to compute a single x coordinate. Cubic splines with 28 knots are then ﬁtted through the (x, y) coordinates to obtain a unique x location of the ﬂame front corresponding to every y. The vector of ﬂame front x-coordinates could be used directly as input observations for inference. We are interested in modelling the variation of the instantaneous heat release rate and ﬂame areas are a better proxy for heat release rates. We therefore divide the domain vertically into 90 horizontal strips and compute the ﬂame area corresponding to the ﬂame segment it contains. Each frame is thus converted into a 90-dimensional vector of ﬂame areas. 10 successive frames from a video recording are stacked to form an observation zi, a 900-long vector from which we need to infer the G-equation parameters θi likely to have generated it. 2.2 Reduced order G-equation model The G-equation is a kinematic ﬂame model that models the ﬂame as an inﬁnitely-thin front propagating into unburnt gases at a ﬁxed speed sL [5]. It uses a level-set formulation where the ﬂame front is deﬁned as the G = 0 contour of the two-dimensional time-varying G-ﬁeld G(x, y, t). The G-equation is written as ∂G ∂t + v.∇G = sL\|∇G\| (1) (1) Here, ﬂame speed sL = sL,u(1 −Mkκ) is a function of the unstretched ﬂame speed sL,u, the ﬂame curvature κ and the Markstein length Mk. The prescribed velocity ﬁeld v = (U(x) + v′)j + u′i is the sum of a Here, ﬂame speed sL = sL,u(1 −Mkκ) is a function of the unstretched ﬂame speed sL,u, the ﬂame curvature κ and the Markstein length Mk. The prescribed velocity ﬁeld v = (U(x) + v′)j + u′i is the sum of a 2 constant parabolic base ﬂow U(x) = U(1 + va −2vax2) and continuity-obeying velocity perturbations v′(x, y, t) = Uϵasin(St(Ky −t)) and v′(x, y, t) = −UϵaKSt β xcos(St(Ky −t)). U is a characteristic ﬂow β speed, va determines the parabolicity of the base ﬂow velocity proﬁle, ϵa is the amplitude and U/K the phase speed of velocity perturbations, St is the Strouhal number and β is the aspect ratio of the unforced ﬂame. Any ﬂame dynamics that can be generated by the G-equation may be uniquely speciﬁed by a set of 6 β speed, va determines the parabolicity of the base ﬂow velocity proﬁle, ϵa is the amplitude and U/K the phase speed of velocity perturbations, St is the Strouhal number and β is the aspect ratio of the unforced ﬂame. Any ﬂame dynamics that can be generated by the G-equation may be uniquely speciﬁed by a set of 6 Figure 2: Left: Experimentally observed dynamics of an acoustically excited ﬂame. Right: A ﬂame generated by the G-equation. Left: Experimentally observed dynamics of an acoustically excited ﬂame. Right: A ﬂame generated equation. parameters which we need to infer from experimental observations: K, ϵa, Mk, va, St and β. To generate our simulation dataset, we sample quasi-randomly from the prior over parameters P(θ), assumed uniform within the hyper-rectangle deﬁned by the following bounds 0 ≤K ≤2.5, 0 ≤ϵa ≤1.0, 0.02 ≤Mk ≤0.08, 0.0 ≤va ≤1.0, 0.5 ≤St ≤125.0, 2.0 ≤β ≤10.0 and 0.08 ≤fs ≤0.20. 2.2 Reduced order G-equation model For each sampled set of parameters θi, the G-equation is solved using the LSGEN2D solver [9] and the solution is time marched until a limit cycle is reached. The simulated ﬂames then undergo the same data pre-processing steps outlined above for the experimental ﬂames. The x-coordinate of the zero level set is identiﬁed for each y, the ﬂame area vectors are computed for each frame and sequences of 10 frames, each separated from adjacent ones by a phase difference so as to match the experimental frame rate fs, are stacked to form observation vectors zi corresponding to θi. We compute limit cycle oscillations for 11800 different θi-s, from which we generate 2.4 million simulated observations zi. 10% of the θi-s (240000 zi-s) are held out in the test set. 3 Inference using heteroscedastic Bayesian Neural Networks e 3: Scatter plots of true parameters (scaled) versus predicted parameter values for simulated test da Figure 4: Experimentally observed ﬂames (black dots) overlaid on re-simulated G-equation ﬂames. Figure 4: Experimentally observed ﬂames (black dots) overlaid on re-simulated G-equation ﬂame igure 4: Experimentally observed ﬂames (black dots) overlaid on re-simulated G-equation ﬂames. 4 Results Results on the test set of simulated observations (Figure 3) indicate that accurate estimates of G equation parameters are recovered by the neural network. The correlation coefﬁcient ρ between true and predicted parameter values are 0.982, 0.994, 0.971, 0.993, 0.976 and 0.990 for K, ϵa, Mk, va, St and β, respectively. Estimates of parameter uncertainty are also well-calibrated, with particularly high uncertainties in K and St predicted for datapoints with low ϵa-s (amplitude of perturbation), which is physically sensible, as it is not possible to recover these parameters from an unperturbed ﬂame. Given a 10-frame observation vector as input, the neural network takes ~0.1 milliseconds on our Tesla K80 GPU to make parameter predictions. Including the pre-processing time for thresholding, spline ﬁtting and area calculation, predictions can therefore be made in less than a millisecond for every few seconds of acquired video data. We use the trained network to predict parameter values for 10 experimental ﬂame videos and re-simulate the ﬂames using mean predicted parameter estimates. The re-simulated ﬂames match the dynamics of the real ﬂames closely (Figure 4). The root mean squared difference between x-coordinates of simulated and real ﬂame fronts, averaged over all y positions and frames, ranges from 0.0171 to 0.068 units (burner radius is 1 units). The mean re-simulation RMSD across all the ﬂame videos is 0.043 units. 3 Inference using heteroscedastic Bayesian Neural Networks We assume that the posterior p(θ\|z) can be modelled as a Bayesian neural network µ(z; w) with heteroscedas- tic Gaussian aleatoric uncertainty σ(z; w). Although the Gaussian assumption may seem restrictive, previous studies as well as our physical intuition indicate that the posterior here is unimodal and weakly correlated. Multi-modality, too, can be dealt with in this framework by using a Bayesian mixture density network instead of a simple neural network, but this has not been considered here. The neural network has a simple MLP architecture of 3 fully connected ReLU layers with 100 units each and a ﬁnal sigmoid layer that forces the output parameters as well as the predicted aleatoric noise to lie between 0 and 1 (input observations and output parameters are both re-scaled into the range [0, 1]). A He normal [10] prior is used for the neural network parameters. The size of the dataset calls for scalable approximate inference techniques for training the neural network. Here we use approximately Bayesian ensembling using randomized maximum a posteriori (MAP) sampling [11]. Ensembling had already been shown [12] to be empirically effective at providing calibrated estimates of uncertainty for neural networks and the randomized MAP sampling approach grounds this in Bayesian theory. For the j-th neural network ensemble member, we draw a sample from the prior distribution over neural network parameters (assumed multivariate normal) wanc,j ∼N(0, Σprior) and compute the MAP estimate corresponding to a prior re-centered at wanc,j. Given ND parameter-observation pairs {θi, zi}, we minimize the following loss function for the j-th neural network Lossj = ND X i=1 (θi −µj(zi))T σj(z)−1(θi −µj(zi)) + ND X i=1 log(\|σj(z)\|) + ∥Σ−1/2 prior(wj −wanc,j)∥2 2 (2) (2) The prediction of a trained ensemble with M neural networks is therefore a mixture of M Gaussians, each centered at µj(zi) with a variance of σj(z). For computational convenience, we approximate this mixture as a single Gaussian with mean 1 M P j µj and variance 1 M P j σj + 1 M P j µ2 j −( 1 M P j µj)2, following similar treatment in [12]. This also allows us to decompose the total predictive uncertainty into an aleatoric component (ﬁrst term) and an epistemic component (second and third terms). 3 Figure 3: Scatter plots of true parameters (scaled) versus predicted parameter values for simulated test data. Broader Impact This work will lead to better-designed high-energy density combustors, such as those found in rocket and aircraft engines, by enabling designers to assimilate data from experiments more quickly, reﬁne thermoacoustics models more accurately, and therefore design out thermoacoustic oscillations more reliably. 5 Conclusions We use a heteroscedastic Bayesian neural network ensemble to calibrate the parameters of the G-equation, a reduced-order model for predicting the dynamics of premixed ﬂames, based on observations. The ensemble was trained on 2.1 million simulated ﬂame observations and results on the test set of simulated ﬂames show that parameters are recovered accurately by the network from only 10 frames of ﬂame data. Reasonable estimates of parameter uncertainties were also obtained. We then use the network to infer parameters from 4 high-speed video footage of acoustically excited Bunsen ﬂames in our lab. Re-simulating the ﬂames using inferred parameters reveals a close agreement between the model output and experimental data, validating our data assimilation. Compared to the Ensemble Kalman ﬁlter used for this task in the literature, our neural network based technique can perform assimilate ﬂame videos much more rapidly and obtain accurate parameter estimates using fewer observations. high-speed video footage of acoustically excited Bunsen ﬂames in our lab. Re-simulating the ﬂames using inferred parameters reveals a close agreement between the model output and experimental data, validating our data assimilation. Compared to the Ensemble Kalman ﬁlter used for this task in the literature, our neural network based technique can perform assimilate ﬂame videos much more rapidly and obtain accurate parameter estimates using fewer observations. There are several promising avenues for future work. We will look at assimilating data from more realistic industrial ﬂame conﬁgurations as well as integrating our calibrated reduced-order ﬂame models into higher- level thermoacoustic network models of an entire combustor. Acknowledgments and Disclosure of Funding This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska- Curie grant agreement number 766264. [12] Balaji Lakshminarayanan, Alexander Pritzel, and Charles Blundell. Simple and scalable predictive uncertainty estimation using deep ensembles. In Advances in neural information processing systems, pages 6402–6413, 2017. References [1] A. Fuller, Z. Fan, C. Day, and C. Barlow. Digital twin: Enabling technologies, challenges and open research. IEEE Access, 8:108952–108971, 2020. [2] Kyle Cranmer, Johann Brehmer, and Gilles Louppe. The frontier of simulation-based inference. Pro- ceedings of the National Academy of Sciences, 2020. ISSN 0027-8424. doi: 10.1073/pnas.1912789117. URL https://www.pnas.org/content/early/2020/05/28/1912789117. [3] Danilo Rezende and Shakir Mohamed. Variational inference with normalizing ﬂows. In International Conference on Machine Learning, pages 1530–1538, 2015. [4] Stefan T Radev, Ulf K Mertens, Andreass Voss, Lynton Ardizzone, and Ullrich Köthe. Bayesﬂow: Learning complex stochastic models with invertible neural networks. arXiv preprint arXiv:2003.06281, 2020. [5] Ann P Dowling. Nonlinear self-excited oscillations of a ducted ﬂame. Journal of ﬂuid mechanics, 346: 271–290, 1997. [6] T Lieuwen. Modeling premixed combustion-acoustic wave interactions: A review. Journal of propulsion and power, 19(5):765–781, 2003. [7] Hans Yu, Thomas Jaravel, Matthias Ihme, Matthew P Juniper, and Luca Magri. Data assimilation and optimal calibration in nonlinear models of ﬂame dynamics. Journal of Engineering for Gas Turbines and Power, 141(12), 2019. [8] Hans Yu, Ushnish Sengupta, Matthew Juniper, and Luca Magri. Time-accurate calibration of a thermoa- coustic model on experimental images of a forced premixed ﬂame. Bulletin of the American Physical Society, 64, 2019. [9] Santosh Hemchandra. Dynamics of turbulent premixed ﬂames in acoustic ﬁelds. PhD thesis, Georgia Institute of Technology, 2009. [10] K. He, X. Zhang, S. Ren, and J. Sun. Delving deep into rectiﬁers: Surpassing human-level performance on imagenet classiﬁcation. In 2015 IEEE International Conference on Computer Vision (ICCV), pages 1026–1034, 2015. [11] Tim Pearce, Mohamed Zaki, Alexandra Brintrup, Nicolas Anastassacos, and Andy Neely. Uncertainty in neural networks: Bayesian ensembling. In International Conference on Artiﬁcial Intelligence and Statistics, 2020. 5 [12] Balaji Lakshminarayanan, Alexander Pritzel, and Charles Blundell. Simple and scalable predictive uncertainty estimation using deep ensembles. In Advances in neural information processing systems, pages 6402–6413, 2017. 6
https://openalex.org/W2920982556	https://scholarlypublications.universiteitleiden.nl/access/item%3A3627669/view	English	null	New indicators and indexes for benchmarking university–industry–government innovation in medical and life science clusters: results from the European FP7 Regions of Knowledge HealthTIES project	Health research policy and systems	2,019	cc-by	11,407	New indicators and indexes for benchmarking university-industry- government innovation in medical and life science clusters: results from the European FP7 Regions of Knowledge HealthTIES project Edmunds, L.D.; Gluderer, S.; Ovseiko, P.V.; Kamerling, R.; Ton, J.; Vis, L.; ... ; Hassan, A.B. New indicators and indexes for benchmarking university-industry- government innovation in medical and life science clusters: results from the European FP7 Regions of Knowledge HealthTIES project Edmunds, L.D.; Gluderer, S.; Ovseiko, P.V.; Kamerling, R.; Ton, J.; Vis, L.; ... ; Hassan, A.B. RESEARCH Open Access Citation Citation Edmunds, L. D., Gluderer, S., Ovseiko, P. V., Kamerling, R., Ton, J., Vis, L., … Hassan, A. B. (2019). New indicators and indexes for benchmarking university-industry-government innovation in medical and life science clusters: results from the European FP7 Regions of Knowledge HealthTIES project. Health Research Policy And Systems, 17. doi:10.1186/s12961-019-0414-5 Edmunds, L. D., Gluderer, S., Ovseiko, P. V., Kamerling, R., Ton, J., Vis, L., … Hassan, A. B. (2019). New indicators and indexes for benchmarking university-industry-government innovation in medical and life science clusters: results from the European FP7 Regions of Knowledge HealthTIES project. Health Research Policy And Systems, 17. doi:10.1186/s12961-019-0414-5 Version: Publisher's Version License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/3129710 Note: To cite this publication please use the final published version (if applicable). Version: Publisher's Version License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/3129710 Version: Publisher's Version License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/3129710 License: Downloaded from: Note: To cite this publication please use the final published version (if applicable). Edmunds et al. Health Research Policy and Systems (2019) 17:10 https://doi.org/10.1186/s12961-019-0414-5 (2019) 17:10 Edmunds et al. Health Research Policy and Systems https://doi.org/10.1186/s12961-019-0414-5 New indicators and indexes for benchmarking university–industry– government innovation in medical and life science clusters: results from the European FP7 Regions of Knowledge HealthTIES project Laurel D. Edmunds1†, Silvia Gluderer2†, Pavel V. Ovseiko1†, Roel Kamerling3, Jacqueline Ton4, Laura Vis5, Mario Jenni6, Gregory Tutton7, Helen Lawton-Smith8, Márta Völgyiné Nadabán9, Máté Rab10, Jon Rees11, John Anson12, Alexander D. Rushforth13, Maxine Allen14, Alastair M. Buchan1, Montserrat Vendrell15, Alex Casta14ˆ, Gábor Mehes16, Pancras C. W. Hogendoorn4, Ernst Hafen17 and A. Bassim Hassan1* Abstract Background: While the European Union is striving to become the ‘Innovation Union’, there remains a lack of quantifiable indicators to compare and benchmark regional innovation clusters. To address this issue, a HealthTIES (Healthcare, Technology and Innovation for Economic Success) consortium was funded by the European Union’s Regions of Knowledge initiative, research and innovation funding programme FP7. HealthTIES examined whether the health technology innovation cycle was functioning differently in five European regional innovation clusters and proposed regional and joint actions to improve their performance. The clusters included BioCat (Barcelona, Catalonia, Spain), Medical Delta (Leiden, Rotterdam and Delft, South Holland, Netherlands), Oxford and Thames Valley (United Kingdom), Life Science Zürich (Switzerland), and Innova Észak-Alföld (Debrecen, Hungary). Methods: Appreciation of the ‘triple helix’ of university–industry–government innovation provided the impetus for the development of two quantifiable innovation indexes and related indicators. The HealthTIES H-index is calculated for disease and technology platforms based on the h-index proposed by Hirsch. The HealthTIES Innovation Index is calculated for regions based on 32 relevant quantitative and discriminative indicators grouped into 12 categories and 3 innovation phases, namely ‘Input’ (n = 12), ‘Innovation System’ (n = 9) and ‘Output’ (n = 11). (Continued on next page) (Continued on next page) * Correspondence: bass.hassan@path.ox.ac.uk †Laurel D. Edmunds, Silvia Gluderer and Pavel V. Ovseiko contributed equally to this work Pancras C. W. Hogendoorn and Ernst Hafen are senior authors ˆDeceased 1Sir William Dunn School, University of Oxford, Oxford, United Kingdom Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. †Laurel D. Edmunds, Silvia Gluderer and Pavel V. Ovseiko contributed equally to this work 1Sir William Dunn School, University of Oxford, Oxford, United Kingdom Full list of author information is available at the end of the article Background and quantifiable data tailored to the biotech, medical technology and pharma sectors are required for effective benchmarking and time dependent analysis [7, 11]. As policy-makers see innovation as a key driver of eco- nomic growth and wealth creation, the European Union (EU) is striving to become the ‘Innovation Union’ [1]. Despite substantial policy focus and investment in re- search and innovation at the regional level, comparing and benchmarking European regional innovation clus- ters is still in its formative stage and remains limited in scope. Benchmarking usually takes the form of observa- tional studies of national innovation systems, sometimes with extended regional scoreboards. For example, the Regional Innovation Scoreboard was developed as an ex- tension of the Innovation Union Scoreboard to measure the innovation performance of European regions on a limited number of indicators [2–4]. The Regional Eco- system Scoreboard places emphasis on the dynamics and conditions that characterise the quality and nature of innovation and entrepreneurship in administrative re- gions, defined according to the Classification of Territor- ial Units for Statistics (NUTS) nomenclature of territorial units for statistical purposes [5]. The Cluster Observatory provides a range of macro- and micro-economic mapping data, reports on clusters and regional competitiveness conditions, information about cluster organisations, as well as educational resources [6]. In addition, a number of EU commissioned reports highlight the varied quantification of the different phases of the innovation cycle, and the identification of critical factors that stimulate clustering from both a ‘bottom-up’ and ‘top-down’ perspective [7, 8]. The primary objective of such scorecards and reports are the relative ranking of countries and large administratively defined regions, but they do not necessarily capture the workings and competitive advantages of actual innovation clusters from a regional co-ordination perspective [9, 10]. More- over, it remains unlikely that they are sensitive to varia- tions between industry sectors, and therefore objective Herein, we focus on actual regional innovation clus- ters, defined as a critical mass of research innovation in academic institutions and companies that are located within a relatively small geographic area [12, 13]. Geo- graphic clustering allows companies to derive competi- tive advantage, not only from their own resources and capabilities, but also from the shared resources and cap- abilities located in their geographical proximity [14]. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Edmunds et al. Health Research Policy and Systems (2019) 17:10 Page 2 of 15 Edmunds et al. Health Research Policy and Systems (Continued from previous page) Results: The HealthTIES regions had developed relatively similar disease and technology platform profiles, yet with distinctive strengths and weaknesses. The regional profiles of the innovation cycle in each of the three phases were surprisingly divergent. Comparative assessments based on the indicators and indexes helped identify and share best practice and inform regional and joint action plans to strengthen the competitiveness of the HealthTIES regions. Conclusion: The HealthTIES indicators and indexes provide useful practical tools for the measurement and benchmarking of university–industry–government innovation in European medical and life science clusters. They are validated internally within the HealthTIES consortium and appear to have a degree of external prima facie validity. Potentially, the tools and accompanying analyses can be used beyond the HealthTIES consortium to inform other regional governments, researchers and, possibly, large companies searching for their next location, analyse and benchmark ‘triple helix’ dynamics within their own networks over time, and to develop integrated public–private and cross-regional research and innovation strategies in Europe and beyond. Keywords: Regional innovation cluster, innovation index, triple helix, university–industry–government innovation, Regions of Knowledge, life sciences, medical sciences, biotechnology, public policy, European Union Keywords: Regional innovation cluster, innovation index, triple helix, university–industry–government innovation, Regions of Knowledge, life sciences, medical sciences, biotechnology, public policy, European Union Background Such geographic clustering is considered to encourage cross-sector networks, spin-out companies, investment, collaborations and regional development, particularly when there is a strong presence of innovative firms from the same industry [15]. Interactions between large com- panies, small- to medium-sized enterprises and research organisations that lead to the sharing and exchange of facilities, knowledge and people, are likely to contribute to technology transfer. Importantly, these interactions may be catalysed through extended networking, personnel and information dissemination within and be- tween clusters [9]. Regions that have developed such clusters, especially those with a strong presence of uni- versities and knowledge-based industry, are considered to have enhanced economic competitiveness, innovation and growth [16]. Given the benefits of clustering, na- tional governments, the European Commission and intergovernmental organisations such as the OECD, ad- vocate creating regional clusters, particularly in the areas that maximise public good [16–21]. The ‘triple helix’ of university–industry–government relations to increase the potential for innovation and wealth creation is now developed through new organisations such as science parks and incubators that cut across the university–in- dustry–government boundaries. Thus, quantitative benchmarking of regional clusters is an essential Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems Page 3 of 15 Page 3 of 15 prerequisite for devising strategies and taking actions to accelerate the impact of regional medical and life science clusters on health and wealth creation. recent study in Health Research Policy and Systems demonstrated that, although significant public resources are invested in European-funded health projects, data and reports from such projects are often unavailable or inaccessible [39]. Globally, billions of dollars in research investment are wasted when full information about health research studies is inaccessible [40]. In line with the objectives of Responsible Research and Innovation [41], which increases the value of European research in- vestment, we herein make the results from the Health- TIES project publicly available in Open Access format. In the field of science, technology and innovation (STI) studies, there are six established sources of data and related approaches [22] that can be potentially used to quantify and measure innovation with a high degree of comparability across a number of diverse regions. First, national-level research and development (R&D) data are used to characterise national contexts and in- puts into the innovation process as well as innovation activities [23]. Background Second, patents and citations provide in- sights into the invention process [24–27]. Third, biblio- metrics help understand and forecast the scientific process underpinning inventions [28, 29]. Fourth, tech- nometrics rely on expert opinion to assess technological change and its policy implications [30, 31]. Fifth, topic-specific databases and innovation surveys provide statistics on collaboration, commercialisation, financing and other innovation activities and opportunities [32– 34]. Finally, composite synthetic indicators use a variety of data sources to assess innovation capabilities and per- formance [35, 36]. Aim and objectives We established the HealthTIES consortium [42] to de- liver on the stated aim of the Regions of Knowledge by examining whether the health technology innovation cycle was functioning differently in five European re- gional innovation clusters, and to propose regional and joint actions to improve their performance. The specific objectives were as follows: develop indicators and indexes for assessing knowledge and innovation capital and the functioning of the innovation cycle; [ ] Building on the established STI approaches, we report and discuss methods and results pertaining to the devel- opment of new HealthTIES (Healthcare, Technology and Innovation for Economic Success) indicators and in- dexes for benchmarking university–industry–govern- ment innovation in European medical and life science clusters. The HealthTIES project was part of the €126 million (2007–2013) Regions of Knowledge initiative supported by the European Commission under the re- search and innovation funding programme FP7 [37]. The aim of the initiative was “to support trans-national mutual learning and cooperation between research- driven clusters, bringing together regional authorities and development agencies, public research organisations, in- dustry and other relevant stakeholders” [37]. The main activities developed by HealthTIES to achieve the aims of Regions of Knowledge were to complete a detailed set of quantitative assessments for the analysis of regional clusters, including the development of a ‘virtual refer- ence cluster’, to develop a mentoring strategy between a highly developed cluster (Life Science Zürich) with one less developed (Debrecen, Hungary), and to develop a set of regional action plans to improve the integration of contributors to regional economies. identify key stakeholders and collect data to operationalise the proposed indicators and indexes; propose joint and regional actions to improve regional performance through benchmarking and mutual learning. Study setting The HealthTIES consortium consisted of five European regional clusters centred around the following cities (Fig. 1): Barcelona (Bioregion of Catalonia, Catalonia), Leiden, Rotterdam, and Delft (Medical Delta, South Holland), Oxford (Oxford and Thames Valley, United Kingdom), Zürich (Life Science Zürich, Switzerland), Debrecen (Innova Észak-Alföld, Hungary). We selected the first four participating clusters on the basis of pre-existing bilateral collaboration between clus- ter members and their proven track record in medical and life sciences research and innovation [38]. Innova Észak-Alföld was selected as a mentoring cluster on the basis of its potential to accelerate research-driven innovation. Hungary has one of the most developed bio- technology sectors among the new EU member states, and Debrecen had attracted significant investments from Gedeon Richter Plc. to build a new biotechnology Given that the primary focus of our project was on the HealthTIES regions, we initially disseminated and used the results of the project to inform actions within the re- gions. Yet, there are a paucity of such research outputs from this and other projects in the public domain and peer-reviewed literature [38], similar to what is observed for other European-funded projects and initiatives. A (2019) 17:10 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Page 4 of 15 Fig. 1 The HealthTIES (Healthcare, Technology and Innovation for Economic Success) consortium Fig. 1 The HealthTIES (Healthcare, Technology and Innovation for Economic Success) consortium production facility that is unique in Central Eastern Eur- ope [43]. generate new knowledge through collaboration, the ‘con- sensus space’ to build relationships, agree on objectives and develop joint actions, and the ‘innovation space’ to imple- ment joint actions by bringing together knowledge, busi- ness expertise and venture capital. The ‘new organisational actors’ or ‘hybrid organisations’, such as science parks, incu- bators and new forms of venture capital, which span trad- itional university–industry–government boundaries, are shown to be effective in creating the aforementioned ‘spaces’ [21, 47, 48]. HealthTIES innovation index In an effort to create a ‘consensus space’ for building re- lationships, agreeing on objectives and developing joint actions under the assumptions of the ‘triple helix’ model, each cluster assembled a regional team, including repre- sentatives from small- to medium-sized enterprises, bio- tech parks, local government, hospitals, technology transfer offices (TTOs) and academia. Initially, we col- lected a wide range of data for each region, but it be- came clear that these data were either not necessarily available for each region, or were not comparable, or were not quantifiable. A workshop was convened to dis- cuss the best data to collect and how to apply this to an index. All regional clusters were represented by 15 members, extending across every strand of the ‘triple helix’. We had discussions about the significance, valid- ity, reliability and feasibility of the potential indicators facilitated by an independent consultant, and we reached consensus on which indicators to select. As a result, we developed a logic model of the innovation cycle consist- ing of three key phases, namely input, innovation system activity and outputs (Fig. 2). p y) We selected these disease fields and technology plat- forms on the basis of the concurrent assessment of the burden of mortality and morbidity in Europe as well as scientific and technological change and by the project’s experts as the most promising for improving the health of European citizens and making European health sys- tems more sustainable in the context of the increasingly ageing population. Cardiovascular disease, cancer, infec- tious diseases and neurodegenerative diseases are the leading causes of mortality and morbidity in Europe. Frequently, they are manifestations of patient/gene–en- vironment interactions. Therefore, interventions directed at environmental risk factors, such as alcohol, obesity and tobacco, are likely to have important but mixed ben- efits depending upon underlying genetic factors and age at exposure. Recent scientific and technological break- throughs in molecular technology and imaging, as well as drug design, development and delivery have opened up new opportunities for primary prevention of these diseases through risk factor identification and secondary prevention through early and accurate diagnosis, as well as more appropriate and effective treatments. We then compiled a list of potential indicators for each phase of the innovation cycle excluding those indi- cators for which no information was available or would not be feasible to collect. Data collection and analysis For each cluster, we collected and analysed the most re- cent data pertaining to universities, industry, govern- ment and new boundary-spanning organisations during the lifespan of the project (2010–2013). Most of our cross-sectional data and analyses refer to 2010 or 2012. We used a variety of well-established STI sources of data and approaches to data analysis. Namely, we collected and analysed R&D data from statistical offices and insti- tutions, bibliometric data, public and commercial data- bases, and a number of readily available composite synthetic indicators. These data and analyses formed the basis of the HealthTIES H-index for disease and technol- ogy platforms and the HealthTIES Innovation index fo- cusing on four disease fields (cardiovascular disease, cancer, neurodegenerative disease, immunology and in- fectious disease) and three technology platforms (mo- lecular technology, imaging, and drug design, development and delivery). Theoretical framework To guide data collection, analysis and actions, this study used the established ‘triple helix’ model of university–in- dustry–government relations based on the theoretical insight that universities, industry and government are be- coming increasingly interdependent and co-evolving, while retaining their institutional identities [17, 21, 44–46]. Im- perative to the functioning of the ‘triple helix’ is the cre- ation of the ‘knowledge space’ to intensify research and Under the assumptions of ‘triple helix’ model, maxi- mising interactions, creating ‘spaces’ and new Edmunds et al. Health Research Policy and Systems (2019) 17:10 Page 5 of 15 Edmunds et al. Health Research Policy and Systems Edmunds et al. Health Research Policy and Systems (2019) 17:10 (2019) 17:10 using the Clarivate Web of Science, formerly Web of Knowledge, research platform (www.webofknowledge.- com) to benchmark our regions in four relevant disease fields and three technology platforms. Our protocol and search terms are shown in Additional file 1: Box S1 and Table S1, respectively. The HealthTIES h-index (HT H-index) for publications in a region in a specific field specifies the number HT of scientific articles for a given keyword (i.e. cardiovascular) for a given address (e.g. Zürich) that have been cited in other articles at least HT times according to Hirsch [49]. As the h-index depends on the length of a person’s scientific career, the HT H-index depends on the volume of research activity within clusters. The HT H-index permits the compari- son of the relative strengths of a given location for spe- cific keyword search terms and thus offers a valuable assessment of the research output of a cluster in a given disease or technology platform. organisations that cut across the university–industry– government boundaries are proposed to increase the po- tential for innovation and wealth creation through free movement of people, knowledge and venture capital be- tween universities, industry and government. HealthTIES innovation index This process resulted in 32 quantitative indicators, which were grouped into 12 cat- egories and three phases of the innovation cycle, namely ‘Input’ (n = 12), ‘Innovation System’ (n = 9) and ‘Output’ (n = 11) (Table 1). We also assigned, to each indicator, a weighting factor reflecting the relative significance of the underlying component in the innovation cycle. We de- termined mean scores for each weighting factor by vot- ing among the 15 members present at the meeting, followed by wider circulation to all the members of the HealthTIES H-index for disease and technology platforms To assess the knowledge and innovation capital in each region we used bibliometric h-indexes [49]. Although most commonly h-indexes are calculated for individual researchers, they could also be calculated for regions, diseases and technologies. We calculated h-indexes Edmunds et al. Health Research Policy and Systems (2019) 17:10 Page 6 of 15 (2019) 17:10 Edmunds et al. Health Research Policy and Systems Fig. 2 A logic model of the HealthTIES (Healthcare, Technology and Innovation for Economic Success) innovation cycle with key indicator categories Fig. 2 A logic model of the HealthTIES (Healthcare, Technology and Innovation for Economic Success) innovation cycle with key indicator categories trials. The latter indicate activity levels in proof of con- cept drug development. regional steering/advisory committees requesting their feedback, amendments and endorsement of the final set of indicators and weighting factors (Table 1). Input indicators There are nine indicators to measure innovation system activity in three categories. First, the existing levels of innovation that were deemed to be reflected in intellec- tual property, university–industry technology transfer and adoption of innovation. Intellectual property and university–industry technology transfer were measured by the numbers of university spin-off companies, granted United States patents, and the number of uni- versity–industry collaborations larger than 5 million euros. The patients WAIT indicator (patients Waiting for Access to Innovative Treatment) was used to meas- ure how fast innovations and new drugs are made avail- able to the public [50]. Second, support for innovation was measured by the number of full-time equivalent (FTEs) posts in university TTOs in a given region and the extent of national government procurement of ad- vanced technology. Third, the quality of innovation in- frastructure was deemed to be reflected within the regional science park capacity and the overall national attractiveness. The capacity of regional science parks was measured by the size of space available in square meters and by the number of support staff in FTEs. The national attractiveness was deemed to be reflected by The final set includes 12 indicators to measure inputs in four categories. First, the numbers of highly cited profes- sors and publications were deemed to be proxy indica- tors for the depth and breadth of the current scientific knowledge base. Given that the h-index reflects the dur- ation of one’s scientific career, an h-index > 30 was chosen as a threshold for highly cited professors to also include younger researchers who had already made a significant impact on their respective fields. Second, the scope of the current research activity was measured through research funding. In doing so, currencies other than the euro were converted into euros using historical exchange rates. Third, both quantity and quality of hu- man capital were considered to be central to current and future discoveries and innovation. We measured the numbers of graduated national and international MSc and PhD students as well as the numbers of European Research Council junior and advanced grants, which serve as a pan-European mark of research quality. Fourth, to approximate research infrastructure for current and future research, we measured research space, research hospital beds, and Phase I and II clinical Edmunds et al. Input indicators the World Economic Forum Global Competitiveness index [51]. the World Economic Forum Global Competitiveness index [51]. economic growth and wealth creation. Second, we mea- sured the number of non-subsidiary independent biotech companies, distinguishing between smaller companies (with fewer than 20 FTE employees) and larger compan- ies (with 20 or more employees). Third, we measured the number of big trade sales (larger than 100 million euros), where small companies financed by venture cap- ital or private equity were bought up by large life Input indicators Health Research Policy and Systems (2019) 17:10 Page 7 of 15 Table 1 HealthTIES innovation indicators, data sources and weighting factors Innovation phase Category Code Indicator General data source Weightin factor Input Scientific knowledge A01 Profs h-index > 30 University index of profs, Web of Science 18 A02 Publications 2001–2010 Web of Science 16 Research funding A03 Research funding Research institutions: annual reports, support staff 24 Human capital A04 International graduated MSc students Research institutions: annual reports, support staff 3 A05 International PhD students Research institutions: annual reports, support staff 3 A06 National graduated MSc students Research institutions: annual reports, support staff 3 A07 National PhD students Research institutions: annual reports, support staff 3 A08 Junior ERC grants 2007–2010 Lists of ERC-funded starting grants 8 A09 Senior ERC grants 2008–2010 Lists of ERC-funded advanced grants 8 Research infrastructure A10 Research space m2 Research institutions: Facility managing staff 3 A11 Research hospital beds Websites and support staff of hospitals 3 A12 Clinical trials phase I and II ClinicalTrials.gov database 8 Innovation System Intellectual property B01 University spin-offs 2007–2010 TTO staff 17 B02 Granted US patents 2007–2010 See-the-Forest Patent Analytics by IP Vision 14 Innovation adoption B03 WAIT indicator for countries EFPIA Patients WAIT indicator 11 Technology transfer B04 University–industry research collaborations > €5 M TTO/support staff of research institutions, EU project database Cordis 11 Support B05 TTO staff FTEs TTO staff 13 B06 National government procurement of advanced technology WEF GCR 2010–2011, pillar 12.05 11 Infrastructure B07 National attractiveness (WEF index) WEF GCR 2010–2011 12 B08 Science parks m2 Science parks 6 B09 Science park staff FTEs Science parks 5 Output Biotech jobs C01 Jobs in biotech companies FTEs Chambers of commerce, statistical offices 21.5 Biotech companies C02 Biotech companies < 20 FTEs Chambers of commerce, statistical offices 5 C03 Biotech companies > 20 FTEs Chambers of commerce, statistical offices 10.5 Big trade sales C04 Big trade sales > €100 M 2001–10 Regional experts 5.5 Products C05 Products on market BiotechGate database 11 C06 Products in clinical trials BiotechGate database 10 C07 Products in discovery phase BiotechGate database 5.5 C08 Medicines available in countries EFPIA Patient’s WAIT indicator 7 Capital C09 Total investments 2007–2010 BiotechGate database 8 C10 Number of investments 2007–2010 BiotechGate database 8 C11 Average Series A investments 2007–2010 BiotechGate database 8 EFPIA European Federation of Pharmaceutical Industries and Associations, ERC European Research Council, FTE full-time equivalent, GCR Global Competitivenes Report, TTO technology transfer office, WAIT Waiting to Access Innovative Therapies, WEF World Economic Forum EFPIA European Federation of Pharmaceutical Industries and Associations, ERC European Research Council, FTE full-time equivalent, GCR Global Competitiveness Report, TTO technology transfer office, WAIT Waiting to Access Innovative Therapies, WEF World Economic Forum EFPIA European Federation of Pharmaceutical Industries and Associations, ERC European Research Council, FTE full-time equivalent, GCR Global Competitiveness Report, TTO technology transfer office, WAIT Waiting to Access Innovative Therapies, WEF World Economic Forum EFPIA European Federation of Pharmaceutical Industries and Associations, ERC European Research Council, FTE full-tim Report, TTO technology transfer office, WAIT Waiting to Access Innovative Therapies, WEF World Economic Forum the World Economic Forum Global Competitiveness index [51]. Output indicators There were 11 indicators to measure direct and more immediate outputs of the innovation system in five cat- egories. First, the number of jobs in biotech companies was deemed to be one of the key indicators for Edmunds et al. Health Research Policy and Systems (2019) 17:10 Page 8 of 15 Page 8 of 15 Edmunds et al. Health Research Policy and Systems science, medical technology, biotech or pharma compan- ies. Fourth, we measured the number of regional biotech companies’ products and the total number of medicines available nationally. The number of regional biotech companies’ products included those on the market, in clinical trials phases I–III and awaiting United States Food and Drug Administration approval, and in the pipeline, including discovery and lead optimisation. Fi- nally, we measured capital as reflected in the total amount of investments (in euros), number of invest- ments and the value of average Series A investments, i.e. the first significant venture capital investments. Given that there was no common publicly available data source for all of the HealthTIES regions, we operationalised products and capital indicators using the BiotechGate database (www.biotechgate.com) established by Venture Valuation Ltd., located in Zürich. BiotechGate is the lar- gest life science company database in Europe and the United States, established primarily for the purposes of supporting partnering meetings. They allowed us to search our specific regions in their BiotechGate database (www.biotechgate.com). Although it is the most compre- hensive database that is currently available, it is not ex- haustive and is dependent on voluntary registration. Therefore, comparison of the indicators derived from this database may have known limitations. with distinctive strengths and weaknesses. For example, the strength of microscopy in Zürich and its weakness in Barcelona (Bioregion of Catalonia; BioCat), or the strength of clinical trials in Oxford and Medical Delta and their weakness in Zürich. We also compared the average HealthTIES disease and technology platform profile with the profile of Boston, which is a renowned metropolitan area in life science innovation [52]. We found that the shape of disease and technology platform profiles was similar, but of signifi- cantly greater magnitude in Boston (Fig. 3). The relative similarity of regional profiles across Europe, especially with respect to cancer and cardiovascular disease, is likely to reflect the relatively similar priorities for health- care funders. Output indicators By contrast, the relatively limited activity in the public domain in chemistry and drug develop- ment is likely to reflect the concentration of this activity in the pharmaceutical industry. Comparison and benchmarking All indicator data were converted into points, on linear scales ranging from 0 to 10, with 10 being the highest aspirational score. The highest actual data value of an in- dicator in any HealthTIES region was set at 7.5 points. This implied that each region may improve to 10 points in the future. This presentation method was applied to both HT Innovation and H-indexes. As stated, each HT Innovation indicator was then attributed by the Health- TIES consortium consensus, a relative weighting factor (Table 1). Overall scores were calculated by multiplying indicator points by the corresponding weighting factors. Category scores were obtained by adding up indicator scores within a category. Radar plots were used to visu- alise and compare indicator and category scores. The original benchmarking results for 2010 and 2012 are re- ported below and in Additional file 1. In support of the utility of the HT Innovation index over time, the changes in data over two periods was de- termined in two regions where we could reassess all in- dicators (Additional file 1: Figure S6). These data strongly suggest that there is a time-dependent dynamic of the investment, innovation and outputs along the innovation cycle, illustrating the need for temporal as- sessments to evaluate fluctuations and (in)stability of the cluster. Over a given time, Life Science Zürich developed both investment and products on the market, whereas Oxford and Thames Valley and Medical Delta have achieved major increases in investment, but with little change in product or outputs in this period. Yet, Oxford and Thames Valley have a number of products in devel- opment, and this probably reflects an emphasis on clin- ical trials. Without a quantifiable HT Innovation index with temporal evaluation, there is a risk that these data could be misinterpreted. For example, the effect of in- creasing external investment in one particular area may not have impact on wealth creation. Likewise, increasing forward intellectual property without correlated develop- ment of investment may not translate to potential im- pacts on healthcare outcomes. Comparative assessment of the innovation cycle using the HealthTIES Innovation Index The profiles obtained for each regional cluster in each of the three phases of the innovation ecosystem were sur- prisingly divergent (Fig. 4 and Additional file 1: Figure S1–S5). Thus, with a single time point or snapshot, it was difficult to determine the exact cause and effect re- lationships between indicators. This could also suggest that no one uniform template for regional innovation clusters exists in Europe, and that variation may be re- gional and context dependent. Comparative assessment of the knowledge and innovation capital Selection of common bibliometric indicators enabled the comparison of the knowledge and innovation capital in regional clusters across operationally independent dis- ease and technology platforms (Fig. 3). Strikingly, all four advanced regions had developed a relatively similar shape of disease and technology platform profiles, yet Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems Page 9 of 15 Fig. 3 A comparative assessment of the knowledge and innovation capital using the HealthTIES H-index for disease and technology platforms, 2010. Data are expressed relative to a maximum of 7.5 in the best performing HealthTIES region Fig. 3 A comparative assessment of the knowledge and innovation capital using the HealthTIES H-index for disease and technology platforms, 2010. Data are expressed relative to a maximum of 7.5 in the best performing HealthTIES region be addressed in the local regions (Additional file 1: Figure S7). In an effort to create the ‘innovation space’ under the assumptions of the ‘triple helix’ model, the initial regional and joint actions following the adoption of the HealthTIES Innovation Indicators included the in- stallation of formal ‘triple helix’ organisations (consisting of academia, industry and government), professionalisa- tion of the academic TTOs, investing in entrepreneur- ship education, combined with incubator facilities and international entrepreneurs in residence programmes (Box 1). A set of joint actions could also lead to coupling and collaboration of some of Europe’s most innovative biotech regions such as combining business develop- ment between regions, public–private R&D and entre- preneurship education. Developing a more detailed regional roadmap for further actions would require a more integrated approach with ‘triple helix’ actors, com- prehensive time-dependent data and prospective evalu- ation following each initiative. HealthTIES indexes inform regional action The indexes were then used to identify the relative strengths, weaknesses and opportunities in each cluster to benchmark regions, and provide a set of common innovation indicators that could be readily applied to any comparable cluster over time. For the gross differences be- tween regions, the strengths, weaknesses, opportunities and threats (SWOT) and gap analyses were internally vali- dated within the HealthTIES consortium. Moreover, as medical and life sciences represent a sizable part of the re- search and innovation system in each of the HealthTIES regions, comparisons of our findings for medical and life science clusters in the HealthTIES regions with the rank- ings of their respective countries and administrative re- gions across all sectors appear to indicate a degree of external prima facie validity of the HealthTIES indicators and indexes. In line with our findings that Life Science Zürich outperforms other HealthTIES regions on the greatest number of indicators, the European Innovation Scoreboard shows that Switzerland is the overall innovation leader in Europe, outperforming all EU mem- ber states [4]. Similar to the Regional Innovation Score- board (RIS) [5] ranking, the Oxford and Thames Valley region was a high innovator and BioCat and Medical Delta (only partial RIS data) were medium-high innovators. The HealthTIES indexes indicate that Oxford and Thames Val- ley performs slightly better than BioCat and Medical Delta. Both the RIS and HealthTIES study revealed that Debrecen is a low innovator. Box 1 Examples of initial regional and joint actions following the adoption of the HealthTIES innovation indicators • BioCat: Improving the technology transfer offices (TTOs) transfer efficiency through roundtables with investors. Increase external cooperation nationally and internationally through partnerships. Improve clinical trial organisation, in particular proposals on how to improve clinical trial management and protocol turnaround. onally and internationally through partnerships. Improve clinical trial organisation, in particular proposals on how to trial management and protocol turnaround. • Észak-Alföld: Improve further public–private investment through Innova regional innovation engine. Student/researcher Mentoring Programme to enhance forward intellectual property and innovation outputs. Improve the attraction and retention of foreign students and leadership e.g. with new European Research Area strategic Chair. • Medical Delta: Develop an innovation ‘Ambition’ programme with an integrated investment and TTO structure. Joint MD professorships and joint education programme across the cluster. Set up an investment fund to support pre-competitive innovation. Cre- ate large R&D facilities for public–private R&D ventures maximising utility of science parks. Study TTO best practice and propose a new TTO format drawing on best practice. • Oxford and Thames Valley: Address a major deficiency in the University’s ‘spin-out’ activity by developing an Oxford incubator. Study best practice in biotech incubation, secure approval from all stakeholders, raise funding from public investment (Oxford City Deal) and gain a planning permission. In Autumn 2015, building started on the Oxford BioEscalator – a new incubation facility to grow small spin- off companies into mid-sized companies. • Life Science Zürich: Address a major deficiency in translational research through a new ‘triple helix’ organisation for translational medicine. Creation of a ripple helix organisation and action agenda for Life Science Zürich is in progress. Consider a potential translational research centre and establish medical translational trials. Conduct a study on successful European Research Council laureate candidates, followed by a proposal on how to improve leadership recruitment. Establish an entrepreneur in residence programme. • Joint actions: HealthTIES partners developed a successful bid to the European Institute for Innovation and Technology (EIT) for a Knowledge and Innovation Community in Health. Participate in joint education in innovation programmes using the EIT Health STELLAR approach (Spark, Transform, Embrace, Lead, Leap, Amplify, Reward). Connect incubators as soft-landing places for exchange scientists, en- trepreneurs and students (EIT Health). that cut across the university–industry–government boundaries. Box 1 Examples of initial regional and joint actions following the adoption of the HealthTIES innovation indicators In three HealthTIES regions (BioCat, Med- ical Delta and Life Science Zürich), bioincubators proved to be successful in stimulating innovation and economic growth by creating new high-tech companies and jobs, and therefore the Oxford regional team carried out site visits and in-depth interviews to study best practice in setting up and running bioincubators with a view to de- veloping a bioincubator for Oxford and Thames Valley. emerged as the most frequently cited weakness of the bioincubators studied. There was a high degree of vari- ation in how different bioincubators raised the invest- ment necessary to build and equip the actual physical environment, attracted start-up companies, and gener- ated revenue from multiple public and private sources over time. As no uniform business model emerged, we summarised a diverse range of best practice in setting up and running bioincubators in a business model can- vas (Additional file 2). Major challenges in learning from international best practice concerned the transferability of findings to the intended setting. First, the functioning of bioincubators appeared to be dependent on laws, taxes and other country-specific institutions. Therefore, to increase the transferability of best practice to the United Kingdom context, in addition to five bioincubators in HealthTIES regions, we studied a further six bioincubators in Eng- land and Scotland. Second, the location and proximity of a bioincubator to major universities, research institu- tions, hospitals and life science companies emerged as the most frequently cited strength of the bioincubators studied. To capitalise on this strength, we studied in-depth BioPartner Centre Leiden, which emulates the proximity of the university and hospitals in Oxford bet- ter than any other bioincubator studied. Third, funding and sustainability in the first ten years of functioning Best practice in bioincubation Identifying and sharing best practice in bioincubation provides an illustrative case of regional actions following the adoption of the HealthTIES innovation indicators. A bioincubator is a physical environment with labs and of- fices where companies can start up, but also have access to experienced entrepreneurs, venture capital vehicles, skills training, mentoring and business services located in close proximity to major universities, research institu- tions, hospitals and life science companies. Most often, bioincubators are new ‘triple helix’ organisational actors Importantly, some of the differences between medical and life science clusters in the HealthTIES regions could Edmunds et al. Health Research Policy and Systems (2019) 17:10 Page 10 of 15 Page 10 of 15 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems nt of the innovation cycle using the HealthTIES Innovation index, 2010 e assessment of the innovation cycle using the HealthTIES Innovation index, 2010 Fig. 4 A comparative assessment of the innovation cycle using the HealthTIES Innovation index, 2010 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Page 11 of 15 Box 1 Examples of initial regional and joint actions following the adoption of the HealthTIES innovation indicators • BioCat: Improving the technology transfer offices (TTOs) transfer efficiency through roundtables with investors. Increase external cooperation nationally and internationally through partnerships. Improve clinical trial organisation, in particular proposals on how to improve clinical trial management and protocol turnaround. • Észak-Alföld: Improve further public–private investment through Innova regional innovation engine. Student/researcher Mentoring Programme to enhance forward intellectual property and innovation outputs. Improve the attraction and retention of foreign students and leadership e.g. with new European Research Area strategic Chair. • Medical Delta: Develop an innovation ‘Ambition’ programme with an integrated investment and TTO structure. Joint MD professorships and joint education programme across the cluster. Set up an investment fund to support pre-competitive innovation. Cre ate large R&D facilities for public–private R&D ventures maximising utility of science parks. Study TTO best practice and propose a new TTO format drawing on best practice. • Oxford and Thames Valley: Address a major deficiency in the University’s ‘spin-out’ activity by developing an Oxford incubator. Study best practice in biotech incubation, secure approval from all stakeholders, raise funding from public investment (Oxford City Deal) and gain a planning permission. Best practice in bioincubation In Autumn 2015, building started on the Oxford BioEscalator – a new incubation facility to grow small spin- off companies into mid-sized companies. • Life Science Zürich: Address a major deficiency in translational research through a new ‘triple helix’ organisation for translational medicine. Creation of a ripple helix organisation and action agenda for Life Science Zürich is in progress. Consider a potential translational research centre and establish medical translational trials. Conduct a study on successful European Research Council laureate candidates, followed by a proposal on how to improve leadership recruitment. Establish an entrepreneur in residence programme. • Joint actions: HealthTIES partners developed a successful bid to the European Institute for Innovation and Technology (EIT) for a Knowledge and Innovation Community in Health. Participate in joint education in innovation programmes using the EIT Health STELLAR approach (Spark, Transform, Embrace, Lead, Leap, Amplify, Reward). Connect incubators as soft-landing places for exchange scientists, en trepreneurs and students (EIT Health). Limitations and future research The HealthTIES indicators and indexes have a number of limitations. First, our study was based on the assump- tions of the ‘triple helix’ model to guide data collection, analysis and actions. Testing the underlying assumptions was beyond the scope of the study, as the ‘triple helix’ was regarded as a starting point. As a result, our analysis and proposed actions focused on the dynamic interac- tions within the ‘triple helix’ to generate a pragmatic, measurable and effective evaluation of the parameters influencing the innovation cycle. It is likely that this ap- proach will have focused activity at the expense of other sectors. For example, taking into the account the appar- ent dependency of the HealthTIES regions on research and drug development in the pharmaceutical industry, incentivising R&D investment in the pharmaceutical in- dustry through tax breaks could be either more or equally advantageous to some of the actions we consid- ered. We intend to repeat data collection and Health- TIES innovation indicator analysis after implementation of the initial regional and joint actions. The next steps in evaluating the utility of the HealthTIES indicators and subsequent actions should include wider evaluation of the outcomes of the actions based on the assumptions of the ‘triple helix’ model, as well as comparison of their advantages and disadvantages with a range of possible alternative actions to accelerate innovation. HealthTIES had regional value, as we used the results from the comparative assessment of the innovation cycle to identify and share best practice and inform regional and joint action plans to increase the competitiveness of the HealthTIES regions. There was also a synergistic ef- fect from involving regional stakeholders across aca- demia, industry and government in gathering and interpreting data. The HealthTIES project also raised awareness and increased commitment to change among the traditional ‘triple helix’ partners (academia, govern- ment, and medical and life science industry), new part- ners such as insurance companies and private investors, and the general public. Second, quantifying and measuring innovation is in- herently challenging because, by definition, it is some- thing novel and the settings within which it occurs are multidimensional and in many ways unique. To ensure comparability and quantitative measurability, our ap- proach uses a variety of indirect innovation indicators (e.g. research funding, publications, patents and invest- ments) as well as quantitative direct innovation indica- tors (e.g. new products on the market, new medicines, big sales). Discussion This study reports the results and outcomes from the European FP7 Regions of Knowledge HealthTIES pro- ject. Specifically, our study provides tools for the meas- urement and benchmarking of university–industry– government innovation in selected European medical and life science clusters. The general outcome is that the HealthTIES indicators and indexes complement the existing generic tools for measuring innovation perform- ance across all sectors at the level of countries and large administratively defined regions by focussing on actual innovation clusters in one specific sector. The very na- ture of this comparative process means that many indi- cators that are potentially informative were subsequently not used because of the lack of universal availability. Page 12 of 15 Page 12 of 15 Page 12 of 15 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems There are therefore limitations to all such tools and indi- cators that are based on what can be measured, rather than on what should be measured. With respect to these caveats, the HealthTIES indicators and indexes have value, as they been co-developed and validated internally within the HealthTIES consortium of regional partners with representation from academia, government and in- dustry, and appear to have a degree of external prima facie validity. revenue and the increasing costs of getting drugs through Phase III, United States Food and Drug Admin- istration and European Medicines Agency approval, mean that many drug development programmes are folding [54]. To counteract this threat in the context of P4 (predictive, preventive, personalised, participatory) medicine, academia and government across Europe may need to play a more active role in basic research and drug development in partnership with industry. y In addition, the HealthTIES indicators and indexes provided ample data for SWOT analysis, which in turn formed the basis of our regional and joint action plans. Our analysis shows that, although HealthTIES innovation regions possess a strong knowledge base and innovation infrastructure in the four mature clusters, and a strong potential for development in the mentoring cluster, they all significantly lag behind the world-leading Boston cluster (Fig. 3). Discussion This is a reflection of Europe be- ing outperformed by the global innovation leaders such as South Korea, Canada, Australia, Japan and the United States [4]. Although the EU still has a clear innovation performance lead over many other countries globally, this lead is declining rapidly and comes under threat from countries such as China, whose innovation per- formance growth is three times that of the EU [4]. Limitations and future research An inherent limitation of such an approach is that it does not focus on the qualitative characteristics of the object of innovation, i.e. innovative products, ser- vices and technologies. Future research may explore al- ternative approaches focusing directly on the object of innovation to investigate whether the new products, ser- vices and technologies are incremental improvements or disruptive innovations, whether they have a potential to With regard to the particular areas of the knowledge and innovation capital, our study shows a major defi- ciency in the area of medical chemistry across all five re- gions. The lack of critical mass in this area in academia and publicly funded research institutes may be compen- sated by a larger volume of research in the pharmaceut- ical industry. All of the regions appear to be dependent on a steady drug development pipeline in the pharma- ceutical industry, including vaccines and biological agents. In view of the changes that are ongoing in the pharmaceutical industry, these dependencies present a potential threat to the continued high-level activity in the HealthTIES clusters. For example, a major fall in rev- enue for pharma is expected as the patent lifetime of many drugs is coming to an end and they approach what has been termed the ‘patent cliff’ [53]. This fall in Page 13 of 15 Page 13 of 15 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems become blockbuster drugs or high-quality services, and whether they have occurred within or outside of the for- mal medical and life science R&D sector. both health and economic outcomes [20, 55, 56]. More- over, women remain under-represented in leadership and management positions in European academic health centres [57], which indicates that research systems may not support the best science or fail to address topics that benefit women and men equitably [58]. Future research may usefully explore qualitative differences between or- ganisations, sectors and regions using theory-building research designs to draw generalisations beyond the current sample of medical and life science clusters. Third, within the inherent limitations of the current approach, there are considerable challenges in obtaining complete, reliable and comparable datasets, particularly for innovation system and output indicators. We were unable to include in our analysis a number of indicators, data for which were not available for all regions, or were not comparable or quantifiable. Additional files Additional file 1: Box S1. Search protocol for HealthTIES (HT) H-indexes for disease and technology platforms, Web of Science. Table S1. Keywords used in the HT H-index analysis. Figure S1. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separately: Biocat (The HealthTIES average is shown in grey). Figure S2. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separ- ately: Észak-Alföld (The HealthTIES average is shown in grey). Figure S3. Radar plots of the HT Innovation index for Input, Innovation System and Output Indi- cators (with weighted scores) for each region separately: Medical Delta (The HealthTIES average is shown in grey). Figure S4. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separately: Oxford and Thames Valley (The HealthTIES average is shown in grey). Figure S5. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separately: Life Science Zurich (The Additional file 1: Box S1. Search protocol for HealthTIES (HT) H-indexes for disease and technology platforms, Web of Science. Table S1. Keywords used in the HT H-index analysis. Figure S1. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separately: Biocat (The HealthTIES average is shown in grey). Figure S2. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separ- ately: Észak-Alföld (The HealthTIES average is shown in grey). Figure S3. Radar plots of the HT Innovation index for Input, Innovation System and Output Indi- cators (with weighted scores) for each region separately: Medical Delta (The HealthTIES average is shown in grey). Figure S4. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separately: Oxford and Thames Valley (The HealthTIES average is shown in grey). Figure S5. Radar plots of the HT Innovation index for Input, Innovation System and Output Indicators (with weighted scores) for each region separately: Life Science Zurich (The Finally, our study did not explore qualitative differ- ences between the clusters with regard to the workforce, business practices and laws, and how they support or prevent innovation. Conclusion Important sectors of the economy, such as medical and life sciences, increasingly revolve around a ‘triple helix’ of university–industry–government relations at both na- tional and regional levels. In light of growing inter- dependencies between previously arms-length actors, it is vital for research and innovation stakeholders and policy-makers to have access to tools for measuring, monitoring and comparing ‘triple helix’ dynamics in key sectors. The HealthTIES indicators and indexes provide useful practical tools for the measurement and bench- marking of university–industry–government innovation in European medical and life science clusters. They have been developed and validated internally within the HealthTIES consortium and display a degree of external prima facie validity. The results from the comparative assessment of the innovation cycle have been used to identify and share best practice and inform regional and joint action plans to strengthen the competitiveness of the HealthTIES regions. Potentially, the tools and ac- companying analyses can be used beyond the Health- TIES consortium to inform other regional governments, researchers and, possibly, large companies searching for their next location, analyse and benchmark ‘triple helix’ dynamics within their own networks over time, and to develop integrated public–private and cross-regional re- search and innovation strategies in Europe and beyond. y Fourth, external validation of the HealthTIES indica- tors and indexes was beyond the scope of the current project. Future research may usefully validate them in other settings using both quantitative and qualitative val- idation techniques. In particular, there may be a problem in using some of the readily available indicators across different settings. For example, the patients WAIT indi- cator typically includes the innovations and new drugs that have undergone market authorisation but are yet to undergo Phase IV clinical trials to determine their long-term effects on large populations, as well as health technology assessment to determine their cost effective- ness. Thus, some innovations and new drugs included in this indicator in some settings may have adverse long-term effects or may not be cost-effective. Our ex- perience of conducting a workshop with a broad range of experts including, among others, front-line clinicians to build a consensus on the selection and feasibility of innovation indicators suggest that member validation and consensus research are likely to be effective means to gather expert opinion and build a consensus on the utility and feasibility of different indicators in the con- text of different regions. Limitations and future research Particularly, there may be limitations to the reliability of some of the data col- lected for the HealthTIES regions. For example, com- panies complete their own entries in the BiotechGate database, and so their entries may vary in terms of qual- ity, consistency and reporting periods. Moreover, the current version of the innovation indicators is based on absolute rather than relative measurements and thus does not take into consideration differences among HealthTIES regions with regards to the size and quality of various sociodemographic characteristics. Future re- search may usefully develop a methodology to account for such differences and to collect more complete, reli- able and comparable data, for example, through compre- hensive and cross-validated innovation surveys. Abbreviations BioCat: Bioregion of Catalonia; ERC: European Research Council; EU: European Union; FTE: full-time equivalent; HealthTIES: Healthcare, Technology and Innovation for Economic Success; HT H-index: HealthTIES h-index; R&D: research and development; RIS: Regional Innovation Scoreboard; STI: science, technology and innovation; SWOT: strengths, weaknesses, opportunities and threats; TTO: technology transfer office; WAIT: patients Waiting for Access to Innovative Treatment Received: 25 July 2018 Accepted: 7 January 2019 Received: 25 July 2018 Accepted: 7 January 2019 Competing interests d f l 18. Potter J, Miranda G. Clusters, Innovation and Entrepreneurship. Paris: OECD; 2009. EH is a director of evalueSCIENCE Ltd., PCWH and ABH are advisors. The other authors declare no financial competing interests. 19. European Commission. Capacities Work Programme: Regions of Knowledge. http://ec.europa.eu/research/participants/data/ref/fp7/132062/q-wp-201301_ en.pdf. Accessed 20 Jun 2018. Funding l h HealthTIES was funded by an EU FP7 Regions of Knowledge award No. 265550. LDE and PVO are supported by the European Union’s Horizon 2020 research and innovation programme award STARBIOS2 under grant agreement No. 709517. ABH, PVO, LDE, ADR and MA are supported by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre, grant BRC-1215-20008 to the Oxford University Hospitals NHS Foun- dation Trust and the University of Oxford. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Depart- ment of Health and Social Care. 6. Center for Strategy and Competitiveness. Cluster Observatory. http://www. clusterobservatory.eu/. Accessed 1 Mar 2017. 6. Center for Strategy and Competitiveness. Cluste clusterobservatory.eu/. Accessed 1 Mar 2017. 7. European Commission. Regional Biotechnology: Establishing a Methodology and Performance Indicators for Assessing Bioclusters and Bioregions Relevant to the KBBE Area. https://www.pwc.de/de/offentliche-unternehmen/assets/ neues-denken/regional-biotech-report.pdf. Accessed 20 Jun 2018. 8. Arundel A, Casali L, Hollanders H. How European public sector agencies innovate: the use of bottom-up, policy-dependent and knowledge-scanning innovation methods. Res Policy. 2015;44(7):1271–82. 9. European Commission. Framework for State Aid for Research and Development and Innovation. http://eur-lex.europa.eu/legal-content/EN/ TXT/?uri=CELEX%3A52014XC0627%2801%29. Accessed 20 Jun 2018. Availability of data and materials All data and analyses are available on reasonable request from the corresponding author (ABH). 10. Saxenian A. Lessons from Silicon Valley. Technol Rev. 1994;97(5):42–51. 11. Malerba F. Sectoral systems of innovation and production. Res Policy. 2002; 31(2):247–64. References 1. European Commission. Innovation Union: A Europe 2020 Initiative. http://ec. europa.eu/research/innovation-union/index_en.cfm. Accessed 20 Jun 2018. 1. European Commission. Innovation Union: A Europe 2020 Initiative. http://ec. europa.eu/research/innovation-union/index_en.cfm. Accessed 20 Jun 2018. Additional files Yet, our previous research in one of the HealthTIES regions shows that innovation, entrepre- neurship and collaboration culture varies between differ- ent healthcare and research organisations and may affect Page 14 of 15 Page 14 of 15 Edmunds et al. Health Research Policy and Systems (2019) 17:10 Edmunds et al. Health Research Policy and Systems Netherlands. 6Bio-Technopark Schlieren-Zürich, Zurich,, Switzerland. 7Cloverton Holdings, Basel, Switzerland. 8Department of Management, Birkbeck, University of London, London, United Kingdom. 9Independent Consultant, Debrecen, Hungary. 10Innonic Group, Debrecen, Hungary. 11Jon Rees Associates Ltd, Oxford, United Kingdom. 12Oxford Gene Technology, Oxford, United Kingdom. 13Nuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, United Kingdom. 14Business Development Team, Medical Sciences Division, University of Oxford, Oxford, United Kingdom. 15BioCat, Bioregion of Catalonia, Barcelona, Spain. 16Faculty of Public Health, University of Debrecen, Debrecen, Hungary. 17Institute of Molecular Systems Biology, ETH Zürich, Zurich, Switzerland. HealthTIES average is shown in grey). Figure S6. Clustered column diagrams of the HT Innovation index over time, 2010 and 2012. Figure S7. Strengths, weaknesses, opportunities and threats (SWOT) analyses for each region. (PDF 1712 kb) Additional file 2: A business model canvas for bioincubators based on best practice in 11 bioincubators in Catalonia, South Holland, Zürich, England and Scotland, 2013. (PDF 192 kb) Acknowledgements 2. Schibany A, Streicher G. The European Innovation Scoreboard: drowning by numbers? Sci Public Policy. 2008;35(10):717–32. We dedicate this paper to the memory of Dr Alex Casta, our co-author and CEO at UPF Ventures, an innovation management company devoted to boost technology transfer and translational innovation, who tragically passed away on 3 December 2018. We are grateful to all those who supported and facilitated this research in each of the five regions of the HealthTIES consor- tium, including those who contributed to the project during its lifespan, or who were members of the advisory boards and steering committees. 3. Foray D, Hollanders H. An assessment of the Innovation Union Scoreboard as a tool to analyse national innovation capacities: the case of Switzerland. Res Eval. 2015;24(2):213–28. 4. European Commission. European Innovation Scoreboard. http://ec.europa. eu/growth/industry/innovation/facts-figures/scoreboards_en. Accessed 20 Jun 2018. 4. European Commission. European Innovation Scoreboard. http://ec.europa. eu/growth/industry/innovation/facts-figures/scoreboards_en. Accessed 20 Jun 2018. 5. European Commission. Regional Innovation Scoreboard. http://ec.europa.eu/ growth/industry/innovation/facts-figures/regional_en. Accessed 20 Jun 2018. 5. European Commission. Regional Innovation Scoreboard. http://ec.europa.eu/ growth/industry/innovation/facts-figures/regional_en. Accessed 20 Jun 2018. 5. European Commission. Regional Innovation Scoreboard. http://ec.europa.eu/ growth/industry/innovation/facts-figures/regional_en. Accessed 20 Jun 2018. 6. Center for Strategy and Competitiveness. Cluster Observatory. http://www. clusterobservatory.eu/. Accessed 1 Mar 2017. Consent for publication 17. Etzkowitz H, Leydesdorff LA. Universities and the Global Knowledge Economy: A Triple Helix of University-Industry-Government Relations. London, New York: Pinter; 1997. Publisher’s Note 20. Ovseiko PV, O'Sullivan C, Powell SC, Davies SM, Buchan AM. Implementation of collaborative governance in cross-sector innovation and education networks: evidence from the National Health Service in England. BMC Health Serv Res. 2014;14:552. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Authors’ contributions 12. Porter ME. Clusters and the new economics of competition. Harv Bus Rev. 1998;76(6):77–90. PCWH, EH, ABH, GM and MV conceived and designed the HealthTIES project. LDE, SG, RK, AC, MVN, MR and ABH participated in data collection and analysis. LDE, SG, EH and ABH wrote the first draft of the report from the HealthTIES project. PVO extensively revised the first draft with input from ADR and prepared the current manuscript for publication as a research article. All authors commented on and approved the final version of the manuscript. 13. Porter ME. Location, competition, and economic development: local clusters in a global economy. Econ Dev Q. 2000;14(1):15–34. in a global economy. Econ Dev Q. 2000;14(1):15–34. 14. Davis CH, Arthurs D, Cassidy E, Wolfe D. What indicators for cluster policies in the 21st century? www.oecd.org/sti/inno/37443546.pdf. Accessed 1 Mar 2017. 15. Beaudry C, Breschi S. Are firms in clusters really more innovative? Econ Innov New Technol. 2003;12(4):325–42. Ethics approval and consent to participate 16. Remoe S, Guinet J. Dynamising National Innovation Systems. Paris: OECD; 2002. Author details 1 ll 21. Etzkowitz H. The Triple Helix: University-Industry-Government Innovation in Action. Abingdon-on-Thames: Routledge; 2008. 1Sir William Dunn School, University of Oxford, Oxford, United Kingdom. 2Astellas Pharma, Zürich, Switzerland. 3Medical Delta, Delft University of Technology, Delft, The Netherlands. 4Leiden University Medical Center, Leiden, The Netherlands. 5Province of South Holland, The Hague, The 22. Smith K. Measuring innovation. In: Fagerberg J, Mowery DC, editors. The Oxford Handbook of Innovation. Oxford: Oxford University Press; 2006. p. 1–38. Page 15 of 15 Edmunds et al. Health Research Policy and Systems (2019) 17:10 (2019) 17:10 23. OECD. Frascati Manual: Guidelines for Collecting and Reporting Data on Research and Experimental Development. http://oe.cd/frascati. Accessed 12 Dec 2018. %20Apifama%20Conference%20-%2013%2006%2028%20w-o%20(2).pdf. Accessed 20 Jun 2018. 51. Schwab K. The Global Competitiveness Report 2010–2011. https://www. weforum.org/reports/global-competitiveness-report-2010-2011. Accessed 20 Jun 2018. 24. Acs ZJ, Anselin L, Varga A. Patents and innovation counts as measures of regional production of new knowledge. Res Policy. 2002;31(7):1069–85. 52. Owen-Smith J, Powell WW. Knowledge networks as channels and conduits: the effects of spillovers in the Boston Biotechnology Community. Organ Sci. 2004;15(1):5–21. 25. Crosby M. Patents, innovation and growth. Econ Rec. 2000;76(234):255–62. 26. Callaert J, Van Looy B, Verbeek A, Debackere K, Thijs B. Traces of prior art: an analysis of non-patent references found in patent documents. Scientometrics. 2006;69(1):3–20. 53. Harrison C. Patent watch. Nat Rev Drug Discov. 2013;12(1):14–5 27. Ovseiko PV, Oancea A, Buchan AM. Assessing research impact in academic clinical medicine: a study using Research Excellence Framework pilot impact indicators. BMC Health Serv Res. 2012;12:478. 54. Nadeau P. Venture capital investment selection: do patents attract investors? Strateg Chang. 2010;19(7–8):325–42. 54. Nadeau P. Venture capital investment selection: do patents a investors? Strateg Chang. 2010;19(7–8):325–42. 55. Ovseiko PV, Buchan AM. Organizational culture in an academic health center: an exploratory study using a Competing Values Framework. Acad Med. 2012;87(6):709–18. 28. Teixeira AAC. Evolution, roots and influence of the literature on National Systems of Innovation: a bibliometric account. Camb J Econ. 2014;38(1):181–214. 56. Ovseiko PV, Melham K, Fowler J, Buchan AM. Organisational culture and post-merger integration in an academic health centre: a mixed-methods study. BMC Health Serv Res. 2015;15:25. 29. Watts RJ, Porter AL, Newman NC. Innovation forecasting using bibliometrics. Competitive Intell Rev. 1998;9(4):11–9. 30. Grupp H. The measurement of technical performance of innovations by technometrics and its impact on established technology indicators. Res Policy. 1994;23(2):175–93. 57. Author details 1 ll Kuhlmann E, Ovseiko PV, Kurmeyer C, Gutiérrez-Lobos K, Steinböck S, von Knorring M, et al. Closing the gender leadership gap: a multi-centre cross- country comparison of women in management and leadership in academic health centres in the European Union. Hum Resour Health. 2017;15:2. y 31. Coccia M. Technometrics: origins, historical evolution and new directions. Technol Forecast Soc Chang. 2005;72(8):944–79. 58. Ovseiko PV, Greenhalgh T, Adam P, Grant J, Hinrichs-Krapels S, Graham KE, et al. A global call for action to include gender in research impact assessment. Health Res Policy Syst. 2016;14:50. 32. Eurostat. Community Innovation Survey (CIS). https://ec.europa.eu/eurostat/ web/microdata/community-innovation-survey. Accessed 12 Dec 2018. 33. BiotechGate. Business development database for the life science industry. https://www.biotechgate.com. Accessed 12 Dec 2018. 34. OECD. Eurostat Oslo Manual 2018; 2018. http://www.oecd.org/sti/inno/oslo- manual-2018-info.pdf. Accessed 21 Jan 2019. 35. Archibugi D, Denni M, Filippetti A. The technological capabilities of nations: The state of the art of synthetic indicators. Technol Forecast Soc Chang. 2009;76(7):917–31. 36. Grupp H, Schubert T. Review and new evidence on composite innovation indicators for evaluating national performance. Res Policy. 2010;39(1):67–78. 37. European Commission. Regions of Knowledge. http://ec.europa.eu/invest-in- research/funding/funding03_en.htm. Accessed 20 Jun 2018. research/funding/funding03_en.htm. Accessed 20 Jun 2018. 38. Lawton Smith H, Bagchi-Sen S, Edmunds L. Innovation cycles and geographies of innovation: a study of healthcare innovation in Europe. Eur Urban Reg Stud. 2017;25(4):405–22. 39. Zander B, Busse R. Is there enough research output of EU projects available to assess and improve health system performance? An attempt to understand and categorise the output of EU projects conducted between 2002 and 2012. Health Res Policy Syst. 2017;15:13. 40. Chan AW, Song F, Vickers A, Jefferson T, Dickersin K, Gotzsche PC, et al. Increasing value and reducing waste: addressing inaccessible research. Lancet. 2014;383(9913):257–66. 41. 2014 Italian Presidency of the Council of the European Union. Rome Declaration on Responsible Research and Innovation in Europe. https://ec. europa.eu/research/swafs/pdf/rome_declaration_RRI_final_21_November. pdf. Accessed 14 Dec 2018. 42. CORDIS. Health-TIES. https://cordis.europa.eu/project/rcn/95846_en.html. Accessed 20 Jun 2018. 43. EuropaBio, Venture Valuation. Biotech in the New EU Member States: An Emerging Sector 2009. http://www.gate2biotech.com/documents/ download/4.pdf. Accessed 1 Mar 2017. 44. Etzkowitz H, Leydesdorff L. The endless transition: a “triple helix” of university-industry-government relations. Minerva. 1998;36(3):203–8. 44. Etzkowitz H, Leydesdorff L. The endless transition: a “triple helix” of university-industry-government relations. Minerva. 1998;36(3):203–8. 45. Etzkowitz H, Leydesdorff L. The dynamics of innovation: from National Systems and “Mode 2” to a Triple Helix of university–industry–government relations. Res Policy. 2000;29(2):109–23. 45. Author details 1 ll Etzkowitz H, Leydesdorff L. The dynamics of innovation: from National Systems and “Mode 2” to a Triple Helix of university–industry–government relations. Res Policy. 2000;29(2):109–23. 46. Greenhalgh T, Ovseiko PV, Fahy N, Shaw S, Kerr P, Rushforth AD, et al. Maximising value from a United Kingdom Biomedical Research Centre: study protocol. Health Res Policy Syst. 2017;15:70. 46. Greenhalgh T, Ovseiko PV, Fahy N, Shaw S, Kerr P, Rushforth AD, et al. Maximising value from a United Kingdom Biomedical Research Centre: study protocol. Health Res Policy Syst. 2017;15:70. 47. Lamine W, Mian S, Fayolle A, Wright M, Klofsten M, Etzkowitz H. Technology business incubation mechanisms and sustainable regional development. J Technol Transf. 2018;43(5):1121–41. 47. Lamine W, Mian S, Fayolle A, Wright M, Klofsten M, Etzkowitz H. Technology business incubation mechanisms and sustainable regional development. J Technol Transf. 2018;43(5):1121–41. 48. Champenois C, Etzkowitz H. From boundary line to boundary space: The creation of hybrid organizations as a Triple Helix micro-foundation. Technovation. 2018;76-77:28–39. 48. Champenois C, Etzkowitz H. From boundary line to boundary space: The creation of hybrid organizations as a Triple Helix micro-foundation. Technovation. 2018;76-77:28–39. 49. Hirsch JE. An index to quantify an individual's scientific research output. Proc Natl Acad Sci U S A. 2005;102(46):16569–72. 49. Hirsch JE. An index to quantify an individual's scientific research output. Proc Natl Acad Sci U S A. 2005;102(46):16569–72. 50. Pickaert M-C. The Value of Innovation. The “Patients W.A.I.T.” Indicator 2013. http://www.apifarma.pt/eventos/Documents/Lisbon%20- 50. Pickaert M-C. The Value of Innovation. The “Patients W.A.I.T.” Indicator 2013. http://www.apifarma.pt/eventos/Documents/Lisbon%20-
https://openalex.org/W1983692789	https://europepmc.org/articles/pmc2842437?pdf=render	English	null	A Camelid Anti-PrP Antibody Abrogates PrPSc Replication in Prion-Permissive Neuroblastoma Cell Lines	PloS one	2,010	cc-by	9,545	A Camelid Anti-PrP Antibody Abrogates PrPSc Replication in Prion-Permissive Neuroblastoma Cell Lines Daryl Rhys Jones1, William Alexander Taylor1, Clive Bate1, Monique David2, Mourad Tayebi1* 1 Department of Pathology & Infectious Diseases, Royal Veterinary College, Hertfordshire, United Kingdom, 2 Multiple Sclerosis Research Center of New York, New York, New York, United States of America Abstract Funding: This study was supported by the UK Royal Society and The Royal Veterinary Colle design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mtayebi@rvc.ac.uk disease, other researchers have demonstrated the effectiveness of antibody-mediated therapy in delaying the onset of disease in vivo [16,17,18,19,20]. The antibody-mediated therapy approach was first investigated in scrapie susceptible neuroblastoma cells (N2a) [12,13], then in transgenic mice with an anti-PrP antibody m-chain [21]. This was followed by vaccinating scrapie-infected mice with rPrP [22], PrP peptides [23], and mucosal vaccination using live attenuated strain of Salmonella typhimurium expressing the mouse PrP gene [24,25]. Crucially and for the first time, we have previously shown that passive transfer of anti-PrP monoclonal antibodies following inoculation of mice with scrapie-infected material via the intraperitoneal route led to inhibition of prion replication in vivo and animals survived throughout their life-span and remained free of detectable prion infection [14]. When the passive antibody transfer was started after onset of clinical signs of disease, all animals succumbed to prion diseases and were not rescued, indicating the inefficiency of these antibodies to transmigrate across the BBB. PLoS ONE \| www.plosone.org Abstract The development of antibodies effective in crossing the blood brain barrier (BBB), capable of accessing the cytosol of affected cells and with higher affinity for PrPSc would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the ‘‘proof-of-principle’’ for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrPC. In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrPSc replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means. Citation: Jones DR, Taylor WA, Bate C, David M, Tayebi M (2010) A Camelid Anti-PrP Antibody Abrogates PrPSc Replication in Prion-Permissive Neuroblastoma Cell Lines. PLoS ONE 5(3): e9804. doi:10.1371/journal.pone.0009804 Editor: Jiyan Ma, Ohio State University, United States of America Received October 29, 2009; Accepted February 26, 2010; Published March 22, 2010 r WA, Bate C, David M, Tayebi M (2010) A Camelid Anti-PrP Antibody Abrogates PrPSc Replication in Prion-Permissive Neuroblastoma Cel 04. doi:10.1371/journal.pone.0009804 Received October 29, 2009; Accepted February 26, 2010; Published March 22, 2010 Copyright: 2010 Jones et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the UK Royal Society and The Royal Veterinary College Internal Grant Scheme to MT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PrioV3 anti-prion antibody binds to prion proteins and peptide For comparison, ICSM35, raised in mouse (positive), was incubated with brain homogenates prepared from RML-infected and normal brain tissue with and with no PK digestion. Figure 1. PrioV3 binds to both native and denatured prion proteins. (A) PrioV3 antibody was screened with 20-mer amino acid sequences spanning the 91–230 region of the prion protein and was shown to bind a region between 170 and 19. ICSM18 that binds to a region between 141 and 160 was used as positive control and normal camel serum (NCS) was used as negative control. Goat anti-llama IgG-HRP was used as secondary detection antibody. Anti-PrP responses were measured in peptide ELISA. Error bars represent the mean antibody level derived from n = 8 wells. (B) PrioV3 antibody bound to native PrPC inside the cytoplasm of N2a cells (orange arrow), in contrast with ICSM35 that strictly stained cell membrane- bound PrPC (blue arrow). Florescence microscopy was performed and images from each source [FITC (450–490 nm), Texas red (510–560 nm) and DAPI (330–380 nm)] were collected. As control, N2a cells were stained with the secondary anti-llama IgG (green) and anti-mouse IgG antibody (red), omitting the primary antibodies. Scale bar = 25 mm (C) PrP wild type, knock-out and RML-infected brain homogenate were digested with PK and tested for reactivity PrioV3. PrioV3 strongly bound both PrPC and PrPSc in brain homogenates (pre- and post-PK). For comparison, ICSM35, raised in mouse (positive), was incubated with brain homogenates prepared from RML-infected and normal brain tissue with and with no PK digestion. doi:10.1371/journal.pone.0009804.g001 Figure 1. PrioV3 binds to both native and denatured prion proteins. (A) PrioV3 antibody was screened with 20-mer amino acid sequences spanning the 91–230 region of the prion protein and was shown to bind a region between 170 and 19. ICSM18 that binds to a region between 141 and 160 was used as positive control and normal camel serum (NCS) was used as negative control. Goat anti-llama IgG-HRP was used as secondary detection antibody. Anti-PrP responses were measured in peptide ELISA. Error bars represent the mean antibody level derived from n = 8 wells. (B) PrioV3 antibody bound to native PrPC inside the cytoplasm of N2a cells (orange arrow), in contrast with ICSM35 that strictly stained cell membrane- bound PrPC (blue arrow). Florescence microscopy was performed and images from each source [FITC (450–490 nm), Texas red (510–560 nm) and DAPI (330–380 nm)] were collected. Introduction Prion diseases also known as transmissible spongiform enceph- alopathies (TSEs) are a group of closely related fatal transmissible neurodegenerative diseases that affect humans and animals [1]. Prion disorders are associated with conversion of the normal cellular prion protein (PrPC) into a disease-associated isoform, PrPSc, that acquires increased b-sheet structure and detergent insolubility [2]. These diseases are characterised by the deposition and aggregation of proteins into highly stable, partially proteinase- resistant plaques and fibrils [3], leading to neuronal cell death and spongiform change of the brain parenchyma [4]. A number of drugs have been assessed for their efficacy in inhibiting prion replication, and these included polyanions [5], Iododoxorubicin, tetracycline [6], Congo red [7], polyene antibiotics [8], and quinacrine [9]. With the exception of an amphotericine analogue that had some effect on disease progression [10], these drugs have been shown to be ineffective in interacting with PrPSc in vivo. Moreover, their capacity to transmigrate across the BBB has not been established. We have previously raised a camelid anti-prion antibody, known as PrioV3, capable of crossing the BBB in vitro and in vivo via receptor-mediated transport (M. Tayebi & J. Greenwood, unpublished data; M. Tayebi et al, presented at the Neuroprion conference, Madrid, September 2008). PrioV3 displayed binding specificity for both PrPC and PrPSc and was believed to bind PrPC in the cytosol of neurons (Tayebi et al, submitted); In marked contrast, conventional anti-prion antibodies produced in mouse Following successful treatment of scrapie-susceptible neuroblas- toma (N2a) cells [11,12,13] and scrapie-infected mice [14], immune-based therapy has become the most promising therapeu- tic approach for the treatment of prion diseases [15]. After the landmark report from White and colleagues [14], showing for the first time efficacy of anti-prion antibodies in treating animal prion- PLoS ONE \| www.plosone.org March 2010 \| Volume 5 \| Issue 3 \| e9804 1 March 2010 \| Volume 5 \| Issue 3 \| e9804 In Vitro Prion Treatment against similar target antigen were unable to enter the neuronal plasma membrane and instead decorated the cell membrane by staining surface PrPC (Tayebi et al, submitted). PrioV3 anti-prion antibody binds to prion proteins and peptide In this report, we show that PrioV3 anti-prion antibody was effective in crossing BBB, reduce peripheral prion replication in vivo and cured chronically scrapie-infected N2a cells and was also able to abolish prion replication. Finally, we also demonstrate here that PrioV3 antibody failed to trigger neurotoxic effects as previously shown with conventional anti-prion antibodies raised in mouse ([26], M. Tayebi and M. David, submitted). In order to confirm specificity to the prion protein and to determine its binding motif, PrioV3 anti-prion antibody was pepscanned (Figure 1A). 20-mer peptide sequences overlapping by 10 amino acids and spanning the 91–230 region of the truncated prion protein were used to epitope-map the PrioV3 antibody and was shown to bind to a linear motif on the C-terminal region of the protein, a sequence that lies between 171 and 190 (Figure 1A). This region was previously shown to bind to the so-called YYR motif used by Paramithiotis and colleagues to generate their PrPSc- specific mAbs [27]. The camelid anti-prion antibodies could potentially form an important tool for the neutralisation/clearance of prions in the cytosol of affected neurons and could be applied for the treatment of prion and other related protein-misfolding diseases. Figure 1. PrioV3 binds to both native and denatured prion proteins. (A) PrioV3 antibody was screened with 20-mer amino acid sequences spanning the 91–230 region of the prion protein and was shown to bind a region between 170 and 19. ICSM18 that binds to a region between 141 and 160 was used as positive control and normal camel serum (NCS) was used as negative control. Goat anti-llama IgG-HRP was used as secondary detection antibody. Anti-PrP responses were measured in peptide ELISA. Error bars represent the mean antibody level derived from n = 8 wells. (B) PrioV3 antibody bound to native PrPC inside the cytoplasm of N2a cells (orange arrow), in contrast with ICSM35 that strictly stained cell membrane- bound PrPC (blue arrow). Florescence microscopy was performed and images from each source [FITC (450–490 nm), Texas red (510–560 nm) and DAPI (330–380 nm)] were collected. As control, N2a cells were stained with the secondary anti-llama IgG (green) and anti-mouse IgG antibody (red), omitting the primary antibodies. Scale bar = 25 mm (C) PrP wild type, knock-out and RML-infected brain homogenate were digested with PK and tested for reactivity PrioV3. PrioV3 strongly bound both PrPC and PrPSc in brain homogenates (pre- and post-PK). PrioV3 anti-prion antibody binds to prion proteins and peptide As control, N2a cells were stained with the secondary anti-llama IgG (green) and anti-mouse IgG antibody (red), omitting the primary antibodies. Scale bar = 25 mm (C) PrP wild type, knock-out and RML-infected brain homogenate were digested with PK and tested for reactivity PrioV3. PrioV3 strongly bound both PrPC and PrPSc in brain homogenates (pre- and post-PK). For comparison, ICSM35, raised in mouse (positive), was incubated with brain homogenates prepared from RML-infected and normal brain tissue with and with no PK digestion. doi:10.1371/journal.pone.0009804.g001 March 2010 \| Volume 5 \| Issue 3 \| e9804 PLoS ONE \| www.plosone.org 2 In Vitro Prion Treatment We have reported that PrPC was widely expressed on the surface of N2a cells when probed with anti-prion mAbs raised against recombinant prion protein [11,28]. Immunofluorescence analysis of PrioV3 antibody displayed strong binding to cellular prion protein expressed on the cell plasma membrane, suggesting its high affinity for PrPC (Figure 1B). Furthermore, co-localisation studies have shown that binding with PrioV3 was also recognised in the cell cytoplasm by immunofluroscence following staining of intact N2a cells (non-fixed and non-permebealised), unlike ICSM35-stain that was strictly restricted to the plasma membrane (Figure 1B). Therefore, PrioV3 binds both surface and cytosolic PrPC and recognizes residues 171 and 190 on the C-terminal region of the truncated PrP27–30. Following PK digestion, PrPC was completely removed, whereas the 33–35 kDa form of PrPSc was shortened to 27–30 kDa, probably as a result of degradation of the amino-terminal segment of residues 23–90 analogous to hamster PrPSc [29]. PrioV3 antibody did not react with Prn-p2/2 brain homogenates. Therefore, PrioV3 was shown to binds both PrPC and PrPSc whether the protein is in native conformation or denatured. PrioV3 antibody is capable of crossing the BBB in vitro and in vivo Transport of PrioV3 and ICSM35 antibodies across the blood- brain barrier was assessed using GPNT rat [30] and HCMEC/D3 human [31] endothelial cells. PrioV3 was shown to cross both BBB cell lines efficiently as measured by ELISA (Figure 2A). In contrast, CD71 antibody, Dextran and ICSM35 antibody were unable to enter the BBB cell lines (Figure 2A). Furthermore, immunohisto- chemistry and immunofluorescence stain of the brain parenchyma of rats following intra-venous injection revealed that PrioV3 antibody was widely distributed, in contrast, ICSM35 did not To assess the ability of PrioV3 antibody to recognise PrPSc, scrapie-infected (RML) or Prn-p2/2 (KO) brain homogenate was treated with PK (QIAGEN) (Figure 1C). Western blot studies displayed similar binding pattern between PrioV3 and ICSM35; with several PrPC bands from normal mice comprising one band at 27 kDa, two 29–32 kDa and 33–35 kDa, and one at 21–22 kDa. Figure 2. Assessment of PrioV transmigration across the BBB in vitro and in vivo. (A) BBB migration of PrioV3 antibody was assessed using GPNT rat and D3 human BBB models. PrioV was shown to cross both BBB cell lines efficiently as measured by ELISA, in contrast with CD71 antibody, Dextran and ICSM35 (con-IgG) that did not cross the BBB; (B) Immunohistochemistry and immunofluorescence stain of PrioV3 (green) in the brain parenchyma following intra-venous injection. PrioV3 antibody was injected i.v. to rats and brain sections were taken 4 h later to assess antibody migration across the BBB in vivo (white arrows). Co-injection with a conventional anti-prion IgG (red) displayed its inability to cross the BBB and staining remained in the kidney (yellow arrow). Scale bar = 5 mm. doi:10.1371/journal.pone.0009804.g002 Figure 2. Assessment of PrioV transmigration across the BBB in vitro and in vivo. (A) BBB migration of PrioV3 antibody was assessed using GPNT rat and D3 human BBB models. PrioV was shown to cross both BBB cell lines efficiently as measured by ELISA, in contrast with CD71 antibody, Dextran and ICSM35 (con-IgG) that did not cross the BBB; (B) Immunohistochemistry and immunofluorescence stain of PrioV3 (green) in the brain parenchyma following intra-venous injection. PrioV3 antibody was injected i.v. to rats and brain sections were taken 4 h later to assess antibody migration across the BBB in vivo (white arrows). Co-injection with a conventional anti-prion IgG (red) displayed its inability to cross the BBB and staining remained in the kidney (yellow arrow). Scale bar = 5 mm. PrioV3 antibody raised against PrP-Dynabeads inhibits accumulation of PrPSc in ScN2a Cells We first assessed the efficiency of PrioV3, an anti-prion mAb antibody raised in camel against PrP-Dynabeads, as an inhibitor of prion replication in ScN2a cells. We have previously shown that treatment of ovine PrP- inducible scrapie-infected Rov cells with a panel of the ICSM mAbs raised against both recombinant a-PrP and b-PrP led to significant reduction of PrPSc accumulation. It was determined that ICSM35 was the most effective in inhibiting PrPSc replication (x 100 to 1500) following treatment of Rov cells over a period 6 weeks [11]. This also suggests that PrioV3 antibody is a more potent inhibitor since it was shown to abrogate PrPSc replication after 24 hours only. In disagreement with previous work [11], PrioV3 antibody did alter PrPC expression, independent of the dose or treatment length (Figure 3 and data not shown), indicating that the antibody has a direct neutralizing effect on PrPC as well as PrPSc. Of note, mAbs with higher affinity for PrPC were shown to be more effective in reducing PrPSc replication in a dose dependent manner [12,13,14]. ScN2a cells were cultured in triplicates and treated for 24 hours or 4 days at 37uC (5% CO2) with PrioV3 antibody. For the 4 day experiment, treatment was renewed daily in order to maintain levels of antibody above PrPC levels, since PrPC has a rapid turnover rate (half-time synthesis 0.1 hour vs. half-time degrada- tion 5 hours) [32]. Normal camel serum (NCS) and no treatment were used as controls. The cells were then lysed and subsequently treated with PK prior to analysis of PrPSc levels by Sandwich ELISA and Western blot. For comparison, ScN2a cells were also treated with 1 or 25 mg ICSM35, an anti-prion mAb antibody raised against human recombinant b-PrP (Figure 3). ICSM35 binds residues 91 and 110 on the N-terminal region of the truncated PrP27–30 and was shown to be useful in delaying disease onset albeit leading to incomplete inhibition of PrPSc replication in the spleen of scrapie-infected mice [14,33]. ICSM35 was also previously shown to disturb cholesterol homeostasis and alter cell signaling (M. Tayebi and M. David, submitted). An isotype matched control for ICSM35, BRIC126 was also used and untreated cells were included in the experiment (Figure 3). ScN2a cells were also treated with 25 mg PrioV3 or ICSM35 over a period of 4 days (Figure 4) with daily treatment renewal in tissue culture medium. PrioV3 antibody is capable of crossing the BBB in vitro and in vivo doi:10.1371/journal.pone.0009804.g002 PLoS ONE \| www.plosone.org March 2010 \| Volume 5 \| Issue 3 \| e9804 3 In Vitro Prion Treatment replication as demonstrated by Sandwich ELISA [(P,0.001), (Figure 3)]. Although 25 mg/ml of PrioV3 antibody led to a significant decrease of PrPSc as assessed by Sandwich ELISA, levels did not differ significantly from the 1 mg/ml of PrioV3 antibody treatment, suggesting the high potency of PrioV3 antibody in inhibiting PrPSc replication [(P,0.001), (Figure 3)]. display staining of the brain parenchyma but was seen in kidneys and liver of these rats (Figure 2B). PrioV3 antibody raised against PrP-Dynabeads inhibits accumulation of PrPSc in ScN2a Cells NCS and BRIC126 treatment were used as controls. Following treatment, cells were lysed and then PK treated as described above. Levels of PrPSc were then analysed in the cell lysates by Sandwich ELISA and Western blotting. Both PrioV3 antibody and ICSM35 completely abrogated PrPSc replication as shown by Western blot. NCS and BRIC126 had no effect on PrPSc levels (Figure 4). As expected, ICSM35 led to a significant dose-dependent reduction in PrPSc accumulation after 24 hour treatment (Figure 3). When compared with PrioV3 antibody, ICSM35 was less potent in inhibiting PrPSc replication in ScN2a cells (Figure 3). ScN2a cells were treated with 1 mg or 25 mg PrioV3 antibody. Here, as little as 1 mg/ml of PrioV antibody abrogated PrPSc It has previously been reported that treatment for three [13] or seven [12] days with anti-PrPC antibodies abolished PrPSc replication as demonstrated by undetectable levels in Western blot, in agreement with the present findings. Figure 3. PrioV3 and ICSM35 antibodies inhibit accumulation of PrPSc in ScN2a cells. ScN2a cells were incubated for 24 hours at 37uC (5% CO2) with 1 or 25 mg PrioV3 and ICSM35. 25 mg NCS (Normal camel serum), and BRIC126-treated as well as untreated cells were also included. The cells were then lysed and subsequently treated with PK prior to analysis of PrPSc levels by Sandwich ELISA in triplicates. Both concentrations of PrioV3 used to treat ScN2a cells show a significant reduction in prion replication (P,0.001) when compared with the NCS control and the untreated cells. PrioV3 has also led to decreased levels of normal prion protein. ICSM35 led to a significant (P,0.001) reduction in prion replication compared to BRIC126-treated or untreated cells. Representative of three experiments. doi:10.1371/journal.pone.0009804.g003 Figure 3. PrioV3 and ICSM35 antibodies inhibit accumulation of PrPSc in ScN2a cells. ScN2a cells were incubated for 24 hours at 37uC (5% CO2) with 1 or 25 mg PrioV3 and ICSM35. 25 mg NCS (Normal camel serum), and BRIC126-treated as well as untreated cells were also included. The cells were then lysed and subsequently treated with PK prior to analysis of PrPSc levels by Sandwich ELISA in triplicates. Both concentrations of PrioV3 used to treat ScN2a cells show a significant reduction in prion replication (P,0.001) when compared with the NCS control and the untreated cells. PrioV3 has also led to decreased levels of normal prion protein. Figure 4. Long term treatment with PrioV3 or ICSM35 anti-prion antibodies abrogate PrPSc accumulation in ScN2a cells. ScN2a cells were incubated for 4 days at 37uC (5% CO2) with 25 mg ICSM35 or of PrioV3 and daily treatment renewal in tissue culture medium. BRIC126- and NCS- treated (Normal camel serum) were also included. The cells were then lysed and subsequently treated with PK prior to analysis of PrPSc levels by Western blot. Both PrioV3 and ICSM35 abrogated PrPSc replication when compared with the BRIC- and NCS-treated cells. Representative of three experiments. doi:10.1371/journal.pone.0009804.g004 medium for another 3 days and levels of PrPSc were assessed by Western blot following PK digestion of cell lysates. In preliminary work, we assessed efficacy of our antibodies to inhibit prion replication in vivo. Here, mice were inoculated with RML scrapie brain homogenate and treated weekly with 2 mg of PrioV3 or isotype control antibody CD71 (IgG) from day 10 post inoculation (p.i.). Western blot analysis of PrPSc at 60 days p.i. showed a marked inhibition of PrPSc accumulation in the spleen (Figure 5 and data not shown). This pilot study confirms the inhibitory effects of PrioV3 on PrPSc inhibition in vivo and experiments are currently underway to assess the effects of PrioV3 on clinical disease in mice. Cell lysates assayed 3 days after removal of PrioV3 antibody failed to display detectable levels of PrPSc as demonstrated by Western blot (Figure 6A) in contrast to ICSM35-treated cells that displayed high expression levels of PrPSc (Figure 6B). Both untreated as well as cells treated with isotype-matched BRIC126 antibody showed high levels of PrPSc replication. Furthermore, 3 days after treatment was discontinued, cell lysates and superna- tants were screened for the presence of PrioV3 or ICSM35 by ELISA. Both PrioV3 and ICSM35 were not detected indicating that PrioV3 inhibitory effect was not the result of residual antibody in the cells but to its potent effect on PrPSc (data not shown). Taken together, these results suggest that only PrioV3 antibody led to permanent depletion of PrPSc in chronically infected N2a cells. Recurrence of PrPSc replication in ICSM35- but not PrioV3-treated cells We have also assessed whether PrPSc accumulation recurs after treatment was ceased in PrioV3- and ICSM35-treated ScN2a cells. ICSM35 but not PrioV3 antibody leads to neurotoxicity PrPC appears to play a crucial role in the process leading to neuronal death [34] as neurotoxic effects associated with prion diseases are not triggered in the absence of PrPC expression [35,36]. Furthermore, a role in signal transduction for PrPC was ScN2a cells were treated over a period of 4 days with daily treatment renewal of antibody in tissue culture medium with 25 mg of PrioV3 or NCS (Figure 6A) and ICSM35 or BRIC126 (Figure 6B). Untreated cells were also included in the experiment. Subsequently, cells were treated with antibody-free tissue culture Figure 5. PrioV3 antibody inhibits PrPSc replication in spleens of antibody-treated mice. Weekly passive transfer of 2 mg PrioV3 antibody for 60 days induced substantial reduction in splenic PrPSc levels when treatment was started at day 10 p.i. CD71-treated as well as untreated mice did not inhibit splenic prion replication and this reduction was not observed. doi:10.1371/journal.pone.0009804.g005 PrioV3 antibody raised against PrP-Dynabeads inhibits accumulation of PrPSc in ScN2a Cells ICSM35 led to a significant (P,0.001) reduction in prion replication compared to BRIC126-treated or untreated cells. Representative of three experiments. doi:10.1371/journal.pone.0009804.g003 March 2010 \| Volume 5 \| Issue 3 \| e9804 PLoS ONE \| www.plosone.org 4 In Vitro Prion Treatment In Vitro Prion Treatment Figure 4. Long term treatment with PrioV3 or ICSM35 anti-prion antibodies abrogate PrPSc accumulation in ScN2a cells. ScN2a cells were incubated for 4 days at 37uC (5% CO2) with 25 mg ICSM35 or of PrioV3 and daily treatment renewal in tissue culture medium. BRIC126- and NCS- treated (Normal camel serum) were also included. The cells were then lysed and subsequently treated with PK prior to analysis of PrPSc levels by Western blot. Both PrioV3 and ICSM35 abrogated PrPSc replication when compared with the BRIC- and NCS-treated cells. Representative of three experiments. doi:10.1371/journal.pone.0009804.g004 Since they are devoid of the light chains, the antigen binding site of camelid antibodies is limited to a single domain. Camelid antibodies are tools of increasing interest for CNS therapy [40,41,42] and biophysical studies have revealed that they possess a number of unique features compared to those of conventional antibody fragments, notably smaller size (,14 kDa), greater solubility and higher stability [40]. In that context, we have produced a camelid anti-prion antibody fragment, known as PrioV3, able to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrPC (Tayebi et al submitted). suggested as binding to anti-PrP antibodies led to PrPC-dependent Fyn activation [37] and modulation of calcium-dependent protein kinase C in mice [38]. Following a recent report by Solforosi and colleagues showing that cross-linking PrPC with specific anti-prion antibodies leads to neuronal apoptosis in vivo [26], we have shown that ICSM35 antibody treatment of neurons alters cell membrane fluidity and leads to disruption of signalling proteins that reside in membrane domains (M. Tayebi and M. David, submitted). To determine whether cross-linking PrPC to PrioV3 antibody could produce neurotoxic effects in vitro, DNA fragmentation was evaluated in N2a cells following treatment with varying concen- trations (0, 5 and 25 mg) of endotoxin-free PrioV3 or ICSM35 antibody. This study showed that ICSM35 caused DNA fragmentation as shown by TUNEL stain in a dose-dependent manner (Figure 7C & D). In contrast, PrioV3 antibody failed to trigger any neurotoxic effect as demonstrated by TUNEL staining by immunofluorescence, even at the highest concentration (Figure 7E & F). Interestingly, only antibodies that recognize epitopes within the 91 to 110 region of PrP are capable of triggering neurotoxic-associated effects following cross-linking with PrPC ([26], [M. Tayebi and M. David, submitted]). While PrioV3 antibody, that binds to a PrP sequence between 171 and 190, failed to elicit neurotoxic effects. In this report, we show that PrioV3 binds to both PrPC and PrPSc and effectively abrogates prion replication in ScN2a cells, but it is unclear whether its inhibitory effects are due to recognition of normal and/or disease-associated isoform of prion protein. However, it was reported that PrPC cleavage with phosphatidy- linositol-specific phospholipase C from the cell surface [13] or stabilization with specific antibodies of PrPC alone is sufficient to inhibit PrPSc replication [12,13,14]. Figure 5. PrioV3 antibody inhibits PrPSc replication in spleens of antibody-treated mice. Weekly passive transfer of 2 mg PrioV3 antibody for 60 days induced substantial reduction in splenic PrPSc levels when treatment was started at day 10 p.i. CD71-treated as well as untreated mice did not inhibit splenic prion replication and this reduction was not observed. doi:10.1371/journal.pone.0009804.g005 March 2010 \| Volume 5 \| Issue 3 \| e9804 March 2010 \| Volume 5 \| Issue 3 \| e9804 March 2010 \| Volume 5 \| Issue 3 \| e9804 PLoS ONE \| www.plosone.org 5 In Vitro Prion Treatment Figure 6. PrioV3 but not ICSM35 anti-prion antibody permanently inhibits accumulation of PrPSc in ScN2a cells following cessation of antibody treatment. ScN2a cells were incubated for 4 days at 37uC (5% CO2) with 25 mg of (A) PrioV3 and (B) ICSM35* and daily treatment renewal in tissue culture medium. NCS- (Normal camel serum) and BRIC126-treated cells were also included. The cells were then further grown for another three with antibody-free tissue culture medium. The cells were then lysed and subsequently treated with PK prior to analysis of PrPSc levels by Western blot. PrioV3 treatment led to permanent inhibition of PrPSc replication but ICSM35-treated cultures displayed de-novo replication of PrPSc; both the BRIC- and NCS-treated cells showed high levels of PrPSc. Representative of three experiments. doi:10.1371/journal.pone.0009804.g006 Figure 6. PrioV3 but not ICSM35 anti-prion antibody permanently inhibits accumulation of PrPSc in ScN2a cells following cessation of antibody treatment. ScN2a cells were incubated for 4 days at 37uC (5% CO2) with 25 mg of (A) PrioV3 and (B) ICSM35* and daily treatment renewal in tissue culture medium. NCS- (Normal camel serum) and BRIC126-treated cells were also included. The cells were then further grown for another three with antibody-free tissue culture medium. The cells were then lysed and subsequently treated with PK prior to analysis of PrPSc levels by Western blot. PrioV3 treatment led to permanent inhibition of PrPSc replication but ICSM35-treated cultures displayed de-novo replication of PrPSc; both the BRIC- and NCS-treated cells showed high levels of PrPSc. Representative of three experiments. doi:10.1371/journal.pone.0009804.g006 Camelids generate functional antibodies consisting of only two heavy chains in comparison with the conventional four-chain antibodies; and differs from the corresponding regions of conventional antibodies in that they lack the CH1 domain [39]. Furthermore, Perrier and colleagues [43] demonstrated that anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation. PLoS ONE \| www.plosone.org Discussion The conversion of normal prion protein (PrPC) into disease- associated PrPSc is a central event underlying the mechanisms of neuronal degeneration in prion diseases. Therefore, targeting both normal and disease-associated isoforms should form the basis for the development of effective therapeutic strategies for prion disorders. We also show in this report that immunidetection of PrPSc from spleens of mice at 60 days p.i. revealed that treatment with PrioV3, but not with CD71 antibody markedly inhibited PrPSc accumu- lation when passive transfer started at day 10. This is in agreement with previous reports [14] demonstrating efficacy of a mouse mAb raised against alpha recombinant protein (i.e. ICSM18) in abrogating splenic prion replication following ip inoculation with RML. Of importance, mice treated with ICSM18 survived indefinitely [14]. Given that human prion diseases are normally diagnosed after onset of disease, it is crucial to develop compounds that can effectively arrest/reverse the disease process even after symptoms have began. Although, antibody-mediated therapy was shown to be effective for the treatment of rodent prion disease, intraperi- toneal passive transfer of anti-PrP mAb did not have a protective effect following CNS invasion/inoculation of prions [14]. Due to their relatively large molecular mass, these anti-PrP mAb were unable to transfer into the CNS across the blood-brain barrier when administered peripherally. Previous reports have shown that binding of anti-PrP antibodies to helix 1 region of PrPC, which is believed to play a crucial role in the conversion process [44,45] inhibits PrPSc accumulation in ScN2a cells by preventing interaction of PrPC with PrPSc [12,13]. However, these antibodies were less efficient in clearing prion infection when used for the treatment of Rov cells [11,12]. Thus, antibodies that bind to a-helix domain are able to abrogate prion March 2010 \| Volume 5 \| Issue 3 \| e9804 6 In Vitro Prion Treatment Figure 7. ICSM35 but not PrioV3 anti-prion antibody leads to neurotoxicity N2a cells following antibody treatment. N2a cells were assessed for neurotoxic effects by immunofluorescence imaging following treatment with PrioV3 or ICSM35. (A) Proprietary positive TUNEL stain and (B) antibody-free tissue culture medium were used as controls. Following treatment with 5 and 25 mg PrioV3 or ICSM35, TUNEL staining was only observed when cells were treated with ICSM35 (C & D). PrioV3 did not elicit DNA fragmentation and toxic effects were not observed (E & F). Cell nuclei were also stained with DAPI. Scale bar = 5 mm. PLoS ONE \| www.plosone.org Discussion In contrast, ICSM35 failed to prevent recurrence of the PrPSc replication in PLoS ONE \| www.plosone.org 7 March 2010 \| Volume 5 \| Issue 3 \| e9804 In Vitro Prion Treatment beads, was then rinsed and resuspended in PBS before immunisation. antibodies that target the N-terminal but not the C-terminal region of the prion protein led to cell membrane disruption in vitro (M. Tayebi and M. David, submitted) and neurotoxic effects in vivo [26]. Whether the binding property of these antibodies to specific sequences is enough to explain these outcomes remain to be resolved. PrioV3 antibody could potentially turn out to be a more useful therapeutic tool for eradicating prion infection since it binds a more C-terminal region of the protein and targets both isoforms of the prion protein. In this case, PrioV3 antibody would be able to block PrPC incorporation into propagating infectious agents and at the same time neutralize the infectious template, PrPSc. Three adult male camels received six subcutaneous injections at weekly intervals of PrP-Dynabeads. For the first injection the antigen was mixed with an equal volume of Freund’s Complete Adjuvant (FCA), and all subsequent boosts were with Freund’s Incomplete Adjuvant (FIA). Forty-five days after the first injection, 50 ml of anticoagulated blood was collected to evaluate the immune response and used to isolate lymphocytes for mRNA extraction [49]. mRNA extraction was followed by cDNA preparation and the final PCR fragments were ligated into a phagemid vector as reported [49]. Ligated material was trans- formed in Escherichia coli cells and specific antibodies against prion proteins (PrioVs) were enriched by three consecutive rounds of in vitro selection. PrioV3 antibody was used in the current study. PrioV3 antibody displayed extensive and diffuse binding of PrPC in the cytoplasm of cells without permebealization. PrioV3 ability to enter and target the cytosol of neurons remains unknown, but we speculate that this is due to the size of the antibody fragment (,14 kDa) as ICSM35 (,175 kDa) was unable to enter the cell. ICSM anti-PrP mAbs (D-Gen) were produced as described [33]. ICSM 18, an IgG1, binds PrPC strongly in its native state but weakly to native PrPSc, and to both isoforms in Western blots. Its epitope lies between codons 144–156. ICSM 35, an IgG2b binds both PrPC and PrPSc from all species tested, whether the protein is in a native conformation or denatured. Its epitope lies between residues 91–110. Peptides and pepscan ELISA 20-mer peptides spanning the truncated prion protein and overlapping by 10 amino acids were made by automated solid phase step-wise synthesis using the Fmoc N terminal protection chemistry as described [48]. Briefly, peptides were cleaved from the solid phase and fully side-chain deprotected using trifluor- oacetic acid with water and tri-isopropylsilane as scavengers. Cleaved peptides were precipitated and washed in ice-cold methyl tertiary butyl ether, dried, dissolved in suitable aqueous solvents and analysed by reverse phase HPLC and MALDI-TOF mass spectrometry. The mechanism(s) of prion-mediated neuronal degeneration are not fully understood but it is believed that this is caused by increased PrPSc neurotoxicity, so-called ‘‘gain of function hypoth- esis’’ as prn-p2/2 mice displayed no obvious phenotype [47], or by loss of PrPC function. It is likely that the apoptotic phenomenon observed after cross-linking of PrPC with ‘toxic’ antibodies mimics the prion-mediated degenerative process and leads to antibody- mediated loss of function. The favourable efficiency and toxicity property observed for PrioV3 together with its ability to cross the BBB and to enter the plasma membrane of affected neurons makes it an attractive weapon in the fight against prion and other related diseases. High binding, 96 well plates (Greiner) were coated with 50 ml/ well of a 10 mg/ml peptide solution in coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6). The plates were incubated for 1 hour at 37uC then washed 3 times with PBS-0.05% Tween 20, and then blocked with SuperBlock (SB; Pierce) for 1 hour at room temperature. After decanting the SB, 50 ml of PrioV3 antibody diluted in PBS-0.05% tween 20 was added and incubated for 1 hour at 37uC. The plates were then washed 3 times with PBS-0.05% tween and a 1/1000 dilution of horseradish-peroxidase (HRP) conjugated goat anti-llama IgG (Bethyl Laboratories) was added for 25 min at 37uC and the plates were again washed 4 times with PBS-0.05% tween. Finally, the plates were developed with OPD buffer (Sigma) until optimum development occurred, when the reaction was stopped with 3 M sulphuric acid prior to spectrophotometric reading at 490 nm. Discussion Although many studies addressed the question of whether antibodies could inhibit PrPSc replication in vitro and in vivo, previous reports have demonstrated that cross-linking anti-prion antibodies with PrPC leads to deleterious effects on neurons in vivo [26] and in vitro (M. Tayebi and M. David, submitted). Solforosi and co-workers used antibodies that specifically bind to epitopes within the N-terminal (95–105) and the C-terminal (133–157) regions of the prion protein. Interestingly, antibodies directed against codons within the 95–105 but not to the 133–157 the region led to apoptosis [26], while antibodies that target a more C- terminal region failed to display neurotoxic effects in prion infected mice and cells [18,26]. Similarly, the current study showed that PrioV3 antibody which also binds to the C-terminal (71–190) region of the prion protein did not trigger neuronal apoptosis. Discussion Representative of three experiments. doi:10.1371/journal.pone.0009804.g007 Figure 7. ICSM35 but not PrioV3 anti-prion antibody leads to neurotoxicity N2a cells following antibody treatment. N2a cells were assessed for neurotoxic effects by immunofluorescence imaging following treatment with PrioV3 or ICSM35. (A) Proprietary positive TUNEL stain and (B) antibody-free tissue culture medium were used as controls. Following treatment with 5 and 25 mg PrioV3 or ICSM35, TUNEL staining was only observed when cells were treated with ICSM35 (C & D). PrioV3 did not elicit DNA fragmentation and toxic effects were not observed (E & F). Cell nuclei were also stained with DAPI. Scale bar = 5 mm. Representative of three experiments. doi:10.1371/journal.pone.0009804.g007 ICSM35-treated neuronal cultures [11]. Bioassay of infectivity from these lysates is in progress. S replication in a murine neuroblastoma cell line (ScN2a; [12]), whereas, their ability to cure an ovine epithelial cell line (Rov cells) infected with prion was shown to be poor [11]. More importantly, ICSM35, which displays higher affinity for PrPSc, also failed to completely rid scrapie-infected mice [14] and ScN2a cells (this study) of PrPSc, but led to efficient prion inhibition in Rov cells [11]. Therefore, anti-PrP mAbs should be chosen on the basis that they selectively target species-specific prion strains. Mechanisms underlying differential outcome of PrPSc inhibition following treatment with anti-PrP antibodies remain unknown and clearly need to be further investigated, since antibodies thought to be effective in eradicating prion infection could in fact only suppress disease transiently. It could be speculated that variable levels of PrPC expression in different cell lines/types would lead to diverse inhibitory effects [11,13]. Further, differences in the binding affinities of antibodies used to inhibit PrPSc replication could also help to explain the difference in treatment outcome, given that PrPC is ubiquitously expressed [46] and, in some cases, higher concentrations would be needed to achieve complete abrogation of prion replication. Binding specific motifs on the prion protein could also explain this dichotomy; for instance, The PrioV3 antibody was able to completely abrogate PrPSc replication in prion-permissive neuronal cultures even after treatment was ceased. This is in agreement with Enari and colleagues that demonstrated that ScN2a exposure to an anti-PrPC mAb not only prevented infection but also cured chronically scrapie-infected cells after treatment was ceased [13]. Production of anti-prion camelid antibodies All procedures involving animals were carried out under a project and personal licence authority issued in accordance with The Animals (Scientific Procedures) Act 1986 and approval by the Royal Veterinary College Institutional Ethics Committee. PLoS ONE \| www.plosone.org Western blot analysis Samples to be electrophoresed were diluted 1:1 in 40 ml sample buffer and boiled for 5 min in eppendorf tubes. The samples were spun for 5 seconds at 14.000 rpm in a microfuge before being loaded on the gel. The gels were electrophoresed at a constant voltage of 200 V for 1 hour. Following electrophoresis, gels were blotted onto PVDF (Invitrogen) at 18 V for 45 min. Following blotting, the membranes were rinsed in PBS-tween (0.05%) before being transferred to blocking solution for 60 min at room temperature. The membranes were again rinsed in PBS-tween (0.05%) to remove all traces of blocking solution. PrioV3, ICSM35 or ICSM18 antibody was added and incubated for 1 hour at room temperature. Following 4 washes of 5 min each, the membranes were then incubated in anti-mouse or anti-llama IgG HRP- conjugated antibody diluted at 1 in 20,000. The membranes were washed as above and developed using the Hybond- chemiluminescence (ECL) system (GE Healthcare), according to the manufacturer’s instructions. Signal development times ranged from 1 second to 30 min. Florescence microscopy was performed with a Leica DM4000B microscope. Images from each source [FITC (450–490 nm), Texas red (510–560 nm) and DAPI (330–380 nm)] were collected by a high resolution DC500 colour camera attached. All images are saved digitally using Leica’s IM500 Image Manager Database software from the same field-of-view. Images were merged using Photoshop 6.0 (Adobe). Assessment of neurotoxicity by TUNEL staining Assessment of neurotoxicity by TUNEL staining Apoptosis was assessed by TUNEL staining using an apoptosis KIT according to manufacturing instructions (Calbiochem). DNA fragmentation was evaluated in ScN2a cells following treatment with varying concentrations of PRIOV3 and ICSM35. Briefly, cells were fixed with 50 ml of LEUCOPERM Reagent A (Serotec) for 10 minutes at room temperature. Cells were then washed with PBS and 100 ml of 5 x TdT Equilibrium Buffer was added (1:5 TdT: H2O) and left to incubate for 10 min. After removing the solution, 30 ml of 1:20 Fluorescein Labelling Reaction Mix: TdT Enzyme was added and incubated for 1 hour at 37uC. The slides were then washed 4 times with PBS for 15 minutes prior to adding 15 ml of DAPI nuclear stain (1:600 DAPI: PBS). The cells were viewed with a LEICA DM4000B fluorescence microscope equipped with a Bio-Rad MRC1024 laser confocal scanning system and a LEICA DC500 camera. Images were layer- combined with Adobe Photoshop. To assess recurrence of PrPSc replication following removal of antibody, 16104 ScN2a cells were incubated for 4 days with daily replacement of antibody-containing tissue culture medium as described above. Following treatment stoppage, ScN2a cultures were continuously fed for another 3 days with tissue culture medium without antibody supplement. Cells were then treated with PK and analysed for the presence of PrPSc by Sandwich ELISA and Western blot. Immunofluorescence staining and imaging g g g For subsequent immunofluorescence staining with anti-prion antibodies (PrioV3 and ICSM35 antibodies), N2a cells were first seeded on glass coverslips in 35-mm dishes and grown to 50% confluence at 37uC in a humidified atmosphere of 5% CO2/95% on air. Coverslips were then rinsed three times in TBS (1 M tris- HCl, 1.5 M NaCl, pH 7.5) and 100 ml of blocking buffer [(1% (v/ v) FBS, 1% BSA (w/v) in TBS] was added. The coverslips were incubated with 100 ml PrioV3 and/or ICSM35 for 1 hour at RT followed by the secondary antisera diluted in TBS [(anti-llama IgG FITC-conjugate, Bethyl; anti-mouse IgG Texas red-conjugate, Sigma)] for 1 hour at RT. After the final wash in TBS, the coverslips were mounted in fluorescence anti-fade solution (Invitrogen) and sealed with clear nail polish to prevent dehydration. 4, 6-dimidino-2-phenylindole (DAPI) (Sigma) was diluted to 2 mg/ml in fluorescence anti-fade solution for nuclear staining and added to coverslips. Tranmigration studies across the blood-brain barrier Rat and human brain microvascular endothelial cell lines [30,31] were used in vitro to assess trancytosis of both PrioV3 and ICSM35 antibodies. The brain microvascular endothelial cell lines were seeded on collagen-coated Falcon tissue culture inserts in the top chamber (1 mm pore size). The bottom chamber of the insert assembly contained appropriate medium (with no cells). The ability of PrioV3 antibody to transmigrate across the in vitro BBB model was assessed by adding 10 or 25 mg of the antibody into the upper compartment and determining the concentration in 200 ml aliquots from the bottom chamber at specified times using by ELISA. Preparation of Dynabead-adsorbed antigen for immunisation was performed as described [48]. Briefly, RML-infected brains from terminally ill scrapie-infected wild type FVB/N mice were used for the preparation of Dynabead-adsorbed antigen [48]. Sheep anti-mouse IgG-coupled immunomagnetic Dynabead particles (Invitrogen) were resuspended with an excess of IgG mAb anti-prion antibody (1–2 mg antibody/107 Dynabeads). The Dynabeads/mAb complex was incubated overnight at 4uC then washed several times with PBS to remove excess mAb. 100 ml of 1% scrapie infected brain homogenate was incubated with 16106 mAb-coated Dynabeads for at least 2 hours at room temperature. The final complex, known as PrP-Dyna- In order to investigate whether PrioV3 retains the ability to cross the BBB in vivo, rats were injected in the tail vein or via PLoS ONE \| www.plosone.org March 2010 \| Volume 5 \| Issue 3 \| e9804 8 In Vitro Prion Treatment intraventricular infusion with PrioV3 antibody or ICSM35. Four hours after injection, the animals were anesthetized. brain, liver and kidney were dissected and processed for histology. 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate and 0.2% sodium dodecyl sulphate (SDS) at 106 cells/ml. AEBSF protease inhibitor (Roche) was added to some cell extracts. Membranes were prepared by repeated passage with a Wheaton homogeniser; nuclei and large fragments were removed. Cell culture Mouse N2a neuroblastoma cultures were plated at 2–46106 in Opti-MEM medium [0.5% (W/V) glucose supplemented with 5% fetal bovine serum (FBS), 50 U/ml penicillin, 50 mg/ml strepto- mycin and 200 mM L-glutamine]. Cultures were maintained at 37uC in 5% CO2 with a change of medium every 48–72 hours. Sandwich ELISA analysis Medium binding, 96 well ELISA plates (Greiner) were coated with 50 ml of a 1 mg/ml ICSM18 antibody solution in coating buffer. The plates were incubated for 1 hour at 37uC then washed 3 times with PBS-0.05% tween, and then blocked with RF10 for 1 hour at room temperature. After decanting the RF10, cell lysates were diluted in PBS-0.05% tween with protease inhibitors (Roche Biochemicals) were added and incubated for 1 hour at 37uC. The plates were then washed 3 times with PBS-0.05% tween and a 1 mg/ml of biotinylated ICSM35 was added for 1 hour at 37uC and the plates were again washed 3 times with PBS-0.05% tween before a 1/1000 dilution of horseradish-peroxidase (HRP) conjugated anti-mouse IgG (Sigma) was added for 25 min at 37uC and the plates were again washed 4 times with PBS-0.05% tween. Finally the plates were developed with OPD buffer until optimum development occurred and the reaction was stopped with 3 M sulphuric acid prior to spectrophotometric reading at 490 nm. Treatment of ScN2a with antibody For subsequent treatment with anti-prion antibodies, ScN2a cells were plated using 6 or 24 well plates (Falcon) at 16104 cells/ well and grown at 37uC in 5% CO2 for a minimum of 24 hours. 4 Appropriate antibody concentrations were added to 16104 ScN2a cells, and left to incubate for 24 hours or 4 days at 37uC (5% CO2). For the 4 day treatment, ScN2a cultures, antibody- containing tissue culture medium was added daily. The cells were then removed from the wells and centrifuged at 800 rpm for 5 minutes. The pellet was lysed with 50 ml lysis buffer followed by 1 mg/ml Proteinase K (PK; Roche) treatment for 25 minutes at 37uC. AEBSF protease inhibitor (Roche) was then added to inhibit PK activity. Cells were then stored at 220uC until further use. Levels of PrPSc in the culture were then analysed by Sandwich ELISA and Western blot. PLoS ONE \| www.plosone.org References 25. Goni F, Prelli F, Schreiber F, Scholtzova H, Chung E, et al. (2008) High titers of mucosal and systemic anti-PrP antibodies abrogate oral prion infection in mucosal-vaccinated mice. Neuroscience 153: 679–686. 1. Prusiner SB (1994) Biology and Genetics of Prion Diseases. Annual Review of Microbiology 48: 655–686. Microbiology 48: 655–686. 2. Prusiner SB (1998) Prions. Proceedings of the National Academy of Sciences of the United States of America 95: 13363–13383. 26. Solforosi L, Criado JR, McGavern DB, Wirz S, Sanchez-Alavez M, et al. (2004) Cross-linking cellular prion protein triggers neuronal apoptosis in vivo. Science 303: 1514–1516. 3. Tayebi M, Bate C, Hawke S, Williams A (2007) A role for B lymphocytes in anti- infective prion therapies? Expert Review of Anti-infective Therapy 5: 631–638. 27. Paramithiotis E, Pinard M, Lawton T, LaBoissiere S, Leathers VL, et al. (2003) A prion protein epitope selective for the pathologically misfolded conformation. Nat Med 9: 893–899. 4. Harris DA (1999) Cellular Biology of Prion Diseases. Clin Microbiol Rev 12: 429–444. 5. Diringer H, Ehlers B (1991) Chemoprophylaxis of scrapie in mice. J Gen Virol 72 (Pt 2): 457–460. 28. Khalili-Shirazi A, Quaratino S, Londei M, Summers L, Tayebi M, et al. (2005) Protein conformation significantly influences immune responses to prion protein. J Immunol 174: 3256–3263. 6. Forloni G, Iussich S, Awan T, Colombo L, Angeretti N, et al. (2002) Tetracyclines affect prion infectivity. Proc Natl Acad Sci U S A 99: 10849–10854. 29. Oesch B, Westaway D, Walchli M, McKinley MP, Kent SB, et al. (1985) A cellular gene encodes scrapie PrP 27–30 protein. Cell 40: 735–746. 7. Caughey B, Ernst D, Race RE (1993) Congo red inhibition of scrapie agent replication. J Virol 67: 6270–6272. 30. Regina A, Romero IA, Greenwood J, Adamson P, Bourre JM, et al. (1999) Dexamethasone regulation of P-glycoprotein activity in an immortalized rat brain endothelial cell line, GPNT. J Neurochem 73: 1954–1963. 8. Adjou KT, Privat N, Demart S, Deslys JP, Seman M, et al. (2000) MS-8209, an amphotericin B analogue, delays the appearance of spongiosis, astrogliosis and PrPres accumulation in the brain of scrapie-infected hamsters. J Comp Pathol 122: 3–8. 31. Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, et al. (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19: 1872–1874. 9. References Korth C, May BC, Cohen FE, Prusiner SB (2001) Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease. Proc Natl Acad Sci U S A 98: 9836–9841. 32. Borchelt DR, Scott M, Taraboulos A, Stahl N, Prusiner SB (1990) Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol 110: 743–752. 10. Adjou KT, Demaimay R, Lasmezas C, Deslys JP, Seman M, et al. (1995) MS- 8209, a new amphotericin B derivative, provides enhanced efficacy in delaying hamster scrapie. Antimicrob Agents Chemother 39: 2810–2812. 33. Beringue V, Mallinson G, Kaisar M, Tayebi M, Sattar Z, et al. (2003) Regional heterogeneity of cellular prion protein isoforms in the mouse brain. Brain 126: 2065–2073. 11. Beringue V, Vilette D, Mallinson G, Archer F, Kaisar M, et al. (2004) PrPSc binding antibodies are potent inhibitors of prion replication in cell lines. J Biol Chem 279: 39671–39676. 34. Chiesa R, Harris DA (2001) Prion diseases: what is the neurotoxic molecule? Neurobiol Dis 8: 743–763. 12. Peretz D, Williamson RA, Kaneko K, Vergara J, Leclerc E, et al. (2001) Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 412: 739–743. 35. Brandner S, Isenmann S, Raeber A, Fischer M, Sailer A, et al. (1996) Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 379: 339–343. 13. Enari M, Flechsig E, Weissmann C (2001) Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. Proc Natl Acad Sci U S A 98: 9295–9299. 36. Mallucci G, Dickinson A, Linehan J, Klohn PC, Brandner S, et al. (2003) Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science 302: 871–874. 14. White AR, Enever P, Tayebi M, Mushens R, Linehan J, et al. (2003) Monoclonal antibodies inhibit prion replication and delay the development of prion disease. Nature 422: 80–83. 37. Mouillet-Richard S, Ermonval M, Chebassier C, Laplanche JL, Lehmann S, et al. (2000) Signal transduction through prion protein. Science 289: 1925–1928. 38. Mazzoni IE, Ledebur HC, Jr., Paramithiotis E, Cashman N (2005) Lymphoid signal transduction mechanisms linked to cellular prion protein. Biochem Cell Biol 83: 644–653. p 15. Tayebi M, Hawke S (2006) Antibody-mediated neuronal apoptosis: therapeutic implications for prion diseases. Immunol Lett 105: 123–126. 39. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, et al. (1993) Naturally occurring antibodies devoid of light chains. Acknowledgments We would like to express our gratitude to Dr. Liam Good for critical discussions. References Nature 363: 446–448. 16. Pilon J, Loiacono C, Okeson D, Lund S, Vercauteren K, et al. (2007) Anti-prion activity generated by a novel vaccine formulation. Neurosci Lett 429: 161–164. 17. Wuertzer CA, Sullivan MA, Qiu X, Federoff HJ (2008) CNS delivery of vectored prion-specific single-chain antibodies delays disease onset. Mol Ther 16: 481–486. 40. Dumoulin M, Dobson CM (2004) Probing the origins, diagnosis and treatment of amyloid diseases using antibodies. Biochimie 86: 589–600. 18. Song CH, Furuoka H, Kim CL, Ogino M, Suzuki A, et al. (2008) Effect of intraventricular infusion of anti-prion protein monoclonal antibodies on disease progression in prion-infected mice. J Gen Virol 89: 1533–1544. 41. Abulrob A, Sprong H, Van Bergen en Henegouwen P, Stanimirovic D (2005) The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells. J Neurochem 95: 1201–1214. 19. Campana V, Zentilin L, Mirabile I, Kranjc A, Casanova P, et al. (2009) Development of antibody fragments for immunotherapy of prion diseases. Biochem J 418: 507–515. 42. Muruganandam A, Tanha J, Narang S, Stanimirovic D (2002) Selection of phage-displayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium. FASEB J 16: 240–242. J 20. Tayebi M, Collinge J, Hawke S (2009) Unswitched immunoglobulin M response prolongs mouse survival in prion disease. J Gen Virol 90: 777–782. blood-brain barrier endothelium. FASEB J 16: 240–242. 43. Perrier V, Solassol J, Crozet C, Frobert Y, Mourton-Gilles C, et al. (2004) Anti- PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation. J Neurochem 89: 454–463. 21. Heppner FL, Musahl C, Arrighi I, Klein MA, Rulicke T, et al. (2001) Prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies. Science 294: 178–182. 44. Morrissey MP, Shakhnovich EI (1999) Evidence for the role of PrP(C) helix 1 in the hydrophilic seeding of prion aggregates. Proc Natl Acad Sci U S A 96: 11293–11298. 22. Sigurdsson EM, Brown DR, Daniels M, Kascsak RJ, Kascsak R, et al. (2002) Immunization delays the onset of prion disease in mice. Am J Pathol 161: 13–17. 45. Speare JO, Rush TS, 3rd, Bloom ME, Caughey B (2003) The role of helix 1 aspartates and salt bridges in the stability and conversion of prion protein. J Biol Chem 278: 12522–12529. 23. Schwarz A, Kratke O, Burwinkel M, Riemer C, Schultz J, et al. Measuring splenic PrPSc levels following treatment of mice with PrioV3 Five FVB/N mice were inoculated via the intraperitoneal (i.p.) route with Rocky Mountain Laboratory (RML) scrapie brain homogenate derived from terminally scrapie-sick mice and treated with PrioV3 or isotype control antibody CD71 by a weekly i.p. injection (2 mg per injection) from 10 day post inoculation (p.i.). Statistical analysis Conceived and designed the experiments: MT. Performed the experi- ments: DRJ WAT MD. Analyzed the data: CB MD MT. Contributed reagents/materials/analysis tools: CB. Wrote the paper: MD MT. Conceived and designed the experiments: MT. Performed the experi- ments: DRJ WAT MD. Analyzed the data: CB MD MT. Contributed reagents/materials/analysis tools: CB. Wrote the paper: MD MT. One-way ANOVA with Dunnett’s post test was performed using GraphPad Prism version 5.00 for Windows, GraphPad Protein extraction from cells ScN2a cells were washed twice in PBS and homogenised in an extraction buffer containing 10 mM Tris-HCl, 100 mM NaCl, PLoS ONE \| www.plosone.org March 2010 \| Volume 5 \| Issue 3 \| e9804 9 In Vitro Prion Treatment Software, San Diego California USA, www.graphpad.com for statistical analysis. Measuring splenic PrPSc levels following treatment of mice with PrioV3 49. Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, et al. (2001) Beta- lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob Agents Chemother 45: 2807–2812. p 48. Tayebi M, Enever P, Sattar Z, Collinge J, Hawke S (2004) Disease-associated prion protein elicits immunoglobulin M responses in vivo. Mol Med 10: 104–111. 47. Bueler H, Fischer M, Lang Y, Bluethmann H, Lipp HP, et al. (1992) Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356: 577–582. 47. Bueler H, Fischer M, Lang Y, Bluethmann H, Lipp HP, et al. (1992) Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356: 577–582. 48. Tayebi M, Enever P, Sattar Z, Collinge J, Hawke S (2004) Disease-associated prion protein elicits immunoglobulin M responses in vivo. Mol Med 10: 104–111. References (2003) Immunisation with a synthetic prion protein-derived peptide prolongs survival times of mice orally exposed to the scrapie agent. Neurosci Lett 350: 187–189. 46. Bendheim PE, Brown HR, Rudelli RD, Scala LJ, Goller NL, et al. (1992) Nearly ubiquitous tissue distribution of the scrapie agent precursor protein. Neurology 42: 149–156. 24. Goni F, Knudsen E, Schreiber F, Scholtzova H, Pankiewicz J, et al. (2005) Mucosal vaccination delays or prevents prion infection via an oral route. Neuroscience 133: 413–421. PLoS ONE \| www.plosone.org March 2010 \| Volume 5 \| Issue 3 \| e9804 March 2010 \| Volume 5 \| Issue 3 \| e9804 10 In Vitro Prion Treatment PLoS ONE \| www.plosone.org March 2010 \| Volume 5 \| Issue 3 \| e9804 11
https://openalex.org/W2320862867	https://europepmc.org/articles/pmc4817152?pdf=render	English	null	Inherently-Forced Tensile Strain in Nanodiamond-Derived Onion-like Carbon: Consequences in Defect-Induced Electrochemical Activation	Scientific reports	2,016	cc-by	8,469	Inherently-Forced Tensile Strain in Nanodiamond-Derived Onion-like Carbon: Consequences in Defect- Induced Electrochemical Activation received: 10 November 2015 accepted: 16 March 2016 Published: 01 April 2016 We analyzed the nanodiamond-derived onion-like carbon (OLC) as function of synthesis temperature (1000~1400 °C), by high-resolution electron microscopy, electron energy loss spectroscopy, visible- Raman spectroscopy, ultraviolet photoemission spectroscopy, impedance spectroscopy, cyclic voltammetry and differential pulse voltammetry. The temperature dependences of the obtained properties (averaged particle size, tensile strain, defect density, density of states, electron transfer kinetics, and electrochemical oxidation current) unanimously coincided: they initially increased and saturated at 1200 °C. It was attributed to the inherent tensile strains arising from (1) the volume expansion associated with the layer-wise diamond-to-graphite transformation of the core, which caused forced dilation of the outer shells during their thermal synthesis; (2) the extreme curvature of the shells. The former origin was dominant over the latter at the outermost shell, of which the relevant evolution in defect density, DOS and electron transfer kinetics determined the electrochemical performances. In detection of dopamine (DA), uric acid (UA) and ascorbic acid (AA) using the OLC as electrode, their oxidation peak currents were enhanced by factors of 15~60 with annealing temperature. Their limit of detection and the linear range of detection, in the post-treatment-free condition, were as excellent as those of the nano-carbon electrodes post-treated by Pt-decoration, N-doping, plasma, or polymer. For a wide variety of the nano-carbon (n-C) materials applications, the defect control is an important issue1. While in many cases one requires defect minimization or healing, in some other cases the opposite is true. For example, the electrochemical performance of graphene, e.g. as the electrochemical electrode, is enhanced by their defects that generate mid-gap states, which accelerate the electron tunneling through the electrode-solution interface2. While the defect generations in graphene had been enabled by such post-treatments as the plasma treatments3, e-beam irradiation4, or doping5, the efforts should be minimized. Thermal activation is a desir- able alternative technique, since it might be autonomously achieved within a thermal synthesis cycle as for the nanodiamond-derived onion-like carbon (OLC)6, apart from its advantage of the simplicity and facile mass-production. Unfortunately, the defects in many n-C materials, e.g. graphene, graphene nanoribbon, carbon nanotubes, are thermally healed, rather than generated (Table S1)7–11 due to their high vacancy formation energy12 in a wide temperature range encompassing up to 3000 °C. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 Inherently-Forced Tensile Strain in Nanodiamond-Derived Onion-like Carbon: Consequences in Defect- Induced Electrochemical Activation p g p g p By contrast, the relevant reports on OLC varied depending on the adopted temperatures: defects genera- tion at relatively low temperatures13,14, in costrast to the defect healing at higher temperatures (Table S1)14–16. Some report was even contradictory to other reports (Table S1)17. Moreover, the origin of the annealing-induced defect density evolution in OLC, relevant to its electrochemical performance, is still far from clarified; here, we investigated this issue, for OLC produced by vacuum annealing of detonation nanodiamond6, at relatively low annealing temperatures of 1000–1400 °C. While the OLC had been actively investigated for the capacitive energy 1Center for Electronic Materials, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea. 2Department of Materials Science and Engineering, Yonsei University, 262 Seongsanno, Seodaemun-Gu, Seoul 120-749, Republic of Korea. 3Indo-Korea Science and Technology Center, Korea Institute of Science and Technology, Bangalore 560064, INDIA. Correspondence and requests for materials should be addressed to W.S.L. (email: wsleemk@gmail.com) Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. HR-TEM images of OLCs synthesized by annealing the detonation nanodiamond at temperatures o (a) 1000 °C, (b) 1100 °C, (c) 1200 °C and (d) 1400 °C. Figure 1. HR-TEM images of OLCs synthesized by annealing the detonation nanodiamond at temperatures of (a) 1000 °C, (b) 1100 °C, (c) 1200 °C and (d) 1400 °C. storage device, the study on its application for electrochemical detection of some important biomolecules such as dopamine (DA), uric acid (UA) and ascorbic acid (AA), is in its infant stage18. Their reported performance was relatively poor compared to those of other n-C-based electrode materials19,20, even though it was additionally post-treated by polymer18. Here we demonstrated that, even without further post-treatments, the performance of the present OLC electrode optimized within the thermal synthesis cycle, was as excellent as those of the various post-treated n-C electrodes. storage device, the study on its application for electrochemical detection of some important biomolecules such as dopamine (DA), uric acid (UA) and ascorbic acid (AA), is in its infant stage18. Their reported performance was relatively poor compared to those of other n-C-based electrode materials19,20, even though it was additionally post-treated by polymer18. Here we demonstrated that, even without further post-treatments, the performance of the present OLC electrode optimized within the thermal synthesis cycle, was as excellent as those of the various post-treated n-C electrodes. Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 Results and Discussion 10~20 HR-TEM pictures were taken for each OLC sample, from which 50–55 clearly isolated particles (See Fig. S4) were selected for the particle size measurement for each OLC sample. See Fig. S5 where the particle size distributions of each OLC samples were given. Analyte Electrode material CV: Individual detection (1 mM) CV: Simultanous detection (0.33 mM, respectively) DPV: Simultanous detection (0.33 mM, respectively) IP (μA) EP (V) IP (μA) EP (V) IP (μA) EP (V) AA OLC-1000 5 0.110 2 0.110 0.3 0.024 OLC-1100 30 − 0.018 10 0.019 0.5 − 0.011 OLC-1200 41 − 0.055 15 − 0.032 20 − 0.054 OLC-1400 42 − 0.053 15 − 0.015 21 − 0.047 DA OLC-1000 44 0.275 15 0.286 10 0.165 OLC-1100 55 0.219 20 0.212 30 0.170 OLC-1200 112 0.210 38 0.207 220.5 0.169 OLC-1400 109 0.209 37 0.208 217.5 0.173 UA OLC-1000 32 0.410 12 0.412 12 0.35 OLC-1100 43 0.355 20 0.362 22 0.319 OLC-1200 143 0.346 50 0.349 197.5 0.331 OLC-1400 143 0.355 53 0.352 195.1 0.335 Table 1. Summary of the oxidation peak currents and potentials for the detection of AA, DA and UA obtained by CV and DPV employing the OLC electrodes. Table 1. Summary of the oxidation peak currents and potentials for the detection of AA, DA and UA obtained by CV and DPV employing the OLC electrodes. annealing at 1200 °C (Fig. S6: HR-TEM picture; Fig. S7: particle size distribution), which was rather similar to the as-received nanodiamond powder (Fig. S1a). It indicated the obvious isothermal volume expansion after 1 hour annealing for the OLC-1200. It strongly suggested that the diamond core, encapsulated by the new-born outer shells, would subsequently undergo additional diamond-to-graphite transformations to generate the inner shells, of which the associated volume expansion should eventually cause the forced radial expansion of the outermost shells i.e. the forced tensile strain in tangential direction. Such process would continue until the diamond core is completely consumed. Due to the action-reaction principle, such forced radial expansion of the outer shells by the new-born inner shells would result in the corresponding radial compression of the inner shells. Such picture was supported by the radially decreasing inter-shell spacing observed at e.g. OLC-1400 (Fig. S2 and Table S2). Results and Discussion Figure 1 shows the HR-TEM pictures of the nanoparticles synthesized by annealing the detonation nanodiamond powder at various temperatures. The core-shell structures of the particles were obvious, with the diamond {111} planes at the core (with interplanar spacing of 0.21 nm) and the approximately concentric graphitic shells; their interplanar spacing varied from 0.34 nm to 0.29 nm (see for example, Fig. S2 and Table S2 for OLC-1400); they decreased with their radial distance from the particle surface, in accordance with the previous reports21. The XRD pattern (Fig. S3) also revealed the coexistence of the diamond peaks (at 2θ = 43.9, 75.3) and the graphite peak (at 2θ = 26.4), e.g. for OLC-1200 sample. The number of shells increased with temperature, at the expense of the diamond core, which almost vanished at 1400 °C (Fig. 1d, see Statement S1 for the naming of the particle). Figure 2 shows the temperature-dependence of the averaged OLC particle diameter. It initially increased with temperature and was eventually started to saturate at 1200 °C. The increasing OLC particle size was attributed to the observation that the interplanar spacing of the newly-generated graphitic shells was about 50% larger than that of the {111} diamond planes at the core, as stated above. It was further supported by the well-known fact that the graphite-to-diamond phase transformation could proceed via the puckering of the graphite basal planes to transform into diamond {111} planes, thereby reducing their interplanar spacing22; it indicated that the reverse process would increase the interplanar spacing as in the present case. Additional possible particle growth mech- anisms will be discussed later.h The consecutive temperature-dependent microstructural variations of the core-shell structure (Fig. 1) sug- gested the analogous isothermal transformation from surface to the core, as reported previously (Kuznetsov et al.23, Pech et al.6: see Fig. S1); it was further supported by the HR-TEM picture of the OLC-1200 after zero-time Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 2 www.nature.com/scientificreports/ Figure 2. The average particle size vs annealing temperature profile of the OLCs. 10~20 HR-TEM pictures were taken for each OLC sample, from which 50–55 clearly isolated particles (See Fig. S4) were selected for the particle size measurement for each OLC sample. See Fig. S5 where the particle size distributions of each OLC samples were given. Figure 2. The average particle size vs annealing temperature profile of the OLCs. Results and Discussion S8a–c and (b) DPV profiles shown in the Fig. S9. Figure 4. Strain of OLC nanoparticles estimated from the shift in 2D peak frequency of Raman spectra, relative to that of the flat multilayer graphene (MLG); the effect of the number of layers on the 2D band peak frequency, for the flat MLG, was included in the calculation30. Figure 4. Strain of OLC nanoparticles estimated from the shift in 2D peak frequency of Raman spectra, relative to that of the flat multilayer graphene (MLG); the effect of the number of layers on the 2D band peak frequency, for the flat MLG, was included in the calculation30. 1000~1200 °C, and were saturated thereafter. Figure S8d shows the CV responses of OLC electrodes from the mixed solution containing 0.33 mM of the three analytes, respectively. Oxidation peak positions did not differ much from those of their individual CV responses, which indicated that the mutual interference was not signif- icant. Furthermore, the annealing-temperature dependence of the peak currents for the simultaneous detection was similar to that of the individual detections. 1000~1200 °C, and were saturated thereafter. Figure S8d shows the CV responses of OLC electrodes from the mixed solution containing 0.33 mM of the three analytes, respectively. Oxidation peak positions did not differ much from those of their individual CV responses, which indicated that the mutual interference was not signif- icant. Furthermore, the annealing-temperature dependence of the peak currents for the simultaneous detection was similar to that of the individual detections. Figure S9 shows the DPV responses from the OLC electrodes for the mixture solution of the three ana- lytes. Figure 3b shows the temperature-dependences of the peak currents. They closely resembled the case of CV (Fig. 3a): they increased by factors as large as 20 (DA), 15 (UA) and 60 (AA) (Table 1) in the range of 1000~1200 °C and were saturated subsequently. Furthermore, the peak separations in the DPV response from the OLC-1200 sample (Table S3), obtained from Fig. S9, were similar to those of other n-C materials post-treated in various ways (see Statement S2 for further comments). While such electrode-material-dependence suggested the relevant rate-determining step of electron transfer at electrode surface, an additional step should be considered also: the analyte diffusion in the solution26. Results and Discussion It was also in accord with the previous reports21, where the focus was on the cumulative radial compression of the inner shells to generate a high pressure and consequent graphite-to-diamond transformation at the core in OLC derived from the e-beam-irradiated amorphous carbon. By contrast, here we focus on the reverse reaction i.e. the diamond-to-graphite transformation at the core to generate the inner shells, which might drive the radial expansion and possibly the consequent tangential tensile strain of the outer shells. Nevertheless, it is important that there is another possible origin of the strain, i.e. the shell curvature which was orders of magnitude greater than the flat graphene layers with mild corrugations24; it might lead to the accordingly large tensile strain in the shells, as previously studied for the graphene nano-bubble25. p y g p Such hypothetical tensile strain in the outer shells might result in the defect generation, which might alter the mid-gap state and the electrochemical activity2. To test the possible effect of such hypothetical strain on the defect formation and consequent electrochemical performances, the OLCs described in Fig. 1 were drop-coated on the glassy carbon electrode for individual electrochemical detection of AA, DA, and UA, employing the cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The resulting CV responses were given in Fig. S8a–c, of which the peak currents and peak potentials were summarized in Table 1. As shown in Fig. 3a, the peak currents initially increased by factors of 2.5 (DA), 4.5 (UA), 8 (AA) (Table 1) with temperature at Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 3 www.nature.com/scientificreports/ Figure 3. The peak current vs annealing temperature profiles of the OLC electrodes corresponding to the (a) CV profiles shown in Fig. S8a–c and (b) DPV profiles shown in the Fig. S9. Figure 3. The peak current vs annealing temperature profiles of the OLC electrodes corresponding to the (a) CV profiles shown in Fig. S8a–c and (b) DPV profiles shown in the Fig. S9. Figure 4. Strain of OLC nanoparticles estimated from the shift in 2D peak frequency of Raman spectra, relative to that of the flat multilayer graphene (MLG); the effect of the number of layers on the 2D band peak frequency, for the flat MLG, was included in the calculation30. Figure 3. The peak current vs annealing temperature profiles of the OLC electrodes corresponding to the (a) CV profiles shown in Fig. Results and Discussion Figure 6. The annealing-temperature dependences of the ID/IG ratio, DOS, and Rct−1 corresponding to Figs S14, S15 and S18. Figure 6. The annealing-temperature dependences of the ID/IG ratio, DOS, and Rct−1 corresponding to Figs S14, S15 and S18. obtained by the impedence spectroscopy (Figs S16, S17 and S18, Table S5 and Statement S4). Furthermore, since the core (diamond) versus the shell (graphite) masses were important for the defect generation mechanism in later discussions, it was analysed by EELS (as the sp2/sp3 ratio) as shown in Fig. 5. The ID/IG ratio, DOS, and Rct−1 were compared in a graph (Fig. 6). It was remarkable that their temperature dependences unanimously agreed among themselves, as well as with those of OLC particle size (Fig. 2), the electrochemical responses (Fig. 3), and the strain (Fig. 4), in the initial increase and the eventual saturation at 1200 °C. It is worth to note here that the defect density (ID/IG ratio in Fig. 6), strain (Fig. 4) and the sp2/sp3 carbon contents (Fig. 5) originate from all the shells of the particle due to the penetration depths of Raman spectroscopy and EELS (Experimental details) which were much larger than the OLC radii. It was in contrast to the electrochemical performances (Fig. 3) and Rct−1 (Fig. S18), which should originate exclusively from the outermost shell, since the electron transfer to the inner shells is unlikely, not only due to the electron interceptions by the acceptor states in the intervening outermost shell but also due to the exponentially decreasing tunneling probability with respect to the tunneling distance32. The DOS data (Fig. S15) is also outermost-shell-specific, due to the short penetration depth of UPS (Experimental details).h The aforementioned unanimous agreement strongly suggested some common underlying origin. To examine it, let us first focus on the possible correlation between the strain (Fig. 4) and the defect density (i.e. the ID/IG ratio: Fig. 6), in the light of the vacancy formation energy (Ev) versus the atom migration energy barrier (Em) of the carbon materials reported previously (Table S6). The lower Ev indicated easier vacancy formation, while the lower Em indicated the easier atomic migrations around the defects and hence possibly their healing. An inter- esting effect of the molecular curvature on Ev and Em was disclosed from Table S6. The Ev (for single vacancy) decreased while the Em increased in the order of graphene, SWCNT, and fullerene, i.e. Results and Discussion Indeed, the further inspecting the CV responses (see Figs S10, S11 and the related Statement S3) indicated that the oxidation reaction was mixed-controlled.h The electron-transfer kinetics depends upon the mid-gap states of which the DOS is enhanced by the defect generation in the graphene electrode2. While plasma treatment27, e-beam irradiation28 or nitrogen doping19 were adopted for defect generations in graphene, it was also enabled by the curvature-induced strain in the graphene nano-bubbles24,25, of which the radii were similar to those of the OLCs in the present study (Fig. 1). It prompted us for the analogous analyses. Figure S12a,b compared the Raman spectra obtained from the present OLC sam- ples. The D/G band peak positions and the peak intensity ratios were summarized in Table S4, along with the 2D band peak positions. The latter resided in the range of 2635~2676 cm−1, which were well below that of the flat, unstressed monolayer graphene (2678 cm−1)25; it indicated the tensile strain in the shells according to the previous report29. Furthermore, since the increasing number of graphene layers is known to cause a blue-shift in the peak frequency of 2D band30, the actual strain of the present multi-shell OLC samples might be even greater than that inferred above (Table S4). Figure 4 shows the temperature dependence of the tensile strain inferred from the aforementioned analyses.h The drastic annealing-induced enhancement in electrochemical responses (Fig. 3) suggested relevant changes in defect density and DOS; the former was analyzed from the D to G band intensity ratio (ID/IG) in Raman spec- tra, which is well known to represent the defects density30,31. It actually increased with the annealing-temperature (Figs S12a and S14, Table S4); the effect of the detonation nanodiamond on the Raman spectra was negligible as shown in Fig. S13 and in its caption. Consequent change in DOS was analyzed by UPS (Figs S12c,d, and S15). The inverse of the charge transfer resistance (Rct−1), which might translate to the charge transfer kinetics, was Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 4 www.nature.com/scientificreports/ Figure 5. (a) EELS profiles of OLC samples (black: OLC-1000, red: OLC-1100, blue: OLC-1200, dark cyan: OLC-1400) and (b) The annealing-temperature dependence of sp2 and sp3 contents corresponding to EELS profiles (a). Figure 5. (a) EELS profiles of OLC samples (black: OLC-1000, red: OLC-1100, blue: OLC-1200, dark cyan: OLC-1400) and (b) The annealing-temperature dependence of sp2 and sp3 contents corresponding to EELS profiles (a). Results and Discussion in the order of their molecular curvatures. It strongly suggested possible effect of the strains (probably induced by the molecular curvature) on Ev; at very large strain (irrespective of the source of the strain), the Ev might be accordingly altered Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 5 www.nature.com/scientificreports/ so that the vacancy generation might be accelerated relative to its healing. We will clarify such possibility for the present OLC samples as follows. p p As our OLC sample were found to be under the inherent tensile strain as shown above, a bond between a given pair of carbon atoms should have been stretched, accordingly closer to the threshold rupture strain which is known to be 6~8% for graphene33. Apart from such inherent tensile strain, additional tensile strain might be imposed on the bond by the thermal vibrations of the bound atoms at the annealing temperatures. The collective thermal motions of the atoms in crystalline solid is governed by the Debye model34, where the phonon modes distribu- tion would obviously shift to the higher energy domain with temperature. It would lead to greater chances of the thermally-triggered rupture of the bonds, provided that the inherent tensile strain had already brought the bond close enough to the rupture threshold. Such possibility was actually supported by a recent works on the thermally facilitated rupture of the mechanically strained polycrystalline graphene in the temperature range encompassing up to 1200 °C (section 3.3 of the reference35). It was also supported by the annealing-induced dangling bond generation at the nanodiamond-derived OLC in a previous report15 in the temperature range including that of present exper- iment; since the vacancy generation was known to generate dangling bonds in the OLC36, it suggested accordingly enhanced vacancy generation. Such conclusion was further supported by the reports by Okotrub et al.37, where they confirmed the thermally-induced void formation in nanodiamond-derived OLC by x-ray emission spectroscopy, although the relevant temperature was higher than that in the present study. Nevertheless, such temperature depend- ence contradicted the thermal healing of the defects reported in other forms of n-Cs, or in OLC at relatively high temperature range (Table S1). The aforementioned considerations concerning Ev and Em suggested that the defect generations/healing might mutually compete, and that their relative dominances might depend on the magnitude of the inherent strain of the molecular bonds. Results and Discussion Indeed, when the strain is not large enough, the thermally-triggered rupture would be negligible even for the same phonon mode distributions; the thermal motions would just suffice for the thermal migration around the defects and consequent healing. g q g How would these two strain sources, i.e. the core volume expansion and the shell curvature, be correlated to the aforementioned temperature-dependences of the wide variety of properties, respectively or collectively? Recall that the electron transfer to the outermost shell dominated the electrochemical performance (Fig. 3) and the Rct−1 (Fig. S18). The UPS-derived DOS (Fig. S15), which is also outermost-shell-specific (Experimental details), is well known to increase with the defect density38. Therefore, the electrochemical performance (Fig. 3) should be attributed to the corresponding defect density variations in the outermost shell. However, among the possible defect-generating strain sources for the outermost shell, the shell curvature should be excluded, since it decreased with temperature (since the particle radii increased accordingly: Figs 1 and 2). Therefore we are left with the other possible origin: the volume expansion of the diamond core on its transformation to the graphitic shells. Such strain should depend upon the extent of the phase transformation, i.e. the cumulative volume of the inner shells generated from the core, which dilated the outermost shell to the eventual OLC particle size at the end of each annealing cycles (Fig. 2). Such line of reasoning reconciled the similarity between the temperature dependence of the particle size (Fig. 2) and those of the electrochemical performance (Fig. 3) and Rct−1 (Fig. S18). Such conclusion was further supported, at least partly, by the similar temperature dependences of the ID/IG ratio (Fig. 6) and the strain (Fig. 4) derived from the Raman spectral signal, although the signal originate from all the shells rather than exclusively from the outermost shell (Experimental details); it will be discussed further later. Nevertheless, some additional processes might have also contributed to the OLC particle size increase dur- ing annealing: the merging of the neighboring OLC shells as actually observed occasionally (Fig. 1d), and the Ostwald ripening39. The effect of these additional processes on the annealing-induced particle size evolution is not well established yet, not only in the present work but also in the previous reports on nanodiamond-derived OLC. Results and Discussion However, these additional processes and the aforementioned core expansion are not mutually exclusive; they should proceed simultaneously, so that the actual contribution from the latter process is unquestionable.h y p y p q Let us extend the analyses to the inner shells, which comprises majority of the OLC particle volume. The aforementioned forced tangential strain should occur not only at the outermost shell but also for the series of other consecutively generated shells, until the complete consumption of the diamond core. As the radial location of the newly-generated inner shells approaches particle center with gradual consumption of diamond core, their radii would decrease, and their curvatures and curvature-induced strains would increase. By contrast, since the incremental volume associated with the new-born inner shell should diminish according to their decreasing radii, the relevant incremental strain in the outer shells should accordingly diminish. Apparently, there should be gradual transition between the contributions from these two strain sources with the radial location of the new-born inner shells; they should be regarded as mutually complementary, i.e. as one of them increases with the radial location, the other decreases accordingly, and vice versa. It implied that the summed-up “strength” of these two strain sources would vary rather more weakly than their respective strengths, with the radial locations of the shells. Such expected tendency was strongly supported by the similar temperature-dependences of the properties inferred from the all-shells-encompassing Raman spectral signals (Figs 4 and 6) and the outermost-shell-specific properties (DOS, electrochemical performances, Rct−1: Figs 3 and 6). p p ( p g ) Now let us estimate the core-expansion-induced strain in the outermost shell, for further discussions. Let us take OLC-1200 (Fig. 1c) as an example. From its averaged particle sizes after zero-time annealing (Fig. S7) and that after 1 hour (Figs 2 and S5c), the tangential linear strain for the outermost shell of OLC-1200 was estimated to be about 25% (let us refer to the large strain of such nature, hereafter, as the total strain). By contrast, the critical rupture strain of the graphene lattice was reported to be only 6~8% (at the temperatures of the present experimental range)33. Therefore the only way to accommodate the 25% strain is the ruptures of the molecular bonds, or the vacancy/void formation, which would involve the plastic deformation of the shell in addition to its elastic deformation. Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 Results and Discussion We already observed that the portion of the shells (relative to the core) in the OLC particle indeed increased with temperature (Fig. 1). Furthermore, the temperature-dependence of the particle size (Fig. 2) indeed suggested similar temperature-dependence in the portion of the generated shell mass. But, would it also follow the same intriguing temperature-dependence mentioned so far, i.e. the initial increase and eventual saturation at 1200 °C? The answer to this question was given by the EELS analysis of the shell/core masses as the sp2 and sp3 carbon contents (Fig. 5)40. The generated shell masse, i.e. the sp2 carbon contents, remarkably replicated the temperature dependence we have been observing so far: the initial increase and saturation at 1200 °C. With the discussions made so far in the preceding paragraphs as the background, such line of reasoning made in the present paragraph readily reconciled the eventual saturations in the various properties relevant to the outermost shell. It also con- firmed the role of particle size (Fig. 2) as the indicator of the forced tensile strain of the outermost shell as men- tioned earlier, although we did not reach the saturation issue at that time. Note that the expanding inner shells were the defect generator while the accordingly strained outer shells were defect accommodator in the preceding analyses. By contrast, for the other strain source (the curvature-induced strain), the roles of defect generator and the defect accommodator are played by the same given shell, so that the relevant analyses is rather more difficult; we put it to the future study. y Finally, for a complete assessment of the OLC’s performance in electrochemical detection, the limit of detec- tion and the linear range of detection should be analyzed. For this purpose, the DPV responses of the OLC-1200 electrode were recorded for varying concentrations of the three analytes, in the potential range between − 0.2 V to 0.6 V, as shown in Fig. 7. The DPV responses for AA, DA, and UA distinctly increased with their respective concen- trations. The oxidation peak currents vs analyte concentration profiles, obtained from Fig. 7, were plotted in Fig. S19; their detection limits, linear range, and linear regression equations, obtained from Fig. S19, were summarized in Table S7. As shown in Fig. Results and Discussion Such argument should be applied also for the OLC samples synthesized at different annealing temperatures. The total strain in the outermost shell should obviously depend upon the number of the generated inner shells during the annealing cycle, and hence on the annealing temperature. Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 6 www.nature.com/scientificreports/ Figure 7. DPV responses from OLC-1200 electrode for 0.1 M PBS containing (a) 1–1200 μM AA, (b) 0.1– 700 μM DA, (c) 0.5–600 μM UA. Figure 7. DPV responses from OLC-1200 electrode for 0.1 M PBS containing (a) 1–1200 μM AA, (b) 0.1– 700 μM DA, (c) 0.5–600 μM UA. However, the measured strain (Fig. 4), derived from Raman spectral signal, was one order of magnitude smaller than the aforementioned total strain of the outermost shell. It was attributed to the two possible reasons. First, the measured strain was derived from the Raman spectral signal that originated from not only from the out- ermost shells but also from the inner shells; the corresponding stains of the inner shells must be smaller than the outermost shell from which we calculated the total strain. Second, the Raman spectral signal originated from the molecular bonds which survived the bond rupture/elimination during the vacancy formation; the non-survived bonds must have been elliminated, thereby prevented from contributing to the Raman spectral signal. Such strain of the surviving bonds after the bond eliminations would be regarded as the residual strain (probably the elastic strain). Since such residual strain is a portion of the total strain, the two of them must share the same origin for their temperature-dependences. Since we showed that the total strain should increase with temperature, so must the residual strain, in agreement with actual observation (Fig. 4).h g g The intriguing eventual saturations in the temperature dependences of the wide variety of properties, which started at 1200 °C, need further clarification. In considering this issue, it was important that the graphite-diamond phase boundary during the phase transformation was quasi-spherical (Fig. 1), which would concentrically con- verge to the particle center with the annealing time, at a given temperature: for a given time, it would converge analogously with the temperature. Since such concentric convergence of phase boundary is a strong geometri- cal limitation, the relevant cumulative mass evolution of the generated shell would be accordingly affected. Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 Methods M t i l Materials. All materials [Detonation nanodiamond powder (average size: 3.82 nm; see Fig. S1 for HR-TEM microstructure and the particle size distribution), Ascorbic acid, Dopamine hydrochloride, Uric acid, potassium ferricyanide, phosphate-buffered saline, and potassium chloride] were purchased from Sigma Aldrich Co. All aqueous solutions of the analytes were prepared with deionized water (resistivity = 18.2 MΩ/cm). The AA, DA and UA solutions were freshly prepared in 0.1 M phosphate buffer solutions (PBS, pH = 7.0) prior to use. The potassium ferricyanide solution was prepared in 1 M potassium chloride (KCl) solution. Synthesis of OLCs. The OLCs were synthesized by vacuum annealing of the detonation nanodiamond pow- der. For the annealing, 0.5 grams of nanodiamond powder was put in the graphite crucible in the vacuum furnace, equipped with the graphite heater thermally insulated by the carbon felt in a water-cooled stainless-steel vacuum chamber, evacuated with rotary pump to 10−3 Torr. The annealing temperature was varied as 1000 °C, 1100 °C, 1200 °C, and 1400 °C; the annealing time was fixed at 1 hour. The synthesized OLC samples were accordingly denoted as OLC-1000, OLC-1100, OLC-1200, and OLC-1400, respectively. For comparison, an additional sample was made by the same annealing process at 1200 °C with zero annealing time. The temperature was elevated at a rate of 10 °C/min. at the ramp stage. The samples were furnace-cooled to room temperature after the annealing. Structural Characterization of OLC. The microstructure and crystalline structure of the OLC were char- acterized by high-resolution transmission electron microscopy (HR-TEM, FEI Co, Titan 300 kV), electron energy loss spectroscopy (EELS, FEI Co, Titan 80 kV, penetration depth41: about 20~30 μm) and x-ray diffraction (XRD, Rigaku Co, D/Max 2500 V). The strain and defect density were analyzed by the visible-Raman spectroscopy (Renishaw Co., inVia Raman spectroscopy, The spectral resolution: 0.5 cm−1), employing the 532 nm Nd:YAG laser; its penetration depth was 10–30 nm42, one order of magnitude larger than the OLC particle diameter. Since the visible-Raman is 50~230 times more sensitive to sp2-carbon than sp3-carbon, the signal arising from the sp3 carbon was negligible43. The density of states (DOS) was characterized by the ultraviolet photoemission spec- troscopy (UPS, Ulvac Co., PHI 5000 Versaprobe), employing a He(I) emission lamp (photon energy: 21.2 eV); the signal collection was made with a 0.01 eV resolution. The electron take-off angle was 90°; the pass energy was 0.585 eV. Gold was used as the reference sample. Results and Discussion S19, the gradient of the oxidation current profiles of DA and UA underwent a tran- sition around 60 μM and 50 μM, respectively (For discussions for the origin of such transition, see Statement S5). Also note that of AA did not undergo such transition. Such differing behaviors were attributed to the differing molec- ular structures of the two parties of the analytes (see Statement S5 for further comments). As shown in Table S7, the linear range of detection for AA, DA, and UA were 1~1200 μM, 0.1~700 μM, and 0.5~600 μM with the measured detection limit of 1000 nM, 100 nM, 500 nM and calculated detection limits of 760 nM, 11 nM, 36 nM, respectively. For the DA detection, the sensitivity (slope of the peak current vs concentration profile), the detec- tion limit and the linear range were summarized in Tables S8 and S9, respectively. It was again remarkable that such performance of the optimized post-treatment-free OLC electrode, OLC-1200, was comparable to those of the n-C electrodes post-treated by nitrogen doping, catalyst metal or oxide decoration, as shown in Table S8 and S9. Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 7 www.nature.com/scientificreports/ Figure S20 shows the DPV response for the AA, DA, and UA detection in the presence of the interference mol- ecules, recorded at the fixed concentrations of the two interference molecules while varying the concentrations of the target molecules. Note that the wide variations in the target molecule concentrations had negligible effect on the peak potentials and peak currents of the interference molecule; they were identical to those shown in Fig. 7 obtained in the absence of the interference molecules, which indicated the excellent selectivity of the present OLC electrodes. The measured detection limits of the present post-treatment-free OLC electrodes were 1000 nM, 250 nM, 500 nM, while their calculated detection limits were 860 nM, 30 nM, 45 nM, respectively for AA, DA, and UA, which were comparable to those of the various n-C electrodes in the previous works, which were post-treated in various ways (see Table S10). Results and Discussion Further analyses by chrono-amperometry also yielded excellent results compara- ble to the post-treated carbon electrodes (see Statement S6, Figs S21, S22, and Table S11).i p ( g ) It was important that the novel inherent strain sources clarified in the present study eliminated the inconven- ience of the post-treatments required for the conventional n-C materials in electrochemical detections of AA, DA and UA, since it enabled the performance optimization within the synthesis cycle. , ( ) 2. Brownson, D. A. C., Kampouris, D. K. & Banks, C. E. Graphene electrochemistry: fundamental concepts through to prominent applications. Chem Soc Rev 41, 6944–6976 (2012). 1. Kholmanov, I. N. et al. Healing of Structural Defects in the Topmost Layer of Graphite by Chemical Vapor Deposition. Adv Mater 23, 1675–1678 (2011). 2. Brownson, D. A. C., Kampouris, D. K. & Banks, C. E. Graphene electrochemistry: fundamental concepts through to prominent applications. Chem Soc Rev 41, 6944–6976 (2012). 1. Kholmanov, I. N. et al. Healing of Structural Defects in the Topmost Layer of Graphite by Chemical Vapor Deposition. Adv M 23, 1675–1678 (2011). Methods M t i l The electron escape depth (or electron mean free path) of the UPS spectra was estimated to be a few Å (section 1.2 of the reference) for the employed photon energy of 21.2 eV44; the UPS signal should have originated largely from the outermost shell. Preparation of working electrodes. Prior to the OLC drop-coating, the glassy carbon electrode (GCE) of 3 mm in diameter was polished successively with 1 μm and 0.05 μm alumina abrasive powders. It was subse- quently rinsed twice with aqueous solution of Isopropyl alcohol for 5 min, respectively, and was dried by blowing nitrogen gas. Two milligrams of synthesized OLC powder were mixed with isopropyl alcohol (0.5 mL) and deion- ized water (0.5 mL), and was subsequently subjected to the ultrasonic agitation. A 5 μL droplet of the suspension was dropped onto the pre-treated GCE surface and was dried at room temperature to form a drop-coated layer of the OLC powder. Electrochemical measurement. All of the electrochemical measurements were performed with a VSP Potentiostat/Galvanostat (Bio-logic Co., France). A conventional three-electrode system was used throughout the measurements. The glassy carbon electrode, modified by the drop-coated OLC powder, was employed as the working electrode. The DPV performance of bare glassy carbon electrode was negligible compared to the OLC drop-coated electrode as shown in Fig. S23. A platinum wire and an Ag/AgCl electrode were used as the counter electrode and the reference electrode, respectively. The experiments were carried out at room temperature. The electrochemical impedance spectroscopy was carried out at 0.3 V from 200 kHz down to 0.1 Hz with 10 mV AC amplitude. The Warburg impedance was analyzed employing the rotating electrode technique with the rotation speed of 500~2500 rpm45. All electrochemical impedance measurements were carried out in a 1 M KCl solution containing 1 mM ferricyanide. References Phys Chem Chem Phys 9, 4018–4025 (2007). 40. Muller, J. O., Su, D. S., Wild, U. & Schlogl, R. Bulk and surface structural investigations of diesel engine soot and carbon black. Phys Chem Chem Phys 9, 4018–4025 (2007). 41. Westsson, E. & Koper, G. J. M. How to Determine the Core-Shell Nature in Bimetallic Catalyst Particles? Catalysts 4, 375–396 (2014). 41. Westsson, E. & Koper, G. J. M. How to Determine the Core-Shell Nature in Bimetallic Catalyst Particles? Catalysts 4, 375–396 (2014). 42. Ray, K. & McCreery, R. L. Spatially resolved Raman spectroscopy of carbon electrode surfaces: Observations of structura chemical heterogeneity. Anal Chem 69, 4680–4687 (1997). chemical heterogeneity. Anal Chem 69, 4680–4687 (1997). 43. Ferrari, A. C. & Robertson, J. Raman signature of bonding and disorder in carbons. Amorphous and Nanostructured Carbon 593, 299–304 (2000). f h l l d l ( ) 43. Ferrari, A. C. & Robertson, J. Raman signature of bonding and disorder in carbons. Amorphous and Nanostructured Carbon 593, 299–304 (2000). H f S Ph l S P l d A l ( ) 44. Hüfner, S. Photoelectron Spectroscopy: Principles and Applications (2003). 44. Hüfner, S. Photoelectron Spectroscopy: Principles and Applications (2003). p py p pp ( ) 45. Carl H. Hamann, A. H. Wolf Vielstich Electrochemistry, Edn. 2nd (Wiley, 2007). 45. Carl H. Hamann, A. H. Wolf Vielstich Electrochemistry, Edn. 2nd (Wiley, 2007). References p 20. Sun, C. L. et al. Microwave-Assisted Synthesis of a Core-Shell MWCNT/GONR Heterostructur of Ascorbic Acid, Dopamine, and Uric Acid. Acs Nano 5, 7788–7795 (2011). 20. Sun, C. L. et al. Microwave-Assisted Synthesis of a Core-Shell MWCNT/GONR Heterostructure for the Electrochemical Detection of Ascorbic Acid, Dopamine, and Uric Acid. Acs Nano 5, 7788–7795 (2011). p 21. Banhart, F. & Ajayan, P. M. Self-compression and diamond formation in carbon onions. Adv Mater 9, 261–263 (1997). 22. Lambrecht, W. R. L. et al. Diamond Nucleation by Hydrogenation of the Edges of Graphitic Precursors. Nature 364, 607–610 (1993). 23. Kuznetsov, V. L., Zilberberg, I. L., Butenko, Y. V., Chuvilin, A. L. & Segall, B. Theoretical study of the formation of closed curved 22. Lambrecht, W. R. L. et al. Diamond Nucleation by Hydrogenation of the Edges of Graphitic Precursors. Nature 364, 607–610 (1993 23 K t V L Zilb b I L B t k Y V Ch ili A L & S ll B Th ti l t d f th f ti f l d W. R. L. et al. Diamond Nucleation by Hydrogenation of the Edges of 22. Lambrecht, W. R. L. et al. Diamond Nucleation by Hydrogenatio y y g g p 23. Kuznetsov, V. L., Zilberberg, I. L., Butenko, Y. V., Chuvilin, A. L. & Segall, B. Theoretical study of the formation of closed c graphite-like structures during annealing of diamond surface. J Appl Phys 86, 863–870 (1999). 24. Lu, J., Bao, Y., Su, C. L. & Loh, K. P. Properties of Strained Structures and Topological Defects in Graphene. Acs Nano 7, 8350– (2013). 25. Lim, C. H. Y. X. et al. A hydrothermal anvil made of graphene nanobubbles on diamond. Nat Commun 4 (2013), doi: 10.1038/ ncomms2579.i 6. Razmi, H. & Azadbakht, A. Electrochemical characteristics of dopamine oxidation at palladium hexacyanoferrate film, electroless plated on aluminum electrode. Electrochim Acta 50, 2193–2201 (2005).fil p 7. Shang, N. G. et al. Catalyst-Free Efficient Growth, Orientation and Biosensing Properties of Multilayer Graphene Nanoflake Film with Sharp Edge Planes. Adv Funct Mater 18, 3506–3514 (2008).i g 28. Teweldebrhan, D. & Balandin, A. A. Modification of graphene properties due to electron-beam irradiation. Appl Phys Le 013101 (2009).h 29. Frank, O. et al. Raman 2D-Band Splitting in Graphene: Theory and Experiment. Acs Nano 5, 2231–2239 (2011). h 30. Ferrari, A. C. & Basko, D. M. References Raman spectroscopy as a versatile tool for studying the properties of graphene. Nat Nanotechn 235–246 (2013).f 1. Cancado, L. G. et al. Quantifying Defects in Graphene via Raman Spectroscopy at Different Excitation Energies. Nano Lett. 11 3190–3196 (2011). 32. Royea, W. J., Hamann, T. W., Brunschwig, B. S. & Lewis, N. S. A comparison between interfacial electron-transfer rate constan metallic and graphite electrodes. J Phys Chem B 110, 19433–19442 (2006). 32. Royea, W. J., Hamann, T. W., Brunschwig, B. S. & Lewis, N. S. A comparison between interfacial electron transfer rate constan metallic and graphite electrodes. J Phys Chem B 110, 19433–19442 (2006). 33. Zhao, H. & Aluru, N. R. Temperature and strain-rate dependent fracture strength of graphene. J Appl Phys 108, 064321 (2010) 34. Kittel, C. Introduction to Solid State Physics, Vol. 8th (Wiley, 2005).f Zhao, H. & Aluru, N. R. Temperature and strain-rate dependent fra 33. Zhao, H. & Aluru, N. R. Temperature and strain rate dependent fracture strength of graphene. J Appl Phys 108, 0643 34. Kittel, C. Introduction to Solid State Physics, Vol. 8th (Wiley, 2005).f 34. Kittel, C. Introduction to Solid State Physics, Vol. 8th (Wiley, 2005 35. Chen, M. Q. et al. Effects of grain size, temperature and strain rate on the mechanical properties of polycrystalline graphene - A molecular dynamics study. Carbon 85, 135–146 (2015). 35. Chen, M. Q. et al. Effects of grain size, temperature and s f molecular dynamics study. Carbon 85, 135–146 (2015). y y ( ) 36. Terrones, M., Terrones, H., Banhart, F., Charlier, J. C. & Ajayan, P. M. Coalescence of single-walled carbon nanotubes. Science 288, 1226–1229 (2000). 7. Okotrub, A. V. et al. X-ray emission studies of the valence band of nanodiamonds annealed at different temperatures. J Phys Chem A 105, 9781–9787 (2001).h 38. Orlikowski, D., Nardelli, M. B., Bernholc, J. & Roland, C. Theoretical STM signatures and transport properties of native defects in carbon nanotubes. Phys Rev B 61, 14194–14203 (2000).hh 38. Orlikowski, D., Nardelli, M. B., Bernholc, J. & Roland, C. Theoretical STM signatures and transport properties of native defects in carbon nanotubes. Phys Rev B 61, 14194–14203 (2000).hh y 39. Voorhees, P. W. The Theory of Ostwald Ripening. J Stat Phys 38, 231–252 (1985). 40. Muller, J. O., Su, D. S., Wild, U. & Schlogl, R. Bulk and surface structural investigations of diesel engine soot and carbon black. References References 1. Kholmanov, I. N. et al. Healing of Structural Defects in the Topmost Layer of Graphite by Chemical Vapor Deposition. Adv Mater 23, 1675–1678 (2011). 2. Brownson, D. A. C., Kampouris, D. K. & Banks, C. E. Graphene electrochemistry: fundamental concepts through to prominent applications. Chem Soc Rev 41, 6944–6976 (2012). 8 Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 www.nature.com/scientificreports/ hf p g p 4. Teweldebrhan, D. & Balandin, A. A. Modification of graphene properties due to electron-beam irradiation. Appl Phys Lett 94, 013101 (2009). ) Evolution of Raman spectra in nitrogen doped graphene. Carbon 6 p g p g p 6. Pech, D. et al. Ultrahigh-power micrometre-sized supercapacitors based on onion-like carbon. Nat Nanotechnol 5, 651–654 (2010) 6. Pech, D. et al. Ultrahigh-power micrometre-sized supercapacitors based on onion-like carbon. Na 6. Pech, D. et al. Ultrahigh-power micrometre-sized supercapacitors based on onion-like carbon. Nat Nanotechnol 5, 651–654 (2010). 7. Xin, G. Q. et al. Advanced Phase Change Composite by Thermally Annealed Defect-Free Graphene for Thermal Energy Storage. Acs 6. Pech, D. et al. Ultrahigh power micrometre sized supercapacitors based on onion like carbon. Nat Nanotechnol 5, 651 654 (2010). 7. Xin, G. Q. et al. Advanced Phase Change Composite by Thermally Annealed Defect-Free Graphene for Thermal Energy Storage. Acs Appl Mater Inter 6 15262 15271 (2014) 7. Xin, G. Q. et al. Advanced Phase Change Composite by Thermally Annealed Defect-Free Graphene for Thermal Energy Storage. Ac Appl Mater Inter 6, 15262–15271 (2014).i pp 8. Ni, Z. H. et al. Tunable stress and controlled thickness modification in graphene by annealing. Acs Nano 2, 1033–1039 (2008) l d l Th l b l d f h bb f l d l f 9. Campos-Delgado, J. et al. Thermal stability studies of CVD-grown graphene nanoribbons: Defect annealing and loop formation Chem Phys Lett 469, 177–182 (2009).i y 0. Andrews, R., Jacques, D., Qian, D. & Dickey, E. C. Purification and structural annealing of multiwalled carbon nanotubes a graphitization temperatures. Carbon 39, 1681–1687 (2001).i g p p 1. Georgakilas, V. et al. Purification of HiPCO carbon nanotubes via organic functionalization. J Am Chem Soc 124, 14318–14319 (2002). ( ) 12. Banhart, F., Kotakoski, J. & Krasheninnikov, A. V. Structural Defects in Graphene. Acs Nano 5, 26–41 (2011). al. Raman spectroscopy study of the nanodiamond-to-carbon onion transformation. Nanotechnology 24, 205703 (2013). Sun, X. Y., Zhang, B. S., Sun, X. Y. & Su, D. S. References Hybrid Nanocarbon as a Catalyst for Direct Dehydrogenation of Propane: f A i d S l i C Sh ll (2)/ (3) N i S Ch E J 20 6324 6331 (2014) J p py y gy , ( ) 14. Wang, R., Sun, X. Y., Zhang, B. S., Sun, X. Y. & Su, D. S. Hybrid Nanocarbon as a Catalyst for Direct Dehydrogenation of Propane: Formation of an Active and Selective Core-Shell sp(2)/sp(3) Nanocomposite Structure. Chem-Eur J 20, 6324–6331 (2014). y y gy 14. Wang, R., Sun, X. Y., Zhang, B. S., Sun, X. Y. & Su, D. S. Hybrid Nanocarbon as a Catalyst for Direct Dehydrogenation of Pro Formation of an Active and Selective Core Shell sp(2)/sp(3) Nanocomposite Structure Chem Eur J 20 6324 6331 (2014) 14. Wang, R., Sun, X. Y., Zhang, B. S., Sun, X. Y. & Su, D. S. Hybrid Nanocarbon as a Catalyst for Direct Dehydrogenation of Propane: Formation of an Active and Selective Core-Shell sp(2)/sp(3) Nanocomposite Structure. Chem-Eur J 20, 6324–6331 (2014). Formation of an Active and Selective Core-Shell sp(2)/sp(3) Nanocomposite Structure. Chem-Eur J 20, 6324–6331 (2014). Formation of an Active and Selective Core-Shell sp(2)/sp(3) Nanocomposite Structure. Chem-Eur J 20, 6324–6331 (2014). 5 Tomita S Sakurai T Ohta H Fujii M & Hayashi S Structure and electronic properties of carbon onions J Chem Phys 114 5. Tomita, S., Sakurai, T., Ohta, H., Fujii, M. & Hayashi, S. Structure and electronic properties of carbon onions. J Chem Phys 114 7477–7482 (2001).l 6. McDonough, J. K. et al. Influence of the structure of carbon onions on their electrochemical performance in supercapacitor electrodes. Carbon 50, 3298–3309 (2012). 17. Portet, C., Yushin, G. & Gogotsi, Y. Electrochemical performance of carbon onions, nanodiamonds, carbon black and multiwalled nanotubes in electrical double layer capacitors. Carbon 45, 2511–2518 (2007). y p 8. Breczko, J., Plonska-Brzezinska, M. E. & Echegoyen, L. Electrochemical oxidation and determination of dopamine in the presence of uric and ascorbic acids using a carbon nano onion and poly(diallyldimethylammonium chloride) composite Electrochim Acta 18. Breczko, J., Plonska-Brzezinska, M. E. & Echegoyen, L. Electrochemical oxidation and determination of dopamine in the presence of uric and ascorbic acids using a carbon nano-onion and poly(diallyldimethylammonium chloride) composite. Electrochim Acta 72, 61–67 (2012). 9. Sheng, Z. H. et al. Electrochemical sensor based on nitrogen doped graphene: Simultaneous determination of ascorbic acid dopamine and uric acid. Biosensors & Bioelectronics 34, 125–131 (2012). Author Contributions Y.-J.K. designed/conducted the experiment and analysed the data. S.-C.L. provided an idea for the origin of the tensile strain in the OLC. J.-M.C., I.K., D.S.J., K.-S.L., J.-K.P., Y.-J.B. and H.-J.C. contributed to the analysis and discussion. W.-S.L. supervised the research and wrote the manuscript. All the authors reviewed the manuscript. Acknowledgementsh g This work was supported by the institutional program grant (2E25670) from Korea Institute of Science and Technology. The authors are grateful to M.K. Cho (in Advanced Analysis Center, KIST) for the comments for HR-TEM characterization, to Dr. K.-W. Chae at HoSeo University for his helps in the vacuum annealing process. Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 9 www.nature.com/scientificreports/ Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Ko, Y.-J. et al. Inherently-Forced Tensile Strain in Nanodiamond-Derived Onion- like Carbon: Consequences in Defect-Induced Electrochemical Activation. Sci. Rep. 6, 23913; doi: 10.1038/ srep23913 (2016). How to cite this article: Ko, Y.-J. et al. Inherently-Forced Tensile Strain in Nanodiamond-Derived Onion- like Carbon: Consequences in Defect-Induced Electrochemical Activation. Sci. Rep. 6, 23913; doi: 10.1038/ srep23913 (2016). How to cite this article: Ko, Y.-J. et al. Inherently-Forced Tensile Strain in Nanodiamond-Derived Onion- like Carbon: Consequences in Defect-Induced Electrochemical Activation. Sci. Rep. 6, 23913; doi: 10.1038/ srep23913 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Scientific Reports \| 6:23913 \| DOI: 10.1038/srep23913 10
https://openalex.org/W4361243696	https://figshare.com/articles/journal_contribution/Supplementary_Figure_Legends_1-2_from_Inhibition_of_Ataxia_Telangiectasia_Mutated_Kinase_Activity_Enhances_TRAIL-Mediated_Apoptosis_in_Human_Melanoma_Cells/22380044/1/files/39825446.pdf	English	null	Supplementary Figure Legends 1-2 from Inhibition of Ataxia Telangiectasia Mutated Kinase Activity Enhances TRAIL-Mediated Apoptosis in Human Melanoma Cells	null	2,023	cc-by	90	Supplemental Figure Legends Supplemental Figure 1. Surface DR4 (red) and DR5 (green) expression in human melanocytes and melanoma cell lines. Immunostaining and FACS analysis was performed. Nonspecific staining- ns. Supplemental Figure 2. Normalized levels of cFLIP-L 0-6 h after treatment of LU1205 melanoma cells with KU55933 (10 μM) or TRAIL (50 ng/ml) in the presence or absence of a general caspase inhibitor, zVAD-fmk (50 μM), and a specific caspase-8 inhibitor, Ac-IETD-CHO (50 μM). Western blot analysis of cFILP and β-Actin levels was performed. Levels of cFLIP were normalized to β-Actin.
W4316672987.txt	https://journals.wlb-stuttgart.de/ojs/index.php/wfr/article/download/4852/4801	de		Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter	Württembergisch Franken	2,023	cc-by	6,659	Das Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter von WOLFGANG DEUTSCH ( Das große Kruzifix in der Haller Katharinenkirche galt noch im Kirchenführer von 1996 als spätgotisch 1, obwohl es durch eine Stifterinschrift in s Jahr 1739 datiert ist. Im neuen Kirchenführer wird es dann dem Öhringer Bildhauer Josef Ritter zugeschrieben 2 • Für diese Angabe soll hier die Begründung nachgeliefert werden. Der 200 cm hohe Gekreuzigte 3 hat einen kräftigen, wohlproportionierten Körper, der - trotz Anlehnung an spätgotische Kruzifixtypen - nach barocker Weise in weichen Übergängen modelliert ist (Abb. 1). Die Rückseite des Leibes ist tief ausgehöhlt. Der Kopf neigt sich nach links (zur rechten Körperseite), die Beine sind nach der gleichen Seite hin angewinkelt. Im Antlitz spiegelt sich die Erlösung nach vollbrachtem Todeskampf, die Augen sind geschlossen. Von dem dunklen Haar fällt eine Strähne nach vorne über die rechte Schulter, eine andere nach hinten über den Nacken. Der kurze Bart umrahmt Mund und Kinn , seine Enden sind eingerollt. Der Brustkorb ist deutlich markiert, am Brustbein durch eine Reihe von Grübchen, an den Flanken durch knorpelige Gebilde. Die Adern überraschen stellenweise durch höchst eigenwillige Formen: flammenartige an der linken Leiste, astartige an der unteren Seite der Unterarme. Von allen fünf Wundmalen ziehen sich Blutspuren abwärts; das Blut der Hände rinnt an der Unterseite der Arme herab , an der rechten Seite dem Körper entlang bis unter das Knie. Das vergoldete Lendentuch wird durch einen Strick gehalten, der an der linken Hüfte sichtbar wird . Es bildet vor dem Körper ein Gefüge von fünf scharfwinkligen Dreiecksfalten und an der linken Hüfte einen Bausch. Die Zipfel des Tuchs sind unterschiedlich groß, der linke kurz, der rechte hängt hinter dem linken Oberschenkel bis zur Kniekehle herab - als Gegengewicht gegen die Ausladung von Kopf und Beinen zur anderen Seite. Die Dornenkrone besteht aus einem lockeren Geflecht knorriger Zweige. Die zwölf vergoldeten Strahlen des Nimbus sind in eine runde Holzscheibe mit 1 B. Dowerk: St. Mi chae l Schwäbisch Hall , Horb-Bittelbronn 1996, S.43. 2 Di e Michaelskirche in Schwäbisch Hall , Lindenberg 2002, S. 52, bzw. '2004, S . 47. 3 Weitere Maße: Spannweite der Arme 170 cm, Tiefe des Korpus 39cm. Die Maße und die übrigen technischen Angaben wurden der Dokumen tation der Restauratorinnen A. Kol/111an11 und A. Gräfin von Schwerin. , Juni/Juli/Oktober 2003, entnommen (Archi v der ev. Gesamtkirchengemeinde Schwäbisch Hall). 204 Wolfgang Deutsch Abb. 1 Chorbogenkreuz der Haller Katharinenkirche (Aufnahme: H. W. Hönes, Schwäbisch Hall) Das Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 205 ringsum verlaufender Nut eingesteckt und verleimt4 . Das INRI-Schild in Form eines seitlich eingerollten Bandes ist holzgeschnitzt und original, ebenso die drei Kreuznägel. Die Fassung des Werkes wechselte mehrmals entsprechend dem jeweiligen Kunstgeschmack und der Vorstellung von historischer Treue. 1844 wurde das Haar schwarz gefärbt und das vergoldete Lendentuch weiß übermalt 5 • Pfarrer Heinrich Merz (1850-63) missbilligte das und ließ 1859, als das Geld dafür durch eine Kollekte beschafft war, dem „Lendentuch statt der weißen Ölfarbe seine ursprüngliche Vergoldung" zurückgeben und das Haar heller färben „nach der geschichtlichen Überlieferung, dass Jesus tiefblondes Haar hatte" 6 • Der beauftragte Maler Carl Weisschedel erledigte das auf seine Weise, indem er Haar und Bart mit Broncefarbe lichter machte7; das Lendentuch, die Strahlen am Kopf und das INRI-Schild vergoldete er8 • - 1898 wurde das Kruzifix von Gottfried Schmidt neu gefaßt und dabei vermutlich das Haar wieder dunkel gefärbt9 . Der Restaurierungsbericht von 2003 vermerkt über „einer sehr dunklen braunen Farbe ... eine grobe Überarbeitung mit etwas hellerem Braun" 10 • - Im Inkarnat liegen laut Befund der Restauratorinnen drei Schichten übereinander; die letzte, ,,gelbliche . .. mit deutlichem und großzügigem Pinselduktus, nass in nass modelliert", stammt vermutlich von 1844. Die „Fassung" von 1898 beschränkte sich auf „kleine lockere Überarbeitungen", die Restaurierung von 2003 auf Konservierung und vorsichtige Retuschierung „störender Fehlstellen" 11 • Das Kreuz selbst misst 309/195 cm und ist hohl: es wurde zur Verminderung des Gewichts aus Brettern zusammengefügt. Querholz und Stamm verbreitern sich 4 Ein auf alten Aufnahmen noch sichtbarer breiterer Strahl , der vor der letzten Restaurierung drei andere, herausgebrochene Strahlen ersetzt hat, gehört nach Befund der Restauratorinnen zum Ep itaph des Johann Jakob Beyschlag und wurde dort wieder angebracht. 5 H. Merz; Die Kirche zu St. Katharina in Hall und ihre Restaurationen, in: Christliches Kunstblatt für Kirche, Schule und Haus, 1858, Nr. 5, S. 36; PfarrA St. Katharina II 7, Beilage zur Pfarrbeschreibung. 6 Merz (ebd.). - A uf welche „ Überlieferung" sich Pfarrer Merz bezog, ließ sich nicht ermitte ln. Nach einer im Spätmittelalter verbreiteten Personenbeschreibung des Lentu lu s, des angeblichen Vorgängers von Pilatus, hatte Jesus vielmeh r dunkles Haar: ,,Er war von ansehnlicher Gestalt. ehrfurchtsgebietendem Ant litz, hatte gelocktes Haupthaar von dunkelglän zender Farbe, in der Mitte des Kopfes nach der Sitte der Nazarener gescheitelt und von der Schulter herabfließend, vollen Bart von der Farbe des Haupthaars, nicht lang, in zwe i Spitzen auslaufend , und gemischtfarbige strahlende Augen." (Lex ikon der christlichen Ikonographie, Bd . 1, 1968, Sp. 418; nach E. v. Dobschütz; Chr istusb ilder, Untersuch un gen zur christl ichen Legende, 1899; J. B. Al(fha11ser: Antike Jes uszeugni sse, 2 1925). 7 PfarrA St. Katharina. Pfarrgemeinderatsprotokoll 1851-1866, S.130, zum 5. Augu st 1859. 8 Ebd. , S. 129, zum 31. Juli 1859, sowie Rechnung Wei sschedel s vom 20. Deze mber 1859. 9 Rechnungsbuch Gottfried Schmidt (Privatbesitz), zum 24. Januar 1898: Kirche zu St. Katharina. 1 großes Cruzifix gefaßt , mit Ergän zung der Schnit zerei M. 40.-; die Angabe Gradmann s, das Kru zifi x sei 1900 ne u bemalt worden , ist demnach ungenau; vgl. E. Gradmann: Die Kun st- und Altertums-Denkmale der Stadt und des Oberamtes Schwäbisch Hall , Ess lin gen 1907, S.45. 10 Dokumentation der Restauratorinnen (w ie Anm. 3), S. 6. 11 Ebd. , S.6 und 11. 206 Wolfgang Deutsch nach außen und oben in sanften Kurven mit konkavem Abschluss an den Seiten. Unten endet der Stamm in einer Verdickung und einer kleeblattförmigen Spitze mit aufgeschraubten Metallverzierungen in Form von Lilien und einer Rosette. Auf der Rückseite des Querbalkens befindet sich eine Inschrift, die heute zum größten Teil durch die Querstange der Aufhängevorrichtung verdeckt ist und daher leicht übersehen wird. Sie lautet nach Gottfried Schmidt 12: Susan(n)a Elisa betha Arnoldin wittib Stifterin; darunter am Kreuzstamm: 1739. Davon heute noch lesbar: .. . wittib [St]ifterin / 17[3]9. Das Kruzifix war ursprünglich - in der wesentlich niedrigeren alten Kirche „unter dem westlichen Thurm- oder Chorbogen frei aufgestellt, unten mit dem Stamm in den Fußboden eingelassen und oben am Spitzbogen mit Eisen befestigt"1 3. Pfarrer Merz, der überzeugt war, das Kreuz sei eigentlich Teil des Altars, ließ es 1851 mit diesem verbinden: Das große zum Altare gehörige und ihn erst abschließende Cruci.fix, das zwischen Chor und Langhaus steht und die Gemeinde im Blicke auf den Altar stört, soll nach der Bestimmung des Pf[arr]G[emeinde]R[aths] wieder an dem Altar, zwischen demselben und der Orgelbrüstung angebracht werden 14 • Als 1880 eine größere Chororgel angeschafft wurde, blieb für das Kruzifix nicht mehr genügend Platz zw ischen Altar und „Klaviaturkasten", und man versetzte es deshalb an die Nordwand des Chors 15. Dies erregte den Unmut von Heinrich Merz, der inzwischen Prälat in Stuttgart und Vorstand des „christlichen Kunstvereins" geworden war: Das Kruzifix ist für den Altaraufsatz ein so guter und schöner Abschluß, daß es nicht von ihm abgelöst werden sollte. Ich gestehe, daß ich lieber den H[errn] Walker [den Orgelbauer] aus dem Chor hinausgeworfen hätte [... ] als den Herrn Christus 16 • Merz setzte durch , dass man das Kreuz wieder an den Altar zurückversetzte und zwischen Orgel und Mensa zwängte nach Abnahme und wesentlicher Verdünnung seines Fußes und Verschiebung des Retabels gegen die Mensamitte 17. Daraus erhellt nebenbei , dass die heutige Verdickung und Ornamentierung des Kreuzfußes eine spätere Zutat ist, und zwar von 1898, als man das Kruzifix nach dem Neubau des Kirchenschiffs in den nun wesentlich höheren Chorbogen hängte. Das Werk wird in der Literatur zu Recht geschätzt, wenn auch ohne Überschwang, wie es seinem tatsächlichen Rang entspricht. Eugen Gradmann nennt es „ein Kruzifix von künstlerischem Wert" (S. 45). Heinrich Merz lobt die „sehr 12 Rec hnungsbuch Schmidt (wie Anm . 9); fast gleichla utend die Angabe bei J. L. Gräter: Kirchliches Jahr-Register 1801/02, III.C, Nr. 6. 13 Merz 1858 (wieAnm.5), S.36. 14 PfarrA St. Katharina , Pfarrgemeinderatsprotokoll 1851-1866, S.3 , 10, Beschluss vom 6. April 1851. 15 Ebd. , Pfarrgemeinderatsprotokoll 1866- 1889, S. 3 13, zum 11. Apri l l 880. 16 Ebd ., Bauakten 1880- 1896, Brief vom 8. April 1880 an Pfarrer Rlihle. 17 Ebd. , Pfarrgemeinderatsprotokol l 1866- 1889, S.3 17, 1, zum 24. Oktober 1880. Das Chorbogenkre uz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 207 kräftigen, wahren Formen" und den „natürlichen Ausdruck" 18, die Figur sei ,,anatomisch richtig und mit kräftigem Gefühle behandelt" 19 • Erstaunlich ist nur, dass ein so eindeutig barockes Werk noch 1996 als spätgotisch galt. Vermutlich handelt es sich hier jedoch um das Ende einer längeren Tradition. Denn ohne Zweifel hat schon Heinrich Merz, auch wenn er es nicht ausdrücklich sagt, das Kruzifix für spätgotisch gehalten. Er hat stets geglaubt, es gehöre zu dem mittelalterlichen Hochaltar, und er ließ es deshalb ja mit diesem vereinen. Ohne das Kreuz sei der Altar „um seine Würde und Zierde gebracht" 20 . Hätte er gewusst, dass der Gekreuzigte aus dem 18. Jahrhundert stammt, aus der Epoche, die er als „die Zeit des künstlerischen und kirchlichen Verfalls" ansah 2 1, er hätte bei der Würdigung der Figur kaum die oben zitierten Worte gebraucht. Weitere Werke Es gibt noch eine Reihe weiterer Kruzifixe, entstanden zwischen 1723 und 1736, die mit dem Haller motivisch und stilistisch weitgehend übereinstimmt. Diese Kruzifixe unterscheiden sich im wesentlichen nur dadurch, dass bei den früheren der Körper Jesu stärker gekrümmt und bewegter modelliert und der Faltenfluss des Lendentuchs weicher, fast teigig ist, während er sich bei den späteren Werken zum Teil geometrischen Formen nähert, zum Beispiel vor dem Körper zu einem Bündel ineinander gesteckter Dreiecke erstarrt. Die einzelnen Werke sind in zeitlicher Folge: J. Das Altarkreuz in der Pfarrkirche zu Spiegelberg (Rems-Murr-Kreis), entstanden vor Oktober 1723, die Christusfigur 95 cm hoch (Abb. 2). Für das Werk wurden „dem Bildhauer von Öhringen auff aufschlag eines gemachten Cruzifix, so der Gesamtrechnung einverleibt, bezahlt I ft [Gulden] 30 x [Kreuzer]" 22 . Bei dem Aufschlag dürfte es sich um die seit dem Mittelalter gebräuchliche Nachbesserung (Gfslac) handeln , die den Künstlern nach Vollendung und Begutachtung ihres Werkes auf Antrag gewährt wurde. Die Spiegelberger Figur war demnach zum Zeitpunkt dieser Zahlung schon vollendet. 2. Das große Steinkreuz im Friedhof von Schwäbisch Hall-Steinbach (Abb. 3). Der Kreuzschaft ist mit Sockel fast 6111 hoch , die Figur überlebensgroß. Auf Fernsicht gearbeitet, wird sie von Gradmann (S. 110) als „derb, aber wirkungs- 18 H. M er ~: Die Kirche St. Katharina in Hall, jenseits Kochers, in : WFr 185 1, S. 81 - 98, hier S.87. 19 M er ~ 1858 (wie Anm. 5), S. 36. 20 M er z 1851 (wie Anm. 18), S . 87. 21 M er ~ 1858 (wie Anm. 19). 22 Pfarr-Registratur. Heiligenrechnungen 15. Dezembe r 1721 - 16. Oktober 1723. Zitiert nach A. Schah/: Die Kun stdenkmäler des Rems- Murr-Kreises. Bd. II, München/B erlin 1983. S . 1060 mit Abb.821. 208 Wolfgan g Deutsch Abb. 2 Altarkreuz in Spiegelberg (Aufnahme : Ingrid Wartenberg, Schwäbisch Hall) Das Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 209 -------.-s.• !1 .,. <:: ~ .~ , --~-- ~ fasser) Abb. 3 Ste;nkreu, ;m foedhof Schwäb;,ch Hali-Sre;nbach (Aufnahme, Ver- 210 Wolfgang Deutsch Abb. 4 Kreuzfragment im Hällisch -Fränkischen Museum Schwäbisch Hall (Aufnahme: Verfasser) voll" bezeichnet. Als Entstehungszeit gilt in der Literatur das Jahr 1725 23 , wohl deshalb, weil laut Gradmann der Friedhof 1725 angelegt wurde. Herta Beutter hat aber nachgewiesen, dass der Friedhof schon 1690 „neu angelegt" und die zugehörige Toten- und Heiliggrabkapelle 1693 gebaut wurde 24 . Dennoch passt 23 Gradmann (wie Anm . 9), S. 110; E. Krüger : Schwäbisch Hall , ein Gang durch Geschichte und Kunst, Schwäbisch Hall 21967, S. 174, 3 1982, S. 162. 24 H. Beutler: Die Heilig-Grabkapelle auf dem Friedhof in Schwäbisch Hall-Steinbach, in: WFr.70 (]986) , S. 147- 150. Das Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 211 das Werk stilistisch gut in die 1720er Jahre. Vielleicht ist die überlieferte Jahreszahl 1725 nicht ganz aus der Luft gegriffen; sie könnte sich auf eine Umgestaltung des Friedhofs oder unmittelbar auf die Errichtung des Kreuzes beziehen. Das schwer beschädigte Werk wurde 1985/86 von der Nördlinger Bauhütte restauriert, dabei der Kreuzschaft völlig erneuert und auch Teile der Figur überai·beitet und ergänzt, ,,zum Beispiel einer der Unterschenkel Christi aus Sandstein nachgemeißelt und in die Figur eingesetzt" 25 . Möglicherweise war auch der Gewandbausch an der linken Hüfte Jesu, den alle anderen der genannten Figuren aufweisen, damals abgewittert und wurde deshalb bei der Restaurierung nicht beachtet. 3. Das Kreuzfragment im Hällisch-Fränkischen Museum (Abb. 4). Im Depot des Museums befindet sich ein 65 cm hohes Sandsteinfragment eines Kruzifixes, das laut Katalog beim Gasthaus „Ilge aus dem Kocher gezogen" wurde 26 . Es könnte vom ehemaligen Johanniterfriedhof stammen. Der untere Teil von Kreuz und Figur ist an den Knien Jesu abgebrochen und verloren gegangen. Das Kreuz selbst hat kurvig geschwungene Konturen mit Kleeblattenden und ist rings von Laubwerk eingefasst. Das obere Kleeblattende des Kreuzstamms mit dem dort anzunehmenden INRI-Schild fehlt ebenfalls. Die unteren Zipfel des Schilds (bzw. Pergamentblatts, vgl. das Högg-Epitaph, s. u.) sind gerade noch erkennbar. Das besonders schöne, leider ziemlich verwitterte Werk dürfte vor dem HöggEpitaph und dem Kruzifix von St. Katharina entstanden sein, denn das Lendentuch bildet vor dem Körper noch nicht die strengen Dreiecksfalten wie bei den späteren Werken . 4. Das Kruzifix auf dem Steinepitaph des Georg Michael Högg (t 1736) im Kirchhof der Steinbacher Pfarrkirche (laut Gradmann früher in der Vorhalle 27 ), 105176cm (Abb. 5) . - Der Hauptteil (das Corpus) des Epitaphs hat die Gestalt eines Torbogens, eingefasst von Pilastern mit Voluten und Blattgehängen und überwölbt von einem profilierten Segmentbogen. Über den Pilastern liegen Totenschädel , über dem Bogen Blattvoluten und im Scheitel steht ein geflügeltes Stundenglas, Symbol der Vergänglichkeit. In der Mitte des Torbogens erhebt sich das Kruzifix, sein oberer Teil vor einem Wolkenhimmel, aus dem Strahlenbündel hervorbrechen. Links vom Kreuzstamm kniet der Verstorbene mit pathetischen Gesten, die Linke beteuernd vor die Brust gelegt, die Rechte zur Seite gestreckt. Sein Gesicht ist weitgehend verwittert. Er trägt eine Perücke und einen kurzen , steifen, vorne offenen und mit Knöpfen besetzten Rock mit weiten Stulpenärmeln. Am Fuß des Kreuzes ist, das Sockelgesims überschneidend, sein 25 Bericht im Hal le r Tagbl att (Kk = Dieter Ka linke): Sandste inkre uz je tzt res tauri ert, Nr. 251 vom 30. Oktobe r 1986. 26 Inventar-Nr. 39. K. Sch auffe/e : Verzeic hni s der Sammlungen des Hi stori sche n Ve re in s für das württemb. Franken, Schwäbisch Hall 1898, S. 3 . 27 Graclmann (wi e Anm. 9), S. 109. 212 Wolfgang Deutsch Abb. 5 Högg-Epitaph im Kirchhof von St. Johannes Baptist, Schwäbisch HallSteinbach (Aufnahme: Verfasser) Das Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 213 Wappen angebracht (im Schild ein springender Hirsch [?], ein aufsteigender in der Heimzier). Der vom Corpus durch ein profiliertes Gesims getrennte Untersatz des Epitaphs enthält in einer Kartusche mit geschweiftem, beiderseits mit Akanthus und großen Voluten besetztem Rahmen die Gedenkschrift. Sie lautet: D[EO] O[PTIMO] M[AXIMO] Siste c[ursum?] Viator et Precato pro Anima Geor / gij Micha[e]lis HÖGG ord[inis] s[anc]ti Jo[hann]is Eqvit[atus] Melit[ensis] Come(n)dce / Rotenburg PP [etc.] Tub[ingensis] Administratoris qvi fra / trem suu(m) visitatur(us) huc veniens cursum con / sumavit (et) ceternitatis iter inivit Comburgi / I 3 iunij 1736 dum vixisset annis 48 men[sibus] 5 d[iebus] 9. · R[EQUIESCAT] · /[N] · P[ACE] · Zu deutsch: Gott dem Besten und Größten (geweiht) Halt inne, Wanderer, und bete für die Seele von Georg Michael Högg, Malteserritter des Johanniterordens der Rothenburger Kommende etc., Tübingischer Administrator, der, als er hierher kam, um seinen Bruder zu besuchen, seinen Lebenslauf vollendete und den Weg in die Ewigkeit antrat zu Comburg am 13. Juni 1736, im Alter von 48 Jahren, 5 Monaten und 9 Tagen. Er ruhe in Frieden. Der Bildhauer Aus der Zahlung für das Spiegelberger Kruzifix (S. 207) erfahren wir, wo der Meister seinen Sitz hatte: in Öhringen. Nach Angabe von Elisabeth Grünenwald28 handelt es sich bei dem „Bildhauer von Öhringen" wahrscheinlich um Josef Ritter. Tatsächlich wird in Öhringen, falls die Quellenlage nicht trügt, damals nur dieser Bildhauer genannt. Er war Öhringer Hofbildhauer, vermutlich aus Ochsenfurt zugezogen, wo - laut einer im Krieg verbrannten Schriftquelle - sein Sohn Nikolaus geboren wurde 29 . In der Hoffnung, über die Autorschaft Ritters Gewissheit zu erlangen, prüfen wir zunächst, was die Schriftquellen über den B ildhauer und seine Werke aussagen. Mehrere Zahlungen an ihn sind überliefert, doch unglücklicherweise ist keines der darin genannten Werke erhalten. - 1726 schuf Josef Ritter die Bildhauerarbeit zum neuen Marktbrunnen in Kün28 29 Mitgeteilt bei Schah/ (wie Anm. 22). M. von Rauch : Heilbronn in der 2. Hälfte des 18. Jahrhunderts, in: Bericht des Historischen Vereins Heilbronn 1906-09, Heilbronn 1909, S. 66; Thieme/Becker: Allgemeines Lexikon der bildenden Künstler[ ... ], Bd. 28 ( 1934), S. 387. - Laut freundlicher Auskunft des Heilbronner Stadtarchivs (Prof. Dr. Christhard Schrenk, Direktor) sind viele Archivalien, d ie Moriz von Rauch ausgewertet hat, im Zweiten Weltkrieg verbrannt, so z. 8. die gesamten Aktenbestände. 214 Wolfgang Deutsch zelsau, der leider Ende l 9. Jahrhundert durch einen gusseisernen ersetzt wurde (1935 abgebrochen) 30. - 1732 schnitzte Ritter aus Lindenholz die Bildwerke an der Orgel der Öhringer Stiftskirche31. Die prächtige Orgel schuf der Ansbacher Orgelbauer Johann Christoph Wiegleben nach einem von dem Öhringer Zimmermeister, Tischler und Ornamentzeichner Peter Schillinger gezeichneten Abriss. Die Zeichnung signiert: Georg Peter Schillinger Fecit - verwahrt das Hohenlohe-Zentralarchiv32. Wenn sich Josef Ritter an Schillingers Visierung gehalten hat, bestand der figürliche Anteil seiner Arbeit aus zwei großen Posaunenengeln und zwei Kinderengeln mit Posaune und Fruchtgehänge (auf den oberen Gesimsen) , zwei Kinderengeln mit Trompete (im Ornamentwerk der Orgelflanken), zwei großen schwebenden , die Orgel stützenden Engeln (unter dem Sockelgesims) und siebzehn geflügelten Engelsköpfen verschiedener Größe in tragender oder schmückender Funktion. - 1729-33 schuf Ritter die Bildhauerarbeiten aus Holz und Stein für die 1730/31 erbaute Pfarrkirche von Kirchberg an der Jagst 33 . Die Kirche und ihre Ausstattung wurden 1929 durch einen Brand zerstört34 . Da es davon aber wenigstens noch Fotos gibt (Abb. 6-8), die wahrscheinlich weiterführen können, seien die Zahlungen an Josef Ritter und seinen mitarbeitenden Sohn hier im Wortlaut wiedergegeben35 : Ausgaab Geldt Denen Bildhauern. 140f [Gulden] An Geldt, nebst 3 M[a]lter Spizen 36 , und 3 M[a]/ter Dinkhel, Hat man dem Bildhauer Joseph Rittern von Öhringen, vor die an den neuen Alltar, Canzel und Orgelwerkh gemachte Bildhauer Arbeiten zu Lohn dem getroffenen Accord nach bezahlt, vermög hieranliegender Specification, vom 6< (en ) 7b[ris] 1729. 327f 20 [Kreuzer] Sind erstgedachtem Bildhauer Rittern zu Öhringen, und seinem Sohn vor über seinen ersten accord noch weiter zu allhießigem Kirchenbau verfertigte Bildhauer arbeiten, und sonder[lich] 12. Steinerne Capitäler, a 6f Eines, die Arbeit an dem herrsch[aftlichen] Standt, Mosßen und Aaron a JOf 30 Werkvertrag vom 1. April 1726, StadtA Künzelsau; vg l. Die Kunstdenkmä ler des ehemaligen Oberamts Künzelsau , 1962, Nachdruck: Frankfurt am Main 1983 , S. 60. 31 StAL B 483 , Depositum im Hohenlohe-Zentralarchiv Neuenstein (HZA) , N-Bü 24. 32 Abgebildet in: Öhringen - Stadt und Stift, hg. von der Stadt Öhringen (FoWFr 3 1), Sigmaringen 1988, Abb. 11 7 . 33 Grundsteinlegung am 7. August 1730, Weihe am 12. Dezember 1731; siehe W. M. Diene/: 250 Jahre Stadtkirche Kirchberg an der Jagst, in: Hohenloher Leben (Beilage des Haller Tag blatts) 12/81 (198 1), S.8. 34 Näheres bei Diene/ (ebd .). 35 HZA, Archiv Kirchberg, Rechnungen Bü 166, vom 6. Oktober 1729 bis 20. Juli 1734. - Fotokopien der einsch lägigen Arch ivali en verdanke ich dem freundlichen Entgegenkommen von Herrn Wilfried Beutler. 36 Der Rückstand beim Enthül sen des Dinkels, der als Schweine- und Hühnerfutter verwende t wu rde. Siehe W Saenger: Die bäuerliche Kulturlandschaft der Hohenloher Ebene und ihre Entw icklung seit dem 16. Jahrhundert, Remagen 1957, S. 90, Anm. I 0. Das Chorbogenkreuz der Haller Katharincnkirche und der Öhringer Bildhauer Josef Ritter 215 Ein, das Hey [lige] Abendtmahl in dem al/tar, die Waappen und lnscriptiones, zu denen 2 großen Logen in die herrsch [aftliche] Crufft, den Tauffstein und Pult, die beede Wappen oben in der Farade 2 große Blendflügel beji der Canzel, die Bildhauer arbeit am alltat; neben dem Abendmahl, 5 dekhel in die klein e Logen in der Crufft, a 4f Ein, den Heji [ligen] Geist in die Canzel und 2 Blendf{ügel , oben am rukh Positiv zu machen, zu Lohn bezahlt worden, vermög zettels, vom 191<en) 8b [ri s] 1733. 9f Sind sein des Bildhauer Ritters Sohn , nachdeme er seines Vatters veraccordirte arbeiten, vollend zu End und Standt gebracht, alß ein e VerEhrung gn [ä]d[i]gst verwilligt worden, weilen ihme sonsten nichts zu gutem kom(m)en ist, lnnh a lt deßwegen ergangenen, und hieranliegenden herrsch[aftlichen] Supplications Bescheidts, vom 261Cen> 8b[ri s] 1733. 5f Hat man ihme jungen Rittern, vor in die neue Kirchen gemachte 2 Capitceler unter die 2 Säulen an dem herrsch[aftlichen] Standt veraccordirter maßen zu Lohn bezahlt, Laut der Beji/aag, vom 3p <en) 8b[ris] 1733. 30f 30 [Kreu zer] Wurden aber ilun e jungen Bildhauer Rittern, vor noch einige außzier Arbeiten an den Herrsch[aftli chen] Standt, Orgel, Can ze /, alltar und Crufft, veraccordirter maßen, zu Verdienst gelohnt, besaag der Anfug vom 201<en) Julii 1734. Summa Denen Bildhauern 51. f 50. [Kreuzer]. Bei den Kirchberger Arbeiten handelt es sich um einen Großauftrag für fast 512 Gulden nebst Naturallei stungen. Er wurde in mehreren Abschnitten ausgeführt. Im ersten Abschnitt entstand eine Reihe Skulpturen für eine dreiteili ge „Kanzelwand", eine Kombination au s Altar, Kanzel und Orgel. Die Werke wurden im September 1729 mit 140 Gu lden und den Naturalien bezahlt, waren also schon vor dem Kirchenbau bestellt und gefertigt worden . Im Wesentlichen dürfte es sich um die Arbeiten handeln , die bei der zweiten Zahlung (s.o.) nicht genannt sind, also um die Hauptfiguren Paulus und Petrus, das Kruzifix und einige Engel3 7• Der zweite Abschnitt wurde erst vier Jahre später, im Oktober 1733, mit 327 1/ 3 Gulden abgerechnet. Er umfasste ei ne größere Zahl von Arbeiten , an denen auch der Sohn des Bildhauers, Nikolaus Ritter, beteiligt war. Im Einzelnen waren es, wenn man die Angaben des Rechnungsbuchs etwas ordnet (vg l. die erhaltenen Fotos): - eine Abendmahlsgruppe im Altarretabel (,,das Heylige Abendtmahl in dem alltar"); - Figuren von Moses und Aaron , zu l O Gulden das Stück (sie fl ankierten das Retabel ). - eine Heilig-Geist-Taube in der Kanzel , wohl unter dem Baldachin (,,den Heyli gen Geist in die Canzel "); - das rahmende Schnitzwerk am Retabel (,,die Bildhauerarbeit am alltar, neben 37 Vg l. die fo lgende Beschreibung. 216 Wolfgang Deutsch dem Abendmahl"; da die dort befindlichen Figuren Moses und Aaron extra aufgeführt sind, kann es sich nur um die Ornamentik handeln); das den Kanzelabschnitt flankierende flügelartige Ornamentwerk (,,2 große Blendflügel bey der Canzel"); das rahmende ornamentale und figürliche Schnitzwerk am Rückpositiv (,,2 Blendflügel, oben am rukh Positiv"); - Taufstein und Pult (sie flankierten die Altarmensa); - Arbeit am gräflichen Gestühl (,,an dem herrschaftlichen Standt"); - Wappen und Inschriften an zwei großen Logen in der gräflichen Gruft (,,die Waappen und Inscriptiones zu denen 2 großen Logen in die herrschaftliche Grufft"); - fünf „Deckel", wohl Baldachine, an die kleinen Logen in der Gruft, zu je 4 Gulden; - zwölf steinerne Kapitelle zu je 6 Gulden (für die Pfeiler und Pilaster der Kirche: Kompositkapitelle mit einzelnen oder doppelten Engelsköpfen 38); - das gräfliche Allianzwappen im Giebel der Kirchenfassade (,,die beede Wappen oben in der Fac;:ade" 39 ). Die Zahlung für den zweiten Arbeitsabschnitt hat wiederum Josef Ritter als Auftragnehmer und Werkstattleiter kassiert. Doch erhielt sein mitarbeitender Sohn Nikolaus, der die Arbeiten zu Ende geführt hat, eine Woche später eine Verehrung von 9 Gulden, da ihm „sonsten nichts zu gutem kommen ist". Außerdem bekam er noch S Gulden für eine eigene Arbeit: er fertigte zwei Kapitelle „unter die 2 Säulen" an dem herrschaftlichen Stand, möglicherweise Konsolen. In einem weiteren Arbeitsabschnitt, für den im Jahr darauf, im Juli 1734, 30 ½ Gulden bezahlt wurden, hat Nikolaus Ritter, der Sohn, noch abschließende „Auszierarbeiten" an der Kanzelwand, am gräflichen Gestühl und in der Gruft vorgenommen. Der Vater war damals in Kirchberg offensichtlich nicht mehr anwesend. Für unsere Zwecke ist von der Kirchberger Arbeit Ritters vor allem die Kanzelwand von Belang, weil es von ihr noch Aufnahmen gibt, mit deren Hilfe sich das Werk stilkritisch an den Haller Gekreuzigten und die mit ihm verbundenen Kruzifixe anschließen läßt40 . Die Fotos erlauben, das Werk wie folgt zu beschreiben. Die Kanzelwand umfasst fünf Stockwerke: Altar, Retabel, Kanzel, Rückpositiv und - oberhalb der Empore entsprechend zurückgesetzt - die Orgel. Der mit geschnitzten Schranken eingefriedete Altartisch wird flankiert von einem Lesepult (vom Beschauer links) und einer Taufkufe (rechts), beide gestützt von 38 Eines der Kapitelle abgebildet bei Diene/ (wie Anm. 33): zwischen den Voluten des Kompositkapitells spannt sich eine Blumengirlande mit e inem geflügelten Engelskopf darüber. 39 Vgl. die Abbildung bei Diene/ (ebd .). 40 Foto im Landesdenkmalamt Baden-Württemberg, Nr. 1712, signiert: Schlossar, Crailsheim ; zwei Postkarten mit Detailaufnahmen der Altar-Kanzel-Partie im evangelischen Pfarramt Kirchberg (Reproduktionen davon verdanke ich der Freundlichkeit von Herrn Pfarrer Alfred Holbein). Das Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 217 Abb. 6 Altar-Kanzel-Wand in Kirchberg an der Jagst (1929 verbrannt) 218 Wo l fgang Deutsch Abb. 7 Altar-Kanzel-Wand in Kirchberg an der Jagst, unterer Teil Das Chorbogenkreuz der Hall er Katharinenkirche und der Öhringer Bildhauer Josef Ritter 219 einem Paar sich umarmender Putten mit lose um den Körper gelegten Manteltüchern. Das niedrige Retabel über der Mensa enthält in einem Kapellenschrein mit Rundbogenfenstern eine Abendmahlsgruppe. Jesus und die Apostel sitzen an einem gedeckten Tisch, acht von ihnen frontal ausgerichtet an der Rückseite, die übrigen vor dem Tisch, von der Seite gesehen und einander zugewandt. Die mit Blattranken geschmückte Vorderwand des Schreins ruht auf zwei Pilastern, flankiert von weit ausladendem, durchbrochenem Ornamentwerk aus eingerollten Akanthusblättern, an denen zwei Blumenbündel hängen. Davor stehen fast lebensgroße Figuren von Moses und Aaron; sie nehmen die ganze Höhe des Retabelgeschosses ein 41 • Moses, links, mit langem Bart, den Blick zum Altar gerichtet, greift mit dem linken Arm am Körper vorbei und stützt die riesigen Gesetzestafeln auf sein hochgestelltes rechtes Bein. Aaron, auf der anderen Seite, in spiegelbildlichem Kontrapost, ist ebenfalls bärtig und am dreifachen Rock des Hohenpriesters und der mitraähnlichen Kopfbedeckung zu erkennen. Sein Brustschild wird durch den Arm verdeckt. Auf der Mensa steht ein Kruzifix, das bis in die Sockelzone des Kanzelbaus hinaufreicht. Sein INRI-Schild ist von Wolken mit geflügelten Engelsköpfen umgeben . Die darüber befindliche Kanzelzone ist mehr als dreimal so hoch wie das Retabel und - entsprechend der Bedeutung des göttlichen Wortes - mit besonderer Pracht gestaltet. Der Kanzelkorb selbst wird von schwebenden Kinderengeln getragen, seine Vorderwand bedeckt eine Kartusche mit einem Bibelspruch (Luc. X.16 / Wer Euch hört/ der häret mich). Den Kanzelbereich rahmen zwei schlanke Säulen , gestützt und bekrönt von blattgeschmückten Gesimsen . Diese kragen an den Säulen vor, das Dachgesims auch in der Mitte; seine Unterseite ist mit Quasten (Lambrequins) geschmückt. Außerhalb der Säulen stehen auf dem Sockelgesims zwei lebensgroße Figuren mit breit gefächertem Strahlennimbus: links Paulus mit Buch und Schwert, rechts Petrus mit Buch und Schlüssel42 . Den seitlichen Abschluss der Kanzelzone bilden, in Fortsetzung der Retabelornamentik, weit ausgreifende, geschweifte Blattformen (die zwei großen „Blendflügel " der Rechnungsakten). In der nächsthöheren Zone sitzen auf den Vorsprüngen des Dachgesimses, über den Säulen, zwei Posaune blasende Kinderengel. Sie flankieren das Rückpositiv, das im Kleinen die eckig gebrochene Form des Kanzelteils wieder aufnimmt. An den Ecken seines Dachgesimses sitzen, in entsprechend kleinerem Maßstab, zwei Horn blasende Engel. Über dem Rückpositiv verläuft gleich einem horizontalen Band die mit Quasten behängte Orgelempore. Sie überschneidet die beiden östlichen Kolossalpilaster der Kirchenarchitektur, deren figürliche Kapitelle ebenfalls die Ritter-Werkstatt 41 42 Es sind ke ine „Engel", wie Diene/ (w ie Anm. 33) an gibt. Diene/ (ebd.) hält di e Fi guren irrtümli c h für „Moses und Aaron " . 220 Wolfgang Deutsch -. .... \ .,. ··~,; ~';,y~ ,., ~ Abb. 8 Altar-Kanzel-Wand in Kirchberg an der Jagst, oberer Teil Das Chorbogenkreuz de r Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 221 schuf (s.o.). Zwischen den Pilastern ist als oberster Abschluss der Kanzel wand die große, ebenfalls reich verzierte Orgel eingebaut. An ihr erkennt man in halber Höhe - im Blattwerk des Seitenschmucks und auf Konsolen im Mittelteil vier Trompete blasende Engel43 . Die Komposition der Kirchberger Kanzelwand ist von erlesener Schönheit. Retabel , Kanzeltrakt und Rückpositiv fügen sich in einen hochovalen Umriss und sind dadurch auch formal zu einer vollendeten Einheit zusammengeschlossen. Leider ist nicht bekannt, wer das Werk entworfen hat. Das System der Kanzelwand ist in den lutherischen Ländern aufgekommen , anscheinend in den l 580er Jahren , nach Einführung der Konkordienformel. Das in der Predigt verkündete Wort Gottes war nach protestantischer Auffassung dem Altarsakrament zumindest gleichwertig. Was lag also näher, als Kanzel und Altar miteinander zu verbinden und sie in der Mittelachse der Kirche an der Ostwand zu errichten - jedenfalls in Neubauten oder Umbauten, wo sich die Möglichkeit dafür bor14 . Eines der ältesten Werke entstand 1587 in Schmalkalden, Thüringen 45 . Von Thüringen und Sachsen aus verbreitete sich die Kanzelwand in der Markgrafschaft Ansbach-Bayreuth, weshalb man sie hierzulande auch als ,,Markgräfler Wand" bezeichnet. Sie kommt aber ebenso im Norden vor, in Mecklenburg46, Braunschweig, Brandenburg und selbst Schleswig-Holstein. Beispiele sind Kissenbrück in Braunschweig (um 1665)-17 , Dobberzin in Brandenburg (1699) 48 und , verbunden mit der Orgel , Ütersen in Schleswig-Holstein (um 1745) 49 . Einen entscheidenden Anstoß für die weite Verbreitung des Kanzelaltars gab der Baumeister und Architekturschriftsteller Joseph Furttenbach d. Ä. mit seiner Schrift „Kirchen-Gebäw" (Augsburg 1649)50 . Dass bei der Verbindung von Kanzel und Altar die Kanzel über dem Altar steht, hat - so Wi ssner - ,,auch mit der Selbsteinschätzung reformatorischer Glaubenserkenntnis, also mit der Wertschätzung des Wortes Gottes vor dem Sakrament zu tun" 51 • Die Höherstellung der Kanzel im Sinne einer Höherbewertung ist sicherlich ein willkommener Nebeneffekt. Der eigentliche Grund für die Reihenfolge dürfte aber praktischer Natur sein. Eine umgekehrte Anordnung wäre schlecht möglich. Der Altar wird vom Kirchenboden aus bedient, und die Kanzel benötigt 43 Sie bl asen di e gerade, me ist a ls Posa une bezeichnete Trompete. 44 Z ur Kan ze lwand im All geme inen und zu We rke n der Sommerwerkstatt im Beso nde ren vg l. M. Wissner: Kanze la ltä re au s der Sommer-We rkstatt, in: F. Kellerma nn (Hg.): Die Künstl erfamilie Somme r, Si g ma rin gen 1988, S. 163 ff. 45 Wissner (ebd .), S. 165. 46 Vgl. C . Stade : Mec kl e nburg ische Kan zel a ltäre, Di ss. Braunschweig 193 1. 47 Abgebilde t im Reallex ikon zur De utsc hen Kun stgeschichte (RDK), Bd . 1, Stuttgart 1937, Sp. 597, Abb. 24. 48 Abgebildet ebd. , Sp. 598, Abb. 25. 49 Abgebildet e bd. , Sp. 602, Abb. 27. 50 RDK, e bd. , Sp. 572 ; sowie Wissn er (wi e Anm.44), S. 165 (dort fal scher Vorname: ,,Johann ") . 5 l Wissner (e bd.), S. 163. 222 Wolfgang Deutsch freie Sicht und Hörbarkeit. Die Kanzeln waren darum schon immer höher angebracht als der Altar. Kommen wir zur Nutzanwendung für die Zuschreibung des Haller Kruzifixes und seiner Verwandten. Wir verfügen dabei über zwei Möglichkeiten: erstens den Direktvergleich mit den Figuren der Kanzelwand, zweitens Vergleiche mit den Werken des Sohnes Nikolaus Ritter, der, wie wir nun wissen, in Kirchberg mit dem Vater zusammengearbeitet, auch sicherlich bei ihm gelernt und freundlicherweise die meisten seiner eigenen Werke signiert hat. Seine Formensprache dürfte der des Vaters nahestehen. Zum Direktvergleich: Obwohl die erhaltenen Fotos der Kirchberger Kanzel wand kaum stilistische Feinheiten erkennen lassen, zeigt sich doch, dass der Gekreuzigte (zwischen Retabel und Kanzel) im Erscheinungsbild mit dem Haller Kruzifix und seinen Verwandten übereinstimmt. Die Form von Körper und Gliedmaßen und die Drapierung des Lendentuchs sind nahezu identisch. Im Vergleich zu Hall ist nur das Haupt etwas weiter herabgesunken, die Beine sind eine Kleinigkeit weiter angewinkelt und die Lendentuchfalten weicher modelliert; d. h. die Figur steht dem Spiegelberger Kruzifix noch einen Grad näher. Sie hält etwa die Mitte zwischen dem frühen Spiegelberger Werk von 1723 und dem Haller von 1739 - wie es ihrer Entstehungszeit, wahrscheinlich 1729, entspricht. - Ein unverkennbares Merkmal des Meisters der Kruzifixe sind die aneinandergeschobenen Faltendreiecke (vgl. S. 203 u. 207); sie finden sich in Kirchberg außer beim Gekreuzigten bei Paulus und Petrus und vor allem - noch schärfer ausgeprägt - bei Moses, der dem zweiten Abschnitt der Kirchberger Arbeiten (bis 1733) angehört und daher den späteren Kruzifixen in Hall ( 1739) und am HöggEpitaph (um 1736) auch zeitlich näher steht. - Die Vergleiche bestätigen also die Zusclu·eibung des Haller Werkes an Josef Ritter. Das Ergebnis lässt sich nun mit Hilfe der Werke von Ritters Sohn Nikolaus absichern. Wir werden uns dabei sinnvollerweise im Frühwerk des Bildhauers umsehen. Seine früheste Arbeit ist der Grabstein des Färbers Johann David Bäuerlin ("!" 21. Oktober 1733), heute außen am Chor der Haller Katharinenkirche angebracht (Abb. 9). Er ist signiert: Nicolaus Ritter Bilthauer5 2 und entstand - nach Bäuerlins Todesjahr zu schließen - wohl zu der Zeit, als der Bildhauer noch in Kirchberg an dem großen Auftrag seines Vaters mitarbeitete. Das Denkmal zeigt außer drei üppig verzierten Inschriftkartuschen vier Figuren: im halbrunden oberen Abschluss zwei schwebende Kinderengel (sie halten das Bäuerlin-Wappen) und im unteren Teil, seitlich vom Wappen der Ehefrau, einer geborenen Schübelin, zwei weibliche Personifikationen, die Hoffnung (Spes), mit einem Anker, und den Glauben (Fides) (Abb. 10), mit einem Kruzifix in der Linken, dessen Kopf leider abgewittert ist. Der Gekreuzigte, einschließlich Lendentuchdrapierung und Strahlenkranz ist mit dem Haller Kruzifix und seinen 52 Vgl. H. W. Hönes: Inschriften der Epitaphe, Grabmale, Gemälde und Stiftertafeln in und an der Katharinenkirche Schwäbisch Hall , Ms. 2004, G 07 , S . 13 f., mit Abb. 30- 35. Das Chorbogenkreuz der Ha lle r Katharin enkirche und der Öhringer Bi ldh auer Josef Ritter Abb. 9 Bäuerlin-Epitaph am Chor der Haller Katharinenkirche Abb. 10 Bäuerlin-Epitaph, Detail (Glaube) (Aufnahmen: H. W Hönes, Schwäbisch Hall) 223 224 Wolfgang Deutsch stilgleichen Vorläufern aufs engste verwandt . Er hat auch das gleiche gelappte INRI-Schild wie das Kreuz am Högg-Epitaph. Also auch ein Vergleich mit dem Frühwerk Nikolaus Ritters bestätigt die Zuschreibung des Haller Kruzifixes an Josef Ritter. In späterer Zeit, etwa bei dem J747 (Abb. 11) vollendeten Kruzifix des PerinyDenkmals in Braunsbach 53, hat Nikolaus Ritter, inzwischen Kirchberger Hofbildhauer, eine eigene Formensprache gefunden. Dennoch ist, besonders beim Braunsbacher Kreuz, die Verwandtschaft mit dem Werk des Vaters auch jetzt noch deutlich zu sehen 54 . Die Kirchberger Kanzelwand mit ihrem Figurenreichtum böte nun die Möglichkeit, dem Meister weitere Skulpturen, nicht nur Kruzifixe, zuzuschreiben. Nur leider hat sich noch kein stilistisch passendes Werk gefunden. So bleibt nur zu hoffen, dass bei künftigen Suchaktionen, z.B. einer Inventarisierung der Hohenloher Kirchen, doch das eine oder andere zum Vorschein kommt. Was lässt sich zur kunstgeschichtlichen Stellung Ritters sagen? Von welchen Vorgängern ließ er sich anregen, welche jüngeren Bildhauer hat er seinerseits angeregt? Von seinen einheimischen Vorgängern - etwa Achilles Kern, dem Öhringer Johann Georg Kern, Johann Michael Hornung, Johann Jakob Betzoldt oder Johann Jakob Sommer - scheint er unberührt zu sein, was kaum erstaunt, kam er doch wahrscheinlich aus Ochsenfurt, da dort sein Sohn Nikolaus geboren wurde (vgl. S. 213). Sein Einfluss auf Spätere - außer seinem Sohn - zeigt sich in einem Fall aber relativ deutlich: bei Johann Andreas Sommer in Künzelsau (1716-1776). Die Kruzifixe Sommers - z.B. in Hollenbach (1748) 55 , Amlishagen (1763) 56 oder auf dem Retabelentwurf im Fürstlich Castellschen Archiv in Castell 57 - stehen Josef Ritter ungleich näher als den entsprechenden Werken der älteren Sommer-Generationen (Beispiel: Johann Jakob Sommers Ki.inzelsauer Triumphkreuz mit seinem füllig gebauschten Lendentuch 58 ). Es kann keinen Zweifel geben, dass Johann Andreas an Josef Ritter anschließt und nicht an seine eigenen Ki.inzelsauer Vorgänger. Die Ähnlichkeit mit Ritter geht so weit, dass bei Geh ilfenarbeiten 53 Ausführlich besprochen bei K. Schumm: Das Kreuz auf dem katholischen Friedhof in Braunsbach und die „ Schlacht bei Übrigshausen", in: Der H aalquell (Beilage des Haller Tagblatts) 8 ( 1956), S.49f. 54 Ein Großteil der Werke Nikolaus Ritters ist aufgezählt bei v. Rauch und Thieme/Becker (wie Anm . 29) . In Schwäbi sch Hall lässt sich Nikolaus Ritter mit Sicherheit noch ein weiteres Frühwerk anhand zweier geflügelter Engelsköpfe und vor allem der Ornamentik zuschreiben: das Denkmal des Pfarrers August Johann Albrecht Kern (gest. 19. Oktober 1734), eines Enkels von Leonhard Kern , heute im Chor der Katharinenkirche (vgl. Hön es. wie Anm. 52, G 02, S. 9 ff. mit Abb. 16, 18-20). 55 Abgebildet bei Kellermann (wie Anm.44) , S. 170; Kunstdenkmäler des ehemaligen Oberamts Künzelsau (wie Anm. 30), S. 161. 56 Kellermann (ebd.), S. 164. 57 Ebd., S. 172; die Entwurfszeichnung ist signiert: Sommer· Sculpl. ä Ciinzelsau; dem fortgeschrinenen Rokokostil nach kommt dafür aber nur Johann Andreas in Frage. 58 Ebd. , S. 154, 160. Das Chorbogenkreuz der Haller Katharinenkirche und der Öhringer Bildhauer Josef Ritter 225 Abb. 11 Grabmal des Rittmeisters Stephanus Periny, Braunsbach (Aufnahme: Verfasser) 226 Wolfgang Deutsch mitunter unklar ist, ob sie aus der Werkstatt Josef Ritters oder Johann Andreas Sommers stammen. Ein Beispiel sind zwei Kruzifixe in der Pfarrkirche von Unterheimbach (l 7S5/57 erbaut). Das Altarkreuz dürfte - ohne seinen Sockel - aus der Vorgängerkirche übernommen und in der Werkstatt Josef Ritters geschnitzt sein, das recht ähnliche Vortragekreuz, das 1759 datiert ist, dagegen in der Werkstatt Sommers. Der künstlerische Rang des Bildhauers hält sich in Grenzen, wenn er sich auch schwer beurteilen lässt. Die Kruzifixe folgen einem festgelegten Werkstattschema, das - wie seit Jahrhunderten - den Sinn hat, auch Gehilfen zu ansprechenden Leistungen zu befähigen. Wir wissen also nicht, wie weit wir es mit Eigenhändigem zu tun haben. Und das Kirchberger Hauptwerk, dessen Figurenreichtum für ein Qualitätsurteil die besten Voraussetzungen böte, ist leider verbrannt. Die vorhandene Gesamtaufnahme und die Postkartenbilder lassen die Formensprache des Meisters kaum erkennen. Fast nur motivische Merkmale geben Qualitätshinweise. Dabei entsteht ein zwiespältiger Eindruck. Während etwa die biedere Gestalt des Petrus in ihrer spannungslosen Haltung eher langweilig wirkt, verraten andere Motive mehr Originalität: die reizvolle Gruppierung der Apostel im Abendmahl; der Paulus, der sein Buch mit gespreizten Fingern gegen den Bart drückt, wobei sich eine Strähne über die Blätter legt; und besonders der Moses, eine kraftvolle Erscheinung mit wallendem Bart, die vor allem durch ihren Kontrapost beeindruckt. Der Patriarch wendet das Haupt nach rechts innen, dem Kruzifix zu, mit den Armen greift er nach links außen und hält mit beiden Händen die Gesetzestafeln, die, so groß wie sein Oberkörper, auf dem Schenkel seines angewinkelten Beines ruhen. Aufschlussreich ist ein Vergleich zwischen Josef Ritter und seinem Sohn Nikolaus, dem späteren Kirchberger Hofbildhauer. Beide haben einen Hang zu krauser Ornamentik mit viel Akanthus. Beim Sohn ist sie jedoch derber und teigiger. Auch Nikolaus ' Figuren sind derber, die beiden Tugenden am Bäuerlin-Grabmal (um 1733) sogar ausgesprochen plump 59 . Der Gekreuzigte des Periny-Denkmals (1747) betont, verglichen mit den Kruzifixen des Vaters, mehr die Merkmale des Leidens - hinfällige Haltung, gekrümmte Arme, vortretende Rippen und Sehnen -, wirkt dabei aber ziemlich ungeschlacht. Die Christusfiguren des Vaters sind dagegen von edler, gleichsam „klassischer" Zurückhaltung, ohne expressive Züge. Alles in allem ist Josef Ritter der Qualitätvollere. Dennoch ist der Sohn der Bekanntere, weil er dank seiner Signaturen und des umfangreichen Schriftwechsels zum Periny-Denkmal 60 zur Ehre des Künstlerlexikons von ThiemeBecker gelangte, während der Vater - bis jetzt - im Dunkel der Anonymität verblieb. 59 60 Abgebildet bei Hö11es (wie Anm. 52), G 07, vor allem Abb. 31. Vgl. den Beitrag Schumm (wie Anm. 53).
W1584358948.txt	https://www.gruppofrattura.it/ocs/index.php/ICF/ICF6/paper/download/3215/8277	en		ENVIRONMENT ASSISTED FRACTURE OF METALLIC GLASSES	Elsevier eBooks	1,984	cc-by	0
https://openalex.org/W3081661727	http://aemjournal.org/index.php/AEM/article/download/1466/508	English	null	Collisional and resonance absorption of electromagnetic waves in a weakly collisional, inhomogeneous magnetoplasma slab	Advanced electromagnetics	2,020	cc-by	4,673	ABSTRACT A ABSTRACT Absorbance of normally incident electromagnetic wave on a cold, weakly collisional, and inhomogeneous magnetoplasma slab is investigated. The plasma density is Budden-like sinusoidal proﬁle, where the inhomogeniety is treated as a multilayered system of homogeneous sub-cells within the transfer matrix technique. For incident wave frequencies much above the ion cyclotron frequency, only right hand circularly polarized waves are relevant for wave propagation parallel to a static magnetic ﬁeld. Calculations are performed in normalized parameters, that make the results suitable for many applications including atmospheric and laboratory plasmas. The presence of the dc-magnetic ﬁeld leads to the formation of two absorption bands explained by plasma collisional dissipation and electron cyclotron resonance in the low frequency branch of the R-wave below the electron cyclotron frequency. The transmittance shows the emergence of the low frequency electron cyclotron wave, which becomes a Whistler mode at very low frequency. More detailed discussion on the effect of plasma collisionality, inhomogeneity, and dc-magnetic ﬁeld on the propagation characteristics is given at the relevant place within the body of the manuscript. INDEX TERMS Magnetoplasma slab, Inhomogeneous plasma, Reﬂectance, Transmittance, Absorbance Collisional and resonance absorption of electromagnetic waves in a weakly collisional, inhomogeneous magnetoplasma slab F. Rawwagah1, Manal M. Al-Ali1, A. M. Al-Khateeb1 and M. S. Bawa’aneh1,2 1Department of Physics, Yarmouk University, Irbid, 211-63, Jordan. 2Curriculum and Assessment Sector, Ministry of Education, United Arab Emirates. Corresponding author: F. Rawwagah (e-mail: frawwag@yu.edu.jo) 1Department of Physics, Yarmouk University, Irbid, 211-63, Jordan. 2Curriculum and Assessment Sector, Ministry of Education, United Arab Emirates. 1Department of Physics, Yarmouk University, Irbid, 211-63, Jordan. 2Curriculum and Assessment Sector, Ministry of Education, United Arab Emirates. Corresponding author: F. Rawwagah (e-mail: frawwag@yu.edu.jo) II. MODEL EQUATIONS where ω is the angular frequency of the incident wave, µm, ϵm, dm, εm are, respectively, the mth sub-slab permeability, permittivity, width, and dielectric function. All necessary in- formation to characterize the propagation of electromagnetic waves across the inhomogeneous plasma slab is contained in equations (1-5). The geometry of the plasma slab is shown in Fig. 1. We assume that waves are normally incident from the left semi- inﬁnite medium (vacuum) into the bound plasma slab of width a. The dielectric permittivity of input (left) and output (right) semi.inﬁnite regions are ϵℓand ϵr, respectively. Assume a linearly polarized plane wave such that ⃗E(x, t) = ˆz Ez(x, t) is incident on an inhomogeneous magnetoplasma slab extending from x = 0 to x = a. The in- cident, reﬂected and transmitted waves are propagating along the x-axis parallel to a background uniform magnetostatic ﬁeld ⃗B = ˆx B0, as shown in Figure 1. The matrix M is the transfer matrix of the whole multilay- ered system, and its matrix elements determine the transmis- sion and the reﬂection amplitudes for the entire system. The complex transmission ˜τ and reﬂection ˜ρ coefﬁcients in terms of global transfer matrix elements are [27], FIGURE 1: Bound inhomogeneous plasma slab in a dc-magnetic ﬁeld. ˜τ = 2γℓ γℓM11 + γℓγrM12 + M21 + γrM22 , (6) ˜ρ = γℓM11 + γℓγrM12 −M21 −γrM22 γℓM11 + γℓγrM12 + M21 + γrM22 , (7) T = √εr √εℓ ˜τ ˜τ ∗, R = ˜ρ˜ρ∗, (8) (6) T = √εr √εℓ ˜τ ˜τ ∗, R = ˜ρ˜ρ∗, (8) (8) where R and T are the corresponding reﬂectance and transmittance. The absorbance (A) is deﬁned as A = 1 − R −T. The paper is organized as follows: In section II we present the model equations used in our numerical calculations. In section III we present numerical results, and ﬁnally, in section IV we discuss the main results and conclusions. E B x=0 = M E B x=Nd , (1) M = N Y m=1 Mm = M11 M12 M21 M22 (2) Mm = cos δm i γm sin δm iγm sin δm cos δm , (3) (1) (2) (3) where Mm stands for the mth sub-slab transfer matrix which connects the electromagnetic ﬁelds at one interface with those at the other, and δm is the phase difference corresponding to one traversal of the mth-layer. For normal incidence, the difference between TM and TE polarized waves disappears, and therefore, the parameters γm and δm are given by γm = √µmϵm = √µ0ϵ0 √εm , (4) δm = ωdm √µmϵm = ωdm √µ0ϵ0 √εm , (5) (4) (5) (5) I. INTRODUCTION EM-waves obliquely incident on an inhomogeneous moving magnetoplasma slab parallel to a magnetic ﬁeld and found an evident correlation between the coefﬁcient of reﬂection and the thickness and the velocity of the moving slab [21]. Lacina et.al. studied numerically the resonant interaction of obliquely incident EM-waves on an inhomogeneous plasma slab and found that a part of the incident electromagnetic energy is transformed into heat and the remaining part into energy of oscillation in the direction of the density gradi- ent [22]. Lin Lie et.al. discussed the dependence of R, T, and A of a plane EM-wave that is normally incident on a homogeneous plasma slab and found that only waves with certain wavelengths can be absorbed, reﬂected or transmitted. The increase of collision frequency of the plasma increases the absorbance and decreases the transmittance and the re- ﬂectance [23]. T HE interaction of electromagnetic waves (EM-Waves) with a plasma slab has drawn the attention of re- searchers for decades for their wide applications in several ﬁelds. Recently, the propagation of EM-waves in cold plasma has gained a major interest due to the vast applications of at- mospheric pressure plasma as reﬂectors or absorbers of EM- waves, which plays a dominant role in many applications that include stealth plasma technology [1]–[4], reentry blackout [5]–[10], communication and space plasma [11]–[15] as well as industrial material switches, absorbers and ﬁlters [16]– [20]. T The behavior of an EM-wave in plasma is controlled by the wave parameters, such as the incident wave frequency, angle of incidence and wave polarization, and by the plasma parameters such as the electron density, electron temperature, thickness of the slab, frequency of collisions, and the magne- tization state. Power transmittance (T), reﬂectance (R), and absorbance (A) of EM-waves incident on a plasma slab were numerically calculated for different conditions of incident waves and the characteristics of plasma slab. Highly collisional and high density plasma with partially linear and sinusoidal electron density proﬁle are found to signiﬁcantly absorb the EM-wave energy over a wide fre- quency band with greatly improved absorption characteristics for slabs with higher thickness [24]. Matrix formalism of the problem of EM-wave propagation in dielectric media [25] Seikai et.al. studied the reﬂection and transmission of 25 VOL. 9, NO. 2, AUGUST 2020 F. Rawwagah et al. can be utilized to overcome the inhomogeneity in the plasma slab medium. Properties of great inﬂuence on the propagation characteristics, such as the medium inhomogeneity, can be modeled by replacing an inhomogeneous layer by a succes- sion of thin discrete homogenous sub-layers [1], [3], [26]. In this paper we investigate the propagation characteristics of a normally incident electromagnetic wave on a cold, weakly collisional, and inhomogeneous magnetoplasma slab. A Budden-like sinusoidal density proﬁle is considered and the plasma slab is treated as a multilayered system of ho- mogeneous sub-slabs within the Max-Born transfer matrix technique [11], [27]. Frequencies of interest are those fre- quencies much above the ion cyclotron frequency so that only the motion of electrons is considered. Consequently, only the right–hand, circularly polarized wave is relevant for wave propagation parallel to a static magnetic ﬁeld. Calculations are performed in normalized parameters, that make the re- sults suitable for many applications including atmospheric as well as laboratory plasma. The paper is organized as follows: In section II we present the model equations used in our numerical calculations. In section III we present numerical results, and ﬁnally, in section IV we discuss the main results and conclusions. ﬁelds at the inner face of the exit layer m = N are related to the ﬁelds at the input interface of incidence by the global (overall) transfer matrix M of the whole multilayered struc- ture [25], [27], namely, can be utilized to overcome the inhomogeneity in the plasma slab medium. Properties of great inﬂuence on the propagation characteristics, such as the medium inhomogeneity, can be modeled by replacing an inhomogeneous layer by a succes- sion of thin discrete homogenous sub-layers [1], [3], [26]. In this paper we investigate the propagation characteristics of a normally incident electromagnetic wave on a cold, weakly collisional, and inhomogeneous magnetoplasma slab. A Budden-like sinusoidal density proﬁle is considered and the plasma slab is treated as a multilayered system of ho- mogeneous sub-slabs within the Max-Born transfer matrix technique [11], [27]. Frequencies of interest are those fre- quencies much above the ion cyclotron frequency so that only the motion of electrons is considered. Consequently, only the right–hand, circularly polarized wave is relevant for wave propagation parallel to a static magnetic ﬁeld. Calculations are performed in normalized parameters, that make the re- sults suitable for many applications including atmospheric as well as laboratory plasma. ωR = ωce 2 + 1 2 q ω2ce + 4ω2pe (10) (10) By increasing the normalized plasma collision frequency, the oscillations of in-plasma interferences in Fig.4 will be washed out. Accordingly, extending the range of wave prop- agation (right shift of reﬂection point) and the removal of the oscillations of in-plasma interferences is either due to collisionality or inhomogeneity of the plasma slab. The mth sub-slab dielectric function for the R-wave is The mth sub-slab dielectric function for the R-wave is ε(m) ± = 1 − ω2 pe,m ω (ω −ωce,m −jνe) , ω2 pe,m = e2ne,m ϵ0me , (11 , (11) where νe is the electron collision frequency with neutral particles. The electrical properties of the plasma regime are embedded within the dielectric function [28]–[31]. In Fig.6 we plot the transmittance T for weakly collisional magnetoplasma slabs for different normalized cyclotron fre- quencies. For either a low density plasma or a high inci- dent wave frequency the plasma is initially transparent, and the wave transmission decreases until it vanishes at cutoff. Within the stop band the transmittance is zero, where the incident wave becomes an evanescent wave, and there will be no wave propagation for a complex plasma index of refraction and complex wave number. In all numerical examples presented below, we use nor- malized quantities and therefore, we initiate the calculations by an arbitrary incident wave frequency ω = ω0. In all calculations we adopt the normalized width of the plasma slab a/λ0 = 5. The normalized electron cyclotron frequency ωce in each plasma slab can take one of the values ωce/ωpe = 0, 0.2, 0.6, 1, 1.4, and the normalized collision frequency of electrons with neutrals νe/ωpe = 0.01 for weak but relevant collisional damping. High collision frequencies add nothing to the understanding of the general problem. At the right boundary of the stop band we observe the emergence of a propagating mode from the stop band which becomes obvious for thinner stop bands when the cyclotron frequency is increased relative to the plasma frequency ωc ≥ ωpe. This mode is the well known low-frequency electron cyclotron wave, which is an important characteristic of the dispersion relation of right hand circular electromagnetic waves (R-wave) in magnetoplasma for parallel propagation. F. Rawwagah et al. the critical plasma density ω0 = ωpe, while the main reﬂec- tion point diffuses to the right for the inhomogeneous slab occuring at frequencies slightly above ωpe. The oscillations in R, T and A curves of Fig.2 result from the in-plasma interferences between waves reﬂected from the inner sides of the plasma interfaces located at x = 0 and x = a. For the homogeneous slab (Fig.3) we observe that the oscillations of the in-plasma interferences are smeared out by the slab inhomogeneity with a slight diffusion of the reﬂection point to the right of ωpe as mentioned above. In addition, the curves of Fig.3 show that the absorbance from collisional dissipation becomes broader due to the slab inhomogeneity. Here n0e is the peak density of plasma electrons. Accord- ing to equation (9) the plasma density increases gradually for zero level (no plasma) at x = 0 and increases until it reaches its peak value at the center of the slab a/2, then it decreases gradually until it disappears completely at x = a. The 5λ wide slab was divided into 50 sub–slabs, where the plasma density function of equation (9) is a smooth function. For high frequency waves, the plasma ions are assumed to be immobile neutralizing background of positive charge immersed in the uniform magnetostatic ﬁeld ⃗B0 = B0ˆx. For wave propagation along the applied magnetic ﬁeld, a magnetoplasma of cold electrons and cold ions supports two transverse right and left circularly polarized electromagnetic waves. Since left circularly polarized waves are off-resonant with the electron motion, only right polarized waves rotating with the same sense as the electrons will be considered. The R-wave has two pass bands separated by a stop band, namely, 0 < ω < ωce and ω > ωR. Here ωR is the cutoff frequency of the R-wave given by In Figs.4 and 5 we plot the reﬂectance R for weakly collisional magnetoplasma slabs for different normalized cyclotron frequencies for the homogeneous and inhomoge- neous cases, respectively. For non-vanishing cyclotron fre- quency, we observe a development of the reﬂection bands, which become narrower with increasing cyclotron frequency. For ωce = 1.4 ωpe, the reﬂection band is in the frequency range ωpe < ωce < ω0. This condition implies that waves are reﬂected either at low plasma frequencies for ﬁxed ω0, or equivalently at higher frequencies for ﬁxed ωpe. III. NUMERICAL RESULTS In the following numerical investigation, we assume an in- homogeneous plasma slab density that may be suitable for ionospheric applications [11], [24]. FIGURE 1: Bound inhomogeneous plasma slab in a dc-magnetic ﬁeld. ne(x) =    n0e sin πx a 0 ≤x ≤a 2 n0e 1 −sin π a x −a 2 a 2 ≤x ≤a (9) We divide the inhomogeneous plasma slab into N sufﬁ- ciently thin homogeneous sub-slabs. The mth layer width is dm+1 −dm, where m = 1, 2, 3, ..., N. The electromagnetic (9) 26 VOL. 9, NO. 2, AUGUST 2020 VOL. 9, NO. 2, AUGUST 2020 VOL. 9, NO. 2, AUGUST 2020 F. Rawwagah et al. around the left boundary of the stop band at ω0 = ωpe. around the left boundary of the stop band at ω0 = ωpe. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 R, T, A ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01, ωce/ωpe = 0 Reﬂectance Transmittance Absorbance FIGURE 3: Reﬂectance, Transmittance, and Absorbance for an inhomogeneous unmagnetized plasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 R, T, A ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01, ωce/ωpe = 0 Reﬂectance Transmittance Absorbance The right absorption band of each curve appears for ωce ̸= 0 and is related to the emergence of the propagating wave at the right boundaries of the stop band. For the cyclotron frequency ωce = 1.4 ωpe, the right absorption band is in the frequency region where the plasma frequency is below both the incident wave and electron cyclotron frequencies. For the cyclotron frequencies ωce ≤ωpe, the right bands move into the low wave frequency or high plasma density regions. q y g p y g The emerging propagating mode in the low branch of the R-wave is the electron-cyclotron wave which moves down from high frequency pass band through the stop band into the low frequency pass band. Accordingly, the wave approaches the resonance point from the incident direction at the end of the stop band (here from left), and obviously, the second absorption right bands are due to the electron- cyclotron resonance at the elctron cyclotron frequency. The results agree with Budden analytical calculations, in which he showed that the wave energy is steadily build up and some is absorbed at the resonance, while some tunnel through to the other side with no reﬂection [11]. The wave energy tunneled to the other side, appear in the low frequency region which is ﬁnally attenuated by the plasma. FIGURE 3: Reﬂectance, Transmittance, and Absorbance for an inhomogeneous unmagnetized plasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Reﬂectance R ωpe ω0 Homogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 FIGURE 4: Reﬂectance for an homogeneous magnetoplasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 R, T, A ωpe ω0 Homogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01, ωce/ωpe = 0 Reﬂectance Transmittance Absorbance FIGURE 2: Reﬂectance, Transmittance, and Absorbance for a homogeneous unmagnetized plasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 R, T, A ωpe ω0 Homogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01, ωce/ωpe = 0 Reﬂectance Transmittance Absorbance FIGURE 4: Reﬂectance for an homogeneous magnetoplasma slab quencies, only transverse right circular mode is resonant with the electrons. Reﬂection bands develop and become narrower with the increase of the magnetic ﬁeld. Waves are reﬂected either at low plasma frequencies for ﬁxed frequency of incident waves, or equivalently at higher frequencies for ﬁxed plasma frequency. FIGURE 2: Reﬂectance, Transmittance, and Absorbance for a homogeneous unmagnetized plasma slab The presence of the dc-magnetic ﬁeld leads to the for- mation of two absorbance (or reﬂection) bands, which are explained by plasma collisional dissipation and electron cy- clotron resonance in the low frequency branch of the right- wave below the electron cyclotron frequency. The transmit- tance shows a low frequency electron cyclotron wave, that is transformed into a Whistler mode at very low frequencies. For obvious characterization of wave propagation in an inhomogeneous magnetoplasma slab, numerical results can be easily understood by selecting the relative electron density to be the independent variable, namely, p n0e/nc = ωpe ω0 = ωpeλ0 2πc , where λ0 is the free space wavelength and nc is the plasma critical density for a plane wave incident on an unmagnetized cold homogenous plasma. Accordingly, the plasma density increases by moving to the right along the ωpe coordinate at ﬁxed wave frequency ω0. Similarly, the wave frequency increases as we move to the left at a ﬁxed plasma density. In Fig. 7 we plot the Absorbance A for weakly collisional magnetoplasma slabs for different normalized cyclotron fre- quencies. The curves for ωce ̸= 0 show two absorption bands; the left absorption band peaks around the corresponding cutoff frequency for each ωce value. These absorption bands are of collisional nature due to dissipation in the vicinity of the high frequency cutoff point ωR. The presence of the mag- netic ﬁeld enhances the collisional absorption peak making it narrower, and shifts it down to the high frequency (or to the low plasma density regions) ωpe ω0 < 1. For unmagnetized plasma ωce = 0, the collisional absorption bands are centered In Figs.2 and 3 we present R, T and A for weakly collisional, unmagnetized plasma slabs for the homogeneous and inhomogeneous cases, respectively. For a homogeneous plasma slab (Fig.2) the incident wave is mainly reﬂected at 27 VOL. 9, NO. 2, AUGUST 2020 REFERENCES [1] Mounir Laroussi and J. Reece Roth, “Numerical calculation of the reﬂec- tion, absorption and transmission of microwaves by a nonuniform plasma slab,” IEEE Transactions on Plasma Science, vol. 21, pp.366–372 (1993). [17] M.A. Abdalla and Z. Hu, “On the study of development of x band meta material radar absorber,” Advanced Electromagnetics, vol. 1, no.3, pp. 94– 98 (2012). [2] Mahdi Rahmanzadeh et. al, “Analytical Investigation of Ultrabroadband Plasma Graphene Radar Absorbing Structure”, IEEE Transactions on Plasma Science,” vol. 45, pp. 945–954, (2017). [18] F. Costa and A. Monorchio, “Electromagnetic absorbers based on high impedance surfaces: From ultra-narrow band to ultra-wide band absorp- tion,” Advanced Electromagnetics, vol. 1, no.3, pp. 7–12 (2012). [3] M. S. Bawa’aneh, A. M. Al-Khateeb and A. S. Sawalha, “Microwave prop- agation in a magnetized inhomogeneous plasma slab using the Appleton- Hartree magneto-ionic theory,” Canadian Joural of Physics, vol. 90, pp.241–247 (2012). [19] Chen Zhou, Mehdi Keshavarz Hedayati and Anders Kristensen, “Mul- tifunctional waveguide interferometer sensor: simultaneous detection of refraction and absorption with size-exclusion function”, Optics Express, vol. 26, pp. 24372–24383 (2019). [4] A. M. Al-Khateeb, M. A. Khasawneh, and M. S. Bawa’aneh, “Forward recursion approach of electromagnetic wave propagation characteristics in a slab of inhomogeneous magnetoplasma”, Advanced Electromagnetics, vol. 8, pp. 44–50 (2019). [20] Aqeel H. Naqvi and Jeong–Heum Park, “Via–monopole based quasi Yagi– Uda antenna for W–band applications using thorough glass silicon via (TGSV) technology”, IEEE Access, vol. 8, pp. 9513–9519 (2020). [21] S. Seikai, K. Tanaka, and T. Shiozawa, “Reﬂection and transmission of obliquely incident electromagneticwaves by an inhomogeneousplasma slab moving parallel to the magnetostatic ﬁeld,” Radio Science, vol. 9, pp. 403–408 (1974). [5] J. P. Rybak, J. P. and R. I. Churchill, “Progress in reentry communications,” IEEE Transactions on Aerospace & Electronic Systems, vol. 7, pp. 879– 894, (1970). [6] M. S. Bawa’aneh, A. M. Al-Khateeb and A. S. Sawalha, “Microwave Prop- agation in Warm, Collisional Magnetoionic Media,” IEEE Transactions on Plasma Science, vol. 41, pp.2496–2500 (2013). [22] J. Lacina and J. Preinhaelter, “The interaction of an electromagnetic wave with an inhomogenbous plasma slab,” Czechoslovak Journal of Physics B, vol. 33, pp. 160–174 (1983). [7] Mohammad A Matin, “Review on Millimeter Wave Antennas- Potential Candidate for 5G”, Advanced Electromagnetics, vol. 5, pp. 98–105 (2016). [23] Lin Lie, Wu Bin, Wu Chengkang, “Propagation Properties of Electromag- netic Wave in a Plasma Slab,” Plasma Science and Technology, vol. 5, pp. 1905–1908 (2003). IV. CONCLUSION 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Reﬂectance R ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 FIGURE 5: Reﬂectance for inhomogeneous magnetoplasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Absorbance A ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 FIGURE 7: Absorbance for inhomogeneous magnetoplasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Absorbance A ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 FIGURE 7: Absorbance for inhomogeneous magnetoplasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Reﬂectance R ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 FIGURE 5: Reﬂectance for inhomogeneous magnetoplasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Absorbance A ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 FIGURE 5: Reﬂectance for inhomogeneous magnetoplasma slab FIGURE 7: Absorbance for inhomogeneous magnetoplasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Transmittance T ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 FIGURE 6: Transmittance for inhomogeneous magnetoplasma slab 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.0 0.5 1.0 1.5 2.0 Transmittance T ωpe ω0 Inhomogeneous plasma slab: a/λ0 = 5, νe/ωpe = 0.01 ωce/ωpe = 0.2 ωce/ωpe = 0.6 ωce/ωpe = 1 ωce/ωpe = 1.4 [10] Kai Yuan, Jiamin Chen, Linfang Shen, Xiaohua Deng, Ming Yao, and Lujun Hong, “Impact of Reentry Speed on the Transmission of Obliquely Incident THz Waves in Realistic Plasma Sheaths”, IEEE Transactions on Plasma Science, vol. 46, pp. 373–379 (2018). [11] K. G. Budden, “Radio waves in the ionosphere”, Cambridge university press (1961). [12] M. IV. CONCLUSION Laroussi, “Interaction of microwaves with atmospheric pressure plas- mas,” Int. J. Infrared Millim. Waves, vol. 16, pp. 2069–2083 (1995). [13] Yunxian Tian, Weizhong Yan, Xiaoliang Gu, Xiaolin Jin, Jianqing Li and Bin Li, “Effect of magnetized plasma on the propagation properties of obliquely incident THz waves,” AIP Advances, vol. 7, pp. 125325-11 (2017). [14] Jiyun Lee, Y. T. Jade Morton, Jinsil Lee, Hee-Seung Moon and Jiwon Seo, “Monitoring and Mitigation of Ionospheric Anomalies for GNSS-Based Safety Critical Systems: A review of up-to-date signal processing tech- niques,” IEEE Signal Processing Magazine, vol. 34, pp. 96–110 (2017). [15] Cagatay Tanil; Samer Khanafseh; Mathieu Joerger; Boris Pervan, “An INS Monitor to Detect GNSS Spoofers Capable of Tracking Vehicle Position,” IEEE Transactions on Aerospace and Electronic Systems, vol. 54, pp. 131– 143 (2018). FIGURE 6: Transmittance for inhomogeneous magnetoplasma slab [16] M. K. Hedayati, et. al, “Photodriven Super Absorber as an Active Metama- terial with a Tunable Molecular Plasmonic Coupling,” Advanced Optical Materials,” vol. 2, pp. 705–710 (2014). IV. CONCLUSION In this paper we present a numerical investigation of the reﬂection, absorption and transmission of a transverse elec- tromagnetic wave normally incident on an inhomogeneous, magnetized, and bound cold plasma slab. The effect of inho- mogeneity of an unmagnetized plasma slab is to smooth out the R, T, and A curves eliminating the in-slab interference effects observed in the curves of the homogeneous slab. A similar effect is observed when collisions are introduced in the slab indicating that interference effects can be washed out either by inhomogeneity of the slab or by the plasma collisionality. Another effect of inhomogeneity is to broaden the absorbance peak of the unmagnetized slab. For wave propagation along a constant magnetic ﬁeld, at high fre- The emerging propagating mode which moves down from high frequency pass band to the low frequency pass band passing through a stop band with the wave approaching the resonance point at the end of the stop band is consistent with Budden analytical calculations. VOL. 9, NO. 2, AUGUST 2020 28 F. Rawwagah et al. REFERENCES [8] Xin Yang, Bing Wei and Weike Yin, “A new method to analyze the EM wave propagation characteristics in the hypersonic sheath”, Optik, vol. 148, pp. 187–195 (2017). [24] C.S. Gurel and E. Oncu, “Interaction of electromagnetic wave and plasma slab with partially linear and sinusoidal electron density proﬁle”, Progress In Electromagnetics Research Letters, vol. 12, pp. 171–181 (2009). [9] M. S. Bawa’aneh, A. M. Al-Khateeb, and Y.-C. Ghim, “Thermal Enhance- ment of Absorption of EM Radiation in a Hot Magnetoplasma Slab,” IEEE Transactions on Antennas and Propagation, vol. 66, No. 12, pp. 6525–6530 (2018). [25] Frank L. Pedrotti, Leno M. Pedrotti, Leno S. Pedrotti, “Introduction to Optics”, 3rd Edition, Cambridge University Press (2017). 29 VOL. 9, NO. 2, AUGUST 2020 F. Rawwagah et al. F. Rawwagah et al. [26] Bin Jie Hu, Gang Wei and Sheng Li Lai, “SMM Analysis of Reﬂec- tion, Absorption and Transmission from Nonuniform, Magnetized Plasma Slab,” IEEE Transactions on Plasma Science, vol. 27, pp.1131–1136 (1999). [27] Max Born and Emil Wolf, “Principles of Optics: Electromagnetic Theory of Propagation, Interference and Diffraction of Light,” seventh edition, Cambridge Press (2003). g ( ) [28] Thomas H. Stix, “Waves in Plasmas”, AIP, New York (1992). [28] Thomas H. Stix, “Waves in Plasmas”, AIP, New York (199 [29] D Gary Swanson, “Plasma Waves”, 2nd Edition, IOP Publishing Ltd (2003). [30] M. S. Bawa’aneh and T. J. M. Boyd, “Enhanced levels of stimulated Brillouin reﬂectivity from non-Maxwellian plasmas”, Journal of Plasma Physics vol. 73, No. 2, pp. 159-166, 2007. [31] Umran Inan and Marek Gokowski, “Principles of Plasma Physics for Engineers and Scientists”, Cambridge University Press, New York (2011). [31] Umran Inan and Marek Gokowski, “Principles of Plasma Physics for Engineers and Scientists”, Cambridge University Press, New York (2011). VOL. 9, NO. 2, AUGUST 2020 30
https://openalex.org/W1421998658	https://www.degruyter.com/document/doi/10.1515/math-2015-0030/pdf	English	null	Properties of k-beta function with several variables	Open Mathematics	2,015	cc-by	9,822	Open Mathematics Research Article Abdur Rehman, Shahid Mubeen, Rabia Safdar, and Naeem Sadiq Properties of k-beta function with several variables Abstract: In this paper, we discuss some properties of beta function of several variables which are the extension of beta function of two variables. We deﬁne k-beta function of several variables and derive some properties of this function which are the extension of k-beta function of two variables, recently deﬁned by Diaz and Pariguan [4]. Also, we extend the formula k.2z/ proved by Kokologiannaki [5] via properties of k-beta function. Keywords: k-Gamma function, k-Beta function, Several variables MSC: 33B15, 33B20, 26B35 DOI 10.1515/math-2015-0030 MSC: 33B15, 33B20, 26B35 DOI 10.1515/math-2015-0030 MSC: 33B15, 33B20, 26B35 DOI 10.1515/math-2015-0030 Received May 26, 2014; accepted February 3, 2015. © 2015 Abdur Rehman et al., licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. © 2015 Abdur Rehman et al., licensee De Gruyter Open. Open Math. 2015; 13: 308–320 Open Access Abdur Rehman: Department of Mathematics, University of Sargodha, Sargodha, Pakistan, E-mail: abdurrehman2007@hotmail.com Corresponding Author: Shahid Mubeen: Department of Mathematics, University of Sargodha, Sargodha, Pakistan, E-mail: smjhanda@gmail.com Rabia Safdar: Department of Mathematics, G.C.University, Faisalabad, Pakistan, E-mail: rabia.safdar1109@gmail.com Naeem Sadiq: Department of Mathematics, G.C.University, Faisalabad, Pakistan, E-mail: naeemsadiq008 @yahoo.com 1 Introduction The beta function or Eulerian integral of the ﬁrst kind with two variables is deﬁned by ˇ.x; y/ D 1 Z 0 tx1.1 t/y1dt ; Re.x/ > 0 ; Re.y/ > 0: (1) (1) The Euler gamma function or Euler integral of the second kind is given by The Euler gamma function or Euler integral of the second kind is given by .x/ D 1 Z 0 ettx1dt ; Re.x/ > 0: (2) (2) The beta function in terms of gamma function is deﬁned in [8] as ˇ.x; y/ D .x/.y/ .x C y/ ; Re.x/ > 0; Re.y/ > 0: ˇ.x; y/ D .x/.y/ .x C y/ ; Re.x/ > 0; Re.y/ > 0: (3) ˇ.x; y/ D .x/.y/ .x C y/ ; Re.x/ > 0; Re.y/ > 0: (3) Also the authors [2] proved some properties of beta function by a simple change of variables as (3) Also, the authors [2] proved some properties of beta function by a simple change of variables as Also, the authors [2] proved some properties of beta function by a simple change of variables as ˇ.x; y/ D 2 =2 Z 0 .cos/2x1.sin/2y1d (4) ˇ.x; y/ D 1 Z 0 tx1 .t C 1/xCy dt: (5) ˇ.x; y/ D 2 =2 Z 0 .cos/2x1.sin/2y1d (4) (4) ˇ.x; y/ D 1 Z 0 tx1 .t C 1/xCy dt: (5) (5) Abdur Rehman: Department of Mathematics, University of Sargodha, Sargodha, Pakistan, E-mail: abdurrehman2007@hotmail.com Corresponding Author: Shahid Mubeen: Department of Mathematics, University of Sargodha, Sargodha, Pakistan, E-mail: smjhanda@gmail.com Rabia Safdar: Department of Mathematics, G.C.University, Faisalabad, Pakistan, E-mail: rabia.safdar1109@gmail.com Naeem Sadiq: Department of Mathematics G C University Faisalabad Pakistan E-mail: naeemsadiq 008 @yahoo com Abdur Rehman: Department of Mathematics, University of Sargodha, Sargodha, Pakistan, E-mail: abdurrehman2007@hotmail.com Corresponding Author: Shahid Mubeen: Department of Mathematics, University of Sargodha, Sargodha, Pakistan, E-mail: smjhanda@gmail.com j g Rabia Safdar: Department of Mathematics, G.C.University, Faisalabad, Pakistan, E-mail: rabia.safdar1109@gmail.com Naeem Sadiq: Department of Mathematics, G.C.University, Faisalabad, Pakistan, E-mail: naeemsadiq008 @yahoo.com © 2015 Abdur Rehman et al., licensee De Gruyter Open. is work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Properties of k-beta function with several variables 309 The collection of most important properties of the beta function of two and more variables is given in [1–3]. The deﬁnition of beta function is extended to three or more variables in [3]. 1 Introduction In general, we denote the standard simplex in Rn by E D En D f.x1; x2; :::; xn/ W xi 0; X xi 1; i D 1; 2; :::; n/g: (7) (7) The points .x1; x2; :::; xn/ of the simplex are (1-1) in correspondence with the .n C 1/-tuples .x1; x2; :::; xn; 1 x1 x2 ::: xn/ of non-negative weights with unit sum. If n is not mentioned , we write E in place of En. The interior of E is denoted by interior of E is denoted by int.E/ D f.x1; x2; :::; xn/ W xi > 0; X xi < 1; i D 1; 2; :::; /g: (8) (8) Let p 2 Cn; pi ¤ 0; jargpij < 2 and n 2.If E D En1 be the standard simplex in Rn1, then integral form of beta function of n variables deﬁned by the relation (9) and is proved in [3]. Let p 2 Cn; pi ¤ 0; jargpij < 2 and n 2.If E D En1 be the standard simplex in Rn1, then integral form of beta function of n variables deﬁned by the relation (9) and is proved in [3]. ˇ.p/ D ˇ.p1; p2; :::; pn/ D Z E xp11 1 :::xpn11 n1 .1 x1 ::: xn1/pn1dx1:::dxn1: (9) (9) 1 Introduction Let p D .p1; p2; :::; pn/ 2 Cn; pi ¤ f0: 1; 2; :::g; n 2. The beta function of n variables is deﬁned as The collection of most important properties of the beta function of two and more variables is given in [1–3]. The deﬁnition of beta function is extended to three or more variables in [3]. Let p D .p1; p2; :::; pn/ 2 Cn; pi ¤ f0: 1; 2; :::g; n 2. The beta function of n variables is deﬁned as ˇ.p/ D ˇ.p1; p2; :::; pn/ D .p1/.p2/:::.pn/ .p1 C p2 C ::: C pn/: (6) (6) For n D 2, the classical beta function is represented by integral (1) over a unit interval 0 t 1. If n D 3, we integrate over a triangular region in .x; y/ plane with vertices (0, 0), (1, 0) and (0, 1). This is the set f.x; y/; x 0; y 0; x C y 1g and is called the standard simplex in R2. The points .x; y/ of the simplex are (1-1) in correspondence with the triples .x; y; 1 x y/ of non-negative weights with unit sum.The standard simplex in R3 is f.x; y; z/; x 0; y 0; z 0; x Cy Cz 1g which is a solid tetrahedron with vertices .0; 0; 0/; .1; 0; 0/.0; 1; 0/ and .0; 0; 1/. In general, we denote the standard simplex in Rn by For n D 2, the classical beta function is represented by integral (1) over a unit interval 0 t 1. If n D 3, we integrate over a triangular region in .x; y/ plane with vertices (0, 0), (1, 0) and (0, 1). This is the set f.x; y/; x 0; y 0; x C y 1g and is called the standard simplex in R2. The points .x; y/ of the simplex are (1-1) in correspondence with the triples .x; y; 1 x y/ of non-negative weights with unit sum.The standard simplex in R3 is f.x; y; z/; x 0; y 0; z 0; x Cy Cz 1g which is a solid tetrahedron with vertices .0; 0; 0/; .1; 0; 0/.0; 1; 0/ and .0; 0; 1/. 310 A. Rehman et al. 310 Theorem 2.2. If there are n variables, then there exists a relation between gamma function and beta function of several variables as r . 1 n/ D n r ˇ 1 n; 1 n; :::; 1 n (14) (14) Proof. Using p1 D p2 D ::: D pn D 1 n in the relation (6), we have ˇ 1 n; 1 n; :::; 1 n D . 1 n/. 1 n/:::. 1 n/ 1 n C 1 n C ::: C 1 n D h . 1 n/ in .1/ ; . 1 n/ D n r ˇ 1 n; 1 n; :::; 1 n : which implies Corollary 2.3. If n D 2 .i.e, there are only two variables we have a classical result (See [2]). Corollary 2.3. If n D 2 .i.e, there are only two variables we have a classical result (See [2]). .1 2/ D r ˇ 1 2; 1 2 D p: Theorem 2.4. If Re.x/ > 0; Re.x C y/ > 0; Re.x C y C z/ > 0 and Re.w/ > 0, then we have the following properties of beta function with three and four variables. Theorem 2.4. If Re.x/ > 0; Re.x C y/ > 0; Re.x C y C z/ > 0 and Re.w/ > 0, then we have the following properties of beta function with three and four variables. ˇ.x; y; z/ D 22 =2 Z 0 =2 Z 0 .cos /2.xCy/2.sin /2.yC2z/2d 2 (15) (15) and ˇ.x; y; z; w/ D 23 =2 Z 0 =2 Z 0 =2 Z 0 .cos /2.xCyCz/3.sin /2.yC2zC3w/3d 3: (16) (16) Proof. From equation (9), we see that the integral form of beta function for three variables is Proof. From equation (9), we see that the integral form of beta function for three variables is ˇ.x; y; z/ D Z E px1qy1.1 p q/z1dpdq; ˇ.x; y; z/ D Z E px1qy1.1 p q/z1dpdq; taking v D 1 p, the above equation takes the form taking v D 1 p, the above equation takes the form ˇ.x; y; z/ D Z px1dp v Z 0 qy1.v q/z1dq: Setting q D vt implies dq D vdt and limits of integration becomes 0 to 1. 2 Properties of beta function of several variables Proposition 2.1. If Re.x/ > 0; Re.x C y/ > 0; Re.x C y C z/ > 0 and Re.w/ > 0, then beta function of four variables can be written as ˇ.x; y/ˇ.x C y; z/ˇ.x C y C z; w/ D ˇ.x; y; z; w/ (10) (10) m the relation (3), we see that for three variables we have Proof. From the relation (3), we see that for three variables we have ˇ.x C y; z/ D .x C y/.z/ .x C y C z/ ; Re.x C y/ > 0; Re.z/ > 0; (11) (11) and for four variables we have and for four variables we have ˇ.x C y C z; w/ D .x C y C z/.w/ .x C y C z C w/ ; Re.x C y C z/ > 0; Re.w/ > 0: (12) Multiplying equations (3), (11) and (12), we get ˇ.x C y C z; w/ D .x C y C z/.w/ .x C y C z C w/ ; Re.x C y C z/ > 0; Re.w/ > 0: (12) (12) ˇ.x; y/ˇ.x C y; z/ˇ.x C y C z; w/ D .x/.y/ .x C y/ .x C y/.z/ .x C y C z/ .x C y C z/.w/ .x C y C z C w/ ) ˇ.x; y/ˇ.x C y; z/ˇ.x C y C z; w/ D .x/.y/.z/.w/ .x C y C z C w/ : (13) (13) g the relation (13) with deﬁnition of beta function of several variables (6) implies the required result. on (13) with deﬁnition of beta function of several variables (6) implies the required result. Remarks. The relation (10) can be extended up to n variables which will be the representation for beta function of several variables. If there are only two variables, we get the classical deﬁnition of beta function. Remarks. The relation (10) can be extended up to n variables which will be the representation for beta function of several variables. If there are only two variables, we get the classical deﬁnition of beta function. A. Rehman et al. 310 A. Rehman et al. ˇ.m1 C 1; m2 C 1; m3 C 1; :::; mn C 1/ D (18) m1m2m3:::mnˇ.m1; m2; m3; :::; mn/ .m1 C ::: C mn/.m1 C ::: C mn C 1/:::.m1 C ::: C mn C n 1/; (19) (19) and and ˇ.m1 C 1; m2; :::; mn/ C ˇ.m1; m2 C 1; :::; mn/ C ::: C ˇ.m1; m2; :::; mn C 1/ D ˇ.m1; m2; :::; mn/: (20) ˇ.m1 C 1; m2; :::; mn/ C ˇ.m1; m2 C 1; :::; mn/ C ::: C ˇ.m1; m2; :::; mn C 1/ D ˇ.m1; m2; :::; mn/: (20) (20) Proof. Using the deﬁnition of beta function of several variables (6), we have Proof. Using the deﬁnition of beta function of several variables (6), we have ˇ.˛; m1 C 1; m2 C 1; :::; mn C 1/ D .˛/.m1 C 1/.m2 C 1/:::.mn C 1/ .˛ C m1 C m2 C ::: C mn C n/ : Using .n C 1/ D nŠ and the property of Pochhammer’s symbol .˛/n D .˛Cn/ .˛/ (See [12]), in the above equation, we ﬁnd the required relation (18). To prove the relation (19), we use the same deﬁnition along with the property of classical gamma function .n C 1/ D n.n/ in the numerator and denominator to conclude the desired result. 310 A. Rehman et al. For (20), we use the relation (6) and proceed as ˇ.m1 C 1; m2; :::; mn/ C ˇ.m1; m2 C 1; :::; mn/ C ::: C ˇ.m1; m2; :::; mn C 1/ D .m1 C 1/.m2/:::.mn/ .m1 C ::: C mn C 1/ C .m1/.m2 C 1/:::.mn/ .m1 C ::: C mn C 1/ C ::: C .m1/.m2/:::.mn C 1/ .m1 C ::: C mn C 1/ ; using .n C 1/ D n.n/ in numerator as well as in denominator, we get D m1.m1/.m2/:::.mn/ .m1 C ::: C mn C 1/ C .m1/m2.m2/:::.mn/ .m1 C ::: C mn C 1/ C ::: C .m1/.m2/:::mn.mn/ .m1 C ::: C mn C 1/ D .m1/.m2/:::.mn/.m1 C m2 C ::: C mn/ .m1 C m2 C ::: C mn/.m1 C m2 C ::: C mn/ D ˇ.m1; m2; :::; mn/: ˇ.m1 C 1; m2; :::; mn/ C ˇ.m1; m2 C 1; :::; mn/ C ::: C ˇ.m1; m2; :::; mn C 1/ D .m1 C 1/.m2/:::.mn/ .m1 C ::: C mn C 1/ C .m1/.m2 C 1/:::.mn/ .m1 C ::: C mn C 1/ C ::: C .m1/.m2/:::.mn C 1/ .m1 C ::: C mn C 1/ ; by using .n C 1/ D n.n/ in numerator as well as in denominator, we get D m1.m1/.m2/:::.mn/ .m1 C ::: C mn C 1/ C .m1/m2.m2/:::.mn/ .m1 C ::: C mn C 1/ C ::: C .m1/.m2/:::mn.mn/ .m1 C ::: C mn C 1/ D .m1/.m2/:::.mn/.m1 C m2 C ::: C mn/ .m1 C m2 C ::: C mn/.m1 C m2 C ::: C mn/ D ˇ.m1; m2; :::; mn/: Corollary 2.6. If 1 is added to any one of the variables, then following results hold ( only three variables are provided here). Corollary 2.6. If 1 is added to any one of the variables, then following results hold ( only three variables are provided here). 310 A. Rehman et al. Thus we have ˇ.x; y; z/ D Z px1dp 1 Z 0 vyCz1qy1.1 q/z1dt D Z px1vyCz1dpˇ.y; z/: Using the similar reasons, we conclude that Using the similar reasons, we conclude that ˇ.x; y; z/ D ˇ.y; z/ 1 Z 0 px1.1 p/yCz1dp D ˇ.y; z/ˇ.x; y C z/: Applying the relation (4) on right hand side of above equation, we get ˇ.x; y; z/ D 2 =2 Z 0 .cos /2y1.sin /2z1d2 =2 Z 0 .cos /2x1.sin /2yC2z1d Properties of k-beta function with several variables 311 Properties of k-beta function with several variables 311 Properties of k-beta function with several variab ) ˇ.x; y; z/ D 22 =2 Z =2 Z .cos /2.xCy/2.sin /2.yC2z/2d 2: 0 0 Similarly, we can prove the relation (16). Similarly, we can prove the relation (16). ve the relation (16). Remarks. We can extend the results of the Theorem 2.4 up to n variables and that can be expressed a Remarks. We can extend the results of the Theorem 2.4 up to n variables and that can be expressed as ˇ.x1; x2; :::; xn/ D 2n1 =2 Z 0 =2 Z 0 ::: =2 Z 0 .cos /2.x1Cx2C:::Cxn1/.n1/.sin /2.x2C2x3C:::C.n1/xn/.n1/d .n1/; (17) (17) which can be proved like the Theorem 2.4. which can be proved like the Theorem 2.4. roved like the Theorem 2.4. which can be proved like the Theorem 2.4. Proposition 2.5. If m1; m2; :::; mn 2 N and ˛ ¤ 0, then we have the following properties of beta function with n variables Proposition 2.5. If m1; m2; :::; mn 2 N and ˛ ¤ 0, then we have the following properties of beta function with n variables. ˇ.˛; m1 C 1; m2 C 1; :::; mn C 1/ D m1Šm2Š:::mnŠ .˛/..m1Cm2C:::Cmn/Cn/ (18) variables. ˇ.˛; m1 C 1; m2 C 1; :::; mn C 1/ D m1Šm2Š:::mnŠ .˛/..m1Cm2C:::Cmn/Cn/ (18) where .˛/n D ˛.˛ C 1/:::.˛ C n 1/, is the Pochhammer’s symbol. 310 A. Rehman et al. pˇ.p; q; r C 1/ D rˇ.p C 1; q; r/ qˇ.p C 1; q; r/ D pˇ.p; q C 1; r/ pˇ.p; q; r C 1/ D rˇ.p C 1; q; r/ qˇ.p C 1; q; r/ D pˇ.p; q C 1; r/ d qˇ.p; q; r C 1/ D rˇ.p; q C 1; r/: pˇ.p; q; r C 1/ D rˇ.p C 1; q; r/ qˇ.p C 1; q; r/ D pˇ.p; q C 1; r/ and and qˇ.p; q; r C 1/ D rˇ.p; q C 1; r/: 312 312 A. Rehman et al. Proof. Just use the deﬁnition (6) along with the result .n C 1/ D n.n/. Proof. Just use the deﬁnition (6) along with the result .n C 1/ D n.n/. Proof. Just use the deﬁnition (6) along with the result .n C 1/ D n.n/. Proof. Just use the deﬁnition (6) along with the result .n C 1/ D n.n/. Proof. Just use the deﬁnition (6) along with the result .n C 1/ D n.n/. 3 Main results properties of k-beta function Recently, Diaz and Pariguan [4] introduced the generalized k-gamma function as Recently, Diaz and Pariguan [4] introduced the generalized k-gamma function as k.x/ D lim n!1 nŠkn.nk/ x k 1 .x/n;k ; k > 0; x 2 C n kZ(21) (21) and also gave the properties of said function. The k is one parameter of deformation of the classical gamma function such that k ! as k ! 1. The k is based on the repeated appearance of the expression of the following form and also gave the properties of said function. The k is one parameter of deformation of the classical gamma function such that k ! as k ! 1. The k is based on the repeated appearance of the expression of the following form ˛.˛ C k/.˛ C 2k/.˛ C 3k/:::.˛ C .n 1/k/: (22) (22) The function of the variable ˛ given by the statement (22), denoted by .˛/n;k, is called the Pochhammer k-symbol. We obtain the usual Pochhammer symbol .˛/n by taking k D 1. The defﬁnition given in (21), is the generalization of .x/ and the integral form of k is given by The function of the variable ˛ given by the statement (22), denoted by .˛/n;k, is called the Pochhammer k-symbol. We obtain the usual Pochhammer symbol .˛/n by taking k D 1. 3 Main results properties of k-beta function The defﬁnition given in (21), is the generalization of .x/ and the integral form of k is given by k.x/ D 1 Z 0 tx1e tk k dt; Re.x/ > 0: (23) (23) k.x/ D k x k 1.x k /: (24) (24) The same authors deﬁned the k-beta function as The same authors deﬁned the k-beta function as The same authors deﬁned the k-beta function as ˇk.x; y/ D k.x/k.y/ k.x C y/ ; Re.x/ > 0; Re.y/ > 0 (25) (25) and the integral form of ˇk.x; y/ is and the integral form of ˇk.x; y/ is and the integral form of ˇk.x; y/ is ˇk.x; y/ D 1 k 1 Z 0 t x k 1.1 t/ y k 1dt: (26) ˇk.x; y/ D 1 k 1 Z 0 t x k 1.1 t/ y k 1dt: (26) (26) From the deﬁnition of ˇk.x; y/ given in (25) and (26), we can easily prove that From the deﬁnition of ˇk.x; y/ given in (25) and (26), we can easily prove that From the deﬁnition of ˇk.x; y/ given in (25) and (26), we can easily prove that ˇk.x; y/ D 1 k ˇ.x k ; y k /: (27) (27) Also, the researchers [5–9] have worked on the generalized k-gamma and k-beta functions and discussed the following properties: Also, the researchers [5–9] have worked on the generalized k-gamma and k-beta functions and discussed the following properties: k.x C k/ D xk.x/ (28) k.x C k/ D xk.x/ (28) .x/n;k D k.x C nk/ k.x/ (29) k.x C k/ D xk.x/ (28) .x/n;k D k.x C nk/ k.x/ (29) k.k/ D 1; k > 0 (30) (28) .x/n;k D k.x C nk/ k.x/ (29) (29) k.k/ D 1; k > 0 (30) k.k/ D 1; k > 0 (30) k..2n C 1/k 2 / D k 2n1 2 .2n/Šp 2nnŠ ; k > 0; n 2 N: (31) (31) In [2], it is proved that gamma function .z/ is analytic on C except the poles at z D 0; 1; 2; ::: and the residue at z D n is equal to .1/n nŠ ; n D 0; 1; 2; :::. 3 Main results properties of k-beta function Recently, Mubeen and Rehman [11] proved that for k > 0, the function k.x/ is analytic on C, except the single poles at x D 0; k; 2k; ::: and the residue at x D nk is 1 .1/nknnŠ. In [2], it is proved that gamma function .z/ is analytic on C except the poles at z D 0; 1; 2; ::: and the residue at z D n is equal to .1/n nŠ ; n D 0; 1; 2; :::. Recently, Mubeen and Rehman [11] proved that for k > 0, the function k.x/ is analytic on C, except the single poles at x D 0; k; 2k; ::: and the residue at x D nk is 1 .1/nknnŠ. Properties of k-beta function with several variables 313 313 Using (25) and (27), we see that, for x; y > 0 and k > 0, the following properties of k-beta function of two variables are satisﬁed by authors (see [5, 6] and [10]): Using (25) and (27), we see that, for x; y > 0 and k > 0, the following properties of k-beta function of two variables are satisﬁed by authors (see [5, 6] and [10]): ˇk.x C k; y/ D x x C y ˇk.x; y/ (32) ˇk.x C k; y/ D x x C y ˇk.x; y/ (32) ˇ . k/ y ˇ . / (33) ˇk.x C k; y/ D x x C y ˇk.x; y/ (32) ˇk.x; y C k/ D y x C y ˇk.x; y/ (33) ˇk.xk; yk/ D 1 k ˇ.x; y/ (34) ˇk.nk; nk/ D Œ.n 1/Š2 k.2n 1/Š; n 2 N (35) ˇk.x; k/ D 1 x ; ˇk.k; y/ D 1 y : (36) (32) ˇk.x; y C k/ D y x C y ˇk.x; y/ (33) ˇk.xk; yk/ D 1 k ˇ.x; y/ (34) (33) ˇk.xk; yk/ D 1 k ˇ.x; y/ (34) (34) ˇk.nk; nk/ D Œ.n 1/Š2 k.2n 1/Š; n 2 N (35) ˇk.x; k/ D 1 x ; ˇk.k; y/ D 1 y : (36) (35) ˇk.x; k/ D 1 x ; ˇk.k; y/ D 1 y : (36) (36) Remarks. When k ! 1 , ˇk.x; y/ ! ˇ.x; y/. Remarks. When k ! 1 , ˇk.x; y/ ! ˇ.x; y/. Now, we deﬁne k-beta function of several variables and prove some properties of the said function. With the help of this deﬁnition, we extend some previous results which will help us to compute the values of ˇk function at particular points. 3.1. Let p D .p1; p2; :::; pn/ 2 Cn; pi ¤ f0: 1; 2; :::g; n 2; k > 0. The k-beta function of n s deﬁned as Deﬁnition 3.1. Let p D .p1; p2; :::; pn/ 2 Cn; pi ¤ f0: 1; 2; :::g; n 2; k > 0. Proof. We denote the integral by In;k D ˇk.p1; :::; pn/. Thus I2;k D ˇk.p1; p2/ which is equivalent to (26). For n 3, ﬁrst we take the case when p 2 Rn,where R is the set of positive real numbers. The integrand is positive and the integral may be evaluated as an iterated integral by Lemma 3.2. Integrating over xn1 and deﬁning v D 1 x1 x2 ::: xn2, we see that In;k D Z En1 x p1 k 1 1 :::x pn2 k 1 n2 dx1:::dxn2: v Z 0 x pn1 k 1 n1 .v xn1/ pn k 1dxn1: (39) (39) The region of integration for outer integral is f.x1; :::xn2/ W x1 0; :::xn2 0; 1 x1 ::: xn2 0g D En2; putting xn1 D vt, the inner integral becomes putting xn1 D vt, the inner integral becomes v pn1Cpn k 1 1 Z 0 t pn1 k 1.1 t/ pn k 1dt D v pn1Cpn k 1:kˇk.pn1; pn/; (40) (40) and using the above result in equation (39), we have In;k D Z En2 x p1 k 1 1 :::x pn2 k 1 n2 dx1:::dxn2v pn1Cpn k 1:kˇk.pn1; pn/ D kˇk.pn1; pn/ Z En2 x p1 k 1 1 :::x pn2 k 1 n2 .1 x1::: xn2/ pn1Cpn k 1dx1:::dxn2 (41) D In1;k.p1; :::; pn2; pn1 C pn/kˇk.pn1; pn/: (41) Now we make the inductive assumption that In1;k is the k-beta function of n 1 variables. So, by the relations (41) and (37), we get In;k D kn2ˇk.p1; :::pn2; pn1 C pn/:kˇk.pn1; pn/ D kn1ˇk.p1; :::; pn/; which is the relation (38) for k > 0; n 3 and p 2 Rn. The same proof holds for the case when p 2 Cn n f0g if the integrand of the equation (39) can be replaced by the absolute values, simply each pi by Re.pi/; i D 1; 2; :::; n. The iterated integral of the absolute value is ﬁnite by the preceding proof for the real case and Lemma 3.2, ensures that the relation (39) is valid for complex case. Now, we prove the symmetry of all arguments. 314 A. Rehman et al. 314 The k-beta function of n variables is deﬁned as ˇk.p/ D ˇk.p1; p2; :::; pn/ D k.p1/k.p2/:::k.pn/ k.p1 C p2 C ::: C pn/ : (37) ˇk.p/ D ˇk.p1; p2; :::; pn/ D k.p1/k.p2/:::k.pn/ k.p1 C p2 C ::: C pn/ : (37) (37) Remarks. For n D 2, we obtain k-beta function represented by (25) and if k D 1, we have the classical beta function. Remarks. For n D 2, we obtain k-beta function represented by (25) and if k D 1, we have the classical beta function. Lemma 3.2. Let and be the positive -ﬁnite or a complex measure on a measure space X and T respectively with jj and jj be the corresponding total variation. Assume that the function f W X T ! C is measurable on the product spaceX T with respect to the product -algebra. If the integral Z X Z T jf .x; t/jdjj.x/djj.t/ Z X Z T jf .x; t/jdjj.x/djj.t/ is ﬁnite when evaluated as an iterated integral in one order or the other, then the value of Z X Z T f .x; t/d.x/d.t/ is independent whether it is evaluated as a double integral or an iterated integral in either order. Remarks. The above Lemma is the Fubini’s Theorem. For more complete and precise detail of Fubini’s Theorem for positive measure (see Rudin 1974 [13]). Proposition 3.3. Let p 2 Cn nf0g; n 2; k > 0 and let E D En1 be the standard simplex in Rn1, then integral form of k-beta function of n variables is deﬁned by ˇk.p/ D ˇk.p1; :::; pn/ D 1 kn1 Z E x p1 k 1 1 :::x pn1 k 1 n1 .1 x1 ::: xn1/ pn k 1dx1:::dxn1: (38) ˇk.p/ D ˇk.p1; :::; pn/ D 1 kn1 Z E x p1 k 1 1 :::x pn1 k 1 n1 .1 x1 ::: xn1/ pn k 1dx1:::dxn1: (38) (38) According to the relation (37), ˇk.p1; :::pn/ is symmetric in p1; :::; pn and it is worth-while to verify directly that the integral on R.H.S of the equation (38) has the same property. Symmetry in p1; :::; pn1 is made evident by simply relabeling the variables of integration, so it sufﬁces to prove symmetry in p1 and pn. We deﬁne (42) xn D 1 x1 ::: xn1 (42) and change the variables from x1; :::; xn1 to x2; :::; xn. The jacobian determinant of the transformation has unit magnitude and the region of integration changes from and change the variables from x1; :::; xn1 to x2; :::; xn. The jacobian determinant of the transformation has unit magnitude and the region of integration changes from f.x1; :::; xn1/ W x1 0; :::; xn1 0; x1 C ::: C xn1 1g (43) (43) to f.x2; :::; xn/ W x2 0; :::; xn 0; x2 C ::: C xn 1g: (44) (44) The last two inequalities in (44) results from the last and ﬁrst in (43). Thus the region of integration is again E in terms of the new variables and integral on R.H.S of the equation (38) becomes 1 kn1 Z E .1 x2 ::: xn/ p1 k 1x p2 k 1 2 :::x pn k 1 n dx2:::dxn: (45) (45) The complete symmetry in 1; :::; n can be exhibited by writing the n1-fold integral in the relation (38) as n-fold integral with a Dirac delta function in the integrand ˇk.p/ D 1 kn1 1 Z 0 1 Z 0 ::: 1 Z 0 x p1 k 1 1 :::x pn k 1 n ı.1 x1 ::: xn/dx1:::dxn: (46) (46) Now we discuss some properties of k-beta function of several variables. Now we discuss some properties of k-beta function of several variables. Proposition 3.4. For Re.x/; Re.y/ > 0 and k > 0, the k-beta function of two variables satisfy the following properties: Proposition 3.4. For Re.x/; Re.y/ > 0 and k > 0, the k-beta function of two variables satisfy the following properties: p p (i) ˇk.x; y/ D R 1 0 s x k 1 .sC1/ xCy k ds p p (i) ˇk.x; y/ D R 1 0 s x k 1 .sC1/ xCy k ds (ii) ˇk.x; y/ D 2 k R =2 0 .cos / 2x k 1.sin / 2y k 1d (iii) k.b a/ xCy k 1ˇk.x; y/ D R b a .s a/ x k 1.b s/ y k p p (i) ˇk.x; y/ D R 1 0 s x k 1 .sC1/ xCy k ds (ii) ˇk.x; y/ D 2 k R =2 0 .cos / 2x k 1.sin / 2y k 1d (iii) k.b a/ xCy k 1ˇk.x; y/ D R b a .s a/ x k 1.b s/ y k 1ds: Proof. By changing the variables as t D s sC1, t D cos2 or t D sin2 and t D sa ba respectively in the relation (26), we have (i), (ii) and (iii). Proof. By changing the variables as t D s sC1, t D cos2 or t D sin2 and t D sa ba respectively in the relation (26), we have (i), (ii) and (iii). Corollary 3.5. For k > 0, we have ˇk. k 2 ; k 2 / D k and hence the values of k-beta function can be computed at particular points. Corollary 3.5. For k > 0, we have ˇk. k 2 ; k 2 / D k and hence the values of k-beta function can be computed at particular points. Proof. Properties of k-beta function with several variables 315 Relabeling xn as x1 changes it to the original form with p1 and pn interchanged. Hence the original integral is symmetric in p1 and pn. We may think of the variables p1; :::; pn as being attached to the vertices of the simplex E D En1 as follows: p1 k to the vertex .1; 0; :::; 0/, p2 k to .0; 1; :::; 0/,..., pn1 k to .0; 0; :::; 1/ and pn k to the .0; 0; :::; 0/. Also, the vertex at the origin is at an equal footing with all other vertices. The points .x1; :::; xn1/ of En1 are in (1-1) correspondence with n-tuples .x1; :::; xn1; 1 x1 ::: xn1/ of non negative weights with unit sum and the last of these weights is the quantity xn deﬁned in (42) which has the value unity at the vertex .0; 0; :::; 0/. The complete symmetry in 1; :::; n can be exhibited by writing the n1-fold integral in the relation (38) as n-fold integral with a Dirac delta function in the integrand Relabeling xn as x1 changes it to the original form with p1 and pn interchanged. Hence the original integral is symmetric in p1 and pn. We may think of the variables p1; :::; pn as being attached to the vertices of the simplex E D En1 as follows: p1 k to the vertex .1; 0; :::; 0/, p2 k to .0; 1; :::; 0/,..., pn1 k to .0; 0; :::; 1/ and pn k to the .0; 0; :::; 0/. Also, the vertex at the origin is at an equal footing with all other vertices. The points .x1; :::; xn1/ of En1 are in (1-1) correspondence with n-tuples .x1; :::; xn1; 1 x1 ::: xn1/ of non negative weights with unit sum and the last of these weights is the quantity xn deﬁned in (42) which has the value unity at the vertex .0; 0; :::; 0/. 316 A. Rehman et al. Remarks. From the corollary 3.5 it seems that k is any natural number. However, we can compute the values of k-beta function for all real numbers k > 0. From the above results, if k D 1, we conclude an important result [2]. ˇ1.1 2; 1 2/ 1 2 D .1 2/ D p: (48) (48) Proposition 3.7. If k > 0; Re.x/ > 0; Re.x C y/ > 0 and Re.z/ > 0, then k-beta function of three variables can be written as Proposition 3.7. If k > 0; Re.x/ > 0; Re.x C y/ > 0 and Re.z/ > 0, then k-beta function of three variables can be written as ˇk.x; y/ˇk.x C y; z/ D ˇk.x; y; z/: (49) (49) Proof. From the relation (25), we see that for three variables Proof. From the relation (25), we see that for three variables ˇk.x C y; z/ D k.x C y/k.z/ k.x C y C z/ ; Re.x C y/ > 0; Re.z/ > 0: (50) (50) Multiplying equations (25) and (50) we have Multiplying equations (25) and (50) we have ˇk.x; y/ˇk.x C y; z/ D k.x/k.y/ k.x C y/ k.x C y/k.z/ k.x C y C z/ D k.x/k.y/k.z/ k.x C y C z/ : (51) (51) Comparing the relation (51) with deﬁnition of k-beta function of several variables (37) implies the desired Comparing the relation (51) with deﬁnition of k-beta function of several variables (37) implies the desired proof. Comparing the relation (51) with deﬁnition of k-beta function of several variables (37) implies the desired proof. Remarks. The relation (49) can be extended up to n variables. Here, we introduced the new notation for beta function of several variables in right hand side of relation (49). If there are only two variables, we get the deﬁnition of k-beta function deﬁned in [4] and if k D 1, the classical one. Theorem 3.8. If there are n variables, then k-gamma and k-beta functions are related as k.k n/ D n r ˇk k n; k n; :::; k n (52) (52) Proof. Using p1 D p2 D ::: D pn D k n in the deﬁnition (37), we have Proof. Using p1 D p2 D ::: D pn D k n in the deﬁnition (37), we have ˇk k n; k n; :::; k n D k. k n/k. k n/:::k. By Proposition 3.4, putting x D y D k 2 , we have Proof. By Proposition 3.4, putting x D y D k 2 , we have ˇk.k 2 ; k 2 / D 2 k =2 Z 0 .cos /0.sin /0d D 2 k : 2 D k : (47) (47) Now, we can compute the values of k-beta function from equation (47) by using k D 1; 2; 3; :::; n as Now, we can compute the values of k-beta function from equation (47) by using k D 1; 2; 3; :::; n as ˇ1.1 2; 1 2/ D ˇ2.1; 1/ D 2 ˇ3.3 2; 3 2/ D 3 ::: ::: ˇn.n 2 ; n 2 / D n ˇ2n.n; n/ D 2n: ˇ1.1 2; 1 2/ D ˇ2.1; 1/ D 2 ˇ3.3 2; 3 2/ D 3 ::: ::: ˇn.n 2 ; n 2 / D n ˇ2n.n; n/ D 2n: Corollary 3.6. Using the relation (25) on left hand side of the relation (47), we get Corollary 3.6. Using the relation (25) on left hand side of the relation (47), we get k. k 2 /k. k 2 / k.k/ D k ) k.k 2 / D r k ; which is a conclusion of the relation (31). which is a conclusion of the relation (31). which is a conclusion of the relation (31). which is a conclusion of the relation (31). A. Rehman et al. 316 Properties of k-beta function with several variables 317 Proof. From Proposition 3.3, we see that the integral form of k-beta function of three variables is Proof. From Proposition 3.3, we see that the integral form of k-beta function of three variables is ˇk.x; y; z/ D 1 k2 Z E p x k 1q y k 1.1 p q/ z k 1dpdq: ˇk.x; y; z/ D 1 k2 Z p x k 1q y k 1.1 p q/ z k 1dpdq: Setting v D 1 p, the above equation takes the form Setting v D 1 p, the above equation takes the form ˇk.x; y; z/ D 1 k2 Z p x k 1dp v Z 0 q y k 1.v q/ z k 1dq; putting q D vt implies dq D vdt and limit of integration becomes 0 to 1. Thus we have ˇk.x; y; z/ D 1 k Z p x k 1dp 1 k 1 Z 0 v yCz k 1q y k 1.1 q/ z k 1dt D 1 k Z p x k 1v yCz k 1dpˇk.y; z/ and by similar arguments, we conclude that ˇk.x; y; z/ D ˇk.y; z/ 1 k 1 Z 0 p x k 1.1 p/ yCz k 1dp D ˇk.y; z/ˇk.x; y C z/: Using the Proposition 3.4 on right hand side of above equation, we have ˇk.x; y; z/ D 2 k =2 Z 0 .cos / 2x k 1.sin / 2yC2z k 1d: 2 k =2 Z 0 .cos / 2y k 1.sin / 2z k 1d ) ˇk.x; y; z/ D 2 k 2 =2 Z 0 =2 Z 0 .cos / 2.xCy/ k 2.sin / 2.yC2z/ k 2d 2: Similarly, we can prove the relation (54). Similarly, we can prove the relation (54). Remarks. We can extend the results of Theorem 3.10 up to n variables and can be expressed as Remarks. 316 A. Rehman et al. k n/ k k n C k n C ::: C k n D h k. k n/ in k.k/ ; ˇk k n; k n; :::; k n D k. k n/k. k n/:::k. k n/ k k n C k n C ::: C k n D h k. k n/ i k.k/ ; and using k.k/ D 1, we have k.k n/ D n r ˇk k n; k n; :::; k n and using k.k/ D 1, we have k.k n/ D n r ˇk k n; k n; :::; k n and using k.k/ D 1, we have and using k.k/ D 1, we have g . / k.k n/ D n r ˇk k n; k n; :::; k n k.k n/ D n r ˇk k n; k n; :::; k n Corollary 3.9. If k D 1; n D 2 .i.e, there are only two variables we have a classical result Corollary 3.9. If k D 1; n D 2 .i.e, there are only two variables we have a classical result .1 2/ D r ˇ 1 2; 1 2 D p Theorem 3.10. If Re.x/ > 0; Re.x C y/ > 0; Re.x C y C z/ > 0 and Re.w/ > 0, then we have the following properties of k-beta function with three and four variables. Theorem 3.10. If Re.x/ > 0; Re.x C y/ > 0; Re.x C y C z/ > 0 and Re.w/ > 0, then we have the following properties of k-beta function with three and four variables. ˇk.x; y; z/ D 2 k 2 =2 Z 0 =2 Z 0 .cos / 2.xCy/ k 2.sin / 2.yC2z/ k 2d 2 (53) (53) and ˇk.x; y; z; w/ D 2 k 3 =2 Z 0 =2 Z 0 =2 Z 0 .cos / 2.xCyCz/ k 3.sin / 2.yC2zC3w/ k 3d 3: (54) (54) We can extend the results of Theorem 3.10 up to n variables and can be expressed as ˇk.x1; x2; :::; xn/ D 2 k n1 =2 Z 0 =2 Z 0 ::: =2 Z 0 ˇk.x1; x2; :::; xn/ D 2 k n1 =2 Z 0 =2 Z 0 ::: =2 Z 0 .cos / 2.x1Cx2C:::Cxn1/ k .n1/.sin / 2.x2C2x3C:::C.n1/xn/ k .n1/d .n1/ (55) .cos / 2.x1Cx2C:::Cxn1/ k .n1/.sin / 2.x2C2x3C:::C.n1/xn/ k .n1/d .n1/ (55) (55) roved like the Theorem 3.10. which can be proved like the Theorem 3.10. which can be proved like the Theorem 3.10. Proposition 3.11. If m1; m2; :::; mn 2 N; k > 0 and ˛ ¤ 0, then we have the following properties of the k-beta function with n variables. Proposition 3.11. If m1; m2; :::; mn 2 N; k > 0 and ˛ ¤ 0, then we have the following properties of the k-beta function with n variables. ˇk.˛; m1 C k; m2 C k; :::; mn C k/ D m1m2:::mn .˛/n;k ˇk.m1; m2; :::; mn; ˛ C nk/ (56) ˇk.˛; m1 C k; m2 C k; :::; mn C k/ D m1m2:::mn .˛/n;k ˇk.m1; m2; :::; mn; ˛ C nk/ (56) (56) where .˛/n;k is the Pochhammer k-symbol deﬁned in the relation (22), where .˛/n;k is the Pochhammer k-symbol deﬁned in the relation (22), ˇk.m1 C k; m2 C k; m3 C k; :::; mn C k/ D ˇk.m1 C k; m2 C k; m3 C k; :::; mn C k/ D m1m2m3:::mn If k is added to any one of the variables, then following results hold (only three variables are provided here). pˇk.p; q; r C k/ D rˇk.p C k; q; r/ qˇk.p C k; q; r/ D pˇk.p; q C k; r/ pˇk.p; q; r C k/ D rˇk.p C k; q; r/ qˇk.p C k; q; r/ D pˇk.p; q C k; r/ and qˇk.p; q; r C k/ D rˇk.p; q C k; r/: qˇk.p; q; r C k/ D rˇk.p; q C k; r/: Just use the deﬁnition 37 along with the result k.x C k/ D xk.x/. Proof. Just use the deﬁnition 37 along with the result k.x C k/ D xk.x/. Here, we give some properties of k-gamma function which will be used to prove some results involving k-beta function of several variables. Lemma 3.13. If r 1 2 N, then we have Lemma 3.13. If r 1 2 N, then we have 1 r 2 r 3 r ::: r 1 r D r 1 2 .2/ .r1/ 2 : (59) (59) (59) Proposition 3.14. If r 1 2 N; k > 0, then we have Proposition 3.14. If r 1 2 N; k > 0, then we have k k r k 2k r k 3k r :::k .r 1/k r D k .1r/ 2 r 1 2 .2/ .r1/ 2 (60) (60) Proof. Replacing x by k r ; 2k r ; :::; .r1/k r and rk r in the relation (24) respectively, we have Proof. Replacing x by k r ; 2k r ; :::; .r1/k r and rk r in the relation (24) respectively, we have Proof. Replacing x by k r ; 2k r ; :::; .r1/k r and rk r in the relation (24) respectively, we have Proof. m1m2m3:::mn m1m2m3:::mn .m1 C ::: C mn/.m1 C ::: C mn C k/:::.m1 C ::: C mn C .n 1/k/ˇk.m1; m2; :::; mn/ (57) (57) and ˇk.m1 C k; m2; :::; mn/ C ˇk.m1; m2 C k; :::; mn/ C ::: C ˇk.m1; m2; :::; mn C k/ D ˇk.m1; m2; :::; mn/ (58) 318 A. Rehman et al. 318 Proof. Using the deﬁnition of k-beta function of several variables (37), we have Proof. Using the deﬁnition of k-beta function of several variables (37), we have ˇk.˛; m1 C k; m2 C k; :::; mn C k/ D k.˛/k.m1 C k/k.m2 C k/:::k.mn C k/ k.˛ C m1 C m2 C ::: C mn C nk/ : Use of k.x C k/ D xk.x/ in R.H.S of the above relation gives D m1m2:::mnk.˛/k.m1/k.m2/:::k.mn/ k.˛ C m1 C m2 C ::: C mn C nk/ : Multiplying the numerator and denominator by k.˛ C nk/ implies D m1m2:::mnk.˛/k.m1/k.m2/:::k.mn/:k.˛ C nk/ k.˛ C m1 C m2 C ::: C mn C nk/k.˛ C nk/ ; and by the property of Pochhammer k- symbol, .˛/n;k D k.˛Cnk/ k.˛/ , the above equation creates the desired result (56). To prove the relation (57), we use the deﬁnition (37) and the relation (28) in the numerator and denominator, and we conclude the required result. For (58), we use the deﬁnition (3.17) and proceed as ˇk.m1 C k; m2; :::; mn/ C ˇk.m1; m2 C k; :::; mn/ C ::: C ˇk.m1; m2; :::; mn C k/ D k.m1 C k/:::k.mn/ k.m1 C ::: C mn C k/ C k.m1/k.m2 C k/:::k.mn/ k.m1 C ::: C mn C k/ C ::: C k.m1/:::k.mn C k/ k.m1 C ::: C mn C k/; using the relation (28), we obtain the required relation (58). ˇk.m1 C k; m2; :::; mn/ C ˇk.m1; m2 C k; :::; mn/ C ::: C ˇk.m1; m2; :::; mn C k/ D k.m1 C k/:::k.mn/ k.m1 C ::: C mn C k/ C k.m1/k.m2 C k/:::k.mn/ k.m1 C ::: C mn C k/ C ::: C k.m1/:::k.mn C k/ k.m1 C ::: C mn C k/ and using the relation (28), we obtain the required relation (58). and using the relation (28), we obtain the required relation (58). Corollary 3.12. If k is added to any one of the variables, then following results hold (only three variables are provided here). Corollary 3.12. m1m2m3:::mn If z 2 C n f0; 1; 2; :::g, then k-beta function of n variables, n 2 satisﬁes the identity n nz k 1ˇk.z; z; :::; z/ D ˇk z; k n ˇk z; 2k n :::ˇk z; .n 1/k n : (61) Theorem 3.16. If z 2 C n f0; 1; 2; :::g, then k-beta function of n variables, n 2 satisﬁes the identity n nz k 1ˇk.z; z; :::; z/ D ˇk z; k n ˇk z; 2k n :::ˇk z; .n 1/k n : (61) orem 3.16. If z 2 C n f0; 1; 2; :::g, then k-beta function of n variables, n 2 satisﬁes the identity n nz k 1ˇk.z; z; :::; z/ D ˇk z; k n ˇk z; 2k n :::ˇk z; .n 1/k n : (61) (61) Proof. Using the relation (25) on R.H.S of above equation (61), we have ˇk z; k n :::ˇk z; .n 1/k n D k.z/k. k n/ k.z C k n/ k.z/k. 2k n / k.z C 2k n / :::k.z/k. .n1/k n / k.z C .n1/k n / D .k.z//nk. k n/k. 2k n /:::k. .n1/k n / k.z/k.z C k n/k.z C 2k n /:::k.z C .n1/k n / : By the Proposition 3.14 and Lemma 3.15, we obtain the required proof. By the Proposition 3.14 and Lemma 3.15, we obtain the required proof. By the Proposition 3.14 and Lemma 3.15, we obtain the required proof. Lately, Kokologiannaki [5] worked on properties and inequalities of generalized k-gamma, k-beta and zeta functions and proved that the function k.x/ satisﬁes the identity Lately, Kokologiannaki [5] worked on properties and inequalities of generalized k-gamma, k-beta and zeta functions and proved that the function k.x/ satisﬁes the identity k.2x/ D r k 22 x k k.x/k.x C k 2 /; for x 2 C and Re.x/ > 0. Here, we extend this result. Theorem 3.17. For x 2 C and Re.x/ > 0, and n 2 N, prove that Theorem 3.17. For x 2 C and Re.x/ > 0, and n 2 N, prove that Theorem 3.17. For x 2 C and Re.x/ > 0, and n 2 N, prove that Theorem 3.17. m1m2m3:::mn Replacing x by k r ; 2k r ; :::; .r1/k r and rk r in the relation (24) respectively, we have k.k r / D k 1 r 1.1 r / ; k.2k r / D k 2 r 1.2 r / k..r 1/k r / D k r1 r 1.r 1 r / ::: k.rk r / D k11.1/ D 1: k.k r / D k 1 r 1.1 r / ; k.2k r / D k 2 r 1.2 r / k.k r / D k 1 r 1.1 r / ; k.2k r / D k 2 r 1.2 r / k..r 1/k r / D k r1 r 1.r 1 r / ::: k.rk r / D k11.1/ D 1: Multiplying all above equations and applying the Lemma 3.13, we get Multiplying all above equations and applying the Lemma 3.13, we get k k r k 2k r :::k .r 1/k r D k 1 r C 2 r C:::C r1 r .r1/.1 r /.2 r /:::.r 1 r / D k 1C2C3C:::C.r1/ r .r1/r 1 2 .2/ .r1/ 2 D k .1r/ 2 r 1 2 .2/ .r1/ 2 : D k 1C2C3C:::C.r1/ r .r1/r 1 2 .2/ .r1/ 2 D k .1r/ 2 r 1 2 .2/ .r1/ 2 : Properties of k-beta function with several variables 319 Lemma 3.15. If r 1 2 N; k > 0, and r 2 C n f0; k; 2k; :::g, then we have k.rx/ D r rx k 1 2 k .r1/ 2 .2/ .1r/ 2 k.x/k.x C k r /k.x C 2k r /:::k.x C .r 1/k r /: Remarks. The above Lemma 3.15 is the k-analogue of Gauss multiplication theorem. If we use r D 2, we have k- analogue of Legendre duplication formula proved in [5]. Also, if k D 1, we have the classical Gauss multiplication and Legendre duplication theorems [12]. Remarks. The above Lemma 3.15 is the k-analogue of Gauss multiplication theorem. If we use r D 2, we have k- analogue of Legendre duplication formula proved in [5]. Also, if k D 1, we have the classical Gauss multiplication and Legendre duplication theorems [12]. Theorem 3.16. m1m2m3:::mn For x 2 C and Re.x/ > 0, and n 2 N, prove that k.2nx/ D r k 2 2nx k 1k.2n1x/k.2n1x C k 2 /: (62) (62) Proof. The integral form of k-beta function in equation (26) is Proof. The integral form of k-beta function in equation (26) is Proof. The integral form of k-beta function in equation (26) is ˇk.x; y/ D 1 k 1 Z 0 t x k 1.1 t/ y k 1dt: ˇk.x; y/ D 1 k 1 Z 0 t x k 1.1 t/ y k 1dt: Replacing x and y by 2n1x, in the above equation, we have Replacing x and y by 2n1x, in the above equation, we have Replacing x and y by 2n1x, in the above equation, we have Replacing x and y by 2n1x, in the above equation, we have ˇk.2n1x; 2n1x/ D 1 k 1 Z 0 t 2n1x k 1.1 t/ 2n1x k 1dt; (63) (63) and changing the variable as t D 1Cs 2 which implies that dt D 1 2ds. Equation (63) gives ˇk.2n1x; 2n1x/ DD 2 k 1 Z 0 .1 s2 22 / 2n1x k 1 1 2ds: By setting s2 D v ) 2sds D dv, we have ˇk.2n1x; 2n1x/ D 1 k 1 2 2nx k 1 1 Z 0 v 1 2 1.1 v/ 2n1x k 1dv D 1 2 2nx k 1 1 k ˇ.2n1x k ; 1 2/: ˇk.2n1x; 2n1x/ DD 2 k 1 Z 0 .1 s2 22 / 2n1x k 1 1 2ds: By setting s2 D v ) 2sds D dv, we have By setting s2 D v ) 2sds D dv, we have By setting s2 D v ) 2sds D dv, we have ˇk.2n1x; 2n1x/ D 1 k 1 2 2nx k 1 1 Z 0 v 1 2 1.1 v/ 2n1x k 1dv D 1 2 2nx k 1 1 k ˇ.2n1x k ; 1 2/: ˇk.2n1x; 2n1x/ D 1 k 1 2 2nx k 1 1 Z 0 v 1 2 1.1 v/ 2n1x k 1dv D 1 2 2nx k 1 1 k ˇ.2n1x k ; 1 2/: A. Rehman et al. 320 A. Rehman et al. 320 320 A. Rehman et al. m1m2m3:::mn We obtain the following expression by using equation (34) We obtain the following expression by using equation (34) ˇk.2n1x; 2n1x/ D 1 2 2nx k 1 ˇk.2n1x; k 2 /: (64) ˇk.2n1x; 2n1x/ D 1 2 2nx k 1 ˇk.2n1x; k 2 /: (64) (64) By applying the deﬁnition of k-beta function as given in equation (25) on both sides of equation (64), we have By applying the deﬁnition of k-beta function as given in equation (25) on both sides of equation (64), we have k.2n1x/:k.2n1x/ k.2nx/ D 1 2 2nx k 1 k.2n1x/:k. k 2 / k.2n1x C k 2 / ; which implies k.2nx/ D 2 2nx k 1k.2n1x/:k.2n1x C k 2 / k. k 2 / : By using the Corollary 3.5, we get k.2nx/ D r k 2 2nx k 1k.2n1x/k.2n1x C k 2 /: Remarks. The Theorem 3.17 is the generalization of the Legendre’s duplication formula of .x/ in [12]. By using n D 1, we ﬁnd a formula k.2x/ which is proved in [5]. Further, if we use k D 1 and n D 0 in the result obtained, we get the classical gamma function and the product form of the above Theorem is given by Remarks. The Theorem 3.17 is the generalization of the Legendre’s duplication formula of .x/ in [12]. By using n D 1, we ﬁnd a formula k.2x/ which is proved in [5]. Further, if we use k D 1 and n D 0 in the result obtained, we get the classical gamma function and the product form of the above Theorem is given by k.2nx/ D . k / n 2 n Y iD1 2 2i x k 1k.x/k.2i1x C k 2 /; n 2 N: Acknowledgement: The authors would like to express profound gratitude to referees for deeper review of this paper and the referee’s useful suggestions that led to an improved presentation of the paper. References [1] Anderson G.D., Vamanmurthy M.K., Vuorinen M.K., Conformal Invarients, Inequalities and Quasiconformal Maps, Wiley, New York, 1997 [1] Anderson G.D., Vamanmurthy M.K., Vuorinen M.K., Conformal Invarients, Inequalities and Quasiconformal Maps, Wiley, New York, 1997 [2] Andrews G.E., Askey R., Roy R., Special Functions Encyclopedia of Mathemaics and its Application 71, Cambridge University Press, 1999 [2] Andrews G.E., Askey R., Roy R., Special Functions Encyclopedia of Mathemaics and its Application 71, Cambridge University Press, 1999 [3] Carlson B.C., Special Functions of Applied Mathemaics, Academic Press, New York, 1977 [4] Diaz R., Pariguan E., On hypergeometric functions and k-Pochhammer symbol, Divulgaciones Mathematics, 200 192 [5] Kokologiannaki C.G., Properties and inequalities of generalized k-gamma, beta and zeta functions, International Journal of Contemp, Math. Sciences, 2010, 5(14), 653-660 [6] Kokologiannaki C.G., Krasniqi V., Some properties of k-gamma function, LE MATHEMATICS, 2013, LXVIII, 13-22 [7] Krasniqi V., A limit for the k-gamma and k-beta function, Int. Math. Forum, 2010, 5(33), 1613-1617 [8] Mansoor M., Determining the k-generalized gamma function k.x/, by functional equations, International Journal Contemp. Math. Sciences, 2009, 4(21), 1037-1042 [9] Mubeen S., Habibullah G.M., An integral representation of some k-hypergeometric functions, Int. Math. Forum, 2012, 7(4), 203 207 [10] Mubeen S., Habibullah G.M., k-Fractional integrals and applications, International Journal of Mathematics and Sc 7(2), 89-94 ( ), [11] Mubeen S., Rehman A., Shaheen F., Properties of k-gamma, k-beta and k-psi functions, Bothalia Journal, 2014, 4, 371-379 [11] Mubeen S., Rehman A., Shaheen F., Properties of k-gamma, k-beta and k-psi functions, Bothalia Journal, 2014, 4, 371-379 [11] Mubeen S., Rehman A., Shaheen F., Properties of k-gamma, k-beta and k-psi functions, Bothalia Journa [12] Rainville E.D., Special Functions, The Macmillan Company, New Yark(USA), 1960 [13] Rudin W., Real and Complex Analysis, 2nd edition McGraw-Hill, New York, 1974
https://openalex.org/W2993015089	https://www.intechopen.com/citation-pdf-url/70321	English	null	Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine	IntechOpen eBooks	2,020	cc-by	5,830	Nabil Sayme Nabil Sayme Abstract Cryopreservation is a worldwide technique that makes it possible to preserve different living cells and tissues, including male and female gametes and embryos, in a structurally intact state using low temperature over time. Since the starting point of the cryopreservation era in 1776, until today, this was one of the most important steps in assisted reproductive techniques. Conventional slow freezing of spermatozoa is commonly used for cryopreservation of both ejaculated and surgi- cally retrieved spermatozoa. The technique of the slow freezing is principally based on dehydration of cells which is performed through slow cooling combined with low concentrations of a cryoprotectant agent for achieving a balance. Besides of slow freezing, for more than a decade, many reports suggest the sperm vitrification tech- nique as an alternative to slow freezing. Contrary to the slow freezing method, with vitrification, the effects of the cryoprotectants in spermatozoa are eliminated since this method is cryoprotectant-free. All of these interesting and promising protocols of vitrification, however, have not been implemented in the lab routine yet, and slow freezing remains the standard cryopreservation method in most laboratories worldwide. Keywords: cryopreservation, vitrification, permeable and non-permeable cryoprotectants, human spermatozoa 1. Introduction Generally, sperm cryopreservation when correctly performed allows long-term storage and usage when needed, which is one of the most essential parts in assisted reproductive techniques. p q Complete cryopreservation as a technique consists three steps: the first one is a collection of the sample, the second is the freezing procedure, and the third is the storage part in liquid nitrogen [9]. For the male gamete cryopreservation, collecting a sample can be divided into collecting ejaculated spermatozoa, collecting epididy- mal spermatozoa, and collecting testicular spermatozoa. The first one, collecting ejaculated spermatozoa, should be always the first option when the patient is capable to provide a sample with adequate sperm viability; since intracytoplasmic sperm injection (ICSI) allows that sperm is directly injected inside of the oocyte, minimum requirement for semen parameters do not exist. On the other hand, the complete absence of sperm in the ejaculate following 2–3 days of abstinence on at least two occasions is the standard used to confirm the diagnosis of azoospermia; in this case, epididymal and testicular collecting of spermatozoa are usual procedures [9, 10]. Percutaneous epididymal sperm aspiration (PESA) does not require a surgi- cal incision. A small needle is going directly into the head of the epididymis through the scrotal skin, and fluid is aspirated and examined for the presence of motile sperm. On the other side, microsurgical epididymal sperm aspiration (MESA) involves dissection of the epididymis under the operating microscope and incision of a single tubule. Fluid spills from the epididymal tubule and then is aspirated. Epididymal spermatozoa are mature and progressively motile, and epididymal aspirates are much cleaner and devoid of the cellular debris that is seen in testicular sperm preparations [10]. For the testicular sperm extraction (TESE) procedures, different techniques have been developed and compared. The microsurgical TESE seems to have the highest sperm retrieval rate and may limit damage to the testicu- lar tissue. Spermatozoa can be retrieved from tubules that are dilated, and this can be visualized with an operating microscope. This technique needs microsurgical skills and general anesthesia is usually required. An open biopsy also allows the excision of a larger tissue mass, allowing access to a greater number of sperm avail- able for freezing [10, 11]. Besides these freezing steps, the thawing procedure is an equivalently important step. Spermatozoa must be allowed to retrieve its normal biological activities. 1. Introduction Cryopreservation, derived from the Greek word κρύος (krýos, “icy cold, chill, frost”), is a worldwide technique that makes it possible to preserve differ- ent living cells and tissues, including male and female gametes and embryos, in a structurally intact state using low temperature over time. It has become an indispensable part of most human-assisted reproductive technology (ART) programs around the world. Since the starting point of the cryopreservation era in 1776, Spallanzani [1] used snow to freeze spermatozoa and assess their motility after thawing. One century later, Mantegazza (reported by Curry [2]) observed that human sperm survived frozen at −17°C for more than 4 days. Since the late 1930–1940s [3, 4], scientists have effectively cryopreserved spermatozoa of several mammalian species, especially bovine and human. In 1947, glycerol was rediscovered as a cryoprotective agent allowing to freeze viable spermatozoa for longer periods. Bernstein and Petropavlovski [3] demonstrated the positive effect 1 Cryopreservation - Current Advances and Evaluations of 1 mol/l glycerol on spermatozoa frozen to −21°C in the rabbit, guinea pig, bull, ram, stallion, and boar. After that in the late 1940s, the results of experiments based on the use of glycerol by Polge et al. [4] in the United Kingdom (1949) were published. The first piglet born from frozen–thawed porcine sperm in 1957 showed that the fertilizing potential could also be preserved and no major harm to the genetic apparatus was done [5]. Some cryopreservation methods developed in the 1950s are in use till now. However, the real success of cryopreservation was achieved in the 1970s with the introduction of dry ice and liquid nitrogen vapor. Following the report published in 1964, for the first time, human spermatozoa were successfully cryopreserved [6]. Definitely, this was one of the most important steps in assisted reproductive techniques, since the preservation of male sample in some conditions is the only opportunity to preserve fertility for some couples. Besides that, sperm cryopreser- vation is also considered as a rescue option for cases prior to radiotherapy and/or chemotherapy [7] in cancer patients, prior to any other medical procedure that may potentially lead to testicular failure or ejaculatory dysfunction as vasectomy [8], as well as in cases of traveling husbands serving in the military or absent partners. Even in pediatric oncologic cases, in which testicular tissue can be preserved, the cryopreservation of immature testicular tissue for later fertilization purposes seems also advisable. 1. Introduction Generally speaking, at the present time, all the cryopreserva- tion protocols use a thawing temperature of 37°C for 10 or 15 min [12]. 2 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 2. The conventional “slow freezing” method g g The cell further downgrades as well through consequent thawing and rewarm- ing that affect their viability by possible excessive osmotic swelling. Chemical and physical damage are also caused affecting the sperm cell membranes as a result of expanded lipid peroxidation, due to the production of reactive oxygen species [20] which also lead to loss of sperm motility [21]. In order to prevent all the previously reported damages, during the time, different cryoprotectants were developed. Cryoprotectants are low-molecular-weight and highly permeable chemicals used to protect spermatozoa from freeze damage by ice crystallization. There are four main well-known cryoprotectants: glycerol, ethylene glycol, dimethyl sulfoxide, and 1,2-propanediol. Cryoprotectants decrease the freezing point of a sample, reduce the amount of salts and solutes present in the liquid phase of the sample, and decrease ice formation within the spermatozoa [22]. Usually, the cryoprotectants are added in an equal volume of semen in a dropwise manner, gently mixed at room temperature, and then placed at 37°C for 10–15 min to allow for proper equilibra- tion between the cells and the medium [23]. Besides the permeable cryoprotectants, there are non-permeable agents, such as raffinose, sucrose, egg yolk citrate, albu- min, polyethylene glycol, and polyvinylpyrrolidone, which are common additives that cannot pass through the plasma membrane but have antioxidant effects and are used to improve post-thaw sperm functional parameters such as post-thaw motility, viability, and reducing DNA damage [12]. Presently, slow freezing techniques have g g The cell further downgrades as well through consequent thawing and rewarm- ing that affect their viability by possible excessive osmotic swelling. Chemical and physical damage are also caused affecting the sperm cell membranes as a result of expanded lipid peroxidation, due to the production of reactive oxygen species [20] which also lead to loss of sperm motility [21]. In order to prevent all the previously reported damages, during the time, different cryoprotectants were developed. p g g y p p Cryoprotectants are low-molecular-weight and highly permeable chemicals used to protect spermatozoa from freeze damage by ice crystallization. There are four main well-known cryoprotectants: glycerol, ethylene glycol, dimethyl sulfoxide, and 1,2-propanediol. Cryoprotectants decrease the freezing point of a sample, reduce the amount of salts and solutes present in the liquid phase of the sample, and decrease ice formation within the spermatozoa [22]. 2. The conventional “slow freezing” method Conventional slow freezing of spermatozoa is commonly used for cryopreser- vation of both ejaculated and surgically retrieved spermatozoa. The technique of slow freezing is principally based on dehydration of cells [13] which is performed through slow cooling combined with low concentrations of a cryoprotectant agent for achieving a balance. Slow freezing of spermatozoa is commonly performed by stepwise manual or continuous programmed freezing of vials or straws, containing a mixture of cryoprotectants and spermatozoa, to subzero temperatures [14]. The manual method is performed by simultaneously decreasing the temperature of the semen while adding a cryoprotectant in a stepwise manner and after immers- ing the samples into liquid nitrogen [15]. Mahadevan et al. [16] reported that the optimal initial cooling rate of the specimen from room temperature to 5°C is 0.5–1°C/min. The sample is then frozen from 5°C to −80°C at a rate of 1–10°C/min and then submerged into liquid nitrogen at −196°C. Despite the fact many research presented successful sperm freezing with manual techniques, the reproducibility of this procedure pointed out some problems. For this reason, programmable freezers have been investigated [17]. These devices generally use a plate to hold the straws; these are cooled by liquid nitrogen held in a storage tank under the plate. Liquid nitrogen is poured into the tank, and the machine, once programmed, uses the software data logging to obtain cooling from 20°C to −80°C at a rate of 1.5°C/min and then at 6°C/min; at completion of the freezing, the straws are removed and stored in liquid nitrogen at −196°C. This takes about 40 min [17]. Software is simple to use and do not require continuous operator intervention and, besides that, have been used to increase the reproducibility of the freezing operations. p y g p However, many research confirmed that the cryopreserved/thawed spermatozoa lose about 50–40% of their pre-freezing motility value, with considerable fluctua- tion among samples [18]. Although, after freezing, the regression of the motility is one of the first affected parameters, the mechanism of sperm impairment and its mechanical, physical, and/or chemical etiology is still the point of discussion. The ice equilibrium in conventional slow freezing is one of the main causes of mechani- cal cell injury, which result in the formation of intra- or extracellular ice crystal, along with the osmotic damage [19]. 3. Vitrification as an alternative: new technique In the past, the first successful vitrification of frog spermatozoa was done by Luyet and Hodapp [26] and fowl spermatozoa 4 years later by Shaffner [27]. In the early 1980s, Rall and Fahy [28] managed to successfully vitrify embryos using high concentrations of permeable cryoprotective agents and a relatively low speed of cooling and warming, and since then, the main approach to vitrifying spermatozoa has been considered the same as the methods used for other types of mammalian cells [29]. Vitrification as a method is based on the ultrarapid increase and decrease of temperatures with or without the use of non-permeable cryoprotectants. During the procedure, water is cooled to a glassy state through extreme increasing of viscosity without intracellular ice crystallization making this procedure less labor- intensive, faster, and presumably safer than traditional slow freezing protocols [30]. intensive, faster, and presumably safer than traditional slow freezing protocols [30]. Contrary to the slow freezing method, the effects of the cryoprotectants in spermatozoa are eliminated since this method is cryoprotectant-free. Using this method, the sperm suspension is plunged directly into liquid nitrogen, and the sperm cells are cooled in an ultrarapid manner, known as kinetic vitrification [25]. What is more, cryoprotectant-free technique avoids the use of the classic toxic cryo- protective agents (CPA) that may have lethal effects of osmotic shock and prevent lethal intracellular ice formation and the harmful effects of high salt concentra- tions during freezing and thawing [25]. The first described cryoprotectant-free vitrification by Nawroth et al. [31] suggested that spermatozoa were located onto copper loops or into standard 0.25 ml insemination straws and plunged directly into liquid nitrogen. During the years of improving technique, different devices have been tested [30, 32, 33]. Isachenko et al. [34] compared the vapor phase and liquid phase for sperm cryopreservation, using the cryoloop, droplet, and open straw methods. Cryoloops with a film of spermatozoa suspension were cooled for 3 min in liquid nitrogen vapor at −160°C and then placed into pre-cooled cryovials (CryoTubesTM, 4.5 ml volume, 92 mm length; Nunc GmbH & Co. KG, Wiesbaden, Germany) and were stored in liquid nitrogen until the time of warming. The second developed method at that time was vitrification in liquid nitrogen vapor using droplets where sperm suspension was located onto aluminum foil previously cooled in liquid nitrogen vapor to −160°C. 2. The conventional “slow freezing” method Usually, the cryoprotectants are added in an equal volume of semen in a dropwise manner, gently mixed at room temperature, and then placed at 37°C for 10–15 min to allow for proper equilibra- tion between the cells and the medium [23]. Besides the permeable cryoprotectants, there are non-permeable agents, such as raffinose, sucrose, egg yolk citrate, albu- min, polyethylene glycol, and polyvinylpyrrolidone, which are common additives that cannot pass through the plasma membrane but have antioxidant effects and are used to improve post-thaw sperm functional parameters such as post-thaw motility, viability, and reducing DNA damage [12]. Presently, slow freezing techniques have 3 Cryopreservation - Current Advances and Evaluations been widely used in sperm cryopreservation with acceptable results for sperm vital- ity and motility after thawing [24]. However, for more than a decade, many reports suggest sperm vitrification technique as an alternative to slow freezing [25]. 3. Vitrification as an alternative: new technique During cooling, the droplet of sperm suspension adopted a spherical form, which was placed into pre-cooled cryovials and stored in liquid nitrogen. With open straw method, sperm suspension was drawn inside the end of open-pulled straws (0.25 ml) (Medical Technology GmbH, Altdorf, Germany) by capillary action [32]. Straws were placed inside other sterile 90 mm straws which were prepared from the standard 0.5 ml insemination straws (Medical Technology GmbH, Altdorf, Germany) and then hermetically closed using a handheld sealer and then plunged into liquid nitrogen. During this pro- cedure, it was described that there was no contact between the wall of the 90 mm straw and the suspension of spermatozoa inside the open-pulled straws, due to the presence of a meniscus in the suspension [34]. At the end, the results report that CPA-free cryopreservation of spermatozoa could occur in a wide range of cooling rates, but the major disadvantage of cryoprotectant-free vitrification was that only small volumes of spermatozoa (≤40 μl) could be vitrified in these systems [32]. Wh i i h i i h f i ifi i S h l p y What is more, in the aim to improve the cryoprotectant-free vitrification, Schulz et al. [35] added some carbohydrate supplements (glucose, sucrose, and trehalose) 4 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 to the sperm suspension and obtained that sucrose has increased spermatozoa motility and viability after thawing. Later on, Isachenko et al. [36] investigates the ability of sucrose to protect sperm motility, viability, mitochondrial membrane potential integrity, spontaneous capacitation, and acrosome reaction. Spermatozoa were cryoprotectant-free vitrified using three different media: human tubal fluid medium (control), human tubal fluid medium with 1% human serum albumin, and fluid medium with 1% human serum albumin and 0.25 M sucrose [36]. Obtained results in this research [36] indicate that the number of progressively motile spermatozoa was significantly higher in the sucrose-supplemented medium group (57%) than with controls (19%). The combination of fluid medium with 1% human serum albumin and sucrose (65%) has a stronger cryoprotective effect on the integrity of mitochondrial membrane potential than with human tubal fluid medium with 1% human serum albumin alone (33%). 3. Vitrification as an alternative: new technique It was concluded that cryo- protectant-free vitrification of human spermatozoa with non-permeable cryopro- tectants such as human serum albumin and sucrose can effectively cryopreserve the cells without significant loss of important physiological parameters. On the other hand, Chen et al. [37] reported that normozoospermic patient samples vitrified by the cryoprotectant-free method with or without the addition of sucrose did not show a significant difference in the sperm recovery rate and motility rate. g p y y During the years, many researchers got their attention into the vitrification of oocytes, embryos, larger cells, and even tissues [38–40]. It was established, at that time, that vitrification for oocyte and embryo could not be achieved without combinations of high concentrations of both permeable and non-permeable cryo- protectants in order to reach stable vitrification and allow using a relatively low rate of cooling and warming. Mainly dimethyl sulfoxide, propanediol, or ethylene glycol were used for ooctyte and embryo vitrification [41–43]. However, these methods also have their limitations, and especially human spermatozoa are intolerant to the high concentrations of cryoprotectants conventionally used in vitrification [44, 45]. Suggested reasons for successful vitrification in the absence of cryoprotectants for spermatozoa are the size of the cells and their relative concentration of soluble mac- romolecules. The shape and size of the sperm head could define the cryo-sensitivity of the cell. Comparative studies [46] have shown a negative correlation between the size of the sperm head and cryo-stability. Oocyte and spermatozoa naturally contain high concentrations of proteins, which help in vitrification. Thus, a higher cryoprotectant concentration is needed for extracellular vitrification than for intra- cellular vitrification [25]. It can be assumed that the amount of osmotically inactive water is also higher in spermatozoa and is combined with several macromolecular structures such as DNA, histones, etc. [47]. Extensive classification of intracellular compounds may also contribute to the successful survival of spermatozoa [48]. p y p In spite of this, vitrification of spermatozoa is still a rather unexplored method- ology, with limited studies showing its efficacy in male gametes [24, 25, 30–32]. In the last two decades, different sperm vitrification protocols have been published, most of them developed by Isachenko [30–32, 34, 36] where not only different combinations of cryoprotection but also different devices have been tested as well. 3. Vitrification as an alternative: new technique These interesting and promising protocols, however, have not been implemented in the IVF lab routine yet, and slow freezing remains the standard cryopreservation method in most laboratories worldwide [49]. 4. Our experience Since there is no optimal accepted algorithm for the vitrification procedure and no evidence has been established regarding the possibility of achieving successful 5 Cryopreservation - Current Advances and Evaluations human spermatozoa vitrification without cryoprotectant, the differences between vitrification of human sperm with and without two non-permeable cryoprotectants (NPC), sucrose (SUC), and trehalose (TRE) were investigated. For that, five human semen samples diagnosed as normal (normozoospermia) were obtained and analyzed. All samples were prepared through 80% one-step density gradient and then were diluted to a defined sperm concentration of 40 × 106 sperms per ml. following the guidelines of the World Health Organization criteria [13]. Motility and vitality of the sperm were recorded in each sample. Each sample was then divided into three parts. Each part of the samples underwent vitrification using three different methods: (i) 150 μl cryostraw filled with the sample was directly plunged into liquid nitrogen, (ii) small drops of the samples were dripped into liquid nitrogen, and (iii) microcapil- laries were plunged directly into liquid nitrogen. Each part was vitrified differently using sucrose and trehalose or without a cryoprotectant. Sucrose and trehalose were diluted in media, 500 mM, after merging it with the sperm solution concentrations till they have reached 250 mM of concentration with 20 × 106 sperms per ml. After a minimum of 24 hours of cryopreservation, samples were thawed in warm water at 37°C for 20 seconds. Then, the samples were washed in 1 ml media (SpermActive®; Gynemed) and incubated at 37°C. Motility and vitality were directly assessed, 30 and 120 min after thawing. It was found that sperms’ overall survival rates ranged from 1.2 to 37.5%. Mean survival rates for each method were as follows: method (i), 15.5 ± 5.3% for sucrose, 16.6 ± 5.2% for trehalose, and 19.8 ± 7.8% without cryoprotectant; method (ii), 47.8 ± 16.6% for sucrose, 53.8 ± 13.4% for trehalose, and 40.4 ± 7.5 without cryoprotectant; and method (iii), 10.3 ± 2.9% for sucrose, 8.2 ± 3.9% for trehalose, and 8.2 ± 3.3% without cryoprotectant (Table 1). Statistical analyses revealed significant differences only between method (ii) and method (iii) (Table 2), with a tendency for better survival rates in method (ii) under all three conditions, especially with trehalose (mean survival rate of 53.8 ± 13.4%; Table 1). Table 1. Mean survival rates for all three methods and conditions. 4. Our experience However, these differences are not representative because of the low survival and motility rate after thawing for method (iii), which might be affected by the heat sealing process (sealing small volume of the sample). No significant differences were observed regarding the addition of sucrose and trehalose or vitrification without a cryoprotectant in terms of overall survival rates (suc. vs. no cryoprotectant n = 5, p > 0.4; tre. vs. no cryoprotectant n = 5, p > 0.3; Table 2). Similarly, no significant differences were assessed between the use of sucrose and trehalose (n = 5, p > 0.7). Mean motility recovery, measured after 120 min of thawing, was 13.7 ± 5.9% for sucrose, 12.8 ± 4.2% for trehalose, and 10.1 ± 4.3% without cryoprotectant, and 6 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Table 2. Comparison of the different methods. Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Comparison of the different methods. between these three groups, there was not a significant difference. However, interestingly, a tendency of improved motility was observed during post-thawing incubation specifically in addition of cryoprotectants (Figure 1). Therefore, the choice of the vitrification method and conditions seems to influence survival rates, motility, and vitality of the sperm, but the significant difference in sperm recovery after vitrification with non-permeable cryoprotectants was not found; the reason Figure 1. Recovery of motility by post-thawing incubation. Figure 1. Figure 1. Recovery of motility by post-thawing incubation. 7 Cryopreservation - Current Advances and Evaluations that influenced the obtained result might be the small sample size. On the other hand, the improvement of the methodology of sperm vitrification could yield in positive additional effects of non-permeable cryoprotectants. Under these condi- tions, obtained data might be encouraging for further studies, to extend on a greater number of normal sperm samples as well as to those patients with reduced semen quality and fertility problems. Moreover, the increase in post-thaw sperm motility is an important aspect in the use of all assisted reproduction techniques and should be also confirmed by further studies. Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Author details Nabil Sayme Team Kinderwunsch Hannover, Hannover, Germany *Address all correspondence to: nsayme5@aol.com © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 5. Conclusion Spermatozoa cryopreservation for males is the standard fertility preservation care in patients undertaking gonadotoxic treatments, such as chemotherapy/ radiotherapy. The conventional cryopreservation, the slow freezing technique, is standardized and commonly used. However, functional sperm parameters includ- ing motility after the thawing are still challenging. New methods that preserve spermatozoa are promising, even though they still need validation before being routinely used in an assisted reproduction program, including the essential use of new cryoprotectants and new antioxidants to improve sperm quality after thawing. Moreover, spermatozoa lyophilization is another method that is still under investi- gation. However, as the spermatozoa are immotile, lyophilized sperm can only be used in ICSI. Hence, future research needs not only to investigate the optimization and safety of methods but also for the health of the offspring. 8 Author details 9 Cryopreservation - Current Advances and Evaluations References Vitro Fertilization. Switzerland: Springer International Publishing; 2019. pp. 625-639. Available from: https://doi. org/10.1007/978-3-319-43011-9_51 Vitro Fertilization. Switzerland: Springer International Publishing; 2019. pp. 625-639. Available from: https://doi. org/10.1007/978-3-319-43011-9_51 Vitro Fertilization. Switzerland: Springer International Publishing; 2019. pp. 625-639. Available from: https://doi. org/10.1007/978-3-319-43011-9_51 Vitro Fertilization. Switzerland: Springer International Publishing; 2019. pp. 625-639. Available from: https://doi. org/10.1007/978-3-319-43011-9_51 [1] Spallanzani L. Osservazioni e spezienze interno ai vermicellispermatici dell’ uomo e degli animali. Modena: Opusculi di Fisica Animalee Vegetabile; 1776 [10] Gangrade BK. Cryopreservation of testicular and epididymal sperm: Techniques and clinical outcomes in assisted conception. Clinics. 2013;68(S1):131-140 [10] Gangrade BK. Cryopreservation of testicular and epididymal sperm: Techniques and clinical outcomes in assisted conception. Clinics. 2013;68(S1):131-140 [2] Curry MR. Cryopreservation of semen from domestic livestock. Day JG, McLellan MR, editors. Methods in Molecular Biology. Cryopreservation and Freeze-Drying Protocols. Vol. 38. New York: Humana Press Inc; 1995. pp. 189-197 [11] Dohle GR, Elzanaty S, van Casteren NJ. Testicular biopsy: Clinical practice and interpretation. Asian Journal of Andrology. 2012;14:88-93 [11] Dohle GR, Elzanaty S, van Casteren NJ. Testicular biopsy: Clinical practice and interpretation. Asian Journal of Andrology. 2012;14:88-93 [3] Bernstein AD, Petropavlovski VV. Influence of non-electrolytes on viability of spermatozoa. Buleten Experimentalnoi Biologii i Medicini. 1937;III:17-28 [12] Di Santo M, Tarozzi N, Nadalini wM, Borini A. Human sperm cryopreservation: Update on techniques, effect on DNA integrity and implications for art. Advances in Urology. 2012;2012:837-854 [4] Polge C, Smith AU, Parkes AS. Revival of spermatozoa after Vitrification and dehydration at low temperatures. Nature. 1949;164(4172):666. Available from: https://doi.org/10.1038/164666a0 [13] Thachil JV, Jewett MAS. Preservation techniques for human semen. Fertility and Sterility. 1921;35(5):546-548 [13] Thachil JV, Jewett MAS. Preservation techniques for human semen. Fertility and Sterility. 1921;35(5):546-548 [5] Hess EA, Teague HS, Ludwick TM, Martig RC. Swine can be bred with frozen semen. Ohio Fm Home Res. 1957;42:100. Available from: https://eurekamag.com/ research/014/216/014216902.php [14] World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th ed. Geneva: World Health Organization Press; 2010 [15] Said TM, Gaglani A, Agarwal A. Implication of apoptosis in sperm cryoinjury. Reproductive Biomedicine Online. 2010;21(4):456-462 [6] Sherman JK. Synopsis of the use of frozen human semen since 1964: State of the art of human semen banking. Fertility and Sterility. 1973;24:397-416 [16] Mahadevan M, Trounson AO. Effect of cooling, freezing and thawing rates and storage conditions on preservation of human spermatozoa. Andrologia. 1984;16(1):52-60 [16] Mahadevan M, Trounson AO. [16] Mahadevan M, Trounson AO. Effect of cooling, freezing and thawing rates and storage conditions on preservation of human spermatozoa. Andrologia. 1984;16(1):52-60 [9] Martins AD, Agarwal A, Henkel R. Sperm cryopreservation. In: Zsolt N, Varghese P, Alex C, Ashok A, editors. References Effect of cooling, freezing and thawing rates and storage conditions on preservation of human spermatozoa. Andrologia. 1984;16(1):52-60 [7] Sanger WG, Olson JH, Sherman JK. Semen cryobanking for men with cancer-criteria change. Fertility and Sterility. 1992;58:1024-1027 [7] Sanger WG, Olson JH, Sherman JK. Semen cryobanking for men with cancer-criteria change. Fertility and Sterility. 1992;58:1024-1027 [7] Sanger WG, Olson JH, Sherman JK. Semen cryobanking for men with cancer-criteria change. Fertility and Sterility. 1992;58:1024-1027 [8] Curry RM. Cryopreservation of semen from domestic livestock. Reviews of Reproduction. 2000;1(5):46-52 [8] Curry RM. Cryopreservation of semen from domestic livestock. Reviews of Reproduction. 2000;1(5):46-52 [17] Holt WV. Basic aspects of frozen storage of semen. Animal Reproduction Science. 2000;62(1-3):3-22 [17] Holt WV. Basic aspects of frozen storage of semen. Animal Reproduction Science. 2000;62(1-3):3-22 [9] Martins AD, Agarwal A, Henkel R. Sperm cryopreservation. In: Zsolt N, Varghese P, Alex C, Ashok A, editors. [9] Martins AD, Agarwal A, Henkel R. Sperm cryopreservation. In: Zsolt N, Varghese P, Alex C, Ashok A, editors. [18] Keel BA, Webster BW, Roberts DK. Semen cryopreservation methodology 10 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 Cryopreservation of Human Spermatozoa: A New Frontier in Reproductive Medicine DOI: http://dx.doi.org/10.5772/intechopen.90152 and results. In: Barratt CLR, Cooke ID, editors. Donor Insemination. Cambridge, UK: Cambridge University Press; 1993. pp. 71-96 [27] Schaffner CS. Longevity of fowl spermatozoa in frozen condition. Science. 1942;96:337 [28] Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature. 1985;313:573-575 [19] Maettner R, Sterzik K, Isachenko V, Strehler E, Rahimi G, Alabart JL, et al. Quality of human spermatozoa: Relationship between high- magnification sperm morphology and DNA integrity. Andrologia. 2013;46:547-555 [19] Maettner R, Sterzik K, Isachenko V, Strehler E, Rahimi G, Alabart JL, et al. Quality of human spermatozoa: Relationship between high- magnification sperm morphology and DNA integrity. Andrologia. 2013;46:547-555 [29] Rall WF. Prospects of the cryopreservation of mammalian spermatozoa by vitrification. In: Johnson LA, Katz D, editors. Boar Semen Preservation. Berlin-Hamburg: Paurey Scientific Publishers; 1991. pp. 125-156 [20] Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: What we ask them to survive. Journal of Andrology. 1990;11:73-88 [30] Katkov II, Isachenko V, Isachenko E, Kim MS, Lulat AG, Mackay AM, et al. Low- and high-temperature vitrification as a new approach to biostabilization of reproductive and progenitor cells. International Journal of Refrigeration. 2006;29:346-357 [21] Baust JG, Gao D, Baust JM. Cryopreservation: An emerging paradigm change. [26] Luyet BJ, Hoddap A. Revival of frog’s spermatozoa vitrified in liquid air. Proceedings of the Meeting of Society for Experimental Biology. 1938;39:433-434 References Organogenesis. 2009;5:90-96 [22] Royere D, Barthelemy C, Hamamah S, Lansac J. Cryopreservation of spermatozoa: A 1996 review. Human Reproduction Update. 1996;2(6):553-559 [31] Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, et al. Vitrification of human spermatozoa without cryoprotectants. Cryo Letters. 2002;23:93-102 [31] Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, et al. Vitrification of human spermatozoa without cryoprotectants. Cryo Letters. 2002;23:93-102 [23] Fabbri R, Ciotti P, Di Tommaso B. Tecniche di crioconservazione riproduttiva. Giornale Italiano di Ostetricia e Ginecologia. 2004;3:33-41 [32] Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, et al. Cryoprotectant free cryopreservation of human spermatozoa by vitrification and freezing in vapor: Effect on motility, DNA integrity, and fertilization ability. Biology of Reproduction. 2004b;71:1167-1173 [24] Riva NS, Ruhlmann C, Iaizzo RS, López CAM, Martínez AG. Comparative analysis between slow freezing and ultra-rapid freezing for human sperm cryopreservation. JBRA Assisted Reproduction. 2018;22(4):331 [33] Vajta G, Booth PJ, Holm PCH. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. Cryo Letters. 1997;18:191-195 [33] Vajta G, Booth PJ, Holm PCH. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. Cryo Letters. 1997;18:191-195 [25] Isachenko E, Isachenko V, Katkov II, Dessole S, Nawroth F. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: From past practical difficulties to present success. Reproductive Biomedicine Online. 2003;6:191-200 [34] Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko A, Nawroth F, et al. Clean technique for cryoprotectant free vitrification of human spermatozoa. Reproductive Biomedicine Online. 2005;10:350-354 [26] Luyet BJ, Hoddap A. Revival of frog’s spermatozoa vitrified in liquid air. Proceedings of the Meeting of Society for Experimental Biology. 1938;39:433-434 11 Cryopreservation - Current Advances and Evaluations in the embryo laboratory? Review on vitrification. Reproductive Biomedicine Online. 2006;12:779-796 [44] Katkov II. The point of maximum cell water volume excursion in case of presence of an impermeable solute. Cryobiology. 2002a;44:193-203 [35] Schulz M, Muñoz M, Risopatrón J, Sánchez R. Cryopreservation of human spermatozoa by vitrification. International Journal of Morphology. 2006;24:31 [35] Schulz M, Muñoz M, Risopatrón J, Sánchez R. Cryopreservation of human spermatozoa by vitrification. International Journal of Morphology. 2006;24:31 in the embryo laboratory? Review on vitrification. Reproductive Biomedicine Online. 2006;12:779-796 [44] Katkov II. The point of maximum cell water volume excursion in case of presence of an impermeable solute. Cryobiology. References 2002a;44:193-203 [44] Katkov II. The point of maximum cell water volume excursion in case of presence of an impermeable solute. Cryobiology. 2002a;44:193-203 [36] Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136:167-173 [45] Katkov II. Symmetrical relativistic permeability approach simplifies optimization of cryobiological protocols. In: Proceedings of the International Institute of Refrigeration (IIR) International Conference, Joined Meeting of the IIR with the Society for low Temperature Biology: Cryopreservation and Safe Keeping of Cells and Tissues. Hradec Kralove, Czech Republic, May 13-1. 2002b [37] Chen Y, Li L, Qian Y, Xu C, Zhu Y, Huang H, et al. Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop. Andrologia. 2015;47:694-699 [38] Tucker M, Wright G, Morton P, Shanguo L, Massey J, Kort H. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Human Reproduction. 1996;11:1513-1515 [38] Tucker M, Wright G, Morton P, Shanguo L, Massey J, Kort H. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Human Reproduction. 1996;11:1513-1515 [46] Nauk VA. Structure and function of spermatozoa from farm animal by cryopreservation. Shtiinca Kishineu;1991 p. 198 [39] Hong SW, Chung HM, Lim JM, Ko JJ, Yoon TK, Yee B, et al. Improved human oocyte development after vitrification: A comparison of thawing methods. Fertility and Sterility. 1999;72:142-146 [39] Hong SW, Chung HM, Lim JM, Ko JJ, Yoon TK, Yee B, et al. Improved human oocyte development after vitrification: A comparison of thawing methods. Fertility and Sterility. 1999;72:142-146 [47] Isachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, et al. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Human Reproduction. 2004;19:932-939 [40] Katayama KP, Stehlik J, Kuwayama M. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertility and Sterility. 2003;80:223-224 [40] Katayama KP, Stehlik J, Kuwayama M. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertility and Sterility. 2003;80:223-224 [40] Katayama KP, Stehlik J, Kuwayama M. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertility and Sterility. 2003;80:223-224 [48] Gao D, Mazur P, Critser J. Fundamental cryobiology of mammalian spermatozoa. In: Karow AM, Critser JK, editors. Reproductive Tissue Banking. London: Academic Press; 1997. pp. 263-328 [41] Ali J, Shelton JN. Design of vitrification solutions for the cryopreservation of embryos. References Journal of Reproduction and Fertility. 1993;99:471-477 [49] Mocé E, Fajardo AJ, Graham JK. Human sperm cryopreservation. EMJ. 2016;1:86-91 [42] Mukaida T, Wada M, Takahashi K. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Human Reproduction. 1998;13:2874-2879 12
https://openalex.org/W3128155714	https://www.duo.uio.no/bitstream/10852/88945/1/How%2bto%2bdo%2bthings%2bwith%2bsigns.pdf	English	null	How to do things with signs. The formulation of directives on signs in public spaces	Journal of pragmatics	2,021	cc-by	11,752	Jan Svennevig a, b a University of Agder, Department of Nordic and Media Studies, P.O. Box 422, Kristiansand, 4604, Norway b University of Oslo, Department of Linguistics and Scandinavian Studies, P.O. Box 1102, Oslo, 0317, Norway a r t i c l e i n f o Article history: Received 13 August 2020 Received in revised form 4 January 2021 Accepted 16 January 2021 Available online 9 February 2021 Keywords: Directives Linguistic landscape Entitlement Public signage Compliance Prohibition Article history: Received 13 August 2020 Received in revised form 4 January 2021 Accepted 16 January 2021 Available online 9 February 2021 Article history: Received 13 August 2020 Received in revised form 4 January 2021 Accepted 16 January 2021 Available online 9 February 2021 This paper analyzes signs and written messages aimed at regulating people's behavior in public spaces. Based on a collection of more than 700 verbal and pictographic signs, the paper focuses on how the formulation of the directives reﬂect and construct the authors' deontic authority, how they account for the social legitimacy of the directive and how they seek to evoke the addressee's goodwill and cooperativeness. The analysis shows that the author's entitlement to perform the directive may be grounded in references to institu- tional authority, or that it may be manifested in the linguistic or pictographic expression itself, such as use of imperative mode, exclamation marks, or threats of sanctions. Enti- tlement may also be established by providing accounts for the legitimacy of the directive. These take the form of reference to rules and norms or information about beneﬁts of the requested action to the recipients or third parties. Finally, the analysis shows how signs may seek to evoke positive attitudes in the addressee by creating afﬁliation or by using humor and poetic devices. By comparing with previous research on directives in conver- sation, the paper identiﬁes request practices that are speciﬁc to this form of written communication. Keywords: Directives Linguistic landscape Entitlement Public signage Compliance Prohibition © 2021 Elsevier B.V. All rights reserved. E-mail address: jan.svennevig@uia.no. E-mail address: jan.svennevig@uia.no. https://doi.org/10.1016/j.pragma.2021.01.016 0378-2166/© 2021 Elsevier B.V. All rights reserved. Journal of Pragmatics 175 (2021) 165e183 Journal of Pragmatics 175 (2021) 165e183 https://doi.org/10.1016/j.pragma.2021.01.016 0378-2166/© 2021 Elsevier B.V. All rights reserved. © 2021 Elsevier B.V. All rights reserved. 1. Introduction Public spaces are full of signs and written notes telling the public what to do and what not to do, such as the omnipresent “No smoking”-signs or the post-it notes commonly found in lunch rooms saying “Clean up after yourself”. Such signs are powerful indices of norms of propriety and social order. Not only do they provide insight into what sorts of actions are deemed mandatory or improper in which locations e and in need of explicit exhortation, they also provide insight into the entitlement various actors claim in regulating the actions of others, and the methods they use to seek compliance. While there is by now a rich literature on the formulation of requests in face-to-face interaction (for an overview, see Drew and Couper- Kuhlen, 2014), there is as of yet hardly any attention devoted to how public signs or other written texts seek to regulate behavior by using different linguistic formulations and pictographic representations. This article is a ﬁrst attempt at iden- tifying some common formats used on such signs and discussing how they display entitlement and seek compliance. Journal of Pragmatics 175 (2021) 165e183 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig 3. Requests in conversation In the ﬁeld of Conversation Analysis, the situation is diametrically opposite. There is a vast amount of studies of directives, but no studies of how they are formulated in written genres. This may not be surprising, given that the ﬁeld of research deals primarily with oral interaction rather than with written text. However, many of the concepts used in CA for describing action formation are clearly relevant and applicable also to written utterances. For instance, Curl and Drew (2008) show how re- quests with modal interrogatives (‘can you…’) claim a higher entitlement to ask for the service than do requests formulated as ‘I wonder if…’. Similarly, imperatives claim higher entitlement than do modal interrogatives (Craven and Potter, 2010). Another dimension of requests is how speakers orient to contingencies (Curl and Drew, 2008), that is, potential obstacles to the accomplishment of the requested action. Interrogatives with modal auxiliaries, such as ‘could you’ and ‘would you’, display an orientation to potential problems related to the recipients' abilities or willingness to complete the requested action (Urbanik and Svennevig, 2019). The study of such practices in conversation is often framed within the ﬁeld of social deontics (Stevanovic and Per€akyl€a, 2012). The term is derived from the semantic term deontic modality, denoting linguistic expressions of necessity, obligation and desirability (Palmer, 2001). Deontic authority may be based on the speakers' deontic status, such as their institutionally established rights to decide about the actions of others. Various degrees of authority may be claimed in conversation by various modal expressions, such as the use of imperative mood (‘Come here!’), modal auxiliaries (‘You must/should/ought to come here’) or modal adverbs and adjectives (‘Smoking forbidden’). By using such expressions, speakers express a deontic stance, that is, a claim to a certain degree of authority to decide about future actions. The relationship between deontic status and stance is not a simple or direct one. Often they will be congruent, so that people with high deontic status will use strong deontic expressions. But sometimes people with high status will downplay their authority by using weak deontic expressions, or conversely, people with low deontic status will try to inﬂate their authority by using strong deontic expressions (Stevanovic and Per€akyl€a, 2012). The current study proposes to use these concepts developed in CA as the analytical apparatus for describing the formulation of directives on signs. 2. Directives in the linguistic landscape There is an established research ﬁeld studying written messages in public spaces, Linguistic Landscape Studies. It emerged within the ﬁeld of sociolinguistics, and a main concern has been the representation of various languages on signs in multilingual societies. As stated in the introduction to the ﬁrst issue of the journal Linguistic Landscape: “The main goal of LL studies is to describe and identify systematic patterns of the presence and absence of languages in public spaces and to un- derstand the motives, pressures, ideologies, reactions and decision making of people regarding the creation of LL in its varied forms.” (Shohamy and Ben-Rafael, 2015:1, emphasis added). Originally dominated by quantitative studies of formal aspects of language, the ﬁeld has increasingly widened the scope to more qualitative approaches, such as ethnography (e.g. Blommaert, 2013) and semiotics (Peck and Stroud, 2015). This has led to an increased interest in multimodality and different material artefacts (Thurlow and Goncalves, 2019). However, up until now, hardly any studies have addressed the pragmatic aspects of signs, especially the linguistic formulation of various classes of actions, such as warnings, informings, prohibitions, requests or offers. Only a few studies touch upon such issues as part of other analytic agendas. One example of a study that partly addresses the formulation of directives is Ferencik (2018), which analyzes practices of politeness in regulatory signs and notices in a tourism site in Slovakia. He notes that in settings where the author has indisputable authority (such as property rights), signs tend to use direct formulations involving imperatives or nominali- zations, thus reﬂecting a traditional Slovak politeness ‘ethos’. Signs in commercial settings, where the customer is assigned more power, conform more to an English or globalized politeness ethos by using conventional indirectness and politeness formulas such as ‘please’ and ‘thank you’. Apart from studies such as this one, there is still a surprising scarcity of studies investigating signs as social actions. 4. Data and method In this way, the collection represents as many different formulations as possible, enabling identiﬁcation of similarities and differences among them and thereby to propose classes of practices with certain fundamental features in common. y p p p The analysis uses the theoretical and conceptual apparatus developed in the study of action formation in the ﬁeld of Conversation Analysis (Levinson, 2013). However, there is one fundamental difference between CA studies and the current one. The study of action formation and ascription in CA is a study of how interlocutors give a certain utterance status as a speciﬁc type of action by a process of interactional negotiation, in which responses and third position moves (responses to responses) are constitutive in determining the action status of an utterance. In Schegloff's (2007) words, action is determined partly by composition e the linguistic and embodied format of the directive e partly by position, that is, its place in a sequence of turns and actions. When studying public signage, the latter aspect becomes problematic. It would in principle be possible to study how the public behave in the presence of signs involving various formulations of directives. However, it would prove difﬁcult to establish the procedural consequentiality of the formulations, that is, to claim that the behavior observed was normatively oriented to the formulation of the directive. Furthermore, representatives of the institution responsible for the sign are seldom present to help clarify the meaning of the sign or to endorse or sanction the public's adherence or non-adherence. Consequently, there are clear limitations to the possibilities of studying signs in a sequential, procedural perspective with the standard methods developed for studying talk-in-interaction. An alternative to investigating the sequential position of a written directive is to focus instead on another type of ‘position’, namely the physical placement of the sign. A directive on a public sign is not primarily designed by reference to a previous utterance or action, but rather to the physical surroundings and their affordances for action. For instance, some sculptures in public parks offer great opportunities for climbing and playing. Signs prohibiting touching the sculptures must therefore be analyzed in relation to the potential for illegitimate action offered by these physical structures. Thus, the analysis does not just investigate the signs themselves, but considers them in light of their physical placement. 4. Data and method The signs included in the study are posted in public spaces, meaning that they are not addressed to speciﬁc individuals but to a broader, unspeciﬁed public. They range from ephemeral signs, such as handwritten post-it notes in lunchrooms, to more permanent ones, such as printed metal signs ﬁxed to walls in public institutions. They all include as their main action a directive, that is, a semiotically mediated action with the purpose of getting the addressees to do something or to refrain from doing something in the interest of the person or institution responsible for the sign or some third party (Drew and Couper- Kuhlen, 2014). They contrast with warnings (such as ‘The ﬂoor is slippery’), where the purpose is to get the addressees to act in their own interest. The class of actions is rather broad, ranging from unconditional orders and prohibitions to weaker forms, such as ‘reminders’ and suggestions. Included are also ‘indirect’ forms, or composite actions (Rossi, 2018), such as “Thank you for not smoking”, where the directive is expressed by means of a vehicle conventionally associated with another class of actions. The study is based on a collection of 700þ photos of signs and notices, taken in the period 2013e2019, mainly in Norway, but some also in various other countries, such as Sweden, Denmark, Germany, Italy, Greece, India, Malaysia and USA. The data 166 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig collection has been conducted with the aim of getting a maximum of variation in wording and other semiotic resources for expressing the directive. The inclusion of signs from different countries also contributes to this aim. This sampling technique differs from many linguistic landscape studies, which have more restricted and coherent sets of data. However, it is motivated by the fact that the study is a qualitative study of types of formulations rather than a quantitative study of frequencies related to contexts of use or types of directives. The inclusion of new instances has been guided by a principle of data saturation, meaning that after a certain formulation has been attested several times (for instance the sign ‘No smoking’ accompanied by a picture of a cigarette with a prohibition sign over it), new instances are no longer added to the collection. 4. Data and method In doing this, the analysis adopts the principles of geosemiotics, a research ﬁeld studying physical locations as holistic, meaningful structures, ‘places’ rather than ‘spaces’ (Scollon and Scollon, 2003). It may also be argued that signs are objects that the members themselves treat as different from turns in conversation. For instance, given that authors of signs do not have any opportunity to clarify their meaning or respond to the audience's re- actions to the signs, we might expect that they will strive for maximal clarity and non-negotiable claims of deontic authority. They will compensate for the possibility to negotiate the meaning of an utterance by reducing ambiguities and other potential sources of misunderstanding. This contributes to giving signs a status as texts ‘speaking for themselves’ (Olson,1996). As such, the composition aspect of the action realized becomes more important. The analysis will thus focus primarily on the linguistic and semiotic conventions drawn upon in the formulation of the individual directive. 5. Analysis The analysis ﬁrst addresses formats that express a high entitlement to perform the directive, either based on the responsible actor's deontic authority (status) or by taking a strong deontic stance. Second, it shows how entitlement is claimed by accounts for the legitimacy of the directive and the requested action. Third, it addresses how signs may seek to create afﬁliation with the recipient and thereby establishing a relation of cooperativeness. Finally, it shows how rhetorical devices may be used to evoke positive attitudes toward the directive. the article, I do not use this term but instead the less technical term ‘author’ to refer to the actor responsible for the sign 5.1. Expressions of authority In public signage, reference to deontic status is sometimes used to signal authority. The social actor responsible for the sign is most commonly an institutional body or a person in an institutional role. These actors are thus ‘principals’ in Goffman's (1979) terminology, that is, the ﬁgure “whose position is established by the words spoken” and “who has committed him- self to what the words say” (Goffman, 1979: 17).1 The actor responsible for the directive is often not identiﬁed on the sign, but occasionally there is some form of a ‘signature’ at the bottom. In such cases, this actor is most commonly an institution or an institutional ﬁgure with authority and high deontic status. In Fig. 1, a sign placed at the entrance to Taj Mahal monument in India, the signature is ‘Superintending archeologist’ plus the institutional body ‘Archeological Survey of India’. In Fig. 2, on a sign posted on the entrance to a building of the University of Oslo, it is the ‘University director’ (Universitetsdirektøren), that is, the head of the administration. Common to these signatures is that they represent the highest ranked function in the 167 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig administrative hierarchy. The fact that the signs only indicate the title (and not the name of the individual, for example) highlights the institutional character of the authority. administrative hierarchy. The fact that the signs only indicate the title (and not the name of the individual, for example) highlights the institutional character of the authority. Fig. 1. Carrying eatables, paan, paan masala, cigarettes, match box, bidis, arm, electronic goods, tripod etc. are strictly prohibited inside the monument. Please do not make the monument dirty. Superintending Archeologist Archeological Survey of India. Fig. 1. Carrying eatables, paan, paan masala, cigarettes, match box, bidis, arm, electronic goods, tripod etc. are strictly prohibited inside the monument. Please do not make the monument dirty. Superintending Archeologist Archeological Survey of India. Fig. 2. Bicycles must not under any circumstances be brought into the buildings of the university. This concerns both employees, students and guests. This is a previously announced prohibition by the University Director in 1997 and has statutory basis in the general stewardship of the Technical department. […]. The University Director Fig. 2. Bicycles must not under any circumstances be brought into the buildings of the university. This concerns both employees, students and guests. 5.1. Expressions of authority This is a previously announced prohibition by the University Director in 1997 and has statutory basis in the general stewardship of the Technical department. […]. The University Director Markers of modality indicating deontic stance are practically omnipresent in signs regulating behavior in public. The majority of signs display a strong deontic stance, claiming unequivocal and complete authority to regulate the actions of others. In the signs in Figs. 1 and 2, we ﬁnd strong modal expressions, such as imperative (‘do not make the monument dirty’), strong modal auxiliaries (‘bicycles must not…’) and verbs indicating institutional authority (‘prohibit’). The deontic stance is furthermore upgraded by various reinforcing adverbs and adverbial phrases (‘strictly prohibited’, ‘under no circumstances’). The physical position of the signs at entrances to public institutions adds to the expression of authority in that the signs acquire a symbolical function as ‘gatekeepers’, regulating the admission to the premises. This tendency to take a strong deontic stance is not restricted to senders with high deontic status. In Fig. 3, from a waste bin in a sports center, we see a similarly strong admonishment, using imperative mood and multiple exclamation marks to upgrade the deontic stance. However, this text is signed by the ‘cleaning dept.’ (renholdsavd.), an institutional body which is not normally associated with the authority to regulate people's handling of waste. Instead, the multiple exclamation marks seem to indicate a different basis for claiming deontic authority, such as personal frustration or moral indignation. Other signs do not have a signature at all, but may still use strong deontic expressions. In Fig. 4 (from the same sports center), the request not to take bikes inside is formulated with the strong modal auxiliary ‘skal’ (shall) and the stance is upgraded by underlining. Who is responsible for this sign is not indicated, but its placement on the booth of the supervision guard may indicate some institutional authority associated with the practical administration of the premises. 168 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig Fig. 3. Do not throw garbage on the ﬂoor!!!!! Use the waste bin!!!! The cleaning dept. J. Svennevig Fig. 4. Bicycles shall not go inside. Fig. 4. Bicycles shall not go inside. Fig. 3. Do not throw garbage on the ﬂoor!!!!! Use the waste bin!!!! The cleaning dept. A power-based strategy for gaining compliance is imposing sanctions on non-compliance. In Fig. 5.1. Expressions of authority 5, the sanction is expulsion and economic compensation, and in Fig. 6 it is conﬁscation. Actually, in the latter case the prohibition is not expressed explicitly at all, so the readers have to infer it from the sanction. Furthermore, the spatial range of the prohibition is left implicit, so it has to be inferred from the position of the sign. In these cases, the authors claim the right to impose the sanction and thereby provide the basis for their deontic authority. The legal basis for the sanction is seldom expressed and it may be questionable in the case of Fig. 6 (but see below on references to legislation). Fig. 5. IMPORTANT The operation of Egnebuloftet is based on sales. Brought-along drinks lead to expulsion for 3 months starting immediately. IMPORTANT Whoever starts a ﬁght in the premises of Egnebuloftet will be expelled for 3 months. If in- ventory is damaged, this will have to be recompensed. Fig. 6. Bicycles will be removed without warning. Bicycle stand beneath the cafe. Fig. 6. Bicycles will be removed without warning. Bicycle stand beneath the cafe. Fig. 5. IMPORTANT The operation of Egnebuloftet is based on sales. Brought-along drinks lead to expulsion for 3 months starting immediately. IMPORTANT Whoever starts a ﬁght in the premises of Egnebuloftet will be expelled for 3 months. If in- ventory is damaged, this will have to be recompensed. 5.2. Accounts for the legitimacy of the directive The author's entitlement to perform the directive may also be established by using accounts, that is, providing reasons for the directive in terms of either causes or motives (Heritage, 1988). Accounts are used when social actors anticipate that their addressees will question or object to their social actions (Scott and Lyman, 1968). The account thus seeks to make the action more understandable or acceptable and thereby to preempt potential resistance from the recipient. Accounts may be explicit 169 J. Svennevig Journal of Pragmatics 175 (2021) 165e183 and take the form of an independent clause or they may be more implicit and embedded in the social action itself (Robinson, 2016). We will here primarily consider explicit accounts formulated in separate clauses. and take the form of an independent clause or they may be more implicit and embedded in the social action itself (Robinson, 2016). We will here primarily consider explicit accounts formulated in separate clauses. Signs requesting or prohibiting some action are often presented as unproblematic and fully legitimate without any ac- counting practices, such as in Fig. 3 above, where there is no account for the cleaning department's right to regulate people's behavior. Others present extensive accounts for the legitimacy of the directive, as in Fig. 2 above: ‘This is a previously announced prohibition by the University Director in 1997 and has statutory basis in the general stewardship (forvalteransvar) of the Technical department.’ The account here provides the institutional and legal basis for the prohibition. Accounts may explain and motivate actions in two ways. First, they may provide causal explanations in relation to already existing norms, conventions, rules or regulations (because-explanations). Second, they may provide functional or teleological explanations with reference to potential, hypothetical or future events or states (in order to-explanations). For instance, the request not to bring bicycles into the building could instead have been motivated by a concern for cleanliness or navigability. We will deal with these two types of accounts in turn. Many causal accounts refer explicitly or implicitly to some type of rule or regulation providing the legal or institutional basis for the directive. In Fig. 7, we ﬁnd explicit references to codiﬁed legislation, with references to speciﬁc paragraphs in the law on “Protection of non-smokers’ health”. Here we ﬁnd a statement of the range of ﬁnes applicable in case of transgression (cf. above on sanctions) and the authority in charge of enforcing the legislation. 5.2. Accounts for the legitimacy of the directive 13, from a street in Agra, India, the account is represented pictographically by a girl holding her ears. This picture conveys the discomfort caused by honking, thereby Fig. 8. Show respect. We start the class by entering quietly and calmly. Take responsibility. We take responsibility that everyone has someone to be with. Show compassion. We show compassion when someone is sad. Keep order. We keep the classroom tidy. Fig. 9. At work we speak NORWEGIAN. Fig. 10. We make a suggestion…. ….you act according to your conscience. The Church Council. Fig. 8. Show respect. We start the class by entering quietly and calmly. Take responsibility. We take responsibility that everyone has someone to be with. Show compassion. We show compassion when someone is sad. Keep order. We keep the classroom tidy. Fig. 9. At work we speak NORWEGIAN. Fig. 8. Show respect. We start the class by entering quietly and calmly. Take responsibility. We take responsibility that everyone has someone to be with. Show compassion. We show compassion when someone is sad. Keep order. We keep the classroom tidy. Fig. 8. Show respect. We start the class by entering quietly and calmly. Take responsibility. We take responsibility that everyone has someone to be with. Show compassion. We show compassion when someone is sad. Keep order. We keep the classroom tidy. Fig. 9. At work we speak NORWEGIAN. Fig. 9. At work we speak NORWEGIAN. Fig. 8. Show respect. We start the class by entering quietly and calmly. Take responsibility. We take responsibility that everyone has someone to be with. Show compassion. We show compassion when someone is sad. Keep order. We keep the classroom tidy. Fig. 9. At work we speak NORWEGIAN. Fig. 10. We make a suggestion…. ….you act according to your conscience. The Church Council. Fig. 10. We make a suggestion…. ….you act according to your conscience. The Church Council. The second class of accounts are the functional or teleological ones. They motivate directives by invoking potential or future consequences of actions for various groups of beneﬁciaries (Clayman and Heritage, 2014). Typically, such consequences are negative, constituting either a hazard or some form of disadvantage to individuals or the general public. In Fig. 11, it is the health risk to the animals and hazard to the public, and in Fig. 12, it is discomfort to allergics. 5.2. Accounts for the legitimacy of the directive In addition to this explicit reference to legislation, we might also note the use of the prohibition sign. This is a more indirect and associative reference to another sort of regulation, namely trafﬁc legislation. Prohibition signs of this sort are extremely common, and are used with a wide range of actions that are not regulated by trafﬁc legislation (as for instance urinating, cf. Fig. 27 below). By using the prohibition sign, senders associate the directive with a formally regulated domain and thereby associatively upgrade their deontic authority. Fig. 7. Smoking forbidden. Law of 16 January 2003 no. 3 art. 51 “Protection of non-smokers’ health”. Law of 11 November 1975 no. 584. Law 28 Dec. 2001 no. 448 e administrative ﬁnes of transgression from 27.50 Euros up to 275.00 Euros. […]. Fig. 7. Smoking forbidden. Law of 16 January 2003 no. 3 art. 51 “Protection of non-smokers’ health”. Law of 11 November 1975 no. 584. Law 28 Dec. 2001 no. 448 e administrative ﬁnes of transgression from 27.50 Euros up to 275.00 Euros. […]. Apart from legal regulations, accounts may also invoke more informal types of norms or conventions. On the sign in Fig. 8, from a classroom in a primary school, we ﬁnd a series of directives, bolstered by accounts underneath in the form of statements of norms of behavior. The norms are formulated in the present tense as descriptions of how ‘we’ behave. The normative regulation is thus disguised as a convention e a generalization about actual behavior. This is common in peda- gogical contexts, but is also found in other settings where the public is treated as in need of some form of guidance or ed- ucation. In Fig. 9, from the lunch room of a renovation company with many multilingual employees, we ﬁnd the same type of 170 J. Svennevig Journal of Pragmatics 175 (2021) 165e183 normative regulation concerning language choice. The placement in the lunch room also seems to imply that having one's lunch break is being ‘at work’, which may be a controversial claim. Actually, in this case the directive is not stated explicitly, but merely implied by the norm formulation, which serves as an account for it. However, the directive intent and exhortative function may be gleaned from the underlining of ‘speak’ and the capitalization of ‘Norwegian’. normative regulation concerning language choice. 5.2. Accounts for the legitimacy of the directive The placement in the lunch room also seems to imply that having one's lunch break is being ‘at work’, which may be a controversial claim. Actually, in this case the directive is not stated explicitly, but merely implied by the norm formulation, which serves as an account for it. However, the directive intent and exhortative function may be gleaned from the underlining of ‘speak’ and the capitalization of ‘Norwegian’. The ‘pedagogical’ tone in these normative statements may be experienced by many as patronizing or condescending, especially by adults who are not in an educational context. A way to avoid this can be to attribute the norm to the addressees themselves. In a sign posted on the entrance to an orthodox church in Greece (Fig. 10), the Church Council motivates the request to wear ‘decent’ clothes by referring to the readers' conscience. The request itself is represented pictographically, by a cross over the kind of attire that is prohibited. The verbal text provides an account which alludes to a norm, without, however, stating it explicitly. The public is thereby invited to take a decision based on their conscience rather than on externally imposed norms (and neither on obedience to authorities, note the weak deontic expression ‘we make a suggestion’). The risk with this line of accounting, especially in a cross-cultural context such as this one (with many foreign tourists visiting), is that religious norms and conventions differ both between individuals and cultural groups, so that it is not at all certain that the foreign tourists' conscience will guide them in questions of what is considered decent attire in places of worship. The second class of accounts are the functional or teleological ones. They motivate directives by invoking potential or future consequences of actions for various groups of beneﬁciaries (Clayman and Heritage, 2014). Typically, such consequences are negative, constituting either a hazard or some form of disadvantage to individuals or the general public. In Fig. 11, it is the health risk to the animals and hazard to the public, and in Fig. 12, it is discomfort to allergics. When the beneﬁciary of the action is represented, it is usually a group ‘worthy of compassion’. For instance, allergics constitute a group disadvantaged by an illness that is not self-inﬂicted. Accounts may be more indirect and subtle than in the cases above. In Fig. 5.2. Accounts for the legitimacy of the directive When the beneﬁciary of the action is represented, it is usually a group ‘worthy of compassion’. For instance, allergics constitute a group disadvantaged by an illness that is not self-inﬂicted. Accounts may be more indirect and subtle than in the cases above. In Fig. 13, from a street in Agra, India, the account is represented pictographically by a girl holding her ears. This picture conveys the discomfort caused by honking, thereby 171 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig providing an account for why the public should comply with the request. This sign is also interesting in that it attributes the request to a representative of the group of beneﬁciaries, namely children. This is conveyed by the highly expressive, colloquial style of the verbal text, with a curse and a request to terminate an action (rather than refrain from it), a formulation normally associated with the expression of annoyance. The picture and the dramatized exclamation thus constitute a vivid repre- sentation of the discomfort caused by the unwanted behavior. And compliance is sought by appealing to the addressee's empathy with children, a group that adults normally have plenty of compassion for. providing an account for why the public should comply with the request. This sign is also interes request to a representative of the group of beneﬁciaries, namely children. This is conveyed by the h Fig. 11. Feeding the animals is not allowed. They may get ill and die, or dangerous situations may occur. Fig. 11. Feeding the animals is not allowed. They may get ill and die, or dangerous situations may occur. Fig. 12. Tobacco-free entrance! Don't smoke here! Show consideration e think about our allergics! Thank you! Fig. 13. For God's sake, STOP HONKING. 172 Fig. 13. For God's sake, STOP HONKING. Fig. 12. Tobacco-free entrance! Don't smoke here! Show consideration e think about our allergics! Thank you! Fig. 13. For God's sake, STOP HONKING. 172 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig However, accounts may also focus on positive consequences of positive behavior. Usually, such consequences are pre- sented as positive to the addressees themselves, thus motivating them to act out of self-interest. On the sign in Fig. 14, the subway authorities in Copenhagen ask passengers to let people out of the train before entering, and motivate the request by claiming that this will make the subway ride better for all. 5.2. Accounts for the legitimacy of the directive And in the university cafeteria in Trondheim, Norway, the request to clean up one's plates is presented as in the interest of the students themselves by giving them a more pleasant cafeteria (see Fig. 15). Fig. 14. First out, then in. Then the subway ride gets better for all. Fig. 14. First out, then in. Then the subway ride gets better for all. Fig. 15. Cleaning duty! Everyone shall clean up after themselves. Show goodwill. Then you [pl] get a more pleasant cafeteria. Fig. 15. Cleaning duty! Everyone shall clean up after themselves. Show goodwill. Then you [pl] get a more pleasant cafeteri Teleological accounts thus differ from causal accounts and authority-based directives in that they appeal to the addressees’ reason, idealism, conscience, compassion, etc. They invite the reader to consider rational or emotional arguments for a certain course of action and take a personal stance. As such, they seek compliance on the basis of personal motivation rather than obedience to an authority or a rule. 5.3. Seeking compliance by establishing afﬁliation An alternative to seeking compliance by appealing to the authority of the author or the legitimacy of the directive is to appeal to the cooperativeness of the addressee. This may be done by establishing afﬁliation, that is, emphasizing the personal relationship between the author of the sign and the addressee. One way of doing this, is using modal interrogatives (such as ‘can/could you’ or ‘would you’) instead of imperatives. These forms display a weaker deontic stance by orienting to contin- gencies, for instance that the interlocutor is not willing or able to perform the requested action (Craven and Potter, 2010). By taking into account contingencies and providing the opportunity to decline, they show respect for the interlocutor's right to self-determination and freedom of action (what is described as ‘negative politeness’ in Brown and Levinson, 1987). Thereby they make compliance contingent upon the interlocutors' goodwill and cooperativeness. 173 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig J. Svennevig Interestingly, in the type of signs studied here, directives with modal interrogatives are extremely rare. However, there are some instances in the corpus. In Fig.16, posted on a reverse vending machine, the modal auxiliary ‘can’ is used in combination with an appeal to the customers' ‘kindness’. In Fig. 17, from a conference hotel room, the chamber maids limit their deontic stance to an expression of appreciation for complying with their request. And on a sign on a cottage serving light meals and refreshments to cross country skiers (Fig. 18), the owners merely express a wish that the visitors will buy the drinks.2 Also the request to leave dogs outside is formulated with a rather weak deontic expression, the performative verb ‘ask’, which focusses metacommunicatively on the act of requesting, thus leaving room for non-compliance. Common to these weak deontic expressions is that they do not take compliance for granted, but instead leave the decision to the addressee (Craven and Potter, 2010). Rather than basing compliance on authority, they appeal to the addressee's ‘kindness’, ‘conscience’ or consideration for the authors' wishes. By doing so, they invoke a logic of solidarity and empathy rather than a logic of power and authority. The more personal tone in these signs is also reﬂected in the use of personal pronouns. Here we ﬁnd expressions of both a ‘we’ (Figs.17 and 18) and a ‘you’ (both in singular (Figs.17 and 18) and plural (Fig. 16). 2 The verb phrase in Norwegian ’ser gjerne’ (literally ’see with pleasure’) is difﬁcult to translate directly, but constitutes a weak expression of a wish or a preference. 5.3. Seeking compliance by establishing afﬁliation This contrasts with most other signs, which avoid reference to either the author or the addressee by the use of imperative mood (as in Figs. 3 and 19), passive voice (as in Figs.1 and 2) or nominalization (as in Figs. 7 and 11). Other features of the more personal tone in these signs are the use of smileys (as in Figs. 16 and 17) and the expressive actions of wishing welcome (Fig. 18) and thanking (Figs. 16 and 17). The scarcity of modal interrogatives and other weak modality markers reveals a distinctive feature about directives in public signage, as opposed to in conversation. The anonymous and non-committing relationship between the parties in sign communication invites authority-based compliance strategies. Only when a more personal relationship is created or implied, more solidarity-based compliance strategies seem to be used. Fig. 16. Dear customers. Can you (pl.) be so kind and remember to empty the bottles before you deposit them? Thanks in advance ☺ F j Fig. 17. Dear guest, We would appreciate that you (sing.) use the luggage stand and just one of the beds. Thanks a lot ☺The Chamber Maids. Fig. 17. Dear guest, We would appreciate that you (sing.) use the luggage stand and just one of the beds. Thanks a lot ☺The Chamber Maids. Fig. 16. Dear customers. Can you (pl.) be so kind and remember to empty the bottles before you deposit them? Thanks in advance ☺ 174 J. Svennevig Journal of Pragmatics 175 (2021) 165e183 Fig. 18. We wish you (sing.) welcome to the NEW GREINE COTTAGE. Here you may take a rest and eat your lunch bag if you like, but we would like you to buy the drinks. We also ask you to keep your four-legged friend in a leash outside the cottage. Bærums Verk Rotary Club. J. Svennevig Journal of Pragmatics 175 (2021) 165e183 3 However, conventional politeness formulae such as this one seems less used in Norwegian signs than in English signs, probably as a result of a general tendency in Norwegian to use such formulae less (Fretheim, 2005). 20 from a poster in a bus. This request practice pushes the logic of cooperativeness even further by portraying the addressee as someone who behaves in line with the transportation company's wishes even without having been asked to do so. The image contributes to this portrayal by showing two young women sitting in the desired position. This is an ‘indirect’ or ‘composite’ action (Rossi, 2018), in which an explicit action format (‘thanking’) is used as a vehicle for a different type of action (‘requesting’). Although this practice might have evolved as an implicature, today it is conventionalized (in Norway and many other societies) as a polite formula for realizing requests, especially in public signage (Terkouraﬁ, 2015). Fig. 21. Are you the last one! Remember to turn out all lights in the assembly hall. Fig. 22. Have you remembered to clean up after yourself? Fig. 22. Have you remembered to clean up after yourself? Fig. 22. Have you remembered to clean up after yourself? Fig. 22. Have you remembered to clean up after yourself? Fig. 21. Are you the last one! Remember to turn out all lights in the assembly hall. Another practice that presupposes compliance and thereby reduces the social sensitivity of imposing on the addressee is presenting the directive as a reminder. In Fig. 21 the directive is formulated this way, and thus does not orient to contingencies related to the addressees’ willingness, only to their ability (to remember). Fig. 22 from the exit from a classroom also ad- dresses memory problems, although here, we might consider this a strategic choice since unwillingness might also be a very real contingency. The placement of these signs on exit doors contributes to their function as memory prompts for checking routine obligations when abandoning a public locale. These signs thus presuppose (and thereby invoke) an attitude of cooperativeness from the recipient. Afﬁliation may also be nurtured by symbolically creating intimacy or an in-group relationship with the addressee (positive politeness in Brown and Levinson's (1987) terms). In the sign in Fig. 23, from a door leading to a terrace outside a bar, such a relation is signaled (and co-created) by means of several indexes of an intimate relationship as friends or buddies (so-called ‘camaraderie’ style, cf. Lakoff,1985). First, there are personal expressions of affection, such as the expression of regret (Sorry, Mac), and an expression of devotion (‘love you all the same’). Fig. 18. We wish you (sing.) welcome to the NEW GREINE COTTAGE. Here you may take a rest and eat your lunch bag if you like, but we would like you to buy the drinks. We also ask you to keep your four-legged friend in a leash outside the cottage. Bærums Verk Rotary Club. One way to deal with potential resistance from the audience is to use conventional politeness formulas, which constitute symbolic markers of respect for the addressee (Brown and Levinson, 1987). In English, the most common politeness marker is ‘please’ (as in Fig. 1 above: ‘Please do not make the monument dirty’). In Norwegian, the corresponding marker is ‘vennligst’ (literally ‘most kindly’), as illustrated in Fig. 19.3 However, unlike directives in conversation, mitigation seems largely restricted to these highly standardized forms of ‘token politeness’. Fig. 20. Thank you for keeping your feet on the ﬂoor. Then we all get a more pleasant journey. Fig. 19. Please place the dishes here. Thank you! Fig. 20. Thank you for keeping your feet on the ﬂoor. Then we all get a more pleasant journey. Fig. 19. Please place the dishes here. Thank you! Fig. 20. Thank you for keeping your feet on the ﬂoor. Then we all get a more pleasant journey. Fig. 19. Please place the dishes here. Thank you! 3 However, conventional politeness formulae such as this one seems less used in Norwegian signs than in English signs, probably as a result of a general tendency in Norwegian to use such formulae less (Fretheim, 2005). 175 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig Another way of underlining interpersonal solidarity as the basis for the directive is thanking the addressee for complying. In the last sign above (Fig. 19), the thanking occurs after the request. In sequential terms, it is a third position action occurring after a ﬁrst position action (Schegloff, 2007). The missing second position action is of course the addressee's granting of the request. The expression of gratitude thus anticipates and presupposes a positive response to the request. This ‘strategic’ anticipation of a positive response may be considered a compliance-gaining practice in that it portrays the addressee as cooperative and willing to help.4 p g p An associated practice is not writing the ﬁrst-position action (the directive) at all, but merely presenting the expression of gratitude as a ﬁrst-position move. This is the case in Fig. 4 The picture here may seem a bit confusing, since it depicts a woman who is doing the opposite of cleaning up, namely scattering the utensils around. The logic seems to be to mock those who do not comply with the request, thereby adding a humorous appeal (more on the use of humor below). Fig. 23. Sorrimækk (Sorry, Mac), not allowed to drink or smoke outside. Love you all the same. 5.4. Rhetorical devices: framing, humor and poetics 5.4. Rhetorical devices: framing, humor and poetics A rather heterogenous category of signs involve various rhetorical devices, such as framing the directive positively, making a humorous point or using poetic devices such as rhyme, rhythm and personiﬁcation. Common to such devices is that they are oriented towards creating an aesthetically or emotionally pleasant experience for the addressee and thereby to generate an attitude of goodwill and cooperativeness. One way of doing this is to bring attention to the positive sides of the message and downplay or ignore the negative ones. This is often referred to as framing, which has been deﬁned as follows: To frame is to select some aspects of a perceived reality and make them more salient in a communicating text, in such a way as to promote a particular problem deﬁnition, causal interpretation, moral evaluation, and/or treatment recommendation for the item described” (Entman, 1993:52). Some signs in the collection seem to use positive framing strategically in that they shift the perspective from what is prohibited to what is permitted or recommended. For instance, instead of telling people where not to park their bike (as in Fig. 6 above), one can tell them where it should be parked (as in Fig. 31 below). Similarly, the sign in Fig. 24 from a recreational area at a conference center does not admonish the public not to throw liquid coffee in the waste bins or leave half-full cups on the tables, instead it presents a solution for what to do with remaining coffee. A clue revealing the double message of this sign is the gaze of the smiley, expressing some type of mischievousness. The sign from a playroom at IKEA in Fig. 25 does not focus on which children are not allowed to enter but instead informs us about who is. Common to these signs is that they avoid the socially sensitive action of prohibiting certain actions or admonishing the recipients. Instead, they convey this implicitly as a possible (but not necessary) inference. Also the practice described above of thanking instead of requesting (Fig. 20) may be considered a positive framing device. Not incidentally, the signs using this device are all taken from commercial organizations, which are dependent on good relations with their customers. Fig. 25. All children 3e7 years old are welcome to play here! Fig. 24. DIDYOUKNOW:youmayemptyhalf-fullcupsinthedrainofthecoffeemachine? Fig. 25. All children 3e7 years old are welcome to play here! 177 The personal tone is underlined by an informal, oral style, such as the Norwegian transcription of the English oral expression “sorrimækk”, the ellipsis of the subject and verb (‘(It is) not allowed’), the verb- based syntax (‘smoke’ instead of ‘smoking’) and the fact that the words are written in dialect (with the use of apocope characteristic of the Trondheim area (‘ittj’ for ikke, ‘røyk’ for røyke and ‘drekk’ for drikke). So, although the directive is formulated with a rather strong deontic stance (‘It is not allowed..’), the imposition is mitigated by presenting the message as addressed to a buddy from the local community, from whom one may expect solidarity, and thus compliance. 4 The picture here may seem a bit confusing, since it depicts a woman who is doing the opposite of cleaning up, namely scattering the utensils around. The logic seems to be to mock those who do not comply with the request, thereby adding a humorous appeal (more on the use of humor below). 176 5 4 Rh i l d i f i h d i Fig. 23. Sorrimækk (Sorry, Mac), not allowed to drink or smoke outside. Love you all the same. J. Svennevig Journal of Pragma J. Svennevig Journal of Pragmatics 175 (2021) 165e183 Fig. 24. DIDYOUKNOW:youmayemptyhalf-fullcupsinthedrainofthecoffeemachine? Fig. 25. All children 3e7 years old are welcome to play here! 177 Fig. 24. DIDYOUKNOW:youmayemptyhalf-fullcupsinthedrainofthecoffeemachine? 177 Fig. 24. DIDYOUKNOW:youmayemptyhalf-fullcupsinthedrainofthecoffeemachine? Fig. 25. All children 3e7 years old are welcome to play here! 177 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig J. Svennevig Another rhetorical device is the use of humor. Humor may take many forms, but a common pattern in the corpus is to exaggerate the punishment for transgression to the point of absurdity. For instance, in Fig. 26 it is that misparked cars will be disassembled and sold, and in Fig. 27 that urinating will be ﬁlmed and published on YouTube. Another ﬁgure is to portray the world as incongruous and absurd, such as people having cigarette stubs in their urine (Fig. 28). Finally, humor may result from altering a conventional ﬁgure of speech into a surprising and incongruous one. An example is Fig. 29, where the conventional exhortation to drive slowly because of hazard to children playing in the street is altered into a warning about hazard to the driver due to scouts playing with axes and knives. As can be seen, this sign is physically altered by taping over and replacing words and by taping on an addition to it (which is in turn altered by adding words in handwriting). Another rhetorical device is the use of humor. Humor may take many forms, but a common pattern in the corpus is to exaggerate the punishment for transgression to the point of absurdity. For instance, in Fig. 26 it is that misparked cars will be disassembled and sold, and in Fig. 27 that urinating will be ﬁlmed and published on YouTube. Another ﬁgure is to portray the world as incongruous and absurd, such as people having cigarette stubs in their urine (Fig. 28). Finally, humor may result from altering a conventional ﬁgure of speech into a surprising and incongruous one. An example is Fig. 29, where the conventional exhortation to drive slowly because of hazard to children playing in the street is altered into a warning about hazard to the driver due to scouts playing with axes and knives. As can be seen, this sign is physically altered by taping over and replacing words and by taping on an addition to it (which is in turn altered by adding words in handwriting). What these humoristic signs have in common is that they seek to activate positive emotions in the readers and thereby to create a positive attitude to the directive. Fig. 24. DIDYOUKNOW:youmayemptyhalf-fullcupsinthedrainofthecoffeemachine? Fig. 25. All children 3e7 years old are welcome to play here! 177 This may in turn have the rhetorical effect of making them more positive to complying with the directive. The humorous effect of the signs lies much in the unexpected, unconventional and often implicit formulation of the directive. If such formulations become conventional they lose much of their humorous and thus rhetorical effect. This can be observed in the phrase “Your mother doesn't work here”, which has become so conventional and worn that it is no longer perceived as humorous. Fig. 26. Parking for visitors to Betania community center. NB: Misparked cars will be disassembled and sold to raise funds for mission in Swaziland. Fig. 27. Pictographic signs prohibiting urinating. Fig. 28. If you regularly suffer from litter in your urine for instance cigarette stubs, cotton pads etc. you ought to see your doctor. with kind regards the plumber. Fig. 27. Pictographic signs prohibiting urinating. Fig. 26. Parking for visitors to Betania community center. NB: Misparked cars will be disassembled and sold to raise funds for mission in Swaziland. Fig. 26. Parking for visitors to Betania community center. NB: Misparked cars will be disassembled and sold to raise funds for mission in Swaziland. Fig. 28. If you regularly suffer from litter in your urine for instance cigarette stubs, cotton pads etc. you ought to see your doctor. with kind regards the plumber. Fig. 27. Pictographic signs prohibiting urinating. Fig. 28. If you regularly suffer from litter in your urine for instance cigarette stubs, cotton pads etc. you ought to see your doctor. with kind regards the plumber. 178 J. Svennevig Journal of Pragmatics 175 (2021) 165e183 Fig. 29. Drive slowly. Scouts are playing. with axe and knife. (Photo: Helene Lane). Fig. 29. Drive slowly. Scouts are playing. with axe and knife. (Photo: Helene Lane). Finally, on some signs, we ﬁnd quasi-literary forms of exposition. A rather common poetic device is personiﬁcation or animation, that is, assigning human characteristics to objects or animals. In Fig. 30, the police horses speak to the public and address their ‘friends’ directly. The image of humanoid, smiling horses contributes to this personiﬁcation, and together, these devices invoke the genre of children's cartoons. By exploiting a genre that is popular among children, the sign implicitly addresses this group of bypassers, and by having the request come directly from the horse's mouth (literally speaking!), it appeals to a form of imagination typical of child culture. Fig. 24. DIDYOUKNOW:youmayemptyhalf-fullcupsinthedrainofthecoffeemachine? Fig. 25. All children 3e7 years old are welcome to play here! 177 Furthermore, the sign is placed rather low on a fence and thus at the eye height of children rather than of adults. In the next Fig. 31, the object of animation is dead objects, namely bicycles. And even though the text does not seem addressed to children, the readers are invited to imagine a ludic, ﬁctional world where bicycles have human emotions and appreciate physical contact. Whether or not such an appeal to imagination is successful as a compliance-gaining device will of course depend on whether or not the audience ﬁnds this playful form of expression intriguing or entertaining. 179 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig Fig. 30. Dear horse-friend! Be so kind not to give us anything to eat! Fat police horses aren't pretty, you know. By the way, we are treated very well here at the police e and get what we need! Fig. 31. Bicycles LOVE to stand together. Park your bike here. Fig. 30. Dear horse-friend! Be so kind not to give us anything to eat! Fat police horses aren't pretty, you know. By the way, we are treated very well here at the police e and get what we need! Fig. 31. Bicycles LOVE to stand together. Park your bike here. Another literary form of exposition is the ﬁctional narrative. There is seldom space enough to present a full narrative on signs such as these, but some signs use ﬁctional characters and events. For instance, in Fig. 32, the text addresses a ﬁctional character, Ingrid, who regularly takes public transportation with a stroller and heavy shopping bags. She is portrayed as an ‘everyday hero’, who upholds values such as respect for the law in spite of the challenges and hardship of life as a mother without a car. In this way, the text invites the readers to identify with this protagonist and to follow her example in paying her ticket (which in Oslo is not enforced by physical barriers or conductors). In Fig. 33, there are also ﬁctional characters, mister and misses Kvist, who risk a ‘sad’ fate if car drivers hit the brakes hard in front of the tram. The address term ‘herr og fru’ (mister and misses) has an archaic ring to it in current Norwegian, and thus invites us to either see the text as an old rhyme or to imagine the couple as elderly. Fig. 24. DIDYOUKNOW:youmayemptyhalf-fullcupsinthedrainofthecoffeemachine? Fig. 25. All children 3e7 years old are welcome to play here! 177 In addition, the text presents itself as a verse, with end rhyme (bilist e trist e Kvist) and a partly regular rhythm, with dactyl as the metrical foot. The ﬁctional universe created here invites the readers to feel compassion for the elderly couple and thus contributes to personalizing the harms that car drivers may cause. These ﬁctional narratives thus seek to create identiﬁcation with story characters and thereby to get the readers to adhere to courses of action that support their interests in the story world, which coincides with the interests of the author in the real world. More generally, common to both humorous and poetic signs is that they seek to move the readers emotionally and thereby to gain their goodwill. 180 J. Svennevig Journal of Pragmatics 175 (2021) 165e183 Fig. 33. If you as a car-driver hit the brakes hard it can onboard become very sad for the passengers, mister and misses Kvist. Fig. 32. Thanks a lot, Ingrid, that you always buy a ticket even though you have more than enough with your stroller and heavy shopping bags. All proﬁt from ticket sales goes back to public transportation. Fig. 33. If you as a car-driver hit the brakes hard it can onboard become very sad for the passengers, mister and misses Kvist. Fig. 32. Thanks a lot, Ingrid, that you always buy a ticket even though you have more than enough with your stroller and heavy shopping bags. All proﬁt from ticket sales goes back to public transportation. 6. Discussion This study has identiﬁed some general practices used in the formulation of directives on signs in public spaces. It has used the theoretical concepts developed within CA in order to describe how entitlement is displayed and how the signs orient to contingencies. The analysis shows that entitlement may be claimed by signatures indicating the institutional authority of the actor responsible for the sign. Such references to deontic status are not common in everyday conversation. A high degree of entitlement is also claimed by the use of strong deontic modality, such as imperatives, high-grade modal auxiliaries (‘must’) and nominalizations expressing categorical prohibition (‘no smoking’). The deontic stance is often also upgraded by in- tensiﬁers of various sorts (‘under no circumstances’). The extended use of strong deontic expressions contrasts with what has been found in everyday conversation, where imperatives are associated with requests for action as part of a common commitment to a joint project and thus partly in the interest of the recipient (Rossi, 2012; Zinken and Ogiermann, 2013). In public signage, imperatives are also used in situations which in conversation are typically associated with the use of modal interrogatives, namely when the requested action is not part of a common project, requires much effort from the recipient and the author does not have special grounds to expect compliance (Urbanik and Svennevig, 2019). An example is the request to clean up after oneself in a cafeteria (Fig. 19). We thus ﬁnd a more extended use of stronger expressions of deontic authority in public signs than what has been reported for everyday conversation. Entitlement to perform the directive may also be claimed by the use of accounts invoking external sources of authority and legitimacy. The analysis has shown that such accounts claim entitlement either by reference to pre-existing rules, norms or conventions, or by reference to future beneﬁts to the recipients or third parties. The ﬁrst type of accounts is associated with strengthening the author's deontic authority in that it grounds the directive in more general and socially established norms. The second type does not in the same way boost the deontic authority of the author but instead grounds the obligation or necessity to act in the recipients' sense of responsibility, empathy or considerateness. The analysis has also shown how signs seek compliance by establishing afﬁliation. Some express appreciation or use personal forms of address and in-group markers. 6. Discussion By using these practices in the impersonal context of anonymous authors addressing a general public, the signs symbolically reduce the social distance between the parties and thereby evoke a logic of 181 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig solidarity and cooperativeness. Especially commercial actors have an interest in maintaining a good relationship with cus- tomers in order to secure a positive image and lay the ground for future transactions. One practice instantiating the more personal tone and a service-minded attitude is conveying the directive by thanking the interlocutor for doing the desired action (‘Thank you for not smoking’). This is a practice that seems generic to signs and is not attested in conversation. Another contrast with directives in conversation is the scarcity of modal interrogatives oriented to the recipients' ability to perform the action (‘Can/could you…’). The practices found on the signs thus seem more oriented towards the recipients' willingness to comply than to their ability. This may reveal an important difference between the two forms of communication. Lack of ability to perform the requested action is a less face-threatening reason for resisting or rejecting a request than lack of willingness. The personal context of face-to-face conversation may thus have developed conventional request practices that offer the participants methods for rejecting a request without threatening the social relation between the parties. In the impersonal context of public signage, by contrast, such face-saving practices are less used. Finally, the analysis has identiﬁed some (but certainly not all) rhetorical devices used in order to counteract the recipients' potential unwillingness to comply and instead create a positive attitude towards the author or the directive. Positive framing seeks to do this by focusing on the possibilities and opportunities rather than the restrictions and prohibitions. Humor and poetic devices seek to do it by giving the recipients an emotionally or aesthetically pleasant experience. These rhetorically ‘embellished’ directives are the class that differs most from what is found in conversation. Here it becomes tangible how the time to plan and design written messages makes a difference for how directives are formulated. p g g In addition to these empirical ﬁndings on how directives are expressed in public signage, the study also contributes theoretically to the ﬁeld of CA by showing how action formation may be studied in written texts. Funding This work was partly ﬁnanced by the Research Council of Norway through its Centres of Excellence funding scheme, project number 223265. Acknowledgements Thanks to Pawel Urbanik for useful comments on a preliminary draft of the article, and to numerous friends and colleagues for sending me photos of signs. 6. Discussion The lack of co-presence and interaction between the author and the addressee has consequences for how directives are formulated and calls for different methods of analysis. As a ﬁxed and non-negotiable text, the utterance must to a larger extent ‘speak for itself’, meaning that it will strive for clarity and a strong deontic stance. This does not, however, mean that they are context-independent, but the relevant context used by addressees in order to interpret them will be material and ‘geographical’ rather than verbal and sequential. The study shows how the placement of signs relative to various objects, locations and the addressees have important semiotic functions in conveying the relevant meaning. In addition, the study aims to contribute to the ﬁeld of Linguistic Landscape studies by placing pragmatic phenomena such as action formation on the agenda. After two decades of sociolinguistic studies that have mainly focused on language forms, it is time to start investigating more in detail the meaning of signs and their status as social actions. The current study aims to take the ﬁrst steps in this direction, but there is a vast amount of other actions worthy of study, such as warnings, informings and invitations. Also more in-depth investigation of linguistic and cultural variation in more focused, comparative data sets is needed in future research. Researchers in pragmatics are hereby invited to enter the ﬁeld and start investigating how to do things with signs. 996. The World on Paper: the Conceptual and Cognitive Implications of Writing and Reading. Cambridge University Press. References Blommaert, Jan, 2013. Ethnography, Superdiversity and Linguistic Landscapes. Multilingual Matters, Clevedon. B P l L i St h 1987 P lit S U i l i L U C b id U i it Blommaert, Jan, 2013. Ethnography, Superdiversity and Linguistic Landscapes. Multilingual Matters, Clevedon. Brown, Penelope, Levinson, Stephen, 1987. Politeness: Some Universals in Language Usage. Cambridge University Press, Cambridge. Clayman, Steven, Heritage, John, 2014. Benefactors and beneﬁciaries: benefactive status and stance in the management of offers and requests. In: Drew, P., Couper-Kuhlen E (Eds ) Requesting in Social Interaction John Benjamins Amsterdam pp 51e82 Brown, Penelope, Levinson, Stephen, 1987. Politeness: Some Universals in Language Usage. Cambridge University Press, Cambridge. Clayman, Steven, Heritage, John, 2014. Benefactors and beneﬁciaries: benefactive status and stance in the management of offers and requests. In: Drew, P. Craven, Alexandra, Potter, Jonathan, 2010. Directives: entitlement and contingency in action. Discourse Stud. 12 (4), 419e442. https://doi.org/10.1177/ 1461445610370126. Craven, Alexandra, Potter, Jonathan, 2010. Directives: entitlement and contingency in action. Discourse Stud. 12 (4), 419e442. https://doi.org/10.1177/ 1461445610370126. ew, Paul, 2008. Contingency and action: a comparison of two forms of requesting. Res. Lang. Soc. Interact. 41 (2), 129e153. h 51810802028613. Curl, Traci, Drew, Paul, 2008. Contingency and action: a comparison of two forms of requesting. Res. Lang. Soc. Interact. 41 (2), 129e153. https://doi.org/10. 1080/08351810802028613. / rew, Paul, Couper-Kuhlen, Elizabeth, 2014. Requesting - from speech act to recruitment. In: Drew, P., Couper-Kuhlen, E Interaction. John Benjamins Publishing Company, Amsterdam/Philadelphia, pp. 1e34. https://doi.org/10.1075/slsi.26.01d Entman, Robert M., 1993. Framing: toward clariﬁcation of a fractured paradigm. J. Commun. 43 (4), 51e58. Fretheim, Thorstein, 2005. Politeness in Norway: how can you be polite and sincere. In: Hickey, Leo, Stewart, Miranda (Eds.), Politeness in Europe. Multilingual Matters. í Entman, Robert M., 1993. Framing: toward clariﬁcation of a fractured paradigm. J. Commun. 43 (4), 51e58. Fretheim, Thorstein, 2005. Politeness in Norway: how can you be polite and sincere. In: Hickey, Leo, Stewart, Miranda (Eds.), Politeness in Europe. Entman, Robert M., 1993. Framing: toward clariﬁcation of a fractured paradigm. J. Commun. 43 (4), 51e58. Fretheim, Thorstein, 2005. Politeness in Norway: how can you be polite and sincere. In: Hickey, Leo, Stewart, Miranda (Eds.), Politeness in Europe. Multilingual Matters. F ík Mil 2018 I / lit th t d f l t di ti i Sl ki ' t f t i J P t 134 183 198 Ferencík, Milan, 2018. lson, David R., 1996. The World on Paper: the Conceptual and Cognitive Implications of Writing and Reading. Cambridge U References Sequence Organization in Interaction: A Primer in Conversation Analysis. Cambridge University Press, Cambridge. Scollon, Ron, Scollon, Suzie W., 2003. Discourses in Place: Language in the Material World. Routledge, London. ossi, Giovanni, 2018. Composite social actions: the case of factual declaratives in everyday interaction. Res. Lang. Soc. chegloff, Emanuel, 2007. Sequence Organization in Interaction: A Primer in Conversation Analysis. Cambridge Univers collon, Ron, Scollon, Suzie W., 2003. Discourses in Place: Language in the Material World. Routledge, London. Scott, Marvin B., Lyman, Stanford M., 1968. Accounts. American Sociological Review, pp. 46e62. Shohamy, E., Ben-Rafael, E., 2015. Introduction: linguistic landscape, a new journal. Linguistic Landscape 1 (1/2 Shohamy, E., Ben-Rafael, E., 2015. Introduction: linguistic landscape, a new journal. Linguistic Landscape 1 (1/2), 1e5. S i M li P €k l€ A i 2012 D i h i i i i h i h d d id R L S I 45 (3) Shohamy, E., Ben-Rafael, E., 2015. Introduction: linguistic landscape, a new journal. Linguistic Landscape 1 (1/2), 1e5. Stevanovic, Melissa, Per€akyl€a, Anssi, 2012. Deontic authority in interaction: the right to announce, propose, and decide. Res. Lang. Soc. Interact. 45 (3 y, , , , g p , j g p ( / ), Stevanovic, Melissa, Per€akyl€a, Anssi, 2012. Deontic authority in interaction: the right to announce, propose, and decid 297e321. https://doi.org/10.1080/08351813.2012.699260. Terkouraﬁ, Marina, 2015. Conventionalization: a new agenda for im/politeness research. J. Pragmat. 86, 11e18. Th l C i i G l K ll 2019 X N h i i li i ti l d Li i ti L d 5 (2) 111 114 Terkouraﬁ, Marina, 2015. Conventionalization: a new agenda for im/politeness research. J. Pragmat. 86, 11e18. Thurlow, Crispin, Goncalves, Kelly, 2019. X-scapes. New horizons in linguistic landscapes. Linguistic Landscape 5 (2), 111e114. Urbanik, Pawel, Svennevig, Jan, 2019. Managing contingencies in requests: the role of negation in Norwegian interrogative directives. J. Pragmat. 139, 109e125. Zinken, J€org, Ogiermann, Eva, 2013. Responsibility and action: invariants and diversity in requests for objects in British E Lang. Soc. Interact. 46 (3), 256e276. Jan Svennevig is Professor of Language and Communication at Agder University and MultiLing e Center for Research on Multilingualism in Society across the Lifespan, University of Oslo, Norway. His research deals with workplace interaction, L2 conversations, pragmatic particles and communication in dementia. Recent publications include “Managing contingencies in requests: The role of negation in Norwegian interrogative requests” (Journal of Pragmatics, 2019, with P. References Im/politeness on the move: a study of regulatory discourse practices in Slovakia's centre of tourism. J. Pragmat. 134, 183e198. Goffman Erving 1979 Footing Semiotica 25 (1 2) 1 30 https://doi org/10 1515/semi 1979 25 1 2 1 Ferencík, Milan, 2018. Im/politeness on the move: a study of regulatory discourse practices in Slovakia's centre of tourism. J. Pragmat. 134, 183e198. Goffman, Erving, 1979. Footing. Semiotica 25 (1e2), 1e30. https://doi.org/10.1515/semi.1979.25.1-2.1. Goffman, Erving, 1979. Footing. Semiotica 25 (1e2), 1e30. https://doi.org/10.1515/semi.1979.25.1-2.1. Heritage, John C., 1988. Explanations as accounts: a conversation analytic perspective. In: Antaki, Charles (Ed.), Analysing Everyday Explanation: A Casebook of Methods. Sage, London, pp. 127e144. 182 Journal of Pragmatics 175 (2021) 165e183 J. Svennevig Palmer, Frank R., 2001. Mood and Modality. Cambridge University Press. Palmer, Frank R., 2001. Mood and Modality. Cambridge University Press. k i d i h ki i i i d ( ) Peck, Amiena, Stroud, Cristopher, 2015. Skinscapes. Linguistic Landscape 1 (1e2), 133e151. Peck, Amiena, Stroud, Cristopher, 2015. Skinscapes. Linguistic Landscape 1 (1e2), 133e151. R bi J ff D 2016 A bili i i l i i I R bi J ff D (Ed ) A Robinson, Jeffrey D., 2016. Accountability in social interaction. In: Robinson, Jeffrey D. (Ed.), Accountability in Social Int Oxford. Rossi, Giovanni, 2012. Bilateral and unilateral requests: the use of imperatives and Mi X? Interrogatives in Italian. Discourse Process 49 (5), 426e458. https://doi.org/10.1080/0163853X.2012.684136. Rossi, Giovanni, 2012. Bilateral and unilateral requests: the use of imperatives and Mi X? Interrogatives in Italian. Discourse Process 49 (5), 426e458. https://doi.org/10.1080/0163853X.2012.684136. Rossi, Giovanni, 2012. Bilateral and unilateral requests: the use of imperatives and Mi X? Interrogatives in Italian. Discourse Process 49 (5), 426e458. https://doi.org/10.1080/0163853X.2012.684136. l h f f l d l d ( ) Rossi, Giovanni, 2018. Composite social actions: the case of factual declaratives in everyday interaction. Res. Lang. Soc. Interact. 51 (4), 379e397. Schegloff, Emanuel, 2007. Sequence Organization in Interaction: A Primer in Conversation Analysis. Cambridge University Press, Cambridge. ossi, Giovanni, 2018. Composite social actions: the case of factual declaratives in everyday interaction. Res. Lang. Soc. Inte chegloff, Emanuel, 2007. Sequence Organization in Interaction: A Primer in Conversation Analysis. Cambridge University P collon Ron Scollon Suzie W 2003 Discourses in Place: Language in the Material World Routledge London Rossi, Giovanni, 2018. Composite social actions: the case of factual declaratives in everyday interaction. Res. Lang. Soc. Interact. 51 (4), 379e397. Schegloff, Emanuel, 2007. References Urbanik), “Pre-empting understanding problems in L1/L2 conversations: Evidence of effectiveness from simulated emergency calls” (Applied Lin- guistics, 2019, with J. Gerwing, B. U. Jensen and M. Allison), and “Accounting for forgetfulness in dementia interaction” (Linguistics Vanguard, 2019, with A. M. Landmark). Jan Svennevig is Professor of Language and Communication at Agder University and MultiLing e Center for Research on Multilingualism in Society across the Lifespan, University of Oslo, Norway. His research deals with workplace interaction, L2 conversations, pragmatic particles and communication in dementia. Recent publications include “Managing contingencies in requests: The role of negation in Norwegian interrogative requests” (Journal of Pragmatics, 2019, with P. Urbanik), “Pre-empting understanding problems in L1/L2 conversations: Evidence of effectiveness from simulated emergency calls” (Applied Lin- guistics, 2019, with J. Gerwing, B. U. Jensen and M. Allison), and “Accounting for forgetfulness in dementia interaction” (Linguistics Vanguard, 2019, with A. M. Landmark). 183
https://openalex.org/W3194742069	https://biblio.ugent.be/publication/8720492/file/8720493.pdf	English	null	Prefrontal tDCS Attenuates Self-Referential Attentional Deployment: A Mechanism Underlying Adaptive Emotional Reactivity to Social-Evaluative Threat	Frontiers in human neuroscience	2,021	cc-by	13,861	Prefrontal tDCS Attenuates Self-Referential Attentional Deployment: A Mechanism Underlying Adaptive Emotional Reactivity to Social-Evaluative Threat Jens Allaert1,2, Maide Erdogan3, Alvaro Sanchez-Lopez4, Chris Baeken1,5,6, Rudi De Raedt2 and Marie-Anne Vanderhasselt1,2 Jens Allaert1,2, Maide Erdogan3, Alvaro Sanchez-Lopez4, Chris Baeken1,5,6, Rudi De Raedt2 and Marie-Anne Vanderhasselt1,2 ORIGINAL RESEARCH published: 17 August 2021 doi: 10.3389/fnhum.2021.700557 Reviewed by: Reviewed by: M. Ali Salehienjad, Leibniz Research Centre for Working Environment and Human Factors (IfADo), Germany Amir Hossien Mehrsafar, University of Tehran, Iran Rodrigo Pegado, Federal University of Rio Grande, Brazil Social-evaluative threat (SET) – a situation in which one could be negatively evaluated by others – elicits profound (psycho)physiological reactivity which, if chronically present and not adaptively regulated, has deleterious effects on mental and physical health. Decreased self-awareness and increased other-awareness are understood to be an adaptive response to SET. Attentional deployment – the process of selectively attending to certain aspects of emotional stimuli to modulate emotional reactivity – is supported by fronto-parietal and fronto-limbic networks, with the dorsolateral prefrontal cortex being a central hub. The primary aim of the current study was to investigate the effects of active (versus sham) prefrontal transcranial direct current stimulation (tDCS) on self and other-attentional deployment during the exposure to a SET context. Seventy-four female participants received active or sham tDCS and were subsequently exposed to a rigged social feedback paradigm. In this paradigm a series of social evaluations were presented together with a photograph of the supposed evaluator and a self- photograph of the participant, while gaze behavior (time to ﬁrst ﬁxation, total ﬁxation time) and skin conductance responses (SCRs; a marker of emotional reactivity) were measured. For half of the evaluations, participants could anticipate the valence (negative or positive) of the evaluation a priori. Analyses showed that participants receiving active tDCS were (a) slower to ﬁxate on their self-photograph, (b) spent less time ﬁxating on their self-photograph, and (c) spent more time ﬁxating on the evaluator photograph. During unanticipated evaluations, active tDCS was associated with less time spent ﬁxating on the evaluation. Furthermore, among those receiving active tDCS, SCRs were attenuated as a function of slower times to ﬁxate on the self-photograph. Taken together, these results suggest that in a context of SET, prefrontal tDCS decreases self-attention Correspondence: Jens Allaert jens.allaert@ugent.be Specialty section: This article was submitted to Cognitive Neuroscience, a section of the journal Frontiers in Human Neuroscience Received: 26 April 2021 Accepted: 12 July 2021 Published: 17 August 2021 Citation: Allaert J, Erdogan M, Sanchez-Lopez A, Baeken C, De Raedt R and Vanderhasselt M-A (2021) Prefrontal tDCS Attenuates Self-Referential Attentional Deployment: A Mechanism Underlying Adaptive Emotional Reactivity to Social-Evaluative Threat. Front. Hum. Neurosci. 15:700557. Citation: Allaert J, Erdogan M, Sanchez-Lopez A, Baeken C, De Raedt R and Vanderhasselt M-A (2021) Prefrontal tDCS Attenuates Self-Referential Attentional Deployment: A Mechanism Underlying Adaptive Emotional Reactivity to Social-Evaluative Threat. Front. Hum. Neurosci. 15:700557. doi: 10.3389/fnhum.2021.700557 1 Ghent Experimental Psychiatry Lab, Department of Head and Skin, Ghent University, University Hospital Ghent (UZ Ghent), Ghent, Belgium, 2 Psychopathology and Affective Neuroscience Laboratory, Department of Experimental Clinical and Health Psychology, Ghent University, Ghent, Belgium, 3 Research in Developmental Disorders Lab, Department of Experimental Clinical and Health Psychology, Ghent University, Ghent, Belgium, 4 Department of Clinical Psychology, Universidad Complutense de Madrid, Madrid, Spain, 5 Department of Psychiatry, Vrije Universiteit Brussel (VUB), Universitair Ziekenhuis Brussel (UZ Brussel), Brussels, Belgium, 6 Department of Electrical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands Edited by: Joukje Oosterman, Radboud University Nijmegen, Netherlands Edited by: Joukje Oosterman, Radboud University Nijmegen, Netherlands INTRODUCTION self-reported negative aﬀect (Ferri et al., 2013, 2016). The prefrontal cortex is essential for executive functioning (e.g., selective attention, working memory, decision making, etc.) and consists mainly of the dorsolateral prefrontal cortex (DLPFC), the medial prefrontal cortex (mPFC) and the orbitofrontal cortex (OFC), with the latter two regions often considered as a relatively uniform structure; the ventromedial prefrontal cortex (vmPFC; Kolb and Whishaw, 2009). The vmPFC and DLPFC are thought to be both involved in emotional processing, with the vmPFC being involved in the generation of emotional reactivity (i.e., emotional arousal) and the DLPFC being more involved in the attribution of positive or negative valence (Nejati et al., 2021). In addition, the vmPFC has been shown to play a central role in the processing of self-referent information (Northoﬀet al., 2006). Furthermore, the DLPFC plays a role in the interplay between anticipatory and “online” regulatory processes (Seo et al., 2014; De Raedt and Hooley, 2016), as prior expectations surrounding an upcoming emotional stimulus can greatly inﬂuence self-regulatory processes when subsequently confronted with that stimulus (Gunther Moor et al., 2010; van der Veen et al., 2019). Taken together, the prefrontal cortex plays an essential role in a wide range of psychological processes related to cognitive ﬂexibility, emotional processing and emotion regulatory processes (Ochsner and Gross, 2005; Kim et al., 2011; Stuss, 2011). Social interactions make up a substantial part of our lives and the need to belong is a fundamental drive in human behavior (Baumeister and Leary, 1995). Situations in which aspects of one’s self could be negatively evaluated by others (social-evaluative threat; SET) elicit profound physiological and psychological reactivity that may – if not adaptively regulated – lead to the development of depression and increase the risk of cardiovascular diseases (Merz et al., 2002; Slavich et al., 2009, 2010; Muscatell et al., 2015). In terms of adaptive reactivity to SET, theories on self-regulation suggest that decreased self-awareness (e.g., attention toward aspects of the self) serves as a self-protective defensive mechanism to attenuate self-referent rejection-related distress (Twenge et al., 2003; Durlik and Tsakiris, 2015). Concurrently, increased other-awareness (e.g., attention toward aspects of others) is suggested to serve as a mechanism promoting prosocial behavior by tuning attention toward the other(s) and taking their perspective, with the aim to repair and maintain social relationships (Hess and Pickett, 2010; Knowles, 2014). INTRODUCTION Given the implications of SET reactivity on both mental and physical health, it is important to further investigate the proposed neurocognitive mechanisms underlying adaptive SET reactivity, and how these adaptive processes can be promoted. The ﬂexible regulation of attention toward relevant and irrelevant information both externally and internally (i.e., selective attention and working memory), is of crucial importance in adaptive behavior and mental health (Gross and Thompson, 2007; Chun et al., 2011; Aldao et al., 2015). An important mechanism underlying depression is assumed to be the presence of attentional biases toward negative self- referent information and diﬃculties to shift attention away from this information, thereby contributing to sustained negative self-referential processing and perpetuating depressive mood (Koster et al., 2011; Joormann and Vanderlind, 2014). Consistent with this view, studies have shown that the ability to disengage attention away from negative information prospectively predicts the onset of depressive symptoms via repetitive negative self- referential thinking (Sanchez-Lopez et al., 2019c; Yaroslavsky et al., 2019). Theories of emotion regulation postulate that attentional deployment is an emotion regulatory process that involves shifting attentional focus toward or away from particular aspects of emotional stimuli, thereby modulating emotional reactivity (Gross and Thompson, 2007). Research employing concurrent fMRI and eye-tracking showed that deploying attention to non-arousing (compared to arousing) aspects of emotional stimuli involves the recruitment of fronto- parietal brain networks and reduces amygdala activity and Studies have shown that the causal modulation of the prefrontal cortex and its associated neural networks, via transcranial direct current stimulation (tDCS), can facilitate processes involved in cognitive ﬂexibility (e.g., selective attention, working memory), modulate the interplay between anticipatory and online self-regulatory processes, and produces a wide array of adaptive cognitive and psychological eﬀects in both depressed and healthy populations (Mondino et al., 2015; Dedoncker et al., 2016; Allaert et al., 2020; Smits et al., 2020). TDCS is a low cost and easy to use form of non-invasive brain stimulation that operates through the delivery of a weak electrical current to the scalp, which modulates the membrane potentials of the underlying neurons and thereby inﬂuences cortical excitability (Stagg and Nitsche, 2011). For example, it has been shown that left prefrontal tDCS can transiently (a) reduce attentional biases to threat (Ironside et al., 2016), (b) improve attentional disengagement abilities from emotional information (Sanchez- Lopez et al., 2018), and (c) attenuate emotional reactivity to SET (Allaert et al., 2020). Reviewed by: doi: 10.3389/fnhum.2021.700557 August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 1 Allaert et al. tDCS Effects on Attentional Deployment while increasing other-attention, and that attenuated self-referential attention speciﬁcally may be a neurocognitive mechanism through which tDCS reduces emotional reactivity. Moreover, the results suggest that tDCS reduces vigilance toward stimuli that possibly convey threatening information, corroborating past research in this area. Keywords: tDCS, self-referential processing, attentional deployment, emotional reactivity, social-evaluative threat, DLPFC Frontiers in Human Neuroscience \| www.frontiersin.org Participants p Seventy-four healthy female individuals (Mage = 20.80, SDage = 2.11) participated in the study. Given the sex diﬀerences in emotional processing and reactivity to social evaluations (McRae et al., 2008; Lithari et al., 2010; Vanderhasselt et al., 2018), only female participants were included, to reduce the sample variability. The sample size was determined based on an a priori power analysis. In lack of clear consensual guidelines for power analyses for the employed statistical method (generalized linear mixed models; see data analytic plan), a power analysis was conducted for linear models instead, using the G∗Power software (Faul et al., 2007). This estimated a total sample size of N = 74 to detect with 80% power the hypothesized between- within interaction eﬀect of a small to moderate magnitude (Cohen’s f = 0.15) using a mixed ANOVA F-test. Participants were recruited in the context of a larger research project investigating the eﬀects of left prefrontal tDCS on the interplay between anticipatory and online emotion regulatory processes to social evaluations (Allaert et al., 2020). They were recruited from the general community via internet postings on social media and posters in public places. Selection criteria were (a) right-handed, (b) normal or corrected to normal vision, (c) no current psychiatric or neurological disorders, (d) no current use of psychiatric drugs, (e) no personal or family history of epilepsy, (f) no recent neurosurgery, (g) not pregnant, and (h) no metal or magnetic objects in or around the scalp. Participants were asked not to smoke or ingest caﬀeine and/or alcohol 2 h prior to the experiment. The study was conducted with the approval of Ghent University’s Medical Ethical Committee and in accordance with the Declaration of Helsinki. Participants provided informed consent at the start of the experiment and received € 15 for participating. y To address these research objectives, an experimental paradigm featuring SET was employed with the aim to simulate the nature of real-life situations (e.g., social interactions). In this paradigm participants are systematically exposed to self-relevant information (e.g., positive and negative social evaluations), evoking strong psychological and physiological responses that are relevant to the pathogenesis of depression (Slavich et al., 2009, 2010; Vanderhasselt et al., 2018). Participants are led to believe that, based on their self-photograph, strangers have formed ﬁrst impressions about them, and that they are presented with these explicit social evaluations. INTRODUCTION However, ecologically valid research that investigates the eﬀects of prefrontal tDCS on the interplay between selective attention processes toward self-referential stimuli and emotional reactivity, and how prior expectations may inﬂuence this, is non-existent. One eye-tracking study August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 2 Allaert et al. tDCS Effects on Attentional Deployment MATERIALS AND METHODS showed that anodal (vs. sham) left prefrontal tDCS was associated with a transient diminished attentional bias (i.e., less gaze ﬁxation) toward negative emotion-eliciting images, and that this was associated with reduced self-reported state anxiety (Chen et al., 2017). Thus, in view of the importance of impairments in attentional processes toward self-referential information underlying depression, and the existing research gap, the goal of the present study was to investigate the eﬀects of prefrontal tDCS on self-referential attentional processes, their interplay with emotional reactivity, and how prior expectations may inﬂuence these. showed that anodal (vs. sham) left prefrontal tDCS was associated with a transient diminished attentional bias (i.e., less gaze ﬁxation) toward negative emotion-eliciting images, and that this was associated with reduced self-reported state anxiety (Chen et al., 2017). Thus, in view of the importance of impairments in attentional processes toward self-referential information underlying depression, and the existing research gap, the goal of the present study was to investigate the eﬀects of prefrontal tDCS on self-referential attentional processes, their interplay with emotional reactivity, and how prior expectations may inﬂuence these. Participants During each exposure to an evaluation, a photograph of the participant and of the supposed evaluator is shown, along with the explicit positive or negative evaluation, while participants’ gaze behavior (time to ﬁrst ﬁxation and total ﬁxation time) to these is measured. Concurrently, emotional reactivity is assessed via the measurement of autonomic changes in skin conductivity (skin conductance responses; SCRs), as SCRs are mediated by amygdala activity and index emotional arousal (Wood et al., 2014; Christopoulos et al., 2019; Allaert et al., 2020). First, based on the self-regulation theories proposing adaptive SET reactivity consists of both decreased self-attention and increased other- attention (Hess and Pickett, 2010; Durlik and Tsakiris, 2015), and given the wide array of research showing adaptive left prefrontal tDCS eﬀects on attention processes and emotional reactivity (Mondino et al., 2015; Smits et al., 2020), we expected that active (in contrast to sham) left prefrontal tDCS would be associated with attenuated self-attention (i.e., slower ﬁrst ﬁxation to and less total ﬁxation time on this information) and increased other-attention (i.e., faster ﬁrst ﬁxation to and more total ﬁxation time on this information). We had no clear expectations whether these eﬀects would be modulated by valence, as previous prefrontal tDCS research has shown both valence-independent (Sanchez-Lopez et al., 2018; Allaert et al., 2020), and valence-dependent tDCS eﬀects (Mondino et al., 2015; Nejati et al., 2021). Second, based on the proposed association between prefrontal-mediated attentional processes and emotional reactivity, we expected the attentional processes that are modulated by tDCS to be more strongly associated with attenuated emotional reactivity (assessed via SCRs) among participants receiving active (versus sham) tDCS. Third, in view of the clinical relevance of the interplay between anticipatory processes and “online” self-regulatory processes, and the role of the DLPFC herein, the last objective was to explore whether tDCS diﬀerentially aﬀected gaze behavior during expected vs. unexpected social evaluations. Frontiers in Human Neuroscience \| www.frontiersin.org Skin Conductance Responses (SCRs) p ( ) Electrodermal activity was recorded at a sample rate of 1000 Hz with the Biopac EDA100c ampliﬁer, in conjunction with the Biopac MP150 (Biopac Systems Inc., Santa Barbara, CA, United States). On the ampliﬁer, the gain was set to 5 µS/V, the low pass ﬁlter set to 10 Hz, and both high pass ﬁlters set to DC mode (oﬀ). Two pre-gelled Ag/AgCL electrodes, connected to the ampliﬁer, were placed on the left, non-dominant hand, on the thenar and hypothenar. The data was collected in the Acqknowledge software on an external computer, together with event triggers that were sent by the E-Prime computer to the Biopac STP100c, via a Cedrus StimTracker Duo device (Biopac Systems Inc., Santa Barbara, CA; Cedrus Corporation, San Pedro, CA, United States). Using the Ledalab 3.4.9 MATLAB toolbox (Karenbach, 2005), the data was downsampled to 50 Hz (to increase processing speed), artifact corrected using spline interpolation, smoothed using a 16 sample moving average, and ﬁltered using a ﬁrst order Butterworth 5 Hz lowpass ﬁlter. Continuous decomposition analysis then extracted the phasic information from the skin conductance signals, based on a SCR detection threshold of 0.01 µS (Boucsein et al., 2012), and computed average SCR amplitude (expressed in µS) for every presentation of a social evaluation, which served as the secondary dependent variable. Transcranial Direct Current Stimulation (tDCS) Transcranial Direct Current Stimulation (tDCS) Transcranial direct current stimulation was applied with a pair of rubber surface electrodes (5 × 7 cm = 35 cm2) covered with electrode gel and delivered with a battery-driven stimulator (DC-Stimulator Plus, neuroConn GmbH). The anodal electrode was vertically positioned over F3 (corresponding to the left DLPFC) according to the 10–20 international EEG system, whereas the cathode was placed over the contralateral supra- orbital area (Fp2). This electrode positioning is in accordance with previous tDCS studies on emotional processing and the level B recommendation for treating major depressive disorders (Nitsche et al., 2008; Dedoncker et al., 2016; Lefaucheur et al., 2017). A current of 2 mA (current density = 0.06), with 30 s of ramp up was applied for 20 min (with ramp down at the end). Half of the participants received active tDCS, whereas the other half received sham tDCS (between-subject design). For sham tDCS (i.e., placebo) the current was directly ramped down after the initial ramp up phase (Nitsche et al., 2008). Social Feedback Paradigm A paradigm was used in which participants are repeatedly confronted with (experimentally rigged) positive and negative social evaluations (Allaert et al., 2020). Participants were led to believe that the study was part of a larger research project in which research groups from diﬀerent universities collaborate to investigate how people form ﬁrst impressions. The participants were briefed that they would have to evaluate participants that were recruited by these other research groups, based on the ﬁrst impression they form when viewing their photograph. In turn, the participants had to provide a self-photograph so that the supposed participants from the other research groups could evaluate them. Before the paradigm, participants were told that the evaluations of these others will be shown to them. During each social evaluation presentation (8000 ms), participants were shown their self-photograph, the photograph of the supposed evaluator and a negative or positive word (e.g., stupid, attractive). The paradigm consisted of 80 trials (i.e., evaluations), divided in 4 blocks of 20 trials with breaks in between. Each trial started with an inter-stimulus interval (ISI; 2500 ms) displaying August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 3 tDCS Effects on Attentional Deployment Allaert et al. a ﬁxation cross. In half of the trials (anticipatory context), participants could correctly anticipate the valence of the social evaluation, prior to its presentation, by means of a cue (8000 ms). In the other half (non-anticipatory context), the evaluation is directly presented after the ISI. Figure 1 shows a visual representation of the experimental sequence for each context. The employed stimuli consisted of 80 unique words (matched on arousal between positive and negative valence) from a validated normative database (see supplementary materials; Moors et al., 2013) and 80 photographs obtained from volunteers (aged 18 – 30) outside the participant pool. To prevent luminance-evoked pupillary responses, all images were gray-scaled and matched on luminance values via the Matlab SHINE toolbox (Willenbockel et al., 2010). During the ISI, gray-scaled placeholder images with the same luminance values were presented on the location where the subsequent photographs appear (Allaert et al., 2020). The order of the speciﬁc combinations of trial features (context, evaluator, gender of evaluator, word, location of the evaluator photograph) was pseudorandom (see Supplementary Materials). During block 1 to 3, the valence of the evaluations was equally distributed, whereas in the last block1 negative feedback was more prevalent (80%). 1This was done to investigate emotional recovery from mainly negative social feedback, however, these results fall outside the scope of the research objectives of this manuscript. Self-Report Measurements To ensure comparable active and sham tDCS groups, an online survey assessing various potential confounders was carried out prior to the experiment. These consisted of age, perceived criticism, self-esteem, resilience, the habitual use of various maladaptive and adaptive emotion regulation strategies, symptoms of depression, anxiety and distress, and self-eﬃcacy. These variables could potentially inﬂuence reactivity to the social feedback paradigm. Table 1 presents an overview of the descriptive and inferential statistics of these variables. Participants were pseudo-randomly assigned to one of the two groups, in order to have comparable groups based on this survey data. This procedure consisted of a random allocation of participants to groups in the ﬁrst half of the participant sample. Halfway through data collection the online survey data of planned participants were monitored and based on this data, scheduled participants were allocated to one of the two groups in order to minimize group diﬀerences on these variables. Independent t-tests showed that the two tDCS groups did not signiﬁcantly diﬀer on any of these variables (all ps > 0.13). Social Feedback Paradigm The paradigm was programmed in E-prime 2.0 Professional (Psychology Software Tools, Pittsburgh, PA, United States). Software Tools, Pittsburgh, PA, United States). A standard 9- point calibration sequence was used to calibrate participants’ eye tracking. Fixations were deﬁned as to be at least 100 ms of duration. Time to ﬁrst ﬁxation (i.e., time elapsed prior to making ﬁrst ﬁxation on a given AOI, in seconds) and total ﬁxation time (i.e., total time spent ﬁxating on a given AOI, in seconds) were computed for every AOI (self, evaluator, feedback) on every trial, using the PhysioData Toolbox 0.5 (Sjak-Shie, 2018), and served as the primary dependent variables. The ﬁrst author and experiment leader received training in eye-tracking methodology from the eye tracker manufacturer (Tobii AB, Stockholm, Sweden). Skin Conductance Responses (SCRs) Figure 2 shows a visualization of the electric ﬁeld simulation of the utilized tDCS montage (Kempe et al., 2014), using Soterix HD-Explore software. The tDCS procedure followed a single- blind design. All involved authors have extensive expertise in the application of tDCS in combination with eye-tracking (Allaert et al., 2019, 2020, 2021). Eye Tracking Every trial started with an ISI and was followed either by an anticipation phase succeeded by an evaluation (anticipatory context), or a direct presentation of an evaluation (non-anticipatory context). During the presentation of an evaluation, a photograph of the participant next to the supposed evaluator was shown. FIGURE 2 \| Electric ﬁeld simulations of the tDCS montage. The electric ﬁeld simulations show the largest electric ﬁeld potentials within prefrontal regions. FIGURE 2 \| Electric ﬁeld simulations of the tDCS montage. The electric ﬁeld simulations show the largest electric ﬁeld potentials within prefrontal regions. FIGURE 2 \| Electric ﬁeld simulations of the tDCS montage. The electric ﬁeld simulations show the largest electric ﬁeld potentials within prefrontal regions. mulations of the tDCS montage. The electric ﬁeld simulations show the largest electric ﬁeld potentials within prefrontal regions. FIGURE 2 \| Electric ﬁeld simulations of the tDCS montage. The electric ﬁeld simulations show the largest electric ﬁeld potentials Self-esteem Self-esteem was measured using the Rosenberg Self- Esteem Scale (RSES; Rosenberg, 1965; Franck et al., 2008), a scale consisting of 10 items (e.g., “I take a positive attitude toward myself ”) rated on a 4-point Likert scale (0 = completely disagree, 3 = completely agree). Eye Tracking Gaze behavior was recorded at a sample rate of 300 Hz with the Tobii TX300 eye tracker (Tobii AB, Stockholm, Sweden) in conjunction with the E-Prime Extensions for Tobii (Psychology 1This was done to investigate emotional recovery from mainly negative social feedback, however, these results fall outside the scope of the research objectives of this manuscript. August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 4 Allaert et al. tDCS Effects on Attentional Deployment FIGURE 1 \| Social feedback paradigm. Every trial started with an ISI and was followed either by an anticipation phase succeeded by an evaluation (anticipatory context), or a direct presentation of an evaluation (non-anticipatory context). During the presentation of an evaluation, a photograph of the participant next to the supposed evaluator was shown. FIGURE 2 \| Electric ﬁeld simulations of the tDCS montage. The electric ﬁeld simulations show the largest electric ﬁeld potentials within prefrontal regions. Perceived criticism Perceived criticism was measured using a 1-item visual analog scale (“How critical do you think people in your nearest environment – such family, friends, partner – are of you?”; Masland and Hooley, 2015), ranging from 0 (not at all) to 100 (a lot). Self-esteem Self-esteem was measured using the Rosenberg Self- Esteem Scale (RSES; Rosenberg, 1965; Franck et al., 2008), a scale consisting of 10 items (e.g., “I take a positive attitude toward myself ”) rated on a 4-point Likert scale (0 = completely disagree, 3 = completely agree). FIGURE 1 \| Social feedback paradigm. Every trial started with an ISI and was followed either by an anticipation phase succeeded by an evaluation (anticipatory context), or a direct presentation of an evaluation (non-anticipatory context). During the presentation of an evaluation, a photograph of the participant next to the supposed evaluator was shown. FIGURE 1 \| Social feedback paradigm. Every trial started with an ISI and was followed either by an anticipation phase succeeded by an evaluation (anticipatory context), or a direct presentation of an evaluation (non-anticipatory context). During the presentation of an evaluation, a photograph of the participant next to the supposed evaluator was shown. FIGURE 2 \| El t i ﬁld i l ti f th tDCS t Th l t i ﬁld i l ti h th l t l t i ﬁld t ti l ithi f t l i FIGURE 1 \| Social feedback paradigm. Perceived criticism Perceived criticism was measured using a 1-item visual analog scale (“How critical do you think people in your nearest environment – such family, friends, partner – are of you?”; Masland and Hooley, 2015), ranging from 0 (not at all) to 100 (a lot). August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org Frontiers in Human Neuroscience \| www.frontiersin.org 5 tDCS Effects on Attentional Deployment Allaert et al. TABLE 1 \| Descriptive statistics and welch two sample t-test statistics. Active tDCS Sham tDCS Variable M (SD) M (SD) t p Age 21.16 (2.42) 20.43 (1.69) 1.50 0.14 Perceived criticism PC 6.62 (1.46) 6.27 (1.88) 0.90 0.37 Self-esteem RSES 18.92 (5.52) 19.59 (5.69) −0.51 0.61 Self-efﬁcacy GSE 28.51 (4.04) 28.76 (5.82) −0.24 0.81 Resilience CD-RISC 25.54 (5.10) 25.19 (5.82) 0.28 0.78 Cognitive reappraisal ERQ 27.35 (5.89) 28.46 (4.77) −0.89 0.38 Expressive suppression ERQ 13.30 (4.29) 13.76 (3.83) −0.49 0.63 Acceptance CERQ 13.81 (2.45) 14.57 (3.45) −1.09 0.28 Positive reappraisal CERQ 13.65 (3.21) 14.41 (3.66) −0.94 0.35 Positive refocus CERQ 11.84 (3.02) 11.14 (3.00) 1.00 0.32 Putting into perspective CERQ 14.78 (3.33) 15.08 (2.92) −0.41 0.68 Refocus on planning CERQ 14.65 (3.43) 15.16 (2.97) −0.69 0.49 Rumination CERQ 13.68 (4.24) 14.08 (4.05) −0.42 0.68 Catastrophizing CERQ 7.54 (2.82) 8.46 (2.98) −1.36 0.18 Self-blame CERQ 13.03 (3.10) 13.16 (2.42) −0.21 0.83 Other-blame CERQ 8.19 (3.06) 8.54 (3.30) −0.47 0.64 General distress MASQ30 24.30 (7.91) 25.46 (7.94) −0.63 0.53 Anhedonic depression MASQ30 25.92 (7.54) 28.92 (9.03) −1.55 0.13 Anxious arousal MASQ30 17.24 (5.96) 18.11 (6.74) −0.58 0.56 Rumination RRS 49.27 (12.98) 48.51 (12.88) 0.25 0.80 Brooding RRS 11.27 (3.34) 10.92 (3.21) 0.46 0.65 Reﬂection RRS 10.84 (3.71) 10.19 (3.64) 0.76 0.45 The scale showed good internal consistency (Cronbach’s α = 0.87). TABLE 1 \| Descriptive statistics and welch two sample t-test statistics. the habitual usage of cognitive reappraisal (e.g., “I control my emotions by changing the way I think about the situation I’m in”) and expressive suppression (e.g., “When I am feeling negative emotions, I make sure not to express them”). The factor expressive suppression (Cronbach’s α = 0.64) showed questionable internal consistency while cognitive reappraisal (Cronbach’s α = 0.76) showed acceptable internal consistency. Resilience Resilience was measured using the Connor-Davidson Resilience Scale (CD-RISC; Connor and Davidson, 2003), a scale consisting of 25 items (e.g., “I am not easily discouraged by failure”) rated on a 5-point Likert scale (0 = not true at all, 4 = true nearly all of the time). The scale showed good internal consistency (Cronbach’s α = 0.86). Self-eﬃcacy G l lf General self-eﬃcacy was measured with the General Self-Eﬃcacy Scale (GSES; Teeuw et al., 1994), a 10-item scale (e.g., “I remain calm when facing diﬃculties because I can rely on my coping abilities”) rated on a 4-Point Likert scale (1 = completely disagree, 4 = completely agree). The scale showed good internal consistency (Cronbach’s α = 0.82). Emotion regulation The habitual use of maladaptive and adaptive emotion regulation strategies was measured with the Ruminative Response Scale (RRS; Nolen-Hoeksema and Morrow, 1991; Raes et al., 2009), the Emotion Regulation Questionnaire (ERQ; Gross and John, 2003), and the Cognitive Emotion Regulation Questionnaire (CERQ; Garnefski and Kraaij, 2007). The RRS is a 22-item scale rated on a 4-point Likert scale (1 = almost never, 4 almost always), assessing 3 types of responses to depressive feelings; rumination (e.g., “I think about how sad I feel”), brooding (e.g., “Thinking about a recent situation, wishing it had gone better”), and reﬂection (e.g., “Analyze my personality to try to understand why I am depressed”). The rumination (Cronbach’s α = 0.91) factor showed excellent internal consistency while the brooding (Cronbach’s α = 0.70) and reﬂection (Cronbach’s α = 0.76) factor showed acceptable internal consistency. The ERQ is a 10-item scale rated on a 7-point Likert scale (1 = completely disagree, 7 = completely agree) and assess Symptoms of depression, anxiety and distress y p f p y Symptoms of depression, anxiety, and stress were measured using the short Mood and Anxiety Symptoms Questionnaire (MASQ-30; Wardenaar et al., 2010). This is a 30-item scale rated on a 5-point Likert scale (1 = not at all, 5 = a lot) assessing anhedonic depression (e.g., “Felt like I had a lot to look forward to”), anxious arousal (e.g., “Heart was racing or pounding”) and general distress (e.g., “Worried a lot about things”). The factors anxious arousal (Cronbach’s α = 0.83) and anhedonic depression (Cronbach’s α = 0.84) showed good internal consistency whereas the factor general distress showed acceptable internal consistency (Cronbach’s α = 0.74). Perceived criticism The CERQ is a 36- item scale rated on a 5-Point Likert scale (1 = almost never, 5 = almost always), assessing 9 types of emotion regulation in response to negative experiences; self-blame (e.g., “I feel that I am the one to blame for it”), acceptance (e.g., “I think that I have to accept the situation”), rumination (e.g., “I dwell upon the feelings the situation has evoked in me”), positive refocus (e.g., “I think of something nice instead of what has happened”), refocus on planning (e.g., “I think of what I can do best”), positive reappraisal (e.g., “I think I can learn something from the situation”), putting into perspective (e.g., “I think that it all could have been much worse”), catastrophizing (e.g., “I often think that what I have experienced is much worse than what others have experienced”), and other-blame (e.g., “I feel that others are responsible for what has happened”). The factors rumination (Cronbach’s α = 0.86), refocus on planning (Cronbach’s α = 0.86), other-blame (Cronbach’s α = 0.87), putting into perspective (Cronbach’s α = 0.81) and positive reappraisal (Cronbach’s α = 0.83) showed good internal consistency, whereas the factors self-blame (Cronbach’s α = 0.71), acceptance (Cronbach’s α = 0.76), positive refocus (Cronbach’s α = 0.76), and catastrophizing (Cronbach’s α = 0.74) showed acceptable internal consistency. The scale showed good internal consistency (Cronbach’s α = 0.87). Mood during the experimental session To assess changes in mood throughout the experimental protocol, self-reported mood was measured at three time points (pre-stimulation, post-stimulation, and post-task), using six visual analog scales (tiredness, vigorousness, angriness, tension, sadness, and happiness; McCormack et al., 1988) presented August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org Frontiers in Human Neuroscience \| www.frontiersin.org 6 tDCS Effects on Attentional Deployment Allaert et al. a generalized linear mixed modeling approach was warranted. Therefore, to ensure the selection of a distribution and link function that best ﬁts the observed data, a series of (G)LMM models were, based on the Akaike Information Criterion (AIC), compared for each dependent variable (time to ﬁrst ﬁxation, total ﬁxation time, SCRs; see Table 2), only diﬀering in their speciﬁed distribution (normal and gaussian) and link (identity, log and inverse) function. The lowest AIC value indicates the best ﬁtting model of the series. The speciﬁc model structure for each of the three dependent variables (time to ﬁrst ﬁxation, total ﬁxation time, SCRs; see further) was based on our hypotheses and continuous predictors were standardized prior to model ﬁtting. Since a gamma distribution is only compatible with data containing positive numbers (excluding zero), SCR data points containing zero (0.75%) were removed to assess the ﬁt under a gamma distribution. Furthermore, prior to model ﬁtting, gaze data points containing (a) more than 50% missing gaze data, or (b) a time to ﬁrst ﬁxation to an AOI faster than 100 ms (i.e., anticipatory saccade; Sanchez-Lopez et al., 2019b) were excluded from subsequent analyses (14.75%). In addition, due to technical malfunctions with the SCR recording equipment and the eye-tracker, the SCR data of 5 subjects were corrupted and excluded from further analysis (6.76%). Based on the AIC and consistent with statistical literature, time to ﬁrst ﬁxation (i.e., reaction time data) was best described by a gamma model with a log-link (Lo and Andrews, 2015; Santhanagopalan et al., 2018), whereas total ﬁxation time (i.e., duration data) was best described by a gamma model with an identity-link (Hardin and Hilbe, 2007). SCR amplitude was best described by a log- gamma model (Braithwaite et al., 2013). The analysis-of-variance tables for these retained models were then computed via the “ANOVA” function of the “car” R package, with the sum of squares estimated using the type III approach (Fox et al., 2012). Mood during the experimental session The statistical signiﬁcance level was set to p < 0.05 and p-values for the ﬁxed eﬀects were estimated with the “lmerTest” R package, using the Satterthwaite approximations to degrees of freedom (Kuznetsova et al., 2017). For the decomposition of interaction eﬀects, follow-up tests were pairwise comparisons of the estimated marginal means (EMMs) or pairwise comparisons of the EMMs of linear trends (i.e., comparison of slopes), via the “emmeans” and “emtrends” function in the “emmeans” R package (Lenth, 2018). To maximize statistical power, follow-up tests for which we had speciﬁc directional hypotheses were one- tailed (self and evaluator), whereas the others were two-tailed. Furthermore, p-values from the follow-up tests were corrected for multiple comparisons using the false discovery rate correction (Benjamini, 2010). To investigate (a) the eﬀects of tDCS on attentional on the computer screen, ranging from “totally not” (0) to “very much” (100). Protocol Participants provided informed consent on a webpage and completed the online survey. They were led to believe that they would take part in a study in which the eﬀects of tDCS on the processing of ﬁrst impressions are investigated. It was stated that they had to ﬁrst evaluate participants recruited by other collaborating universities, based on the ﬁrst impression they experience when viewing a photograph of them. In turn, these other students would then evaluate them, based on their self- photograph. On the webpage, participants were presented a series of 20 pictures of strangers along with 4 evaluative descriptive words (2 negative and 2 positive words, obtained from a validated database of Dutch words; Moors et al., 2013). For each picture, participants were asked to indicate which word corresponded the most with the ﬁrst impression they had formed about the stranger. Afterward, participants could upload a self-photograph. At the end, participants could schedule the experiment. In the laboratory, participants were seated in front of a computer screen and were connected to the physiological recording equipment. Participants underwent active or sham tDCS, and mood states were assessed before and after the stimulation session. Then, the social feedback paradigm started. Finally, mood states were assessed, and participants were debriefed and paid. Frontiers in Human Neuroscience \| www.frontiersin.org Data Analytic Plan All data were analyzed in R 3.6.1 (R Core Team, 2013) using (generalized) linear mixed models [(G)LMM]s ﬁtted via the “lmer” and “glmer” functions of the “lme4” R package (Bates et al., 2014). These mixed models can handle unbalanced data, account for inter-individual variability in psychophysiological reactivity and oﬀer higher control of type I errors compared to classic (general) linear models (Cnaan et al., 1997; Bagiella et al., 2000; Mallinckrodt et al., 2001; Boisgontier and Cheval, 2016). The implementation of GLMMs requires to specify an underlying distribution of the observed data (e.g., normal, gamma), instead of relying on the assumption that the data follows a normal distribution as in classic linear models (e.g., ANOVA, ANCOVA, repeated measures ANOVA; Jiang and Nguyen, 2021). In addition, when using GLMMs, a link function needs to be speciﬁed; this speciﬁes the type of relationship between the independent variables and the dependent variable, such as an identity (linear), logarithmic or inverse relationship. By specifying a link function, data transformations that are often employed in classic linear models are not required. This is advantageous as data transformations alter the original scale of the measurement and may potentially lead to misleading conclusions (Lo and Andrews, 2015). Taken together, GLMMs oﬀer increased ﬂexibility to model data by allowing diﬀerent types of data distributions and relationships between the predictors and outcome (e.g., linear, logarithmic, and inverse). To investigate (a) the eﬀects of tDCS on attentional deployment during social evaluations, and (b) whether the possibility of prior anticipation aﬀected these, two GLMMs were ﬁtted with time to ﬁrst ﬁxation (i.e., elapsed time until ﬁrst ﬁxation on an AOI) and total ﬁxation time [i.e., total time spent ﬁxating on an area of interest (AOI)] as respective dependent variables. These GLMMs both featured group (active tDCS, sham tDCS), AOI (self, evaluator, and feedback), valence (negative, positive), type (anticipated, non-anticipated) as ﬁxed factors, and subject as random intercept. First, a visual inspection of the histogram of each of the three dependent variables (Time to ﬁrst ﬁxation; see Figure 3A, Total ﬁxation time; see Figure 3B, SCRs, see Figure 3C) suggested that none of the variables followed a normal distribution, and that August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 7 Allaert et al. tDCS Effects on Attentional Deployment FIGURE 3 \| Histograms of time to ﬁrst ﬁxation, total ﬁxation time, and skin conductance responses. Data Analytic Plan The histograms of the three employed dependent variables [time to ﬁrst ﬁxation; (A), total ﬁxation time; (B), SCRs, (C)] suggest that none follow a normal distribution and that a GLMM approach is warranted. FIGURE 3 \| Histograms of time to ﬁrst ﬁxation, total ﬁxation time, and skin conductance responses. The histograms of the three employed dependent variables [time to ﬁrst ﬁxation; (A), total ﬁxation time; (B), SCRs, (C)] suggest that none follow a normal distribution and that a GLMM approach is warranted. RESULTS To investigate how tDCS aﬀects the relationship between tDCS-aﬀected attentional processes and emotional reactivity to social evaluations, one GLMM was ﬁtted with SCR amplitude as the dependent variable. This model featured group (active tDCS, sham tDCS), valence (negative, positive), type (anticipated, non-anticipated) as ﬁxed factors, and subject as random intercept. Furthermore, based on the results of the previous gaze analyses, the speciﬁc observed tDCS eﬀects on the various attentional indices (see results; i.e., group × self-ﬁxation time, group × evaluator-ﬁxation time, group × time to ﬁrst self-ﬁxation, group × type × time to ﬁrst feedback-ﬁxation) were entered as predictors in this GLMM. Participants were blind to their allocation to the tDCS groups as they were unable to correctly guess whether they received active or sham tDCS; the proportion of incorrect guesses (80%) was signiﬁcantly higher than chance level, (0.50), p < 0.001. No adverse eﬀects of tDCS (e.g., headache, fatigue, itching; Matsumoto and Ugawa, 2017) were reported. In addition, there were no signiﬁcant diﬀerences in the time of day of the experimental sessions between the active and sham tDCS groups, t = −0.59, p = 0.56. DISCUSSION (vs. sham) tDCS was associated with a slower time to ﬁxate on the feedback word, b = 0.12, SE = 0.06, z = 2.07, p = 0.04, whereas this was not the case during anticipated evaluations, b = 0.05, SE = 0.06, z = 0.82, p = 0.41. This indicates that the tDCS eﬀect on time to ﬁxate on the feedback word was modulated by type. For the evaluator-photograph, no signiﬁcant diﬀerences were present between active and sham tDCS during neither anticipated, b = 0.06, SE = 0.06, z = 1.06, p = 0.85, or non- anticipated evaluations, b = −0.06, SE = 0.06, z = −1.08, p = 0.14. All remaining tDCS-implicated eﬀects (i.e., group, group × type, group × valence, group × type × valence, group × valence × AOI, group × type × valence × AOI) were non-signiﬁcant (all ps > 0.06). (vs. sham) tDCS was associated with a slower time to ﬁxate on the feedback word, b = 0.12, SE = 0.06, z = 2.07, p = 0.04, whereas this was not the case during anticipated evaluations, b = 0.05, SE = 0.06, z = 0.82, p = 0.41. This indicates that the tDCS eﬀect on time to ﬁxate on the feedback word was modulated by type. For the evaluator-photograph, no signiﬁcant diﬀerences were present between active and sham tDCS during neither anticipated, b = 0.06, SE = 0.06, z = 1.06, p = 0.85, or non- anticipated evaluations, b = −0.06, SE = 0.06, z = −1.08, p = 0.14. All remaining tDCS-implicated eﬀects (i.e., group, group × type, group × valence, group × type × valence, group × valence × AOI, group × type × valence × AOI) were non-signiﬁcant (all ps > 0.06). The aim of this study was to investigate the eﬀects of prefrontal tDCS on self- versus other-attention (assessed via eye-tracking) during the confrontation with social evaluations, and how the attentional processes aﬀected by tDCS are linked to emotional reactivity (assessed via SCRs). Additionally, it was explored whether the possibility to anticipate the valence of a social evaluation modulated the tDCS eﬀects. First, the results showed that participants who received active (versus sham) tDCS were a) slower to ﬁxate on their self- photograph, b) spent less time ﬁxating on their self-photograph, and c) spent more time ﬁxating on the photograph of the evaluator. Effects of tDCS on Total Fixation Time Effects of tDCS on Total Fixation Time This GLMM showed a group × AOI interaction (see Figure 4B), χ2(2) = 19.98, p < 0.001. Follow-up pairwise comparisons probing the eﬀect of group for each AOI (self, evaluator, feedback) showed that active (versus sham) tDCS was associated with a shorter total ﬁxation time on the self-photograph, b = −0.17, SE = 0.08, z = −2.15, p = 0.02, and a longer total ﬁxation time on the evaluator-photograph, b = 0.15, SE = 0.09, z = 1.65, p = 0.04. Active (versus sham) tDCS was not associated with a signiﬁcant diﬀerence on total ﬁxation time to the feedback word, b = −0.04, SE = 0.070.07, z = −0.58, p = 0.56. All remaining tDCS-implicated eﬀects (i.e., group, group × type, group × valence, group × type × valence, group × type × AOI, group × valence × AOI, group × type × valence × AOI) were non-signiﬁcant (all ps > 0.17). Effects of tDCS on the Relationship Between Attentional Indices and Emotional Reactivity This GLMM showed a group × time to ﬁrst self-ﬁxation interaction (see Figure 6), χ2(1) = 14.12, p < 0.001. Follow- up tests showed that in the active tDCS group, time to ﬁrst self-ﬁxation was negatively associated with SCR amplitude to social evaluations, b = −0.19, SE = 0.03, z = −6.62, p < 0.001, whereas this association was non-signiﬁcant in the sham tDCS group, b = −0.04, SE = 0.030.03, z = −1.34, p = 0.18. Thus, during active (compared to sham) tDCS slower times to self- ﬁxate were associated with attenuated SCRs. All remaining tDCS eﬀects where attentional deployment indices were implicated (group × self-ﬁxation time, group × evaluator-ﬁxation time, group × time to ﬁrst feedback-ﬁxation, group × type × time to ﬁrst feedback-ﬁxation) were non-signiﬁcant (all ps > 0.24). For an overview of the tDCS eﬀects on SCRs in which attentional deployment was not investigated, see Allaert et al. (2020). Regarding the impact of social feedback expectations on tDCS eﬀects, the results showed that during unanticipated evaluations, participants receiving active (versus sham) tDCS were slower to ﬁxate on the feedback word, whereas this was not the case during anticipated evaluations. Generally, participants tended to ﬁrst ﬁxate on the feedback word when presented with an evaluation, as this conveys the speciﬁc content (e.g., intelligent and pretentious) of the social evaluation. During unanticipated evaluations, participants did not know in advance whether the evaluation would be negative or positive, and by looking at the feedback word they could ascertain whether or not the presented evaluation was self-threatening (Sherman and Cohen, 2006). Following this reasoning, the current result Effects of tDCS on Time to First Fixation Finally, to check whether the observed tDCS eﬀects are independent of changes in mood throughout the experimental procedure, six LMMs (for every mood state; tiredness, vigorousness, angriness, tension, sadness, and happiness) were ﬁtted with group (active tDCS and sham tDCS), and time (pre-stimulation, post-stimulation, and post-task) as ﬁxed factors, and subject as random intercept. Effects of tDCS on Time to First Fixation This GLMM showed a group × AOI interaction (see Figure 4A), χ2(2) = 18.54, p < 0.001. Follow-up pairwise comparisons probing the eﬀect of group for each AOI (self, evaluator, feedback) showed that active (versus sham) tDCS was associated with a slower time to ﬁxate on the self-photograph, b = 0.10, SE = 0.05, z = 1.91, p = 0.03. There were no signiﬁcant diﬀerences in time to ﬁrst ﬁxation between active and sham tDCS for the evaluator-photograph, b = −0.0003, SE = 0.050.05, z = −0.010.01, p = 0.500.50, or for the feedback word, b = 0.080.08, SE = 0.06, z = 1.52, p = 0.13. Furthermore, a higher-order group × AOI × type interaction (see Figure 5) was observed, χ2(2) = 13.74, p = 0.001. Follow-up pairwise comparisons investigating the eﬀect of group on each AOI (self, evaluator, feedback) and type (anticipated and non-anticipated) showed that active (vs. sham) tDCS was associated with a slower time to ﬁxate on the self-photograph during both the presentation of anticipated, b = 0.11, SE = 0.06, z = 1.91, p = 0.03, and non-anticipated evaluations, b = 0.10, SE = 0.06, z = 1.71, p = 0.04. This indicates that the tDCS eﬀect on time to ﬁxate on the self-photograph was not modulated by type. However, during the presentation of non-anticipated evaluations, active Results of the 3 main analyses where tDCS was not implicated are reported in the Supplementary Materials, as these fall outside the scope of the research objectives. TABLE 2 \| Model ﬁt (AIC) for dependent variables. Dependent variable Normal Log-gamma Identity-gamma Inverse-gamma Time to ﬁrst ﬁxation 40915 22004 22245 22216 Total ﬁxation time 49468 44054 43598* 44128 SCR amplitude −23682 −32396* *lowest value; lower values indicate better model ﬁt. August 2021 \| Volume 15 \| Article 700557 8 tDCS Effects on Attentional Deployment Allaert et al. DISCUSSION These ﬁndings show that in a context of SET, prefrontal tDCS attenuates attentional processes toward self- referential stimuli, while increasing attention toward other- referential stimuli. These results are in line with previous studies showing that prefrontal tDCS can modulate selective attention to (emotional) stimuli (Mondino et al., 2015; Sanchez-Lopez et al., 2018). Second, analyses investigating the relationship between these attentional processes and emotional reactivity showed that among participants receiving active (versus sham tDCS), emotional reactivity decreased in function of slower times to ﬁxate on their self-photograph. This might suggest that delayed attention toward self-referent information may be a neurocognitive mechanism through which prefrontal tDCS adaptively reduces emotional reactivity in situations featuring SET, as was observed in prior studies (Antal et al., 2014; Allaert et al., 2020; Carnevali et al., 2020). Moreover, this would be in line with past research showing that improved cognitive ﬂexibility (e.g., selective attention and working memory) mediates the eﬀects of tDCS on emotional reactivity to negative or stressful stimuli (Chen et al., 2017). Furthermore, It has been suggested that the attenuation of self- referential processes plays an underlying role in the adaptive eﬀects of prefrontal tDCS (Vanderhasselt et al., 2015a; De Raedt et al., 2017; Hoebeke et al., 2021). Taken together, the current results extend previous ﬁndings regarding the link between cognitive ﬂexibility and emotional reactivity (Vanderhasselt et al., 2013; Chen et al., 2017), by more directly measuring processes of clinical interest (i.e., attentional processes in the context of SET) in an ecologically valid experimental paradigm featuring natural responding in absence of task instructions. August 2021 \| Volume 15 \| Article 700557 Effects of tDCS on Mood Throughout the Experimental Procedure The LMMs on the mood measures (tiredness, vigorousness, angriness, tension, sadness, and happiness) yielded non- signiﬁcant eﬀects of group and group × time (all Fs < 0.88, all ps > 0.42), indicating that tDCS did not inﬂuence reported mood (across time). August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 9 Allaert et al. tDCS Effects on Attentional Deployment FIGURE 4 \| tDCS effects on self- vs. other-attention. The group receiving active (vs. sham) tDCS were slower to ﬁxate on their self-photograph (A), and ﬁxated less on it (B). Furthermore, the active (vs. sham) tDCS group ﬁxated more on the evaluator-photograph (B). FIGURE 4 \| tDCS effects on self- vs. other-attention. The group receiving active (vs. sham) tDCS were slower to ﬁxate on their self-photograph (A), and ﬁxated less on it (B). Furthermore, the active (vs. sham) tDCS group ﬁxated more on the evaluator-photograph (B). FIGURE 5 \| tDCS effect on early attention to unanticipated feedback. The group receiving active (vs. sham) tDCS were slower to ﬁxate on their self-photograph during both anticipated and unanticipated evaluations. During unanticipated evaluations, the group receiving active (vs. sham) tDCS were slower to ﬁxate on the feedback word, whereas this was not the case during anticipated evaluations. FIGURE 5 \| tDCS effect on early attention to unanticipated feedback. The group receiving active (vs. sham) tDCS were slower to ﬁxate on their self-photograph during both anticipated and unanticipated evaluations. During unanticipated evaluations, the group receiving active (vs. sham) tDCS were slower to ﬁxate on the feedback word, whereas this was not the case during anticipated evaluations. of both the anode (left DLPFC) and cathode (right supra- orbital area) electrode (Bikson et al., 2010) and the strongest electrical ﬁeld is suggested to be present somewhere in the middle (mPFC) between the anode and cathode (De Witte et al., 2018). Therefore, it is highly likely that multiple brain regions and networks were modulated by tDCS, including the frontoparietal network (FPN; DLPFC, and posterior parietal cortex), which is involved in the ﬂexible regulation of attention toward relevant and irrelevant environmental information and tuning behavior accordingly (Ptak, 2012; Zanto and Gazzaley, 2013), and the default mode network (DMN; mPFC, posterior cingulate cortex, may suggest that prefrontal tDCS decreased attentional bias toward potential threatening stimuli. Frontiers in Human Neuroscience \| www.frontiersin.org Effects of tDCS on Mood Throughout the Experimental Procedure However, in order to clearly elucidate the neural circuitry involved in these tDCS eﬀects, the application of neuro-imaging methods such as functional magnetic resonance imaging or high-density electroencephalogram is warranted. on valence. Such reasoning would be consistent with research showing that neuro-stimulation of the DLPFC contributed to sustained cognitive resource allocation (Pulopulos et al., 2020), and the DLPFC and associated neural networks being implicated in the implementation of proactive regulatory mechanisms (Vanderhasselt et al., 2009). The results of the current study could be clinically relevant as reactivity to (perceived) social evaluative threat plays a signiﬁcant role in mental health (Slavich et al., 2009, 2010). Furthermore, impairments in attentional processes have been identiﬁed as mechanisms underlying perpetual negative self- referential processing (i.e., rumination) and pose a vulnerability factor for aﬀective disorders (Koster et al., 2011; Sanchez- Lopez et al., 2019c; Yaroslavsky et al., 2019). Given the therapeutic value of prefrontal tDCS, and its eﬀects on attentional processes and emotional reactivity (De Raedt et al., 2015; Mondino et al., 2015; Smits et al., 2020), the current study suggest that attenuated attention toward self-referent information (e.g., a self-photograph) potentially reﬂects a mechanism of action underlying the adaptive eﬀects of prefrontal tDCS on SET reactivity. There is a growing body of research suggesting that training emotional attentional ﬂexibility can promote adaptive emotion regulatory processes (e.g., cognitive reappraisal; cognitive reframing of a stimuli or situation to change its meaning and emotional valence), while undermining maladaptive regulatory processes (e.g., rumination; Sanchez- Lopez et al., 2016, 2019a,b). Moreover, combining prefrontal tDCS with attention training shows the largest adaptive eﬀects on cognitive reappraisal and rumination, thereby highlighting the importance of multimodal interventions (Sanchez-Lopez et al., 2020). In future studies the eﬀects of prefrontal tDCS combined with attentional training (i.e., focusing less on yourself) on SET reactivity could be investigated. Taken together, in view of the clinical relevance of SET reactivity and the important role of ﬂexible cognition in mental health, the It should be noted that, under normal conditions, the valence (negative, positive) of social evaluations diﬀerentially aﬀects self- and other-attentional deployment patterns (Vanderhasselt et al., 2015b, 2018). However, in the current study, tDCS applied to the prefrontal cortex did not reveal a valence speciﬁc eﬀect. Effects of tDCS on Mood Throughout the Experimental Procedure This would be in line with past research (a) implicating the DLPFC in the regulation of attention toward threatful stimuli (Öhman, 2005; Sagliano et al., 2016), and (b) showing prefrontal tDCS eﬀects in attenuating vigilance toward threatening stimuli (Heeren et al., 2015; Ironside et al., 2016, 2017). Transcranial direct current stimulation is known to modulate large-scale brain networks rather than brain regions in isolation (Peña-Gómez et al., 2012; Stagg et al., 2013). Furthermore, the electric ﬁeld distribution is inﬂuenced by the placement August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 10 Allaert et al. tDCS Effects on Attentional Deployment FIGURE 6 \| tDCS effect on the relationship between time to ﬁrst self-ﬁxation and emotional reactivity. Within the group receiving active tDCS, slower times to ﬁxate on the self-photograph were associated with smaller SCR amplitudes, whereas this was not the case among the group receiving sham tDCS. FIGURE 6 \| tDCS effect on the relationship between time to ﬁrst self-ﬁxation and emotional reactivity. Within the group receiving active tDCS, slower times to ﬁxate on the self-photograph were associated with smaller SCR amplitudes, whereas this was not the case among the group receiving sham tDCS. precuneus, and inferior parietal cortex), which is involved in self-referential processing (Raichle et al., 2001; Davey et al., 2016). This would be in line with past research showing that prefrontal tDCS increases connectivity within the FPN while reducing connectivity within the DMN (Keeser et al., 2011; Peña-Gómez et al., 2012). Interestingly, depression is characterized by excessive self-referential processing associated with hyperconnectivity of the DMN, and impaired cognitive ﬂexibility associated with attenuated FPN connectivity (Qin et al., 2014; Li et al., 2018; Schultz et al., 2018). In addition, in view of the observed tDCS- associated attenuation of SCRs linked to attentional processes, prefrontal tDCS could have modulated (a) neural activity of the mPFC, since mPFC activity has been shown to be associated with SCRs (Critchley et al., 2000; Nagai et al., 2004; Zhang et al., 2012), and (b) fronto-limbic networks, as attentional deployment involves the recruitment of the FPN and an attenuation of amygdala activity (Ferri et al., 2016), which is also associated with SCRs (Wood et al., 2014; Christopoulos et al., 2019). Frontiers in Human Neuroscience \| www.frontiersin.org Effects of tDCS on Mood Throughout the Experimental Procedure Past research has shown mixed results regarding the role of valence on tDCS eﬀects, with some studies showing valence- dependent tDCS eﬀects (Mondino et al., 2015; Nejati et al., 2021), and others showing valence-independent tDCS eﬀects (Sanchez-Lopez et al., 2018). Possibly, in the current study, tDCS could have promoted the employment of a general and sustained attentional strategy to a broader context of social evaluative threat, instead of trial-by-trial adjustments based August 2021 \| Volume 15 \| Article 700557 11 Allaert et al. tDCS Effects on Attentional Deployment FUNDING This research was supported by the Research Foundation Flanders (G044019N and G044016N awarded to M-AV and RD), the Special Research Fund Ghent University (BOFSTA2017002501 awarded to M-AV), and the Ghent University Multidisciplinary Research Partnership “the integrative neuroscience of behavioral control.” This research was supported by the Research Foundation Flanders (G044019N and G044016N awarded to M-AV and RD), the Special Research Fund Ghent University (BOFSTA2017002501 awarded to M-AV), and the Ghent University Multidisciplinary Research Partnership “the integrative neuroscience of behavioral control.” In conclusion, this is the ﬁrst study to investigate the eﬀects of left prefrontal tDCS on self- and other-attentional deployment (via eye-tracking) in a context featuring SET, and how these are linked to autonomic emotional reactivity. The results suggest that prefrontal tDCS promotes adaptive regulatory attentional processes (reduced self-attention and increased other- attention), and that delayed self-attention may be a speciﬁc mechanism of action via which tDCS attenuates emotional reactivity to SET. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnhum. 2021.700557/full#supplementary-material ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ghent University Medical Ethical Committee. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identiﬁable images or data included in this article. DATA AVAILABILITY STATEMENT current results suggest that prefrontal tDCS may promote ﬂexible regulatory processes in response to situations featuring SET. The data and analysis script are available online (https://osf.io/ 3xjcw/). Besides that this study has several strengths, such as the direct assessment of attentional processes through eye-tracking in an ecologically valid paradigm of social evaluation, some limitations must be acknowledged. Past research has shown that (a) the phase of the menstrual cycle (Horvath et al., 2014; Rudroﬀet al., 2020), and (b) habitual nicotine use (Mclaren et al., 2018), can inﬂuence tDCS eﬀects, and this was not explicitly controlled for in the experimental protocol. Given the employed relatively large sample size, it could be argued that these potential eﬀects were balanced out between the two tDCS groups. Furthermore, although participants were instructed to look at the center ﬁxation cross of the screen prior to the presentation of an evaluation, the paradigm was not explicitly programmed to wait for a ﬁxation on this cross before presenting the evaluation. Consequentially, participants could already be looking at the location of any given AOI prior to its presentation, thereby introducing the possibility of confounding anticipatory gaze behavior. To solve this issue, data points indicative of these anticipatory responses were discarded from subsequent analyses, thereby removing a considerable portion of the observed data. Another limitation is that a between- subject design was employed, with no baseline assessment of attentional deployment prior to either active or sham tDCS. However, this design was chosen to prevent habituation and desensitization to the paradigm, which would be present with a pre-post design. To have a comparable participant pool in the active and sham tDCS group, groups were matched on a series of potential confounders. Furthermore, the usage of GLMMs allowed modeling the eﬀects of inter-individual diﬀerences via the inclusion of random eﬀects. Finally, the sample only consisted of females, limiting the generalizability of the ﬁndings. AUTHOR CONTRIBUTIONS JA designed the experiment with guidance from M-AV and RD, collected the data, performed statistical analyses with support from ME, and wrote the original manuscript with feedback from M-AV, RD, CB, AS-L, and ME. JA and M-AV revised the manuscript with feedback from RD, CB, AS-L, and ME. All authors contributed to the article and approved the submitted version. REFERENCES Allaert, J., Sanchez-Lopez, A., De Raedt, R., Baeken, C., and Vanderhasselt, M.-A. (2019). Inverse eﬀects of tDCS over the left versus right DLPC on emotional processing: a pupillometry study. PLoS One 14:e0218327. doi: 10.1371/journal. pone.0218327 Aldao, A., Sheppes, G., and Gross, J. J. (2015). Emotion regulation ﬂexibility. Cogn. Ther. Res. 39, 263–278. doi: 10.1007/s10608-014- 9662-4 Antal, A., Fischer, T., Saiote, C., Miller, R., Chaieb, L., Wang, D. J. J., et al. (2014). Transcranial electrical stimulation modiﬁes the neuronal response to psychosocial stress exposure. Hum. Brain Mapp. 35, 3750–3759. doi: 10.1002/ hbm.22434 Allaert, J., De Raedt, R., Sanchez, A., Baeken, C., and Vanderhasselt, M. A. (2020). Mind the social feedback: eﬀects of tDCS applied to the left DLPFC on psychophysiological responses during the anticipation and reception of social evaluations. Soc. Cogn. Aﬀect. Neurosci. doi: 10.1093/scan/nsaa066 [Epub ahead of print]. Bagiella, E., Sloan, R. P., and Heitjan, D. F. (2000). Mixed-eﬀects models in psychophysiology. Psychophysiology 37, 13–20. doi: 10.1111/1469-8986. 3710013 Allaert, J., De Raedt, R., Van Der Veen, F. M., Baeken, C., and Vanderhasselt, M.-A. (2021). Prefrontal tDCS attenuates counterfactual thinking in female individuals prone to self-critical rumination. Sci. Rep. 11:11601. doi: 10.1038/ s41598-021-90677-7 Bates, D., Mächler, M., Bolker, B., and Walker, S. (2014). Fitting linear mixed-eﬀects models using lme4. arXiv [Preprint]. Available online at: https://www.jstatsoft. org/article/view/v067i01 (accessed February 28, 2020). August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org Frontiers in Human Neuroscience \| www.frontiersin.org 12 Allaert et al. tDCS Effects on Attentional Deployment Baumeister, R. F., and Leary, M. R. (1995). The need to belong: desire for interpersonal attachments as a fundamental human motivation. Psychol. Bull. 117, 497–529. doi: 10.1037/0033-2909.117.3.497 Durlik, C., and Tsakiris, M. (2015). Decreased interoceptive accuracy following social exclusion. Int. J. Psychophysiol. 96, 57–63. doi: 10.1016/j.ijpsycho.2015. 02.020 Faul, F., Erdfelder, E., Lang, A.-G., and Buchner, A. (2007). G∗Power 3: a ﬂexible statistical power analysis program for the social, behavioral, and biomedical Benjamini, Y. (2010). Discovering the false discovery rate. J. R. Stat. Soc. Ser. B 72, 405–416. doi: 10.1111/j.1467-9868.2010.00746.x Faul, F., Erdfelder, E., Lang, A.-G., and Buchner, A. (2007). G∗Power 3: a ﬂexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39, 175–191. doi: 10.3758/BF03193146 Bikson, M., Datta, A., Rahman, A., and Scaturro, J. (2010). Electrode montages for tDCS and weak transcranial electrical stimulation: role of “return” electrode’s position and size. Clin. Neurophysiol. 121, 1976–1978. REFERENCES doi: 10.1177/109442811668 1073 Hardin, J. W., and Hilbe, J. M. (2007). Generalized Linear Models and Extensions. College Station, TX: Stata Press. Chun, M. M., Golomb, J. D., and Turk-Browne, N. B. (2011). A taxonomy of external and internal attention. Annu. Rev. Psychol. 62, 73–101. doi: 10.1146/ annurev.psych.093008.100427 Heeren, A., Baeken, C., Vanderhasselt, M.-A., Philippot, P., and De Raedt, R. (2015). Impact of anodal and cathodal transcranial direct current stimulation over the left dorsolateral prefrontal cortex during attention bias modiﬁcation: an eye-tracking study. PLoS One 10:e0124182. doi: 10.1371/journal.pone. 0124182 Cnaan, A., Laird, N. M., and Slasor, P. (1997). Using the general linear mixed model to analyse unbalanced repeated measures and longitudinal data. Stat. Med. 16, 2349–2380. doi: 10.1002/(sici)1097-0258(19971030)16:20<2349::aid-sim667> 3.0.co;2-e Hess, Y. D., and Pickett, C. L. (2010). Social rejection and self- versus other- awareness. J. Exp. Soc. Psychol. 46, 453–456. doi: 10.1016/j.jesp.2009.12.004 Connor, K. M., and Davidson, J. R. (2003). Development of a new resilience scale: the Connor-Davidson Resilience Scale (CD-RISC). Depress. Anxiety 18, 76–82. doi: 10.1002/da.10113 Hoebeke, Y., Desmedt, O., Özçimen, B., and Heeren, A. (2021). The impact of transcranial Direct Current stimulation on rumination: a systematic review of the sham-controlled studies in healthy and clinical samples. Compr. Psychiatry 106:152226. doi: 10.1016/j.comppsych.2021.152226 Critchley, H. D., Elliott, R., Mathias, C. J., and Dolan, R. J. (2000). Neural activity relating to generation and representation of galvanic skin conductance responses: a functional magnetic resonance imaging study. J. Neurosci. 20, 3033–3040. doi: 10.1523/jneurosci.20-08-03033.2000 Horvath, J. C., Carter, O., and Forte, J. D. (2014). Transcranial direct current stimulation: ﬁve important issues we aren’t discussing (but probably should be). Front. Syst. Neurosci. 8:2. doi: 10.3389/fnsys.2014.00002 Davey, C. G., Pujol, J., and Harrison, B. J. (2016). Mapping the self in the brain’s default mode network. Neuroimage 132, 390–397. doi: 10.1016/j.neuroimage. 2016.02.022 Ironside, M., Browning, M., Ansari, T. L., Harvey, C. J., Sekyi-Djan, M. N., Bishop, S. J., et al. (2017). Prefrontal cortex regulates amygdala response to threat in trait anxiety. bioRxiv [Preprint]. doi: 10.1101/215699 De Raedt, R., and Hooley, J. M. (2016). The role of expectancy and proactive control in stress regulation: a neurocognitive framework for regulation expectation. Clin. Psychol. Rev. 45, 45–55. doi: 10.1016/j.cpr.2016. 03.005 Ironside, M., O’Shea, J., Cowen, P. J., and Harmer, C. J. (2016). Frontal cortex stimulation reduces vigilance to threat: implications for the treatment of depression and anxiety. Biol. Psychiatry 79, 823–830. doi: 10.1016/j.biopsych. REFERENCES doi: 10.1016/j.clinph. 2010.05.020 Ferri, J., Schmidt, J., Hajcak, G., and Canli, T. (2013). Neural correlates of attentional deployment within unpleasant pictures. Neuroimage 70, 268–277. doi: 10.1016/j.neuroimage.2012.12.030 Boisgontier, M. P., and Cheval, B. (2016). The anova to mixed model transition. Neurosci. Biobehav. Rev. 68, 1004–1005. doi: 10.1016/j.neubiorev.2016.05.034 Ferri, J., Schmidt, J., Hajcak, G., and Canli, T. (2016). Emotion regulation and amygdala-precuneus connectivity: focusing on attentional deployment. Cogn. Aﬀect. Behav. Neurosci. 16, 991–1002. doi: 10.3758/s13415-016-0447-y j g amygdala-precuneus connectivity: focusing on attentional deployment. Cogn. Aﬀect. Behav. Neurosci. 16, 991–1002. doi: 10.3758/s13415-016-0447-y Boucsein, W., Fowles, D. C., Grimnes, S., Ben-Shakhar, G., Roth, W. T., Dawson, M. E., et al. (2012). Publication recommendations for electrodermal measurements. Psychophysiology 49, 1017–1034. doi: 10.1111/j.1469-8986.2012. 01384.x Fox, J., Weisberg, S., Adler, D., Bates, D., Baud-Bovy, G., Ellison, S., et al. (2012). Package ‘car’. Vienna: R Foundation for Statistical Computing. Franck, E., De Raedt, R., Barbez, C., and Rosseel, Y. (2008). Psychometric properties of the dutch rosenberg self-esteem scale. Psychol. Belg. 48, 25–34. doi: 10.5334/pb-48-1-25 Braithwaite, J. J., Watson, D. G., Jones, R., and Rowe, M. (2013). A guide for analysing electrodermal activity (EDA) & skin conductance responses (SCRs) for psychological experiments. Psychophysiology 49, 1017–1034. Garnefski, N., and Kraaij, V. (2007). The cognitive emotion regulation questionnaire. Eur. J. Psychol. Assess. 23, 141–149. Carnevali, L., Pattini, E., Sgoifo, A., and Ottaviani, C. (2020). Eﬀects of prefrontal transcranial direct current stimulation on autonomic and neuroendocrine responses to psychosocial stress in healthy humans. Stress 23, 26–36. doi: 10. 1080/10253890.2019.1625884 Gross, J. J., and John, O. P. (2003). Individual diﬀerences in two emotion regulation processes: implications for aﬀect, relationships, and well-being. J. Pers. Soc. Psychol. 85, 348. doi: 10.1037/0022-3514.85.2.348 Chen, N. T. M., Basanovic, J., Notebaert, L., MacLeod, C., and Clarke, P. J. F. (2017). Attentional bias mediates the eﬀect of neurostimulation on emotional vulnerability. J. Psychiatr. Res. 93, 12–19. doi: 10.1016/j.jpsychires.2017.05.008 Gross, J. J., and Thompson, R. A. (2007). “Emotion regulation: conceptual foundations,” in Handbook of Emotion Regulation, ed. J. J. Gross (New York, NY: Guilford Press), 3–24. Gunther Moor, B., Crone, E. A., and van der Molen, M. W. (2010). The heartbrake of social rejection:heart rate deceleration in response to unexpected peer rejection. Psychol. Sci. 21, 1326–1333. doi: 10.1177/0956797610379236 Christopoulos, G. I., Uy, M. A., and Yap, W. J. (2019). The body and the brain: measuring skin conductance responses to understand the emotional experience. Organ. Res. Methods 22, 394–420. REFERENCES Neuroscientist 18, 502–515. doi: 10.1177/1073858411409051 Pulopulos, M., Allaert, J., Vanderhasselt, M.-A., Sanchez-Lopez, A., De Witte, S., Baeken, C., et al. (2020). Eﬀects of HF-rTMS over the left and right DLPFC on proactive and reactive cognitive control. Soc. Cogn. Aﬀect. Neurosci. doi: 10.1093/scan/nsaa082 [Epub ahead of print]. Lo, S., and Andrews, S. (2015). To transform or not to transform: using generalized linear mixed models to analyse reaction time data. Front. Psychol. 6:1171. doi: 10.3389/fpsyg.2015.01171 Qin, J., Wei, M., Liu, H., Yan, R., Luo, G., Yao, Z., et al. (2014). Abnormal brain anatomical topological organization of the cognitive-emotional and the frontoparietal circuitry in major depressive disorder. Magn. Reson. Med. 72, 1397–1407. Mallinckrodt, C. H., Clark, W. S., and David, S. R. (2001). Type I error rates from mixed eﬀects model repeated measures versus ﬁxed eﬀects ANOVA with missing values imputed via last observation carried forward. Drug Information J. 35, 1215–1225. doi: 10.1177/009286150103500418 Masland, S. R., and Hooley, J. M. (2015). Perceived criticism: a research update for clinical practitioners. Clin. Psychol. 22, 211–222. doi: 10.1111/cpsp.12110 R Core Team (2013). R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing. Matsumoto, H., and Ugawa, Y. (2017). Adverse events of tDCS and tACS: a review. Clin. Neurophysiol. Pract. 2, 19–25. doi: 10.1016/j.cnp.2016.12.003 Raes, F., Schoofs, H., Hoes, D., Hermans, D., Van Den Eede, F., and Franck, E. (2009). ‘Reﬂection’ en ‘brooding’ als subtypes van rumineren: een herziening van de ruminative response scale. [Reﬂection and brooding as subcomponents of rumination: a revision of the ruminative response scale.]. Gedragstherapie 42, 205–214. McCormack, H. M., de, L., Horne, D. J., and Sheather, S. (1988). Clinical applications of visual analogue scales: a critical review. Psychol. Med. 18, 1007–1019. doi: 10.1017/S0033291700009934 Mclaren, M. E., Nissim, N. R., and Woods, A. J. (2018). The eﬀects of medication use in transcranial direct current stimulation: a brief review. Brain Stimul. 11, 52–58. doi: 10.1016/j.brs.2017.10.006 Raichle, M. E., Macleod, A. M., Snyder, A. Z., Powers, W. J., Gusnard, D. A., and Shulman, G. L. (2001). A default mode of brain function. Proc. Natl. Acad. Sci. U.S.A. 98, 676–682. doi: 10.1073/pnas.98.2.676 Rosenberg, M. (1965). Society and the Adolescent Self-Image. Princeton, NJ: Princeton University Press. McRae, K., Ochsner, K. N., Mauss, I. B., Gabrieli, J. J. D., and Gross, J. J. (2008). Gender diﬀerences in emotion regulation: an fMRI study of cognitive reappraisal. Group Process Intergroup Relat. 11, 143–162. REFERENCES G., Wassermann, E. M., Priori, A., Lang, N., Antal, A., et al. (2008). Transcranial direct current stimulation: state of the art 2008. Brain Stimul. 1, 206–223. doi: 10.1016/j.brs.2008.06.004 Kolb, B., and Whishaw, I. Q. (2009). Fundamentals of Human Neuropsychology. Basingstoke: Macmillan. Koster, E. H. W., De Lissnyder, E., Derakshan, N., and De Raedt, R. (2011). Understanding depressive rumination from a cognitive science perspective: the impaired disengagement hypothesis. Clin. Psychol. Rev. 31, 138–145. doi: 10.1016/j.cpr.2010.08.005 Nolen-Hoeksema, S., and Morrow, J. (1991). A prospective study of depression and posttraumatic stress symptoms after a natural disaster: the 1989 Loma Prieta earthquake. J. Pers. Soc. Psychol. 61, 115–121. doi: 10.1037/0022-3514.61.1.115 Northoﬀ, G., Heinzel, A., de Greck, M., Bermpohl, F., Dobrowolny, H., and Panksepp, J. (2006). Self-referential processing in our brain—A meta-analysis of imaging studies on the self. Neuroimage 31, 440–457. doi: 10.1016/j. neuroimage.2005.12.002 Kuznetsova, A., Brockhoﬀ, P. B., and Christensen, R. H. B. (2017). lmerTest package: tests in linear mixed eﬀects models. J. Stat. Softw. 82, 1–26. Lefaucheur, J. P., Antal, A., Ayache, S. S., Benninger, D. H., Brunelin, J., Cogiamanian, F., et al. (2017). Evidence-based guidelines on the therapeutic use of transcranial direct current stimulation (tDCS). Clin. Neurophysiol. 128, 56–92. doi: 10.1016/j.clinph.2016.10.087 Ochsner, K. N., and Gross, J. J. (2005). The cognitive control of emotion. Trends Cogn. Sci. 9, 242–249. doi: 10.1016/j.tics.2005.03.010 Öhman, A. (2005). The role of the amygdala in human fear: automatic detection of threat. Psychoneuroendocrinology 30, 953–958. doi: 10.1016/j.psyneuen.2005. 03.019 Lenth, R. (2018). Emmeans: Estimated Marginal Means, Aka Least-Squares Means. R package version, 1.6.2-1. Available online at: https://cran.r-project.org/web/ packages/emmeans/index.html (accessed March 5, 2020). Peña-Gómez, C., Sala-Lonch, R., Junqué, C., Clemente, I. C., Vidal, D., Bargalló, N., et al. (2012). Modulation of large-scale brain networks by transcranial direct current stimulation evidenced by resting-state functional MRI. Brain Stimul. 5, 252–263. doi: 10.1016/j.brs.2011.08.006 Li, B. J., Friston, K., Mody, M., Wang, H. N., Lu, H. B., and Hu, D. W. (2018). A brain network model for depression: from symptom understanding to disease intervention. CNS Neurosci. Ther. 24, 1004–1019. doi: 10.1111/cns.12998 252–263. doi: 10.1016/j.brs.2011.08.006 Lithari, C., Frantzidis, C. A., Papadelis, C., Vivas, A. B., Klados, M. A., Kourtidou- Papadeli, C., et al. (2010). Are females more responsive to emotional stimuli? A neurophysiological study across arousal and valence dimensions. Brain Topogr. 23, 27–40. doi: 10.1007/s10548-009-0130-5 Ptak, R. (2012). The frontoparietal attention network of the human brain:action, saliency, and a priority map of the environment. REFERENCES 2015.06.012 De Raedt, R., Remue, J., Loeys, T., Hooley, J. M., and Baeken, C. (2017). The eﬀect of transcranial direct current stimulation of the prefrontal cortex on implicit self-esteem is mediated by rumination after criticism. Behav. Res. Ther. 99, 138–146. doi: 10.1016/j.brat.2017.10.009 Jiang, J., and Nguyen, T. (2021). Linear and Generalized Linear Mixed Models and their Applications. New York, NY: Springer. Joormann, J., and Vanderlind, W. M. (2014). Emotion regulation in depression:the role of biased cognition and reduced cognitive control. Clin. Psychol. Sci. 2, 402–421. De Raedt, R., Vanderhasselt, M.-A., and Baeken, C. (2015). Neurostimulation as an intervention for treatment resistant depression: from research on mechanisms towards targeted neurocognitive strategies. Clin. Psychol. Rev. 41, 61–69. doi: 10.1016/j.cpr.2014.10.006 Karenbach, C. (2005). Ledalab – A Software Package for the Analysis of Phasic Electrodermal Activity. Internal Report, Allgemeine Psychologie, Institut für Psychologie, Institut ftir Psychologie, Technical Report. Available online at: http://www.ledalab.de/ (accessed June 22, 2019). De Witte, S., Klooster, D., Dedoncker, J., Duprat, R., Remue, J., and Baeken, C. (2018). Left prefrontal neuronavigated electrode localization in tDCS: 10–20 EEG system versus MRI-guided neuronavigation. Psychiatry Res. Neuroimaging 274, 1–6. doi: 10.1016/j.pscychresns.2018.02.001 Keeser, D., Meindl, T., Bor, J., Palm, U., Pogarell, O., Mulert, C., et al. (2011). Prefrontal transcranial direct current stimulation changes connectivity of resting-state networks during fMRI. J. Neurosci. 31, 15284–15293. doi: 10.1523/ jneurosci.0542-11.2011 Dedoncker, J., Brunoni, A. R., Baeken, C., and Vanderhasselt, M. A. (2016). A systematic review and meta-analysis of the eﬀects of transcranial direct current stimulation (tDCS) over the dorsolateral prefrontal cortex in healthy and neuropsychiatric samples: inﬂuence of stimulation parameters. Brain Stimul. 9, 501–517. doi: 10.1016/j.brs.2016.04.006 Kempe, R., Huang, Y., and Parra, L. C. (2014). Simulating pad-electrodes with high- deﬁnition arrays in transcranial electric stimulation. J. Neural Eng. 11:026003. doi: 10.1088/1741-2560/11/2/026003 August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 13 Allaert et al. tDCS Effects on Attentional Deployment Kim, C., Johnson, N. F., Cilles, S. E., and Gold, B. T. (2011). Common and distinct mechanisms of cognitive ﬂexibility in prefrontal cortex. J. Neurosci. 31, 4771–4779. doi: 10.1523/jneurosci.5923-10.2011 Nejati, V., Majdi, R., Salehinejad, M. A., and Nitsche, M. A. (2021). The role of dorsolateral and ventromedial prefrontal cortex in the processing of emotional dimensions. Sci. Rep. 11:1971. doi: 10.1038/s41598-021- 81454-7 Knowles, M. L. (2014). Social rejection increases perspective taking. J. Exp. Soc. Psychol. 55, 126–132. doi: 10.1016/j.jesp.2014.06.008 Nitsche, M. A., Cohen, L. REFERENCES doi: 10.1177/ 1368430207088035 Rudroﬀ, T., Workman, C. D., Fietsam, A. C., and Kamholz, J. (2020). Response variability in transcranial direct current stimulation: Why sex matters. Front. Psychiatry 11:585. doi: 10.3389/fpsyt.2020.00585 Merz, C. N. B., Dwyer, J., Nordstrom, C. K., Walton, K. G., Salerno, J. W., and Schneider, R. H. (2002). Psychosocial stress and cardiovascular disease: pathophysiological links. Behav. Med. 27, 141–147. doi: 10.1080/ 08964280209596039 Sagliano, L., D’Olimpio, F., Panico, F., Gagliardi, S., and Trojano, L. (2016). The role of the dorsolateral prefrontal cortex in early threat processing: a TMS study. Soc. Cogn. Aﬀect. Neurosci. 11, 1992–1998. doi: 10.1093/scan/nsw105 Mondino, M., Thiﬀault, F., and Fecteau, S. (2015). Does non-invasive brain stimulation applied over the dorsolateral prefrontal cortex non-speciﬁcally inﬂuence mood and emotional processing in healthy individuals? Front. Cell. Neurosci. 9:399. doi: 10.3389/fncel.2015.00399 Sanchez-Lopez, A., De Raedt, R., Puttevils, L., Koster, E. H. W., Baeken, C., and Vanderhasselt, M.-A. (2020). Combined eﬀects of tDCS over the left DLPFC and gaze-contingent training on attention mechanisms of emotion regulation in low-resilient individuals. Prog. Neuropsychopharmacol. Biol. Psychiatry 108:110177. doi: 10.1016/j.pnpbp.2020.110177 Moors, A., De Houwer, J., Hermans, D., Wanmaker, S., van Schie, K., Van Harmelen, A. L., et al. (2013). Norms of valence, arousal, dominance, and age of acquisition for 4,300 Dutch words. Behav. Res. Methods 45, 169–177. doi: 10.3758/s13428-012-0243-8 Sanchez-Lopez, A., De Raedt, R., van Put, J., and Koster, E. H. W. (2019a). A novel process-based approach to improve resilience: eﬀects of computerized mouse-based (gaze)contingent attention training (MCAT) on reappraisal and rumination. Behav. Res. Ther. 118, 110–120. doi: 10.1016/j.brat.2019.04.005 Muscatell, K. A., Dedovic, K., Slavich, G. M., Jarcho, M. R., Breen, E. C., Bower, J. E., et al. (2015). Greater amygdala activity and dorsomedial prefrontal–amygdala coupling are associated with enhanced inﬂammatory responses to stress. Brain Behav. Immun. 43, 46–53. doi: 10.1016/j.bbi.2014.06.201 Sanchez-Lopez, A., Everaert, J., and Koster, E. H. (2016). Attention training through gaze-contingent feedback: eﬀects on reappraisal and negative emotions. Emotion 16, 1074–1085. doi: 10.1037/emo0000198 Nagai, Y., Critchley, H. D., Featherstone, E., Trimble, M. R., and Dolan, R. J. (2004). Activity in ventromedial prefrontal cortex covaries with sympathetic skin conductance level: a physiological account of a “default mode” of brain function. Neuroimage 22, 243–251. doi: 10.1016/j.neuroimage.2004.01.019 Sanchez-Lopez, A., Everaert, J., Van Put, J., De Raedt, R., and Koster, E. H. W. (2019b). Eye-gaze contingent attention training (ECAT): examining the causal role of attention regulation in reappraisal and rumination. Biol. Psychol. 142, 116–125. REFERENCES doi: 10.1016/j.biopsycho.2019.01.017 August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org Frontiers in Human Neuroscience \| www.frontiersin.org 14 Allaert et al. tDCS Effects on Attentional Deployment self-referent thoughts by neuromodulation of emotional working memory. Neuropsychologia 51, 2581–2589. doi: 10.1016/j.neuropsychologia.2013. 08.011 self-referent thoughts by neuromodulation of emotional working memory. Neuropsychologia 51, 2581–2589. doi: 10.1016/j.neuropsychologia.2013. 08.011 Sanchez-Lopez, A., Koster, E. H. W., Van Put, J., and De Raedt, R. (2019c). Attentional disengagement from emotional information predicts future depression via changes in ruminative brooding: a ﬁve-month longitudinal eye-tracking study. Behav. Res. Ther. 118, 30–42. doi: 10.1016/j.brat.2019.03. 013 Vanderhasselt, M. A., De Raedt, R., and Baeken, C. (2009). Dorsolateral prefrontal cortex and Stroop performance: tackling the lateralization. Psychon. Bull. Rev. 16, 609–612. doi: 10.3758/PBR.16.3.609 Sanchez-Lopez, A., Vanderhasselt, M.-A., Allaert, J., Baeken, C., and De Raedt, R. (2018). Neurocognitive mechanisms behind emotional attention: inverse eﬀects of anodal tDCS over the left and right DLPFC on gaze disengagement from emotional faces. Cogn. Aﬀect. Behav. Neurosci. 18, 485–494. doi: 10.3758/ s13415-018-0582-8 Vanderhasselt, M. A., De Raedt, R., Namur, V., Lotufo, P. A., Bensenor, I. M., Boggio, P. S., et al. (2015a). Transcranial electric stimulation and neurocognitive training in clinically depressed patients: a pilot study of the eﬀects on rumination. Prog. Neuropsychopharmacol. Biol. Psychiatry 57, 93–99. doi: 10. 1016/j.pnpbp.2014.09.015 Santhanagopalan, M., Chetty, M., Foale, C., Aryal, S., and Klein, B. (2018). Modeling neurocognitive reaction time with gamma distribution. Paper Presented at the Australasian Computer Science Week Multiconference, Brisband. doi: 10.1145/3167918.3167941 Vanderhasselt, M. A., De Raedt, R., Nasso, S., Puttevils, L., and Mueller, S. C. (2018). Don’t judge me: psychophysiological evidence of gender diﬀerences to social evaluative feedback. Biol. Psychol. 135, 29–35. doi: 10.1016/j.biopsycho.2018.02. 017 Schultz, D. H., Ito, T., Solomyak, L. I., Chen, R. H., Mill, R. D., Anticevic, A., et al. (2018). Global connectivity of the fronto-parietal cognitive control network is related to depression symptoms in the general population. Netw. Neurosci. 3, 107–123. doi: 10.1162/netn_a_00056 Vanderhasselt, M. A., Remue, J., Ng, K. K., Mueller, S. C., and De Raedt, R. (2015b). The regulation of positive and negative social feedback: a psychophysiological study. Cogn. Aﬀect. Behav. Neurosci. 15, 553–563. doi: 10.3758/s13415-015- 0345-8 Seo, D., Olman, C. A., Haut, K. M., Sinha, R., MacDonald, A. W., and Patrick, C. J. (2014). Neural correlates of preparatory and regulatory control over positive and negative emotion. Soc. Cogn. Aﬀect. Neurosci. 9, 494–504. doi: 10.1093/ scan/nst115 Wardenaar, K. REFERENCES J., van Veen, T., Giltay, E. J., de Beurs, E., Penninx, B. W., and Zitman, F. G. (2010). Development and validation of a 30-item short adaptation of the Mood and Anxiety Symptoms Questionnaire (MASQ). Psychiatry Res. 179, 101–106. doi: 10.1016/j.psychres.2009.03.005 Sherman, D. K., and Cohen, G. L. (2006). “The psychology of self-defense: self- aﬃrmation theory,” in Advances in Experimental Social Psychology, Vol. 38, ed. M. P. Zanna (San Diego, CA: Academic Press), 183–242. doi: 10.1016/s0065- 2601(06)38004-5 Willenbockel, V., Sadr, J., Fiset, D., Horne, G. O., Gosselin, F., and Tanaka, J. W. (2010). Controlling low-level image properties: the SHINE toolbox. Behav. Res. Methods 42, 671–684. doi: 10.3758/brm.42.3.671 Sjak-Shie, E. E. (2018). PhysioData Toolbox (Version 0.4). Available online at: https://PhysioDataToolbox.leidenuniv.nl (accessed January 12, 2020) Wood, K. H., Ver Hoef, L. W., and Knight, D. C. (2014). The amygdala mediates the emotional modulation of threat-elicited skin conductance response. Emotion 14, 693–700. doi: 10.1037/a0036636 Slavich, G. M., O’Donovan, A., Epel, E. S., and Kemeny, M. E. (2010). Black sheep get the blues: a psychobiological model of social rejection and depression. Neurosci. Biobehav. Rev. 35, 39–45. doi: 10.1016/j.neubiorev.2010.01.003 Yaroslavsky, I., Allard, E. S., and Sanchez-Lopez, A. (2019). Can’t look Away: Attention control deﬁcits predict Rumination, depression symptoms and depressive aﬀect in daily Life. J. Aﬀect. Disord. 245, 1061–1069. doi: 10.1016/ j.jad.2018.11.036 Slavich, G. M., Thornton, T., Torres, L. D., Monroe, S. M., and Gotlib, I. H. (2009). Targeted rejection predicts hastened onset of major depression. J. Soc. Clin. Psychol. 28, 223–243. doi: 10.1521/jscp.2009.28.2.223 j j Zanto, T. P., and Gazzaley, A. (2013). Fronto-parietal network: ﬂexible hub of cognitive control. Trends Cogn. Sci. 17, 602–603. doi: 10.1016/j.tics.2013. 10.001 Smits, F. M., Schutter, D. J., van Honk, J., and Geuze, E. (2020). Does non- invasive brain stimulation modulate emotional stress reactivity? Soc. Cogn. Aﬀect. Neurosci. 15, 23–51. doi: 10.1093/scan/nsaa011 Zhang, S., Hu, S., Chao, H. H., Luo, X., Farr, O. M., and Li, C.- S. R. (2012). Cerebral correlates of skin conductance responses in a cognitive task. Neuroimage 62, 1489–1498. doi: 10.1016/j.neuroimage.2012. 05.036 Stagg, C. J., Lin, R. L., Mezue, M., Segerdahl, A., Kong, Y., Xie, J., et al. (2013). Widespread modulation of cerebral perfusion induced during and after transcranial direct current stimulation applied to the left dorsolateral prefrontal cortex. J. Neurosci. 33, 11425–11431. REFERENCES doi: 10.1523/jneurosci.3887-12 .2013 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Stagg, C. J., and Nitsche, M. A. (2011). Physiological basis of transcranial direct current stimulation. Neuroscientist 17, 37–53. doi: 10.1177/1073858410386614 Stuss, D. T. (2011). Functions of the frontal lobes: relation to executive functions. J. Int. Neuropsychol. Soc. 17, 759–765. doi: 10.1017/s1355617711000695 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Teeuw, B., Schwarzer, R., and Jerusalem, M. (1994). Dutch General Self-Eﬃcacy Scale. Available online at: http://userpage.fu-berlin.de/~gesund/publicat/ehps_ cd/health/dutch.htm (accessed May 7, 2021). Twenge, J. M., Catanese, K. R., and Baumeister, R. F. (2003). Social exclusion and the deconstructed state: time perception, meaninglessness, lethargy, lack of emotion, and self-awareness. J. Pers. Soc. Psychol. 85, 409–423. doi: 10.1037/ 0022-3514.85.3.409 Copyright © 2021 Allaert, Erdogan, Sanchez-Lopez, Baeken, De Raedt and Vanderhasselt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. van der Veen, F. M., Burdzina, A., and Langeslag, S. J. E. (2019). Don’t you want me, baby? Cardiac and electrocortical concomitants of romantic interest and rejection. Biol. Psychol. 146:107707. doi: 10.1016/j.biopsycho.2019.05.007 Vanderhasselt, M.-A., Brunoni, A. R., Loeys, T., Boggio, P. S., and De Raedt, R. (2013). Nosce te ipsum–Socrates revisited? Controlling momentary ruminative August 2021 \| Volume 15 \| Article 700557 Frontiers in Human Neuroscience \| www.frontiersin.org 15
https://openalex.org/W2032392236	https://bmcgenomics.biomedcentral.com/counter/pdf/10.1186/1471-2164-10-646	English	null	Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing	BMC genomics	2,009	cc-by	10,969	BioMed Central BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-2164/10/646 © 2009 Lee et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Genomics Open Access Published: 31 December 2009 Published: 31 December 2009 BMC Genomics 2009, 10:646 doi:10.1186/1471-2164-10-646 BMC Genomics 2009, 10:646 doi:10.1186/1471-2164-10-646 BMC Genomics 2009, 10:646 doi:10.1186/1471-2164-10-646 This article is available from: http://www.biomedcentral.com/1471-2164/10/646 Methodology article Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing Hane Lee1, Brian D O'Connor1, Barry Merriman1, Vincent A Funari2, Nils Homer1, Zugen Chen1, Daniel H Cohn2 and Stanley F Nelson1 Address: 1Department of Human Genetics, University of California, Los Angeles, California, USA and 2Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA Email: Hane Lee - hanelee@ucla.edu; Brian D O'Connor - boconnor@ucla.edu; Barry Merriman - barrym@ucla.edu; Vincent A Funari - FunariV@cshs.org; Nils Homer - nilshomer@ucla.edu; Zugen Chen - zugenchen@mednet.ucla.edu; Daniel H Cohn - dan.cohn@cshs.org; Stanley F Nelson - snelson@ucla.edu * Corresponding author Received: 18 February 2009 Accepted: 31 December 2009 Received: 18 February 2009 Accepted: 31 December 2009 Published: 31 December 2009 Background specificity of the capture was very good, only ~11,000 exons were captured. specificity of the capture was very good, only ~11,000 exons were captured. g Sequencing capacity has greatly advanced over the years and took a major leap with the commercialization of two new platforms for next-generation sequencers. Currently, three major platforms are actively being used including Applied Biosystems (ABI) SOLiD (Sequencing by Oligo Ligation and Detection), Illumina Genome Analyzer (GA) and Roche 454 Sequencing System [1-3]. Further, proofs of principle experiments with the Helicos system and Pacific Biosciences system have been published [4-6]. These sequencer technologies differ in their sequencing methods and hence they vary in number of reads sequenced, read length, error characteristics. However, all rely on the generation of shotgun libraries for sequencing. With these technologies, a single machine can generate in the range of 0.5-2 gigabases (Gb) of sequence reads per day. While advancement in these technologies is certain, the use of these technologies for sequencing targeted regions of the genome has been limited based on the effi- ciency of methods to enrich regions of the genome for analysis which are matched to the capacity. The rapid advancement in genotyping technology made possible by the advent of DNA microarrays has resulted in a flood of linkage and whole genome association studies for various disorders, and now the community is overwhelmed with genomic regions of interest for which additional targeted sequence analysis is key bottleneck. Most recently, several studies on exonic capture for broad based sequencing of the amino acid coding portion of the genome have shown successful identification of rare mutations/alleles involved in rare genetic disorders and yielded insights in applying the technique to searching for common variants as well as de novo cancer mutations [7-10]. At the same time when Porreca et al. published, Albert et al. and Okou et al. described methods for using DNA arrays to capture large genomic loci directly [15-17]. In the following month, Hodges et al. demonstrated success at capturing 'all' exons using the same methods with better balance of coverage (uniformity) and specificity relative to any other previous capture assays, and it was the least labor intensive and the most cost-effective method [18]. However, the performance of these assays varied across the sample types and array types used. Albert et al. targeted a total ~5 Mb of sequences for 660 genes dispersed across the genome [16]. Background They reported the specificity varied with the same array design from 38% to 76% depending on the samples captured. For their tiling arrays encompassing from 200 Kb to 5 Mb around the BRCA1 gene, the fraction of the reads mapped to the intended targets varied from 14% to 64%. The Nimblegen 385 K custom array was used for all capture protocols while the 454 GLX sequencer was used for sequencing. Since the 454 sequencers produce longer individual reads than those from ABI or Illumina sequencers, their sequences are easier to map to the correct genomic loca- tion and this should be factored in when comparing cap- ture technologies. Hodges et al. targeted 'all' human exons using seven Nim- blegen arrays and sequenced the captured DNA with the Illumina sequencer [18]. They first hybridized 500 bp genomic fragments to all seven arrays, and the fraction of the reads mapped to the targeted regions varied from 36% to 55%. When they extended the definition of the targeted region to include 300 bp upstream and downstream of each exon, the targeting efficiency was increased to 55- 85%. Next, they used 100-200 bp fragments to hybridize to one of the arrays in an attempt to tighten the sequenced region around the targets. However, the specificity of the intended targets was reduced three-fold with the exon cov- erage rate up to 99%. For both studies, no detailed inter- pretation was described for the specificity variations across different array designs and sample types. Several groups have attempted to capture regions of inter- est by multiplex amplification [11-14]. For the target regions, primer pairs are systematically designed and, as the target regions are amplified by PCR, only the frag- ments with the right primer pairs are enriched. Reports have demonstrated successful amplification of hundreds of ~200 bp sized fragments but with substantial bias in the amplification between the different fragments. This method may work well for targeting tens of genes but beyond that scale, it requires more effort to design unique primers and optimize the PCR amplification process to ensure uniformity across all fragments. Also, the high cost of primer design and amplification will not compare favo- rably with that of sequencing as sequencing costs have been reduced significantly. To overcome the cost and effort of a primer design process, Porreca et al. Abstract Background: The emergence of next-generation sequencing technology presents tremendous opportunities to accelerate the discovery of rare variants or mutations that underlie human genetic disorders. Although the complete sequencing of the affected individuals' genomes would be the most powerful approach to finding such variants, the cost of such efforts make it impractical for routine use in disease gene research. In cases where candidate genes or loci can be defined by linkage, association, or phenotypic studies, the practical sequencing target can be made much smaller than the whole genome, and it becomes critical to have capture methods that can be used to purify the desired portion of the genome for shotgun short-read sequencing without biasing allelic representation or coverage. One major approach is array-based capture which relies on the ability to create a custom in-situ synthesized oligonucleotide microarray for use as a collection of hybridization capture probes. This approach is being used by our group and others routinely and we are continuing to improve its performance. Results: Here, we provide a complete protocol optimized for large aggregate sequence intervals and demonstrate its utility with the capture of all predicted amino acid coding sequence from 3,038 human genes using 241,700 60-mer oligonucleotides. Further, we demonstrate two techniques by which the efficiency of the capture can be increased: by introducing a step to block cross hybridization mediated by common adapter sequences used in sequencing library construction, and by repeating the hybridization capture step. These improvements can boost the targeting efficiency to the point where over 85% of the mapped sequence reads fall within 100 bases of the targeted regions. Conclusions: The complete protocol introduced in this paper enables researchers to perform practical capture experiments, and includes two novel methods for increasing the targeting efficiency. Coupled with the new massively parallel sequencing technologies, this provides a powerful approach to identifying disease-causing genetic variants that can be localized within the genome by traditional methods. Page 1 of 12 (page number not for citation purposes) BMC Genomics 2009, 10:646 http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 Baseline capture Initial data demonstrated a specificity of the intended cap- ture intervals of 35% with exon hit rate of 99% using 100- 200 bp genomic fragments and protocols similar to the published results and Agilent custom 244 K oligo arrays. These results were comparable with the Hodges et al. data using shorter fragments (29% specificity). With these ini- tial data as baseline, we attempted to improve the capture efficiency by changing the hybridization protocol. Modified capture protocol with hybridization repeat The other protocol modification tested was to repeat the hybridization step with the notion that each round of suc- cessive hybridization will further enrich for the target sequence as a substantially simplified amplicon is hybrid- ized in the second round. In 2005, Bashiardes et al. reported in Nature Methods on capturing genomic loci using bacterial artificial chromosome (BAC) in solution [19]. They performed two rounds of hybridization to enhance targeting and achieved 50% specificity. We incor- porated this idea and repeated the hybridization step after the 2nd PCR. The targeted specificity was successfully increased up to 90% (Table 1). This two step modified protocol was independently repli- cated externally. Total of 2 Mb was targeted for 7,475 non- overlapping exon intervals under 10 linkage regions across the genome. The capture was done by Agilent cus- tom 244 K oligo arrays strictly following the presented protocols except for reducing the 2nd hybridization time to 24 hrs and re-using the same capture array for the 2nd hybridization. The 36 bp single end sequencing was per- formed using Illumina GA I in the authors' laboratory and the sequences were aligned to the whole genome using MAQ (Mapping and Assembly with Qualities). With the We tried two different approaches to overcome this non- specific pull down issue. First, to remove complementary adapter strands from the hybridization mix, we separated the two strands of genomic fragments and used one of the strands for the hybridization. To accomplish this, we biotinylated only one of the PCR primers (primer1.1) used in the generation of the genomic library. After the Table 1: Summary of the results. Primers Single strand separation Double Hyb Specificity Exons with reads* Bases Covered* Baseline - - - 35% 98% 75% Single Strand - Yes - 54% 99% 92% Primer Block Yes - - 62% (63%†) 99% (99%†) 88% (94%†) Double Hybridization Yes - Yes 90% (82%†) 98% (98%†) 84% (89%†) Table 1: Summary of the results. Table 1: Summary of the results. Primers Single strand separation Double Hyb Specificity Exons with reads* Bases Covered* Baseline - - - 35% 98% 75% Single Strand - Yes - 54% 99% 92% Primer Block Yes - - 62% (63%†) 99% (99%†) 88% (94%†) Double Hybridization Yes - Yes 90% (82%†) 98% (98%†) 84% (89%†) * Not considering flanking regions † Results from replication using the tumor genomic DNA. Modified capture protocol to block adapter-adapter hybridization The first change we made to the Agilent hybridization pro- tocol was to block the adapters ligated at the end of every genomic amplicon in the hybridization mix. We assumed that possible hybridization between different genomic targets based on adapter-adapter hybridization will lead to the inadvertent and non-specific enrichment of off-tar- get fragments. This is due to the fact that all of the genomic fragments in the hybridization mix are flanked with the same Illumina adapters (52 nt and 34 nt), which have comparable length to the intended genome location specific target probes (45-60 nt). Thus, the melting tem- perature between the adapter hybridizations will be simi- lar to that between the appropriate genomic fragment and its specific probe. Moreover, the effective concentration in the hybridization of the adapter sequence is approxi- mately 107 fold higher than the genome specific sequences. Thus, these adapter mediated hybridization may dominate the hybridization process. * Not considering flanking regions Results PCR step, the amplicon was bound to streptavidin beads, and the non-biotinylated strand was collected and hybrid- ized to the array. The second approach we tried was mech- anistically easier to prepare than separating the two strands. For this method, we added 10 fold molar excess of Illumina primers to the hybridization mix assuming that the primers will bind to all of the adapters flanking the genomic fragments and block them from hybridizing to other adapter sequences on different genomic frag- ments. It was shown that both approaches increased the specificity to ~60% with more even coverage resulting from the simpler blocking approach, which is the pre- ferred protocol (Table 1). Background have devel- oped an assay that uses a microarray to design the oligo- nucleotides in parallel [15]. Using a modification of MIP (molecular inversion probes) assay, 55,000 exons of sizes varying from 60 to 191 bp were targeted and although the Here, we concentrate on improving the capture specificity using consistent sample and array design. Throughout the experiment, paired genomic DNA of both cancer and nor- mal tissues were used from a cancer patient. We approached with two different methods to specifically block the adapters while generating the genomic library for hybridization and investigated two sequential rounds of hybridization. These changes resulted in improvements in our measured specificity of the targeted genomic DNA from the same sample and the same array design. Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 † p g g 4 μg of genomic library was used for all experiments. The hybridization mix contained 50 μg of Human Cot-1, 52 μl of Agilent 10× Blocking agent and 260 μl of Agilent 2× hybridization buffer. Specificity is calculated by dividing sequence counts within the targeted region by total sequence counts mapable to the human genome uniquely for each run. Targeted region is defined as the targeted exon +/- 100 bp. g g g † Results from replication using the tumor genomic DNA. * Not considering flanking regions Not considering flanking regions † Results from replication using the tumor genomic DNA. * Not considering flanking regions Modified capture protocol with hybridization repeat 4 μg of genomic library was used for all experiments. The hybridization mix contained 50 μg of Human Cot-1, 52 μl of Agilent 10× Blocking agent and 260 μl of Agilent 2× hybridization buffer. Specificity is calculated by dividing sequence counts within the targeted region by total sequence counts mapable to the human genome uniquely for each run. Targeted region is defined as the targeted exon +/- 100 bp. † Results from replication using the tumor genomic DNA. 4 μg of genomic library was used for all experiments. The hybridization mix contained 50 μg of Human Cot-1, 52 μl of Agilent 10× Blocking agent and 260 μl of Agilent 2× hybridization buffer. Specificity is calculated by dividing sequence counts within the targeted region by total sequence counts mapable to the human genome uniquely for each run. Targeted region is defined as the targeted exon +/- 100 bp. Page 3 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 C Genomics 2009, 10:646 http://www.biomedcentral.com/1471-2164/10 ping of sequences relative to probe position in the genome ure 1 pping of sequences relative to probe position in the genome. a) Sequence coverage distribution averaged acros eted regions captured by basal capture protocol and b) sequence coverage distribution averaged across all targeted reg tured by double hybridization (modified) protocol show that the sequence reads are tightly limited around the targete ons. Here, a targeted region is not necessarily a targeted exon but a probeset composed of multiple probes that are < part to each other. The y axis plots the relative abundance and the x axis is the base position relative to the probes p Mapping of sequences relative to probe position in the genome Figure 1 Mapping of sequences relative to probe position in the genome. a) Sequence coverage distribution averaged across all targeted regions captured by basal capture protocol and b) sequence coverage distribution averaged across all targeted regions captured by double hybridization (modified) protocol show that the sequence reads are tightly limited around the targeted regions. Here, a targeted region is not necessarily a targeted exon but a probeset composed of multiple probes that are < 200 bp apart to each other. The y axis plots the relative abundance and the x axis is the base position relative to the probes posi- tions. Modified capture protocol with hybridization repeat Mapping of sequences relative to probe position in the genome Figure 1 Mapping of sequences relative to probe position in the genome. a) Sequence coverage distribution averaged across all targeted regions captured by basal capture protocol and b) sequence coverage distribution averaged across all targeted regions captured by double hybridization (modified) protocol show that the sequence reads are tightly limited around the targeted regions. Here, a targeted region is not necessarily a targeted exon but a probeset composed of multiple probes that are < 200 bp apart to each other. The y axis plots the relative abundance and the x axis is the base position relative to the probes posi- tions. use of blockers, the specificity resulted in 44% and by repeating the hybridization with the blockers, the specifi- city increased to 84%, which are comparable to the data presented. ated (Figure 1). It has been shown in the previous reports that SNPs (single nucleotide polymorphisms) can be reli- ably detected [16-18]. For validation of the variant calls, we compared the captured data to the Illumina 1 M Duo genotyping array data of the same sample. There were 5,746 dbSNP129 SNPs that were present on both the 1 M Duo genotyping array and within the targeted amplicons. The amplicons were sequenced an average of 6× for single hybridization and 9× for double hybridization. 6.3% and 9% of the polymorphic positions were not sequenced in single and double hybridization, respectively. Excluding Page 4 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 The sample was also known to have DNA amplification of EGFR, "epidermal growth fac- tor receptor", and a focused observation indicated ~25× fold more reads mapping to the EGFR exons in tumor sample than in normal sample (normalized to average coverage of all targeted exons across the whole genome) when single hybridization capture protocol was applied to both samples. We note that this is substantially higher than the SNP based copy number data which indicated a 2 fold increase at EGFR and may indicate higher dynamic range from the capture approach followed by sequencing than a microarray approach which has a limited dynamic range due to fluorophor measurement. In addition, the background noise for the ~1 Mb flanking region of EGFR also showed the ~25× fold amplification compared to elsewhere on the same chromosome reflecting that this 1 Mb region containing the EGFR gene is amplified itself at the same ratio (Figure 3). these positions, the false negative rate (missing the variant allele from capture data while detecting it by the Illumina genotype data) was calculated to be 7.1% and 8.4% for the single and double hybridization, respectively. The false positive rate (detecting the correct variant allele according to the HapMap Caucasian data when Illumina genotype data calls it homozygous reference) was less than 0.1% in both experiments. Although random sam- pling effect was observed, the range of the variant allele detection ratio at the polymorphic positions was nar- rowed as the coverage increased for both experiments (Additional file 1). In addition to base substitutions, we have detected small (< 3 bp) insertions and deletions in our dataset that are described in the dbSNP129. Further, novel indels have been discovered and validated from cancer samples that will be described more completely in another publication. We also gathered information about the frequency of sequence observations and their correlation with copy number in the targeted genome post single hybridization based pull down. Since we used both cancer and normal tissue samples from a single cancer patient, we compared the copy number differences between the two (Figure 2) based on the relative frequency of reads mapping to spe- cific chromosomes. In this experiment, the cancer sample was trisomic for chromosome 7 which had been previ- ously determined by whole genome SNP typing (data not shown). http://www.biomedcentral.com/1471-2164/10/646 The mean number of counts normalized for the physical length of chromosome in the tumor tissue was 1.4 relative to that in the normal tissue. Further, the cancer sample had a loss of one chromosome, which we observed to have 0.65 the number of reads as the same chromosome in the normal tissue. These results indicate the capture method in aggregate preserves the copy number state of the original genomic DNA, and may be useful for copy number detection even when using the capture method. This is important for the identification of http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 larger deletions using sequencing based approaches. Out of 18 places in the genome that showed regional copy number changes in the cancer sample by whole genome SNP typing, 6 of them harbored captured gene(s) and all except for one place agreed between the two datasets (Table 2). However, considering that there was no SNP placed by Affymetrix 250 K array within the disagreed genic region and the low resolution of the Affymetrix 250 K array for detecting copy number changes, it is likely that the copy number changes detected by the captured sequencing may be true. The sample was also known to have DNA amplification of EGFR, "epidermal growth fac- tor receptor", and a focused observation indicated ~25× fold more reads mapping to the EGFR exons in tumor sample than in normal sample (normalized to average coverage of all targeted exons across the whole genome) when single hybridization capture protocol was applied to both samples. We note that this is substantially higher than the SNP based copy number data which indicated a 2 fold increase at EGFR and may indicate higher dynamic range from the capture approach followed by sequencing than a microarray approach which has a limited dynamic range due to fluorophor measurement. In addition, the background noise for the ~1 Mb flanking region of EGFR also showed the ~25× fold amplification compared to elsewhere on the same chromosome reflecting that this 1 Mb region containing the EGFR gene is amplified itself at the same ratio (Figure 3). larger deletions using sequencing based approaches. Out of 18 places in the genome that showed regional copy number changes in the cancer sample by whole genome SNP typing, 6 of them harbored captured gene(s) and all except for one place agreed between the two datasets (Table 2). However, considering that there was no SNP placed by Affymetrix 250 K array within the disagreed genic region and the low resolution of the Affymetrix 250 K array for detecting copy number changes, it is likely that the copy number changes detected by the captured sequencing may be true. Analysis of the sequence data For both the basal and modified capture protocols, only a few percent of sequences mapped beyond 100 bp upstream or downstream from the end oligo sequence boundaries reflecting the sharpness of the capture which was determined by the fragment library size initially cre- Page 4 of 12 (page number not for citation purposes) Page 4 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 specific amplicons. For example, the fold difference observed for EGFR gene was weakened by 2.5-fold when the double hybridization capture protocol was applied, suggesting saturation of the hybridization step effectively normalizing the yield from each amplicon. The overall correlation coefficient between the single hybridization experiment and the double hybridization experiment after excluding the ~100 exons that were outlier was 0.82. This interferes some with the ability to reliable call copy number state of individual exons from the pull down sequence data. Two-round hybridization should be used with caution when copy number detection is critical. The array designed for our current experiments and those in previous reports were all masked for repeats. To test whether including the repeat regions would affect the cap- ture, we have attempted to tile every 15 bp across a 4 Mb region of a single chromosome using Agilent 244 K cus- tom array without vigorously masking for the repeats. The specificity was significantly reduced to 15~30% even with the addition of the primer blockers and increased human cot-1 DNA in the hybridization mix (data not shown). improved capture protocol with a troubleshooting guide (Table 3) that should facilitate the preparation of enriched genomic libraries given access to either Agilent or Nimble- gen hybridization equipment and any of the next genera- tion sequencers and be applicable to other genomes. Two simple optimizations of the hybridization protocol have improved the capture performance significantly. First, by blocking the adapter sequences flanking each of the genomic fragments, we reduced the non-specific pull down through adapter-adapter hybridization. Blocking the nonspecific DNA is an old trick to reduce the back- ground when microarray experiments are performed, with human cot-1 being the most commonly used reagent to block repetitive sequences [20]. Recently, Hodges et al. has shown similar results with the same approach, vali- dating our experimental protocol [21]. Secondly, we repeated the hybridization step to further enrich the genomic fragment pool. While the specificity was enhanced up to 90%, this step introduced ~1% of variant loss and some degree of bias in the relative abundance of EGFR DNA amplification event is preserved in sequence data Figure 3 EGFR DNA amplification event is preserved in sequence data. Discussion Both Nimblegen and Agilent have released their commer- cial products for capture. However, Nimblegen's protocol is specific for the Roche 454 sequencer and no details of the hybridization mix contents are provided. Agilent's protocol uses solution based oligos and although the pro- tocol can be adjusted for either Illumina GA or ABI SOLiD sequencing, it is not cost effective yet for small number of samples. Here, we described a complete instruction of the Copy number fold differences between the normal and tumor tissues per chromosome using single hybridization capture pro- tocol with blockers Figure 2 Copy number fold differences between the normal and tumor tissues per chromosome using single hybridiza- tion capture protocol with blockers. The cancer specimen used in these experiments was known to have a chromosome 7 copy number gain and a chromosome 10 deletion. The normalized counts per chromosome are plotted for all chromosomes and are markedly different for the two chromosomes at altered copy numbers. Copy number fold differences between the normal and tumor tissues per chromosome using single hybridization capture pro tocol with blockers Figure 2 Copy number fold differences between the normal and tumor tissues per chromosome using single hybridiza- tion capture protocol with blockers. The cancer specimen used in these experiments was known to have a chromosome 7 copy number gain and a chromosome 10 deletion. The normalized counts per chromosome are plotted for all chromosomes and are markedly different for the two chromosomes at altered copy numbers. Page 5 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 Table 2: Genomic intervals with regional aberration detected by SNP array were examined by capture array and the results were compared. Genomic Interval SNP array Capture array chr1:141510591-142763403 Gain None captured chr1:92652000-93425291 Gain Gain chr2:42528749-43214954 Loss None captured chr3:52074481-55997844 Gain None captured chr6:144171325-144331154 Gain (4 copies) Gain (25 copies) chr6:164267664-164772727 Loss Loss chr6:169375842-169748900 Loss None captured chr6:56774326-56953414 Loss Loss chr7:54464822-55373694 Gain None captured chr9:14995594-24802191 Gain None captured chr14:68301617-68906063 Loss None captured chr15:38336909-38504594 Gain Loss chr16:2652029-2764985 Loss None captured chr17:5541586-5968563 Gain No Change chr20:39176664-39815654 Gain None captured chr20:61246745-61325513 Gain None captured chrX:13343420-151922021 Gain None captured chrX:1674881-1838306 Loss None captured Table 2: Genomic intervals with regional aberration detected by SNP array were examined by capture array and the results were compared. arrays or one Agilent 1 M custom arrays. Figure 4 shows the proportion of 8 million targeted bases covered at var- ious minimum coverage for different mean coverage within the targeted regions. For example, 76% of the tar- geted bases were considered completely sequenced with sequence depth of 20× or more when the mean coverage within the targeted regions was 55×. From these data, we can project how many sequence reads are required to comprehensively sequence all RefSeq exons. In this report, we used 36 bp of single end sequence reads gener- ated by Illumina GA I. Currently, longer reads of 76 bp paired end sequence reads can be generated and are of suf- ficient quality for resequencing by Illumina GA IIx. This improvement not only increases the total sequences read by one channel of a flowcell, but also facilitates the align- ment to the genome significantly. On average, 2.5 Gb of sequences are generated by one channel of Illumina GA IIx run. Of this, about half of the sequences are mapped uniquely to the human genome and assuming 60-85% specificity of capture, we will be able to generate 0.75-1.06 Gb of sequences within the targeted region. If targeting 33 Mb of the human genome for all RefSeq coding exons, it will require 2 channels (quarter a machine run) of sequencing with Illumina Genome Analyzer (GA) IIx to achieve 20× or more coverage on ~80% of the targeted sequences for one sample: or four samples can be sequenced with each machine run. Conclusions Capturing genomic regions for sequencing has a wide scope of application. Many genetic studies with linkage or association signals will benefit immensely as it becomes possible to reliably and inexpensively capture the region of interest and perform high throughput, shotgun sequencing. Additionally, improvements in exonic enrichment protocols will usher in an era of cost effective sequencing of all the amino acid coding bases of genomes. This will lead to more rapid identification of the causative genes in many disorders. This phenomenon should be taken into account when it is unavoidable to target the repeat regions. Throughout the experiments, the sequence reads gener- ated were tightly mapped nearby the intended probe regions. For each probe, the local sequence coverage will extend out in relation to the length of genomic fragment library initially created. Without any major variations in the genomic fragments that could interrupt the hybridiza- tion to the probe, the sequence coverage will peak within the probe region and decrease with increased distance. http://www.biomedcentral.com/1471-2164/10/646 Alternatively, each run of the ABI SOLiD 3 Plus instrument can generate up to 1 billion 50 base paired end reads, and a total of 40 Gb of mapped genomic sequence, such that 12 exomes can be resequenced at comparable coverage with each machine run (S. Nelson, unpublished results). Thus, whole tran- scriptome resequencing is economically feasible on the current generation of capture tools and sequencing devices, and, in principle, can be performed for under $2000 per genome. http://www.biomedcentral.com/1471-2164/10/646 A 200 Kb sized moving average of the interval flanking a) known EGFR amplification event are plotted in genomic position and b) for reference another genomic interval around the FOXP2 gene also on chromosome 7 is shown demonstrating the more typical coverage. The EGFR region is amplified 25× in average compared to the region outside of EGFR. EGFR DNA amplification event is preserved in sequence data Figure 3 EGFR DNA amplification event is preserved in sequence data. A 200 Kb sized moving average of the interval flanking a) known EGFR amplification event are plotted in genomic position and b) for reference another genomic interval around the FOXP2 gene also on chromosome 7 is shown demonstrating the more typical coverage. The EGFR region is amplified 25× in average compared to the region outside of EGFR. EGFR DNA amplification event is preserved in sequence data Figure 3 EGFR DNA amplification event is preserved in sequence data. A 200 Kb sized moving average of the interval flanking ) k EGFR lifi ti t l tt d i i iti d b) f f th i i t l d th EGFR DNA amplification event is preserved in sequence data Figure 3 EGFR DNA amplification event is preserved in sequence data. A 200 Kb sized moving average of the interval flanking a) known EGFR amplification event are plotted in genomic position and b) for reference another genomic interval around the FOXP2 gene also on chromosome 7 is shown demonstrating the more typical coverage. The EGFR region is amplified 25× in average compared to the region outside of EGFR. Page 6 of 12 (page number not for citation purposes) Solution Both forward and reverse strands of each probe region were spotted on the array and 3 of the genes (150 exons) of higher interest were spotted at 12× (6× for each strand). Instead of using Nimblegen arrays as other studies have, we used Agilent 244 K custom 60 mer arrays. Probe design was performed using Agilent e-array system http:// www.Agilent.com with the repeat mask function on. This resulted in losing 155 exons completely and ~27% of the exons were partially covered (see Additional file 2). DNA was extracted from the blood sample of the same patient. The collections were approved by the UCLA IRB and the samples were processed at the Biological Samples Processing Core at UCLA using Autopure LS™ nucleic acid purification instrument from Gentra Systems. Both the normal and tumor samples were run on the Affymetrix GeneChip Human Mapping 250 K array for global genomic examination and comparison. Both chromo- somal and regional copy number aberrations were detected in the tumor sample with the hallmarks of gliob- lastoma like EGFR amplification and chromosome 10 loss observed. The detailed description of the mutational land- scape and the chromosomal abnormalities of this cancer sample is in preparation (Lee et al.) As we were sequenc- ing with the Illumina Genome Analyzer, we followed the Illumina library generation protocol version 2.3. Five μg of high molecular weight whole genomic DNA from each sample were diluted in 150 μl water as the starting mate- rial. The DNA was sheared using a sonicator (Bioruptor, Diagenode) for 1 hr at high power level to generate short fragments. The size of the sheared product ranged between 150 bp to 400 bp with the median size around 200-250 bp. The sample was concentrated in 30 μl of elu- tion buffer (EB) after purification using QIAquick PCR Purification Kit (Qiagen). To repair 3' or 5' overhangs and Solution Possible Reason Purify the DNA. We used QIAGEN PCR Purification Kit, eluted in EB to have it work. It is good to check the gel to see if the DNA is present after ~10 min run when the DNA is not smeared at a wide range. Even though nothing is visible, it is highly possible that the DNA is still present. Proceed to the next step regardless and see if PCR amplifies anything. When the starting amount of DNA is small or there is significant DNA loss during the process for various reasons, it is possible that the DNA is smeared over a wide range after an hour of electrophoresis and not visible on the gel. Gasket slide was re-used. The array slide was lifted up too quickly. Different buffer was used for the 95C stripping process. Do not re-use the gasket slide. The solution can be flushed to a collection boat and collected. When using multi-array slides like 2×105 K, 4×44 K or 8×15 K, it is still possible to run the capture protocol as indicated. After the stripping, the array slide should be slowly lifted up to prevent contamination. The solution tends to stay within the gaskets. Cannot collect ~400 ul after the stripping step. Stripping was not efficient. Stripping was not efficient. Not enough DNA amplified after the first stripping. Not enough DNA amplified after the first stripping. Another stripping process can be done and checked if there were left over genomic fragments hybridized on the probes. Since it does not matter if the stripped solution contains contaminants as long as the contaminants do not have adapters ligated at the end, it is possible to thoroughly continue the stripping process until no products get amplified. bined[22]. Boundaries for exons and UTRs (untranslated regions) were retrieved from RefSeq database and any intervals that overlapped were merged so that non-redun- dant contiguous intervals were generated. In total, 3,038 genes (31,678 exons) spanning 8.4 Mb were included in the list. Based on the preliminary capture experience data (not shown), we tiled the probes every 120 bp on average so that the distance between the start positions of the two consecutive probes is ~120 bp and the regions between the probes are covered by the two flanking probes result- ing in the same coverage as the regions within the probes. Array Design We chose to capture exonic sequence of ~3,000 cancer genes. Two cancer gene lists, 'cancer gene census list' and 'CGP (cancer genome project) planned studies list' were retrieved from Wellcome Trust COSMIC (catalogue of somatic mutations in cancer) database and com- There are ~18,000 genes in RefSeq database composed of ~33 Mb of coding sequences. To tile every 30 bp, ~914 K probes are needed to be designed which is possible to accomplish on a total set of four Agilent 244 K custom Page 7 of 12 (page number not for citation purposes) Page 7 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 http://www.biomedcentral.com/1471-2164/10/646 Table 3: Troubleshooting Guide Problem Possible Reason Solution Genome is not fragmented after sonication. Buffer condition is not adaptable for sonication. Purify the DNA. We used QIAGEN PCR Purification Kit, eluted in EB to have it work. Nothing is visible on the gel after 1 hr electrophoresis during library generation. When the starting amount of DNA is small or there is significant DNA loss during the process for various reasons, it is possible that the DNA is smeared over a wide range after an hour of electrophoresis and not visible on the gel. It is good to check the gel to see if the DNA is present after ~10 min run when the DNA is not smeared at a wide range. Even though nothing is visible, it is highly possible that the DNA is still present. Proceed to the next step regardless and see if PCR amplifies anything. Cannot collect ~400 ul after the stripping step. Gasket slide was re-used. The array slide was lifted up too quickly. Different buffer was used for the 95C stripping process. Do not re-use the gasket slide. The solution can be flushed to a collection boat and collected. When using multi-array slides like 2×105 K, 4×44 K or 8×15 K, it is still possible to run the capture protocol as indicated. After the stripping, the array slide should be slowly lifted up to prevent contamination. The solution tends to stay within the gaskets. Not enough DNA amplified after the first stripping. Stripping was not efficient. Another stripping process can be done and checked if there were left over genomic fragments hybridized on the probes. Since it Do not re-use the gasket slide. The solution can be flushed to a collection boat and collected. When using multi-array slides like 2×105 K, 4×44 K or 8×15 K, it is still possible to run the capture protocol as indicated. After the stripping, the array slide should be slowly lifted up to prevent contamination. The solution tends to stay within the gaskets. Page 8 of 12 (page number not for citation purposes) Sample Preparation We used a paired normal and tumor whole genomic DNA as the starting materials. The tumor DNA was extracted from the glioblastoma (GBM) specimen and the normal Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) BMC Genomics 2009, 10:646 http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 http://www.biomedcentral.com/1471-2164/10/64 t bl t d f ll th d bl t d d f t b ) ith th t d t i li t d th t l t Percentage of targeted bases sequenced at various minimum coverage for different mean coverages Figure 4 Percentage of targeted bases sequenced at various minimum coverage for different mean coverages. X-axis represents the coverage per base level and the corresponding y-axis represents the percentage of targeted bases that were covered at greater or equal with certain coverage. Table legends describe the detail of each line shown. Percentage of targeted bases sequenced at various minimum coverage for different mean coverages Figure 4 Percentage of targeted bases sequenced at various minimum coverage for different mean coverages. X-axis represents the coverage per base level and the corresponding y-axis represents the percentage of targeted bases that were covered at greater or equal with certain coverage. Table legends describe the detail of each line shown. Percentage of targeted bases sequenced at various minimum coverage for different mean coverages Figure 4 Percentage of targeted bases sequenced at various minimum coverage for different mean coverages. X-axis represents the coverage per base level and the corresponding y-axis represents the percentage of targeted bases that were covered at greater or equal with certain coverage. Table legends describe the detail of each line shown. bp) with the correct adapter pairs ligated, the templates were purified and size fractionated on a 2% agarose gel for 1 hr at 120 V. A band from 150-250 bases (including the adaptors) was excised, gel extracted using Gel Extraction Kit (Qiagen) and eluted in 30 μl of EB. The template was PCR amplified with 1 μl of template DNA, 25 μl Phusion DNA polymerase, 1 μl of Illumina primer 1.1, 1 μl of Illu- mina primer 2.1 and 22 μl of water. Four replicates of PCR reactions were performed to generate sufficient genomic amplicon of ~4 ug for successful hybridization. Sample Preparation Total of 18 cycles of PCR with the following cycle conditions was performed: 30 sec at 98°C, [10 sec at 98°C, 30 sec at 65°C, 30 sec at 72°C] × 18 cycles, 5 min at 72°C and hold at 4°C. Samples were purified, eluted in 50 μl of EB and quantified with spectrophotometer (Nanodrop). generate blunt ends for all the double stranded fragments, 5 μl of T4 DNA polymerase, 1 μl of Klenow DNA polymer- ase, 5 μl of T4 PNK, 4 μl of dNTP mix, 10 μl of T4 DNA ligase buffer with 10 mM ATP and 45 μl water were added to the DNA solution and incubated at 20°C for 30 min and purified to be eluted in 32 μl of EB. Single 'A' base was added to the fragments in a reaction with 3 μl of Klenow exo- (3' to 5' exo minus), 10 μl of dATP and 5 μl of Klenow buffer and incubated at 37°C for 30 min. After purifica- tion with MinElute PCR purification kit (QIAGEN) and elution in 10 μl of EB, the Illumina supplied adapters were ligated to the genomic fragments. Five μl of DNA ligase, 10 μl of adapter oligo mix, 25 μl of DNA ligase buffer were used and the sample was incubated for 15 min at room temperature. To select a size-range of templates (100-200 Page 9 of 12 (page number not for citation purposes) Page 9 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 BMC Genomics 2009, 10:646 BMC Genomics 2009, 10:646 http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 Pipette was used to collect the solution and transfer to a 1.5 mL microtube. This step needed to be done promptly as the solution was heated to 95°C and it started evapo- rating quickly. Collected solution was approximately 400 μl. The sample was aliquoted into 4 tubes so that when added with the Illumina primer pair (final concentration 0.1 μM), enzyme (final concentration 0.5×), and dNTPs (final concentration 250 μM each), the final volume were 100 μl. The stripped DNA was amplified by 15 cycles of PCR as described previously. The samples were purified with QIAquick PCR purification kit, consolidating and eluting the sample in 50 μl of EB. Washing After hybridization, the arrays were washed according to the Agilent CGH wash procedure A protocol with the 2nd wash extended to 5 minutes for increased stringency. The chamber was disassembled and the array slide was sepa- rated from the gasket slide in a glass dish filled with Oligo aCGH wash buffer 1 (Agilent). The array slide was placed on a slide-rack in another glass dish filled with Oligo aCGH wash buffer 1 and washed for 5 min at room tem- perature with stirring on a magnetic stirring plate. The slide rack was carefully moved to a glass dish filled with Agilent Oligo aCGH wash buffer 2 that was pre-heated to 37°C in a water bath and was washed for 5 min. After washing, the arrays were transferred back to a glass dish with Agilent Oligo aCGH wash buffer 1 until the next step was ready. Sequencing h ll i The Illumina flowcell was strictly prepared according to the manufacturer's protocol and the clusters were sequenced on the Genome Analyzer using standard man- ufacturer's recommended protocols. The image data pro- duced were converted to intensity files and the sequential image data were processed through the "Firecrest" and "Bustard" algorithms provided by Illumina were used to call the individual sequence reads. Hybridization Hybridization was performed according to Agilent CGH 244 K array protocol. 4 μg of amplified products were mixed with 50 μg of Human Cot-1 DNA (Invitrogen, Carlsbad, CA), 52 μl of Agilent 10× Blocking Agent, 260 μl of Agilent 2× Hybridization Buffer (Agilent) and 8 μl of each of the Illumina primer stock (100 μM each) in a final volume of 520 μl. Blockers were designed to be comple- mentary to the Illumina supplied primers. The sequences provided by Illumina are shown in Table 4 (Oligonucle- otide sequences© 2006 Illumina, Inc. All rights reserved) and the two primers were separately ordered from IDT (Integrated DNA technologies). The hybridization mix was denatured at 95°C for 3 min and pre-hybridized at 37°C for 30 min. 490 μl of the prehybridized amplicons were dispensed to a gasket slide settled in a hybridization chamber base (Agilent), then covered with the array slide and tightly locked with the cover chamber. Hybridization occurred in a rotating oven (Robbins Scientific) for 65 hours at 65°C, 20 rpm. Sample Preparation The concentration of DNA was measured and the size was checked on a 2% aga- rose gel to confirm that the size matched the size extracted from the gel in the previous step. Hybridization, washing and stripping steps were repeated. The stripped DNA was amplified under the same condition as before. After checking the concentration and template size again, the sample was diluted in a final concentration of 10 nM, which is the working concentration for cluster generation. Alignment d h We used the Blat-like Fast Accurate Search Tool (BFAST, in submission) to map each sequence read back to its loca- tion in the reference genome. Here we used the NCBI human genome build 36 as our reference genome [23]. Ten different genome indexes (Table 5) were built for use in the BFAST alignment process to be robust to errors and variants in the short (typically 36 base pairs) reads used throughout this project. For each read, the potential genome locations identified by BFAST were evaluated using a standard local alignment algorithm and ranked by score (see http://genome.ucla.edu/bfast). The best scoring alignment for each read was chosen, while reads with multiple top-scoring alignments or no alignments were discarded. MAQ was run on the same dataset to ensure we do not see any bias introduced by the alignment program. The aligned reads were post filtered so that the reads with Additional file 1 The variant allele ratios for each polymorphic position that were called by Illumina 1 M duo genotype data. The x-axis is the coverage per base and the y-axis is the variant allele ratio. Each spot is a polymor- phic position that the capture data intersected with the Illumina genotype data. Different colors represent either homozygous reference (blue), homozygous non-reference (red) or heterozygous (black). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-646-S1.BMP] Authors' contributions HL and BM conceived and designed the experiments. HL performed the experiments and wrote the paper. HL and BO analyzed the data. VAF and DHC contributed the rep- lication dataset. NH contributed the analysis tools. ZC contributed the reagents and supervised the sequencing run. SN supervised the project as PI on the grant. All authors read and approved the final manuscript. Additional file 3 Summary table. Summary Table of the Illumina Runs for both BFAST and MAQ alignment. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-646-S3.RTF] [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-646-S3.RTF] Additional file 2 Oligonucleotide Capture Probes. The table lists the sequence and the chromosomal locations of the oligonucleotide capture probes used. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2164-10-646-S2.CSV] Additional material mapping quality 0 that are aligned to multiple places in the genome were removed. The difference of the number of reads uniquely mapped to the whole genome (paired t- test p-value: 0.14) and the specificities for each experi- ment (paired t-test p-value: 0.13) were not significant between the BFAST aligned data and MAQ aligned data (Additional file 3). Two summary reports were created once the most likely alignment for each read was identi- fied by BFAST. The first report was a BED file describing mismatches between the Illumina sequence and their cor- responding genomic sequence. The second report was a Wiggle (WIG) file describing sequence coverage at each position along the chromosomes. These file formats were used because they are compatible with the popular UCSC genome browser. The open source SeqWare project (in submission), which provides a LIMS tool for tracking samples (SeqWare LIMS) and a pipeline for sequence analysis (SeqWare Pipeline), http://seqware.source forge.net was used throughout this work. It streamlined the sequence processing by running the Illumina-pro- vided image analysis and base-calling tools, BFAST, and the report generation code, which itself is part of the SeqWare project. The BED file was further mapped to the dbSNP129 database to filter out known SNPs from de novo variants. Stripping l 490 μl of 1× Titanium Taq PCR Buffer (Clontech) pre- heated to 95°C was dispensed to a new gasket slide and covered with the array slide that was washed. After securely locking the arrays in the chamber, the array was incubated in the 95°C rotating oven for 10 min at 20 rpm. After this stripping process, the chamber was disassem- bled and the array slide was carefully lifted up from one side so that the solution converged on the gasket slide. Table 4: PCR primer sequences used to design primers for blocking the adapters in the hybridization mix. Primer 1.1 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT Primer 2.1 5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT Oligonucleotide sequences© 2006 Illumina, Inc. All rights reserved. Page 10 of 12 (page number not for citation purposes) Table 4: PCR primer sequences used to design primers for blocking the adapters in the hybridization mix. Primer 1.1 5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT Primer 2.1 5' CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT Oligonucleotide sequences© 2006 Illumina, Inc. All rights reserved. Table 4: PCR primer sequences used to design primers for blocking the adapters in the hybridization mix Page 10 of 12 (page number not for citation purposes) http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 Table 5: Masks that define each index for sequence alignment. Index Masks 1 111111111111111111 2 11110100110111101010101111 3 11111111111111001111 4 1111011101100101001111111 5 11110111000101010000010101110111 6 1011001101011110100110010010111 7 1110110010100001000101100111001111 8 1111011111111111111 9 11011111100010110111101101 10 111010001110001110100011011111 downstream from the final oligo intended to capture each exon interval. To compare the copy number of each chro- mosome between the normal and tumor sample from a patient, sequence counts were first tallied for each target interval and divided by total sequence counts within all target intervals for normalization. Mean of sequence counts per chromosome was calculated and compared between the normal and tumor sample. To compare the background noise between flanking region of EGFR and FOXP2 genes on chromosome 7, only the coverage in the non-targeted region (excluding targeted region +/- 100 bp) was considered. 200 Kb moving average of the cover- age was calculated across each region. References ( ) 16. Albert TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song X, Richmond TA, Middle CM, Rodesch MJ, Packard CJ, Weinstock GM, Gibbs RA: Direct selection of human genomic loci by micro- array hybridization. Nat Methods 2007, 4(11):903-905. 1. Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosen- baum AM, Wang MD, Zhang K, Mitra RD, Church GM: Accurate multiplex polony sequencing of an evolved bacterial genome. Science 2005, 309(5741):1728-1732. y y ( ) 17. Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME: Microarray-based genomic selection for high-throughput resequencing. Nat Methods 2007, 4(11):907-909. genome. Science 2005, 309(5741):1728 1732. 2. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM: Genome sequencing in microfabricated high-density picolitre reac- tors. Nature 2005, 437(7057):376-380. 18. Hodges E, Xuan Z, Balija V, Kramer M, Molla MN, Smith SW, Middle CM, Rodesch MJ, Albert TJ, Hannon GJ, McCombie WR: Genome- wide in situ exon capture for selective resequencing. Nat Genet 2007, 39(12):1522-1527. ( ) 19. Bashiardes S, Veile R, Helms C, Mardis ER, Bowcock AM, Lovett M: Direct genomic selection. Nat Methods 2005, 2(1):63-69. 20. Landegent JE, Jansen in de Wal N, Dirks RW, Baao F, Ploeg M van der: Use of whole cosmid cloned genomic sequences for chromo- somal localization by non-radioactive in situ hybridization. Hum Genet 1987, 77(4):366-370. ( ) 3. Bennett S: Solexa Ltd. Pharmacogenomics 2004, 5(4):433-438. 3. Bennett S: Solexa Ltd. Pharmacogenomics 2004, 5(4):433 438. 4. References Nature 2001, 409(6822):860-921. ( ) 5. Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Braslavsky I, Cau- sey M, Colonell J, Dimeo J, Efcavitch JW, Giladi E, Gill J, Healy J, Jarosz M, Lapen D, Moulton K, Quake SR, Steinmann K, Thayer E, Tyurina A, Ward R, Weiss H, Xie Z: Single-molecule DNA sequencing of a viral genome. Science 2008, 320(5872):106-109. g , ( ) 6. Pushkarev D, Neff NF, Quake SR: Single-molecule sequencing of an individual human genome. Nat Biotechnol 2009, 27(9):847-52. 7. Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloglu A, Ozen S, Sanjad S, Nelson-Williams C, Farhi A, Mane S, Lifton RP: Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA 2009, 106(45):19096-19101. 8. Ng SB, Buckingham KJ, Lee C, Bigham AW, Tabor HK, Dent KM, Huff CD, Shannon PT, Jabs EW, Nickerson DA, Shendure J, Bamshad MJ: Exome sequencing identifies the cause of a mendelian disor- der. Nat Genet 2009 in press. p 9. Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, Shaffer T, Wong M, Bhattacharjee A, Eichler EE, Bamshad M, Nicker- son DA, Shendure J: Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009, 461(7261):272-276. ( ) 10. Reversade B, Escande-Beillard N, Dimopoulou A, Fischer B, Chng SC, Li Y, Shboul M, Tham PY, Kayserili H, Al-Gazali L, Shahwan M, Bran- cati F, Lee H, O'Connor BD, Schmidt-von Kegler M, Merriman B, Nel- son SF, Masri A, Alkazaleh F, Guerra D, Ferrari P, Nanda A, Rajab A, Markie D, Gray M, Nelson J, Grix A, Sommer A, Savarirayan R, Janecke AR, Steichen E, Sillence D, Hausser I, Budde B, Nurnberg G, Nurnberg P, Seemann P, Kunkel D, Zambruno G, Dallapiccola B, Schuelke M, Robertson S, Hamamy H, Wollnik B, Van Maldergem L, Mundlos S, Kornak U: Mutations in PYCR1 cause cutis laxa with progeroid features Nat Genet 2009 41(9):1016-1021 e M, Robertson S, Hamamy H, Wollnik B, Van Maldergem L, progeroid features. Nat Genet 2009, 41(9):1016-1021. p g ( ) 11. Dahl F, Gullberg M, Stenberg J, Landegren U, Nilsson M: Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments. Nucleic Acids Res 2005, 33(8):e71. ( ) 12. http://www.biomedcentral.com/1471-2164/10/646 http://www.biomedcentral.com/1471-2164/10/646 BMC Genomics 2009, 10:646 and the National Institute of Mental Health (R01 MH071852). The replica- tion study was funded by grant D22657 and DE019567. and the National Institute of Mental Health (R01 MH071852). The replica- tion study was funded by grant D22657 and DE019567. 15. Porreca GJ, Zhang K, Li JB, Xie B, Austin D, Vassallo SL, LeProust EM, Peck BJ, Emig CJ, Dahl F, Gao Y, Church GM, Shendure J: Multiplex amplification of large sets of human exons. Nat Methods 2007, 4(11):931-936. References Eid J, Fehr A, Gray J, Luong K, Lyle J, Otto G, Peluso P, Rank D, Bay- bayan P, Bettman B, Bibillo A, Bjornson K, Chaudhuri B, Christians F, Cicero R, Clark S, Dalal R, Dewinter A, Dixon J, Foquet M, Gaertner A, Hardenbol P, Heiner C, Hester K, Holden D, Kearns G, Kong X, Kuse R, Lacroix Y, Lin S, Lundquist P, Ma C, Marks P, Maxham M, Murphy D, Park I, Pham T, Phillips M, Roy J, Sebra R, Shen G, Soren- son J, Tomaney A, Travers K, Trulson M, Vieceli J, Wegener J, Wu D, Yang A, Zaccarin D, Zhao P, Zhong F, Korlach J, Turner S: Real- Time DNA Sequencing from Single Polymerase Molecules. Science 2008, 323(5910):133-8. 21. Hodges E, Rooks M, Xuan Z, Bhattacharjee A, Benjamin Gordon D, Brizuela L, Richard McCombie W, Hannon GJ: Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing. Nat Protoc 2009, 4(6):960-974. 22. Forbes SA, Bhamra G, Bamford S, Dawson E, Kok C, Clements J, Menzies A, Teague JW, Futreal PA, Stratton MR: The Catalogue of Somatic Mutations in Cancer (COSMIC). Curr Protoc Hum Genet 2008, Chapter 10(Unit 10):11. 23. , p ( ) 23. References Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McK- ernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Ray- mond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ains- cough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mun- gall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chin- walla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette- Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Feder- spiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, John- son LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ: Initial sequencing and analysis of the human genome. References Nature 2001, 409(6822):860-921. 3. References Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McK- ernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Ray- mond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ains- cough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mun- gall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chin- walla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette- Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Feder- spiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, John- son LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ: Initial sequencing and analysis of the human genome. Acknowledgements We counted the sequences that were mapped back to the targeted region to calculate the specificity. From the WIG file for each chromosome, the base positions that mapped within the target intervals were filtered, and the sequence counts for each base position were summed. The target intervals were defined as extending 100 bp upstream and g We thank Jason Liu and Felipe Cervantes for technical assistance and the members of the Nelson Lab for guidance and input. Funding was provided from the Dani Saleh Cancer Fund, the Henry Singleton Brain Cancer Research Fund, and a grant to support the UCLA Site of the National Insti- tutes of Health Neuroscience Microarray Consortium (NS U24NS052108) Page 11 of 12 (page number not for citation purposes) Page 11 of 12 (page number not for citation purposes) References Dahl F, Stenberg J, Fredriksson S, Welch K, Zhang M, Nilsson M, Bick- nell D, Bodmer WF, Davis RW, Ji H: Multigene amplification and massively parallel sequencing for cancer mutation discovery. Proc Natl Acad Sci USA 2007, 104(22):9387-9392. ( ) 13. Fredriksson S, Baner J, Dahl F, Chu A, Ji H, Welch K, Davis RW: Mul- tiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector. Nucleic Acids Res 2007, 35(7):e47. g y ( ) 14. Stenberg J, Dahl F, Landegren U, Nilsson M: PieceMaker: selection of DNA fragments for selector-guided multiplex amplifica- tion. Nucleic Acids Res 2005, 33(8):e72. Page 12 of 12 (page number not for citation purposes)
https://openalex.org/W4362625932	https://aacr.figshare.com/articles/journal_contribution/Supplementary_S2_from_Combined_Targeting_of_PARG_and_Wee1_Causes_Decreased_Cell_Survival_and_DNA_Damage_in_an_S-Phase_Dependent_Manner/22526369/1/files/39989237.pdf	English	null	Supplementary S2 from Combined Targeting of PARG and Wee1 Causes Decreased Cell Survival and DNA Damage in an S-Phase–Dependent Manner	null	2,023	cc-by	512	Supplemental Figure 2 B) Time-dependent statistical analysis was performed using tumor volumes across experimental conditions. Data represent Time-Series Cubic Growth regression analysis comparing experimental conditions to PARG WT + vehicle as the reference control, *P < 0.001. Supplemental Figure 2 Supplemental Figure 2 Supplemental Figure 2 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + Wee1i PARG WT + V 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + V PARG KO + V 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + V PARG KO + Wee1i 0 4 8 12 16 20 24 28 3 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + Wee1i PARG KO + Wee1i 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG KO + V PARG KO + Wee1i Supplemental Figure 2 B A 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + V PARG KO + V A A 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + Wee1i PARG WT + V 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + Wee1i PARG KO + Wee1i Tumor Volume (mm3) Day Post Treatment Day Post Treatment Day Post Treatment 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG WT + V PARG KO + Wee1i 0 4 8 12 16 20 24 28 32 36 40 0 250 500 750 1000 1250 1500 Day Post Treatment Tumor Volume (mm3) PARG KO + V PARG KO + Wee1i 1 1 Tumor Volume (mm3) Day Post Treatment Day Post Treatment B B Supplementary Figure 2 A) Tumor volumes were plotted via spaghetti plot for each experimental group ending at the last day of the vehicle treated PARG WT experimental arm (day 36). Comparison of two experimental arms are plotted per graph for comparison. Supplementary Figure 2 A) Tumor volumes were plotted via spaghetti plot for each experimental group ending at the last day of the vehicle treated PARG WT experimental arm (day 36). Comparison of two experimental arms are plotted per graph for comparison. B) Time-dependent statistical analysis was performed using tumor volumes across experimental conditions. Data represent Time-Series Cubic Growth regression analysis comparing experimental conditions to PARG WT + vehicle as the reference control, *P < 0.001.
https://openalex.org/W4392383425	https://aacr.figshare.com/articles/journal_contribution/Table_S4_from_ERBB4-Mediated_Signaling_Is_a_Mediator_of_Resistance_to_PI3K_and_BTK_Inhibitors_in_B-cell_Lymphoid_Neoplasms/25337696/1/files/44837807.pdf	English	null	Table S4 from ERBB4-Mediated Signaling Is a Mediator of Resistance to PI3K and BTK Inhibitors in B-cell Lymphoid Neoplasms	null	2,024	cc-by	79	Table S4. Sequences of sgRNAs used for induction of ERBB4 transcription by CRISPR activation technique. single guide RNA sequence ERBB4 sgRNA 3 TAAGATTTCCCTTTGGAAGG (1) ERBB4 sgRNA 5 CAATAAGATTTCCCTTTGGA (1) Negative control GCTGAAAAAGGAAGGAGTTGA Table S4. Sequences of sgRNAs used for induction of ERBB4 transcription by CRISPR activation technique. single guide RNA sequence ERBB4 sgRNA 3 TAAGATTTCCCTTTGGAAGG (1) ERBB4 sgRNA 5 CAATAAGATTTCCCTTTGGA (1) Negative control GCTGAAAAAGGAAGGAGTTGA able S4. Sequences of sgRNAs used for induction of ERBB4 transcription by CRISPR activation chnique
https://openalex.org/W3160516192	http://www.scielo.br/pdf/eagri/v34n2/a09v34n2.pdf, http://www.scielo.br/j/eagri/a/FM74XQm5GFjbspzM5dYMmmb/?format=pdf&lang=es, http://pdfs.semanticscholar.org/15ea/46a8481dfe98fc7f85b0ab9ff733db6af969.pdf	es		OEM/ODM 100ml de alcohol al 75% de mano de jabón líquido Gel higienizadorresistance to penetration	null	2,014	cc-by	5,957	DEMANDA TRACTIVA EN FUNCIÓN DE LA RESISTENCIA MECÁNICA DEL SUELO A LA PENETRACIÓN PEDRO H. M. BORGES1, ALOÍSIO BIANCHINI2, JOÃO C. S. MAIA3, HAROLDO C. FERNÁNDES4, ZAÍRA M. S. H. MENDOZA5 RESUMO: No presente estudo, foram elaborados modelos empíricos para determinar a força de tração demandada por arados de discos, escarificadores e semeadoras adubadoras, em função da resistência mecânica do solo à penetração. A média da referida resistência, determinou-se até as profundidades de 25; 35 e 15 cm, em correspondência com as regulagens do arado de discos, escarificador e semeadora adubadora. O ajuste foi verificado de acordo com o coeficiente de determinação, gráficos de dispersão, análise residual e teste t (Student). Com base nessas análises, confirmou-se a normalidade dos resíduos, e foram estabelecidos intervalos de confiança com 95% de probabilidade. Os valores estimados e obtidos experimentalmente não discreparam significativamente. Concluiu-se que as funções exponenciais associadas apresentaram adequada precisão e confiabilidade para predizer a força de tração, considerando-se a resistência mecânica do solo à penetração e que os modelos podem ser uma ferramenta útil para o planejamento e gestão de operações agrícolas mecanizadas em solos com textura similar às desta pesquisa. PALAVRAS-CHAVE: desempenho de máquinas, modelos empíricos, modelos não lineares. TRACTIVE DEMAND AS FUNCTION OF SOIL MECHANICAL RESISTANCE TO PENETRATION ABSTRACT: In this study, empirical models were developed to determine the tensile force demanded by disc plows, harrows and planters fertilizers, as function of soil mechanical resistance to penetration. The mean of the referred resistance was determined until 25, 35 and 15 cm depths, according to the regulations for the disc plow, springs harrow and fertilizer seeder. The adjustment was verified based on the coefficient of determination, dispersion graphics, residual analysis and t test (Student). It was confirm the normality of the residuals and to establish intervals of confidence with 95% of probability. The estimated and experimentally obtained values did not differ significantly. It was concluded that the associated exponential functions presented appropriate precision and reliability to predict the traction force being considered the soil mechanical resistance to penetration and that the models can be a useful tool for planning and management of mechanized farming operations in soils with texture similar to those of this research. KEYWORDS: machine performance; empirical models; nonlinear models. INTRODUCCIÓN Para implantar una cultura agrícola, se puede preparar el terreno convencionalmente o adoptando métodos conservacionistas. En el preparo convencional existe elevada movilización del suelo causada por arados y gradas. Por otro lado, los métodos conservacionistas tienen por finalidad disminuir el número de operaciones, como es el sistema reducido utilizándose un escarificador o aún eliminar totalmente esas actividades como es en el plantío directo, realizado solamente con una sembradora abonadora apropiada. Sin embargo, independientemente del método de preparo, para ___________________________________________ 1 Ing. Agrícola, Prof. Doctor, Departamento de Suelos e Ingeniería Rural, UFMT/Cuiabá-MT, Teléfono: 65 3615 8433, pborges@ufmt.br 2 Ing. Agrónomo, Prof. Associado, Departamento de Suelos e Ingeniería Rural, UFMT/Cuiabá-MT, bianchi@ufmt.br 3 Ing. Agrónomo, Prof. Asociado, Departamento de Suelos e Ingeniería Rural, UFMT/Cuiabá-MT, jotace@terra.com.br 4 Ing. Agrícola, Prof. Asociado, Departamento de Ingeniería Agrícola, UFV/Viçosa-MG, haroldo@ufv.br 5 Inga Forestal, Profª. Adjunta, Departamento de Ingeniería Forestal, UFMT/Cuiabá-MT, zaira@ufmt.br Recebido pelo Conselho Editorial em: 17-10-2012 Aprovado pelo Conselho Editorial em: 25-11-2013 Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Pedro H. M. Borges, Aloísio Bianchini, João C. S. Maia et al. 274 elevar los rendimientos de las culturas es necesario seleccionar y dimensionar correctamente los tractores de acuerdo con las máquinas e implementos correspondientes. Establecer una relación adecuada entre el tractor y el implemento o máquina, significa formar racionalmente un conjunto mecanizado, lo que implica en disminuir los costos de producción y los impactos ambientales como por ejemplo excesiva erosión o compactación de los suelos. La formación racional de conjuntos mecanizados requiere el conocimiento de la demanda tractiva en la operación. En este sentido, ASAE (2003) desarrolló modelos empíricos que determinan la referida fuerza con base en parámetros de las máquinas e implementos y del suelo. No obstante, esos modelos son muy limitados, pues no presentan variables para considerar el estado actual del terreno en relación al contenido de agua, grado de compactación y tipo de cobertura, entre otros. Debemos destacar que los modelos propuestos por la ASAE (2003) fueron establecidos en condiciones experimentales, probablemente, muy distintas a las presentes en los suelos brasileños, llevando a la determinación de valores de fuerza significativamente diferentes. Este aspecto fue abordado por SANTOS (2010), quien comprobó que esos modelos subestimaron la fuerza requerida por escarificadores y sembradoras abonadoras. MODOLO et al. (2004, 2005), SILVEIRA et al. (2005 a, b), FURLANI et al. (2007), MACHADO et al. (2007), FERNANDES & GAMERO (2010), SILVEIRA et al. (2011) estudiaron la fuerza de tracción requerida por diversos equipos agrícolas. No obstante, los resultados obtenidos por los referidos autores no pueden ser extrapolados con seguridad para condiciones distintas de las citadas en sus experimentos. Una variable muy utilizada para representar el estado actual del terreno es la resistencia mecánica media del suelo a la penetración para determinada profundidad. MOLIN et al. (2006) verificaron que la mencionada resistencia es influenciada principalmente por el contenido de agua y la densidad del suelo, siendo eficiente para indicar su compactación. También, BEUTLER & CENTURION (2004); BEUTLER et al. (2006); PÉREZ (2006); ACOSTA (2008); BONNIN et al. (2010) emplearon esa variable como principal indicativo para evaluar la compactación del terreno. Sin embargo, en esos estudios no se desarrollaron dependencias funcionales para relacionar la fuerza de tracción de implementos y máquinas agrícolas, considerándose el estado de compactación del suelo. Esta investigación se fundamentó en la hipótesis de que la referida fuerza varía con la resistencia del terreno, teniendo como objetivo estimarla para arado de discos, escarificadores y sembradoras abonadoras, en función de la resistencia mecánica media del suelo a la penetración, de acuerdo con la profundidad de trabajo. MATERIAL Y MÉTODOS El experimento fue conducido durante dos años consecutivos, en un área perteneciente a la Universidad Federal de Viçosa, situada en la ciudad de Viçosa-MG, definida por las coordenadas geográficas 20º 45' 14” de latitud Sur y 42º 52' 53” de longitud Oeste y altitud media de 650 m. En la región predomina el relevo montañoso (aproximadamente el 85%). El terreno fue sometido a la siembra directa durante los últimos cinco años con los cultivos de maíz y frijol de forma alternada. La temperatura anual media varía entre 14,0º C y 26,1º C. El clima fue clasificado como Cwa, templado húmedo con veranos calientes e inviernos secos (KÖEPPEN & GEIGER, 1928) y el suelo como Latosuelo rojo-amarillo de textura franco-arcillosa (EMBRAPA, 1999). Las características climáticas de la región permiten definir en el año tres épocas: húmeda (enero, febrero, noviembre y diciembre), intermedia (marzo, abril, septiembre y octubre) y seca (mayo, junio, julio y agosto). Los respectivos contenidos de agua (base volumétrica) en el suelo varían de 35 a 45%, de 25 a 35% y de 15 a 25%, para los períodos húmedo, intermedio y seco. En la Tabla 1 se presentan los valores medios de las variables utilizadas para caracterizar el área experimental. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Demanda tractiva en función de la resistencia mecánica del suelo a la penetración 275 TABLA 1. Valores medios de las variables utilizadas para caracterizar el área experimental. Mean values of the variables used to characterize the experimental area. Variable y unidad Masa seca de la cobertura vegetal (t ha-1) Densidad del suelo (g cm-3) Contenido de agua en el suelo (% bv) Porosidad del suelo (%) Textura del suelo (%) Período húmedo Período intermedio Período seco Macro porosidad Micro porosidad Porosidad total Arcilla Limo Arena gruesa Arena fina Valor medio 5,59 1,53 42,78 28,62 19,97 12,83 38,64 51,47 48,06 26,93 7,19 17,82 Para realizar esta investigación se utilizaron dos tractores de ruedas con tracción delantera auxiliar (4x2 TDA). El primero, modelo MF 265, fabricado por la Massey Ferguson, con potencia nominal a 2200 rpm de 47,8 kW y masa con lastre de 3700 Kg. El segundo, modelo 7630, fabricado por la Valtra, con potencia nominal a 2200 rpm de 75,8 kW y masa con lastre de 6196 Kg. Como implementos y máquinas, montados en el sistema de levante hidráulico del tractor, se emplearon un arado reversible con 3 discos de 29”, un escarificador de muelles constituido por 6 astas parabólicas de ancho 30 mm con punteras de tipo común sin alas y una sembradora-abonadora para plantío directo, modelo PC 2123, fabricada por la SEED MAX, compuesta por tres unidades pantográficas separadas a 450 mm con discos de corte liso de diámetro 406 mm, dosificador de semillas y fertilizantes, surcadores de discos dobles y ruedas compactadoras lisas en forma de “V”. También, fueron necesarios otros equipos y herramientas computacionales de soporte como el penetrómetro electrónico, modelo PNT 2000, fabricado por la DLG Automación Industrial Ltda, la celda de carga, marca SODMEX y modelo N400, con sensibilidad de 2,156 mV V-1 y escala nominal de 50 kN, computadora portátil, modelo ASPIRE 5920, fabricado pela ACER, con sistema operacional WINDOWS XP, planilla electrónica EXCEL de la empresa Microsoft, programa para ajuste de curvas CurveExpert 1.4 y programa estadístico STATISTICA 8.0. El tractor MF 265 se destinó para el montaje de los equipos agrícolas en su sistema de levante hidráulico, mientras el tractor Valtra 7630 para remolcar el conjunto mecanizado y entre los dos se acopló la celda de carga. El arado, el escarificador y la sembradora abonadora fueron regulados para las profundidades de 25, 35 y 15 cm, respectivamente. Durante las lecturas de fuerza de tracción en la celda de carga, el tractor remolcador operó en segunda marcha reducida con el motor a 1700 rpm, siendo que inicialmente los implementos estaban suspendidos y después en la posición de trabajo correspondiente a las regulaciones de profundidad. Luego, la fuerza de tracción requerida por el implemento fue determinada por la diferencia entre las dos lecturas. Para planificar la colecta de los datos, el terreno de forma rectangular con 140 m de largo y 110 m de ancho fue dividido en dos partes, siendo una para cada año. En cada mitad del terreno se definieron 9 áreas, destinadas a los 3 equipos agrícolas (arado, escarificador y sembradora abonadora) y a los 3 períodos del año. Finalmente en esas áreas fueron demarcadas 20 unidades experimentales con 5 m de largo y 3 m de ancho, correspondiendo 10 parcelas para cada mes y equipo agrícola en todo el trabajo. En las parcelas experimentales se realizaron inicialmente 5 lecturas aleatorias de la resistencia mecánica media del suelo a la penetración para la profundidad correspondiente al implemento o máquina y 5 de la fuerza de tracción en la barra, calculándose posteriormente la media entre los dos años de investigación. Luego, los meses se representaron por 5 observaciones, totalizando 60 pares Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Pedro H. M. Borges, Aloísio Bianchini, João C. S. Maia et al. 276 de valores para cada implemento o máquina. Los modelos empíricos para describir analíticamente la fuerza requerida para la tracción de los equipos agrícolas, en función de la referida resistencia media se elaboraron con ayuda de los módulos disponibles en los programas computacionales. La calidad del ajuste se evaluó con base en el coeficiente de determinación, gráficos de dispersión, análisis residual y Test t, conforme sugerido por MONTGOMERY et al. (2004), LEVINE et al. (2005) y WEISBERG (2005). La citación de marcas no significa recomendación de los autores, sino la caracterización de los equipos utilizados en esta investigación. RESULTADOS Y DISCUSIÓN Dentro de las diversas expresiones matemáticas que podían representar los valores de fuerza de tracción, considerándose el índice de cono, se optó por la función exponencial asociada para todos los equipos agrícolas. Ese modelo empírico tiene como características el inicio en el origen del sistema de coordenadas, el aumento de la fuerza en la barra de tracción con el incremento del índice de cono y la definición de un valor asintótico para a variable dependiente. La tendencia de los datos experimentales concuerdan adecuadamente con las características del referido modelo, cuya forma general es dada por: (1) en que, Ftr – Fuerza de tracción requerida, cuja unidad depende del implemento, así se tiene: - específica por área trabajada para el arado (N cm-2) y - por línea de trabajo para el escarificador y sembradora (kN asta-1 o línea-1); ICp - Índice de cono a la profundidad alcanzada por el equipo agrícola (MPa); A – Parámetro del modelo que representa el valor asintótico de la fuerza requerida, luego: - específica por área trabajada para el arado (N cm-2) y - por línea de trabajo para el escarificador y sembradora (kN asta-1 o línea-1); k - Parámetro del modelo que indica la velocidad en el aumento de fuerza (adimensional). Los valores, unidades y estadísticas de los parámetros para las ecuaciones ajustadas, así como los coeficientes de determinación (R2) correspondientes a cada equipo agrícola se encuentran en la Tabla 2. A pesar de los elevados valores del coeficiente de determinación, este no es el único índice para evaluar el ajuste, pues se trata de un modelo no lineal. Los bajos valores de probabilidad indican más del 99% de confianza para aceptar los parámetros de los modelos obtenidos (Tabla 2). TABLA 2. Valores, unidades y estadísticas de los parámetros del modelo ajustado, bien como los respectivos coeficientes de determinación (R2) para cada equipo agrícola.Values, units and statistics of the parameters of the adjusted models, as well as the respective determination coefficient (R2) for each farm equipment. Tipo de información Valor Unidad Desviación estándar Valor t Valor de probabilidad IC95% R2 Arado de discos A k 9,6142 0,6164 N cm-2 0,9289 0,1332 10,3501 4,6276 0,0023 0,0004 ±1,8587 ±0,2665 0,9817 Escarificador de muelles Sembradora abonadora A k A k 5,6612 0,7169 4,5828 0,6992 kN asta-1 kN línea-1 0,7039 0,0956 0,8908 0,0720 8,0409 7,4999 5,1448 9,2000 0,0017 0,0009 0,0038 0,0012 ±1,4085 ±0,1913 ±1,7825 ±0,1521 0,9725 0,9411 Obs. IC95% : Intervalo de confianza para un 95% de probabilidad. A, k: Parámetros del modelo ajustado. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Demanda tractiva en función de la resistencia mecánica del suelo a la penetración 277 En las Figuras 1, 2 y 3 se pueden observar los gráficos de dispersión con los datos experimentales, la curva de la ecuación ajustada para la fuerza requerida en la barra de tracción, en función del índice de cono correspondiente a los implementos estudiados y los límites del intervalo de confianza para 95% de probabilidad. Se puede constatar un aumento de la referida fuerza con el incremento del índice de cono, siendo más acentuado en el inicio (Figuras 1, 2 y 3). De acuerdo con esas Figuras, la curva ajustada acompaña adecuadamente la tendencia presentada por los valores experimentales y todos estos variaron dentro del intervalo de confianza establecido. Fuerza de Tracción específica (N cm -2) 9,0 8,0 7,0 6,0 Datos experimentales 5,0 Ecuación ajustada Intervalo de confianza para 95% 4,0 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Resistencia mecánica media del suelo a la penetración (MPa) FIGURA 1. Datos experimentales y curva de la ecuación ajustada para el arado de discos. Experimental data and curve of the adjusted equation for the disk plow. Fuerza de Tracción (kN asta -1) 5,5 5,0 4,5 4,0 Datos experimentales 3,5 Ecuación ajustada Intervalo de Confianza para 95% 3,0 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Resistencia mecánica media del suelo a la penetración (MPa) FIGURA 2. Datos experimentales y curva de la ecuación ajustada para el escarificador de muelles. Experimental data and curve of the adjusted equation for the springs harrow. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Pedro H. M. Borges, Aloísio Bianchini, João C. S. Maia et al. 278 Fuerza de Tracción (kN línea -1) 4,5 4,0 3,5 3,0 Datos experimentales Ecuación ajustada Intervalo de confianza para 95% 2,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Resistencia mecánica media del suelo a la penetración (MPa) FIGURA 3. Datos experimentales y curva de la ecuación ajustada para la sembradora abonadora. Experimental data and curve of the adjusted equation for the fertilizer seeder. Conforme las Figuras 1, 2 y 3, la resistencia mecánica media del suelo a la penetración varió entre 1,35 y 3,50 MPa. El límite inferior en torno de 1,35 MPa se encuentra próximo al estimado por MOLIN et al. (2006). Esos autores evaluando la referida resistencia en un suelo con histórico de manejo, contenido de arcilla y contenido de agua semejantes a las de nuestra investigación obtuvieron 1,50 MPa como mínimo. No obstante, el valor máximo presentado por los referidos autores de 3,06 MPa es inferior al de este estudio. También, el límite superior de este trabajo está próximo al determinado por ASSIS & LANÇAS (2005) de 3,39 MPa en condiciones experimentales similares y fue superior a los estimados por ASSIS et al. (2009) de 2,98 MPa, CARVALHO et al. (2006) de 2,8 MPa y CUNHA et al. (2009) de 2,6 MPa. Esas diferencias pueden ser originadas por la mayor amplitud del contenido de agua considerado en esta investigación, lo que representa un margen de seguridad para los modelos. Los limites para la resistencia mecánica media del suelo a la penetración de este trabajo fueron superiores a los determinados por VENIALGO et al. (2004), que variaron entre 0,61 y 2,67 MPa. Esas discrepancias se deben, probablemente, a la rotación de culturas en ese estudio, incluyendo el algodón que posee un sistema radicular más profundo, el cual podría favorecer la disminución de la resistencia del suelo a la penetración en etapas posteriores. El intervalo de la resistencia mecánica establecido en nuestra investigación incluye los rangos de valores obtenidos por BEUTLER et al. (2006), WEIRICH et al. (2006), respectivamente, de 2,0 a 3,0 MPa y de 2,0 a 2,2 MPa. La mayor amplitud de nuestro intervalo de valores les confiere adecuada confiabilidad a los modelos propuestos para estimar la fuerza de tracción en situaciones extremas relativas al tipo de suelo y contenido de agua. De acuerdo con los límites de confianza de la Figura 1, podemos afirmar con un nivel de significancia de 5% que la fuerza de tracción específica requerida por el arado de discos varió aproximadamente de 4,5 a 8,5 N cm-2. Ese intervalo comprende los valores obtenidos por MELLO & MAGALHÃES (1995) entre 6,37 y 7,22 N cm-2, bien como la media determinada por FERNANDES & GAMERO (2010) de 7,16 N cm-2. Debemos destacar que en la mayoría de los trabajos consultados, se constataron valores de resistencia mecánica del suelo a la penetración entre 2 y 3 MPa. En ese intervalo la fuerza de tracción específica varió de 6,5 a 7,5 N cm-2 (Figura 1), valores próximos a los estimados en los estudios comentados. Esos resultados indican adecuada predicción del modelo. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Demanda tractiva en función de la resistencia mecánica del suelo a la penetración 279 Conforme la Figura 2, la fuerza de tracción por asta demandada por el escarificador de muelles, varió en una faja de valores entre 3,4 y 5,4 kN asta-1 con el 95% de probabilidad. Ese rango se encuentra dentro del intervalo sugerido por ASAE (2003) de 3,08 a 3,6 kN asta-1 y el determinado por SANTOS (2010) entre 3,8 y 4,7 kN asta-1. Sin embargo, SALVADOR et al. (2008) estimaron valores de 2,7 a 4,2 kN asta-1. Esas discrepancias pueden ser debidas al preparo utilizado por esos autores, antes de realizar la escarificación, lo que contribuyó para disminuir la resistencia del suelo a la penetración. De todas formas, aunque las ecuaciones sobreestimen la fuerza de tracción para bajos valores de la mencionada resistencia, esto serviría como margen de seguridad, confiriéndole confiabilidad al modelo empírico propuesto. Considerándose los limites de confianza para un 95% de probabilidad de la Figura 3 se deduce que la fuerza de tracción por línea de siembra fluctuó aproximadamente entre 2,6 y 4,25 kN línea-1. Ese intervalo supera la faja de valores establecida por ASAE (1996) de 1,1 a 2 kN línea-1, los obtenidos por FURLANI et al. (2004) de 2,15 a 2,42 kN línea-1 y por MERCANTE et al. (2005) entre 2,1 y 2,5 kN línea-1, así como también el valor medio determinado por MODOLO et al. (2005) de 2,1 kN línea-1. No obstante, el intervalo definido en este estudio contiene el rango indicado por LEVIEN et al. (1999) entre 3,24 y 3,64 kN línea-1 y el valor medio estimado por FURLANI et al. (2007) de 2,97 kN línea-1. La mayor amplitud de valores observada en el presente experimento puede ser debida a las condiciones extremas del contenido de agua, aspecto determinante en las fluctuaciones de la resistencia del suelo a la penetración. Por otro lado, el límite inferior de 2,6 kN línea-1 del presente trabajo pertenece al intervalo verificado por ALMEIDA et al. (2010), cuyos valores oscilaron entre 2,26 y 2,51 kN línea-1. También, el límite superior correspondiente a esta investigación de 4,25 kN línea-1 se encuentra dentro del rango obtenido por SILVEIRA et al. (2005 b) de 3,90 a 4,53 kN línea-1, respectivamente, para la menor y mayor profundidad de trabajo. Estas discrepancias pueden estar influenciadas por los mecanismos para abrir y cerrar los surcos, cortar la paja, distribuir semillas y fertilizantes, bien como estar relacionadas con el tipo y estado de la cobertura del terreno, los cuales son factores que interfieren en la resistencia del suelo. A pesar de las diferencias se considera el modelo confiable. Para verificar el ajuste de los modelos empíricos para cada implemento, se elaboraron los diagramas de dispersión con los valores de la fuerza en la barra de tracción observada, en función de los estimados (Figura 4). En esa Figura, también se incluyó la bisectriz del cuadrante con la finalidad de identificar la relación entre los valores teóricos y reales. Se puede constatar que la mayoría de los puntos están distribuidos uniformemente encima y debajo de la bisectriz para los tres equipos agrícolas (Figura 4). También, esos puntos se encuentran próximos de la referida recta, indicando que los modelos obtenidos pueden ser apropiados para determinar la fuerza en la barra de tracción, considerándose la resistencia mecánica media del suelo a la penetración. La premisa de normalidad de las diferencias entre los valores teóricos y reales para cada equipo agrícola es un aspecto muy importante para definir criterios sobre el ajuste de los modelos empíricos elaborados y se puede evaluar con base en los histogramas de residuos presentados en la Figura 5. Conforme esa Figura se puede deducir que las diferencias mencionadas se distribuyeron con adecuada simetría y muy próximas de la curva normal, es decir, la mayoría de pequeños residuos concentrados en el centro y una menor cantidad en los extremos. Aunque la distribución de los residuos se aproximó a la curva normal, el análisis de los histogramas se considera un procedimiento subjetivo. Así, se optó por comprobar analíticamente la normalidad mediante las pruebas de Kolmogorov-Smirnov, Lilliefors y Shapiro-Wilk. Los resultados correspondientes a esas pruebas se presentan en la Tabla 3. Los valores de probabilidad obtenidos muy superiores al 5% permiten afirmar que realmente la premisa de normalidad de los residuos para los tres implementos fue satisfecha. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Pedro H. M. Borges, Aloísio Bianchini, João C. S. Maia et al. 280 9,0 -2 Fuerza de Tracción observada (N cm ) A 8,0 7,0 6,0 5,0 4,0 4,0 5,0 6,0 7,0 8,0 9,0 5,0 5,5 Fuerza de Tracción estimada (N cm-2) B -1 Fuerza de Tracción observada (kN asta ) 5,5 5,0 4,5 4,0 3,5 3,0 3,0 3,5 4,0 4,5 Fuerza de Tracción estimada (kN asta-1) C -1 Fuerza de Tracción observada (kN línea ) 4,5 4,0 3,5 3,0 2,5 2,5 3,0 3,5 4,0 4,5 Fuerza de Tracción estimada (kN línea-1) FIGURA 4. Bisectriz y valores observados de la fuerza en la barra de tracción, en función de los estimados: a) arado de discos; b) escarificador de muelles; c) sembradora abonadora.Bisector and observed values of the force on the drawbar, as function of the estimates: a) disk plow; b) springs harrow; c) fertilizer seeder. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Demanda tractiva en función de la resistencia mecánica del suelo a la penetración 281 16 A 14 12 Curv a normal 10 8 6 4 Frecuencia ( 2 0 -0,10 -0,08 -0,06 -0,04 -0,02 0,00 0,02 0,04 0,06 0,08 0,10 Valores de los Resíduos (N cm -2) 18 B 16 Curva normal 14 12 10 8 6 4 Frecuencia (ad 2 0 -0,10 -0,08 -0,06 -0,04 -0,02 0,00 0,02 0,04 0,06 0,08 0,10 Valores de los Resíduos (kN asta -1) 20 18 C 16 14 12 Curv a normal 10 8 6 4 Frecuencia ( 2 0 -0,10 -0,08 -0,06 -0,04 -0,02 0,00 0,02 0,04 0,06 0,08 0,10 Valores de los Resíduos (kN línea -1) FIGURA 5. Histograma de los residuos: a) arado de discos; b) escarificador de muelles; c) sembradora abonadora. Histogram of residues: a) disk plow; b) springs harrow; c) fertilizer seeder. TABLA 3. Resultados de las pruebas de normalidad para los tres equipos agrícolas. Results of normality tests for the three agricultural equipments. Prueba de normalidad Equipo Agrícola Kolmogorov-Smirnov Lilliefors Shapiro-Wilk Arado de discos p>0,20 (ns) p>0,20 (ns) p=0,87 (ns) Escarificador de muelles p>0,20 (ns) p>0,20 (ns) p=0,52 (ns) Sembradora-abonadora p>0,20 (ns) p>0,20 (ns) p=0,54 (ns) Obs. p: Valor de probabilidad; ns: no significativo Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Pedro H. M. Borges, Aloísio Bianchini, João C. S. Maia et al. 282 En la Figura 6 se presentan los gráficos de residuos estandarizados para cada equipo agrícola, en función de la resistencia mecánica media del suelo a la penetración. De acuerdo con esos gráficos se deduce que no hay relación entre los residuos y las variables de los modelos elaborados, pues los puntos se sitúan aleatoriamente a lo largo del eje horizontal y no existen evidencias de algún padrón de dependencia funcional. Esa distribución se considera por MONTGOMERY et al. (2004), LEVINE et al. (2005) y WEISBERG (2005) como condición ideal en la elaboración de modelos empíricos. Resíduos estandarizados (Adimensional) 3 A 2 1 0 -1 -2 -3 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Resistencia mecánica media del suelo a la penetración (MPa) Resíduos estandarizados (Adimensional) 3 B 2 1 0 -1 -2 -3 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Resistencia mecánica media del suelo a la penetración (MPa) Resíduos estandarizados (Adimensional) 3 C 2 1 0 -1 -2 -3 1,0 1,5 2,0 2,5 3,0 3,5 4,0 Resistencia mecánica media del suelo a la penetración (MPa) FIGURA 6. Residuos estandarizados en función de la resistencia mecánica media del suelo: a) arado de discos; b) escarificador; c) sembradora abonadora. Standardized residuals as function of mean soil mechanical resistance to penetration: a) disk plow; b) springs harrow; c) fertilizer seeder. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Demanda tractiva en función de la resistencia mecánica del suelo a la penetración 283 En la Figura 6A se puede verificar que tres puntos se encuentran fuera del intervalo de residuos estandarizados de -2 a 2. Sin embargo, esos puntos no superan el 5% del total y por tanto son observaciones atípicas que no alteran significativamente el ajuste de la ecuación (Figura 6). Para el caso del escarificador, se constata que solamente un punto se encuentra fuera de los límites del 95% de confianza, teniendo poca importancia para la validación del modelo (Figura 6B). La Figura 6C indica sobreestimación de la fuerza, en función de la resistencia mecánica media del suelo a la penetración entre 2,0 y 2,5 MPa. Más, como son pocos valores y un pequeño intervalo, considerase que no afecta la eficiencia en la predicción. Para confirmar analíticamente la adecuación de los modelos ajustados, los valores teóricos y experimentales de la fuerza de tracción fueron comparados estadísticamente, aplicándose la prueba t de Student. Los resultados obtenidos muestran que no hubo diferencias significativas entre los referidos valores (Tabla 4). Luego, se puede afirmar que las ecuaciones desarrolladas presentan adecuada confiabilidad, justificándose su aplicación, pues son apropiadas para determinar la fuerza de tracción requerida por los respectivos equipos agrícolas, en función de la resistencia mecánica media del suelo a la penetración. Tabla 4. Resultados de la prueba t-Student para los tres equipos agrícolas. Results of the t-Student test for the three agricultural equipments. Equipo Agrícola Arado de discos Escarificador de muelles Sembradora-abonadora Calculado 0,1566 0,0332 0,3632 Obs. α: Nivel de probabilidad; ns: no significativo Valores de t Tabulado α=5% α=1% 1,9944 2,6479 Significancia ns ns ns CONCLUSIONES Las funciones exponenciales asociadas fueron apropiadas para estimar la fuerza de tracción requerida por equipos agrícolas, considerándose la resistencia mecánica del suelo a la penetración. Las dependencias funcionales desarrolladas presentaron adecuada confiabilidad en las predicciones, lo que justifica su aplicación. Los modelos obtenidos pueden ser una herramienta útil para el planeamiento y gestión de operaciones agrícolas mecanizadas en suelos con textura similar a las de esta investigación. REFERENCIAS ACOSTA, J. J. B. Índice de cone, capacidade de suporte de carga e teor de água de quatro solos da fazenda Lageado, Botucatu/SP: correlações e mapas de isocompactação. 2008. 145 f. Tese (Doutorado em Agronomia) - Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, Botucatu, 2008. ALMEIDA, R. A. S.; TAVARES-SILVA, C. A.; SILVA, S. L. Desempenho energético de um conjunto trator-semadora em função do escalonamento de marchas e rotações do motor. Revista Agrarian, Dourados, v.3, n.7, p.63-70, 2010. ASAE. American Society of Agricultural Engineers. Agricultural machinery management data. In: ASAE Standards. St. Joseph, 2003. p.373-380. (ASAE D-497.4). ASSIS, R. L.; LANÇAS, K. P. Avaliação dos atributos físicos de um nitossolo vermelho distroférrico sob sistema plantio direto, preparo convencional e mata nativa. Revista Brasileira de Ciência do Solo, Viçosa - MG, v.29, n.4, p.515-522, 2005. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Pedro H. M. Borges, Aloísio Bianchini, João C. S. Maia et al. 284 ASSIS, R. L.; LAZARINI, G. D.; LANÇAS, K. P.; CARGNELUTTI FILHO, A. Avaliação da resistência do solo à penetração em diferentes solos com a variação do teor de água. Engenharia Agrícola, Jaboticabal, v.29, n.4, p.558-568, out./dez. 2009. BEUTLER, A. N.; CENTURION, J. F. Compactação do solo no desenvolvimento radicular e na produtividade de soja. Pesquisa Agropecuária Brasileira, Brasília, v.39, n.6, p.581-8, 2004. BEUTLER, A. N.; CENTURION, J. F.; SILVA, A. P.; BARBOSA, J. C. Intervalo hídrico ótimo e produtividade de cultivares de soja. Revista Brasileira de Engenharia Agrícola & Ambiental, Campina Grande, v.10, n.3, p.639-645, 2006. BONNIN, J. J.; MIRÁS-ÁVALOS, J. M.; LANÇAS, K. P.; GONZÁLEZ, A. P.; VIEIRA, S. R. Spatial variability of soil resistance influenced by season of sampling. Bragantia, Campinas, v.69, Suplemento, p.163-173, 2010. CARVALHO, G. J.; CARVALHO, M. P.; FREDDI, O. S.; MARTINS, M. V. Correlação da produtividade do feijão com a resistência à penetração do solo sob plantio direto. Revista Brasileira de Engenharia Agrícola & Ambiental, Campina Grande, v.10, n.3, p.765-771, 2006. CUNHA, J. P. A. R.; CARVALHO JÚNIOR, P. C.; SOUZA, J. V.; BORGES, E. N.; REIS, E. F. Compactação do solo sob sistemas de manejo convencionais e conservacionistas. Revista Engenharia na Agricultura, Viçosa - MG, v.17, n.2, p. 155-162, 2009. EMBRAPA. Empresa Brasileira de Pesquisa Agropecuária. Centro Nacional de Pesquisa dos solos. Sistema brasileiro de classificação dos solos. Brasília, 1999. 412 p. FERNANDES, J. C.; GAMERO, C. A. Avaliação do desempenho das máquinas agrícolas na implantação da cultura do girassol. Revista Energia na Agricultura, Botucatu, v.25, n.2, p.74-87, 2010. FURLANI, C. E. A.; GAMERO, C. A.; LEVIEN, R.; LOPES, A. Desempenho operacional de uma semeadora-adubadora de precisão, em função do preparo do solo e do manejo de cobertura de inverno. Engenharia Agrícola, Jaboticabal, v.24, n.2, p.388-395, maio/ago. 2004. FURLANI, C. E. A.; PAVAN JÚNIOR, A.; LOPES, A.; SILVA, R. P.; GROTTA, D. C. C.; CORTEZ, J. W. Desempenho operacional de semeadora-adubadora em diferentes manejos da cobertura e da velocidade. Engenharia Agrícola, Jaboticabal, v. 27, n. 2, p.456-462, maio/ago, 2007. KÖEPPEN, W.; GEIGER, R. Klima der Erde. Gotha: Verlag Justus Perthes. 1928. LEVIEN, R.; MARQUES, J.P.; BENEZ, S.H. Desempenho de uma semeadora-adubadora de precisão, em semeadura de milho (Zea mays L.), sob diferentes formas de manejo do solo. In: CONGRESSO BRASILEIRO DE ENGENHARIA AGRÍCOLA, 28. 1999, Pelotas. Anais... Pelotas: Sociedade Brasileira de Engenharia Agrícola, 1999. 1 CD-ROM. LEVINE, D.M.; STEPHAN, D.; KREHBIEL, T.C.; BERENSON, M.L. Estatística – Teoria e aplicações usando-se o Microsoft EXCEL em português. Rio de Janeiro: Livros Técnicos e Científicos Editora, 2005. 819 p. MACHADO, R. L. T.; FERREIRA, M. F.; MACHADO, A. L. T.; REIS, A. V.; BUTZKE, H. P. Força de tração de semeadoras para plantio direto: informações de campo x informações de catálogo. Tecno-Lógica, Santa Cruz do Sul, v.11, n.1 e 2, p.15-17, jan./dez. 2007. MELLO, R. C.; MAGALHÃES, P. S. G. Desempenho operacional de um arado de discos. Bragantia, Campinas, v.54, n.2, p.447-454, 1995. MERCANTE, E.; SILVA, S. L.; MODOLO, A. J.; SILVEIRA, J. C. M. Demanda energética e distribuição de sementes de milho em função da velocidade de duas semeadoras. Revista Brasileira de Engenharia Agrícola & Ambiental, Campina Grande, v.9, n.3, p.424-428, 2005. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014 Demanda tractiva en función de la resistencia mecánica del suelo a la penetración 285 MODOLO, A. J.; SILVA, S. L.; SILVEIRA, J. C. M.; MERCANTE, E. Avaliação do desempenho de duas semeadoras-adubadoras de precisão em diferentes velocidades. Engenharia na Agricultura, Viçosa, MG, v.12, n.4, p.298-306, out./dez. 2004. MODOLO, A. J.; GABRIEL FILHO, A.; SILVA, S. L.; GNOATTO, E. Força de tração necessária em função do número de linhas de semeadura utilizadas por uma semeadora-adubadora de precisão. Engenharia Agrícola, Jaboticabal, v. 25, n. 2, p.465-473, maio/ago. 2005. MOLIN, J. P.; MAGALHÃES, R. P.; FAULIN, G. D. Análise espacial da ocorrência do índice de cone em área sob semeadura direta e sua relação com fatores do solo. Engenharia Agrícola, Jaboticabal, v. 26, n. 2, p.442-452, maio/ago. 2006. MONTGOMERY, D. C.; RUNGER, G. C.; HUBELE, N. F. Estatística aplicada à engenharia. Rio de Janeiro: LTC, 2004. 335 p. PÉREZ, M. Rendimiento de un tractor agrícola en función del sistema de labranza y la carga. I. Características de la tracción. Revista Bioagro, Barquisimeto, v.18, n.1, p.49-55, 2006. SALVADOR, N.; BENEZ, S. H.; MION, R. L. Consumo de combustível na operação de subsolagem realizada antes e depois de diferentes sistemas de preparo periódico do solo. Engenharia Agrícola, Jaboticabal, v.28, n.2, p.256-262, abr./jun., 2008. SANTOS, P. M. Modelagem do desempenho em tração de conjuntos mecanizados visando ao dimensionamento do trator. 2010. 162 f. Tese (Doutorado em Engenharia Agrícola) – Universidade Federal de Santa Maria, Centro de Ciências Rurais, Santa Maria, 2010. SILVEIRA, J. C. M.; GABRIEL FILHO, A.; SECCO, D. Demanda de potência e força de tração de uma semeadora na implantação do milho safrinha sob plantio direto. Engenharia na Agricultura, Viçosa, v.13, n.4, p. 256-267, Out./Dez., 2005 a. SILVEIRA, J. C. M.; MODOLO, A. J.; SILVA, S. L.; GABRIEL FILHO, A. Força de tração e potência de uma semeadora em duas velocidades de deslocamento e duas profundidades de deposição de sementes. Revista Brasileira de Engenharia Agrícola & Ambiental, Campina Grande, v.9, n.1, p.125-128, 2005 b. SILVEIRA, J. C. M.; FERNANDES, H. C.; MODOLO, A. J.; SILVA, S. L.; TROGELLO, E. Furrow depth, soil disturbance area and draft force of a seeder-fertilizer at different seeding speeds. Revista Ceres, Viçosa - MG, v.58, n.3, p.293-298, 2011. VENIALGO, C. A.; INGARAMO, O.; IBALO, S.; ROLDÁN, M. F.; BANZHAF, G.; GUTIERREZ, N. Índice de cono, humedad presente y densidad aparente en diferentes labranzas y rotaciones. Corrientes: Comunicaciones Científicas y Tecnológicas, Universidad Nacional del Nordeste de Argentina, A-076, 2004. WEIRICH NETO, P. H.; BORGHI, E.; SVERZUT, C. B.; MANTOVANI, E. C.; GOMIDE, R. L.; NEWES, W. L. C. Análise multivariada da resistência do solo à penetração sob plantio direto. Ciência Rural, Santa Maria, v.36, n.4, p.1186-1192, jul-ago, 2006. WEISBERG, S. Applied linear regression. New York: John Wiley & Sons, 2005. 330 p. Eng. Agríc., Jaboticabal, v.34, n.2, p.273-285, mar./abr. 2014
https://openalex.org/W4368375852	https://www.mdpi.com/1422-0067/24/9/8148/pdf?version=1683857673	English	null	Multi-Targeting Neuroprotective Effects of Syzygium aromaticum Bud Extracts and Their Key Phytocompounds against Neurodegenerative Diseases	International journal of molecular sciences	2,023	cc-by	21,957	Citation: Sharma, H.; Kim, D.Y.; Shim, K.H.; Sharma, N.; An, S.S.A. Multi-Targeting Neuroprotective Effects of Syzygium aromaticum Bud Extracts and Their Key Phytocompounds against Neurodegenerative Diseases. Int. J. Mol. Sci. 2023, 24, 8148. https://doi.org/ 10.3390/ijms24098148 Keywords: Alzheimer’s disease; Syzygium aromaticum; H2O2 induced stress; anti-acetylcholine esterase activity; anti-glycation; MDS; oxidative stress Himadri Sharma †, Dan Yeong Kim † Department of Bionano Technology, Gachon Bionano Research Institute, Gachon University, 1342 Seongnam-daero, Sujeong-gu, Seongnam-si 461-701, Gyeonggi-do, Republic of Korea * Correspondence: nitisharma@gachon.ac.kr (N.S.); seongaan@gachon.ac.kr (S.S.A.A.); Tel.: +82-31-750-8591 (N.S.); +82-31-750-8755 (S.S.A.A.) † Authors made equal contribution to the manuscript. Department of Bionano Technology, Gachon Bionano Research Institute, Gachon University 1342 Seongnam-daero, Sujeong-gu, Seongnam-si 461-701, Gyeonggi-do, Republic of Korea Abstract: Alzheimer’s disease (AD) is a neurodegenerative disease that causes a gradual loss of normal motor and cognitive function. The complex AD pathophysiology involves various factors such as oxidative stress, neuroinﬂammation, amyloid-beta (Aβ) aggregation, disturbed neurotransmission, and apoptosis. The available drugs suffer from a range of side effects and are not able to cover different aspects of the disease. Therefore, ﬁnding a safer therapeutic approach that can affect multiple targets at a time is highly desirable. In the present study, the underlying neuroprotective mechanism of an important culinary spice, Syzygium aromaticum (Clove) extract, and major bioactive compounds were studied in hydrogen peroxide-induced oxidative stress in human neuroblastoma SH-SY5Y cell lines as a model. The extracts were subjected to GC-MS to identify important bioactive components. The extracts and key bio-actives reduced reactive oxygen species (ROS), restored mitochondrial membrane potential (MMP), and provided neuroprotection from H2O2-induced oxidative stress in cell-based assays due to the antioxidant action. They also reduced lipid peroxidation signiﬁcantly and restored GSH content. Clove extracts have also displayed anti-acetylcholinesterase (AChE) activity, anti-glycation potential, and Aβ aggregation/ﬁbrilization inhibition. The multitarget neuroprotective approach displayed by Clove makes it a potential candidate for AD drug development. International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences International Journal of Molecular Sciences Multi-Targeting Neuroprotective Effects of Syzygium aromaticum Bud Extracts and Their Key Phytocompounds against Neurodegenerative Diseases Sharma †, Dan Yeong Kim † , Kyu Hwan Shim , Niti Sharma * and Seong Soo A. An * 1. Introduction Neurodegenerative diseases (NDDs) are a group of diseases affecting various regions of the brain and causing progressive deterioration of intellectual faculties such as learning, cognition, memory, and motor skills. Alzheimer’s disease (AD) is one of the most preva- lent NDDs, affecting millions all over the world. The aggregation of amyloid-beta (Aβ) peptide, tau protein hyperphosphorylation, lower levels of acetylcholine, neuroﬁbrillary tangles, and neuronal damage from oxidative stress are important characteristics of AD pathophysiology [1]. The oxidative stress further generates a vicious cycle by generating more reactive oxygen species (ROS), superoxide radicals (O2•−), peroxynitrite (NO3−), and other reactive nitrogen species (RNS), affecting enzymes, proteins, and ion channels [2,3]. Academic Editor: Anna Kozłowska Received: 5 April 2023 Revised: 20 April 2023 Accepted: 26 April 2023 Published: 2 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). g p g y p The existing therapies for improving AD symptoms work on a speciﬁc target, for example, acetylcholine esterase inhibitors (Donepezil, Galantamine, and Rivastigmine), anti-amyloid antibodies (Aduhelm), and NMDA receptor antagonists (Memantine). Un- fortunately, these drugs exhibit side effects ranging from dizziness to amyloid-related imaging defects [4]. However, as AD is a multifaceted disease, a multitarget neuropro- tective approach is required to ameliorate the symptoms. Therefore, there is an urgent need to identify compounds with better efﬁcacy and minimal side effects for the treatment https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 8148. https://doi.org/10.3390/ijms24098148 Int. J. Mol. Sci. 2023, 24, 8148 2 of 24 of AD. The therapeutic role of plant extracts has been long-established, and numerous phytocompounds have been identiﬁed with multiple neuroprotective properties [4,5]. of AD. The therapeutic role of plant extracts has been long-established, and numerous phytocompounds have been identiﬁed with multiple neuroprotective properties [4,5]. The dried bud of Syzygium aromaticum (Clove; Family Myrtaceae) is a notable culinary spice that has been used for ages in folk medicine for its antiviral [6], anti-inﬂammatory [7], anti-nociceptive [8], anti-oxidant [9], anti-allergic [10], anti-microbial [11], anti-cancer [12], hepatoprotective [13], and neuroprotective [14] properties. Recent studies indicated the importance of exercise and clove oil supplementation in reversing memory deﬁcits, apop- tosis, and maintaining mitochondrial homeostasis in the hippocampus region of the AD brain [15]. 2.1. Phytochemical Estimation and Antioxidant Potential of Clove Extract 2.1. Phytochemical Estimation and Antioxidant Potential of Clove Extract Total phenolics (TPC) and ﬂavonoids (TFC) were estimated in the extracts using colorimetric assays. Phenols and ﬂavonoids are secondary metabolites with an impor- tant role in growth, communication, and plant defense [19,20]. The phenolic content in CL–H (Clove–Hexane) and CL–EA (Clove–Ethyl Acetate) extracts was estimated to be 91.24 ± 0.21 mg GAE/g and 48.75 + 0.10 mg GAE/g, respectively. The ﬂavonoid content in the CL–EA (10.55 + 0.59 mg QE/g) was signiﬁcantly higher than that in the CL–H (1.15 + 0.09 mg QE/g). For the determination of the antioxidant potential of the extracts, DPPH, ABTS, and FRAP assays were conducted. In the DPPH assay, the percent radical scavenging activity observed was 72.3 + 1.19% and 61 + 0.47% for CL–H and CL–EA, respectively. The FRAP assay is an electron transfer (ET)-based method that measures the reduction of Fe3+ to Fe2+ in the presence of antioxidants. CL–H exhibited a better FRAP value (214.95 + 0.007 µM Fe2+/g) compared to CL–EA (68 + 0.003 µM Fe2+/g). The better antioxidant potential of CL–H compared to CL–EA can be correlated with the higher phenolic content of CL–H. A positive correlation between phenolic content and antioxidant potential has already been reported [21]. 1. Introduction It also modulated the seizures and alleviated neuronal death in the CA1 region of the hippocampus [16]. One of the major factors in NDDs is the generation of reactive oxygen species (ROS). Hydrogen peroxide (H2O2)-induced stress in the cultured cells generates ROS and acceler- ates other associated pathogenic reactions such as inﬂammation, mitochondrial dysfunction, tau hyperphosphorylation, the generation of neuroﬁbrillary tangles, and apoptosis [17,18]. Therefore, protection against oxidative stress is an important therapeutic strategy for NDDs. The present study has been designed to study the effect of hexane and ethyl acetate extracts of clove bud and bioactive compounds on H2O2-induced oxidative stress in SH- SY5Y neuronal cells. Additionally, other aspects of neuroprotection were explored by studying acetylcholine esterase (AChE) inhibitory potential, anti-oligomerization and ﬁbrillation activity, and the ability to inhibit the formation of advanced glycation end products (AGEs) to elucidate the mechanism of neuroprotection by clove extract. 2.2. GC–MS Analysis The GC–MS chromatogram of hexane and ethyl acetate extracts of Syzygium aro- maticum identiﬁed the important bioactive compounds in each extract. GC–MS recorded a total of 28 and 14 peaks for CL–EA and CL–H, respectively, based on retention time, peak area (%), height (%), and mass spectral fragmentation compared to those of the known com- pounds in the NIST library (Supplementary Figure S1). The main components identiﬁed in CL–EA were eugenol (33.98%), caryophyllene (16.12%), caryophyllene oxide (9.55%), and 2′,3′,4′-trimethoxy acetophenone (8.92%) (Supplementary Figure S1A). On the other hand, CL–H had eugenol (48.93%), acetyl eugenol (20.13%), and caryophyllene (18.69%) (Supplementary Figure S1B). The proﬁle of bioactive components in CL–EA and CL–H depends on the polarity of the solvent, as the EA fraction is moderately polar while hexane is non-polar. As a result, non-polar compounds become extracted in the hexane fraction ﬁrst as a major percentage of eugenol, acetyl eugenol, and caryophyllene compared to the Int. J. Mol. Sci. 2023, 24, 8148 3 of 24 EA fraction. However, the EA fraction is considered safer and more active compared to the hexane fraction [22]. hexane is non-polar. As a result, non-polar compounds become extracted in the hexane fraction ﬁrst as a major percentage of eugenol, acetyl eugenol, and caryophyllene com- pared to the EA fraction However the EA fraction is considered safer and more active EA fraction. However, the EA fraction is considered safer and more active compared to the hexane fraction [22]. hexane is non-polar. As a result, non-polar compounds become extracted in the hexane fraction ﬁrst as a major percentage of eugenol, acetyl eugenol, and caryophyllene com- pared to the EA fraction However the EA fraction is considered safer and more active Eugenol is a compound with versatile pharmacological properties such as antidiabetic, neuroprotective, anti-cancer, antioxidant, anti-inﬂammatory, and analgesic. It has also been generally recognized as safe (GRAS) by the World Health Organization (WHO) [23]. Both caryophyllene and caryophyllene oxide possess antioxidant, anticancer, and analgesic properties. They are also approved as ﬂavoring agents by the Food and Drug Admin- istration (FDA) and the European Food Safety Authority (EFSA) [24]. Eugenyl acetate possesses anti-AChE and antioxidant activity [25,26]. The neuroprotective potential of other smaller (%) components identiﬁed in the extract, such as Humulene, Copane [27], Anethol [28], Vanillin [29], and Cubebene [30], has already been reported. 2.3. In Vitro Anti-AGE Activity of Clove Extract u e io e i a a e u a i e iga io A BSA–AGE ﬂuorescence assay was used to evaluate the anti-glycation potential of Clove extracts and eugenol, β-caryophyllene, and 2′,3′,4′-trimethoxy acetophenone (TMA) identiﬁed by GC–MS in the extracts (Supplementary Figure S1). For the study, D-glucose was chosen as it is the key glycating sugar compared to other sugars. CL–EA potentially inhibited the glycation with an IC50 value of 83.09 µg/mL, comparable to the positive control, aminoguanidine hydrochloride (IC50 53.27 µg/mL). In a previous report, aminoguanidine displayed an IC50 of 59.2 µg/mL [31], which is in agreement with the value reported by us. CL–H also exhibited good anti-AGE activity (IC50 274.2 µg/mL) (Figure 1). Earlier, the hydroalcoholic, aqueous, and methanolic extracts of Clove inhibited AGE formation (88%, 91.5%, and 72.8%, respectively) in the BSA-glucose model [32–34]. Eugenol exhibited anti-AGE activity (IC50 152 µg/mL), while β-caryophyllene and TMA did not show any signiﬁcant inhibition. Similar results were obtained previously where eugenol exhibited anti-AGE properties (IC50 10 mM) [35], while β-caryophyllene failed to do so [36]. 2.3. In Vitro Anti‐AGE Activity of Clove Extract A BSA–AGE ﬂuorescence assay was used to evaluate the anti-glycation potential of Clove extracts and eugenol, β-caryophyllene, and 2′,3′,4′-trimethoxy acetophenone (TMA) identiﬁed by GC–MS in the extracts (Supplementary Figure S1). For the study, D-glucose was chosen as it is the key glycating sugar compared to other sugars. CL–EA potentially inhibited the glycation with an IC50 value of 83.09 µg/mL, comparable to the positive con- trol, aminoguanidine hydrochloride (IC50 53.27 µg/mL). In a previous report, aminoguan- idine displayed an IC50 of 59.2 µg/mL [31], which is in agreement with the value reported by us. CL–H also exhibited good anti-AGE activity (IC50 274.2 µg/mL) (Figure 1). Earlier, the hydroalcoholic, aqueous, and methanolic extracts of Clove inhibited AGE formation (88%, 91.5%, and 72.8%, respectively) in the BSA-glucose model [32–34]. Eugenol exhib- ited anti-AGE activity (IC50 152 µg/mL), while β-caryophyllene and TMA did not show any signiﬁcant inhibition. Similar results were obtained previously where eugenol exhib- ited anti-AGE properties (IC50 10 mM) [35], while β-caryophyllene failed to do so [36]. Figure 1. The anti-glycation activity exhibited by Clove extracts and eugenol. IC50 values were estimated using the Glucose-BSA model. Data are means of triplicate. Results were calculated using GraphPad Prism 9.5. Abbreviations: AG—Aminoguanidine; CL–H—Clove–Hexane; CL-EA—Clove– Ethyl Acetate. Figure 1. The anti-glycation activity exhibited by Clove extracts and eugenol. IC50 values were estimated using the Glucose-BSA model. Data are means of triplicate. 2.2. GC–MS Analysis Meanwhile, 2′,3′,4′—trimethoxy acetophenone is a heterocyclic thioether derivative of pyrogallic acid, and the literature on its therapeutic action is dearth. Hence, out of curiosity, we selected this compound along with the major phytocompounds, eugenol and β-caryophyllene, for further biochemical and cellular investigations. pared to the EA fraction. However, the EA fraction is considered safer and more active compared to the hexane fraction [22]. Eugenol is a compound with versatile pharmacological properties such as antidia- betic, neuroprotective, anti-cancer, antioxidant, anti-inﬂammatory, and analgesic. It has also been generally recognized as safe (GRAS) by the World Health Organization (WHO) [23]. Both caryophyllene and caryophyllene oxide possess antioxidant, anticancer, and an- algesic properties. They are also approved as ﬂavoring agents by the Food and Drug Ad- ministration (FDA) and the European Food Safety Authority (EFSA) [24]. Eugenyl acetate possesses anti-AChE and antioxidant activity [25,26]. The neuroprotective potential of other smaller (%) components identiﬁed in the extract, such as Humulene, Copane [27], Anethol [28], Vanillin [29], and Cubebene [30], has already been reported. Meanwhile, 2’,3’,4’—trimethoxy acetophenone is a heterocyclic thioether derivative of pyrogallic acid, and the literature on its therapeutic action is dearth. Hence, out of curiosity, we selected this compound along with the major phytocompounds, eugenol and β-caryophyllene, for f th bi h i l d ll l i ti ti 2.3. In Vitro Anti-AGE Activity of Clove Extract u e io e i a a e u a i e iga io Results were calculated using GraphPad Prism 9.5. Abbreviations: AG—Aminoguanidine; CL–H—Clove–Hexane; CL-EA—Clove– Ethyl Acetate. Figure 1. The anti-glycation activity exhibited by Clove extracts and eugenol. IC50 values were estimated using the Glucose-BSA model. Data are means of triplicate. Results were calculated using GraphPad Prism 9.5. Abbreviations: AG—Aminoguanidine; CL–H—Clove–Hexane; CL-EA—Clove– Ethyl Acetate. 2.4. In Vitro Acetylcholinesterase Inhibitory Activity Acetylcholinesterase (AChE; E.C.3.1.1.7) is an acetylcholine (ACh) hydrolyzing en- zyme with an important role in the cholinergic signaling pathway. Inhibition of AChE is desirable in the treatment of AD to maintain ACh levels. Therefore, the extracts were Int. J. Mol. Sci. 2023, 24, 8148 4 of 24 rolyzing n of ACh initially screened for anti-AChE activity at 100 µg/mL, where CL–H and CL–EA displayed 50.97% and 56.64% inhibition, respectively. Galantamine hydrobromide was used as an inhibitor control for the assay, and it exhibited 96.75% inhibition at 10 µg/mL. The IC50 values (half maximal inhibitory concentration) of the extracts, eugenol, β-caryophyllene, and the positive control have been shown in Figure 2. y y µg p 50.97% and 56.64% inhibition, respectively. Galantamine hydrobromide was used a inhibitor control for the assay, and it exhibited 96.75% inhibition at 10 µg/mL. The values (half maximal inhibitory concentration) of the extracts, eugenol, β-caryophyll and the positive control have been shown in Figure 2. initially screened for anti-AChE activity at 100 µg/mL, where CL–H and CL–EA displayed 50.97% and 56.64% inhibition, respectively. Galantamine hydrobromide was used as an inhibitor control for the assay, and it exhibited 96.75% inhibition at 10 µg/mL. The IC50 values (half maximal inhibitory concentration) of the extracts, eugenol, β-caryophyllene, and the positive control have been shown in Figure 2. 50.97% and 56.64% inhibition, respectively. Galantamine hydrobromide was used a inhibitor control for the assay, and it exhibited 96.75% inhibition at 10 µg/mL. The values (half maximal inhibitory concentration) of the extracts, eugenol, β-caryophyll and the positive control have been shown in Figure 2. Fi 2 IC f Cl t t l d β C h ll ith i hibit t l (G Figure 2. IC50 curves of Clove extracts, eugenol, and β-Caryophyllene with inhibitor control (Galan- tamine) against AChE. The IC50 values were calculated using GraphPad Prism 9.5. Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate. Fi 2 IC f Cl l d β C h ll i h i hibi l (G Figure 2. IC50 curves of Clove extracts, eugenol, and β-Caryophyllene with inhibitor control (Galan- tamine) against AChE. The IC50 values were calculated using GraphPad Prism 9.5. Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate. tamine) against AChE. The IC50 values were calculated using GraphPad Prism 9.5. Abbreviat CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate. The IC50 values of extracts and compounds were 345.1 µg/mL (CL-EA), 268.3 µg (CL-H), 5.6 µg/mL (eugenol), and 113 µg/mL (β-caryophyllene). 2.4. In Vitro Acetylcholinesterase Inhibitory Activity However, TMA did show any signiﬁcant anti-AChE activity (28% at 1 mg/mL). The IC50 value of galantam hydrobromide was calculated as 1 02 µg/mL similar to the previously reported valu The IC50 values of extracts and compounds were 345.1 µg/mL (CL-EA), 268.3 µg/mL (CL-H), 5.6 µg/mL (eugenol), and 113 µg/mL (β-caryophyllene). However, TMA did not show any signiﬁcant anti-AChE activity (28% at 1 mg/mL). The IC50 value of galantamine hydrobromide was calculated as 1.02 µg/mL, similar to the previously reported value of 1.45 µg/mL [37]. The IC50 value for eugenol obtained in our experiment was comparable to the earlier reported value of 40.32 µM (i.e., 6.62 µg/mL) [38]. hydrobromide was calculated as 1.02 µg/mL, similar to the previously reported valu 1.45 µg/mL [37]. The IC50 value for eugenol obtained in our experiment was compar to the earlier reported value of 40.32 µM (i.e., 6.62 µg/mL) [38]. The mode of inhibition of CL-H, CL-EA, eugenol, and β-caryophyllene was analy by a Lineweaver-Burk plot. The kinetic parameter values attained by extracts and p compounds have been summarized in Table 1. Competitive inhibition was observed the tested samples (Figure 3), where Vmax remained unaﬀected (~3.9 µmol/min/ while Km increased (17.64, 12.28, 14.19, and 17.87 mM) for CL-H, CL-EA, eugenol, an p µ ( , µg ) [ ] The mode of inhibition of CL-H, CL-EA, eugenol, and β-caryophyllene was analyzed by a Lineweaver-Burk plot. The kinetic parameter values attained by extracts and pure compounds have been summarized in Table 1. Competitive inhibition was observed for the tested samples (Figure 3), where Vmax remained unaffected (~3.9 µmol/min/mg) while Km increased (17.64, 12.28, 14.19, and 17.87 mM) for CL-H, CL-EA, eugenol, and β-caryophyllene, respectively. In a previous study, eugenol inhibited snails’ AChE by a competitive-non-competitive mechanism (mixed inhibition) [39]. caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization Vmax (µmole/min/mg) Km (mM) Type of Inhibition No Inhibitor 3.90 ± 0.09 10.49 ± 0.02 CL-H 3.90 ±0.17 17.64 ± 0.04 Competitive CL-EA 4.24 ± 0.06 12.28 ±0.01 Competitive Eugenol 4.01 ± 0.14 14.19 ± 0.03 Competitive β-Caryophyllene 4.21 ± 0.19 17.87 ± 0.05 Competitive 3.90 ±0.17 17.64 ± 0.04 Competitive 4.24 ± 0.06 12.28 ±0.01 Competitive 4.01 ± 0.14 14.19 ± 0.03 Competitive 4.21 ± 0.19 17.87 ± 0.05 Competitive Table 1. Kinetic parameters. Vmax (µmole/min/mg) Km (mM) Type of Inhibition No Inhibitor 3.90 ± 0.09 10.49 ± 0.02 CL-H 3.90 ±0.17 17.64 ± 0.04 Competitive CL-EA 4.24 ± 0.06 12.28 ±0.01 Competitive Eugenol 4.01 ± 0.14 14.19 ± 0.03 Competitive β-Caryophyllene 4.21 ± 0.19 17.87 ± 0.05 Competitive 3.90 ±0.17 17.64 ± 0.04 Competitive 4.24 ± 0.06 12.28 ±0.01 Competitive 4.01 ± 0.14 14.19 ± 0.03 Competitive 4.21 ± 0.19 17.87 ± 0.05 Competitive Table 1. Kinetic parameters. Vmax (µmole/min/mg) Km (mM) Type of Inhibition No Inhibitor 3.90 ± 0.09 10.49 ± 0.02 CL-H 3.90 ±0.17 17.64 ± 0.04 Competitive CL-EA 4.24 ± 0.06 12.28 ±0.01 Competitive Eugenol 4.01 ± 0.14 14.19 ± 0.03 Competitive β-Caryophyllene 4.21 ± 0.19 17.87 ± 0.05 Competitive CL-H 3.90 ±0.17 17.64 ± 0.04 Competitive CL-EA 4.24 ± 0.06 12.28 ±0.01 Competitive Eugenol 4.01 ± 0.14 14.19 ± 0.03 Competitive β-Caryophyllene 4.21 ± 0.19 17.87 ± 0.05 Competitive Figure 3. Lineweaver-Burk plot of AChE in the presence of (A) CL-EA, (B)) CL-H, (C) eugenol, and (D) β-Caryophyllene. Experiments were performed in triplicate. The graph was plotted using GraphPad Prism 9.5. Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate. Figure 3. Lineweaver-Burk plot of AChE in the presence of (A) CL-EA, (B)) CL-H, (C) eugenol, and (D) β-Caryophyllene. Experiments were performed in triplicate. The graph was plotted using GraphPad Prism 9.5. Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate. Figure 3. Lineweaver-Burk plot of AChE in the presence of (A) CL-EA, (B) CL-H, (C) eugenol, and (D) β-Caryophyllene. Experiments were performed in triplicate. The graph was plotted using GraphPad Prism 9.5. Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate. 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization The MDS was used to evaluate the Aβ oligomerization inhibition eﬀect of the clove extract and the pure compounds. To conﬁrm the oligomerization inhibition potential, the samples were reacted with Aβ1–42 for 0, 2, and 4 h (Figure 4A–C). caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization p p ( ) [ ] Table 1. Kinetic parameters. Vmax (μmole/min/mg) Km (mM) Type of Inhibition The MDS was used to evaluate the Aβ oligomerization inhibition effect of the clove extract and the pure compounds. To conﬁrm the oligomerization inhibition potential, the samples were reacted with Aβ1–42 for 0, 2, and 4 h (Figure 4A–C). MDS results were calcu- lated based on each sample’s 0 hr value; hence, all sample signals were at 1.0 (Figure 4A). At 2 h, oligomerization reduction was signiﬁcant in CL-EA (** p < 0.01) and TMA (* p < 0.05) (Figure 4B). Eugenol also decreased the oligomerization at 2 h, but the p value was non- signiﬁcant. As the time of incubation progressed, CL-H also decreased oligomerization signiﬁcantly (* p <0.05) as did CL-EA; however, more signiﬁcant reduction was shown by TMA (** p <0.01) going from 2 h to 4 h (Figure 4C). Considering the overall Aβ1–42 oligomerization tendency at every time point, the area under the curve (AUC) was calcu- lated. As a result, CL-EA (* p <0.05) and TMA ( p <0.01) showed a signiﬁcant decrease in Aβ1-42 oligomerization tendency with TMA performing better than CL–EA (Figure 4D). The possible explanation could be that as AUC is calculated over a period of time (0–4 h), depending on the kinetics of each compound, the reaction time with Aβ is different. In other words, since each compound may have a different phase of inhibiting Aβ, the effect of inhibiting Aβ appears at different times. The compound can degrade with time, but this Int. J. Mol. Sci. 2023, 24, 8148 5 of 24 degradation cannot be monitored by MDS. Hence, to summarize the results, TMA and CL- EA are effective in inhibiting Aβ oligomerization compared to CL-H and β-caryophyllene, which slightly decreased the oligomerization without a signiﬁcant p-value, and eugenol was like the negative control (Figure 4D). R REVIEW 5 of 26 3.90 ± 0.09 10.49 ± 0.02 Table 1. Kinetic parameters. caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization MDS results were calcu- lated based on each sample’s 0 hr value; hence, all sample signals were at 1.0 (Figure 4A). At 2 h, oligomerization reduction was signiﬁcant in CL-EA ( p < 0.01) and TMA (* p < 0.05) (Figure 4B). Eugenol also decreased the oligomerization at 2 h, but the p value was To access the Aβ ﬁbrilization inhibition activity of the samples, a ThT assay was con- ducted. ThT is a ﬂuorescent dye that interacts with the β-sheet of the ﬁbrils; consequently, it is used to monitor Aβ ﬁbril formation. The ﬂuorescence is increased upon binding of ThT to the amyloid ﬁbrils. The extracts and compounds were screened (100 µg/mL) for Aβ ﬁbrilization inhibition using phenol red as a positive control. ThT ﬂuorescence was reduced signiﬁcantly in the extracts compared to the control (buffer + Aβ). Phenol red displayed 71.34 ± 2.9% inhibition (* p < 0.001) at 50 µM similar to the previously reported value [40]. Both CL-H and CL-EA exhibited potent inhibitory activity (* p < 0.001) with 73.49 ± 5.53% and 86.59 ± 0.38%, respectively. On the other hand, eugenol, β-caryophyllene, and TMA displayed lower inhibition (48.38 ± 0.33%, * p < 0.001; 18.44 ± 3.54%, p < 0.01; 9.13 ± 0.38%, * p < 0.05, respectively) compared to the extracts (Figure 5). To summa- rize the results, TMA, and CL-EA effectively reduced Aβ oligomerization, while CL-EA and CL-H inhibited ﬁbrilization to a greater extent compared to others. Previously, the aqueous extract of clove inhibited amyloid ﬁbril formation by 90% in a concentration- dependent manner [41]. Int. J. Mol. Sci. 2023, 24, 8148 6 of 24 aryo- , and Figure 4. Inhibition eﬀect of Aβ oligomerization by Clove extracts (CL-H and CL-EA), eugenol, β- Caryophyllene, and TMA. The relative amount of Aβ oligomers at 0 (A), 2 (B), and 4 h (C) and the value of Aβ oligomerization area under the curve (D). All data are presented as mean ± SEM (n = 3). A signiﬁcant diﬀerence * (p < 0.05) and (p < 0.01) using one-way ANOVA followed by Dunnett’s post-hoc was observed in the reduction in oligomerization vs. the negative control (no treatment). Figure 4. Inhibition effect of Aβ oligomerization by Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA. caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization T ThT to the amyloid ﬁbrils. The extracts and ﬁbrilization inhibition using phenol red as duced signiﬁcantly in the extracts compare played 71.34 ± 2.9% inhibition (* p < 0.001 value [40]. Both CL-H and CL-EA exhibited Figure 4. Inhibition eﬀect of Aβ oligomerization by Clove extracts (CL-H and CL-EA), eugenol, β- Caryophyllene, and TMA. The relative amount of Aβ oligomers at 0 (A), 2 (B), and 4 h (C) and the value of Aβ oligomerization area under the curve (D). All data are presented as mean ± SEM (n = 3). A signiﬁcant diﬀerence * (p < 0.05) and ** (p < 0.01) using one-way ANOVA followed by Dunnett’s post-hoc was observed in the reduction in oligomerization vs. the negative control (no treatment). Figure 4. Inhibition effect of Aβ oligomerization by Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA. The relative amount of Aβ oligomers at 0 (A), 2 (B), and 4 h (C) and the value of Aβ oligomerization area under the curve (D). All data are presented as mean ± SEM (n = 3). A signiﬁcant difference * (p < 0.05) and (p < 0.01) using one-way ANOVA followed by Dunnett’s post- hoc was observed in the reduction in oligomerization vs. the negative control (no treatment). Abbrevi- ations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. 73.49 ± 5.53% and 86.59 ± 0.38%, respectively. On the other hand, eugenol, β-caryo- phyllene, and TMA displayed lower inhibition (48.38 ± 0.33%, * p < 0.001; 18.44 ± 3.54%, ** p < 0.01; 9.13 ± 0.38%, p < 0.05, respectively) compared to the extracts (Figure 5). To summarize the results, TMA, and CL-EA eﬀectively reduced Aβ oligomerization, while CL-EA and CL-H inhibited ﬁbrilization to a greater extent compared to others. Previously, the aqueous extract of clove inhibited amyloid ﬁbril formation by 90% in a concentration- dependent manner [41]. Figure 5. Fibrilization inhibition in the presence of Clove extracts (CL-H and CL-EA), eugenol, β- Caryophyllene, and TMA. The values are expressed as the mean ± SD (n = 3). Phenol Red (50 µM) was used as a positive control. A signiﬁcant diﬀerence (p < 0.05), (p < 0.01), and (* p < 0.001) using one-way ANOVA followed by Dunnett’s post hoc was observed in the reduction in oligomer- ization vs. the negative control (buﬀer + Aβ). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove- Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone. 2.6. caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization The relative amount of Aβ oligomers at 0 (A), 2 (B), and 4 h (C) and the value of Aβ oligomerization area under the curve (D). All data are presented as mean ± SEM (n = 3). A signiﬁcant difference * (p < 0.05) and (p < 0.01) using one-way ANOVA followed by Dunnett’s post- hoc was observed in the reduction in oligomerization vs. the negative control (no treatment). Abbrevi- ations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. R REVIEW 7 of 26 Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Aceto- phenone. To access the Aβ ﬁbrilization inhibition activity of the samples, a ThT assay was con- ducted. ThT is a ﬂuorescent dye that interacts with the β-sheet of the ﬁbrils; consequently, it is used to monitor Aβ ﬁbril formation. The ﬂuorescence is increased upon binding of ThT to the amyloid ﬁbrils. The extracts and compounds were screened (100 µg/mL) for Aβ ﬁbrilization inhibition using phenol red as a positive control. ThT ﬂuorescence was re- duced signiﬁcantly in the extracts compared to the control (buﬀer + Aβ). Phenol red dis- played 71.34 ± 2.9% inhibition (* p < 0.001) at 50 µM similar to the previously reported value [40]. Both CL-H and CL-EA exhibited potent inhibitory activity (* p < 0.001) with 73.49 ± 5.53% and 86.59 ± 0.38%, respectively. On the other hand, eugenol, β-caryo- phyllene, and TMA displayed lower inhibition (48.38 ± 0.33%, * p < 0.001; 18.44 ± 3.54%, ** p < 0.01; 9.13 ± 0.38%, p < 0.05, respectively) compared to the extracts (Figure 5). To summarize the results, TMA, and CL-EA eﬀectively reduced Aβ oligomerization, while CL-EA and CL-H inhibited ﬁbrilization to a greater extent compared to others. Previously, the aqueous extract of clove inhibited amyloid ﬁbril formation by 90% in a concentration- dependent manner [41]. 7 of 26 Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Aceto- ivity of the samples, a ThT assay was con- ith the β-sheet of the ﬁbrils; consequently, ﬂuorescence is increased upon binding of pounds were screened (100 µg/mL) for Aβ ositive control. ThT ﬂuorescence was re- the control (buﬀer + Aβ). Phenol red dis- 50 µM similar to the previously reported tent inhibitory activity (** p < 0.001) with R REVIEW Abbreviations: CL-H: Clove-Hexane; CL-EA: Cl phenone. To access the Aβ ﬁbrilization inhibition ducted. ThT is a ﬂuorescent dye that interac it is used to monitor Aβ ﬁbril formation. caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization Noncytotoxic Eﬀect of Clove Extracts on the SH‐SY5Y Cell Line Th ll l i bilit ti t d [42] i th bl t ll li (SH SY5Y) Figure 5. Fibrilization inhibition in the presence of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA. The values are expressed as the mean ± SD (n = 3). Phenol Red (50 µM) was used as a positive control. A signiﬁcant difference * (p < 0.05), (p < 0.01), and (* p < 0.001) using one-way ANOVA followed by Dunnett’s post hoc was observed in the reduction in oligomerization vs. the negative control (buffer + Aβ). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. Figure 5. Fibrilization inhibition in the presence of Clove extracts (CL-H and CL-EA), eugenol, β- Caryophyllene, and TMA. The values are expressed as the mean ± SD (n = 3). Phenol Red (50 µM) was used as a positive control. A signiﬁcant diﬀerence * (p < 0.05), (p < 0.01), and (* p < 0.001) using one-way ANOVA followed by Dunnett’s post hoc was observed in the reduction in oligomer- ization vs. the negative control (buﬀer + Aβ). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove- Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone. 2.6. Noncytotoxic Eﬀect of Clove Extracts on the SH‐SY5Y Cell Line Figure 5. Fibrilization inhibition in the presence of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA. The values are expressed as the mean ± SD (n = 3). Phenol Red (50 µM) was used as a positive control. A signiﬁcant difference * (p < 0.05), (p < 0.01), and (* p < 0.001) using one-way ANOVA followed by Dunnett’s post hoc was observed in the reduction in oligomerization vs. the negative control (buffer + Aβ). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. 7 of 24 Int. J. Mol. Sci. 2023, 24, 8148 2.6. Noncytotoxic Effect of Clove Extracts on the SH-SY5Y Cell Line The cellular viability was estimated [42] in the neuroblastoma cell line (SH-SY5Y) after 24 h of treatment with different concentrations of the extracts (1, 10, 25, 50 µg/mL) using WST-8 dye. No cytotoxicity was observed up to 25 µg/mL for CL-H and CL-EA with cell viability over 95%. However, at 50 µg/mL, the cell viability decreased to 88.4% (* p < 0.05) and 86.7% (** p < 0.01) for CL-H and CL-EA, respectively (Figure 6). caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization Therefore, subsequent cell culture experiments were conducted using concentrations below 50 µg/mL to minimize cell death due to the extract’s toxicity. Similar results were obtained in a previous study where no cytotoxicity was observed up to 20 µg/mL when treating SH-SY5Y cells with the clove essential oil [43]. The pure compounds were tested at lower concentrations (0.01, 0.1, 1, 10 µg/mL), where eugenol and TMA displayed signiﬁcant (* p < 0.05) decreases in cell viability (89.9% and 87.2%, respectively) compared to the control at 10 µg/mL. While the cell viability was over 93% for β-caryophyllene. PEER REVIEW 8 of 26 cells with the clove essential oil [43]. The pure compounds were tested at lower concen- trations (0.01, 0.1, 1, 10 µg/mL), where eugenol and TMA displayed signiﬁcant (* p < 0.05) decreases in cell viability (89.9% and 87.2%, respectively) compared to the control at 10 µg/mL. While the cell viability was over 93% for β-caryophyllene. Figure 6. Cytotoxicity assay of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA on the SH-SY5Y cells. The cells were treated for 24 h with varying extract concentrations (1, 10, 25, and 50 µg/mL). The cell viability was reported as the percentage of the control group (100%). All data are presented as mean ± SEM (n = 3). A signiﬁcant diﬀerence * (p < 0.05) and ** (p < 0.01) using one-way ANOVA followed by Dunnett’s post hoc was observed in the % of cell viability vs. the control group (no treatment). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone. Figure 6. Cytotoxicity assay of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA on the SH-SY5Y cells. The cells were treated for 24 h with varying extract concentrations (1, 10, 25, and 50 µg/mL). The cell viability was reported as the percentage of the control group (100%). All data are presented as mean ± SEM (n = 3). A signiﬁcant difference * (p < 0.05) and ** (p < 0.01) using one-way ANOVA followed by Dunnett’s post hoc was observed in the % of cell viability vs. the control group (no treatment). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. Figure 6. Cytotoxicity assay of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA on the SH-SY5Y cells. The cells were treated for 24 h with varying extract concentrations (1, 10, 25, and 50 µg/mL). caryophyllene, respectively. In a previous study, eugen petitive-non-competitive mechanism (mixed inhibition 2.5. Clove Extract Reduced Aβ Oligomerization and Fibrilization The cell viability was reported as the percentage of the control group (100%). All data are presented as mean ± SEM (n = 3). A signiﬁcant diﬀerence * (p < 0.05) and ** (p < 0.01) using one-way ANOVA followed by Dunnett’s post hoc was observed in the % of cell viability vs. the control group (no treatment). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone. Figure 6. Cytotoxicity assay of Clove extracts (CL-H and CL-EA), eugenol, β-Caryophyllene, and TMA on the SH-SY5Y cells. The cells were treated for 24 h with varying extract concentrations (1, 10, 25, and 50 µg/mL). The cell viability was reported as the percentage of the control group (100%). All data are presented as mean ± SEM (n = 3). A signiﬁcant difference * (p < 0.05) and ** (p < 0.01) using one-way ANOVA followed by Dunnett’s post hoc was observed in the % of cell viability vs. the control group (no treatment). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. 2.7. Clove Extracts Protected against H2O2‐Induced Oxidative Stress The neuroprotective eﬀects of the extracts were assessed by H2O 2.7. Clove Extracts Protected against H2O2-Induced Oxidative Stress 2.7. Clove Extracts Protected against H2O2‐Induced Oxidative Stress The neuroprotective eﬀects of the extracts were assessed by H2O 2.7. Clove Extracts Protected against H2O2-Induced Oxidative Stress The neuroprotective eﬀects of the extracts were assessed by H2O2-induced oxidative stress in the cells. In our experiment, H2O2 at 100 µM resulted in 50% cell survival after 6 h of treatment. Hence, this H2O2 concentration was used to induce stress in SH-SY5Y cells pre-treated with the extracts for 12 h. CL-H performed better in protecting the cells against oxidative damage and signiﬁcantly (## p < 0.01) increased the cell viability at 0.1–1 µg/mL and 10-30 µg/mL (# p < 0.05) as compared to H2O2 control. CL-EA signiﬁcantly (# p < 0.05) i d th ll i bilit t 0 1 1 / L d t H O t l ft hi h it l The neuroprotective effects of the extracts were assessed by H2O2-induced oxidative stress in the cells. In our experiment, H2O2 at 100 µM resulted in 50% cell survival after 6 h of treatment. Hence, this H2O2 concentration was used to induce stress in SH-SY5Y cells pre-treated with the extracts for 12 h. CL-H performed better in protecting the cells against oxidative damage and signiﬁcantly (## p < 0.01) increased the cell viability Int. J. Mol. Sci. 2023, 24, 8148 8 of 24 at 0.1–1 µg/mL and 10-30 µg/mL (# p < 0.05) as compared to H2O2 control. CL-EA signiﬁcantly (# p < 0.05) increased the cell viability at 0.1-1 µg/mL as compared to H2O2 control, after which it almost remained constant. In our experiment, pre-treatment with eugenol at lower concentrations (0.01 and 0.1 µg/mL) did not show neuroprotection; hence, higher concentrations (2.5 and 5 µg/mL) were used to study the effect. On the other hand, as β-caryophyllene displayed signiﬁcant (### p < 0.001) neuroprotection at 1 µg/mL, lower concentrations (0.01 and 0.1 µg/mL) were also used. TMA provided better neuroprotection (### p < 0.001) than the others, even at the lower concentrations (0.1 µg/mL) (Figure 7). Hence, the neuroprotective effect shown by Clove extract is due to the major bioactive components. Additionally, the better neuroprotection displayed by CL-H could be due to a higher content of eugenol and eugenyl acetate [44] as compared to CL-EA. The neuroprotective mechanism was further explored by measuring ROS and MMP. ER REVIEW 9 of 26 displayed signiﬁcant (### p < 0.001) neuroprotection at 1 µg/mL, lower concentrations (0.01 and 0.1 µg/mL) were also used. 2.7. Clove Extracts Protected against H2O2‐Induced Oxidative Stress The neuroprotective eﬀects of the extracts were assessed by H2O 2.7. Clove Extracts Protected against H2O2-Induced Oxidative Stress untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone. Figure 7. Neuroprotective effects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, and TMA in H2O2-induced neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate % cell viability vs. the control cells mean ± SEM (n = 3). A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), and /### (p < 0.001), using one-way ANOVA followed by Dunnett’s test, was observed in the % of cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. racts (CL H and CL EA) Eugenol β Caryophyllene xtracts (CL–H and CL–EA), Eugenol, β-Caryophyllen Figure 7. Neuroprotective eﬀects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, and TMA in H2O2-induced neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate % cell viability vs. the control cells mean ± SEM (n = 3). A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), using one-way ANOVA followed by Dunnett’s test, was observed in the % of cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone. Figure 7. Neuroprotective effects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, and TMA in H2O2-induced neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate % cell viability vs. the control cells mean ± SEM (n = 3). A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), using one-way ANOVA followed by Dunnett’s test, was observed in the % of cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. 2.7. Clove Extracts Protected against H2O2‐Induced Oxidative Stress The neuroprotective eﬀects of the extracts were assessed by H2O 2.7. Clove Extracts Protected against H2O2-Induced Oxidative Stress TMA provided better neuroprotection (### p < 0.001) than the others, even at the lower concentrations (0.1 µg/mL) (Figure 7). Hence, the neuropro- tective eﬀect shown by Clove extract is due to the major bioactive components. Addition- ally, the better neuroprotection displayed by CL-H could be due to a higher content of eugenol and eugenyl acetate [44] as compared to CL-EA. The neuroprotective mechanism was further explored by measuring ROS and MMP. Figure 7. Neuroprotective eﬀects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, and TMA in H2O2-induced neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate % cell viability vs. the control cells mean ± SEM (n = 3). A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), using one-way ANOVA followed by Dunnett’s test, was observed in the % of cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone. Figure 7. Neuroprotective effects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, and TMA in H2O2-induced neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate % cell viability vs. the control cells mean ± SEM (n = 3). A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), using one-way ANOVA followed by Dunnett’s test, was observed in the % of cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone. Figure 7 Neuroprotective eﬀects of Clove extracts (CL H and CL EA) Eugenol β Caryophyllene Figure 7. Neuroprotective effects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, Figure 7. Neuroprotective eﬀects of Clove extracts (CL–H and CL–EA), Eugenol, β-Caryophyllene, and TMA in H2O2-induced neuroblastoma SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate % cell viability vs. the control cells mean ± SEM (n = 3). A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), using one-way ANOVA followed by Dunnett’s test, was observed in the % of cell viability vs. 2.8. Clove Extracts Prevented H2O2‐Induced ROS Generation 2.8. Clove Extracts Prevented H2O2-Induced ROS Generation 2.8. Clove Extracts Prevented H2O2‐Induced ROS Generation 2.8. Clove Extracts Prevented H2O2-Induced ROS Generation Oxidative stress is one of the important factors in the pathogenesis of AD. To access the intracellular reactive oxygen species (ROS) scavenging activity of the extracts, the SH- SY5Y cells were pre-treated with the extracts for 12 h, followed by 4 h exposure to H2O2 (100 µM). The ROS production in the cells was monitored by a ﬂuorescent dye (H2DCFDA), which is oxidized to DCF by ROS. The SH-SY5Y cells treated with H2O2 gen- erated 154.8 ± 2.5% ROS compared to the untreated cells (100%). Pre-treatment of the cells with extracts resulted in a dose-dependent decrease in ROS production. The CL-H was more eﬀective and signiﬁcant (131.7 ± 0.6% at 1 µg/mL, p < 0.01; 122.5 ± 6.5% at 10 µg/mL; Oxidative stress is one of the important factors in the pathogenesis of AD. To access the intracellular reactive oxygen species (ROS) scavenging activity of the extracts, the SH-SY5Y cells were pre-treated with the extracts for 12 h, followed by 4 h exposure to H2O2 (100 µM). The ROS production in the cells was monitored by a ﬂuorescent dye (H2DCFDA), which is oxidized to DCF by ROS. The SH-SY5Y cells treated with H2O2 generated 154.8 ± 2.5% ROS compared to the untreated cells (100%). Pre-treatment of the cells with extracts resulted in a dose-dependent decrease in ROS production. The CL-H was Int. J. Mol. Sci. 2023, 24, 8148 Int J Mol Sci 2023 24 x 9 of 24 more effective and signiﬁcant (131.7 ± 0.6% at 1 µg/mL, p < 0.01; 122.5 ± 6.5% at 10 µg/mL; and 115.7 ± 2.5% at 25 µg/mL) in reducing ROS compared to CL-EA (150.7 ± 7.4% at 1 µg/mL; 134.5 ± 0.7% at 10 µg/mL; and 140.8 ± 2.5% at 25 µg/mL) Figure 8. Similarly, both eugenol and β-caryophyllene attenuated increased ROS levels signiﬁcantly (## p < 0.01) at 0.01 µg/mL and 0.03–0.3 µg/mL (### p < 0.001), whereas TMA showed a signiﬁcant dose-dependent decrease in ROS at 0.1–5 µg/mL (### p < 0.001) (Figure 8). A possible explanation for better ROS scavenging activity by CL-H could be due to the higher content of eugenol, acetyl eugenol, and β-caryophyllene. 2.8. Clove Extracts Prevented H2O2‐Induced ROS Generation 2.8. Clove Extracts Prevented H2O2-Induced ROS Generation and 115.7 ± 2.5% at 25 µg/mL) in reducing ROS compared to CL-EA (150.7 ± 7.4% at 1 µg/mL; 134.5 ± 0.7% at 10 µg/mL; and 140.8 ± 2.5% at 25 µg/mL) Figure 8. Similarly, both eugenol and β-caryophyllene attenuated increased ROS levels signiﬁcantly (## p < 0.01) at 0.01 µg/mL and 0.03–0.3 µg/mL (### p < 0.001), whereas TMA showed a signiﬁcant dose- dependent decrease in ROS at 0.1–5 µg/mL (### p < 0.001) (Figure 8). A possible explanation for better ROS scavenging activity by CL-H could be due to the higher content of eugenol, acetyl eugenol, and β-caryophyllene. Figure 8. Eﬀect of Clove extracts, eugenol, β-Caryophyllene, and TMA on H2O2-induced ROS pro- duction in SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate the % of the ROS level vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA fol- lowed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), /### (p < 0.001), and **/#### (p < 0.0001) was observed in the % ROS vs. untreated cells () and H2O2 treated cells (#). Ab- breviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophe- none; ROS: Reactive oxygen species. ROS—reactive oxygen species; TMA—2’,3’,4’-Trimethoxy Acetophenone. Figure 8. Effect of Clove extracts, eugenol, β-Caryophyllene, and TMA on H2O2-induced ROS production in SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h followed by 6 h of H2O2 (100 µM) treatment. The results indicate the % of the ROS level vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one- way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01) /### (p < 0.001), and */#### (p < 0.0001) was observed in the % ROS vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA 2′,3′,4′-Trimethoxy Acetophenone; ROS: Reactive oxygen species. Fi 8 Eﬀ t f Cl t t l β C h ll d TMA H O i d d ROS Figure 8. Effect of Clove extracts, eugenol, β-Caryophyllene, and TMA on H2O2-induced ROS Figure 8. Eﬀect of Clove extracts, eugenol, β-Caryophyllene, and TMA on H2O2-induced ROS pro- duction in SH-SY5Y cells. 2.8. Clove Extracts Prevented H2O2‐Induced ROS Generation 2.8. Clove Extracts Prevented H2O2-Induced ROS Generation The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate the % of the ROS level vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA fol- lowed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), /### (p < 0.001), and */#### (p < 0.0001) was observed in the % ROS vs. untreated cells () and H2O2 treated cells (#). Ab- breviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophe- none; ROS: Reactive oxygen species. ROS i i TMA 2’ 3’ 4’ T i h A h Figure 8. Effect of Clove extracts, eugenol, β-Caryophyllene, and TMA on H2O2-induced ROS production in SH-SY5Y cells. The SH-SY5Y cells were preincubated with the extracts for 12 h, followed by 6 h of H2O2 (100 µM) treatment. The results indicate the % of the ROS level vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one- way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), /### (p < 0.001), and */#### (p < 0.0001) was observed in the % ROS vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; ROS: Reactive oxygen species. ROS reactive oxygen species; TMA 2 ,3 ,4 Trimethoxy Acetophenone. f h d l b l b l E 2.9. Restoration of Mitochondrial Membrane Potential by Clove Extract 2.9. Restoration of Mitochondrial Membrane Potential by Clove Extract Mitochondrial membrane potential (∆Ψm) is reduced due to oxidative damage to the cell. In the present study, H2O2 was used to induce oxidative stress in SH-SY5Y cells. The H2O2 concentration and time of induction were optimized using a ﬂuorescent dye (TMRE), which has an aﬃnity for active mitochondria. Depolarized mitochondria have decreased Mitochondrial membrane potential (∆Ψm) is reduced due to oxidative damage to the cell. In the present study, H2O2 was used to induce oxidative stress in SH-SY5Y cells. The H2O2 concentration and time of induction were optimized using a ﬂuorescent dye (TMRE), which has an afﬁnity for active mitochondria. Depolarized mitochondria have decreased membrane potential and cannot hold the dye. In our experiment, at a 200 µM H2O2 concentration, the MMP reduced to 50% of the untreated cells; hence, this condition was used for further examination. Pre-treatment of cells with CL-EA for 12 h followed by H2O2 (200 µM) treatment for 2 h displayed a dose-dependent increase in MMP with full recovery at 10 and 25 µg/mL (### p < 0.001). Pre-treatment with CL-H also restored MMP to 10 µg/mL (p < 0.01), but it decreased afterward (## p < 0.01) (Figure 9) which might be due to toxicity. Pre-treating the cells with eugenol, β-caryophyllene, and TMA Int. J. Mol. Sci. 2023, 24, 8148 10 of 24 mL city. 10 of 24 mL ity. also resulted in a dose-dependent restoration of MMP. Eugenol at 0.1 µg/mL displayed a signiﬁcant effect (# p < 0.05), and at 1 and 10 µg/mL, it completely reversed the effect of H2O2 (### p <0.001) and established MMP as in the untreated cells. β-caryophyllene and TMA shared the same trend and displayed highly signiﬁcant recovery (### p <0.001) at 1 and 10 µg/mL for β-caryophyllene, and 0.1–10 µg/mL for TMA (Figure 9). p g µg p y g ( p 0.05), and at 1 and 10 µg/mL, it completely reversed the eﬀect of H2O2 (### p <0.001) and established MMP as in the untreated cells. β-caryophyllene and TMA shared the same trend and displayed highly signiﬁcant recovery (### p <0.001) at 1 and 10 µg/mL for β- caryophyllene, and 0.1–10 µg/mL for TMA (Figure 9). Figure 9. Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 µM H2O2 for 2 h after 12 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. ROS reactive oxygen species; TMA 2 ,3 ,4 Trimethoxy Acetophenone. f h d l b l b l E 2.9. Restoration of Mitochondrial Membrane Potential by Clove Extract Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; ∆Ψm: Mitochondrial membrane potential. 2.10. Pre‐Treatment of Clove Extract and Bioactive Compounds Restored Oxidative Stress Markers altered by H2O2‐Induced Oxidative Stress 2.10. Pre-Treatment of Clove Extract and Bioactive Compounds Restored Oxidative Stress Markers altered by H2O2-Induced Oxidative Stress Next, we studied the effect of clove extract, eugenol, β-caryophyllene, and TMA on the H2O2-induced oxidative stress-affected parameters (GSH, GSSG, and MDA) in the SH-SY5Y cells. ROS reactive oxygen species; TMA 2 ,3 ,4 Trimethoxy Acetophenone. f h d l b l b l E 2.9. Restoration of Mitochondrial Membrane Potential by Clove Extract The results indicate % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), was observed in the % cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone.; ∆Ψm: Mitochondrial membrane potential. 2.10. Pre‐Treatment of Clove Extract and Bioactive Compounds Restored Oxidative Stress Figure 9. Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 µM H2O2 for 2 h after 12 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), */### (p < 0.001), and #### (p < 0.0001) was observed in the % cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; ∆Ψm: Mitochondrial membrane potential. 2.10. Pre-Treatment of Clove Extract and Bioactive Compounds Restored Oxidative Stress Markers Figure 9. Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 µM H2O2 for 2 h after 12 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), was observed in the % cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone.; ∆Ψm: Mitochondrial membrane potential. Figure 9. Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 µM H2O2 for 2 h after 12 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), */### (p < 0.001), and #### (p < 0.0001) was observed in the % cell viability vs. ROS reactive oxygen species; TMA 2 ,3 ,4 Trimethoxy Acetophenone. f h d l b l b l E 2.9. Restoration of Mitochondrial Membrane Potential by Clove Extract untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; ∆Ψm: Mitochondrial membrane potential. Figure 9. Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 µM H2O2 for 2 h after 12 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), d */### ( < 0 001) b d i th % ll i bilit t t d ll () d H O t t d ll Figure 9. Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 µM H2O2 for 2 h after 12 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), */### ( 0 001) d #### ( 0 0001) b d i th % ll i bilit t t d ll () Figure 9 Mitocho d ial e b a e pote tial i SH SY5Y cells exposed to 200 M H O fo 2 h afte Figure 9. Mitochondrial membrane potential in SH-SY5Y cells exposed to 200 µM H2O2 for 2 h after p , g , β y p y , % ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001), was observed in the % cell viability vs. untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone.; ∆Ψm: Mitochondrial membrane potential. ∆Ψm vs. the control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), */### (p < 0.001), and #### (p < 0.0001) was observed in the % cell viability vs. untreated cells () and H2O2 treated cells (#). 2.10.1. Restoration of Glutathione Levels Reduced glutathione content in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after Figure 10. Reduced glutathione content in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀer- ence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone; GSH: reduced glutathione. Figure 10. Reduced glutathione content in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove- Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; GSH: reduced glutathione. 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀer- ence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone; GSH: reduced glutathione. 24 h of pre treatment with Clove extracts, eugenol, β Caryophyllene, and TMA. The results indicate GSH (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove- Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; GSH: reduced glutathione. 2.10.1. Restoration of Glutathione Levels As shown in Figure 10, pre-treatment of SH-SY5Y cells with varying concentrations of clove extract (1, 10, and 30 µg/mL) for 24 h before 6 h incubation with H2O2 (100 µM) resulted in a dose-dependent increase in GSH level, where CL-EA displayed a signiﬁcant increase (# p < 0.05) at 10 µg/mL and a complete recovery at 30 µg/mL (# p < 0.001). On the other hand, CL-H also increased GSH levels to 30 µg/mL, though the effect was non-signiﬁcant. Additionally, among the three pure compounds, only β-caryophyllene (0.1, Int. J. Mol. Sci. 2023, 24, 8148 11 of 24 the sig 11 of 24 the sig 1, and 10 µg/mL) showed a slight dose-dependent increase in GSH levels, but that too was non-signiﬁcant. 10 µg/mL) showed a slight dose-dependent increase in GSH levels, but that too was non- signiﬁcant. 1, and 10 µg/mL) showed a slight dose-dependent increase in GSH levels, but that too was non-signiﬁcant. 10 µg/mL) showed a slight dose-dependent increase in GSH levels, but that too was non- signiﬁcant. Figure 10. Reduced glutathione content in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀer- ence /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone; GSH: reduced glutathione. Figure 10. Reduced glutathione content in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove- Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; GSH: reduced glutathione. Figure 10 Reduced glutathione content in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after Figure 10. 2.10.1. Restoration of Glutathione Levels 2023, 24, x FOR PEER REVIEW 14 of 26 indicate GSSG (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence of /# (p < 0.05), and /## (p < 0.01) was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone.; GSSG: oxidized glutathione. The GSH/GSSG ratio is an indicator of a cell’s health. In our study, only CL-EA ex- erted a dose-dependent increase in the GSH/GSSG ratio towards the normal state signiﬁ- indicate GSSG (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference of /# (p < 0.05), and /## (p < 0.01) was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; GSSG: oxidized glutathione. Mol. Sci. 2023, 24, x FOR PEER REVIEW 14 of 26 cantly at 30 µg/mL ( p < 0.05). CL H displayed a signiﬁcant eﬀect at a lower concentration of 1 µg/mL (# p < 0.05), whereas eugenol and TMA had a non-signiﬁcant moderate eﬀect and no eﬀect of β-Caryophyllene (Figure 12). Figure 12. GSH/GSSG ratio in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre- treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH/GSSG ratio in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence of /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’- Trimethoxy Acetophenone; GSH: reduced glutathione; GSSG: oxidized glutathione. 2.10.2. Attenuation of MDA Levels by Clove Extract MDA arises from the peroxidation of polyunsaturated fatty acids present in the mem- brane and forms toxic adducts with proteins and nucleic acids. MDA levels increase with Figure 12. GSH/GSSG ratio in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. 2.10.1. Restoration of Glutathione Levels The oxidized glutathione levels were also measured (Figure 11). The H2O2-induced stress elevated intracellular GSSG levels. In our study, CL-H signiﬁcantly (# p < 0.05) re- duced GSSH levels at 1 µg/mL; however, the decrease was non-signiﬁcant at higher con- centrations (10 and 30 µg/mL). Meanwhile, CL-EA displayed a dose-dependently signiﬁ- cant (# p < 0.05) decrease in GSSG at 30 µg/mL. Eugenol also helped in decreasing the The oxidized glutathione levels were also measured (Figure 11). The H2O2-induced stress elevated intracellular GSSG levels. In our study, CL-H signiﬁcantly (# p < 0.05) reduced GSSH levels at 1 µg/mL; however, the decrease was non-signiﬁcant at higher concentrations (10 and 30 µg/mL). Meanwhile, CL-EA displayed a dose-dependently signiﬁcant (# p < 0.05) decrease in GSSG at 30 µg/mL. Eugenol also helped in decreasing the elevated GSSG and displayed a signiﬁcant (# p < 0.05) effect at 0.1 µg/mL, after which the decrease was non-signiﬁcant. β-Caryophyllene modestly decreased GSSG levels, while on the contrary, TMA restored GSSG levels at 0.1 and 1 µg/mL (# p < 0.05). The increased GSSG at 10 µg/mL might be due to toxicity at this concentration. The GSH/GSSG ratio is an indicator of a cell’s health. In our study, only CL-EA exerted a dose-dependent increase in the GSH/GSSG ratio towards the normal state signiﬁcantly at 30 µg/mL (# p < 0.05). CL-H displayed a signiﬁcant effect at a lower concentration of 1 µg/mL (# p < 0.05), whereas eugenol and TMA had a non-signiﬁcant moderate effect and no effect of β-Caryophyllene (Figure 12). Int. J. Mol. Sci. 2023, 24, 8148 12 of 24 Figure 11. Oxidized glutathione content in the SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSSG (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence of /# (p < 0.05), and /## (p < 0.01) was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone.; GSSG: oxidized glutathione. The GSH/GSSG ratio is an indicator of a cell’s health. 2.10.1. Restoration of Glutathione Levels In our study, only CL-EA ex- erted a dose-dependent increase in the GSH/GSSG ratio towards the normal state signiﬁ- cantly at 30 µg/mL (# p < 0 05) CL H displayed a signiﬁcant eﬀect at a lower concentration Figure 11. Oxidized glutathione content in the SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSSG (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference of /# (p < 0.05), and /## (p < 0.01) was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; GSSG: oxidized glutathione. . Mol. Sci. 2023, 24, x FOR PEER REVIEW 14 of 26 Figure 11. Oxidized glutathione content in the SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSSG (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence of /# (p < 0.05), and /## (p < 0.01) was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone.; GSSG: oxidized glutathione. The GSH/GSSG ratio is an indicator of a cell’s health. In our study, only CL-EA ex- erted a dose-dependent increase in the GSH/GSSG ratio towards the normal state signiﬁ- Figure 11. Oxidized glutathione content in the SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSSG (µM/µg protein) levels in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference of /# (p < 0.05), and /## (p < 0.01) was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone.; GSSG: oxidized glutathione. Mol. Sci. 2.10.2. Attenuation of MDA Levels by Clove Extract Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone; MDA: Malondialdehyde. Figure 13. Malondialdehyde (MDA) content in SH-SY5Y cells lysate exposed to 100 µM H2O2 for 6 h after 24 h pre-treatment with Clove extracts, Eugenol, β-Caryophyllene, and TMA. The results indicate MDA (µM) in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by One-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05 was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; MDA: Malondialdehyde. Figure 13. Malondialdehyde (MDA) content in SH-SY5Y cells lysate exposed to 100 µM H2O2 for 6 h after 24 h pre-treatment with Clove extracts, Eugenol, β-Caryophyllene, and TMA. The results indicate MDA (µM) in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by One-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05 was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone; MDA: Malondialdehyde. Figure 13. Malondialdehyde (MDA) content in SH SY5Y cells lysate exposed to 100 µM H2O2 for 6 h after 24 h pre-treatment with Clove extracts, Eugenol, β-Caryophyllene, and TMA. The results indicate MDA (µM) in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by One-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05 was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; MDA: Malondialdehyde. 2.10.1. Restoration of Glutathione Levels The results indicate GSH/GSSG ratio in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference of /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; GSH: reduced glutathione; GSSG: oxidized glutathione. y µg/ ( p ) p y g of 1 µg/mL (# p < 0.05), whereas eugenol and TMA had a non-signiﬁ and no eﬀect of β-Caryophyllene (Figure 12). Figure 12. GSH/GSSG ratio in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre- treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH/GSSG ratio in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence of /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’- Trimethoxy Acetophenone; GSH: reduced glutathione; GSSG: oxidized glutathione. 2.10.2. Attenuation of MDA Levels by Clove Extract MDA arises from the peroxidation of polyunsaturated fatty acids present in the mem- b d f i dd i h i d l i id MDA l l i i h Figure 12. GSH/GSSG ratio in SH-SY5Y cell lysate exposed to 100 µM H2O2 for 6 h after 24 h of pre-treatment with Clove extracts, eugenol, β-Caryophyllene, and TMA. The results indicate GSH/GSSG ratio in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by one-way ANOVA followed by Dunnett’s test. A signiﬁcant difference of /# (p < 0.05), /## (p < 0.01), and */### (p < 0.001) was observed in comparison to untreated cells () and H2O2 treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; GSH: reduced glutathione; GSSG: oxidized glutathione. Int. J. Mol. Sci. 2023, 24, 8148 13 of 24 13 of 24 2.10.2. Attenuation of MDA Levels by Clove Extract 2.10.2. Attenuation of MDA Levels by Clove Extract MDA arises from the peroxidation of polyunsaturated fatty acids present in the membrane and forms toxic adducts with proteins and nucleic acids. MDA levels increase with stress; hence, they are used as markers of oxidative damage. In the present study, clove extracts decreased MDA content in a dose-dependent manner, though the results were non-signiﬁcant (Figure 13). The decrease in MDA levels was signiﬁcant (# p < 0.05) in eugenol (1 and 10 µg/mL) and TMA (10 µg/mL). However, β-caryophyllene exhibited the most potent response by reducing levels of MDA signiﬁcantly (# p < 0.05) at all the concentrations tested. ER REVIEW 15 of 26 Figure 13. Malondialdehyde (MDA) content in SH-SY5Y cells lysate exposed to 100 µM H2O2 for 6 h after 24 h pre-treatment with Clove extracts, Eugenol, β-Caryophyllene, and TMA. The results indicate MDA (µM) in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by One-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05 was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2’,3’,4’-Trimethoxy Acetophenone; MDA: Malondialdehyde. Figure 13. Malondialdehyde (MDA) content in SH-SY5Y cells lysate exposed to 100 µM H2O2 for 6 h after 24 h pre-treatment with Clove extracts, Eugenol, β-Caryophyllene, and TMA. The results indicate MDA (µM) in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by One-way ANOVA followed by Dunnett’s test. A signiﬁcant difference /# (p < 0.05 was observed in comparison to untreated cells () and H2O2-treated cells (#). Abbreviations: CL-H: Clove-Hexane; CL-EA: Clove-Ethyl Acetate; TMA: 2′,3′,4′-Trimethoxy Acetophenone; MDA: Malondialdehyde. Figure 13 Malondialdehyde (MDA) content in SH SY5Y cells lysate exposed to 100 µM H2O2 for 6 Figure 13. Malondialdehyde (MDA) content in SH-SY5Y cells lysate exposed to 100 µM H2O2 Figure 13. Malondialdehyde (MDA) content in SH-SY5Y cells lysate exposed to 100 µM H2O2 for 6 h after 24 h pre-treatment with Clove extracts, Eugenol, β-Caryophyllene, and TMA. The results indicate MDA (µM) in treated and control cells (untreated cells). Values are mean ± SEM (n = 3). The data were analyzed by One-way ANOVA followed by Dunnett’s test. A signiﬁcant diﬀerence /# (p < 0.05 was observed in comparison to untreated cells () and H2O2-treated cells (#). 3. Discussion 3. Discussion In addition to being an important spice, Syzygium aromaticum has also been used in traditional medicine to cure several diseases. In the present study, the underlying neuro- protective mechanism of S. aromaticum (Clove) extract and its major bioactive compounds were studied under hydrogen peroxide-induced oxidative stress in human neuroblastoma SH-SY5Y cell lines as a model. I the e e t tudy hyto he i al a aly i fo TPC a d TFC alo ith a tio i In addition to being an important spice, Syzygium aromaticum has also been used in traditional medicine to cure several diseases. In the present study, the underlying neuro- protective mechanism of S. aromaticum (Clove) extract and its major bioactive compounds were studied under hydrogen peroxide-induced oxidative stress in human neuroblastoma SH-SY5Y cell lines as a model. In the present study, phytochemical analysis for TPC and TFC, along with antioxi- dant potential, was carried out. Phenols and ﬂavonoids are secondary metabolites with an important role in growth, communication, and plant defense [19,20]. As per the GC- MS, CL-H has a higher content of eugenol and eugenol acetate, which possess strong an- tioxidant activities by hydrogen/electron transfer or directly trapping the free radicals [45]. β-Caryophyllene, a sesquiterpene detected in GC-MS of Clove extract, is also a mod-t In the present study, phytochemical analysis for TPC and TFC, along with antioxidant potential, was carried out. Phenols and ﬂavonoids are secondary metabolites with an important role in growth, communication, and plant defense [19,20]. As per the GC-MS, CL-H has a higher content of eugenol and eugenol acetate, which possess strong antioxidant activities by hydrogen/electron transfer or directly trapping the free radicals [45]. β- Int. J. Mol. Sci. 2023, 24, 8148 14 of 24 14 of 24 Caryophyllene, a sesquiterpene detected in GC-MS of Clove extract, is also a moderate antioxidant [46,47]. Likewise, the better antioxidant potential of CL-H compared to CL-EA observed in the antioxidant assays can be correlated to the higher phenolic content in CL-H. A positive correlation between phenolic content and antioxidant potential has already been reported [21]. Caryophyllene, a sesquiterpene detected in GC-MS of Clove extract, is also a moderate antioxidant [46,47]. Likewise, the better antioxidant potential of CL-H compared to CL-EA observed in the antioxidant assays can be correlated to the higher phenolic content in CL-H. A positive correlation between phenolic content and antioxidant potential has already been reported [21]. 3. Discussion 3. Discussion The involvement of AGEs in the pathology of NDDs is evident [48]. The AGEs are gen- erated from the irreversible non-enzymatic glycation (Maillard reaction) between proteins and reducing sugars [49], which leads to the formation of cross-linked β structures that eventually form amyloid ﬁbrils, causing protein dysfunction/enzyme deactivation. The binding of AGEs to RAGE (receptors of AGEs) recruits cellular signaling, triggering nuclear factors and leading to the increased expression of inﬂammatory cytokines and oxidative stress. As observed in our study, Eugenol exhibited anti-AGE activity (IC50 152 µg/mL), while β-caryophyllene and TMA failed to display signiﬁcant inhibition. Based on the structure-activity relationship (SAR) study, the presence of a hydroxyl/nitro group and halogen atoms in the phenol ring enhances antiglycation activity; on the other hand, alkyl, triﬂuoromethyl, and bulky groups reduce the activity. Likewise, the activity is lost by phenyl group replacement by hetaryl or nonaromatic groups or if a blend of various scaf- foldings is present in a single molecule [50]. Additionally, methoxy groups decrease the inhibition [51], as observed for TMA. Eugenol is an allyl-substituted guaiacol-derived phenylpropanoid. It has a phenolic hydroxyl group, a methoxy group, and an allylic double bond. Whereas β-caryophyllene is a bicyclic sesquiterpene having a cyclobutene ring and a trans-double bond in a 9-membered ring. Hence, due to structural complexity and the absence of a hydroxy group in β-caryophyllene, it is unable to display signiﬁcant anti-glycation activity. Furthermore, the Clove reduced protein carbonyl content and thiol oxidation by scavenging hydroxy and superoxide radicals generated during oxidative reactions (auto-oxidation of sugar, degradation of Amadori products) in the AGE forma- tion [34]. The higher anti-glycation potential of CL-EA compared to a single metabolite, eugenol, suggests the synergistic effect of other compounds present in the extract for anti- glycation action [35]. Since eugenol and β-caryophyllene are common in both extracts, CL-EA showed better inhibition than CL-H, indicating the synergistic action of the phy- tocompounds exclusively present in CL-EA in boosting anti-AGE activity. Additionally, higher ﬂavonoid content in CL-EA can also result in higher glycation inhibition, as a strong correlation between the two has already been reported [52]. Consequently, CL-EA can be considered a new potential antiglycation agent of natural origin with reduced toxicity for the development of anti-AGE drugs in the management of NDDs. We have also investigated the anti-AChE potential and mode of inhibition by Clove extracts and their components in an in vitro system. 3. Discussion 3. Discussion The hydroxyl and methoxy groups in the molecule are involved in multiple non-covalent bindings with globular proteins [58]. Additionally, the 4′-hydroxyl group of eugenol speciﬁcally interacts with the lysine residue of the protein [33], which might prevent/delay ﬁbrillization. Eugenol can easily cross the BBB and is c apable of reducing the hippocampal Aβ plaques in animal models [59,60]. Therefore, it is speculated that aromatic compounds in CL-EA and CL-H interfere with β sheet structure of the protein through π-stacking or hydrophobic interaction, impairing Aβ ﬁbrillization. The lower inhibitory activity displayed by eugenol and β-caryophyllene compared to the extract in our study suggests the synergistic or additive effects of phytocompounds present in clove extracts for the anti-amyloidogenic property. y g p p y Oxidative stress is one of the important factors in the pathogenesis of AD. Overpro- duction of ROS induces oxidative stress, which further hampers normal cellular activity by damaging proteins/enzymes and other cellular components, including mitochondria. Therefore, it is important to identify compounds that can reduce intracellular ROS. The dose-dependent reduction in ROS observed in our study indicates the antioxidant nature of the extracts and compounds tested. Both eugenol and β-caryophyllene are the main components of clove extract, which are well-known for their antioxidant action at low concentrations by preventing lipid peroxidation and preserving the levels of antioxidative enzymes [58–63] via maintaining oxidative balance through the sirtuin-1 (SIRT1) path- way [62]. Antioxidant activity is increased by the presence of the methoxy group and the number of hydroxyl groups in the phenol ring [59]. Eugenol scavenges free radicals by donating the phenolic hydroxyl group [23]. Much literature is unavailable on TMA, but Paeonol (2′-Hydroxy-4′-methoxyacetophenone), a structurally similar compound to TMA, has shown neuroprotection in vivo and in vitro by reducing oxidative stress [64,65]. A previous study suggested that eugenol, being lipophilic, inﬂuences the redistribution of protons across the membrane, thereby affecting membrane potential [66]. Aspirin eugenol ester exerted a shielding effect on oxidative stress generated on H2O2-induced PC12 cells by scavenging free radicals, reducing intracellular ROS, stabilizing mitochondrial membranes, and increasing the ∆Ψm by regulating the PI3K/Akt signal pathway [67,68]. β-caryophyllene has also been reported to inhibit ROS production and recovery of MMP via Cannabinoid type 2 (CB2) receptor-dependent nuclear factor erythroid 2–related factor 2 (Nrf2) activation [69,70]. 3. Discussion 3. Discussion The extracts and components displayed potential inhibition against AChE (electric eel) with a competitive mode of inhibition. Better IC50 values of pure compounds compared to the extract can be explained based on the presence of various metabolites in the extract, which might have diluted the effect. The electric eel AChE has Trp286, Phe297, Tyr337, Tyr341, and His447 at the active site [53], and β-caryophyllene has been shown to interact with Phe297 and Trp286 [54] at the active site. The anti-AChE activity of eugenol derivatives was found to be dependent on substitution at the hydroxyl group, as the p-substituted ethyl group had better activity than the methyl derivative [55]. These results suggest that both β-caryophyllene and eugenol bind to the active site of the AChE enzyme as competitive inhibitors. As indicated by the kinetic parameters, Vmax (the maximal reaction velocity when the enzyme is saturated with its substrate) remains unchanged, but Km (the concentration of the substrate that permits the enzyme to achieve half Vmax) increases from normal, indicating a competitive inhibition. Km is also an indicator of the binding afﬁnity for the enzyme (the lower the Km, the higher the afﬁnity). As the competitive inhibitors can bind to E and not to the ES complex (hence cannot change the catalysis in ES), the Vmax is unaffected. The apparent Km for the substrate is increased in the presence of competitive inhibitors, as a higher concentration of substrate will be required to overcome the inhibitory effect. In the present study, CL-EA Int. J. Mol. Sci. 2023, 24, 8148 15 of 24 15 of 24 exhibited the lowest Km value, followed by eugenol, CL-H, and caryophyllene, indicating weak binding and hence less competition for the enzyme. g p y Under normal conditions, Aβ is present in soluble form in cerebrospinal ﬂuid (CSF) and blood. However, in AD pathology, it is ﬁbrillated as the key component of amyloid plaques. Aβ has been known to boost free radical production, resulting in oxidative stress and neuroinﬂammation, ultimately leading to cognitive and behavioral decline [56]. The π-stacking interactions between aromatic side chains of Aβ are responsible for amyloid aggregation [57]. Eugenol has been reported to suppress individual aggregation as well as coaggregation and exert a dual role in preventing amyloid formation by stabilizing the native protein conformation and delaying ﬁbril formation [56]. Additionally, a compound (2′-Hydroxy-4′-methoxyacetophenone) similar to TMA alleviated Aβ deposition in APP/S1 mice [57]. 4.1. Chemicals Acetylcholinesterase (Electrophorus electricus, Type VI-S), 6,6′-dinitro-3,3′-dithiodibenzoic acid, bis(3-carboxy-4-nitrophenyl) disulﬁde (DTNB), acetyl thiocholine chloride, galan- tamine, gallic acid, ascorbic acid, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), Folin– Ciocalteu reagent (FCR), eugenol, β-caryophyllene, 2′,3′,4′-trimethoxy acetophenone, 2′,7′- dichloroﬂuorescin diacetate (DCFDA), tetramethylrhodamine, ethyl ester (TMRE), hydro- gen peroxide, thioﬂavin T (ThT), bovine serum albumin (BSA), sodium azide, aminoguani- dine, dextrose, thiobarbituric acid (TBA), trichloroacetic acid (TCA), malondialdehyde (MDA), oxidized glutathione (GSSG), reduced glutathione (GSH), O-phthaldehyde (OPT), sodium hydroxide, N-ethylmaleimide (NEM), Triton X-100 were purchased from Sigma- Aldrich (St. Louis, MO, USA). The BCA protein estimation kit was from Thermo Scientiﬁc (Waltham, MA, USA). Aβ1–42 (Aggresure™) was procured from AnaSpec (Fremont, CA, USA). The WST-8 kit was purchased from Roche Diagnostics GmbH (Mannheim, Ger- many). Aβ1–42 for MDS was obtained from GenicBio Inc. (Shanghai, China). Phosphate Buffered Saline (PBS) containing 0.05% Tween, and 3,3′,5,5′-Tetramethylbenzidine solution (TMB) were purchased from Thermo Fisher Scientiﬁc (Waltham, MA, USA). The puriﬁed anti-β-Amyloid, 1–16 antibody, and the horseradish peroxidase (HRP)-conjugated W0-2 monoclonal antibody was purchased each from Biolegend (San Diego, CA, USA) and Peoplebio Inc. (Seongnam, Republic of Korea). All organic solvents of HPLC grade were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4.2. Plant Material and Extraction The dried clove buds were purchased from the Expat Mart in Seoul, Republic of Korea. The pre-weighted samples were powdered using a pestle and mortar. The powder was extracted successively using n-hexane and ethyl acetate. The extracted fractions were dried, weighed, and kept at 4 ◦C until further experiments. 3. Discussion 3. Discussion Likewise, a close relative of TMA, Paeonol, attenuated oxidative stress and mitochondrial dysfunction in streptozotocin (STZ) mice in a model of sporadic Alzheimer’s disease (sAD) [71]. GSH is a potent endogenous antioxidant that protects cells from oxidative stress. These results indicate that CL-EA is most effective in restoring levels of GSH that were dropped due to oxidative stress induced by H2O2 treatment. Even though individual compounds were not able to increase GSH levels on their own, it can be suggested that the higher efﬁciency of CL-EA is the result of the synergistic action of the bioactive components. GSH restoration by CL-EA is supported by earlier ﬁndings where ﬂavonoids protected the cells against oxidative stress by upregulating the GSH-redox system [72]. The improved GSH/GSSG ratio is an indicator of the effective conversion of GSSG to GSH catalyzed by glutathione reductase (GR) in the presence of clove extract, especially CL-EA, and is Int. J. Mol. Sci. 2023, 24, 8148 16 of 24 16 of 24 supported by a previous study on the recovery of neuronal oxidative stress by GSH/GSSG conditioning in the presence of an antioxidant [73]. Eugenol and eugenol acetate inhibit lipid peroxidation by donating their phenolic hydrogen atoms to trap peroxyl radicals that cause lipid peroxidation [74,75]. Hence, it can be concluded that clove extract can reduce lipid peroxidation due to the synergistic effects of the key bioactives present. Our results are also supported by a previous ﬁnding that clove extract (hydroalcoholic) improved antioxidant status in animal studies [76]. To summarize, the extracts have a variety of components in different concentrations that have an impact on various cellular pathways either by synergistic/additive or an- tagonistic effects. CL-H is showing better anti-AChE activity, anti-ﬁbrilization potential, better neuroprotection, and ROS scavenging, whereas CL-EA displayed a superior proﬁle in Aβ oligomerization and ﬁbrilization, anti-glycation, and restoring MMP, glutathione, and MDA levels. The difference in action can be explained based on the concentration of phytocompounds present in them, the mechanism of action, and the affected pathways. where Ab = Absorbance of the blank and As = Absorbance of the extract. 4.6.2. Free Radical Scavenging by 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid) [ABTS] Radical 4.6.2. Free Radical Scavenging by 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid) [ABTS] Radical The free radical scavenging capacity of extracts was measured with minor modiﬁca- tions to the method described earlier [80]. Equal volumes of ABTS (0.7 mM) and potassium persulfate (2.45 mM) were mixed and kept in the dark at RT for 30 min to generate ABTS radicals. Later, to the extract, an ABTS radical solution was added and kept in the dark for 30 min at RT. The absorbance was measured at 734 nm using the microplate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). Ascorbic acid (100 µg/mL) was used as a standard. The percentage of inhibition of ABTS+• was calculated as: % RSA = [(Ab −As/Ab)] × 100 where Ab = Absorbance of the blank and As = Absorbance of the extract. where Ab = Absorbance of the blank and As = Absorbance of the extract. 4.3. Gas Chromatography-Mass Spectrometry (GC-MS) Method The sample was separated on a fused-silica capillary column (DB-5ms UI, 30 m × 0.25 mm i.d., ﬁlm thickness 0.25 µm, Agilent, Santa Clara, CA, USA) installed on GCMS-QP2020 (Shimadzu, Kyoto, Japan). The oven temperature was programmed as isothermic at 60 ◦C for 2 min, 100 ◦C at 4 ◦C/min, 290 ◦C at 10 ◦C/min, and ﬁnally isothermic for 10 min. The split injection mode (1:10) was used. The carrier gas was helium at a constant ﬂow rate of 1 mL/min. The injection port, ion source, and interface temperatures were 280, 280, and 150 ◦C, respectively. The energy of ionization was 70 eV. The mass spectra were obtained in full scan mode (40-700 AMU). The hexane and ethyl acetate fractions (1 µL, 1 mg/mL) were injected into the GC/MS via an auto-injector. The unknown compounds were identiﬁed Int. J. Mol. Sci. 2023, 24, 8148 17 of 24 17 of 24 by matching known compounds in the National Institute of Standards and Technology (NIST) library. by matching known compounds in the National Institute of Standards and Technology (NIST) library. 4.4. Determination of Total Phenolic Content The total phenolic concentration in the extracts was determined as per the Folin−Ciocalteu method [77], with modiﬁcations for a 96-well format. The extract was incubated with Folin−Ciocalteu reagent (1N) for 5 min at room temperature (RT), followed by the addition of sodium carbonate (100 g/L in deionized water). The plate was incubated in the dark for 2 h at RT, and the absorbance was measured at 765 nm using a microplate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). Gallic acid (10–200 mg/L) was used as a standard for calibration. The experiment was carried out in triplicate, and the results are expressed as mg gallic acid equivalent (GAE)/g of extract. 4.5. Determination of Total Flavonoids Content The total ﬂavonoids were estimated using a previously described method [78], with modiﬁcations. To the extract, 10% aluminum chloride and 150 µL of 96% ethanol and sodium acetate (1M) were added. The reagents were mixed, and the plate was incubated at RT for 40 min in the dark. The absorbance was measured at 415 nm using a microplate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). The standard curve of quercetin (10–100 µg/mL) was used to estimate ﬂavonoids in the extract and was expressed as mg quercetin equivalents per gram of sample (mg/g). 4.6. Determination of Antioxidant Capacity 4.6.1. Free Radical Scavenging by 2,2-diphenyl-1-picrylhydrazylhydrate (DPPH) Radical The DPPH radical scavenging capacity of extracts was determined using a method [79] with minor modiﬁcations. The diluted extract was mixed with ethanolic DPPH (120 µM). The plate was incubated in the dark for 30 min at RT, and the absorbance was monitored at 515 nm using a plate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). Ascorbic acid was used as a standard (0.1–10 µg/mL). Radical scavenging activity (RSA) was calculated using the following formula: % RSA = [(Ab −As/Ab)] × 100 where Ab = Absorbance of the blank and As = Absorbance of the extract. where Ab = Absorbance of the blank and As = Absorbance of the extract. 4.7. Advanced Glycation End-Product (AGE) Inhibition Activity The bovine serum albumin (BSA) glycation reaction was carried out as described earlier [35] by incubating the extract with BSA (50 mg/mL) in phosphate buffer (100 mM, pH 7.4), dextrose monohydrate (0.5 M), and sodium azide (5 mM) at 37 ◦C for 14 days. Aminoguanidine was used as a positive control. The ﬂuorescence (Ex 370 nm; Ems 440 nm) was measured (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA), and the percent inhibition of glycation was calculated as: Inhibition (%) = [(C −T)/C × 100] where C and T are ﬂuorescence intensity in the absence and presence of the sample, respectively. The IC50 values were determined by GraphPad Prism 9.5. where C and T are ﬂuorescence intensity in the absence and presence of the sample, respectively. The IC50 values were determined by GraphPad Prism 9.5. 4.9. Thioﬂavin T (ThT) Assay A previously reported method was followed with slight modiﬁcation [40]. The assay was performed using 5 µM Aβ1–42 (Aggresure™AnaSpec) in PBS and incubated with or without extract (100 µg/mL) at 37 ◦C for 24 h. Later, 100 µM ThT was added, and the plate was incubated for 15 min at 37 ◦C, after which the ﬂuorescence (Ex 450 nm; Ems 490 nm) was measured (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). Phenol red (50 µM) served as the inhibitor control. The aggregation inhibition was calculated as: The aggregation inhibition was calculated as: Percent Inhibition (%) = [(1 −Fi/Fc) × 100%] where Fi and Fc are the ﬂuorescence intensity with and without the inhibitors, respectively 4.8. Acetylcholinesterase Inhibitory Activity AChE activity was monitored with slight modiﬁcations to Ellman’s method [82]. The extracts were incubated with AChE (4 ng) and ATCC (10 mM) in phosphate buffer (100 mM, pH 7.6) at 37 ◦C for 15 min. The reaction was terminated by DTNB (15 mM) and incubated for 10 min. The absorbance was measured at 412 nm using the plate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). Galantamine served as a positive control. The percent inhibition was calculated as: Percent Inhibitory activity (I%) = [(A1 −A2) −(B1 −B2)]/(A1 −A2) × 100 where A1 is the absorbance without inhibitor; A2 is the negative control without inhibitor; B1 is the absorbance with inhibitor and B2 is the negative control with inhibitor. where A1 is the absorbance without inhibitor; A2 is the negative control without inhibitor; B1 is the absorbance with inhibitor and B2 is the negative control with inhibitor. The IC50 values were determined by GraphPad Prism 9.5. For inhibition kinetics, substrate concentration was varied (0.1–1.6 mM), keeping other conditions the same as described above. Km and Vmax were calculated from the Lineweaver–Burk plot. 4.6.3. Ferric Reducing Antioxidant Potential (FRAP) Assay The ability of extracts to chelate metal ions was determined by the FRAP assay, which was carried out by modifying a previously described method [81]. The working FRAP reagent was prepared by mixing 10:1:1 volumes of 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) in 40 mM hydrochloric acid, and 20 mM ferric chloride. A standard curve was prepared using FeSO4.7H2O at various concentrations (1 mM). For Int. J. Mol. Sci. 2023, 24, 8148 18 of 24 the assay, the extract was mixed with 300 µL of FRAP reagent, and the reduction of ferric tripyridyltriazine to a ferrous complex by the extract was monitored at 593 nm (microplate reader, Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA) after 30 min of incubation at RT in the dark. FRAP values were expressed as µM Fe2+/g of the sample. All measurements were carried out in triplicate. 4.7. Advanced Glycation End-Product (AGE) Inhibition Activity Cytotoxicity% = (Ac −At)/(Ac) × 100 where Ac = absorbance of the control cells and At = Absorbance of the treated cells. The plot of percent cytotoxicity versus sample concentration was used to calculate the extract concentration that killed 50% of the cells (IC50). 4.11.3. Measurement of Intracellular Reactive Oxygen Species (ROS) The ROS was measured using 2′,7′-dichlorodihydroﬂuorescein diacetate (H2DCFDA) as previously described [84]. The cells (1 × 104 cells/well) were seeded in a 96-well plate and incubated for 18–24 h after which they were pre-treated with the extract for 12 h, followed by 4 h of treatment with H2O2 (100 µM). Later, 25 µM H2DCFDA was added, and the cells were incubated for another 2 h in the dark at 37 ◦C. Fluorescence intensity (Ex 495 nm, Ems 520 nm) was measured by a microplate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). The ROS was calculated as a percentage of the untreated control cells (100%) in triplicate measurements. 4.10. Multiple Detection System (MDS) MDS was conducted as in the previous report [83]. In brief, Aβ1–42 (GenicBio Inc., Hong Kong, China) was reconstituted in PBS and stored at −80 ◦C. The samples were collected at 0 h, 2 h, and 4 h during incubation with the extracts and Aβ1–42 at RT. The collected samples were stored at −80 ◦C until further use. The clear 96-well plate (Grenier Bio-One, Kremsmünster, Austria) was coated with anti-β-Amyloid, which targeted Aβ, and incubated overnight at 4 ◦C. After incubation, the Int. J. Mol. Sci. 2023, 24, 8148 19 of 24 19 of 24 plate was washed three times with PBS containing 0.05% Tween (PBST). The 2% bovine serum albumin was added for blocking and incubated at RT for 2 h. The prepared samples were incubated at RT for 1 h after diluting 4 times. The detection solution containing horseradish peroxidase (HRP)-conjugated W0-2 monoclonal antibody was incubated at RT for 30 min. 3,3′,5,5′-Tetramethylbenzidine solution (TMB) was applied as a color-changing reagent and incubated at RT for 15 min. The optical density was measured at 450 nm wavelength using a microplate reader (Victor3, PerkinElmer) after adding a stop solution. h d h ( ) l l d g g p g p The area under the curve (AUC) was calculated as: The area under the curve (AUC) was calculated as: 4.11. Cell Culture Human neuroblastoma SH-SY5Y cells (ATCC CRL-2266) were maintained in Dul- becco’s modiﬁed Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS), 1% Kanamycin, and 1% Penicillin (Thermo Fisher Scientiﬁc, Waltham, MA, USA) at 37 ◦C in a 95% humidiﬁed atmosphere containing 5% CO2 in the incubator. The cells were passaged twice per week, and the experiments were performed at 80–90% conﬂuency. 4.11.2. Neuroprotective Activity Assay The neuroprotective activity in H2O2-induced oxidative stress in SH-SY5Y was per- formed as previously described [40]. The cells (1 × 104 cells/well) were seeded in a 96-well plate and incubated for 18–24 h. After stabilization, cells were pre-treated with the extracts for 24 h before 6 h of incubation with H2O2 (100 µM). A solvent control, H2O2 alone, and extract alone treatments were also included. After incubation, the % of cell viabilities were determined using the WST-8 reagent in triplicate experiments. 4.11.3. Measurement of Intracellular Reactive Oxygen Species (ROS) 4.11.1. Cell Viability Assay Brieﬂy, 1 × 104 cells were seeded per well in sterile 96-well plates and exposed to different concentrations of extracts for 24 h. The cells were washed twice with PBS (1X) and incubated in the fresh medium with 10% WST-8 reagent (Roche, Germany) for 2 h. The absorbance was measured at 450 nm in a multiplate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). The percent cytotoxicity was calculated as: Cytotoxicity% = (Ac −At)/(Ac) × 100 Estimation of Glutathione The concentrations of GSH (reduced) and GSSG (oxidized) in the lysate were measured ﬂuorometrically using a previously described method [87] with modiﬁcations [88], and the ﬂuorescence was recorded at 350/420 (Ex/Ems) in a microplate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). Estimation of Malondialdehyde (MDA) MDA levels were measured by modiﬁcations to the method of Heath and Packer [89]. In the reaction, MDA reacts with two molecules of thiobarbituric acid (TBA) to give a pink pigment that absorbs at 532 nm. The standard curve of MDA (1–100 µM) was used to calculate lipid peroxidation in the lysate. 4.11.5. Antioxidant Parameters in Cell Lysate The cells (5 × 104 cells/well) were seeded in a 6-well plate and incubated for 18–24 h. After stabilization, cells were pre-treated with the extracts for 24 h before 6 h of incubation with H2O2 (100 µM). A solvent control, H2O2 alone, and extract alone treatments were also included. The culture media was removed, and the cells were washed with cold PBS (1X). Placed the cells on ice and incubated with pre-chilled phosphate buffer (100 mM, pH 7.4) containing 1% Triton X-100 for 30 min. Remove the lysate in microfuge tubes and spin at 20,000× g for 10 min at 4 ◦C (Labogene 1730R, BMS, Paju-si, Republic of Korea) [86]. The supernatant was collected and stored at −80 ◦C. Protein Estimation The protein concentration in the samples was measured using the BCA protein estima- tion kit (Thermo Scientiﬁc, Waltham, MA, USA). The BSA standard (10–1000 µg/mL) was used to calculate protein concentration in the unknown samples. 4.12. Statistical Analysis Statistical analysis was established by one-way ANOVA followed by Dunnett’s post hoc test. The data are registered as the mean ± SD of at least three experiments. The symbols ###, * represent p < 0.001; ##, represent p < 0.01; and #, * represent p < 0.05. The symbol # indicates signiﬁcance compared to H2O2 control, while * indicates signiﬁcance compared to untreated control. The IC50 values were determined using non-linear regression, and Lineweaver–Burk plots were drawn using linear regression analysis by GraphPad Prism 9.5. 4.11.4. Mitochondrial Membrane Potential (∆Ψm) Assay The mitochondrial membrane potential was measured using the tetramethyl rho- damine, methyl ester (TMRE) staining method [85]. The cells (1 × 104 cells/well) were seeded in a 96-well plate and incubated for 18–24 h after which they were pre-treated with the extract for 12 h, followed by 2 h of treatment with H2O2 (200 µM). The cells were Int. J. Mol. Sci. 2023, 24, 8148 20 of 24 20 of 24 incubated for 1 h with 1 µM TMRE at 37 ◦C. The ﬂuorescence (Ex 549 nm, Ems 575 nm) was read in a microplate reader (Synergy-H1 BioTek, Agilent, Santa Clara, CA, USA). The ∆Ψm was calculated as a percentage of the untreated control cells (100%) in triplicate measurements. 4.11.5. Antioxidant Parameters in Cell Lysate References 1. Podtelezhnikov, A.A.; Tanis, K.Q.; Nebozhyn, M.; Ray, W.J.; Stone, D.J.; Loboda, A.P.; Loboda, A.P. Molecular insights into the pathogenesis of Alzheimer’s disease and its relationship to normal aging. PLoS ONE 2011, 6, e29610. [CrossRef] [PubMed] 2. Ono, K.; Hamaguchi, T.; Naiki, H.; Yamada, M. Anti-amyloidogenic effects of antioxidants: Implications for the prevention and 1. Podtelezhnikov, A.A.; Tanis, K.Q.; Nebozhyn, M.; Ray, W.J.; Stone, D.J.; Loboda, A.P.; Loboda, A.P. Molecular insights into the pathogenesis of Alzheimer’s disease and its relationship to normal aging. PLoS ONE 2011, 6, e29610. [CrossRef] [PubMed] p g p g g 2. Ono, K.; Hamaguchi, T.; Naiki, H.; Yamada, M. Anti-amyloidogenic effects of antioxidants: Implications for the prevention and therapeutics of Alzheimer’s disease. Biochim. Et Biophys. Acta (BBA)-Mol. Basis Dis. 2006, 1762, 575–586. [CrossRef] [PubMed] 3. Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.; Butterﬁeld, D.A.; Giuffrida Stella, A.M. Nitric ox system: Neuroprotection versus neurotoxicity. Nat. Rev. Neurosci. 2007, 8, 766–775. [CrossRef] [PubMe 3. Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.; Butterﬁeld, D.A.; Giuffrida Stella, A.M. Nitric oxide in the central nervous system: Neuroprotection versus neurotoxicity. Nat. Rev. Neurosci. 2007, 8, 766–775. [CrossRef] [PubMed] 4. Sharma, N.; Tan, M.A.; An, S.S.A. Mechanistic aspects of Apiaceae family spices in ameliorating Alzheimer’s Disease. Antioxidants 3. Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.; Butterﬁeld, D.A.; Giuffrida Stella, A.M. Nitric oxide in the central nervous system: Neuroprotection versus neurotoxicity. Nat. Rev. Neurosci. 2007, 8, 766–775. [CrossRef] [PubMed] , ; , ; , ; , ; , ; , system: Neuroprotection versus neurotoxicity. Nat. Rev. Neurosci. 2007, 8, 766–775. [CrossRef] [PubMed] 4. Sharma, N.; Tan, M.A.; An, S.S.A. Mechanistic aspects of Apiaceae family spices in ameliorating Alzheimer’s Disease. Antioxidants 2021, 10, 1571. [CrossRef] N.; An, S.S.A. Phyto-Carbazole Alkaloids from the Rutaceae Family as Potential Protective Agents against Diseases. Antioxidants 2022, 11, 493. [CrossRef] 5. Tan, M.A.; Sharma, N.; An, S.S.A. Phyto-Carbazole Alkaloids from the Rutaceae Family as Potential P Neurodegenerative Diseases. Antioxidants 2022, 11, 493. [CrossRef] g 6. Vicidomini, C.; Roviello, V.; Roviello, G.N. Molecular basis of the therapeutical potential of clove (Syzygium aromaticum L.) and clues to its anti-COVID-19 utility. Molecules 2021, 26, 1880. [CrossRef] nti-COVID-19 utility. Molecules 2021, 26, 1880. [CrossR y 7. Rosarior, V.L.; Lim, P.S.; Wong, W.K.; Yue, C.S.; Yam, H.C.; Tan, S.A. Antioxidant-rich Clove Extract, A Strong Antimicrobial Agent against Urinary Tract Infections-causing Bacteria in vitro. Trop. Life Sci. Res. 2021, 32, 45. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24098148/s1. Author Contributions: Conceptualization, N.S. and S.S.A.A.; Experimentation and data analysis, H.S., D.Y.K., K.H.S., and N.S.; writing—original draft preparation, N.S.; writing—review and editing, N.S. and S.S.A.A.; funding acquisition, S.S.A.A., H.S., and D.Y.K. made equal contribution to the experiments, data analysis and the preparation of the manuscript as co-authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Research Foundation of Korea by the Korean Government (2021R1A2C1093218 and 2021R1A6A1A03038996). Funding: This research was funded by the National Research Foundation of Korea by the Korean Government (2021R1A2C1093218 and 2021R1A6A1A03038996). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are contained within the article. Additional information is provided in Supplementary Materials. Data Availability Statement: Data are contained within the article. Additional information is provided in Supplementary Materials. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions Herein, various cell-based and biochemical studies were conducted to evaluate the neuroprotective potential of Syzygium aromaticum (Clove) extracts. Oxidative stress was induced in human neuroblastoma SH-SY5Y cell lines using H2O2 as it is the key ROS generator and mediator of oxidative damage in AD pathogenesis. Oxidative stress hastens abnormal protein aggregation (Aβ, Tau) which in turn initiates an avalanche of reactions leading to microglia activation, neuroinﬂammation, and neuronal apoptosis. The clove ex- tract not only provided neuroprotection against H2O2-induced oxidative stress by reducing ROS, restoring mitochondrial membrane potential, increasing GSH, and decreasing lipid peroxidation in the cells, but also displayed anti-acetylcholinesterase activity, anti-glycation potential, and Aβ oligomerization/ﬁbrilization inhibition. Additionally, the neuroprotec- tive effect of 2’,3’,4’-Trimethoxy acetophenone has been reported for the ﬁrst time in this study. It is speculated that the neuroprotective mechanism involves a synergistic effect of the bioactive components present in the clove extract, and it would be no exaggeration Int. J. Mol. Sci. 2023, 24, 8148 21 of 24 21 of 24 to mention clove as “Nature’s cure for AD”. This multitarget neuroprotective approach displayed by Clove makes it a promising candidate for AD drug development. We tend to proceed with in vivo studies with Clove in the near future. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24098148/s1. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24098148/s1. References β-caryophyllene and β-caryophyllene oxide—Natural compounds of anticancer and analgesic properties. Cancer Med. 2016, 5, 3007–3017. [CrossRef] g p p 25. de Oliveira, M.S.; da Cruz, J.N.; Silva, S.G.; da Costa, W.A.; de Sousa, S.H.B.; Bezerra, F.W.F.; de Carvalho Junior, R.N. Phytochem- ical proﬁle, antioxidant activity, inhibition of acetylcholinesterase and interaction mechanism of the major components of the Piper divaricatum essential oil obtained by supercritical CO2. J. Supercrit. Fluids 2019, 145, 74–84. [CrossRef] p y p p 26. Shoaib, S.; Ansari, M.A.; Fatease, A.A.; Safhi, A.Y.; Hani, U.; Jahan, R.; Alomary, M.N.; Ansari, M.N.; Ahmed, N.; Wahab, S. Plant-Derived Bioactive Compounds in the Management of Neurodegenerative Disorders: Challenges, Future Directions and Molecular Mechanisms Involved in Neuroprotection. Pharmaceutics 2023, 15, 749. [CrossRef] p K.H.; Sharma, N.; An, S.S.A. Mechanistic Insights into the Neuroprotective Potential of Sacred Ficus Trees. Nutrient sRef] 27. Shim, K.H.; Sharma, N.; An, S.S.A. Mechanistic Insights into the Neuroprotective Potential of Sacred Ficus [CrossRef] 28. Vastegani, S.M.; Khoshnam, S.E.; Mansouri, E.; Hajipour, S.; Ghafouri, S.; Bakhtiari, N.; Sarkaki, A.; Farbood, Y. Neuroprotective effect of anethole against rotenone induced non-motor deﬁcits and oxidative stress in rat model of Parkinson’s disease. Behav. Brain Res. 2023, 437, 114100. [CrossRef] 29. Iannuzzi, C.; Liccardo, M.; Sirangelo, I. Overview of the Role of Vanillin in Neurodegenerative Diseases and Neuropathophysio- logical Conditions. Int. J. Mol. Sci. 2023, 24, 1817. [CrossRef] 30. Park, S.Y.; Jung, W.J.; Kang, J.S.; Kim, C.M.; Park, G.; Choi, Y.W. Neuroprotective effects of α-iso-c induced damage in the HT22 hippocampal neuronal cell line. Int. J. Mol. Med. 2015, 35, 525–532. [C 30. Park, S.Y.; Jung, W.J.; Kang, J.S.; Kim, C.M.; Park, G.; Choi, Y.W. Neuroprotective effects of α iso cubebene against glutamate induced damage in the HT22 hippocampal neuronal cell line. Int. J. Mol. Med. 2015, 35, 525–532. [CrossRef] 31. Yokozawa, T.; Nakagawa, T. Inhibitory effects of Luobuma tea and its components against glucose-mediated protein damage. Food Chem. Toxicol. 2004, 42, 975–981. [CrossRef] g pp p 31. Yokozawa, T.; Nakagawa, T. Inhibitory effects of Luobuma tea and its components against glucose-mediated protein damage. Food Chem. Toxicol. 2004, 42, 975–981. [CrossRef] 32. Starowicz, M.; Zieli´nski, H. Inhibition of advanced glycation end-product formation by high antioxidant-leveled spices commonly used in European cuisine. Antioxidants 2019, 8, 100. [CrossRef] 33. Perera, H.; Wijetunge, D. Strong protein glycation inhibitory potential of clove and coriander. Br. J. Pharm. Res. 2015, 6, 306–312. [CrossRef] 34. References N-stearoyl-l-Tyrosine inhibits the cell senescence and a HEK293/Tau cells via the CB2 receptor. Chem.-Biol. Interact. 2017, 272, 135–144. [CrossRef] ; Zhou, K.Y.; Wang, Z.J.; Lu, Y.; Yin, M. N stearoyl l Tyrosine inhibits the cell senescence and 93/Tau cells via the CB2 receptor. Chem.-Biol. Interact. 2017, 272, 135–144. [CrossRef] p 18. Somayajulu, M.; McCarthy, S.; Hung, M.; Sikorska, M.; Borowy-Borowski, H.; Pandey, S. Role of mitochondria in neuronal cell death induced by oxidative stress; neuroprotection by Coenzyme Q10. Neurobiol. Dis. 2005, 18, 618–627. [CrossRef] death induced by oxidative stress; neuroprotection by Coenzyme Q10. Neurobiol. Dis. 2005, 18, 618 627. [CrossRef] 19. Kumar, S.; Abedin, M.M.; Singh, A.K.; Das, S. Role of phenolic compounds in plant-defensive mechanisms. In Plant Phenolics in Sustainable Agriculture; Springer: Berlin/Heidelberg Germany 2020; pp 517 532 y p y y 19. Kumar, S.; Abedin, M.M.; Singh, A.K.; Das, S. Role of phenolic compounds in plant-defensive mechanisms. In Plant Phenolics in Sustainable Agriculture; Springer: Berlin/Heidelberg, Germany, 2020; pp. 517–532. g p g g y pp 20. Mathesius, U. Flavonoid functions in plants and their interactions with other organisms. Plants 2018, 7, 30. [CrossRef] 20. Mathesius, U. Flavonoid functions in plants and their interactions with other organisms. Plants 2018, 7 21. González-Palma, I.; Escalona-Buendía, H.B.; Ponce-Alquicira, E.; Téllez-Téllez, M.; Gupta, V.K.; Díaz-Godínez, G.; Soriano-Santos, J. Evaluation of the antioxidant activity of aqueous and methanol extracts of Pleurotus ostreatus in different growth stages. Front. Microbiol. 2016, 7, 1099. [CrossRef] 22. Lohani, U.C.; Fallahi, P.; Muthukumarappan, K. Comparison of ethyl acetate with hexane for oil extraction from various oilseeds. J. Am. Oil Chem. Soc. 2015, 92, 743–754. [CrossRef] Nisar, M.F.; Khadim, M.; Raﬁq, M.; Chen, J.; Yang, Y.; Wan, C.C. Pharmacological properties and health b 23. Nisar, M.F.; Khadim, M.; Raﬁq, M.; Chen, J.; Yang, Y.; Wan, C.C. Pharmacological properties and heal comprehensive review Oxidative Med Cell Longev 2021 2021 2497354 [CrossRef] m, M.; Raﬁq, M.; Chen, J.; Yang, Y.; Wan, C.C. Pharmacological properties and health beneﬁts of eugenol: A 23. Nisar, M.F.; Khadim, M.; Raﬁq, M.; Chen, J.; Yang, Y.; Wan, C.C. Pharmacological properties comprehensive review. Oxidative Med. Cell. Longev. 2021, 2021, 2497354. [CrossRef] , ; , ; q, ; , J ; g, ; , g p p comprehensive review. Oxidative Med. Cell. Longev. 2021, 2021, 2497354. [CrossRef] p g [ ] 24. Fidyt, K.; Fiedorowicz, A.; Strz ˛adała, L.; Szumny, A. References [CrossRef] 8. Tanko, Y.; Mohammed, A.; Okasha, M.A.; Umah, A.; Magaji, R. Anti-nociceptive and anti-inﬂammatory activities of ethanol extract of Syzygium aromaticum ﬂower bud in wistar rats and mice. Afr. J. Tradit. Complement. Altern. Med. 2008, 5, 209–212. [CrossRef] 9. Trifan, A.; Zengin, G.; Brebu, M.; Skalicka-Wo´zniak, K.; Luca, S.V. Phytochemical Characterization and Evaluation of the Antioxidant and Anti-Enzymatic Activity of Five Common Spices: Focus on Their Essential Oils and Spent Material Extractives. Plants 2021, 10, 2692. [CrossRef] 10. DjidjouTagne, A.M.; Rosari, S.; Silalahi, R.L. Anti-Allergic Effect of Clove. Proceeding of 5th International Conference on Agro-Industry “Agroindustry 4.0—Digital Transformation on Agro-Food Value Chains”, Bali, Indonesia, 26–27 September 2018. 10. DjidjouTagne, A.M.; Rosari, S.; Silalahi, R.L. Anti-Allergic Effect of Clove. Proceeding of 5th International Conference on Agro-Industry “Agroindustry 4.0—Digital Transformation on Agro-Food Value Chains”, Bali, Indonesia, 26–27 September 2018. 11. Mejía-Argueta, E.; Santillán-Benítez, J.G.; Canales-Martinez, M.M.; Mendoza-Medellín, A. Antimicrobial activity of Syzygium aromaticum L. essential oil on extended-spectrum beta-lactamases-producing Escherichia coli. Bull. Natl. Res. Cent. 2020, 44, 201. [CrossRef] g y g y g g p 11. Mejía-Argueta, E.; Santillán-Benítez, J.G.; Canales-Martinez, M.M.; Mendoza-Medellín, A. Antimicrobial activity of Syzygium aromaticum L. essential oil on extended-spectrum beta-lactamases-producing Escherichia coli. Bull. Natl. Res. Cent. 2020, 44, 201. [CrossRef] [ ] 12. Nirmala, M.J.; Durai, L.; Gopakumar, V.; Nagarajan, R. Anticancer and antibacterial effects of a clove bud essential oil-based nanoscale emulsion system. Int. J. Nanomed. 2019, 14, 6439. [CrossRef] y 13. El-Hadary, A.E.; Ramadan Hassanien, M.F. Hepatoprotective effect of cold-pressed Syzygium aromaticum oil against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Pharm. Biol. 2016, 54, 1364–1372. [CrossRef] [PubMed] p y 14. Halder, S.; Mehta, A.K.; Kar, R.; Mustafa, M.; Mediratta, P.K.; Sharma, K.K. Clove oil reverses learning and memory deﬁcits in scopolamine-treated mice. Planta Med. 2011, 77, 830–834. [CrossRef] [PubMed] 15. Panahzadeh, F.; Mirnasuri, R.; Rahmati, M. Exercise and Syzygium aromaticum reverse memory deﬁcits, apoptosis and mitochondrial dysfunction of the hippocampus in Alzheimer’s disease. J. Ethnopharmacol. 2022, 286, 114871. [CrossRef] [PubMed] Int. J. Mol. Sci. 2023, 24, 8148 22 of 24 22 of 24 16. Parvizi, F.; Mehrabi, S.; Naghizadeh, A.; Goudarzi, S. Comparison of intranasal and intraperitoneal administration of Eugenia caryophyllata (clove) essential oil on spatial memory, anxiety-like behavior and locomotor activity in a pilocarpine-induced status epilepticus rat model. BMC Complement. Med. Ther. 2022, 22, 231. [CrossRef] p p p 17. Hu, Y.; Zhou, K.Y.; Wang, Z.J.; Lu, Y.; Yin, M. 40. Tan, M.A.; Zakharova, E.; An, S.S.A. Diaportheone A Analogues Instigate a Neuroprotective Effect by Protecting Neuroblastoma SH-SY5Y Cells from Oxidative Stress. Biology 2021, 10, 199. [CrossRef] References jfpp A comparison of antioxidant activities by eugenyl acetate and eugenyl butyrate at frying temperature. J. Food Process. Preserv. 2021, 45, e15320. [CrossRef] 46. Nogueira Sobrinho, A.C.; Morais, S.M.D.; Souza, E.B.D.; Albuquerque, M.R.J.R.; Santos, H.S.D.; Cavalcante, C.S.D.P.; Fontenelle, R.O.D.S. Antifungal and antioxidant activities of Vernonia chalybaea Mart. ex DC. essential oil and their major constituent β-caryophyllene. Braz. Arch. Biol. Technol. 2020, 63, e20190177. [CrossRef] β y p y [ ] 47. Gushiken, L.F.S.; Beserra, F.P.; Hussni, M.F.; Gonzaga, M.T.; Ribeiro, V.P.; De Souza, P.F.; Pellizzon, C.H. Beta-caryophyllene as an antioxidant, anti-inﬂammatory and re-epithelialization activities in a rat skin wound excision model. Oxid. Med. Cell. Longev. 2022, 2022, 9004014. [CrossRef] 48. Takeuchi, M.; Kikuchi, S.; Sasaki, N.; Suzuki, T.; Watai, T.; Iwaki, M.; Yamagishi, S.I. Involvement of advanced glycation end-products (AGEs) in Alzheimer’s disease. Curr. Alzheimer Res. 2004, 1, 39–46. [CrossRef] 49. Maillard, L.C. Action des acides amines sur les sucres: Formation des melanoidines par voie methodique. CR Acad. Sci. 1912, 154, 66–68. 50. Savateev, K.V.; Spasov, A.A.; Rusinov, V.L. Small synthetic molecules with antiglycation activity. Structure–activity relationship. Russ. Chem. Rev. 2022, 91, RCR5041. [CrossRef] p q 50. Savateev, K.V.; Spasov, A.A.; Rusinov, V.L. Small synthetic molecules with antiglycation activity. Structure–activity relationship. Russ. Chem. Rev. 2022, 91, RCR5041. [CrossRef] 51. Taha, M.; Alkadi, K.A.; Ismail, N.H.; Imran, S.; Adam, A.; Kashif, S.M.; Khan, K.M. Antiglycation and antioxidant potential of novel imidazo[4,5-b]pyridine benzohydrazones. Arab. J. Chem. 2019, 12, 3118–3128. [CrossRef] py y 52. Grzegorczyk-Karolak, I.; Goł ˛ab, K.; Gburek, J.; Wysoki´nska, H.; Matkowski, A. Inhibition of Advanced Glycation End-Product Formation and Antioxidant Activity by Extracts and Polyphenols from Scutellaria alpina L. and S. altissima L. Molecules 2016, 21, 739. [CrossRef] [ ] 53. Loh, Z.H.; Kwong, H.C.; Lam, K.W.; Teh, S.S.; Ee, G.C.L.; Quah, C.K.; Mah, S.H. New 3-O-substituted xanthone derivatives as promising acetylcholinesterase inhibitors. J. Enzym. Inhib. Med. Chem. 2021, 36, 627–639. [CrossRef] [PubMed] wong, H.C.; Lam, K.W.; Teh, S.S.; Ee, G.C.L.; Quah, C.K.; Mah, S.H. New 3-O-substituted xanthone deriva 54. Hung, N.H.; Quan, P.M.; Satyal, P.; Dai, D.N.; Hoa, V.V.; Huy, N.G.; Giang, L.D.; Ha, N.T.; Huong, L.T.; Hien, V.T.; et al. N. Acetylcholinesterase Inhibitory Activities of Essential Oils from Vietnamese Traditional Medicinal Plants. Molecules 2022, 27, 7092. [CrossRef] [PubMed] 55. Zamli, K.M.; Asari, A.; Khaw, K.Y.; Murugaiyah, V.; Al-Rashida, H.M.; Yusoff, H.M.; Hasnah, N.H.A.W. Cholinesterase Inhibition Activity and Molecular Docking Study of Eugenol Derivatives. Sains Malays. 2021, 50, 1037–1045. References [CrossRef] 56. Chauhan, A.; Chauhan, V. Beneﬁcial effects of walnuts on cognition and brain health. Nutrients 202 56. Chauhan, A.; Chauhan, V. Beneﬁcial effects of walnuts on cognition and brain health. Nutrients 2020, 12, 550. [CrossRef] 57. Gazit, E. Mechanisms of amyloid ﬁbril self-assembly and inhibition: Model short peptides as a key research tool. FEBS J. 2005, 272, 5971–5978. [CrossRef] 57. Gazit, E. Mechanisms of amyloid ﬁbril self-assembly and inhibition: Model short peptides as a key research tool. FEBS J. 2005, 272, 5971–5978. [CrossRef] 58. Dahham, S.S.; Tabana, Y.M.; Iqbal, M.A.; Ahamed, M.B.; Ezzat, M.O.; Majid, A.S.; Majid, A.M. The Anticancer, Antioxidant and Antimicrobial Properties of the Sesquiterpene β-Caryophyllene from the Essential Oil of Aquilaria crassna. Molecules 2015, 20, 11808–11829. [CrossRef] 59. Gülçin, ˙I. Antioxidant activity of eugenol: A structure–activity relationship study. J. Med. Food 2011, 14, 975–985. [CrossRef] 59. Gülçin, ˙I. Antioxidant activity of eugenol: A structure–activity relationship study. J. Med. Food 2011, 14, 975–985. [CrossRef] 60. Gaspar, E.M.; Duarte, R.; Santana, J.C. Volatile composition and antioxidant properties of clove products. Biomed. J. Sci. Tech. Res. 2018, 9, 7270–7276. [CrossRef] 60. Gaspar, E.M.; Duarte, R.; Santana, J.C. Volatile composition and antioxidant properties of clove products. Biomed. J. Sci. Tech. Res. 2018, 9, 7270–7276. [CrossRef] 61. Ullah, H.; Di Minno, A.; Santarcangelo, C.; Khan, H.; Daglia, M. Improvement of oxidative stress and m by β-caryophyllene: A focus on the nervous system. Antioxidants 2021, 10, 546. [CrossRef] 62. Shekhar, S.; Yadav, Y.; Singh, A.P.; Pradhan, R.; Desai, G.R.; Dey, A.B.; Dey, S. Neuroprotection by ethanolic extract of Syzygium aromaticum in Alzheimer’s disease like pathology via maintaining oxidative balance through SIRT1 pathway. Exp. Gerontol. 2018, 110, 277–283. [CrossRef] 63. Amir Rawa, M.S.; Mazlan, M.K.N.; Ahmad, R.; Nogawa, T.; Wahab, H.A. Roles of Syzygium in Anti-Cholinesterase, Anti-Diabetic, Anti-Inﬂammatory, and Antioxidant: From Alzheimer’s Perspective. Plants 2022, 11, 1476. [CrossRef] 64. Latif, S.; Choi, S.-H.; Gyawali, A.; Hyeon, S.J.; Kang, Y.-S.; Ryu, H. Antioxidant and Neuroprotecti Oxidative Stress and Altered Carrier-Mediated Transport System on NSC-34 Cell Lines. Antioxidan 64. Latif, S.; Choi, S.-H.; Gyawali, A.; Hyeon, S.J.; Kang, Y.-S.; Ryu, H. Antioxidant and Neuroprotective Effects of Paeonol against Oxidative Stress and Altered Carrier-Mediated Transport System on NSC-34 Cell Lines. Antioxidants 2022, 11, 1392. [CrossRef] 65. Adki, K.M.; Kulkarni, Y.A. Neuroprotective effect of paeonol in streptozotocin-induced diabetes in rats. Life Sci. 2021, 271, 119202. [CrossRef] 65. Adki, K.M.; Kulkarni, Y.A. References Suantawee, T.; Wesarachanon, K.; Anantsuphasak, K.; Daenphetploy, T.; Thien-Ngern, S.; Thilavech, T.; Adisakwattana, S. Protein glycation inhibitory activity and antioxidant capacity of clove extract. J. Food Sci. Technol. 2015, 52, 3843–3850. [CrossRef] [PubMed] [ ] 35. Singh, P.; Jayaramaiah, R.H.; Agawane, S.B.; Vannuruswamy, G.; Korwar, A.M.; Anand, A.; Giri, A.P. Potential dual role of eugenol in inhibiting advanced glycation end products in diabetes: Proteomic and mechanistic insights. Sci. Rep. 2016, 6, 18798. [CrossRef] [PubMed] [ ] [ ] 36. Rafey, A.; Amin, A.; Kamran, M.; Aziz, M.I.; Athar, V.; Niaz, S.I.; Pieters, L. Evaluation of Major Constituents of Medicinally Important Plants for Anti-Inﬂammatory, Antidiabetic and AGEs Inhibiting Properties: In Vitro and Simulatory Evidence. Molecules 2022, 27, 6715. [CrossRef] 37. Balkrishna, A.; Pokhrel, S.; Tomer, M.; Verma, S.; Kumar, A.; Nain, P.; Gupta, A.; Varshney, A. Anti-acetylcholinesterase activities of mono-herbal extracts and exhibited synergistic effects of the phytoconstituents: A biochemical and computational study. Molecules 2019, 24, 4175. [CrossRef] 38. Farag, M.A.; Ezzat, S.M.; Salama, M.M.; Tadros, M.G.; Serya, R.A. Anti-acetylcholinesterase activity of essential oils and their major constituents from four Ocimum species. Z. Für Nat. C 2016, 71, 393–402. [CrossRef] 39. Kumar, P.; Singh, V.; Singh, D. Kinetics of enzyme inhibition by active molluscicidal agents ferulic acid, umbelliferone, eugenol and limonene in the nervous tissue of snail Lymnaea acuminata. Phytother. Res. Int. J. Devoted Pharmacol. Toxicol. Eval. Nat. Prod. Deriv. 2009, 23, 172–177. 40. Tan, M.A.; Zakharova, E.; An, S.S.A. Diaportheone A Analogues Instigate a Neuroprotective Effect by Protecting Neuroblastoma SH-SY5Y Cells from Oxidative Stress. Biology 2021, 10, 199. [CrossRef] 23 of 24 23 of 24 Int. J. Mol. Sci. 2023, 24, 8148 41. Kotormán, M.; Varga, A.; Kasi, P.B.; Nemcsók, J. Inhibition of the formation of amyloid-like ﬁbrils with spices, especially cloves. Acta Biol. Hung. 2018, 69, 385–394. [CrossRef] g 42. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef] 43. Hamdi, D.; Ouachikh, O.; Ouchchane, L.; Omara-Reda, H.; Messaoud, C.; Haﬁdi, A. The neuroprotective effect of Clove essential oil against 6-OHDA-induced cell death in SH-SY5Y and a rat model of Parkinson’s disease. Res. Sq. 2023. [CrossRef] 44. Prasad, S.N.; Muralidhara. Neuroprotective efﬁcacy of eugenol and isoeugenol in acrylamide-induced neuropathy in rats: Behavioral and biochemical evidence. Neurochem. Res. 2013, 38, 330–345. [CrossRef] 45. Keramat, M.; Golmakani, M.T.; Durand, E.; Villeneuve, P.; Hosseini, S.M.H. p p y p 67. Zhang, Z.-D.; Yang, Y.J.; Liu, X.W.; Qin, Z.; Li, S.H.; Bai, L.X.; Li, J.Y. The Protective Effect of Aspirin Eugenol Ester on Oxidative Stress to PC12 Cells Stimulated with H2O2 through Regulating PI3K/Akt Signal Pathway. Oxid. Med. Cell. Longev. 2021, 2021, 5527475. [CrossRef] References Enzymol.; Elsevier: Amsterdam, The Netherlands, 1999; Volume 299, pp. 152–178. 78. Ribarova, F.; Atanassova, M.; Marinova, D.; Ribarova, F.; Atanassova, M. Total phenolics and ﬂavonoids in Bulgarian fruits and vegetables. JU Chem. Metal 2005, 40, 255–260. 78. Ribarova, F.; Atanassova, M.; Marinova, D.; Ribarova, F.; Atanassova, M. Total phenolics and ﬂavonoids in Bulgarian fruits and vegetables. JU Chem. Metal 2005, 40, 255–260. g J , , 79. Koleva, I.I.; Van Beek, T.A.; Linssen, J.P.; Groot, A.D.; Evstatieva, L.N. Screening of plant extracts for antioxidant activity: A comparative study on three testing methods. Phytochem. Anal. Int. J. Plant Chem. Biochem. Tech. 2002, 13, 8–17. [CrossRef] 80. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef] g J 79. Koleva, I.I.; Van Beek, T.A.; Linssen, J.P.; Groot, A.D.; Evstatieva, L.N. Screening of plant extracts for antioxidant activity: A comparative study on three testing methods. Phytochem. Anal. Int. J. Plant Chem. Biochem. Tech. 2002, 13, 8–17. [CrossRef] 80 Re R ; Pellegrini N ; Proteggente A ; Pannala A ; Yang M ; Rice-Evans C Antioxidant activity applying an improved ABTS p y g y 80. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity app radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef] 80. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef] 81 Akt k A Z i G G l G O C k k YS D A A ti id t t ti l d ti h li t ti iti f 81. Aktumsek, A.; Zengin, G.; Guler, G.O.; Cakmak, Y.S.; Duran, A. Antioxidant potentials and antich methanolic and aqueous extracts of three endemic Centaurea L. species. Food Chem. Toxicol. 2013, 55, 29 82. Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; Featherstone, R.M. A new and rapid colorimetric determination of acetyl- cholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef] 83. An, S.S.A.; Shim, K.H.; Kang, S.; Kim, Y.K.; Subedi, L.; Cho, H.; Kim, S. The potential anti-amyloidogenic candidate, SPA1413, for Alzheimer’s disease. Br. J. Pharmacol. 2022, 179, 1033–1048. [CrossRef] Ñ 84. PeÑalver, P.; Zodio, S.; Lucas, R.; de-Paz, M.V.; Morales, J.C. References Neuroprotective effect of paeonol in streptozotocin-induced diabetes in rats. Life Sci. 2021, 271, 119202. [CrossRef] 66. Usta, J.; Kreydiyyeh, S.; Bajakian, K.; Nakkash-Chmaisse, H. In vitro effect of eugenol and cinnamaldehyde on membrane potential and respiratory chain complexes in isolated rat liver mitochondria. Food Chem. Toxicol. 2002, 40, 935–940. [CrossRef] 67. Zhang, Z.-D.; Yang, Y.J.; Liu, X.W.; Qin, Z.; Li, S.H.; Bai, L.X.; Li, J.Y. The Protective Effect of Aspirin Eugenol Ester on Oxidative Stress to PC12 Cells Stimulated with H2O2 through Regulating PI3K/Akt Signal Pathway. Oxid. Med. Cell. Longev. 2021, 2021, 5527475. 66. Usta, J.; Kreydiyyeh, S.; Bajakian, K.; Nakkash-Chmaisse, H. In vitro effect of eugenol and cinnamaldehyde on membrane potential and respiratory chain complexes in isolated rat liver mitochondria. Food Chem. Toxicol. 2002, 40, 935–940. [CrossRef] potential and respiratory chain complexes in isolated rat liver mitochondria. Food Chem. Toxicol. 2002, 40, 935–940. [CrossRef] 67. Zhang, Z.-D.; Yang, Y.J.; Liu, X.W.; Qin, Z.; Li, S.H.; Bai, L.X.; Li, J.Y. The Protective Effect of Aspirin Eugenol Ester on Oxidative Stress to PC12 Cells Stimulated with H2O2 through Regulating PI3K/Akt Signal Pathway. Oxid. Med. Cell. Longev. 2021, 2021, 5527475. [CrossRef] 67. Zhang, Z.-D.; Yang, Y.J.; Liu, X.W.; Qin, Z.; Li, S.H.; Bai, L.X.; Li, J.Y. The Protective Effect of Aspirin Eugenol Ester on Oxidative Stress to PC12 Cells Stimulated with H2O2 through Regulating PI3K/Akt Signal Pathway. Oxid. Med. Cell. Longev. 2021, 2021, 5527475. [CrossRef] Int. J. Mol. Sci. 2023, 24, 8148 24 of 24 68. Mesole, S.B.; Alfred, O.O.; Yusuf, U.A.; Lukubi, L.; Ndhlovu, D. Apoptotic inducement of neuronal cells by aluminium chloride and the neuroprotective effect of eugenol in wistar rats. Oxid. Med. Cell. Longev. 2020, 2020, 8425643. [CrossRef] p g g 69. Assis, L.; Straliotto, M.R.; Engel, D.; Hort, M.A.; Dutra, R.C.; De Bem, A.F. β-Caryophyllene protects the C6 glioma cells against glutamate-induced excitotoxicity through the Nrf2 pathway. Neuroscience 2014, 279, 220–231. [CrossRef] 70. Wang, G.; Ma, W.; Du, J. β-Caryophyllene (BCP) ameliorates MPP+ induced cytotoxicity. Biomed. Pharmacother. 2018, 103, 1086–1091. [CrossRef] 71. Tayanloo-Beik, A.; Kiasalari, Z.; Roghani, M. Paeonol Ameliorates Cognitive Deﬁcits in Streptozotocin Murine Model of Sporadic Alzheimer’s Disease via Attenuation of Oxidative Stress, Inﬂammation, and Mitochondrial Dysfunction. J. Mol. Neurosci. 2022, 72, 336–348. [CrossRef] , [ ] 72. Boadi, W.Y.; Stevenson, C.; Johnson, D.; Mohamed, M.A. Flavonoids reduce lipid peroxides and increase glutathione levels in pooled human liver microsomes (HLMs). Adv. Biol. Chem. 2021, 11, 283–295. References [CrossRef] 73. Kim, S.H.; Smith, A.J.; Tan, J.; Shytle, R.D.; Giunta, B. MSM ameliorates HIV-1 Tat induced neuronal oxidative stress via rebalance of the glutathione cycle. Am. J. Transl. Res. 2015, 7, 328–338. [PubMed] g y J [ ] a, A.R.; Vijayakumar, M.; Rao, C.V.; Mathela, C.S. In vitro and in vivo antioxidant properties and DNA dam 74. Verma, A.R.; Vijayakumar, M.; Rao, C.V.; Mathela, C.S. In vitro and in vivo antioxidant properties and DNA damage protective activity of green fruit of Ficus glomerata. Food Chem. Toxicol. 2010, 48, 704–709. [CrossRef] [PubMed] 75. Khalil, A.A.; ur Rahman, U.; Khan, M.R.; Sahar, A.; Mehmood, T.; Khan, M. Essential oil eugenol: Sou and nutraceutical perspectives. RSC Adv. 2017, 7, 32669–32681. [CrossRef] p p 76. Adegbola, M.V.; Anyim, G.; Ntwasa, M.; Ayeleso, A.O.; Oyedepo, T.A. Potential Effect of Syzygium aromaticum (Cloves) Extract on Serum Antioxidant Status and Lipid Proﬁles in Wistar Rats with Artesunate Toxicity. Appl. Sci. 2022, 12, 8216. [CrossRef] 76. Adegbola, M.V.; Anyim, G.; Ntwasa, M.; Ayeleso, A.O.; Oyedepo, T.A. Potential Effect of Syzygium aromaticum (Cloves) Extract 76. Adegbola, M.V.; Anyim, G.; Ntwasa, M.; Ayeleso, A.O.; Oyedepo, T.A. Potential Effect of Syzygium aromaticum (Cloves) Extract S A ti id t St t d Li id P ﬁl i Wi t R t ith A t t T i it A l S i 2022 12 8216 [C R f] 76. Adegbola, M.V.; Anyim, G.; Ntwasa, M.; Ayeleso, A.O.; Oyedepo, T.A. Potential Effect of Syzygium aromaticum (Cloves) Extract on Serum Antioxidant Status and Lipid Proﬁles in Wistar Rats with Artesunate Toxicity. Appl. Sci. 2022, 12, 8216. [CrossRef] 77. Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In Meth. Enzymol.; Elsevier: Amsterdam, The Netherlands, 1999; Volume 299, pp. 152–178. on Serum Antioxidant Status and Lipid Proﬁles in Wistar Rats with Artesunate Toxicity. Appl. Sci. 2022, 12, 8216. [CrossRef] 77. Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin ciocalteu reagent In Meth Enzymol ; Elsevier: Amsterdam The Netherlands 1999; Volume 299 pp 152 178 p y pp 77. Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In Meth. Enzymol.; Elsevier: Amsterdam, The Netherlands, 1999; Volume 299, pp. 152–178. by means of folin-ciocalteu reagent. In Meth. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. References Neuroprotective and anti-inﬂammatory effects of pterostilbene metabolites in human neuroblastoma SH-SY5Y and RAW 264.7 macrophage cells. J. Agric. Food Chem. 2020, 68, 1609–1620. [CrossRef] 85. Alvariño, R.; Alonso, E.; Lacret, R.; Oves-Costales, D.; Genilloud, O.; Reyes, F.; Botana, L.M. Caniferolide A, a macrolide from Streptomyces caniferus, attenuates neuroinﬂammation, oxidative stress, amyloid-beta, and tau pathology in vitro. Mol. Pharm. 2019, 16, 1456–1466. [CrossRef] 86. Harlow, E.; Lane, D. Lysing tissue-culture cells for immunoprecipitation. Cold Spring Harb. Protoc. 2006, 2006, pdb.prot4531. [CrossRef] 87. Hissin, P.J.; Hilf, R. A ﬂuorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 1976, 74, 214–226. [CrossRef] 88. Singh, V.; Gera, R.; Purohit, M.P.; Patnaik, S.; Ghosh, D. Fluorometric estimation of glutathione in cultured microglial cell lysate. Bio-Protoc. 2017, 7, e2304. [CrossRef] 89. Heath, R.L.; Packer, L. Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 1968, 125, 189–198. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W2969965412	https://www.sci-notes.ru/jour/article/download/557/pdf_140	Russian	null	PREVENTION OF PURULENT CHOLANGITIS AFTER PERCUTANEOUS ENDOBILIARY TRANSPAPILLARY DRAINAGE IN PATIENTS WITH OBSTRUCTIVE JAUNDICE	Učënye zapiski Sankt-Peterburgskogo gosudarstvennogo medicinskogo universiteta im. Akad. I.P. Pavlova/Učënye zapiski Sankt-Peterburgskogo gosudarstvennogo medicinskogo universiteta imeni akademika I. P. Pavlova	2,019	cc-by	5,284	УЧЕНЫЕ ЗАПИСКИ СПбГМУ им. акад. И. П. ПАВЛОВА The Scientific Notes of Pavlov University journal homepage: www.scinotes.ru УЧЕНЫЕ ЗАПИСКИ СПбГМУ им. акад. И. П. ПАВЛОВА The Scientific Notes of Pavlov University journal homepage: www.scinotes.ru © СС Коллектив авторов, 2019 УДК 616.36-008.5-089-06:616.361-002.3-084 DOI: 10.24884/1607-4181-2019-26-1-35-41 © СС Коллектив авторов, 2019 УДК 616.36-008.5-089-06:616.361-002.3-084 DOI: 10.24884/1607-4181-2019-26-1-35-41 А. Гуня1, А. А. Ребров1, Д. Ю. Cеменов2, В. В. Мельников1, А. А. Ваганов1 З. А. Гуня1, А. А. Ребров1, Д. Ю. Cеменов2, В. В. Мельников1, А. А. Ваганов1 1 Федеральное государственное бюджетное образовательное учреждение высшего образования «Первый Санкт-Петербургский государственный медицинский университет имени академика И. П. Павлова» Министерства здравоохранения Российской Федерации, Санкт-Петербург, Россия 2 Государственное бюджетное учреждение здравоохранения Московской области «Московский областной научно-исследовательский клинический институт имени М. Ф. Владимирского», Москва, Россия 1 Федеральное государственное бюджетное образовательное учреждение высшего образования «Первый Санкт-Петербургский медицинский университет имени академика И. П. Павлова» Министерства здравоохранения Российской Федерации, Санкт-П 2 Государственное бюджетное учреждение здравоохранения Московской области «Московский областной научно-исследовательс институт имени М. Ф. Владимирского», Москва, Россия ПРОФИЛАКТИКА ГНОЙНОГО ХОЛАНГИТА ПРИ ЧРЕСКОЖНОЙ ЭНДОБИЛИАРНОЙ ТРАНСПАПИЛЛЯРНОЙ ДЕКОМПРЕССИИ У БОЛЬНЫХ С МЕХАНИЧЕСКОЙ ЖЕЛТУХОЙ Поступила в редакцию 22.10.18 г.; принята к печати 21.05.19 г. Резюме Введение. При выполнении транспапиллярного наружно-внутреннего эндобилиарного дренирования частота развития холангитов составляет от 9 до 33 %. С целью профилактики гнойных осложнений при проксимальном уров- не билиарного блока широко используются методы супрапапиллярной установки наружно-внутреннего дренажа, что технически невозможно при дистальном уровне блока. Проблема выбора первичного наружного или транспа- пиллярного наружно-внутреннего дренирования при дистальных блоках желчевыводящих протоков неоднозначна. Цель работы – улучшение результатов лечения больных с механической желтухой при низком уровне билиарного блока после первичной антеградной эндобилиарной декомпрессии. Материал и методы. 82 пациентам с дистальным блоком желчевыводящих протоков (ЖВП) опухолевого генеза в качестве первичной декомпрессии выполнены чрескожные чреспеченочные эндобилиарные вмешательства под УЗ- и Rg-контролем. 30 пациентам выполнено наружно-внутреннее транспапиллярное дренирование, 21 пациенту декомпрессию ЖВП осуществляли с помощью наружного желчеотведения, у 31 пациента применяли оригинальную методику комбинированного наружно-внутреннего дренирования. Результаты. Интраоперационные и ранние послеоперационные осложнения зарегистрированы у 23 (28 %) человек. Осложнения в группе наружного эндобилиарного дренирования отмечены у 4 (19 %) пациентов. В группе наружно-внут реннего дренирования частота осложнений составила 53,3 % (16 пациентов). При этом инфекционные осложнения от- мечены в 30 % (9 человек) случаев. При выполнении комбинированного наружно-внутреннего дренирования гнойные осложнения не были диагностированы ни у одного пациента. При лечении всех осложнений были успешно использованы мини-инвазивные вмешательства. Выводы. Полученные результаты показали преимущества предлагаемой методики комбинированного эндоби- лиарного дренирования ввиду отсутствия риска инфекционных осложнений в послеоперационном периоде с одно временным обеспечением транспапиллярного доступа. Ключевые слова: обструктивная желтуха, минимально инвазивные вмешательства, чрескожные эндобилиарные вмешательства, осложнения Для цитирования: Гуня З. А., Ребров А. А., Cеменов Д. Ю., Мельников В. В., Ваганов А. А. Профилактика гнойного холангита при чрескожной эндобилиарной транспапиллярной декомпрессии у больных с механической желтухой. Ученые записки СПбГМУ им. акад. И. П. Павлова. 2019;26(1):35–41. DOI: 10.24884/1607-4181-2019-26-1-35-41. * Автор для связи: Зинаида Анатольевна Гуня, ФГБОУ ВО «ПСПбГМУ им. И. П. Павлова» МЗ РФ, 197022, Россия, Санкт-Петербург, д. 6-8. E-mail: zina727@list.ru. © СС Composite authors, 2019 UDC 616.36-008.5-089-06:616.361-002.3-084 DOI: 10.24884/1607-4181-2019-26-1-35-41 © СС Composite authors, 2019 UDC 616.36-008.5-089-06:616.361-002.3-084 DOI: 10.24884/1607-4181-2019-26-1-35-41 PREVENTION OF PURULENT CHOLANGITIS AFTER PERCUTANEOUS ENDOBILIARY TRANSPAPILLARY DRAINAGE IN PATIENTS WITH OBSTRUCTIVE JAUNDICE R i d 22 10 18 d 21 35 Гуня З. А. и др. / Ученые записки СПбГМУ им. акад. И. П. Павлова Т. XXVI № 1 (2019) С. 35–41 Summary Introduction. The incidence of cholangitis varied from 9 % to 33 % after the insertion of transpapillary external-internal endobiliary drainage. In case of proximal bile obstruction, the method of suprapapillary insertion of external-internal drainage were widely used for prevention of purulent complications. But it was impossible to use this method in case of distal bile obstruction. The choice of initial decompression method for distal bile duct obstruction (external only or transpapillary external-internal drainage) was unclear. The objective was to improve results of primary percutaneous transhepatic biliary drainage in patients with jaundice and distal bile duct obstruction. Material and methods. As a primary decompression method, percutaneous transhepatic endobiliary interventions under ultrasound and X-ray control were performed in 81 patients with distal bile duct obstruction. External-internal transpapillary drainage were performed in 30 patients, only external drainage – in 21 patients and in 31 patients we used original combined technique (compilation of external and external-internal transpapillary drainage). Results. Intraoperative and early postoperative complications were noticed in 23 patient (28 %). Complications in the group of external endobiliary drainage were observed in 4 (19 %) patients. In the group of external-internal drainage, complication rate was 53.3 % (16 patients). At the same time, purulent complications were noted in 30 % of cases (9 people). In case of using combined external-internal drainage, no purulent complications was diagnosed. In the treatment of all complications, minimally invasive methods were successfully used. Conclusion. Our results showed advantages of the original combined technique due to the absence of the risk of purulent complications, and ensuring transpapillary access at the same time. Keywords: obstructive jaundice, minimally invasive interventions, percutaneous endobiliary intervention, complications Keywords: obstructive jaundice, minimally invasive interventions, percutaneous endobiliary intervention, c For citation: Gunya Z. A., Rebrov A. A., Semenov D. Yu., Mel’nikov V. V., Vaganov A. A. Prevention of purulent ch percutaneous endobiliary transpapillary drainage in patients with obstructive jaundice. The Scientific Notes of IPP-SPSM 35–41. (In Russ.). DOI: 10.24884/1607-4181-2019-26-1-35-41. For citation: Gunya Z. A., Rebrov A. A., Semenov D. Yu., Mel’nikov V. V., Vaganov A. A. Prevention of purulent cholangitis afte percutaneous endobiliary transpapillary drainage in patients with obstructive jaundice. The Scientific Notes of IPP-SPSMU. 2019;26(1) 35–41. (In Russ.). DOI: 10.24884/1607-4181-2019-26-1-35-41. * Corresponding author: Zinaida A. Gunya, Pavlov University, 6-8 L’va Tolstogo street, Saint-Petersburg, Russia, 197022. E-mail: zina7 МАТЕРИАЛ И МЕТОДЫ ИССЛЕДОВАНИЯ ляющем большинстве случаев использовали дрена- жи типа «pigtail» с фиксацией, диаметром 10,2 Fr. ляющем большинстве случаев использовали дрена- жи типа «pigtail» с фиксацией, диаметром 10,2 Fr. Оригинальную методику чрескожного комби- нированного эндобилиарного дренирования также выполняли под УЗ- и Rg-контролем. Первый этап операции не отличался от стандартного выполне- ния чрескожного наружно-внутреннего эндобили- арного дренирования. Вторым этапом после бужи- рования доступа по проводнику в желчные протоки проксимальнее стриктуры вводили 2-канальный расширитель (диаметр – 10,2 Fr): центральный порт использовали для первичного проводника, а через второй порт вводили дополнительный проводник, дистальный конец которого устанавливали в желч- ных протоках проксимальнее стриктуры (рис. 1). Проанализированы результаты лечения 82 па- циентов с механической желтухой за период с 2007 по 2018 г., получавших лечение на кафедре хирургии общей с клиникой НИИ хирургии и не- отложной медицины ПСПбГМУ им. И. П. Павлова. В исследование включены пациенты с дистальным блоком ЖВП опухолевого генеза. В качестве пер- вичной декомпрессии ЖВП выполнены чрескож- ные чреспеченочные эндобилиарные вмешатель- ства под УЗ- и Rg-контролем. Оригинальную методику чрескожного комби- нированного эндобилиарного дренирования также выполняли под УЗ- и Rg-контролем. Первый этап операции не отличался от стандартного выполне- ния чрескожного наружно-внутреннего эндобили- арного дренирования. Вторым этапом после бужи- рования доступа по проводнику в желчные протоки проксимальнее стриктуры вводили 2-канальный расширитель (диаметр – 10,2 Fr): центральный порт использовали для первичного проводника, а через второй порт вводили дополнительный проводник, дистальный конец которого устанавливали в желч- ных протоках проксимальнее стриктуры (рис. 1). В зависимости от способа первичной эндобили- арной декомпрессии пациенты были разделены на 3 группы. В 1-ю группу вошли 30 пациентов, кото- рым выполняли наружно-внутреннее транспапил- лярное дренирование; во 2-ю группу – 21 паци- ент, которым декомпрессию ЖВП осуществляли с помощью наружного желчеотведения; 3-я груп- па – 31 пациент, которым была выполнена ори- гинальная методика комбинированного наружно- внутреннего дренирования. После установления 2 проводников расширитель удаляли и третьим этапом выполняли установку дре- нажей. По первичному проводнику (установленно- му через стриктуру) в тонкую кишку устанавливали тонкопросветный дренаж (3 Fr), а выше стриктуры по второму проводнику устанавливали второй на- ружный эндобилиарный дренаж (10,2 Fr) (рис. 2). у р др р Все 3 группы были сопоставимы по уровню бло- ка ЖВП, гипербилирубинемии и ее длительности до дренирования, тяжести печеночной недостаточно- сти. У всех пациентов обструкция желчных путей носила злокачественный генез. Средний возраст исследуемых больных составил (65,6±1,4) года. Про- должительность желтухи на момент поступления со- ставляла (16,7±3,49) суток. Средний уровень общего билирубина крови составлял (263,4±161,2) мкмоль/л. МАТЕРИАЛ И МЕТОДЫ ИССЛЕДОВАНИЯ Все пациенты поступали в экстренном порядке в среднетяжелом и тяжелом состоянии. Оценку тяже- сти механической желтухи осуществляли по клас- сификации Э. И. Гальперина, согласно которой, при поступлении 24 (29,3 %) пациента были отнесены к классу С, а остальные 58 (70,7 %) – к классу В тяжести механической желтухи. Тяжесть состояния по шкале SAPS II составляла (36,7±0,6) балла, что соответствует 18,5 %-й вероятной летальности в группе. Консерва- тивная терапия в предоперационном периоде была направлена на коррекцию водно-электролитного баланса, гипокоагуляции, печеночной дисфункции (гепатопротекторы), проводили антисекреторную терапию, антибактериальную профилактику, при- меняли прокинетики (обязательно при наружно- внутреннем транспапиллярном дренировании). Наружный эндобилиарный дренаж, диаметром 10,2 Fr, который устанавливали проксимальнее стриктуры, использовали в дальнейшем исключи- тельно для декомпрессии желчевыводящей системы. Второй тонкопросветный дренаж (3 Fr), уста- новленный через стриктуру в просвете тонкой кишки, имеет отверстие только на дистальном кон- це и используется в дальнейшем для проведения проводника с целью протезирования стриктуры (как правило, для стентирования стриктуры жел- чевыводящих протоков). Помимо этого, данный дренаж в ближайшем послеоперационном пери- оде применяли для возврата желчи в желудочно- кишечный тракт (ЖКТ) и нутритивной поддержки в рамках комплексного лечения пациентов. Статистическая обработка данных выполнена с помощью программы «Statistica 10.0». Для оценки данных использовали дескриптивный анализ. Для проверки значимости отличия процента осложне- ний в разных группах использовали точный крите- рий Фишера. Статистически значимыми считали различия при р<0,05. ВВЕДЕНИЕ составляет 9–33 % [12–14], что, в первую оче- редь, связано с дуоденобилиарным рефлюксом. Развитие холангиогенного сепсиса наблюдается у 42,5 % больных с холангитами, летальность при этом составляет 3,9 % [15]. Особой формой ин- фекционных осложнений чрескожных чреспече- ночных вмешательств (ЧЧВ) являются холангио- генные абсцессы печени, которые наблюдаются в 1,2 % случаев [4] и чаще встречаются также после наружно-внутреннего эдобилиарного дренирова- ния. Данный факт вынуждает минимизировать применение данного вида декомпрессии желче- выводящих путей, ограничиваясь наложением симптоматической наружной холангиостомы, что в большинстве случаев является обременитель- ным для больных, технически утяжеляет прове- дение дальнейших эндобилиарных вмешательств, направленных на протезирование желчных про- токов. С целью профилактики гнойных осложне- ний при проксимальном уровне билиарного блока в последнее время широко используются методы супрапапиллярной установки наружно-внутрен- него дренажа [4], что технически невозможно при дистальном уровне блока. Анализ литературных данных показывает, что выбор первичного на- ружного или транспапиллярного наружно-вну- треннего дренирования при дистальных блоках желчевыводящих протоков неоднозначен. Дан- ный вопрос требует более широкого анализа и поиска методов безопасного первичного эндоби- лиарного дренирования. Чрескожное эндобилиарное дренирование – ведущий метод декомпрессии желчевыводящих путей при механической желтухе опухолевого генеза, а в ряде случаев и холелитиаза. Несмотря на накопленный опыт и отработанную методоло- гию, данные вмешательства имеют свои специфи- ческие осложнения. По данным литературы [1–3], общая частота осложнений чрескожных эндоби- лиарных вмешательств составляет 0,5–32,7 %. Послеоперационная летальность регистрируется на уровне 0,4–13,8 % при лечении механической желтухи [4–8], а при наличии тяжелых сопут- ствующих заболеваний (в том числе печеночно- почечной недостаточности, сепсиса и др.) может достигать 31 % [4]. Особое место в структуре осложнений после антеградных эндобилиарных операций занимают инфекционные осложнения – от острого холан- гита до развития холангиогенного сепсиса. Они встречаются в 15 % наблюдений, а без адекватной антибиотикопрофилактики их частота возраста- ет до 40–58 %, что обусловлено уже нередко име ющейся обсемененностью желчных протоков до дренирования [9, 10]. Частота развития инфекци- онных осложнений пропорциональна продолжи- тельности существования механической желтухи [11]. Основной предпосылкой для их развития яв- ляется длительная механическая желтуха, а непо- средственной причиной – неадекватное дрени- рование [11]. Цель работы – улучшение результатов лече- ния больных с механической желтухой при низ- ком уровне билиарного блока после первичной антеградной эндобилиарной декомпрессии. При выполнении транспапиллярного наруж- но-внутреннего эндобилиарного дренирования частота развития холангитов, вне зависимости от уровня блока желчевыводящих протоков (ЖВП), 36 Gunya Z. A. et al. / The Scientific Notes of Pavlov University Vol. XXVI № 1 (2019) P. 35–41 РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Схема чрескожного комбинированного эндобили- арного дренирования при дистальном блоке ЖВП – в желчных протоках 2-канальный расширитель с провод- никами (1–2-канальный расширитель; 2 – дополнительный проводник находится проксимальнее стриктуры; 3 – первич- ный проводник заведен через стриктуру в двенадцатиперстную кишку; 4 – опухолевая стриктура) Рис. 2. Схема чрескожного комбинированного эндоби- лиарного дренирования при дистальном блоке ЖВП – установлено 2 эндобилиарных дренажа (1 – наружный декомпрессионный эндобилиарный дренаж (10,2 Fr); Рис. 1. Схема чрескожного комбинированного эндобили- арного дренирования при дистальном блоке ЖВП – в желчных протоках 2-канальный расширитель с провод- никами (1–2-канальный расширитель; 2 – дополнительный проводник находится проксимальнее стриктуры; 3 – первич- ный проводник заведен через стриктуру в двенадцатиперстную кишку; 4 – опухолевая стриктура) 2 – дополнительный тонкопросветный дренаж (3 Fr), проведенный через стриктуру в двенадцатиперстную кишку; 3 – опухолевая стриктура) Fig. 2. Scheme of the original technique of the combined external-internal endobiliary drainage in distal biliary Fig. 2. Scheme of the original technique of the combined external internal endobiliary drainage in distal biliary Fig. 1. Scheme of the original technique of the combined external-internal endobiliary drainage in distal biliary block 2-channel dilatator with introducers in bile ducts (1–2-chan- nel dilatator; 2 – additional introducer is installed up to the stricture level; 3 – primary introducer is installed behind the stricture in duo- denum; 4 – tumor stricture) y g y block – 2 endobiliary drainage was installed (1 – external decompression endobiliary drainage (10.2 Fr); 2 – additional thin lumen drainage (3 Fr) installed behind the stricture in duodenum; 3 – tumor stricture) вила 53,3 % (16 пациентов). При этом в основном встречались инфекционные осложнения в виде послеоперационного холангита, обусловленного транспапиллярным дренированием и, как следст- вие, дуоденобилиарным рефлюксом – отмечены в 30 % случаев (9 человек). У 3 (10 %) пациентов течение холангита осложнилось формированием холангиогенных абсцессов печени. РЕЗУЛЬТАТЫ ИССЛЕДОВАНИЯ И ИХ ОБСУЖДЕНИЕ Наружное и наружно-внутреннее эндобили- арное дренирование осуществляли по класси- ческой методике. Под местной анестезией, под УЗ-контролем выполняли пункцию желчного протока правой доли с последующим выполнени- ем Rg-холангиоскопии (определение уровня бло- ка ЖВП, причины и протяженности стриктуры), проведением проводника, бужированием доступа, выполнением дренирования по Сельдингеру – на- ружного или наружно-внутреннего. При наружно- внутреннем дренировании с учетом уровня блока дренажи устанавливали транспапиллярно. В подав- Для сравнительной оценки результатов лечения больных 3 групп было проведено исследование следующих параметров: 1) клиническое и лабораторное купирование желтухи; 2) купирование печеночной дисфункции; 3) частота и характер хирургических осложне- ний, способы их коррекции. Анализ результатов лечения показал лучшие темпы декомпрессии, нормализации печеночной дисфункции у больных с наружным и комбини- 37 Гуня З. А. и др. / Ученые записки С рованным эндобилиарным дренирование объясняется методиками операции, позвол ми контролировать темп и качество декомп ЖВП, отсутствием предрасположенности тия восходящей инфекции вследствие реф кишечного содержимого. Темпы декомп при различных способах антеградного др вания отражены на рис. 3. Интраоперационные и ранние послеоп онные осложнения зарегистрированы у 23 человек. Анализ частоты развития послеоп онных осложнений при разных видах антег эндобилиарной декомпрессии показал, что нения после наружно-внутреннего транспап ного дренирования встречались значительн (р=0,0004). Достоверных различий между ча возникновения осложнений в группах наруж комбинированного дренирования не было (р В структуре хирургических осложнений транспапиллярных вмешательств преоб осложнения инфекционного характера, к диагностированы в 40 % случаев (n=12). С ра хирургических осложнений приведена в Осложнения в группе наружного эндоб ного дренирования отмечены у 4 (19 %) пац Они были представлены геморрагическими нениями (9,5 %) и желчеистечением, как пр обусловленным дислокацией дренажа (9,5 Во 2-й группе послеоперационные ос ния наблюдали наиболее часто. Их частота Рис. 1. Схема чрескожного комбинированного эн арного дренирования при дистальном блоке Ж в желчных протоках 2-канальный расширитель с никами (1–2-канальный расширитель; 2 – дополнит проводник находится проксимальнее стриктуры; 3 – ный проводник заведен через стриктуру в двенадцатип кишку; 4 – опухолевая стриктура) Fig. 1. Scheme of the original technique of the com external-internal endobiliary drainage in distal bilia 2-channel dilatator with introducers in bile ducts (1– nel dilatator; 2 – additional introducer is installed up to th level; 3 – primary introducer is installed behind the strictu denum; 4 – tumor stricture) Гуня З. А. и др. / Ученые записки СПбГМУ им. акад. И. П. Павлова Т. XXVI № 1 (2019) С. 35–41 Рис. 1. вила 53,3 % (16 пациентов). При этом в основном встречались инфекционные осложнения в виде послеоперационного холангита, обусловленного транспапиллярным дренированием и, как следст- вие, дуоденобилиарным рефлюксом – отмечены в 30 % случаев (9 человек). У 3 (10 %) пациентов течение холангита осложнилось формированием холангиогенных абсцессов печени. рованным эндобилиарным дренированием, что объясняется методиками операции, позволяющи- ми контролировать темп и качество декомпрессии ЖВП, отсутствием предрасположенности разви- тия восходящей инфекции вследствие рефлюкса кишечного содержимого. Темпы декомпрессии при различных способах антеградного дрениро- вания отражены на рис. 3. вила 53,3 % (16 пациентов). При этом в основном встречались инфекционные осложнения в виде послеоперационного холангита, обусловленного транспапиллярным дренированием и, как следст- вие, дуоденобилиарным рефлюксом – отмечены в 30 % случаев (9 человек). У 3 (10 %) пациентов течение холангита осложнилось формированием холангиогенных абсцессов печени. Интраоперационные и ранние послеопераци- онные осложнения зарегистрированы у 23 (28 %) человек. Анализ частоты развития послеопераци- онных осложнений при разных видах антеградной эндобилиарной декомпрессии показал, что ослож- нения после наружно-внутреннего транспапилляр- ного дренирования встречались значительно чаще (р=0,0004). Достоверных различий между частотой возникновения осложнений в группах наружного и комбинированного дренирования не было (р=0,42). В структуре хирургических осложнений после транспапиллярных вмешательств преобладают осложнения инфекционного характера, которые диагностированы в 40 % случаев (n=12). Структу- ра хирургических осложнений приведена в табл. 1. При выполнении комбинированного наружно- внутреннего дренирования гнойные осложнения, дислокация дренажной системы не были диагно- стированы ни у одного пациента. У 1 (3,2 %) паци- ента выявлена гемобилия, и у 2 (6,6 %) – желче истечение по типу желчного плеврита. При лечении всех осложнений были успешно использованы мини-инвазивные вмешательства. Структура осложнений с точки зрения их лечения приведена в табл. 2. Во 2-й группе больных при выявлении инфек- ционных осложнений тактика лечения предусмат ривала налаживание адекватного антеградного желчеоттока, которое осуществляли путем посто- янного (или фракционного) проточного промыва- ния эндобилиарного дренажа, продолжительного проведения антибактериальной терапии. Поло- жительная динамика в виде купирования холан- гита, регресса желтухи отмечена у 9 пациентов. Осложненное течение холангитов с формирова- нием холангиогенных абсцессов печени возникло у 3 пациентов и потребовало перевода наружно- Осложнения в группе наружного эндобилиар- ного дренирования отмечены у 4 (19 %) пациентов. Они были представлены геморрагическими ослож- нениями (9,5 %) и желчеистечением, как правило, обусловленным дислокацией дренажа (9,5 %). Во 2-й группе послеоперационные осложне- ния наблюдали наиболее часто. Их частота соста- 38 Gunya Z. A. et al. / The Scientific Notes of Pavlov University Vol. XXVI № 1 (2019) P. 35–41 0 50 100 150 200 250 300 1 7 Уровень билирубина, мкмоль/л Сутки после дренирования Наружное дренирование Наружно-внутреннее дренирование Комбинированное дренирование Рис. 3. Темпы декомпрессии при разных способах антеградного эндобилиарного дренирования Fig. 3. The rate of decompression with different methods of transhepatic endobiliary drainage папиллярного наружно-внутреннего дренирования при дистальных блоках желчевыводящих протоков неоднозначен. вила 53,3 % (16 пациентов). При этом в основном встречались инфекционные осложнения в виде послеоперационного холангита, обусловленного транспапиллярным дренированием и, как следст- вие, дуоденобилиарным рефлюксом – отмечены в 30 % случаев (9 человек). У 3 (10 %) пациентов течение холангита осложнилось формированием холангиогенных абсцессов печени. Данные методы антеградной эндо- билиарной декомпрессии имеют свои положитель- ные и отрицательные стороны. Главным плюсом первичного наружно-внутреннего дренирования является факт прохождения стриктуры, что спо- собствует в дальнейшем технически простому протезированию ЖВП, обеспечению поступления желчи в просвет ЖКТ. Отрицательной стороной данной методики является отсутствие контроля за темпом декомпрессии желчных путей и характером отделяемого, что важно в случае развития ослож- папиллярного наружно-внутреннего дренирования при дистальных блоках желчевыводящих протоков неоднозначен. Данные методы антеградной эндо- билиарной декомпрессии имеют свои положитель- ные и отрицательные стороны. Главным плюсом первичного наружно-внутреннего дренирования является факт прохождения стриктуры, что спо- собствует в дальнейшем технически простому протезированию ЖВП, обеспечению поступления желчи в просвет ЖКТ. Отрицательной стороной данной методики является отсутствие контроля за темпом декомпрессии желчных путей и характером отделяемого, что важно в случае развития ослож- внутреннего эндобилиарного дренажа в наружную холангиостому. Помимо предотвращения реф- люкса, обеспечения адекватного наружного жел- чеотведения, с целью санации абсцессов печени также выполняли пункционно-дренирующие вме- шательства: в 1 случае – пункционные санации, в 2 – чрескожное дренирование гнойных очагов. В связи с септическим состоянием данные паци- енты получали лечение в отделении реанимации и интенсивной терапии, 1 больная погибла от нара- стающей полиорганной недостаточности. внутреннего эндобилиарного дренажа в наружную холангиостому. Помимо предотвращения реф- люкса, обеспечения адекватного наружного жел- чеотведения, с целью санации абсцессов печени также выполняли пункционно-дренирующие вме- шательства: в 1 случае – пункционные санации, в 2 – чрескожное дренирование гнойных очагов. В связи с септическим состоянием данные паци- енты получали лечение в отделении реанимации и интенсивной терапии, 1 больная погибла от нара- стающей полиорганной недостаточности. Анализируя собственные результаты, мы соглас- ны, что выбор первичного наружного или транс- Т а б л и ц а 1 Структура осложнений у больных с дистальным блоком ЖВП после первичных эндобилиарных вмешательств T a b l e 1 Structure of complications in patients with distal biliary block after primary percutaneous endobiliary drainage Осложнения Вид эндобилиарной декомпрессии наружное (n=21) наружно-внутреннее (n=30) комбинированное (n=31) Геморрагические Гемобилия 1 (4,8) 1 (3,3) 1 (3,2) Поддиафрагмальная гематома 1 (4,8) – – Гемоперитонеум – 1 (3,3) – Желчеистечение Отграниченный желчный затек 1 (4,8) – – Желчный плеврит – 1 (3,3) 2 (6,5) Диффузный желчный перитонит 1 (4,8) 1 (3,3) – Инфекционные Холангит – 9 (30,0) – Абсцедирующий холангит – 3 (10,0) – В с е г о (n=23, 28,0 %) 4 (19,0) 16 (53,3) 3 (9,7) П р и м е ч а н и е: здесь и далее в скобках – %. вила 53,3 % (16 пациентов). При этом в основном встречались инфекционные осложнения в виде послеоперационного холангита, обусловленного транспапиллярным дренированием и, как следст- вие, дуоденобилиарным рефлюксом – отмечены в 30 % случаев (9 человек). У 3 (10 %) пациентов течение холангита осложнилось формированием холангиогенных абсцессов печени. Т а б Структура осложнений у больных с дистальным блоком ЖВП после первичных эндобилиарных вмеша Т а б л и ц а 1 ожнений у больных с дистальным блоком ЖВП после первичных эндобилиарных вмешательств T a b l e 1 а б л ц а Структура осложнений у больных с дистальным блоком ЖВП после первичных эндобилиарных вмешательств T a b l e 1 Structure of complications in patients with distal biliary block after primary percutaneous endobiliary drainage 39 Гуня З. А. и др. / Ученые записки СПбГМУ им. акад. И. П. Павлова Т. XXVI № 1 (2019) С. 35–41 Т а б л и ц а 2 Структура послеоперационных осложнений и их лечения T a b l e 2 Structure of postoperative complications and their treatment Осложнение Вид лечения Всего консервативная терапия чрескожные операции* эндовидеохирургические операции (общая анестезия) Гемобилия 3 – – 3 Поддиафрагмальная гематома – 1 – 1 Гемоперитонеум – 1 – 1 Отграниченный желчный затек – 1 – 1 Желчный плеврит – 3 – 3 Диффузный желчный перитонит – 1 1 2 Холангит 9 – – 9 Холангиогенные абсцессы – 3 – 3 Степень осложнений по Clavien – Dindo I (52,2) IIIa (43,5) IIIb (4,3) 23 (100) * – операции, выполненные под местной анестезией, не требующие анестезиологического пособия. Т а б л и ц а 2 Структура послеоперационных осложнений и их лечения нений послеоперационного периода. Но главным сдерживающим моментом выполнения наружно- внутреннего дренирования является возможность развития гнойного холангита и других септических осложнений, в патогенезе которого ведущую роль играет дуоденобилиарный рефлюкс содержимого через билиарный дренаж. По данным литературы [16, 17], частота этих осложнений возрастает при нарушении моторики двенадцатиперстной кишки и небольшой продукции желчи. Высокий риск раз- вития гнойно-септических осложнений, как прави- ло, заставляет хирургов первично выполнять только наружное дренирование ЖВП. исключить инфекционные осложнения, связанные с дуоденобилиарным рефлюксом. С другой сторо- ны, наличие внутреннего дренажа обеспечивает возможность возврата желчи в пищеварительный тракт и проведение эндобилиарных вмешательств в дальнейшем (стентирование, фотодинамическая терапия и т. д.), минимизирует риск дислокации дренажных систем. Соответствие нормам этики Авторы подтверждают, что соблюдены права людей, принимавших участие в исследовании, включая получе- ние информированного согласия в тех случаях, когда оно необходимо, и правила обращения с животными в случаях их использования в работе. Подробная информация со- держится в Правилах для авторов. Оригинальная методика комбинированного антеградного эндобилиарного дренирования, на наш взгляд, сочетает лучшие стороны наружного и наружно-внутреннего методов. С одной стороны, обеспечение желчеотведения через наружный дре- наж позволяет контролировать темпы и качество декомпрессии ЖВП (в том числе при геморрагиче- ских осложнениях по типу гемобилии), полностью Conflict of interest Conflict of interest Authors declare no conflict of interest. Authors declare no conflict of interest. ВЫВОДЫ 1. Полученные результаты показали преимуще- ства предлагаемой методики комбинированного эндобилиарного дренирования. В отличие от наружно-внутреннего эндоби- лиарного дренирования, при наружном имеется возможность контроля темпа и качества деком- прессии, отсутствует возможность восходящей инфекции вследствие рефлюкса кишечного содер- жимого. Отрицательными сторонами этого вида операций являются, во-первых, необходимость прохождения стриктуры в дальнейшем при про- должении мини-инвазивного чрескожного лече- ния, что не всегда технически возможно при про- грессии заболевания. Во-вторых, после наружной эндобилиарной декомпрессии всегда возникает необходимость возврата желчи в пищеваритель- ный тракт, что, как правило, требует дополнитель- ных (и не всегда возможных) вмешательств (эндо- скопическая постановка интестинального зонда, эндоскопическая гастростомия). 2. Отсутствие риска инфекционных осложне- ний в послеоперационном периоде с одновремен- ным обеспечением транспапиллярного доступа позволяет рекомендовать данное вмешательство при первичной антеградной эндобилиарной де- компрессии в случаях дистального блока. Конфликт интересов Конфликт интересов Авторы заявили об отсутствии потенциального конф ликта интересов. REFERENCES 1. Гальперин Э. И., Котовский А. Е. Темп декомпрес- сии желчных протоков при механической желтухе опу- холевой этиологии // Хирургия. – 2011. – № 8. – С. 33–42. 1. Gal’perin Je. I., Kotovskij A. E. Temp dekompressii zhelchnyh protokov pri mehanicheskoj zheltuhe opuholevoj jetiologii. Khirurgija. 2011;(8):33–42. (In Russ.). 2. Каримов Ш. И., Хакимов М. Ш., Адылходжаев А. А. и др. Лечение осложнений чреспеченочных билиарных вмешательств при механической желтухе, обусловленной периампулярными опухолями // Анн. хирург. гепатол. – 2015. – № 3. – С. 68–74. 2. Karimov Sh. I., Hakimov M. Sh., Adylhadzhaev F. F, Rah- manov S. U., Hasanov V. R. Lechenie oslozhnenij chreskozhnyh chrespechenochnyh jendobiliarnyh vmeshatel’stv pri mehanich- eskoj zheltuhe, obuslovlennoj periampuljarnymi opuholjami. Annaly khirurgicheskoj gepatologii. 2015;(3):68–74. (In Russ.). 3. Кубачев К. Г., Борисов А. Е., Изудинов А. С. Выбор способа дренирования желчных протоков при механиче- ской желтухе опухолевого генеза // Анн. хирург. гепатол. – 2009. – Т. 14, № 3. – С. 56–62. 3. Kubachev K. G., Borisov A. E., Izudinov A. S. Vybor sposoba drenirovaniya zhelchnykh protokov pri mekhanich- eskoy zheltukhe opukholevogo geneza. Annaly khirurgich- eskoy gepatologii. 2009;14(3):56–62. (In Russ.). 4. Кулезнева Ю. В., Израилов Р. Е., Капустин В. И. Такти- ка антеградной билиарной декомпрессии при механической желтухе опухолевого генеза // Вестн. Нац. медико-хирург. центра им. Н. И. Пирогова. – 2010. – Т. 5, № 2. – С. 24–28. 4. Kulezneva Yu. V., Izrailov R. E., Kapustin V. I. Takti- ka antegradnoy biliarnoy dekompressii pri mekhanicheskoy zheltukhe opukholevogo geneza. Vestnik Natsional’no- go mediko-khirurgicheskogo tsentra im. N. I. Pirogova. 2010;5(2):24–28. (In Russ.). 5. Лукичев О. Д., Ившин В. Г., Макаров Ю. И. и др. Сравнительный анализ различных методик чрескожного чреспеченочного желчеотведения у больных с механиче- ской желтухой опухолевой этиологии // Вестн. РОНЦ им. Н. Н. Блохина РАМН. – 2004. – Т. 15, № 1–2. – C. 121–125. 5. Lukichev O. D., Ivshin V. G., Makarov Ju. I. i dr. Sravni- tel’nyj analiz razlichnyh metodik chreskozhnogo chrespeche- nochnogo zhelcheotvedenija u bol’nyh s mehanicheskoj zhel- tuhoj opuholevoj jetiologii. Vestnik RONC im. N. N. Blohina RAMN. 2004;15(1–2):121–125. (In Russ.). 6. Рыбачков В. В., Дряженков Т. Г., Кабанов Е. Н. При- чины эндогенной интоксикации при гнойном холангите // Анн. хирург. гепатол. – 2009. – Т. 14, № 2. – С. 28–32. 6. Rybachkov V. V., Dryazhenkov T. G., Kabanov E. N. Prichiny endogennoy intoksikatsii pri gnoynom kholangite. An- naly khirurgicheskoy gepatologii. 2009;14(2):28–32. (In Russ.). 7. REFERENCES Диагностика и хирургическая тактика при синдроме механической желтухи / Ю. Л. Шевченко, П. С. Ветшев, Ю. М. Стойко, А. Л. Левчук // Вестн. Нац. медико-хирург. центра им. Н. И. Пирогова. – 2008. – Т. 13, № 4. – С. 96–105. 7. Shevchenko Yu. L., Vetshev P. S., Stoyko Yu. M., Levchuk A. L. Diagnostika i khirurgicheskaya taktika pri sindrome mekhanicheskoy zheltukhi. Vestnik Natsional’no- go mediko-khirurgicheskogo tsentra im. N. I. Pirogova. 2008;13(4):96–105. (In Russ.). 8. Fang Y., Gurusamy K. S., Wang Q. et al. Preoperative biliary drainage fore obstructive jaundice // Cochrane Data- base Syst. Rev. – 2012. – № 12. – Р. 9. 8. Fang Y., Gurusamy K. S., Wang Q. et al. Preoperative biliary drainage fore obstructive jaundice. Cochrane Database Syst Rev. 2012;(12):9. 9. Rosch T., Trihtard A., Chigogize N. еt al. Bacteriobilia in percutaneous transhepatic biliary drainage occurrence over time and clinical sequeial. A prospective observational study // Scanal. J. Gastroenterol. – 2003. – Vol. 38, № 11. – Р. 1162–1168. 9. Rosch T., Trihtard A., Chigogize N. et al. Bacteriobilia in percutaneous transhepatic biliary drainage occurrence over time and clinical sequeial. A prospective observational study. Scanal J Gastroenterol. 2003;38(11):1162–1168. 10. Kucukav F., Okten R. S., Cumhur T. Percutaneous biliary intervention for primary sclerosing cholangitis in a patient with situs inversus totalis // Turk. J. Gastroenterol. – 2011. – Vol. 22, № 6. – Р. 636–640. 10. Kucukav F., Okten R. S., Cumhur T. Percutaneous biliary intervention for primary sclerosing cholangitis in a patient with situs inversus totalis. Turk J Gastroenterol. 2011;22(6):636–640. 11. Brody L. A., Brown K. T., Getrajdman G. I. et al. Clinical factors associated with positive bile cultures during primary percutaneous biliary drainage // Journ. of Vascular and Inter- ventional Radiology. – 1998. – Vol. 9, № 4. – Р. 572–578. 11. Brody L. A., Brown K. T., Getrajdman G. I. et al. Clinical factors associated with positive bile cultures during primary percutaneous biliary drainage. Journal of Vascular and Interventional Radiology. 1998;9(4):572–578. 12. Salmiuen P., Laine S., Gullichsen R. Severe and fatal complicaitions after ERCP: analysis of 2555 procedures in a single experienced center // Surg. Endoscopy. – 2008. – Vol. 22, № 9. – Р. 1965–1970. 12. Salmiuen P., Laine S., Gullichsen R. Severe and fatal complicaitions after ERCP: analysis of 2555 procedures in a single experienced center. Surg Endoscopy. 2008;22(9):1965–1970. 13. Compliance with ethical principles The authors confirm that they respect the rights of the people participated in the study, including obtaining informed consent when it is necessary, and the rules of treatment of animals when they are used in the study. Author Guidelines contains the detailed information. 40 Gunya Z. A. et al. / The Scientific Notes of Pavlov University Vol. XXVI № 1 (2019) P. 35–41 REFERENCES British society of interventional radiolodgy: Billiary drainage and stenting resistry (BDCR) / R. Uberoi, N. Bas, J. Moss, I. Robertson // Cardiovasc. Intervent. Radiol. – 2012. – № 35. – Р. 127–138. 13. Uberoi R., Bas N., Moss J., Robertson I. British society of interventional radiolodgy: Billiary drainage and stenting resistry (BDCR). Cardiovasc Intervent Radiol. 2012;(35):127–138. 14. Winikc A. B., Waybill P. N., Venbrux A. C. Complicai- tions of transhepatic biliary interventions // Tech. Vasc. Interv. Radiol. – 2001. – № 4. – Р. 200–206. 14. Winikc A. B., Waybill P. N., Venbrux A. C. Compli caitions of transhepatic biliary interventions. Tech Vasc Interv Radiol. 2001;(4):200–206. 15. Malignant biliary obstruction: can we predict imme- diate postprosedural cholangitis after percutaneous biliary drainage? / Abn Shin, Lee Yoon-Seon, R. S. Lion, S. L. Lee // Surgeon Care Cancel. – 2013. – № 21. – Р. 2321–2326. 15. Shin Abn, Yoon-Seon Lee, Lion R. S., Lee S. L. Malignant biliary obstruction: can we predict immediate postprosedural cholangitis after percutaneous biliary dra inage? Surgeon Care Cancel. 2013;(21):2321–2326. 16. Кулезнева Ю. В., Бруслик С. В., Мусаев Г. Х. и др. Антеградные методы декомпрессии желчных протоков: эволюция и спорные вопросы // Анн. хирург. гепатол. – 2011. – Т. 16, № 3. – С. 35–41. 16. Kulezneva Yu. V., Bruslik S. V., Musaev G. H., Izrai lov R. E., Kirillova M. S. Antegradnye metody dekompressii zhelchnyh protokov: evolyuciya i spornye voprosy. Annaly khirurgicheskoj gepatologii. 2011;16(3):35–41. (In Russ.). 17. Борисова Н. А., Борисов А. Е., Кареев А. В. Эндоби- лиарные методы коррекции механической желтухи: руко- водство по хирургии печени и желчевыводящих путей. Т. 2 / под ред. А. Е. Борисова. – СПб.: Скифия, 2003. – С. 254–281. 17. Borisova N. A., Borisov A. E., Kareev A. V. Endobiliarnye metody korrekcii mekhanicheskoj zheltuhi: Rukovodstvo po hirurgii pecheni i zhelchevyvodyashchih putej. T. 2. Pod red. Borisova A. E. SPb., Skifiya, 2003:254–281. (In Russ.). 41
W2462665058.txt	http://www.periodicos.letras.ufmg.br/index.php/cesp/article/download/6641/5641	fr		Outra era a vez: reescrevendo Chapeuzinho Vermelho	Revista do Centro de Estudos Portugueses	2,006	cc-by	3,695	Velha história em roupa nova: revisitando Chapeuzinho Vermelho Ana Maria Clark Peres Universidade Federal de Minas Gerais O riginários de antigas lendas e mitos, e de início transmitidos oralmente de geração em geração, os contos de fadas vêm seduzindo há séculos crianças dos mais diversos países, além de constituírem objeto de estudo de inúmeros pesquisadores. Dentre os contos adaptados e registrados em livro por Charles Perrault no final do século XVII, na França, e pelos irmãos Grimm no início do século XIX, na Alemanha, há longos anos venho destacando um em especial: Chapeuzinho Vermelho.1 Inicialmente, interessei-me em investigar as variadas traduções brasileiras do conto francês e também da versão alemã, tendo mais tarde procurado analisar algumas recriações livres da antiga narrativa, destinadas ou não às crianças, como a de Chico Buarque, a de Guimarães Rosa e a do teatrólogo Antunes Filho. Instigada pelo fato de a história não cessar de chamar a atenção de crianças e adultos ainda hoje, mais recentemente optei por verificar como vem se apresentando o antigo conto em suportes 1 Vale ressaltar que “Chapeuzinho Vermelho” é, originalmente, um conto admonitório, que recebeu um tratamento literário por parte de Perrault. De acordo com estudiosos de linha francesa, ao adaptar por escrito diversas narrativas populares e orais, Perrault inaugura o “gênero literário” conto de fadas, o qual não necessita obrigatoriamente apresentar fadas em seu enredo. Com relação a esse “gênero”, destaque-se, por exemplo, a cuidadosa publicação dirigida por Olivier Piffault, Il était une fois... les contes de fées, lançada por ocasião da exposição de mesmo nome organizada pela Biblioteca Nacional da França em 2001. O site da referida exposição está disponível em <http://expositions.bnf.fr/contes/index.htm>. 191 Velha história em roupa nova:... Ana Maria Clark Peres – p.191-200 tecnológicos atuais: CD, CD-ROM, fita VHS, DVD e e-book. As seguintes questões iniciais nortearam minha nova investigação: ao ser transmitida por meio desses novos suportes, a velha história da menininha que atravessa um bosque para ir visitar sua avó e no meio do caminho encontra um lobo mau, teria sofrido alterações inventivas em sua estrutura narrativa que visassem a adequá-la às especificidades das crianças contemporâneas? Ou, diferentemente, foram mantidos fielmente os enredos clássicos de Perrault e dos irmãos Grimm? A essas questões, outras se somaram no desenrolar da pesquisa, quais sejam: por que o velho conto ainda interessaria tanto o público infantil e mesmo o adulto, continuando a ser oferecido às crianças do século XXI? Que ideal de criança subjaz às produções brasileiras contemporâneas, comparado ao ideal de criança dos séculos XVII, XVIII e XIX na Europa, ocasião em que versões escritas de Chapeuzinho Vermelho fizeram tanto sucesso? Novos recursos tecnológicos seriam sempre capazes de provocar inovações significativas de histórias tradicionais, constituindo, de fato, um espaço de reinvenção? Buscando responder a essas questões, opto por focalizar inicialmente alguns pontos das adaptações escritas de Perrault e Grimm, que, sem dúvida, provocaram as novas versões. Com relação ao texto de Perrault, graças a pesquisas de Paul Delarue e Marie-Louise Tenèze, podemos ter hoje uma idéia das versões orais que inspiraram o escritor francês, das quais destaco as seguintes invariantes: a menina que leva pão e leite a sua avó, a mando da mãe, não usa capuz vermelho ou de qualquer outra cor; cenas violentas se apresentam na narrativa (o animal mata a avó, despeja seu sangue numa garrafa e corta sua carne em fatias, colocando tudo em uma travessa sobre uma arca), incluindo-se aí cenas de canibalismo (o lobo sugere à menina que coma aquele “alimento” e tome aquele “vinho”, no que é prontamente atendido); ao chegar à casa da avó, a garota recebe logo um convite para tirar a roupa e se deitar na cama com o animal; o que acontece em seguida pode ser considerado um verdadeiro striptease da menina, que, após desvencilhar-se de cada peça de roupa, pergunta ao lobo onde colocá-la, escutando dele a indicação de que deve jogá-la no fogo; finalmente, uma vez deitada na cama com o animal, a menina lhe faz perguntas que refletem seu estranhamento quanto aos pêlos, os ombros, as unhas e os dentes do lobo, que acaba por comê-la após a última pergunta.2 2 Cf. DARNTON, 1986, p. 21-22. 192 Revista do CESP – v. 26, n. 35 – jan.-jun. 2006 Perrault suprime os elementos mais “chocantes” dessas versões orais, por exemplo, a cena em que a criança é convidada a comer a carne e o sangue da avó. Omite, igualmente, o lento strip-tease da menina diante do lobo, mantendo, contudo, uma boa dose de erotismo em sua narrativa, já que Chapeuzinho, “a mais linda” de quantas já se viram, se deita, nua, na cama em que está o animal e logo faz referência a suas pernas. O objetivo do conto é, sem dúvida, alertar as crianças contra os “perigos” do sexo. Senão, vejamos a moral após o relato: que as meninas, sobretudo as mais bonitinhas, não parem para escutar estranhos, principalmente os lobos mais doces e gentis, que, dentre todos, são os mais perigosos. Não podemos nos esquecer de que, nessa época, uma nova concepção de infância começa a predominar na sociedade francesa: consideradas como seres inocentes (assexuados), ingênuos, frágeis, débeis, irracionais, as crianças vão sendo descobertas, particularizadas e isoladas do mundo adulto capaz de “corrompê-las”. É para essas crianças – para educá-las e distraí-las – que escreve Perrault (recordemos que, em sua narrativa, Chapeuzinho, além de “linda”, é também uma “pobre menina” que não sabia como é perigoso parar para escutar um lobo). Apoiando-se nessa visão da infância, o autor teria identificado a mentalidade popular à mentalidade infantil, ambas pouco desenvolvidas – a primeira, devido à condição social; a segunda, à idade – e é considerado um dos criadores da literatura infantil.3 O principal acréscimo introduzido em sua narrativa concerne ao chapeuzinho vermelho usado pela menina com tanta freqüência, que passa a nomeá-la. Mais de cem anos após a publicação de Perrault, começam a surgir na Alemanha, em 1812, as adaptações de contos populares feitas pelos irmãos Grimm, filólogos e folcloristas que se preocuparam em fixar as narrativas orais de seu país. Se compararmos a versão alemã de Chapeuzinho Vermelho com a francesa, encontraremos várias diferenças, dentre as quais ressalto as seguintes: a menina não é “a mais linda”, mas “pequena e meiga”; entregando à filha um bolo e uma garrafa de vinho para serem levados à avó (e não o potinho de manteiga, como em Perrault), a mãe de Chapeuzinho Vermelho lhe dá inúmeras lições de bom comportamento, inexistentes na versão francesa; ao chegar à casa da avó, a menina não se deita na cama com o lobo 3 A esse respeito, remeto o leitor para os trabalhos de Philippe Ariès, História social da criança e da família, e de Marc Soriano, Contes de Perrault: culture savante et traditions populaires. 193 Velha história em roupa nova:... Ana Maria Clark Peres – p.191-200 (tampouco é convidada a fazê-lo); no diálogo com o animal, ela não menciona as pernas do lobo; o desenlace não é trágico, como na versão francesa, mas feliz, com a intervenção providencial de um caçador, que salva Chapeuzinho e sua avó; o lobo morre, e a menina promete nunca mais desobedecer à mãe. Os Grimm publicaram também um outro conto, que relata uma nova visita de Chapeuzinho Vermelho à avó: chegando à floresta, mais um lobo tenta desviá-la do caminho, mas ela não lhe dá atenção, pois, de fato, aprendera a lição; já na casa da avó, as duas armam um plano para destruir o animal. Observemos que a versão dos irmãos Grimm, bem menos elaborada que a de Perrault, incorpora à narrativa lições explícitas a serem transmitidas às crianças, além de atenuar as cenas mais “eróticas” do conto francês. Tudo isso em nome de um ideal de criança, inocente, cuja “pureza”, “ingenuidade” e “fragilidade” devem ser preservadas (lembremos que a moral de Perrault é fora da história e comumente não aparece nas traduções do conto no Brasil). A partir dessas duas adaptações clássicas, Chapeuzinho Vermelho recebeu, nos mais diversos países, inúmeras traduções e adaptações. Numa pesquisa anterior, em que considerei quarenta dessas traduções, publicadas no Brasil de 1953 a 1998, cheguei a levantar as seguintes insistências nos textos traduzidos ou adaptados: omissão de partes da narrativa, sobretudo de cenas que envolvem a maldade do lobo, bem como o convite que faz à menina para se deitar com ele, além da morte da avó e de Chapeuzinho; acréscimo de informações as mais variadas (muita ação, diálogos e descrições extras, intensificação da afetividade, através do uso de diminutivos e adjetivos – clichês de linguagem); ênfase no tom moralizador.4 Posteriormente, como já foi dito, analisei algumas recriações de Chapeuzinho Vermelho, destinadas ou não às crianças, a saber: Chapeuzinho Amarelo, de Chico Buarque, “Fita verde no cabelo (Nova velha estória)”, de Guimarães Rosa, e a peça “Nova velha história”, de Antunes Filho. Dialogando com proposições lacanianas sobre os registros do Simbólico, do Imaginário e do Real, intentei abordar a especial relação que essas recriações mantêm com o Real, tendo em vista especificidades temporais e culturais. Surpreenderam-me, igualmente, as conclusões desse estudo, pois, nele, pude perceber que as produções que trazem um nível mais radical de invenção (e que são capazes de tocar em fragmentos de Real, no fora de sentido) não são inicialmente endereçadas às crianças, como é o caso do conto de Guimarães Rosa e da peça de Antunes Filho. 4 Cf. PERES, 2000, p. 186-187. 194 Revista do CESP – v. 26, n. 35 – jan.-jun. 2006 Mas, lidando ainda com Chapeuzinho Vermelho, o objetivo deste trabalho, insisto, foi verificar como vem se apresentando o antigo conto em suportes tecnológicos atuais. Para a escolha do corpus da investigação, utilizei um site de procura da internet e lojas virtuais, onde adquiri três CDs, dois CD-ROMs, uma fita VHS e um DVD. De início, destaco que as imagens dos CD-ROMs se fazem acompanhar de um texto escrito. O e-book, por sua vez, foi baixado gratuitamente de um site especializado. Busquei ter acesso, pois, àquele material que mais facilmente chega às mãos dos interessados, sejam eles crianças ou adultos, numa facilidade que o mundo contemporâneo, com sua tecnologia avançada, pôs à nossa disposição. A análise dessas oito novas versões do “velho” conto continuou me surpreendendo, e é um apanhado do trabalho que apresento a seguir. Das oito versões, cinco (apresentadas em dois CDs, dois CD-ROMs e no DVD) não indicam se se trata de adaptação de Perrault ou dos irmãos Grimm, mas o final feliz presente em todas elas aproxima-as, de alguma maneira, da versão alemã. Duas afirmam ser originárias de Perrault (fita VHS e e-book). Destas, uma, a do e-book, mistura elementos do conto francês e do alemão: o começo é de Perrault, e o final é dos Grimm; a outra, aproximase apenas do conto dos Grimm. Considerando que a história é apresentada em suportes tecnológicos inovadores, e já que não se mantêm intactos os enredos clássicos de Perrault e Grimm, era de se esperar que houvesse também uma reinvenção da trama do relato, mas essa expectativa acabou não se confirmando. É bem verdade que algumas inovações puderam ser percebidas, e são elas que destaco inicialmente. Em certas versões, por exemplo, ao encontrar-se com Chapeuzinho na floresta, o lobo apresenta-se à menina disfarçado, fato inexistente nos contos originais, sendo que ele se disfarça de viajante, de lobo vegetariano ou declara ser médico e chamar-se Dr. Vilas Lobo (isso em CDs). Já num CDROM, o lobo, escondido no meio de folhagens, afirma ser o Anjo da Floresta. Na fita de VHS, sua primeira aparição acontece atrás de uma árvore; semiescondido, tem uma pele de cordeiro sobre os ombros. Logo a seguir, vemos o animal fantasiado de lenhador e, mais tarde, de velho, com barbas brancas. Já na fita de DVD, ele se fantasia de borboleta, e tem jeito de bebê, na imagem. Vários desses disfarces do lobo visam a algum tipo de adequação ao mundo contemporâneo e também à tentativa de introdução do humor nas histórias. O CD cuja história é narrada por Lilia Cabral, atriz da Rede Globo de Televisão, é o que mais enfatiza esses aspectos. Nele, o lobo é roqueiro, 195 Velha história em roupa nova:... Ana Maria Clark Peres – p.191-200 “metaleiro”, usa gírias com freqüência (“Pintava no pedaço”; “Cheguei, macacada”; “E aí, gata, está a fim de curtir um som da pesada?” etc). Diz-se que ele tem uma longa cabeleira oxigenada e veste uma camiseta onde se lê: “Fera radical”. Usa também jaqueta de couro, pulseira em todas as patas, brincos nas orelhas, no nariz e no rabo, bem como óculos escuros espelhados. Toca guitarra e tem um walkman “martelando rock pauleira a todo volume”. Ao ser indagado por Chapeuzinho sobre sua voz, afirma, mais no final da história: “Minha voz continua a mesma, mas os meus cabelos...”, remetendo-nos a uma antiga propaganda de xampu apresentada na televisão. Quando é ameaçado pelos lenhadores, o animal pede-lhes que não sejam violentos, “pois tem criança no recinto”. Ainda nesse CD, encontramos, igualmente, um apelo à ecologia: a avó, vegetariana, havia se mudado para o campo em razão da poluição das cidades grandes. Mais uma inovação encontrada nas versões analisadas é a tentativa de contextualização da história, com a intromissão de motivos brasileiros. Por exemplo, em algumas delas, Chapeuzinho leva à avó biscoitos de polvilho, compota, pão de queijo. Em outra, um bem-te-vi, que é o narrador, bem como os caçadores que aparecem no final têm sotaque do interior paulista ou mineiro, apresentando-se em cena com música caipira ao fundo. Em determinada versão, quando a Chapeuzinho desaparece porque foi comida pelo animal, a mãe acredita que a filha foi seqüestrada. Uma outra novidade presente na história narrada pela atriz global acontece quando, após se afirmar que a menina não seguiu os conselhos da mãe, acrescenta-se: “Não é só porque era esquecida, mas se fizesse tudo certinho, não haveria história para ser contada.” Outras inovações, estas dos CD-ROMs, são os jogos interativos que acompanham a história, dos mais variados tipos: quebra-cabeça; jogo da memória; cruzadinha; jogo dos 5 erros etc. É importante ressaltar que o CDROM da Coleção Disquinho apresenta um acabamento muito superior aos demais, e a voz de Chapeuzinho é mesmo de uma criança; menos artificial, portanto. Apesar dessas novas propostas, a trama do conto nas diversas versões mantém-se apoiada, insisto, em Grimm, ainda que a referência ao nome dos folcloristas alemães seja suprimida. Se os eixos norteadores da trama permanecem inalterados, curiosamente há o reforço de alguns itens já presentes nas adaptações clássicas, além de determinados acréscimos, ambos não exatamente inventivos. Senão, vejamos. A idealização que marca os contos tradicionais (menina linda, meiga, inocente; floresta repleta de flores e borboletas etc) é sublinhada nas versões 196 Revista do CESP – v. 26, n. 35 – jan.-jun. 2006 contemporâneas. Estereótipos povoam as histórias, em textos e imagens. Por exemplo, insistentes e irritantes pios de passarinho são, com freqüência, o fundo “musical” nos CDs e CD-ROMs. Chapeuzinho continua a ser descrita como “uma linda menina”, e mais: “de olhos muito azuis, pele clarinha e faces coradas”. Por vezes, canções ao fundo louvam a beleza da mata na primavera, alertando ao mesmo tempo para seus perigos, sobretudo o representado pelo lobo mau. No CD-ROM da Coleção “Contos clássicos”, a imagem é animada: fica “tremelicando” e apresenta a menina abrindo e fechando os olhos, os passarinhos piando e fazendo ligeiros movimentos repetitivos. A página não se altera sozinha e, se não providenciamos logo sua mudança, a repetição estridente de sons beira o insuportável. A voz da menina é imbecilizada; os recursos são pobres, repetitivos. Nesses casos, a nova tecnologia não evitou, portanto, os estereótipos; ao contrário, só fez aguçá-los em alguns aspectos. Na fita de VHS, as imagens do desenho animado são também altamente clicherizadas. A história é repleta de animaizinhos (apelo ao “infantil”?): há veadinhos, passarinhos, castores, além dos intermináveis (e inevitáveis, pelo visto) pios de passarinho como “música” de fundo. Uma cantiga fala em “criança adorável”, em “raio de sol [que] cintila no espelho”, na “beleza de menina [que] me faz feliz”, em “todo amor e carinho” etc. Em outras versões, encontramos também muitos diminutivos, excesso de adjetivos, imagens de coraçõezinhos e demais recursos “melosos”. O apelo ao “infantil” aparece, igualmente, na referência a determinadas comidas e bebidas que costumam ser atraentes às crianças: doces, tortas, suco de maçã, bolo de chocolate, katchup. A música do compositor João de Barrro (Braguinha) é reproduzida na maioria das histórias: “Pela estrada afora, eu vou bem sozinha, levar estes doces para a vovozinha...” Considerando que as adaptações de Perrault e Grimm já pressupõem ensinamentos diversos a serem assimilados pelo público infantil, note-se que as versões em suportes tecnológicos atuais, por sua vez, intensificam também esse aspecto. Um exemplo paradigmático pode ser encontrado na capa da fita de VHS, onde se lê: “Esta divertida aventura vai manter as crianças atentas na história e nas cativantes canções; aprendendo lições importantes sobre o respeito aos ensinamentos e a ajuda ao próximo”. Lições diversas perpassam, de fato, não apenas esta, mas várias outras versões da história, a saber: os conselhos e instruções da mãe são consideravelmente ampliados e detalhados; mesmo a menina não se furta a dar lições e prestar ajuda aos animaizinhos que encontra no caminho; o final, já bastante moralizador no texto dos Grimm, é normalmente realçado, com a introdução de lembretes 197 Velha história em roupa nova:... Ana Maria Clark Peres – p.191-200 os mais variados às crianças. A preocupação em instrui-las aparece inclusive num dos jogos propostos em um CD-ROM, que visa a esclarecer a origem do cultivo de certos legumes e frutas. Se nas versões contemporâneas o lobo ganha roupas novas, mais em sintonia com a moda atual adotada por alguns jovens, Chapeuzinho continua vestindo sua velha capinha vermelha e se comportando como a garotinha ingênua, inocente, aquela que “não tem maldade alguma no coração”. Normalmente, a menina acredita piamente em todos os disfarces precários do lobo (até quando ele se fantasia de borboleta) e em todas as incongruências ditas por ele. Com freqüência, a voz da garota, reproduzida artificialmente por intérpretes adultos, é imbecilizada. Por vezes, também a ingenuidade da avó (“tolinha como ela só”) é reforçada. Para dar ênfase aos ensinamentos morais, há sempre conversas e explicações sobre a vida familiar de Chapeuzinho (inclusive sobre o pai da garota), que só retardam a narrativa. Na maior parte do tempo, Chapeuzinho passeia pela floresta, ou conversa com o lobo, ou este conversa com a avó, ou ainda os lenhadores conversam entre si, e essas conversas chegam a ser exaustivamente longas. Acontece, igualmente, muita ação, correria, brigas. Seria esse excesso mais um apelo ao “infantil”, partindo-se do pressuposto de que criança só suportaria história com muita, muita ação? Nas correrias intermináveis, há gritos e tentativas de humor, como já foi assinalado, mas freqüentemente, de um “humor-pastelão”. Ao lado desses acréscimos, encontramos também omissões no que concerne às adaptações originais. Por exemplo, além de comumente o lobo deixar de ser mau para tornar-se supostamente cômico, não é raro a avó e/ ou a menina não serem comidas por ele. Tudo isso para que não se apresentem enredos “pesados” às “débeis” criancinhas de hoje? O que concluir desse breve apanhado? Primeiramente, não podemos deixar de constatar que a persistência do conto Chapeuzinho Vermelho sinaliza para a sua perene atualidade. Considerado por muitos como a história mais popular do mundo, creio que ele continua a nos tocar precisamente por trazer, de forma tão escancarada, o Real: do sexo, do mal, da morte. Curiosamente, ainda que tocados, ao que tudo indica, por esse Real, os adaptadores contemporâneos tentam desconsiderá-lo, omitindo as cenas em que ele se insinua, como se às crianças o Real não fizesse questão. Além disso, as alterações pouco inventivas efetuadas nos enredos clássicos e o reforço de tantos estereótipos evidenciam que as versões atuais, em sua grande maioria, mesmo apelando para novas tecnologias, pressupõem 198 Revista do CESP – v. 26, n. 35 – jan.-jun. 2006 uma criança ainda mais débil, inocente, ingênua e incapaz do que a dos séculos passados. Note-se que adultos “zelosos” continuam se sentindo na obrigação de alertá-la contra os tantos “perigos” que a rodeiam, esquecendo-se, contudo, de um outro perigo: o de subestimar a infância, oferecendo-lhe justamente o que há de mais ultrapassado na velha história, que se reapresenta em cena de roupa nova, talhada segundo a moda de artifícios chamativos, mas superficiais e vazios. Referências bibliográficas ARIÈS, Philippe. História social da criança e da família. Trad. Dora Flaksman. 2ª ed. Rio de Janeiro: Zahar, 1981. DARNTON, Robert. O grande massacre de gatos: e outros episódios da história cultural francesa. Trad. Sonia Coutinho. Rio de Janeiro: Graal, 1986. PERES, Ana Maria Clark. La traduction des contes des fées: l’enfant entre la tradition et l’avenir. In: BEEBY, Allison; ENSINGER, Doris; PRESAS, Marisa (Coord.). Investigating translation: select papers from the 4th International Congress on Translation, Barcelona, 1998. Amsterdam: John Benjamins Publishing Company, 2000. p. 186-187. PIFFAULT, Olivier (Dir.) Il était une fois... les contes de fées. Paris: Seuil/ Bibliothèque nationale de France, 2001. SORIANO, Marc. Les contes de Perrault: culture savante et traditions populaires. Paris: Gallimard, 1968. Documentos eletrônicos <http://expositions.bnf.fr/contes/index.htm>. Acesso em 30 de novembro de 2004. CHAPEUZINHO Vermelho. Disponível em <hhttp:// virtualbooks.terra.com.br/freebook/infantis/chapeuzinho_vermelho.htm>. Acesso em 05 de março de 2005. CHAPEUZINHO Vermelho, Manaus, Sonpress, [s.d.], 1 CD, digital audio. CHAPEUZINHO Vermelho, Manaus, Angels Recors, [s.d]. 1 CD, digital audio (Eternos contos, 1). 199 Velha história em roupa nova:... Ana Maria Clark Peres – p.191-200 CHAPEUZINHO Vermelho, [s.l.], Bandeirantes, Artes Gráficas e Editora Sesil, [s.d.]. 1 CD, digital audio. Acompanha livreto. (Contos clássicos). CHAPEUZINHO Vermelho, [Manaus], Goodtimes, 1995. 1 fita de vídeo (45 minutos), VHS, son., color., dublado. CHAPEUZINHO Vermelho, Manaus, [s.l.], Cooperdisc, [s.d.]. 1 DVD (30 min.), color., dublado. (Vídeo brinquedo). LILIA Cabral conta Chapeuzinho Vermelho, Manaus, Sonopress, [s.d.]. 1 CD, digital audio. (Coleção infantil Olha quem está contando). O CHAPEUZINHO Vermelho, Manaus, Warner Musica Brasil, 2003. 1 CDROM. Acompanha livreto. (Disquinho). Resumo Este estudo tem como objetivo analisar a apresentação do antigo conto Chapeuzinho Vermelho em CD, CD-ROM, VHS, DVD e e-book. Résumé Il s’agit dans cette étude d’analyser la présentation de l’ancien récit Le Petit Chaperon rouge en CD, CD-ROM, VHS, DVD et e-book. 200
https://openalex.org/W2204245756	https://europepmc.org/articles/pmc4673502?pdf=render	English	null	A coherent transcriptional feed-forward motif model for mediating auxin-sensitive PIN3 expression during lateral root development	Nature communications	2,015	cc-by	14,091	ARTICLE Received 3 Jan 2015 \| Accepted 7 Oct 2015 \| Published 18 Nov 2015 A coherent transcriptional feed-forward motif model for mediating auxin-sensitive PIN3 expression during lateral root development Qian Chen1,2,3, Yang Liu1,2,4, Steven Maere1,2, Eunkyoung Lee5, Gert Van Isterdael1,2, Zidian Xie6, Wei Xuan1,2, Jessica Lucas5, Valya Vassileva1,2,7, Saeko Kitakura1,2,8, Peter Marhavy´9,10, Krzysztof Wabnik1,2,9, Niko Geldner10, Eva Benkova´9, Jie Le11, Hidehiro Fukaki12, Erich Grotewold6, Chuanyou Li3, Jirˇı´ Friml1,2,9, Fred Sack5,z, Tom Beeckman1,2 & Steffen Vanneste1,2 Multiple plant developmental processes, such as lateral root development, depend on auxin distribution patterns that are in part generated by the PIN-formed family of auxin-efﬂux transporters. Here we propose that AUXIN RESPONSE FACTOR7 (ARF7) and the ARF7-regulated FOUR LIPS/MYB124 (FLP) transcription factors jointly form a coherent feed-forward motif that mediates the auxin-responsive PIN3 transcription in planta to steer the early steps of lateral root formation. This regulatory mechanism might endow the PIN3 circuitry with a temporal ‘memory’ of auxin stimuli, potentially maintaining and enhancing the robustness of the auxin ﬂux directionality during lateral root development. The cooperative action between canonical auxin signalling and other transcription factors might constitute a general mechanism by which transcriptional auxin-sensitivity can be regulated at a tissue-speciﬁc level. 1 Department of Plant Systems Biology, VIB, Ghent 9052, Belgium. 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent 9052, Belgium. 3 State Key Laboratory of Plant Genomics, National Centre for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. 4 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China. 5 Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada. 6 Center for Applied Plant Sciences (CAPS) and Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, USA. 7 Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Academik Georgi Bonchev Street, Soﬁa 1113, Bulgaria. 8 Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka 560-0043 Japan. 9 Institute of Science and Technology Austria, Klosterneuburg 3400, Austria. 10 Department of Plant Molecular Biology, UNIL-Sorge, University of Lausanne, Lausanne 1015, Switzerland. 11 Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. 12 Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Kobe 657-8501, Japan. Correspondence and requests for materials should be addressed to T.B. (email: tobee@psb.vib-ugent.be) or to S.V. (email: stnes@psb.vib-ugent.be). zDeceased on June 30, 2015. 1 NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Results FOUR L 1e). Together with its early, SLR/IAA14-ARF7-dependent expression, we hypothesized that FLP could be a direct target of ARF7. Therefore, we performed ChIP analyses using anti-GR antibodies on proARF7::ARF7-GR/ arf7 arf19. In the absence of DEX, none of the fragments were signiﬁcantly enriched (Fig. 1f). By contrast, the FLP_P2 amplicon became signiﬁcantly enriched when plants were treated with DEX, while the enrichment of neither FLP_P1 nor ACTIN2 changed signiﬁcantly (Fig. 1f). Together these data demonstrate that FLP is a direct target of ARF7. Auxin transporters of the PIN-formed (PIN) family16 contribute to nearly every step of these regulatory auxin ﬂows6,12,13. Prominently among the LR-regulating PINs is PIN3, which is transiently expressed in the endodermis cells overlaying FCs to promote their transition to LR initiation12, and is later in development also expressed in LR primordium- overlaying tissues to facilitate LR emergence6. These examples demonstrate the importance of dynamic PIN expression patterns in LR development. p The canonical auxin signalling pathway has been implicated as the main mechanism for regulating PIN expression6,17. This pathway is deﬁned by the auxin-induced proteolysis of transcriptional repressor of the Aux/IAA family, thereby derepressing auxin response transcription factors (ARFs)8. Expression of different combinations of members of both protein families within a single cell is believed to deﬁne different auxin responsive transcriptional outputs18–20. Detailed analyses of dynamic transcriptional changes associated with auxin-induced and gravity-induced LR formation start revealing the underlying gene regulatory networks21. Besides the canonical auxin signalling pathway, several other transcription factors have been implicated in transcriptional control over PINs; the MADS- box XAANTAL2 (XAL2/AGL14) and INDETERMINATE DOMAIN (IDD) (IDD14, IDD15, IDD16) transcription factors were recently identiﬁed as direct regulators of PIN1 and/or PIN4 through direct binding to the respective promoters22,23. However, almost nothing is known about how such regulators bind to cis-elements in the PIN regulatory regions and how they are integrated with the canonical auxin signalling pathway to regulate PIN expression during organogenetic processes such as LR development. We previously demonstrated that PIN3 shows remarkably dynamic expression during the patterning of stomatal complexes in the leaf epidermis24. We thus investigated whether the mechanisms controlling PIN3 expression are similar during stomatal and LR development. Two genes involved in the late steps of stomatal development encode the transcription factors FOUR LIPS (FLP)/MYB124 and its closest paralogue MYB88 (ref. 25). Results FOUR L The plant hormone auxin commonly plays a master role in organogenesis and morphogenesis8, as is explicit for LR development9. In the lateral root cap, the auxin precursor indole-3-butyric acid is converted to indole-3-acetic acid (IAA), feeding auxin into the root clock, thereby stimulating prebranch site formation10. Auxin accumulation drives the acquisition of FC identity in pericycle cells11, and the subsequent transition from FC to LR initiation requires stabilization of auxin signalling in these cells12. Subsequent LR patterning and morphogenesis are orchestrated by dynamic auxin ﬂows6,13,14 as well as by complex interactions with surrounding tissues4,5,7,15. FOUR LIPS is a direct target of ARF7. While FLP is best known for its role in the late stages of stomatal development25, we observed prominent FLP promoter activity in developing LR primordia (Fig. 1a; Supplementary Fig. 1a). Given the importance of auxin in triggering LR development, we probed whether FLP expression is induced by auxin. In auxin-treated roots, transcripts of PIN3, as well as the expression of GATA23 and ACR4, which are hallmarks of LR initiation29,30, were rapidly upregulated (Fig. 1b). Similarly, transcripts of FLP, but not of MYB88, were also strongly auxin responsive (Fig. 1b). This auxin-activated FLP expression can be found in xylem pole pericycle cells, the cell type where LRs originate from (Supplementary Fig. 1b). The auxin signalling pathway that is central to LR induction depends largely upon the presence of the Auxin Response transcription Factor7 (ARF7) and SOLITARY ROOT/IAA14 (SLR)9. Together, these proteins deﬁne a canonical auxin pathway that regulates trans- criptional changes associated with auxin-induced LR initiation. High auxin levels activate ARF7 transcriptional activity by destabilizing its interaction partner SLR/IAA14 (ref. 31). A gain-of-function mutation in SLR/IAA14 (slr-1), or loss-of- function mutations in the ARF7 and ARF19 genes disrupt auxin signalling to the extent that the respective (primary) roots are completely devoid of LRs32,33. We then tested whether the auxin inducible expression of FLP depends upon SLR/IAA14-ARF7- based auxin signalling. In slr-1 and in proARF7::ARF7-GR/arf7 arf19 mutants, FLP expression was reduced and almost com- pletely insensitive to auxin treatment (Fig. 1c). Moreover, when ARF7 functionality was restored to proARF7::ARF7-GR/arf7 arf19 via dexamethasone (DEX/NAA co-treatment), FLP transcription regained auxin-responsiveness. A nearly identical expression proﬁle was found for PIN3 (Fig. 1d), which suggests it represents a direct target of this auxin signalling pathway6,17. FLP contains two canonical AuxREs in the 50 region upstream of its start codon (included in FLP_P2 amplicon) (Fig. ARTICLE ARTICLE P lant growth involves the reiterative formation of post- embryonic organs such as lateral roots (LRs), tailoring the root architecture to environmentally imposed limitations. Typically, LRs are regularly spaced along the main root. The mechanism that controls this regular spacing involves oscillatory gene expression in the elongation zone of the primary root meristem1, also called the root clock2. This mechanism deﬁnes groups of cells that are competent to form LR primordia (prebranch sites). Xylem pole pericycle cells within such prebranch sites can become LR founder cells (FCs) that subsequently can be activated to initiate a LR primordium3. Throughout subsequent developmental stages, the nascent LR primordium communicates with the overlaying root tissues to facilitate LR emergence4–7. P l r auxin responsive, downstream of ARF7, and that FLP binds directly to the PIN3 promoter as part of the molecular mechanism that controls its auxin-sensitive expression. Together, FLP and ARF7 could deﬁne a coherent feed-forward motif that controls PIN3 expression. Mathematical modelling reveals that such transcriptional circuit could generate a temporal ‘memory’ of auxin stimuli, potentially enhancing the robustness of the auxin ﬂux patterns. Consistently, the corresponding cis- regulatory modules in the PIN3 promoter are required for normal auxin-responsive PIN3-YFP expression that can complement the LR defect in pin3 mutants. The feed-forward transcriptional circuit that we propose for auxin-sensitive PIN3 transcription thus seems critical for PIN3-controlled LR development. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited All rights reserved Results FOUR L Scale bar, 50 mm. (b) qRT-PCR analysis of GATA23, ACR4, PIN3, FLP and MYB88 expression during a 12 h auxin (10 mM NAA) time course from roots of 6-day-old WT seedlings. Expression levels were normalized to those of ACTIN2. (c,d) Relative auxin-inducibility (6 h, 10 mM NAA) of (c) FLP and (d) PIN3 in WT (±10 mM DEX for 6 h), slr-1, and proARF7::ARF7-GR/arf7 arf19 (±10 mM DEX for 6 h). Samples with different letters are signiﬁcantly different: P o0.05 (Fisher’s LSD mean separation test). (e) Schematic presentation of the FLP promoter (3.0 kb upstream of the translational start site at position ( þ 1) with indication of the regions targeted for ChIP analysis (FLP_P1 and FLP_P2), and the presence of auxin response elements (AuxREs, black triangles) (f) Enrichment of the indicated DNA fragments (FLP_P1, and FLP_P2) following ChIP using anti-GR antibodies with proARF7::ARF7-GR/arf7 arf19 treated for 6 h with or without DEX. A fragment from the ACTIN2 promoter was tested as a negative control. n ¼ 3. Data are means ± s.d. *Po0.001 (Student’s t-test). developmental stages. These phenotypes were more pronounced in the ﬂp-1 myb88 and in the ﬂp-7 myb88 double mutants. Our results thus reveal a key role for FLP in LR initiation, with MYB88 playing only a minor role, ﬁndings that echo the absence of obvious stomatal defects in the single myb88 mutants25, and the lack of persistent MYB88 expression in LR primordia (Supplementary Fig. 1c). consistent with LR initiation defects in cyca2;234 and ﬂp-7 myb88 arising via distinct pathways, suggesting that the ﬂp-7 myb88 LR defect is at least in part independent of the misregulated proliferation seen in cyca2;234. Next, we addressed whether perturbation of LR development resulted from impaired auxin signalling. Therefore, we intro- gressed the synthetic auxin-response output reporter proDR5:: GUS into ﬂp-7 myb88 (Fig. 2b). Whereas normally this reporter is prominently expressed in developing LRs, the proDR5::GUS signal was only weakly detectable in LR primordia of ﬂp-7 myb88 double mutants (Fig. 2b). Because this reduced proDR5::GUS signal might have resulted from altered auxin signalling and/or distribution, we analysed the auxin responsiveness of proDR5:: GUS in WT and ﬂp-7 myb88. A 6h auxin treatment equalized the GUS expression in both the WT and in ﬂp-7 myb88 (Fig. 2b). Moreover, both genotypes showed near-identical auxin- induced increases of IAA19, GATA23 and ACR4 transcripts (Supplementary Fig. 4). Results FOUR L I II IV E VI V a I II IV E VI V 70 60 50 40 30 20 10 0 0 NAA: 0.5 1 2 4 6 12 (Hours) GATA23 ACR4 FLP PIN3 MYB88 Relative expression Relative expression Relative expression FLP 18 16 14 12 10 8 6 4 2 0 a a MS NAA c c d d e d b DEX: (10 µM) DEX: (10 µM) Col-0 Col-0 – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 PIN3 7 6 5 4 3 2 1 0 a a MS NAA c c ded e d – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 a b c d a b a 70 60 50 40 30 20 10 0 0 NAA: 0.5 1 2 4 6 12 (Hours) GATA23 ACR4 FLP PIN3 MYB88 Relative expression b b a E VI V 20 10 0 0 NAA: 0.5 1 2 4 6 12 (Hours) FLP PIN3 MYB88 Rela Relative expression Relative expression FLP 18 16 14 12 10 8 6 4 2 0 a a MS NAA c c d d e d b DEX: (10 µM) DEX: (10 µM) Col-0 Col-0 – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 PIN3 7 6 5 4 3 2 1 0 a a MS NAA c c ded e d – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 c d a b a Relative expression DEX: (10 µM) Col-0 R PIN3 7 6 5 4 3 2 1 0 a a MS NAA c c ded e d – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 d a b Relative expression Relative expression FLP 18 16 14 12 10 8 6 4 2 0 a a MS NAA c c d d e d b DEX: (10 µM) DEX: (10 µM) Col-0 Col-0 – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 PIN3 7 6 5 4 3 2 1 0 a a MS NAA c c ded e d – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 c d a b a c Relative enrichment 5 4 3 2 1 0 FLP_P1 FLP_P2 ACTIN2 MS DEX f FLP promoter 1 2 + 1 FLP_P1 FLP_P2 1 2 AuxRE1: GAGACA AuxRE2: GAGACA e e Figure 1 \| FLP is a direct target of ARF7. (a) proFLP::NLS-GFP expression in different stages of LR development. Results FOUR L FLP and MYB88 act redundantly to restrict guard mother cell proliferation, in part by repressing the expression of cell cycle genes26–28. Here, we propose that FLP transcription is A role for FLP and MYB88 in PIN3-regulated LR development. To evaluate the relative contributions of FLP and MYB88 expression to LR development, we analysed the LR phenotypes of corresponding single and double mutants (Fig. 2a; Supplementary Fig. 2a,b). Although myb88 single mutants showed a wild-type (WT) root phenotype, the ﬂp-1 and ﬂp-7 alleles each exhibited reduced numbers of LRs, including a reduction in the earliest NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 2 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 I II IV E VI V 70 60 50 40 30 20 10 0 0 NAA: 0.5 1 2 4 6 12 (Hours) GATA23 ACR4 FLP PIN3 MYB88 Relative expression Relative expression Relative enrichment Relative expression FLP 18 16 14 12 10 8 6 4 2 0 a a MS NAA c c d d e d b DEX: (10 µM) DEX: (10 µM) Col-0 Col-0 – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 FLP promoter 1 2 + 1 FLP_P1 FLP_P2 1 2 AuxRE1: GAGACA AuxRE2: GAGACA PIN3 7 6 5 4 3 2 1 0 a a MS NAA c c ded e d – + – – + slr-1 proARF7::ARF7-GR arf7 arf19 5 4 3 2 1 0 FLP_P1 FLP_P2 ACTIN2 MS DEX a b c d f e a b a Figure 1 \| FLP is a direct target of ARF7. (a) proFLP::NLS-GFP expression in different stages of LR development. Scale bar, 50 mm. (b) qRT-PCR analysis of GATA23, ACR4, PIN3, FLP and MYB88 expression during a 12 h auxin (10 mM NAA) time course from roots of 6-day-old WT seedlings. Expression levels were normalized to those of ACTIN2. (c,d) Relative auxin-inducibility (6 h, 10 mM NAA) of (c) FLP and (d) PIN3 in WT (±10 mM DEX for 6 h), slr-1, and proARF7::ARF7-GR/arf7 arf19 (±10 mM DEX for 6 h). Samples with different letters are signiﬁcantly different: P o0.05 (Fisher’s LSD mean separation test). Results FOUR L (e) Schematic presentation of the FLP promoter (3.0 kb upstream of the translational start site at position ( þ 1) with indication of the regions targeted for ChIP analysis (FLP_P1 and FLP_P2), and the presence of auxin response elements (AuxREs, black triangles) (f) Enrichment of the indicated DNA fragments (FLP_P1, and FLP_P2) following ChIP using anti-GR antibodies with proARF7::ARF7-GR/arf7 arf19 treated for 6 h with or without DEX. A fragment from the ACTIN2 promoter was tested as a negative control. n ¼ 3. Data are means ± s.d. Po0.001 (Student’s t-test). & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 M ill P bli h Li it d All i ht d NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 2.5 2.0 1.5 1.0 0.5 0 LRP density (/cm) a a a bb cccc I II III IV V VI VII E LR a a a bb cc cc Col-0 flp-7 flp-1 myb88 flp-7 myb88 flp-7 myb88 pin3–4 Ler myb88 flp-1 pin3–4 a proDR5::GUS proDR5::GUS/flp-7 myb88 Stage I Stage VII Stage I Stage VII MS NAA NAA NAA 6h NAA 6 h 6 h MS MS MS 6 h b 8 6 4 2 0 LRP/cm proDR5::GFP / WT proDR5::GFP / flp-1 myb88 6 5 4 3 2 1 0 FC/cm 10 8 6 4 2 0 FC + LRP/cm c d e Figure 2 \| Lateral root phenotypic analysis. (a) Densities of the different LR stages in 6-day-old seedlings of Col-0, Ler, ﬂp-1, ﬂp-7, myb88, pin3-4, ﬂp-1 myb88, ﬂp-7 myb88 and ﬂp-7 myb88 pin3-4. E ¼ just emerged, not yet mature LRs; LR ¼ mature LRs. Data shown are means ± s.d. of at least three independent experiments, each time sampling (nZ20). Samples in each stage with different letters are signiﬁcantly different: P o0.05 (Fisher’s LSD mean separation test). (b) Expression pattern of the auxin response output reporter proDR5::GUS in different LR stages of WTand ﬂp-7 myb88 (±10 mM NAA for 6 h). Scale bar, 50 mm. (c–e) Densities of (c) initiated LR primordia, (d) FCs and (e) the sum of FCs and initiated LR primordia in WT and ﬂp-1 myb88 as visualized by proDR5::GFP. Data shown are means ± s.e.m. and are representative of at least three independent experiments. (For each experiment, n ¼ 10). Po0.01 and Po0.001 (Student’s t-test). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 2.5 2.0 1.5 1.0 0.5 0 LRP density (/cm) a a a bb cccc I II III IV V VI VII E LR a a a bb cc cc Col-0 flp-7 flp-1 myb88 flp-7 myb88 flp-7 myb88 pin3–4 Ler myb88 flp-1 pin3–4 a b b proDR5::GUS proDR5::GUS/flp-7 myb88 Stage I Stage VII Stage I Stage VII MS NAA NAA NAA 6h NAA 6 h 6 h MS MS MS 6 h b 8 6 4 2 0 LRP/cm proDR5::GFP / WT proDR5::GFP / flp-1 myb8 6 5 4 3 2 1 0 FC/cm 10 8 6 4 2 0 FC + LRP/cm c d e d 8 6 4 2 0 LRP/cm proDR5::GFP / WT proDR5::GFP / flp-1 myb88 6 5 4 3 2 1 0 FC/cm 10 8 6 4 2 0 FC + LRP/cm c d e c e proDR5::GFP / WT proDR5::GFP / WT proDR5::GFP / flp-1 myb88 proDR5::GFP / flp-1 myb88 proDR5::GFP / flp-1 myb88 Figure 2 \| Lateral root phenotypic analysis. (a) Densities of the different LR stages in 6-day-old seedlings of Col-0, Ler, ﬂp-1, ﬂp-7, myb88, pin3-4, ﬂp-1 myb88, ﬂp-7 myb88 and ﬂp-7 myb88 pin3-4. E ¼ just emerged, not yet mature LRs; LR ¼ mature LRs. Data shown are means ± s.d. of at least three independent experiments, each time sampling (nZ20). Samples in each stage with different letters are signiﬁcantly different: P o0.05 (Fisher’s LSD mean separation test). (b) Expression pattern of the auxin response output reporter proDR5::GUS in different LR stages of WTand ﬂp-7 myb88 (±10 mM NAA for 6 h). Scale bar, 50 mm. (c–e) Densities of (c) initiated LR primordia, (d) FCs and (e) the sum of FCs and initiated LR primordia in WT and ﬂp-1 myb88 as visualized by proDR5::GFP. Data shown are means ± s.e.m. and are representative of at least three independent experiments. (For each experiment, n ¼ 10). Po0.01 and *Po0.001 (Student’s t-test). Figure 2 \| Lateral root phenotypic analysis. (a) Densities of the different LR stages in 6-day-old seedlings of Col-0, Ler, ﬂp-1, ﬂp-7, myb88, pin3-4, ﬂp-1 myb88, ﬂp-7 myb88 and ﬂp-7 myb88 pin3-4. E ¼ just emerged, not yet mature LRs; LR ¼ mature LRs. Data shown are means ± s.d. of at least three independent experiments, each time sampling (nZ20). Results FOUR L These results argue against the idea that pp y g Given FLPs function in restricting proliferation in the stomatal cell lineage, and its necessity for cell proliferation during early LR development, we analysed the genetic interaction between ﬂp-7 myb88, and a triple mutant in A2-type cyclins (cyca2;234) at the level of LR formation (Supplementary Fig. 3). Consistent with the inherent proliferation requirement for LR formation, the cell proliferation-impaired cyca2;234 mutants26 exhibited a lower LR density than the WT, mostly resulting from a reduced level of LR initiation, a defect comparable to that of ﬂp-7 myb88. Strikingly, the quintuple mutant ﬂp-7 myb88 cyca2;234 developed much less LR initiation sites compared to cyca2;234 or ﬂp-7 myb88 alone, 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 Samples in each stage with different letters are signiﬁcantly different: P o0.05 (Fisher’s LSD mean separation test). (b) Expression pattern of the auxin response output reporter proDR5::GUS in different LR stages of WTand ﬂp-7 myb88 (±10 mM NAA for 6 h). Scale bar, 50 mm. (c–e) Densities of (c) initiated LR primordia, (d) FCs and (e) the sum of FCs and initiated LR primordia in WT and ﬂp-1 myb88 as visualized by proDR5::GFP. Data shown are means ± s.e.m. and are representative of at least three independent experiments. (For each experiment, n ¼ 10). Po0.01 and *Po0.001 (Student’s t-test). the LR defect in ﬂp myb88 double mutants is due to a general reduction in auxin sensitivity. Instead, the altered proDR5::GUS expression level might be the result of abnormal auxin distribution in ﬂp myb88 double mutants. cumulated densities of FCs and initiated LRPs together that were comparable to WT (Fig. 2e). An identical LR defect with less initiated LRPs but more FCs was recently also described for pin3-4 (ref. 12). This striking resemblance prompted us to test for genetic interactions between ﬂp-7 myb88 and pin3-4. Analysis of different stages of LR development conﬁrmed that pin3-4 and ﬂp-7 myb88 LR defects were highly similar, with the most prominent defects appearing during initiation (Fig. 2a; Supplementary Fig. 2). Importantly, the LR phenotype in the ﬂp-7 myb88 pin3-4 triple mutants showed near-identical reductions in LR initiation as the parental mutants. The absence of clear additive effects suggests that FLP, MYB88 and PIN3 act collectively in regulating LR initiation. To further dissect the contribution of defects in auxin transport to the LR phenotype in ﬂp myb88, we focussed on the earliest visible stages of LR formation; namely LR FCs (undivided pericycle cells that exhibit proDR5::GFP expression)11 and LR initiation sites (FC that have undergone asymmetric cell division). Consistent with our other analyses (Fig. 2a), the densities of initiated LRPs were reduced in ﬂp-1 myb88 compared with the WT (Fig. 2c). In contrast, the FC densities in ﬂp-1 myb88 had dramatically increased compared to WT (Fig. 2d), resulting in NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 4 & 2015 Macmillan Publishers Limited. All rights reserved ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 Five different promoter fragments of the PIN3 promoter (FR1-5) were tested for their ability to interact with FLP via yeast-one- hybrid assays (Fig. 4a). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 Because both the expression level of FLP and PIN3 are relatively rapidly induced by auxin and depend upon SLR/IAA14-ARF7 signalling, we explored whether FLP might be a downstream effector of the auxin signalling pathway by directly regulating PIN3 expression. However, it has been reported that auxin-inducible PIN3 expression does not require de novo protein synthesis6,17, proPIN3::PIN3-GFP proPIN3::PIN3-GFP/ flp-1 myb88 proFLP::FLP-GR flp-1 myb88 proFLP::FLP-GR flp-1 myb88 255 0 255 0 Stage I MS Stage I MS Stage VII MS Stage VII MS Stage I NAA 6 h Stage I NAA 6 h Stage VII NAA 6 h Stage VII NAA 6 h 8 a c b d a b b b b a a MS NAA 6 h MS NAA 6 h a a a a b c c c d PIN1 PIN3 Relative expression Relative expression 7 6 5 4 3 2 1 0 7 6 5 4 3 2 1 0 – – + – – + + + Col–0 Col–0 DEX: (10 µM) DEX: (10 µM) Figure 3 \| FLP controls PIN3 expression. (a,b) Expression pattern of proPIN3::PIN3-GFP in (a) WT and (b) ﬂp-1 myb88 in early stage (LR initiation, left panel) and later stage (stage VII, right panel) of LR with or without 6 h auxin treatments (10 mM NAA). Images were taken using identical confocal settings for all samples. Scale bar, 50 mm. Relative intensities are colour coded. (c,d) Relative auxin-inducible expression (6 h of 10 mM NAA) of (c) PIN1 and (d) PIN3 determined by qRT-PCR from roots of WT and proFLP::FLP-GR/ﬂp-1 myb88 seedlings (± 10 mM DEX for 6 h). Data shown are means±s.d. and are representative of at least three independent experiments. Samples with different letters are signiﬁcantly different: P o0.05 (Fisher’s LSD mean separation test). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 Only the fragment FR2 was found to generate Aureobasidin A (AbA)-resistant yeast growth, suggest- ing that this fragment can recruit FLP (Fig. 4b). This fragment contains two sites (FBS1 ¼ AGCCG, FBS2 ¼ TACCC) that meet the experimentally determined [A/T/G][A/T/G]C[C/G][C/G] consensus sequence for FLP binding27. Using short fragments containing either FBS, only the construct containing FBS1 could render the yeast resistant to AbA (Fig. 4b). Moreover, no such AbA-resistance was found when the FBS1 sequence was changed to AATTA (mFBS1) within this construct. These data suggest that FLP can bind directly to the PIN3 promoter via FBS1. PIN3 expression depends on FLP. Since the expression of both FLP and PIN3 are strongly auxin-inducible (Fig. 1b) and downstream of a common auxin signalling module (Fig. 1c,d), we tested whether auxin-induced PIN3 expression requires functional FLP protein. Indeed, proPIN3::PIN3-GFP showed reduced auxin-inducible expression in LR primordia when introgressed into ﬂp-1 myb88 (Fig. 3a,b). In addition, we analysed PIN3 expression in proFLP::FLP-GR/ﬂp-1 myb88 lines in which the LR defect, but not the stomatal phenotype, could be com- pletely complemented by DEX-treatment (Supplementary Fig. 5). In this line, PIN1 expression levels exhibited auxin responsive amplitudes that were comparable to those of the WT, indepen- dently of DEX treatment (Fig. 3c). By contrast, in the absence of DEX, the auxin inducibility of PIN3 in roots was severely reduced (Fig. 3d). Importantly, DEX-treatment restored the auxin responsiveness of PIN3 to WT levels in a proFLP::FLP-GR/ﬂp-1 myb88 background (Fig. 3d). These results show that FLP is required for auxin-induced PIN3 expression, as was also shown for PIN3 expression in the columella of the primary root meristem34. y p We then tested whether this interaction also occurs in planta using ChIP-PCR analysis with anti-GR antibodies (Fig. 4d). FLP activity was controlled conditionally in the proFLP::FLP-GR/ﬂp-1 myb88 line, and analysed for a differential enrichment at two different regions in the PIN3 promoter (amplicons PIN3_P1 and PIN3_P2) (Fig. 4a) as well as one in the unrelated ACTIN2 promoter. Compared to the mock treatment, a 6-h DEX treatment strongly enriched the amplicon closest to FBS1 (PIN3_P1), whereas DEX had no impact on the enrichment of PIN3_P2 and ACTIN2 (Fig. 4d). These data further conﬁrm that FLP is a direct regulator of PIN3 expression, a ﬁnding that was corroborated by Wang et al.34. PIN3 is directly regulated by FLP and ARF7. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 NATURE COMMUNICATIONS \| 6 8821 \| DOI 10 1038/ 9821 \| t / t i ti 5 proPIN3::PIN3-GFP/ flp-1 myb88 255 0 Stage I MS Stage VII MS Stage I NAA 6 h Stage VII NAA 6 h b proPIN3::PIN3-GFP 255 0 Stage I MS Stage VII MS Stage I NAA 6 h Stage VII NAA 6 h a b proPIN3::PIN3-GFP/ flp-1 myb88 b a proFLP::FLP-GR flp-1 myb88 d MS NAA 6 h a a a b c c c d PIN3 Relative expression 7 6 5 4 3 2 1 0 – – + + Col–0 DEX: (10 µM) proFLP::FLP-GR flp-1 myb88 8 c a b b b b a a MS NAA 6 h a PIN1 Relative expression 7 6 5 4 3 2 1 0 – + – + Col–0 DEX: (10 µM) d c Figure 3 \| FLP controls PIN3 expression. (a,b) Expression pattern of proPIN3::PIN3-GFP in (a) WT and (b) ﬂp-1 myb88 in early stage (LR initiation, left panel) and later stage (stage VII, right panel) of LR with or without 6 h auxin treatments (10 mM NAA). Images were taken using identical confocal settings for all samples. Scale bar, 50 mm. Relative intensities are colour coded. (c,d) Relative auxin-inducible expression (6 h of 10 mM NAA) of (c) PIN1 and (d) PIN3 determined by qRT-PCR from roots of WT and proFLP::FLP-GR/ﬂp-1 myb88 seedlings (± 10 mM DEX for 6 h). Data shown are means±s.d. and are representative of at least three independent experiments. Samples with different letters are signiﬁcantly different: P o0.05 (Fisher’s LSD mean separation test). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 PIN3 promoter FR1 FR2 FR3 FR4 FR5 –1,764 –1,293 –794 –494 PIN3_P1 PIN3_P2 –242 + 1 1231 2 AuxRE1: GAGACA, –1,128 / –1,123 AuxRE2: TGTCTC, –1,032 / –1,027 AuxRE3: TGTCTC, –1,016 / –1,011 FBS1: AGCCG, –902 / –898 FBS2: TACCC, –863/ –859 1,000 100 10 1 FLP + FR1 FLP + FR2 FLP + FR3 FLP + FR4 FLP + FR5 FLP + FBS1 FLP + mFBS1 FLP + FBS2 –Leu –Leu + AbA (100 ng ml–1) –Leu –Leu + AbA (100 ng ml–1) ARF7 + FR2 ARF7 + AuxRE1 ARF7 + AuxRE2 ARF7 + AuxRE3 ARF7 + mAuxRE3 ARF7 + mAuxRE2 ARF7 + mAuxRE1 1,000 100 10 1 a b c 1 2 3 1 2 c MS DEX Relative enrichment PIN3_P2 PIN3_P1 ACTIN2 proARF7::ARF7-GR/arf7 arf19 6 5 4 3 2 1 0 e (100 ng ml ) MS DEX 5 4 3 2 1 0 Relative enrichment PIN3_P1 PIN3_P2 ACTIN2 proFLP::FLP-GR/flp-1 myb88 d d Figure 4 \| FLP and ARF7 are direct regulators of PIN3 expression. (a) Schematic presentation of the PIN3 promoter (1.8 kb upstream of the translational start site at position ( þ 1) with indication of the regions targeted for ChIP analysis (PIN3_P1 and PIN3_P2), the fragments used for yeast-one-hybrid (FR1-5) and the presence of auxin response elements (AuxREs, black triangles) and FLP binding sites (FBS, grey triangles) in fragment FR2. (b, c) Yeast-one-hybrid analysis of the interaction of (b) FLP with PIN3 promoter fragments (FR1-5), FBS1, a mutated FBS1 (mFBS1) and FBS2, and (c) of ARF7 with PIN3 promoter fragment FR2, its internal AuxREs (AuxRE1-3) and corresponding mutated versions (mAuxRE1-3). Left is yeast grown on SD–Leu medium in absence of Aureobasidin A (AbA). Right are the corresponding yeast strains in a dilution series grown on SD–Leu medium in the presence of 100 ng ml 1 AbA, a concentration where no autoactivation was detected for any of the strains. (d,e) Enrichment of the indicated DNA fragments (PIN3_P1, and PIN3_P2) following ChIP using anti-GR antibodies with (d) proFLP::FLP-GR/ﬂp-1 myb88 or (e) proARF7::ARF7-GR/arf7 arf19 treated for 6 h with or without DEX. A fragment from the ACTIN2 promoter was tested as a negative control. Data are presented as means ± s.d. Po0.01, *Po0.001 (Student’s t-test). The experiment was repeated three times with similar results. regulation of PIN3 on LR development. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications 201 M ill P bli h Li i d All i h 5 NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 PIN3 promoter FR1 FR2 FR3 FR4 FR5 –1,764 –1,293 –794 –494 PIN3_P1 PIN3_P2 –242 + 1 1231 2 AuxRE1: GAGACA, –1,128 / –1,123 AuxRE2: TGTCTC, –1,032 / –1,027 AuxRE3: TGTCTC, –1,016 / –1,011 FBS1: AGCCG, –902 / –898 FBS2: TACCC, –863/ –859 1,000 100 10 1 FLP + FR1 FLP + FR2 FLP + FR3 FLP + FR4 FLP + FR5 FLP + FBS1 FLP + mFBS1 FLP + FBS2 –Leu –Leu + AbA (100 ng ml–1) –Leu –Leu + AbA (100 ng ml–1) ARF7 + FR2 ARF7 + AuxRE1 ARF7 + AuxRE2 ARF7 + AuxRE3 ARF7 + mAuxRE3 ARF7 + mAuxRE2 ARF7 + mAuxRE1 1,000 100 10 1 MS MS DEX DEX 5 4 3 2 1 0 Relative enrichment Relative enrichment PIN3_P1 PIN3_P2 PIN3_P2 PIN3_P1 ACTIN2 ACTIN2 proFLP::FLP-GR/flp-1 myb88 proARF7::ARF7-GR/arf7 arf19 6 5 4 3 2 1 0 a b d c e 1 2 3 1 2 Figure 4 \| FLP and ARF7 are direct regulators of PIN3 expression. (a) Schematic presentation of the PIN3 promoter (1.8 kb upstream of the translational start site at position ( þ 1) with indication of the regions targeted for ChIP analysis (PIN3_P1 and PIN3_P2), the fragments used for yeast-one-hybrid (FR1-5) and the presence of auxin response elements (AuxREs, black triangles) and FLP binding sites (FBS, grey triangles) in fragment FR2. (b, c) Yeast-one-hybrid analysis of the interaction of (b) FLP with PIN3 promoter fragments (FR1-5), FBS1, a mutated FBS1 (mFBS1) and FBS2, and (c) of ARF7 with PIN3 promoter fragment FR2, its internal AuxREs (AuxRE1-3) and corresponding mutated versions (mAuxRE1-3). Left is yeast grown on SD–Leu medium in absence of Aureobasidin A (AbA). Right are the corresponding yeast strains in a dilution series grown on SD–Leu medium in the presence of 100 ng ml 1 AbA, a concentration where no autoactivation was detected for any of the strains. (d,e) Enrichment of the indicated DNA fragments (PIN3_P1, and PIN3_P2) following ChIP using anti-GR antibodies with (d) proFLP::FLP-GR/ﬂp-1 myb88 or (e) proARF7::ARF7-GR/arf7 arf19 treated for 6 h with or without DEX. A fragment from the ACTIN2 promoter was tested as a negative control. Data are presented as means ± s.d. Po0.01, **Po0.001 (Student’s t-test). The experiment was repeated three times with similar results. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 9 8 7 6 5 4 3 2 1 0 Relative expression MS NAA PIN3 promoter WT mA mF mA + mF AuxREs FBS1 proPIN3::PIN3-YFP/pin3–4 proPIN3_3xmAuxREs::PIN3-YFP/pin3–4 proPIN3_mFBS1::PIN3-YFP/pin3–4 proPIN3_3xmAuxREs_mFBS1::PIN3-YFP/pin3–4 mA mF mA + mF 3.5 3 2.5 2 1.5 1 Lateral root densities (cm–1) Col-0 pin3–4 pin3–4 (YFP neg.) WT WT mA mF mA + mF a b e 5 \| In planta assessment of the relevance of ARF7 and FLP recruitment on auxin-inducibility of PIN3 and PIN3-dependent LR development. elative auxin-inducibility (10 mM NAA for 6 h) of PIN3-YFP driven by the indicated proPIN3 variants. Roots of YFP positive plants were used for PCR assay. Scheme of PIN3 promoter illustrating by colour-coding the variants of the PIN3 constructs used to complement the pin3-4 mutant. are means ± s.d. of at least three independent experiments. Asterisks denote Student’s t-test signiﬁcance: Po0.05, Po0.01 and Po0.001. uantiﬁcation of the LR densities in 8-day-old seedlings of Col-0, pin3-4, YFP-negatives segregating among transgenic plants (proPIN3_WT), N3_WT, proPIN3_mA, proPIN3_mF and proPIN3_mA þ mF. Box plot description: centre lines show the medians of the averages of ﬁve independent genic lines, calculated for at least 20 individual plants per line; box limits indicate the 25th and 75th percentiles as determined by R software; ers extend to minimum and maximum values. n ¼ 5. 9 8 7 6 5 4 3 2 1 0 Relative expression MS NAA PIN3 promoter WT mA mF mA + mF AuxREs FBS1 proPIN3::PIN3-YFP/pin3–4 proPIN3_3xmAuxREs::PIN3-YFP/pin3–4 proPIN3_mFBS1::PIN3-YFP/pin3–4 proPIN3_3xmAuxREs_mFBS1::PIN3-YFP/pin3–4 mA mF mA + mF WT a 3.5 3 2.5 2 1.5 1 Lateral root densities (cm–1) Col-0 pin3–4 pin3–4 (YFP neg.) WT mA mF mA + mF b b p (YFP neg.) Figure 5 \| In planta assessment of the relevance of ARF7 and FLP recruitment on auxin-inducibility of PIN3 and PIN3-dependent LR development. (a) Relative auxin-inducibility (10 mM NAA for 6 h) of PIN3-YFP driven by the indicated proPIN3 variants. Roots of YFP positive plants were used for qRT-PCR assay. Scheme of PIN3 promoter illustrating by colour-coding the variants of the PIN3 constructs used to complement the pin3-4 mutant. Data are means ± s.d. of at least three independent experiments. Asterisks denote Student’s t-test signiﬁcance: Po0.05, Po0.01 and *Po0.001. (b) Quantiﬁcation of the LR densities in 8-day-old seedlings of Col-0, pin3-4, YFP-negatives segregating among transgenic plants (proPIN3_WT), proPIN3_WT, proPIN3_mA, proPIN3_mF and proPIN3_mA þ mF. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 For this purpose, we designed proPIN3 variants of 1.8 kb, containing speciﬁc muta- tions predicted to abrogate activation by FLP (mF) or by ARF7 (mA), then fused them to PIN3-YFP and transformed them into pin3-4 (Fig. 5). Five independent lines were selected per construct, to minimize user-biased pre-selection for transgenic lines, and YFP positive individuals were analysed for auxin-responsive PIN3-YFP amplitude and LR density. In lines expressing PIN3-YFP from the reference PIN3 promoter (WT) a 5–8-fold higher PIN3-YFP expression was measured after auxin treatment, an amplitude within the order of magnitude of what is typically observed for endogenous PIN3 (Fig. 5a). Although strongly reduced, the proPIN3::PIN3-YFP variants impaired in either FLP or ARF7 recruitment (mF or mA) still showed signiﬁcant auxin responsive activity. When recruitment of both FLP and ARF7 was impaired (mF þ mA), auxin could no longer up-regulate PIN3- YFP expression signiﬁcantly. These results are consistent with a model where FLP and ARF7 constitute the core mechanism by which auxin activates PIN3 expression. suggesting that PIN3 is a primary auxin-responsive gene, thus not requiring FLP as an intermediate regulator. Interestingly, the PIN3 promoter contains three ‘canonical’ auxin responsive elements (AuxREs)35,36, all of which are located within FR2, in close proximity to FBS1 (Fig. 4a). Using yeast-one-hybrid assays we found that ARF7 can interact with FR2 (Fig. 4c). In addition, short fragments spanning the individual AuxREs, also interacted with ARF7, and these interactions were lost when the AuxRE sequences were mutated (mAuxRE). We then tested whether ARF7 interacts with the PIN3 promoter in planta via ChIP-PCR in proARF7::ARF7-GR/arf7 arf19. In the absence of DEX, PIN3_P1 was only mildly enriched compared with PIN3_P2 and ACTIN2 (Fig. 4e). By contrast, the PIN3_P1 amplicon became strongly enriched when plants were treated with DEX, but DEX treatment did not stimulate the enrichment of PIN3_P2 and ACTIN2, conﬁrming that PIN3 is indeed a primary auxin responsive gene. Thus our data demonstrate that PIN3 is a direct target of both ARF7 and FLP, two transcription factors that are required for auxin-responsive PIN3 transcription. These lines thus allowed us to probe the functional importance of FLP- and/or ARF7- regulated PIN3 expression for LR development. Therefore, we analysed the ability of these PIN3-driven LR development requires ARF7 and FLP binding. Next, we evaluated the role of FLP- and/or ARF7-mediated NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited All rights reserved NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 To probe how the joint activity of ARF7 and FLP impacts on auxin-regulated PIN3 transcription, we developed a mathematical model that integrates ARF7 and FLP into a coherent feed-forward motif (FFM) regulating PIN3 transcription and PIN3-mediated 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 dicating that PIN3 response delay upon auxin stimulus a robust feature of the circuit incorporating the In the present context, we speculate that the FFM reg auxin signalling provides a temporary memory of cell Ext. auxin Ext. auxin Int. auxin Int. auxin ARF7 PIN3 PIN3 FLP ARF7 0.03 0.025 0.02 0.015 0.01 0.005 0 0 5 10 15 20 25 30 35 40 Time (h) Time (h) Time (h) Time (h) Time (h) Time (h) PIN3 mRNA (µM) FLP mRNA (µM) Intracellular auxin (µM) 0.015 0.01 0.005 0 0 5 10 15 20 25 30 35 40 0 0 5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 Total PIN3 protein (µM) FLP protein (µM) 3.5 3 2.5 2 1.5 1 0.5 0 0 5 10 15 20 25 30 35 40 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 WT flp Active ARF7 (µM) a b d c e f h g mulation of ARF7 and FLP-regulated PIN3 dynamics. (a) The coherent feed-forward motif (FFM) scheme and (b) the no-FFM el auxin-induced PIN3 transcription. (c–h) Simulations of a PIN3-dependent auxin transport model subject to extracellular auxin were performed on model systems with (blue) and without (green) a transcriptional FFM involving FLP (see Methods), using th n in Supplementary Table 1. Two-hour long 1 mM auxin pulses were given to the system at t ¼ 4 h, t ¼ 12 h and t ¼ 26 h. The subplot amics of PIN3 mRNA (c), total PIN3 protein, that is, the sum of intracellular and membrane-bound PIN3 (d), FLP mRNA (e), FLP auxin (g) and active ARF7 (h) concentrations over time. The model simulations indicate that the FLP feed-forward circuit cause al activation of PIN3, resulting in an enhanced memory effect by which auxin stimulation reduces the impact of subsequent extrac RF7 activity and downstream signalling for a prolonged period of time. The system dynamics are qualitatively independent of th ettings used (see Supplementary Figs 6-10). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 LE NATURE COMMUNICATIONS \| DOI: 10.1038/n Ext. auxin Ext. auxin Int. auxin Int. auxin ARF7 PIN3 PIN3 FLP ARF7 0.03 0.025 0.02 0.015 0.01 0.005 0 0 5 10 15 20 25 30 35 40 Time (h) Time (h) PIN3 mRNA (µM) 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 Total PIN3 protein (µM) a b d c Ext. auxin Int. auxin PIN3 ARF7 b Ext. auxin Int. auxin ARF7 PIN3 FLP a Time (h) 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 Total PIN3 protein (µM) d 0.03 0.025 0.02 0.015 0.01 0.005 0 0 5 10 15 20 25 30 35 40 Time (h) PIN3 mRNA (µM) c 0.03 0.025 0.02 0.015 0.01 0.005 0 0 5 10 15 20 25 30 35 40 Time (h) Time (h) PIN3 mRNA (µM) 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 Total PIN3 protein (µM) d c d Time (h) Time (h) FLP mRNA (µM) 0.015 0.01 0.005 0 0 5 10 15 20 25 30 35 40 e Time (h) FLP protein (µM) 3.5 3 2.5 2 1.5 1 0.5 0 0 5 10 15 20 25 30 35 40 f f e Time (h) Intracellular auxin (µM) 0 0 5 10 15 20 25 30 35 40 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 g Time (h) 5 10 15 20 25 30 35 40 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 WT flp Active ARF7 (µM) h g g h Figure 6 \| Simulation of ARF7 and FLP-regulated PIN3 dynamics. (a) The coherent feed-forward motif (FFM) scheme and (b) the no-FFM scheme used to model auxin-induced PIN3 transcription. (c–h) Simulations of a PIN3-dependent auxin transport model subject to extracellular auxin pulses. Simulations were performed on model systems with (blue) and without (green) a transcriptional FFM involving FLP (see Methods), using the parameter settings given in Supplementary Table 1. Two-hour long 1 mM auxin pulses were given to the system at t ¼ 4 h, t ¼ 12 h and t ¼ 26 h. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 Box plot description: centre lines show the medians of the averages of ﬁve independent transgenic lines, calculated for at least 20 individual plants per line; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend to minimum and maximum values. n ¼ 5. auxin transport (Fig. 6a). In parallel, we tested a model that lacks FLP function (mimicking a ﬂp mutation) (Fig. 6b). A full description of the model and the parameter settings used can be found in the Methods section. Our simulation results indicate that the FFM may function to prolong and amplify PIN3 expression in response to auxin stimuli (Fig. 6c–h). In contrast to the system lacking the FFM, PIN3 in the FFM model continues to be actively transcribed for several hours upon removal of the auxin stimulus. Such a delay in response shutdown after stimulus removal is thought to be a key feature of coherent OR-type feed- forward circuits37. Simulations with other parameter settings gave qualitatively similar results (see Supplementary Figs 6–9). To test the sensitivity of the observed PIN3 response delay to variations in the model parameters, we screened the ratio of the total PIN3 protein concentration response time upon auxin stimulus removal for model circuits with and without FFM as a function of single parameter changes (see Methods and Supplementary Fig. 10). For none of the parameters screened, the FFM/no FFM response time ratio drops below 1 in the parameter range constructs to complement the LR defect in pin3-4. When PIN3-YFP was expressed under the control of the reference PIN3 promoter (WT), the LR density was restored to WT (Col-0) levels, whereas segregating YFP negative plants still showed the pin3-4 LR defect (Fig. 5b). In contrast, none of the mutant PIN3 promoter constructs were able to rescue the pin3-4 LR defect. Importantly, while mutations in either FLP or ARF7 binding sites still showed some PIN3-YFP auxin-mediated upregulation, this auxin-responsive PIN3-YFP expression was not sufﬁcient to complement the LR defect in pin3-4. These results demonstrate that the regulation of auxin-sensitive PIN3 expression as deﬁned by FLP as well as ARF7 is a crucial parameter for LR development. Model of ARF7- and FLP-dependent PIN3 transcription. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications acmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 The subplots depict the resulting dynamics of PIN3 mRNA (c), total PIN3 protein, that is, the sum of intracellular and membrane-bound PIN3 (d), FLP mRNA (e), FLP protein (f), intracellular auxin (g) and active ARF7 (h) concentrations over time. The model simulations indicate that the FLP feed-forward circuit causes prolonged transcriptional activation of PIN3, resulting in an enhanced memory effect by which auxin stimulation reduces the impact of subsequent extracellular auxin stimuli on ARF7 activity and downstream signalling for a prolonged period of time. The system dynamics are qualitatively independent of the exact parameter settings used (see Supplementary Figs 6-10). In the present context, we speculate that the FFM regulation of auxin signalling provides a temporary memory of cellular auxin levels to speciﬁc specialized cells, potentially enhancing the robustness of auxin ﬂux patterns and focussing auxin maxima during post-embryonic patterning processes, such as LR initiation and development. In addition, our simulations indicate that delayed PIN3 down-regulation might temporarily capacitate the system for mitigating the effects of subsequent auxin stimuli on ARF7 activity and downstream auxin signalling (Fig. 6h). Together our data represent a ﬁrst example of a mechanism by which plant endogenous cues such as auxin can become proﬁled, indicating that PIN3 response delay upon auxin stimulus removal is a robust feature of the circuit incorporating the FLP FFM. As expected, the response time ratio approximates 1 when FFM parameters approach values that make the FFM non-functional, for example, very-low FLP mRNA or protein synthesis rates (aFLP or bFLP), very-high FLP mRNA or protein decay rates (gFLP or dFLP), or very-high half-max constants for FLP-dependent activation of PIN3 transcription or ARF7-dependent activation of FLP transcription (KFP or KAF). Long PIN3 mRNA or protein half-lives (small gPIN3 or dPIN3) also appear to offset the effect of the FFM (see Supplementary Fig. 10). proﬁled, indicating that PIN3 response delay upon auxin stimulus removal is a robust feature of the circuit incorporating the FLP FFM. As expected, the response time ratio approximates 1 when FFM parameters approach values that make the FFM non-functional, for example, very-low FLP mRNA or protein synthesis rates (aFLP or bFLP), very-high FLP mRNA or protein decay rates (gFLP or dFLP), or very-high half-max constants for FLP-dependent activation of PIN3 transcription or ARF7-dependent activation of FLP transcription (KFP or KAF). Discussion GUS staining and microscopy. The b-glucuronidase (GUS) assays were performed as follows: seedlings were incubated in GUS staining buffer (0.1 M Tris pH7.5 containing 2.9 mg ml 1 NaCl, 6.6 mg ml 1 K3Fe(CN)6) at 37 C over- night49. For microscopic analysis, samples were cleared by mounting in lactic acid (Acros Organics, Geel, Belgium) or were transferred to 0.24 N HCl in 20% at 57 C for 15 min. This solution was replaced with 7% NaOH, 7% hydroxylamine-HCl in 60% ethanol for 15 min at room temperature. Roots were then rehydrated for 5 min each in 40, 20 and 10% ethanol, and inﬁltrated for 15 min in 5% ethanol, 25% glycerol. Roots were mounted in 50% glycerol on glass microscope slides50. All samples were analysed using a BX53 Olympus microscope. Fluorescence images were acquired by a Zeiss 710 confocal laser scanning microscope equipped with a C-Apochromat 40 water immersion objective. If needed for visualization purposes, further processing was done using Image J software by uniformly changing brightness and contrast to the entire image per channel. For the images in Fig. 1a, the smoothing function was applied to both channels. Local auxin accumulation serves an essential role in triggering transcriptional changes associated with developmental transitions during plant organogenesis, as is reﬂected during LR development. A remarkable feature of auxin transport orchestrat- ing organogenesis and regeneration, is the multi-level feed-back regulation of PIN-mediated auxin transport ﬂows that are reinforced by the hormone itself8. Prominently among them is the auxin-induced transcription of PINs17, stimulating the cellular auxin transport capacity. Although not studied in detail, it has been generally accepted that the auxin-sensitive expression of several PINs, including PIN3, is directly activated by the canonical auxin signalling6,17. Our data further elaborate this notion by demonstrating that direct binding of ARF7 to the PIN3 promoter is critical for auxin-sensitive transcription of PIN3. Phenotyping and statistics. To analyse stages of LR development, about 20 roots from 6-day-old seedlings were processed per data point and genotype. Plant material was cleared following the more elaborate protocol as described above50. Root lengths were measured with the Image J software (NIH; http:// rsb.info.nih.gov/ij). LRPs were counted with a differential interference contrast microscope (BX53; Olympus). LRP density was calculated as the ratio of the total number of LRP over the sum of root length. Phenotypic analysis of FC and LRP was done as described12. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 reporter lines were crossed into ﬂp-1 myb88 and ﬂp-7 myb88. proFLP::FLP-GR/ﬂp-1 myb88, proFLP::NLS-GFP, proPIN3::PIN3-YPF/pin3-4 lines were generated through Agrobacterium-mediated ﬂoral dip transformation48. ‘memorized’ to temporarily sustain transcriptional auxin signalling in selected cells for speciﬁc transcripts. Discussion The resulting promoters were cloned into pDONRTMP4-P1R, and together with PIN3-YFP in pDONRTM221 (ref. 45), subcloned into pB7m24GW52,53. The above-mentioned primers are listed in Supplementary Table 2. Our data suggest that this auxin-sensing circuitry of PIN3 expression is involved LR initiation. In this developmental process, the high auxin concentrations that promote the progression from FC to LRI depends in part on PIN3-mediated auxin transport12, as illustrated by the diminished DR5 expression in ﬂp myb88 LR primordia. Similar transcriptional architectures could underlie the auxin-sensitive expression of other PINs, including PIN1 and PIN4 that were found to be directly controlled by the auxin-inducible MADS-box XAL2/AGL14 (ref. 22), and PIN7 that was also found to be directly regulated by FLP binding to its promoter34. Gene Expression Analysis by qRT-PCR. For qRT-PCR analysis, 6-day-old seedlings were treated with NAA (10 mM) and/or DEX (10 mM) at different time points and roots were harvested for RNA extraction. RNA was extracted with an RNeasy kit (QIAGEN). Poly(dT) cDNA was prepared from 1 mg of total RNA with SuperScript III reverse transcriptase (Invitrogen) and analysed on a LightCycler 480 apparatus (Roche Diagnostics) with the SYBR Green I Master kit (Roche Diagnostics) according to the manufacturer’s instructions. Primer pairs were designed with Beacon Designer 4.0 (Premier Biosoft International) (Supplementary Table 2). All individual reactions were performed as biological triplicates. Data was analysed with qBase54. Expression levels were normalized to those of ACTIN2. The statistical signiﬁcance was evaluated by Student’s t-test analysis for pair-wise comparisons and by Fisher’s LSD means separation test (SPSS) for multiple comparisons. Discussion In brief, 10 roots of 5-day-old seedling (grown at 21C 16 h light 8 h dark) were analysed per experiment. The counting of FCs and LRP was performed in the direction from root tip towards the root base. Zeiss LSM 710 confocal microscope with a 40 (water immersion) objective with GFP settings (excitation 488 nm, emission 507 nm) were used to identify and count DR5rev::GFP-positive signals in the pericycle. GFP signals accompanied with nuclear divisions were scored as LRP, whereas FCs were counted when no cell divisions were observed. The data are represented as averages of three independent experiments, unless indicated differently. The statistical signiﬁcance was evaluated by Student’s t-test analysis. For multiple comparisons, an analysis of variance followed by Fisher’s Least Signiﬁcant Difference (LSD) mean separation test (SPSS) was performed on the data. Samples with different letters are signiﬁcantly different at Po0.01 or Po0.05. Boxplots were generated using BoxPlotR51. p Such auxin-dependent regulation might mainly act to amplify the complex, developmentally wired cis-regulatory architectures that underlie the tissue-speciﬁc expression patterns of the different PINs38. Here, we propose that the auxin-sensitive expression of PIN3 depends on binding of both ARF7 and FOUR LIPS, a MYB transcription factor previously characterized for its role in guard cell development. In addition, we found that ARF7 also controls auxin-sensitive FLP expression, thus deﬁning a coherent FFM for auxin-induced PIN3 expression. Mathematical simulations revealed that this conﬁguration could amplify the auxin-response output of PIN3 transcription and also temporarily sustain elevated PIN3 transcription after the auxin-stimulus is removed. This could allow for depletion of cellular auxin below the levels that were initially needed to activate PIN3 transcription, and for temporal buffering of random auxin ﬂuctuations. This enhanced auxin transport capacity seems to stimulate FCs to proceed into LR initiation. Such transcriptional control mechanisms could also be relevant for developmental processes that are associated with auxin depletion, such as fruit valve speciﬁcation21,39 and stomatal development and patterning24. Interestingly, coherent FFMs of the type described here are abundantly found in transcriptional networks in yeast, animals and plants40–43, suggesting that this motif might represent an evolutionarily favoured solution to obtain a response delay upon stimulus shutdown. Cloning. To construct proFLP::FLP-GR, a FLP promoter region (±3 kb upstream of translational start) was ampliﬁed with primers proFLP-F and proFLP-R and cloned into pDONRTMP4-P1R. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 Long PIN3 mRNA or protein half-lives (small gPIN3 or dPIN3) also appear to offset the effect of the FFM (see Supplementary Fig. 10). NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunicat & 2015 Macmillan Publishers Limited. All rights reserved. 8 & 2015 Macmillan Publishers Limited. All rights reserved Discussion The FLP cDNA was ampliﬁed with primers FLP-F and FLP-R and cloned into pDONRTM221; The above-mentioned pDONR vectors were subcloned together with a Glucocorticoid ORF containing (pDONRTMP2R- P3) into pB7m34GW52,53 with MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen). cDNA of ARF7 was PCR ampliﬁed with primers ARF7-F and ARF7-R, and cloned into pDONRTM221. proFLP in pDONRTMP4- P1R was subcloned into pEX-K7SNFm14GW to construct proFLP::NLS-GFP. A promoter 1.8 kb upstream of the PIN3 translational start was cloned into pDONRTMP4-P1R with primers proPIN3_FL_F and proPIN3_FL_R. Site-directed mutagenesis was performed using PCR to mutate AuxREs and FBS. The resulting promoters were cloned into pDONRTMP4-P1R, and together with PIN3-YFP in pDONRTM221 (ref. 45), subcloned into pB7m24GW52,53. The above-mentioned primers are listed in Supplementary Table 2. Cloning. To construct proFLP::FLP-GR, a FLP promoter region (±3 kb upstream of translational start) was ampliﬁed with primers proFLP-F and proFLP-R and cloned into pDONRTMP4-P1R. The FLP cDNA was ampliﬁed with primers FLP-F and FLP-R and cloned into pDONRTM221; The above-mentioned pDONR vectors were subcloned together with a Glucocorticoid ORF containing (pDONRTMP2R- P3) into pB7m34GW52,53 with MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen). cDNA of ARF7 was PCR ampliﬁed with primers ARF7-F and ARF7-R, and cloned into pDONRTM221. proFLP in pDONRTMP4- P1R was subcloned into pEX-K7SNFm14GW to construct proFLP::NLS-GFP. Cloning. To construct proFLP::FLP-GR, a FLP promoter region (±3 kb upstream of translational start) was ampliﬁed with primers proFLP-F and proFLP-R and cloned into pDONRTMP4-P1R. The FLP cDNA was ampliﬁed with primers FLP-F and FLP-R and cloned into pDONRTM221; The above-mentioned pDONR vectors were subcloned together with a Glucocorticoid ORF containing (pDONRTMP2R- P3) into pB7m34GW52,53 with MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen). cDNA of ARF7 was PCR ampliﬁed with primers ARF7-F and ARF7-R, and cloned into pDONRTM221. proFLP in pDONRTMP4- P1R was subcloned into pEX-K7SNFm14GW to construct proFLP::NLS-GFP. A promoter 1.8 kb upstream of the PIN3 translational start was cloned into pDONRTMP4-P1R with primers proPIN3_FL_F and proPIN3_FL_R. Site-directed mutagenesis was performed using PCR to mutate AuxREs and FBS. The resulting promoters were cloned into pDONRTMP4-P1R, and together with PIN3-YFP in pDONRTM221 (ref. 45), subcloned into pB7m24GW52,53. The above-mentioned primers are listed in Supplementary Table 2. p p A promoter 1.8 kb upstream of the PIN3 translational start was cloned into pDONRTMP4-P1R with primers proPIN3_FL_F and proPIN3_FL_R. Site-directed mutagenesis was performed using PCR to mutate AuxREs and FBS. & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 In addition, the form of the auxin-dependent saturation characteristic was reverted to the form used earlier by Mitchison et al.58, which includes an additional KA factor in the numerator of the Michaelis-Menten relation, to restore the unit balance. In equations (6) and (7), the formulation of auxin-dependent PIN3 membrane transport and PIN3 membrane localization has been changed to reﬂect the observation that auxin inhibits the internalization of membrane-bound PIN3 rather than stimulating PIN3 delivery to the plasma membrane60,61, and to better reﬂect the membrane concentration nature of PIN3mem (mmol dm 2). precipitated without antibody was used as negative control, while the isolated chromatin before precipitation was used as input control55,56. The enrichment of DNA fragments was determined by quantitative PCR using the following primer pairs: proACT2_F and proACT2_R; proFLP(P1)_F, proFLP(P1)_R, proFLP(P2)_F, proFLP(P2)_R, proPIN3(P1)_F and proPIN3(P1)_R; proPIN3(P2)_F and proPIN3(P2)_R (Supplementary Table 2). Yeast-one hybrid. Yeast-one-hybrid assays were performed with the kit provided by Clontech (Matchmaker One-Hybrid Library Construction and Screening kit) using the Y1HGold yeast strain according to the manufacturer’s instructions. This system detects binding events by resistance against the antibiotic Aureobasidin A (AbA). We complemented this auxin transport model with equations describing the dynamics of the transcriptional feed-forward circuit impacting PIN3 expression (Equations (2)–(6)). Assuming a constant ARF7 protein concentration (ARF7 ¼ 1 mM), ARF7 activity kinetics are modelled as a combination of basal, auxin independent ARF7 activity (oARF7) and auxin-dependent ARF7 activity (Equation (2)). Equation (3) describes how FLP mRNA levels are inﬂuenced by ARF7act-dependent transcription and mRNA degradation, while equation (4) describes FLP protein concentration changes as a function of protein production and degradation. Equation (5) incorporates the effects of active ARF7 and FLP on PIN3 transcription, as well as an mRNA degradation term. Equation (6) incorporates PIN3 protein production and degradation terms, in addition to the membrane transport-related terms discussed above. A protein degradation term was also added to equation (7), under the assumption that membrane-bound PIN3 is also earmarked for degradation. ( ) The primers used for cloning the related cDNAs or promoter DNAs are listed in Supplementary Table 2. The cDNA of ARF7 and FLP in pDONRTM221 were subcloned into pGADT7. The promoter fragments of PIN3 were cloned into the SacI/SalI sites of pAbAi vector, and efﬁciently integrated into the genome of the Y1H Gold yeast strain by homologous recombination on SD-U medium. ARTICLE Matchmakert Insert Check PCR Mix was used to verify positive colonies. Autoactivation tests for the promoter were conducted in the range from 0 to 500 ng ml 1 AbA, and background activation was detected up to 50 ng ml 1. An AbA concentration of 100 ng ml 1 was used to screen positive colonies. Then pGADT7 prey vectors harboring ORF of ARF7 or FLP were transformed into the above-mentioned yeast strain that have already integrated the promoter fragments of PIN3 or FLP. After having grown on SD-Leu for 2–3 days, the colonies able to grow on the selective medium were transferred to 100 ml of sterile ultrapure water and spotted as 5 ml droplets onto SD-Leu þ AbA plates. Plates were incubated at 28 C for 3–4 days. The primers used for cloning the related cDNAs or promoter DNAs are listed in Supplementary Table 2. The cDNA of ARF7 and FLP in pDONRTM221 were subcloned into pGADT7. The promoter fragments of PIN3 were cloned into the SacI/SalI sites of pAbAi vector, and efﬁciently integrated into the genome of the Y1H Gold yeast strain by homologous recombination on SD-U medium. h k t h k d f l g Simulations were performed in MATLAB R2013b, using an implicit numerical solver with adaptable time step used for solving initial value problems for stiff ordinary differential equations (ode23s.m function). The MATLAB scripts are available from http://www.psb.ugent.be/esb/ESB/FLP_FFM_SI.html. The default simulations (Fig. 6c–h) were performed using the parameter settings described in Supplementary Table 1. mRNA and protein degradation rate constants were set to reﬂect typical mRNA and protein half-lives of B1 h. In the default settings, FLP was set to have a shorter protein half-life (30 min) than PIN3 (2 h) and the half-max constants for FLP- and ARF7-dependent activation of PIN3 transcription (KFP and KAP) were set to 0.6 and 0.3 mM, respectively, in order to achieve a B50% drop in PIN3 expression level as observed in the ﬂp mutant after 6 h (see Fig. 3d). Initial auxin, PIN3 and FLP levels were set to the steady-state levels obtained for a constant extracellular auxin concentration (Aext) of 0.1 mM. Simulations of the ﬂp mutant were performed by setting aFLP, bFLP, gFLP, dFLP and the initial FLP mRNA and protein concentrations to zero. PIN3 response delay upon stimulus removal to parameters. Although the with Aint and Aext the intra- and extracellular auxin concentrations, respectively, ARF7act the concentration of active ARF7 protein, FLPmRNA and FLP the ARTICLE In all simulations, a background extracellular auxin concentration of 0.1 mM was assumed, and 2 h-long 1 mM extracellular auxin pulses were simulated at t ¼ 4–6 h, t ¼ 12–14 h and t ¼ 26–28 h. Simulations were performed in MATLAB R2013b, using an implicit numerical solver with adaptable time step used for solving initial value problems for stiff ordinary differential equations (ode23s.m function). The MATLAB scripts are available from http://www.psb.ugent.be/esb/ESB/FLP_FFM_SI.html. The default simulations (Fig. 6c–h) were performed using the parameter settings described in Supplementary Table 1. mRNA and protein degradation rate constants were set to reﬂect typical mRNA and protein half-lives of B1 h. In the default settings, FLP was set to have a shorter protein half-life (30 min) than PIN3 (2 h) and the half-max constants for FLP- and ARF7-dependent activation of PIN3 transcription (KFP and KAP) were set to 0.6 and 0.3 mM, respectively, in order to achieve a B50% drop in PIN3 expression level as observed in the ﬂp mutant after 6 h (see Fig. 3d). Initial auxin, PIN3 and FLP levels were set to the steady-state levels obtained for a constant extracellular auxin concentration (Aext) of 0.1 mM. Simulations of the ﬂp mutant were performed by setting aFLP, bFLP, gFLP, dFLP and the initial FLP mRNA and protein concentrations to zero. In all simulations, a background extracellular auxin concentration of 0.1 mM was assumed, and 2 h-long 1 mM extracellular auxin pulses were simulated at t ¼ 4–6 h, t ¼ 12–14 h and t ¼ 26–28 h. Modelling the effects of FLP on PIN3 and auxin dynamics. We mathematically modelled the impact of the FLP FFM on PIN3 expression dynamics starting from an earlier model by Jo¨nsson et al.57. PIN3 response delay upon stimulus removal to parameters. Although the PIN3 response delay upon stimulus removal to parameters. Although the parameters listed in Supplementary Table 1 are within biologically reasonable ranges, the resulting model cannot be taken to be quantitatively accurate, as this would require quantitative measurements of all model parameters and absolute molecular concentration ranges in vivo. To test the robustness of our modelling- based conclusions, we performed a series of simulations screening the effects of parameter changes on the qualitative dynamics of the FFM versus no-FFM circuits. In each simulation, one parameter was changed from its default value listed in Supplementary Table 1, while the other parameters were kept at their default values. Hill coefﬁcients n1, n2, n3, n4, n5 were screened in the range [1, 3], oARF7 was screened in the range [0, 1] and the other parameters were screened in a range encompassing one order of magnitude above and below the default values listed in Supplementary Table 1 (with the exception of bA, the midrange value of which (on log scale) was set to 5 10 6 s 1 instead of 0 s 1 for screening purposes). The resulting alternatively parameterized circuits were simulated with and without FFM under a 1 mM extracellular auxin stimulus until steady-state was reached, after which the auxin stimulus was shut down and the PIN3 protein response time was measured, deﬁned as the time it takes the system to bridge half of the PIN3 protein concentration difference between the auxin-stimulated and unstimulated steady states. FFM/no FFM response time ratios 41 indicate that the circuit with FFM exhibits a delayed PIN3 protein response upon auxin stimulus shutdown. dFLP dt ¼ bFLPFLPmRNA dFLPFLP ð4Þ ð6Þ dPIN3mem dt ¼ V a k1PIN3 k2 Kn5 Kn5 þ An5 ext PIN3mem dPIN3PIN3mem ð7Þ with Aint and Aext the intra- and extracellular auxin concentrations, respectively, ARF7act the concentration of active ARF7 protein, FLPmRNA and FLP the concentrations of FLP mRNA and protein, PIN3mRNA the concentration of PIN3 mRNA, and PIN3 and PIN3mem the concentrations of intracellular and membrane- localized PIN3 protein. a and b parameters denote mRNA and protein (or auxin) production rates, respectively, and g and d parameters denote the corresponding degradation rates. K0s indicate half-maximal activation constants, and n0s are activation curve Hill coefﬁcients. Detailed parameter deﬁnitions and dimensions, as well as the default parameter values used, are presented in Supplementary Table 1 (refs 57–59). Methods Pl t t Plant materials and growth conditions. Arabidopsis seeds were surface-sterilized for 15 min in 10% bleach, washed four times with sterile water, and plated on half-strength Murashige and Skoog medium (0.8% agar). Plants were stratiﬁed at 4 C for 2 days in darkness and then transferred to a growth chamber at 22 C under continuous illumination (light intensity 120 mmol m 2 s 1). Arabidopsis thaliana ecotypes Columbia (Col-0), and Ler were used as controls. The following lines/seeds/constructs were used in this study: ﬂp-1 (ref. 44), ﬂp-7 (ref. 25), myb88 (SALK_068691)25, ﬂp-1 myb88 (ref. 25), ﬂp-7 myb88 (ref. 25), proFLP::GUS-GFP25, cyca2;234 (ref. 26), ﬂp-7 myb88 cyca2;234 (ref. 26), slr-1 (ref. 32), proARF7::ARF7-GR/nph4-1(arf7)arf19-1 (ref. 33), pin3-4 (SALK_038609)45, proDR5::GUS46, proDR5rev::GFP47 proPIN3::PIN3-GFP45. Published mutants and ChIP-qPCR assay. Eight-day-old seedlings of proFLP::FLP-GR/ﬂp-1 myb88 or proARF7::ARF7-GR/arf7 arf19 were treated with 10 mM DEX for 6 h. One gram root material per sample was used for ChIP experiments. Plant materials were cross-linked in 1% formaldehyde and their chromatin isolated. Anti-GR antibody ab3580 (abcam) (1:500) was used to immunoprecipitate the protein-DNA complex, and the precipitated DNA was puriﬁed using a PCR puriﬁcation kit (Qiagen) for qRT-PCR analysis. The ChIP experiments were performed three times. Chromatin ChIP-qPCR assay. Eight-day-old seedlings of proFLP::FLP-GR/ﬂp-1 myb88 or proARF7::ARF7-GR/arf7 arf19 were treated with 10 mM DEX for 6 h. One gram root material per sample was used for ChIP experiments. Plant materials were cross-linked in 1% formaldehyde and their chromatin isolated. Anti-GR antibody ab3580 (abcam) (1:500) was used to immunoprecipitate the protein-DNA complex, and the precipitated DNA was puriﬁed using a PCR puriﬁcation kit (Qiagen) for qRT-PCR analysis. The ChIP experiments were performed three times. Chromatin 9 NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications 9 & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE The revised model is deﬁned by the following ordinary differential equations: dAint dt ¼ bA dAAint þ a V pAH f wall AH Aext f cell AH Aint þ pA PIN3mem RP f wall A Ninflux KAAext KA þ Aext f cell A Nefflux KAAint KA þ Aint ARF7act ¼ ARF7 oARF7 þ An1 int Kn1 ARF7 þ An1 int dFLPmRNA dt ¼ aFLP ARF7n2 act Kn2 AF þ ARF7n2 act gFLPFLPmRNA dFLP dt ¼ bFLPFLPmRNA dFLPFLP dAint dt ¼ bA dAAint þ a V pAH f wall AH Aext f cell AH Aint þ pA PIN3mem RP f wall A Ninflux KAAext KA þ Aext f cell A Nefflux KAAint KA þ Aint ð1Þ ARF7act ¼ ARF7 oARF7 þ An1 int Kn1 ARF7 þ An1 int ð2Þ dFLPmRNA dt ¼ aFLP ARF7n2 act Kn2 AF þ ARF7n2 act gFLPFLPmRNA ð3Þ dFLP dt ¼ bFLPFLPmRNA dFLPFLP ð4Þ dPIN3mRNA dt ¼ aPIN3 ARF7n3 act=Kn3 AP þ FLPn4=Kn4 FP 1 þ ARF7n3 act=Kn3 AP þ FLPn4=Kn4 FP gPIN3PIN3mRNA ð5Þ dPIN3 dt ¼ bPIN3PIN3mRNA dPIN3PIN3 k1PIN3 þ a V k2 Kn5 Kn5 þ An5 ext PIN3mem ð6Þ dPIN3mem d ¼ V k1PIN3 k2 Kn5 n PIN3mem dPIN3PIN3mem ð7Þ ð1Þ ARTICLE qBase relative quantiﬁcation framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19 (2007). 22. Garay-Arroyo, A. et al. The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression. EMBO J. 32, 2884–2895 (2013). 55. Gendrel, A. V., Lippman, Z., Martienssen, R. & Colot, V. Proﬁling histone modiﬁcation patterns in plants using genomic tiling microarrays. Nat. Methods 2, 213–218 (2005). p 23. Cui, D. et al. The Arabidopsis IDD14, IDD15, and IDD16 cooperatively regulate lateral organ morphogenesis and gravitropism by promoting auxin biosynthesis and transport. PLoS Genet. 9, e1003759 (2013). 56. Kaufmann, K. et al. Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP). Nat. Protoc. 5, 457–472 (2010). p 24. Le, J. et al. Auxin transport and activity regulate stomatal patterning and development. Nat. Commun. 5, 3090 (2014). 57. Jo¨nsson, H., Heisler, M. G., Shapiro, B. E., Meyerowitz, E. M. & Mjolsness, E. An auxin-driven polarized transport model for phyllotaxis. Proc. Natl Acad. Sci. USA 103, 1633–1638 (2006). 25. Lai, L. B. et al. The Arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage. Plant Cell 17, 2754–2767 (2005). 58. Mitchison, G. J. The dynamics of auxin transport. Proc. R. Soc. B Biol. 209, 489–511 (1980). 26. Vanneste, S. et al. Developmental regulation of CYCA2s contributes to tissue-speciﬁc proliferation in Arabidopsis. EMBO J. 30, 3430–3441 (2011). 59. Grieneisen, V. A., Xu, J., Maree, A. F., Hogeweg, P. & Scheres, B. Auxin transport is sufﬁcient to generate a maximum and gradient guiding root growth. Nature 449, 1008–1013 (2007). 27. Xie, Z. et al. Regulation of cell proliferation in the stomatal lineage by the Arabidopsis MYB FOUR LIPS via direct targeting of core cell cycle genes. Plant Cell 22, 2306–2321 (2010). 60. Paciorek, T. et al. Auxin inhibits endocytosis and promotes its own efﬂux from cells. Nature 435, 1251–1256 (2005). 28. Yang, K. et al. Requirement for A-type cyclin-dependent kinase and cyclins for the terminal division in the stomatal lineage of Arabidopsis. J. Exp. Bot. 65, 2449–2461 (2014). 61. Robert, S. et al. ABP1 mediates auxin inhibition of clathrin-dependent d t i i A bid i C ll 143 111 121 (2010) 61. Robert, S. et al. ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis. Cell 143, 111–121 (2010). 29. ARTICLE g y p p expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9, 1963–1971 (1997). 14. Swarup, K. et al. The auxin inﬂux carrier LAX3 promotes lateral root emergence. Nat. Cell Biol. 10, 946–954 (2008). emergence. Nat. Cell Biol. 10, 946–954 (2008). 47. Friml, J. et al. Efﬂux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature 426, 147–153 (2003). 15. Pe´ret, B. et al. Auxin regulates aquaporin function to facilitate lateral root emergence. Nat. Cell Biol. 14, 991–998 (2012). p 48. Clough, S. J. & Bent, A. F. Floral dip: a simpliﬁed method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998). 16. Petra´sˇek, J. et al. PIN proteins perform a rate-limiting function in cellular auxin efﬂux. Science 312, 914–918 (2006). 49. Beeckman, T. & Engler, G. An easy technique for the clearing of histochemically stained plant tissue. Plant Mol. Biol. Rep. 12, 37–42 17. Vieten, A. et al. Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression. Development 132, 4521–4531 (2005). 50. Malamy, J. E. & Benfey, P. N. Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124, 33–44 (1997). 18. Vernoux, T. et al. The auxin signalling network translates dynamic input into robust patterning at the shoot apex. Mol. Syst. Biol. 7, 508 (2011). 51. Spitzer, M., Wildenhain, J., Rappsilber, J. & Tyers, M. BoxPlotR: a web tool for generation of box plots. Nat. Methods 11, 121–122 (2014). p g p y 19. Han, M. et al. Structural basis for the auxin-induced transcriptional regulation by Aux/IAA17. Proc. Natl Acad. Sci. USA 111, 18613–18618 (2014). 52. Karimi, M., Depicker, A. & Hilson, P. Recombinational cloning with plant gateway vectors. Plant Physiol. 145, 1144–1154 (2007). 20. Pierre-Jerome, E., Jang, S. S., Havens, K. A., Nemhauser, J. L. & Klavins, E. Recapitulation of the forward nuclear auxin response pathway in yeast. Proc. Natl Acad. Sci. USA 111, 9407–9412 (2014). 53. Hilson, P. et al. Versatile gene-speciﬁc sequence tags for Arabidopsis functional genomics: transcript proﬁling and reverse genetics applications. Genome Res. 14, 2176–2189 (2004). 21. Lavenus, J. et al. Inference of the Arabidopsis lateral root gene regulatory network suggests a bifurcation mechanism that deﬁnes primordia ﬂanking and central zones. Plant Cell 27, 1368–1388 (2015). 54. Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. & Vandesompele, J. Author contributions 36. Boer, D. R. et al. Structural basis for DNA binding speciﬁcity by the auxin-dependent ARF transcription factors. Cell 156, 577–589 (2014). S.V., T.B and F.S. conceived the project. S.V., Q.C., Y.L., G.V.I., P.M., Z.X., P.M., E.L., V.V., S.K. performed general experiments. Y.L. performed yeast-one hybrid assays. S.M. and K.W. performed mathematical modelling analyses. H.F., S.M., E.G., J.L. and J.F. helped analysing the data. All authors contributed to writing the manuscript. 37. Mangan, S. & Alon, U. Structure and function of the feed-forward loop network motif. Proc. Natl Acad. Sci. USA 100, 11980–11985 (2003). 38. Tanaka, H., Dhonukshe, P., Brewer, P. B. & Friml, J. Spatiotemporal asymmetric auxin distribution: a means to coordinate plant development. Cell. Mol. Life Sci. 63, 2738–2754 (2006). ARTICLE De Rybel, B. et al. A novel Aux/IAA28 signaling cascade activates GATA23- dependent speciﬁcation of lateral root founder cell identity. Curr. Biol. 20, 1697–1706 (2010). Acknowledgements 30. De Smet, I. et al. Receptor-like kinase ACR4 restricts formative cell divisions in the Arabidopsis root. Science 322, 594–597 (2008). This work was supported by the National Basic Research Program of China (2015CB942900) (to Q.C. and C.L.) and (2011CB710902) (to J.L.), a European This work was supported by the National Basic Research Program of China (2015CB942900) (to Q.C. and C.L.) and (2011CB710902) (to J.L.), a European 31. Fukaki, H., Nakao, Y., Okushima, Y., Theologis, A. & Tasaka, M. Tissue-speciﬁc expression of stabilized SOLITARY-ROOT/IAA14 alters lateral root development in Arabidopsis. Plant J. 44, 382–395 (2005). (2015CB942900) (to Q.C. and C.L.) and (2011CB710902) (to J.L.), a European Community FP7 Marie Curie Fellowship (PIIF-GA-2012-330972) (to Q.C.), grants of the Research Foundation-Flanders (to S.V. and (FWO G.0088.12) to S.M.), of the European Research Council (project ERC-2011-StG-20101109-PSDP) (to J.F.), of the USA National Science Foundation (NSF MCB-0418891) (to E.G.), a Natural Sciences and Engineering Research Council of Canada Discovery Grant #22R92904 (to F. S.), a Grant-in-Aid for Scientiﬁc Research on Priority Areas (19060006) from The Ministry of Education, Culture, Sports, Science and Technology, Japan (to H.F.), a FEBS long-term fellowship (to P.M.) and work in the lab of T.B. was supported by grants of the Interuniversity Attraction Poles Programme (IAP VI/33 and IUAP P7/29 ‘MARS’) initiated by the Belgian Science Policy Ofﬁce, the Research Foundation-Flanders and Ghent University Special Research Fund. p p 32. Fukaki, H., Tameda, S., Masuda, H. & Tasaka, M. Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J. 29, 153–168 (2002). 33. Okushima, Y., Fukaki, H., Onoda, M., Theologis, A. & Tasaka, M. ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis. Plant Cell 19, 118–130 (2007). p 34. Wang, H. et al.Transcriptional regulation of PINs by FOUR LIPS and MYB88 during Arabidopsis root gravitropism. Nat. Commun. 6, 8822 (2015). Belgian Science Policy Ofﬁce, the Research Foundation-Flanders and Ghent University Special Research Fund. g p g p ( ) 35. Ulmasov, T., Liu, Z. B., Hagen, G. & Guilfoyle, T. J. Composite structure of auxin response elements. Plant Cell 7, 1611–1623 (1995). 35. Ulmasov, T., Liu, Z. B., Hagen, G. & Guilfoyle, T. J. Composite structure of auxin response elements Plant Cell 7 1611 1623 (1995) ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 8. Vanneste, S. & Friml, J. Auxin: a trigger for change in plant development. Cell 136, 1005–1016 (2009). 41. Alon, U. Network motifs: theory and experimental approaches. Nat. Rev. Genet. 8, 450–461 (2007). 42. Le, D. H. & Kwon, Y. K. A coherent feedforward loop design principle to sustain robustness of biological networks. Bioinformatics 29, 630–637 (2013). 9. Lavenus, J. et al. Lateral root development in Arabidopsis: ﬁfty shades of auxin. Trends Plant Sci. 18, 450–458 (2013). 10. Xuan, W. et al. Root cap-derived auxin pre-patterns 10. Xuan, W. et al. Root cap-derived auxin pre-patterns th Arabidopsis root. Curr. Biol. 25, 1381–1388 (2015). 10. Xuan, W. et al. Root cap-derived auxin pre-patterns the longitudinal axis of the Arabidopsis root. Curr. Biol. 25, 1381–1388 (2015). 43. Carrera, J., Rodrigo, G., Jaramillo, A. & Elena, S. F. Reverse-engineering the Arabidopsis thaliana transcriptional network under changing environmental conditions. Genome Biol. 10, R96 (2009). Arabidopsis root. Curr. Biol. 25, 1381–1388 (2015) p Dubrovsky, J. G. et al. Auxin acts as a local morphogenetic trig 11. Dubrovsky, J. G. et al. Auxin acts as a local morphogenetic trigger to specify lateral root founder cells. Proc. Natl Acad. Sci. USA 105, 8790–8794 (2008). 44. Yang, M. & Sack, F. D. The too many mouths and four lips mutations affect stomatal production in Arabidopsis. Plant Cell 7, 2227–2239 (1995). 12. Marhavy´, P. et al. Auxin reﬂux between the endodermis and pericycle promotes lateral root initiation. EMBO J. 32, 149–158 (2013). 12. Marhavy´, P. et al. Auxin reﬂux between the endodermi lateral root initiation. EMBO J. 32, 149–158 (2013). y lateral root initiation. EMBO J. 32, 149–158 (2013). p p 45. Za´dnı´kova´, P. et al. Role of PIN-mediated auxin efﬂux in apical hook d l f b d h l l ( ) 45. Za´dnı´kova´, P. et al. Role of PIN-mediated auxin efﬂux in apical hook development of Arabidopsis thaliana. Development 137, 607–617 (2010). a´dnı´kova´, P. et al. Role of PIN-mediated auxin efﬂux in apical ho 13. Benkova´, E. et al. Local, efﬂux-dependent auxin gradients as a common module for plant organ formation. Cell 115, 591–602 (2003). p p p 46. Ulmasov, T., Murfett, J., Hagen, G. & Guilfoyle, T. J. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9, 1963–1971 (1997). References 1. De Smet, I. et al. Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis. Development 134, 681–690 (2007). p p 2. Moreno-Risueno, M. A. et al. Oscillating gene expression determines competence for periodic Arabidopsis root branching. Science 329, 1306–1311 (2010). 3. Van Norman, J. M., Xuan, W., Beeckman, T. & Benfey, P. N. To branch or not to branch: the role of pre-patterning in lateral root formation. Development 140, 4301–4310 (2013). As in Jo¨nsson et al.57, the intracellular auxin concentration in our model is inﬂuenced by intracellular auxin production and degradation, and by passive and active auxin transport across the cell membrane. In contrast to Jo¨nsson et al.57, the auxin dynamics in the cell wall are not modelled explicitly. Instead, the temporal proﬁle of the extracellular auxin concentration (Aext) is predeﬁned for each simulation, in terms of a constant background concentration and auxin pulses during speciﬁc epochs (see below). In equation (1), the saturability of PIN3mem- mediated active auxin transport is modelled slightly differently from the original model57. In particular, PIN3mem/RP (with RP a reference PIN3mem concentration) is used as a factor to modulate the membrane permeability pA , instead of limiting the maximum membrane PIN concentration to 1.0 mmol per unit area as in ref. 57. 4. Vermeer, J. E. et al. A spatial accommodation by neighboring cells is required for organ initiation in Arabidopsis. Science 343, 178–183 (2014). 5. Lucas, M. et al. Lateral root morphogenesis is dependent on the mechanical properties of the overlaying tissues. Proc. Natl Acad. Sci. USA 110, 5229–5234 (2013). 6. Pe´ret, B. et al. Sequential induction of auxin efﬂux and inﬂux carriers regulates lateral root emergence. Mol. Syst. Biol. 9, 699 (2013). 7. Kumpf, R. P. et al. Floral organ abscission peptide IDA and its HAE/HSL2 receptors control cell separation during lateral root emergence. Proc. Natl Acad. Sci. USA 110, 5235–5240 (2013). 10 & 2015 Macmillan Publishers Limited. All rights reserved. Additional information 39. Sorefan, K. et al. A regulated auxin minimum is required for seed dispersal in Arabidopsis. Nature 459, 583–586 (2009). 39. Sorefan, K. et al. A regulated auxin minimum is required for seed dispersal in Arabidopsis. Nature 459, 583–586 (2009). Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 40. Lee, T. I. et al. Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804 (2002). 40. Lee, T. I. et al. Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804 (2002). Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications 11 & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms9821 NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ How to cite this article: Chen, Q. et al. A coherent transcriptional feed-forward motif model for mediating auxin-sensitive PIN3 expression during lateral root development. Nat. Commun. 6:8821 doi: 10.1038/ncomms9821 (2015). Nat. Commun. 6:8821 doi: 10.1038/ncomms9821 (2015). 12 NATURE COMMUNICATIONS \| 6:8821 \| DOI: 10.1038/ncomms9821 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved.
https://openalex.org/W2523766120	http://dspace.mit.edu/bitstream/1721.1/106174/1/12864_2016_Article_3109.pdf	English	null	Erratum to: Comparative genomics and transcriptomics of Pichia pastoris	BMC genomics	2,016	cc-by	437	MIT Open Access Articles The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Version: Final published version: final published article, as it appeared in a journal, conference proceedings, or other formally published context Terms of use: Creative Commons Attribution © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Erratum In the original publication of this article [1], the accession numbers for the genomes and transcriptome listed are incorrect. The correct details of the NCBI accession numbers can be found below: Erratum to: Comparative genomics and transcriptomics of Pichia pastoris Kerry R. Love1†, Kartik A. Shah1†, Charles A. Whittaker2†, Jie Wu2, M. Catherine Bartlett1, Duanduan Ma2, Rachel L. Leeson1, Margaret Priest3, Jonathan Borowsky2, Sarah K. Young3 and J. Christopher Love1,3* Author details 1 1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 76-253, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. 2The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 3The Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA. 3The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Received: 6 September 2016 Accepted: 21 September 2016 Love et al. BMC Genomics (2016) 17:762 DOI 10.1186/s12864-016-3109-0 Open Access Availability of data and materials The genomic sequencing data and assembled and annotated genomes are deposited at NCBI under bio- project accession numbers PRJNA304627 (K. pastoris), PRJNA304977 (K. phaffii wildtype), and PRJNA304976 (K. phaffii GS115). RNA-seq data are deposited at NCBI under the bioproject accession numbers PRJNA311606. In addition to this, please find the direct links to the data below: Transcriptome study: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA311606 http://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_ sra_all&from_uid=311606 Genome assemblies: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA304627 http://www.ncbi.nlm.nih.gov/bioproject/PRJNA304977 http://www.ncbi.nlm.nih.gov/bioproject/PRJNA304976 Reference 1. Love KR, et al. Comparative genomics and transcriptomics of Pichia pastoris. BMC Genomics. 2016;17:550. doi:10.1186/s12864-016-2876-y. * Correspondence: clove@mit.edu †Equal contributors 1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 76-253, 77 Massachusetts Avenue, Cambridge, MA 02139, USA 3The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA * Correspondence: clove@mit.edu †Equal contributors 1Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 76-253, 77 Massachusetts Avenue, Cambridge, MA 02139, USA 3The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
https://openalex.org/W4249714081	https://amt.copernicus.org/articles/2/423/2009/amt-2-423-2009.pdf	English	null	Stratospheric isotopic water profiles from a single submillimeter limb scan by TELIS	null	2,009	cc-by	13,157	Stratospheric isotopic water proﬁles from a single submillimeter limb scan by TELIS A. de Lange, J. Landgraf, and R. Hoogeveen SRON Netherlands Institute for Space Research, Sorbonnelaan 2, Utrecht, The Netherlands Received: 30 January 2009 – Published in Atmos. Meas. Tech. Discuss.: 18 March 2009 Revised: 23 July 2009 – Accepted: 23 July 2009 – Published: 5 August 2009 Abstract. Around 490 GHz relatively strong HDO and H218O emission lines can be found in the submillimeter thermal-emission spectrum of the Earth’s atmosphere, along with lines of the principal isotopologue of water vapour. These can be used for remote sensing of the rare/principal isotope ratio in the stratosphere. A sensitivity study has been performed for retrieval simulations of water isotopologues from balloon-borne measurements by the limb sounder TELIS (TErahertz and submillimeter LImb Sounder). The study demonstrates the capability of TELIS to determine, from a single limb scan, the proﬁles for H218O and HDO between 20 km and 37 km with a retrieval error of ≈3% and a spatial resolution of 1.5 km, as determined by the width of the averaging kernel. In addition HDO can be retrieved in the range of 10–20 km, albeit with a strongly deteriorated retrieval error. Expected uncertainties in instrumental param- eters have only limited impact on the retrieval results. which results in freeze drying of tropospheric air, as was al- ready proposed by Brewer (1949) to explain the stratospheric aridity. Although this explanation has been accepted as the general dehydration process of incoming tropospheric air, the actual mechanisms and locations of the inﬂux have been subject of discussions ever since, and are still not fully re- solved (Rosenlof, 2003). Several mechanisms of dehydra- tion have been recognised; convective overshooting (Sher- wood and Dessler, 2000), gradual dehydration (Holton and Gettelman, 2001), and ice lofting (Danielsen, 1982; Moyer et al., 1996; Smith et al., 2006). The reaction constants of the various processes differ for different water isotopologues, and therefore, the isotope ratios of water will provide addi- tional insight in the different pathways of dehydration of tro- pospheric air. In situ measurements by Webster and Heyms- ﬁeld (2003) have shown that these processes can indeed be discerned by isotope signatures. More recently, Read et al. (2008) have shown how water isotopologue data from satel- lites can contribute in understanding the roles of convection, mixing, and freeze drying. Atmospheric Measurement Techniques Atmospheric Measurement Techniques Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. Atmospheric Measurement Techniques 1 Introduction To shed light on the manifold of processes of inﬂux, local production, and the various chemical reactions of water, water isotopologues are a useful tool. Vertical stratospheric proﬁles of water isotopo- logues can contribute to solving these long-standing issues. TELIS had its maiden ﬂight in June 2008 in Teresina, Brazil. It was mounted on the MIPAS-B2 balloon gondola and ﬂew together with the MIPAS instrument (Michelson Interferometer for Passive Atmospheric Sounding) (Friedl- Vallon et al., 2004) and mini-DOAS (Differential Optical Absorption Spectroscopy) (Weidner et al., 2005). Dur- ing this ﬂight all subsystems were separately successfully tested (Yagoubov et al., 2008). The ﬁrst scientiﬁc ﬂight is foreseen in spring 2009 in Kiruna, Sweden. In natural abundance 99.7% of water consists of the prin- cipal isotopologue H216O, and hence retrieving concentra- tions of the rare isotopologues with remote sensing tech- niques is challenging. Currently operational satellite instru- ments that provide stratospheric water isotopologue proﬁles are MIPAS (Steinwagner et al., 2007), Odin (Zelinger et al., 2006; Urban et al., 2007), ACE-FTS (Nassar et al., 2007), and, up to 100 hPa (≈17 km), also TES (Worden et al., 2006). However, due to low S/N ratios, averaging is required, at the expense of spatial and/or temporal resolution. In view of the aforementioned science cases, a signiﬁcant step forward would be the possibility of the retrieval of water isotopologue proﬁles from a single measurement and thereby improving on spatial and temporal resolution. Heterodyne detection is based on measuring the difference frequency of the atmospheric signal and a reference signal. The reference signal is generated in the so-called local os- cillator (LO). It is crucial to keep the LO frequency stable and well deﬁned, and in the 480–650 GHz channel this is achieved by either a phase lock loop or by frequency lock- ing. Both methods result in a different shape of the power spectrum. Frequency locking results in a Lorentzian lineshape of the LO with a full width half maximum (FWHM) of 2–10 MHz, depending on LO frequency. The central frequency, how- ever, may drift by ≈2 MHz. With the phase lock loop this drift is suppressed and the central frequency is ﬁxed within 1 kHz. The lineshape in this case is less well de- ﬁned, but can be measured separately. Most of the power is generated in a delta-like peak around the locking frequency (FWHM<1 kHz). 1 Introduction A second source of stratospheric water is in situ oxidation of methane and molecular hydrogen. This water is enriched in the rare, heavy isotopes with respect to water transported from the troposphere. As oxidation becomes more important for higher altitudes, this leads to an enrichment of rare iso- topes with altitude (Moyer et al., 1996; Johnson et al., 2001; Ridal et al., 2001; Zahn et al., 2006). Water is one of the most important species in the Earth’s at- mosphere, which occurs not only in the humid troposphere but also in the relative arid stratosphere. There it plays a vi- tal role in the radiative budget over a wide spectral range as well as in chemical reactions, both homogeneous and hetero- geneous. Water dynamics become even more complex if chem- istry is also accounted for. Water is directly linked to the HOx family, and thereby to numerous chemical processes in the stratosphere, including ozone-hole chemistry. Strato- spheric ozone is relatively more enriched in 17O than in 18O with respect to other oxygen containing species, dubbed as the ozone anomaly. If stratospheric water vapour shows The main source of stratospheric water is the inﬂux of hu- mid air from the troposphere in tropical regions. Upon entry into the stratosphere the air crosses the very cold tropopause, Correspondence to: A. de Lange (a.de.lange@sron.nl) Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 424 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS deviations in the relative depletion of the rare-oxygen iso- topes with respect to natural abundances, this may give addi- tional information on how water is interconnected to ozone chemistry and how important exchange reactions are (Franz and R¨ockmann, 2005). tunable 480–650 GHz channel (SRON). The instrument ei- ther records a 4 GHz spectrum with the 500 GHz chan- nel or records simultaneously two 2 GHz spectra with the 1.8 THz and 480–650 GHz channels. The spectral resolution is 2.2 MHz. The latter two channels will both be employed to retrieve proﬁles of water isotopologues. The presented ca- pacity study pertains to the 480–650 GHz channel by SRON. All in all, the stratospheric hydrological cycle is very com- plex and is still far from understood. 1 Introduction A part, however, is stored in two side lobes, located 10–15 MHz from the central peak. It is noted that further signal processing results in an additional uncer- tainty of the LO frequency of ≈1 MHz, independent of the chosen locking technique. With a low spectral resolution spectrometer, the spectral features of the rare isotopologues will most likely be dwarfed by the absorption and emission lines of the principal isotopo- logue, if not other species. A high resolution spectrome- ter, with the resolving power of individual lines, is therefore a promising route to obtain the stratospheric isotopic water composition, as the weak lines may be isolated from strong lines. In this paper the possibilities of isotopic water re- trieval from the 480–650 GHz channel of the balloon borne instrument TELIS (TErahertz and submillimeter LImb Sounder) (Hoogeveen et al., 2005; Yagoubov et al., 2008) are investigated by retrieval simulations. The outline of this pa- per is as follows. In Sects. 2–4 TELIS, the radiative transfer code and the retrieval algorithm are introduced respectively. Then in Sect. 5 proﬁle retrieval of water isotopologues are investigated for the ideal case with perfect knowledge of the instrument. Finally, in Sect. 6, the impact of uncertainties in instrument parameters on these retrievals are studied. The two different locking modes can be selected in-ﬂight and in this paper frequency locking is assumed with an FWHM=2 MHz. Note that the pressure broadening of the at- mospheric lines results in linewidths >10 MHz for altitudes <37 km (ﬂight altitude of TELIS), and the actual locking mechanism, and thus the spectral lineshape of the LO is of minor importance. The atmospheric signal is measured against the abso- lute difference frequency with respect to the local oscilla- tor. Hence, recorded signal may come from either side of the local oscillator frequency νLO, or sideband. In double- sideband mode no sideband is suppressed, and the measured spectrum is the superposition of two spectra covering the fre- quency ranges νLO+νIF respectively νLO−νIF, where νIF is the measured difference frequency, or the intermediate fre- quency (IF). For the 480–650 GHz channel νIF=5–7 GHz. 2 The TELIS instrument TELIS is a cryogenic heterodyne balloon sounder developed in a collaboration of three institutes: Deutsches Zentrum f¨ur Luft- und Raumfahrt (DLR), Germany, Rutherford Ap- pleton Laboratory (RAL), United Kingdom, and SRON – Netherlands Institute for Space Research, the Netherlands. Each institute develops one channel; a miniaturised 500 GHz channel (RAL), a tunable 1.8 THz channel (DLR), and a TELIS is currently operating in double-sideband mode. The sideband ratio (SBR) represents the relative magnitude Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS 425 5 5.5 6 6.5 7 IF [GHz] 0 50 100 150 Intensity [K] H2 18O H2O LO = 495.040 GHz 5 5.5 6 6.5 7 IF [GHz] 0 50 100 150 Intensity [K] HDO H2O LO = 496.880 GHz Fig. 1. Noise free spectra as function of difference frequency νIF for a local oscillator frequency νLO=495.040 GHz in the upper panel and νLO=496.880 GHz in the lower panel. The spectra are for TELIS, recording in double-sideband mode with a sideband ra- tio of 1.0. The ﬂight altitude is 37 km and the probed tangent height is 27.5 km. In grey is indicated which part of the spectrum is taken for retrieval of the mentioned molecule. 5 5.5 6 6.5 7 IF [GHz] 0 50 100 150 Intensity [K] H2 18O H2O LO = 495.040 GHz 5 5.5 6 6.5 7 IF [GHz] 0 50 100 150 Intensity [K] HDO H2O LO = 496.880 GHz of the two sidebands in the recorded spectrum. In the follow- ing this ratio is deﬁned as s=γUSB/γLSB, where γUSB and γLSB are respectively the gain of the upper sideband (USB) and the lower sideband (LSB). The recorded spectrum I is the weighted superposition of the spectra of the two side- bands, 5 5.5 6 6.5 7 IF [GHz] 0 50 100 150 Intensity [K] H2 18O H2O LO = 495.040 GHz I = γUSBIUSB + γLSBILSB γUSB + γLSB = s s + 1IUSB + 1 s + 1ILSB, (1) (1) where IUSB and ILSB are respectively the spectra of the upper and lower sideband. In this study a sideband ratio of s=1.0 is assumed for convenience and hence the weighting factors for both sidebands are 50%. In Sect. 6.2 deviations in the sideband ratio from unity are addressed. 2 The TELIS instrument In the 480–650 GHz channel the receiver antenna, the lo- cal oscillator, and the mixer that generates the difference frequency are all situated on a 4×4 mm2 chip, called the Super-conducting Integrated Receiver (SIR). This chip is de- veloped in collaboration with Institute of Radio Engineering and Electronics (IREE), Moscow, and is a major step in the miniaturisation of submillimeter receiver systems. For de- tails we refer to Hoogeveen et al. (2005); Koshelets et al. (2007). In the 480–650 GHz frequency window, there are a hand- ful relatively strong isotopic water emission lines. Unfor- tunately, most of these lines are blended by strong ozone or water lines. Notable exceptions are the H218O line at 489.054 GHz and the HDO line at 490.597 GHz which are also the lines probed by the Odin satellite (Urban et al., 2007). It is noted that the HDO line shows a small ozone feature in its wing. Fig. 1. Noise free spectra as function of difference frequency νIF for a local oscillator frequency νLO=495.040 GHz in the upper panel and νLO=496.880 GHz in the lower panel. The spectra are for TELIS, recording in double-sideband mode with a sideband ra- tio of 1.0. The ﬂight altitude is 37 km and the probed tangent height is 27.5 km. In grey is indicated which part of the spectrum is taken for retrieval of the mentioned molecule. In selecting the local oscillator frequency, one should take care that the lines aimed for do not overlap with lines from the other sideband. With the local oscillator set to respec- tively 495.040 GHz and 496.880 GHz, the H218O and HDO lines are isolated from lines of the other sideband. More- over, at these frequencies there is a water line of the prin- cipal isotopologue in each spectrum, such that the ratios H218O/H216O and HDO/H216O can each be determined from a single measurement. It is noted that the main water isotopo- logue line lies in the same sideband in the case of H218O and in the opposite sideband in case of HDO. www.atmos-meas-tech.net/2/423/2009/ 2 The TELIS instrument where Tsys=250 K is the noise temperature of the instru- ment in the frequency range of the rare water isotopo- logues (Yagoubov et al., 2008), 1ν=2.2 MHz is the spec- tral resolution, and t=1.5 s is the integration time, which re- sults in an RMS (Root Mean Squares) measurement noise 1T =0.13 K, corresponding to a signal to noise ratio of S/N=10/0.13≈75 for a single spectrum with a tangent point at 27.5 km. In Fig. 1 typical spectra are depicted for the measurement windows of HDO and H218O for a tangent height of 27.5 km, meaning the lowest point in altitude along a light path. From these spectra it can be seen that the signal levels of the rare water isotopologues are ≈10 K. The vertical resolution is determined by the ﬁeld of view of the instrument in conjunction with the spacing between probed tangent heights and the spectral features in the spec- trum. The ﬁeld of view of the 480–650 GHz channel is al- most Gaussian shaped with an FWHM in the vertical direc- tion of 13 arc min at 490 GHz (Yagoubov et al., 2008), which translates to ≈1.5 km at a tangent point of 26 km which is the middle of the limb sequence. The ﬁeld of view in the The measurement noise per spectral sampling point can be expressed as 1T = Tsys √ 1ν t , 1T = Tsys √ 1ν t , (2) Atmos. Meas. Tech., 2, 423–435, 2009 426 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS horizontal direction is twice as large, but horizontal gradients in the atmosphere are much smaller than vertical gradients and can be neglected on this scale. due to the pressure and temperature dependence of the index of refraction and is included in the model. To account for instrument characteristics, the modelled at- mospheric spectra are ﬁrst convolved with the instrumental lineshape, as determined by the locking mechanism of the local oscillator. Then the spectra are convolved with the ﬁeld of view (FOV) of the telescope. Finally the absolute differ- ence frequency with the local oscillator is taken, effectively mapping the two sidebands on top of each other. The latter is true in double sideband mode, but also in single sideband mode if the suppressed sideband still gives rise to residual signal. 4 Retrieval and regularisation The aim of the forward model is to simulate the measure- ments by TELIS for a given state of the atmosphere and viewing geometry. For this purpose the radiative transfer equation is solved, which gives the intensity I(ν, s) of ra- diation with frequency ν at location s in a light path. For a non-scattering atmosphere in local thermal equilibrium the radiative transfer equation can be integrated along the instru- ment’s line of sight With the forward model a spectrum for a speciﬁc atmo- sphere and geometry is calculated that is representative for the TELIS measurement. In the following the forward model will be denoted by F and the discrete set of atmospheric pa- rameters by the vector x. A measurement r can be simulated by r = F(x) + ey, (6) r = F(x) + ey, (6) I(ν, s) = I(ν, s0)e−τ(ν,s0,s) + Z s s0 B(ν, T (s′))κ(ν, s′)e−τ(ν,s′,s)ds′, (3) where ey is the measurement error including noise and bi- ases. (3) The aim of retrieval is to invert Eq. (6) to obtain the atmo- spheric condition x from the measurement r. As the forward model is nonlinear a Gauss–Newton iterative scheme is de- ployed to solve the inversion problem. In every iteration the forward model F is linearised by means of a Taylor expan- sion around a reference state xi−1 and solved for xi, where the index i refers to the iteration number. This results in where B(T ) is the Planck function at temperature T , and I(ν, s0) is the background intensity at the start of the line of sight. The optical density τ is given by τ(ν, s′, s) = Z s s′ κ(ν, s′′)ds′′, (4) (4) and the absorption coefﬁcient κ is deﬁned as and the absorption coefﬁcient κ is deﬁned as (7) y = Kxi + ey, (7) κ(ν, s) = X m km(ν, s)nm, (5) (5) where K is the Jacobian matrix and y=r−F(xi−1)+Kxi−1. where K is the Jacobian matrix and y=r−F(xi−1)+Kxi−1. A least squares ﬁt minimises the residue of the modelled spectrum with respect to the measured spectrum, A least squares ﬁt minimises the residue of the modelled spectrum with respect to the measured spectrum, with km the absorption cross section per molecule of molecule m and nm its number density. 4 Retrieval and regularisation In general km is a summation over many lines, each with different cen- tral frequency, line strength, and line proﬁle. To ac- count for random, collision-induced, phase shifts the van Vleck–Weisskopf correction is applied to the Voigt line pro- ﬁles (Vleck and Weisskopf, 1945). The actual proﬁles are based on line parameters taken from the HITRAN 2004 database (Rothman et al., 2005). xlsq = min x ∥(Kx −y)∥2. (8) (8) Within a limb scan each recorded spectrum probes predomi- nately the atmospheric state at a different tangent height, and therefore all spectra contain different information about the atmosphere. At ﬁrst glance one might therefore believe that a least squares ﬁt with a retrieval grid equal to the probed tangent heights would give good results. However, the at- mosphere above the instrument, where no tangent points are possible, may not be neglected in the forward model and will be of inﬂuence for the retrieval. Below the lowest tan- gent point the atmosphere is probed due to the extended ﬁeld of view. As a result Eq. (8) becomes an ill-posed problem, implying that different atmospheric proﬁles of temperature, pressure, and molecular concentrations, reproduce the mea- surement within noise limits. Within a limb scan each recorded spectrum probes predomi- nately the atmospheric state at a different tangent height, and therefore all spectra contain different information about the atmosphere. At ﬁrst glance one might therefore believe that a least squares ﬁt with a retrieval grid equal to the probed tangent heights would give good results. However, the at- mosphere above the instrument, where no tangent points are possible, may not be neglected in the forward model and will be of inﬂuence for the retrieval. Below the lowest tan- gent point the atmosphere is probed due to the extended ﬁeld of view. As a result Eq. (8) becomes an ill-posed problem, implying that different atmospheric proﬁles of temperature, pressure, and molecular concentrations, reproduce the mea- surement within noise limits. In the 480–650 GHz frequency range water shows some strong emission lines, the most notable around 557 GHz. Even far from line centre the wings contribute signiﬁcantly to the spectrum, acting as an offset or continuum. The cal- culated line proﬁles show small discrepancies with the actual line proﬁles. To account for this, the empirical Liebe model for the continuum is adopted (Liebe, 1989). The total path integral of the radiative transfer equation (Eq. 2 The TELIS instrument The limb sequence for water isotopologue measurements covers the altitude range of 10–36.5 km. Below 20 km the tangent height spacing is set to 1 km and above 20 km to 1.5 km resulting in a limb sequence of 22 spectra. The ratio- nale behind this is that the range below 20 km is of relevance for the inﬂux of tropospherical water into the stratosphere. The total time required for a single limb scan will be ≈1 min, including radiometric calibration cycles. 4 Retrieval and regularisation 3) is numerically solved. This path is slightly curved Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ 427 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS The least squares solution will then predominately be de- termined by the noise on the measurement, rather than the spectral features. To address this problem Tikhonov regular- isation (Tikhonov, 1963; Phillips, 1962; Twomey, 1963) is employed in this study. with Sy the measurement noise covariance matrix and I the unity matrix. Due to the measurement error, a retrieval noise error is imposed on the retrieved vector. This error propagation is also governed by the contribution matrix, With Tikhonov regularisation the least squares condition is minimised in addition to a side constraint, sensitive to the retrieval noise. The minimisation equation becomes Sx = DSyDT, (12) Sx = DSyDT, (12) where Sx is the retrieval noise covariance matrix. where Sx is the retrieval noise covariance matrix. The retrieved vector xγ is not an estimate of the true at- mospheric state vector xtrue, but rather a weighted average. By employing Eqs. (7) and (10) the retrieved vector can be expressed as function of the true vector as follows xγ = min x ∥(Kx −y)∥2 + γ 2∥Lx∥2 , (9) (9) where γ is the regularisation parameter, balancing the con- tributions of the least square solution and the side constraint, and L is a suitably chosen matrix. In its standard form, the side constraint is the norm of x, ∥x∥2, and subsequently L is the identity matrix. Other choices of L include the ma- trix representation of the nth derivative of x. In this study, however, Tikhonov regularisation is employed in its standard from. xγ = Axtrue + ex, (13) (13) where A=DK is the averaging kernel and ex=Dey the error in the state vector caused by measurement errors. It is noted here that xγ may contain more than one atmo- spheric proﬁle and also other parameters to be retrieved. As the norm is used as as side constraint, it may be that this norm is mainly determined by only a subset of these parameters, if these are much larger than others. This is circumvented by a rescaling of the elements of the state vector which is em- ployed in the current study where appropriate. 4 Retrieval and regularisation It is clear that a good choice of the regularisation param- eter γ is of crucial importance for the solution. It should be chosen large enough that the side constraint ﬁlters out noise contributions. However, if γ is chosen too large, the least square residual norm will not be minimised anymore, and the spectrum associated with the retrieved vector will depart from the measurement. By setting the regularisation parameter γ , and thereby the contribution matrix D, a retrieval produces three quantities; the retrieved state vector xγ , the averaging kernel A, and the covariance matrix Sx. In the following the xγ is given in units of volume mixing ratio (VMR) and the retrieval error, or the diagonal of the covariance matrix, in percent of the true proﬁle. The averaging kernel is associated with the spatial resolution of the retrieval. To interpret the kernel as a mea- sure of resolution, the FWHM is calculated for every row in kilometres. A successful approach to set the regularisation parameter γ is the use of the so-called L-curve as proposed by Hansen (1992). The L-curve is a parametric plot of the logarithms of the two terms in the minimisation function of Eq. (9), i.e. the norm ∥x∥2 vs the least squares norm ∥(Kx −y)∥2. For small values of γ the least squares residue will be minimised, and the norm will be large. If γ is slowly increased, the norm of x will sharply drop while the residual norm will almost stay ﬁxed at its minimal value. This means that the spectrum associated with this state vector x still represents the mea- surement, while the noise contributions become suppressed. There is a rather well deﬁned point where further increment of γ will result in an increase of the least squares norm, in- dicating a departure from the measurement, without much change in the contribution of the side constraint. This point is the bend in the L-curve and the corresponding γ balances the two norms, resulting in a state vector xγ that is mostly determined by the atmospheric condition as opposed to the noise on the measurement. We refer to Hansen (1992) for the algorithm to locate this point. 5 Water isotopologue retrieval 0 2e-08 4e-08 [H2 18O] [VMR] 10 15 20 25 30 35 40 Altitude [km] xtrue Axtrue xγ H2 18O 0 3e-09 6e-09 [HDO] [VMR] 10 15 20 25 30 35 40 Altitude [km] HDO Table 1. Instrument parameters used for the generation of synthetic spectra. LO stands for local oscillator, FWHM for full width half maximum, and FOV for ﬁeld of view. LO frequency 495.040 GHz (H218O) 496.880 GHz (HDO) LO shape Lorentzian LO FWHM 2 MHz Sideband ratio 1.0 FOV FWHM 13 arc minutes Integration time 1.5 seconds RMS noise 0.13 K Limb sequence 10–20 km @ 1.0 km 20–36.5 km @ 1.5 km Flight altitude 37 km Fig. 2. Typical example of state vectors for H218O (left panel) and HDO (right panel) for TELIS. The retrieved proﬁle (solid) is shown along with the retrieval error (grey), the true proﬁle (dashed), and Axtrue (dotted). The results are obtained from a single limb sequence with 22 spectra recorded at different tangent points and an integration time of 1.5 s with perfect knowledge of the instrument parameters. be seen that the averaging kernel peaks to 1.0 over an alti- tude range of 15–35 km in the case of H218O and 20–35 km in the case of HDO resulting in an FWHM equal to the tan- gent spacing, i.e. 1.5 km above 20 km and 1.0 km below. The retrieval error is 2–3% over a wide range of altitudes, peaking at 17–19 km at values of 6% and 11% for respectively HDO and H218O. For HDO there is a clear correlation between the broadening of the averaging kernel, effectively averaging the proﬁles over a wider range of altitudes, and the decrease of the retrieval error for the lower part of the limb scan. It is noted that the absolute retrieval error increases with decreas- ing altitude below 20 km for both isotopologues as both the volume mixing ratio and the air density increase. to higher retrieval errors. This depletion is a function of al- titude and lessens with increasing altitude due to methane oxidation. The depletion of stratospheric H218O is less than 10%. Examples of noise-free simulated spectra are depicted in Fig. 1 for both HDO and H218O. The parts indicated in grey are used for retrieval. 5 Water isotopologue retrieval Above 37 km the retrieval error increases for both H218O and HDO in conjunction with a broadening of the averaging kernel as information in the spectra is limited for these alti- tudes. This is because no tangent heights are possible above the ﬂight altitude of 37 km. Although the radiative transfer of this altitude range is of importance for the retrieval at lower altitudes, the actual retrieved values are of limited value only. In this study the temperature, pressure, O2, N2O, and ClO proﬁles are assumed to be known and set to their a priori values. The H218O line occurs in the wing of a strong water line (just outside the spectrum of Fig. 1) acting as a large baseline offset for low tangent heights. Therefore, H2O is retrieved simultaneously with H218O. The purpose of this retrieval is not to get a proper water proﬁle, but rather to account for the offset such that its inﬂuence on the H218O retrieval is minimised. For this retrieval, the O3 proﬁle is set to the a priori values. Another aspect to consider are cross-correlations between different proﬁles. In Fig. 3 only the part of the averaging kernel is depicted that links the retrieved proﬁle to the true proﬁle of that particular species. The cross-averaging ker- nel links the retrieved proﬁle to the true proﬁle of another species, i.e. H2O in case of H218O retrieval and O3 in case of HDO. Ideally these values are close to zero, which would indicate that the retrieved isotopic water proﬁle is not mixed with water, respectively ozone. For HDO, this is indeed the case and the retrieved proﬁle is not intermingled with the ozone proﬁle. For H218O, however, the cross-averaging ker- nel departs from zero in the range 10–20 km, as is illustrated in the left panel of Fig. 4. This means that for H218O signiﬁ- cant amounts of H2O is mixed into the isotopic water proﬁle below 20 km. In the right panel the contributions of the aver- aging kernel and the cross-averaging kernel to the retrieved H218O proﬁle are both shown. Below 20 km the contribu- tion of the cross-averaging kernel increases with decreasing In the wing of the HDO line a O3 feature resides. To in- vestigate how well HDO can be retrieved without a priori knowledge of ozone, the O3 proﬁle is retrieved simultane- ously with HDO. 5 Water isotopologue retrieval Retrieval simulations have been performed for the rare water isotopologues H218O and HDO in the 480–650 GHz channel of TELIS. The retrieval results are all for a single limb scan, without further averaging. For this study synthetic spectra are generated with instrument parameters as given in Sect. 2 and are for convenience also listed in Table 1. Besides the rare isotopologues of water, the species O3, O2, H2O, N2O, and ClO are taken into account in the forward model in this study. The atmospheric conditions are set to the AFGL US standard atmosphere (Anderson et al., 1986), with the tropopause of 217 K at ≈12 km. However, An- derson et al. does not provide proﬁles for different isotopo- logues. For the current study the proﬁles for the rare isotopo- logues are constructed by taking the proﬁle of the principal isotopologue H216O which is then corrected for the natural abundance of the rare isotopologue. It is noted that the pro- ﬁle of HDO may be depleted in the stratosphere by 30–50% which would lead to lower signal strengths and consequently Once the regularisation parameter γ is set, the solution can be written as xγ = Dy, (10) (10) xγ = Dy, where D is the pseudo-inverse of K or the contribution ma- trix, D = KTK + γ 2I −1 KTS −1 2 y , (11) (11) www.atmos-meas-tech.net/2/423/2009/ Atmos. Meas. Tech., 2, 423–435, 2009 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS 428 0 2e-08 4e-08 [H2 18O] [VMR] 10 15 20 25 30 35 40 Altitude [km] xtrue Axtrue xγ 0 3e-09 6e-09 [HDO] [VMR] 10 15 20 25 30 35 40 Altitude [km] H2 18O HDO Fig. 2. Typical example of state vectors for H218O (left panel) and HDO (right panel) for TELIS. The retrieved proﬁle (solid) is shown along with the retrieval error (grey), the true proﬁle (dashed), and Axtrue (dotted). The results are obtained from a single limb sequence with 22 spectra recorded at different tangent points and an integration time of 1.5 s with perfect knowledge of the instrument parameters. 5 Water isotopologue retrieval The diagonal elements of the retrieved covariance matrices (left panel), the widths of the retrieved averaging kernels (centre panel), and the averaging kernels (right panel) for a typical HDO retrieval, respectively, H218O retrieval with TELIS. In Fig. 2 the retrieved state vectors are illustrated. -0.2 0 0.2 0.4 0.6 0.8 1 A cross 10 15 20 25 30 35 40 Altitude [km] -2.5e-08 0 2.5e-08 5e-08 [H2 18O] [VMR] 10 15 20 25 30 35 40 Altitude [km] AxH2 18O A crossxH2 16O H2O for H2 18O retrieval Fig. 4. The cross-averaging kernel of H2O acting on H218O is shown in the left panel. In the right panel Axtrue is depicted for both contributions of H2O (orange line) and H218O (black line). -2.5e-08 0 2.5e-08 5e-08 [H2 18O] [VMR] 10 15 20 25 30 35 40 Altitude [km] AxH2 18O A crossxH2 16O -0.2 0 0.2 0.4 0.6 0.8 1 A cross 10 15 20 25 30 35 40 Altitude [km] H2O for H2 18O retrieval 15 20 or [%] H2 18O HDO 0 1 2 3 FWHM A [km] - A Fig. 3. The diagonal elements of the retrieved covariance matrices (left panel), the widths of the retrieved averaging kernels (centre panel), and the averaging kernels (right panel) for a typical HDO retrieval, respectively, H218O retrieval with TELIS. In Fig. 2 the retrieved state vectors are illustrated. Fig. 4. The cross-averaging kernel of H2O acting on H218O is shown in the left panel. In the right panel Axtrue is depicted for both contributions of H2O (orange line) and H218O (black line). with ST the ECMWF temperature covariance matrix, D the contribution matrix, KT the Jacobian of the temperature, and Sx the covariance matrix of the retrieved state vector (Rodgers, 2000). The result of this analysis is depicted in Fig. 5. As can be seen, the error is in the range of the tangent heights only ≈0.1%, which is less than the retrieval error (see Fig. 3) and is therefore of only minor consequence. Alterna- tively, the temperature proﬁle may be determined from the measurements from the 16O18O line at 508.996 GHz. altitude, contaminating the retrieved isotopic water proﬁle and hampering a straightforward interpretation. In conclusion the retrieval for HDO can be done over the whole 10–37 km altitude range of the limb sequence, with a retrieval error of 1–6% in the undepleted case. 5.1 Temperature error propagation The assumed temperature proﬁle has only limited accuracy and may therefore have an impact on the retrieval error. The temperature proﬁle will be taken from the analysis data by the ECMWF (European Centre for Medium-Range Weather Forecasts). The accuracies for March 2009 in Northern Scan- dinavia is on the order of 1 K up to the maximum validated pressure level of 10 mbar which corresponds to an altitude of ≈32 km (K. Fielding, private communication, eCMWF, 2009). A slight increase can be discerned above ≈27 km. The accuracies are in the following analysis set to 1 K for altitudes up to 27 km and then a linear increase with altitude to 2 K at 46 km. To assess the error propagation of this un- certainty onto the retrieved rare water isotopologue proﬁles the following formula is employed: 6 Sensitivity to instrumental parameters In the results presented above, perfect knowledge of instru- mental parameters is assumed. Unlike with synthetic data this is obviously not the case with actual measurements. A sensitivity study has been performed to see how imperfect knowledge inﬂuences the retrieval results. The considered error sources are pointing knowledge, sideband ratio, LO fre- quency, ﬁeld of view, and the instrumental lineshape. 5 Water isotopologue retrieval In case HDO is depleted the retrieval error ranges from ≈2–8%. The spa- tial resolution for HDO, as determined by the averaging ker- nel widths, is 1.5 km above 20 km and below 20 km 1.5– 2.5 km. For H218O the retrieval results in an uncontaminated pro- ﬁle above 20 km, with a retrieval error of ≈3% and an aver- aging kernel FWHM of 1.5 km. The retrieval below 20 km is hampered by a strong water line and the retrieval results are hard to interpret as the proﬁle becomes mixed with the proﬁle of H2O. 5 Water isotopologue retrieval For HDO retrieval, the H2O proﬁle is set to the a priori value. In Fig. 2 the retrieved state vector (solid line) with its re- trieval error (grey area), the true proﬁle (dashed line), and Axtrue (dotted line) are plotted in Fig. 2 for H218O (left panel) and HDO (right panel) for the case that only receiver noise is considered. As ﬁrst guess, the true state vector is taken with a scaling factor. The scaling factor has no inﬂu- ence on the retrieval results in the range of 10% to the upper limit of 400%. In Fig. 3 the retrieval error, the averaging ker- nel and its FWHM are shown for both isotopologues. It can Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS 429 0 5 10 15 20 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] H2 18O HDO 0 1 2 3 FWHM A [km] -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] Fig. 3. The diagonal elements of the retrieved covariance matrices (left panel), the widths of the retrieved averaging kernels (centre panel), and the averaging kernels (right panel) for a typical HDO retrieval, respectively, H218O retrieval with TELIS. In Fig. 2 the retrieved state vectors are illustrated. -0.2 0 0.2 0.4 0.6 0.8 1 A cross 10 15 20 25 30 35 40 Altitude [km] -2.5e-08 0 2.5e-08 5e-08 [H2 18O] [VMR] 10 15 20 25 30 35 40 Altitude [km] AxH2 18O A crossxH2 16O H2O for H2 18O retrieval Fig. 4. The cross-averaging kernel of H2O acting on H218O is shown in the left panel. In the right panel Axtrue is depicted for both contributions of H2O (orange line) and H218O (black line). 0 5 10 15 20 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] H2 18O HDO 0 1 2 3 FWHM A [km] -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] Fig. 3. Sx = DKT ST KT T DT, 6.1 Pointing The retrieval error, the FWHM of the averaging kernels, and xγ −Axtrue for HDO retrieval with a systematic pointing error. For reference, the retrieval results with no offset is illustrated as well for the case that pointing information is not retrieved (dashed black line) and that pointing information is simultaneously retrieved (solid black line). The pointing offset covers 1–10 arc min, which corresponds to a shift of the tangent height of 200 m to 2 km for the lowest tangent points. In the right panel the solid black line refers to the retrieval error in case of no pointing offset. g. 5. The error propagation of the limited accuracy on the temperature on the retrieved HDO and H2 oﬁles is depicted. The error is given in % relative to the true state vector and can thus directly be compa ainst the retrieval error. Fig. 5. The error propagation of the limited accuracy on the tem- perature on the retrieved HDO and H218O proﬁles is depicted. The error is given in % relative to the true state vector and can thus di- rectly be compared against the retrieval error. It is investigated what the effect of a pointing error is for both a systematic offset and a random pointing error. 22 If pointing is not accounted for, the spectra corresponding to the retrieved proﬁles show systematic residuals with re- spect to the measurement, implying that pointing information is contained in the measurements. In contrast to the retrieval in Sect. 5, in the following the pointing angle is retrieved for each spectrum in the limb sequence in addition to the molec- ular proﬁles. respect to the retrieval without an offset. In fact, this is true over a wide range of pointing offsets. Above 20 km, how- ever, the retrieval error increases, with increasing pointing error. For 3 arc min the retrieval error is still comparable to a retrieval with perfect instrument knowledge for 23 km and above, but below 23 km the error becomes signiﬁcantly larger. This can be explained by the fact that a pointing offset corresponds to an increasing offset in kilometers at tangent point for decreasing altitude. The impact on the retrieval is therefore highest for the lowest tangent heights. For increas- ing pointing offsets, the affected range extends to higher al- titudes. 6.1 Pointing For an offset of 10 arc min, the retrieval error is de- teriorated over the whole altitude range with respect to the retrieval without pointing offset. The retrieval results for HDO for a systematic pointing off- set are depicted in Fig. 6. In this ﬁgure, the dashed black line refers to the retrieval in Sect. 5, i.e. a retrieval with perfect knowledge of the instrument and no instrumental parame- ters are retrieved. The solid black line in the left and centre panels corresponds to the case where no pointing offset is ap- plied, but nevertheless pointing information is retrieved. In the range 20–35 km the dashed and solid black lines coincide in the centre panel, indicating that the averaging kernel in this altitude range is virtually unaffected. Below 20 km, however, the averaging kernel narrows when pointing information is retrieved. This corresponds to the observed increase in the retrieval error in this altitude range. The increase in retrieval error is by a factor of 6, up to 35%. Apart from the retrieval error, of a stochastic nature, a sys- tematic error to the retrieved proﬁle may be induced by a pointing offset. This can be investigated by comparing the re- trieved proﬁle with the smoothed true proﬁle, or xγ −Axtrue. This effect is shown in the right panel of Fig. 6. The solid black line refers to the retrieval error without pointing offset. As can be seen, no systematic error is introduced over the whole altitude range, even up to a pointing error of 10 arc min. This is due to the fact that the HDO line is pressure broad- ened at low altitudes. A pointing error results in ﬁrst order, at low altitudes, in an intensity shift of the spectrum. The sensitivity of the spectrum to the HDO proﬁle and the point- ing angle show therefore in both cases broad spectral features and no distinction can be made anymore between contribu- tions from one or the other. The addition of extra parameters to the state vector results then in the increase of the retrieval error. In conclusion, HDO retrieval is feasible even for the most severe pointing offsets. The retrieval error, however, be- comes large below 20 km, such that a retrieval from a sin- gle limb sequence will have only limited information in this altitude range. 6.1 Pointing The error propagation of the limited accuracy on the tem- perature on the retrieved HDO and H218O proﬁles is depicted. The error is given in % relative to the true state vector and can thus di- rectly be compared against the retrieval error. 0 25 50 75 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0’ 0’ 1’ 3’ 5’ 10’ -50 -25 0 25 50 xγ - Axtrue [%] 10 15 20 25 30 35 40 Altitude [km] 0 1 2 3 FWHM A [km] Fig. 6. The retrieval error, the FWHM of the averaging kernels, and xγ −Axtrue for HDO retrieval with a systematic pointing error. For reference, the retrieval results with no offset is illustrated as well for the case that pointing information is not retrieved (dashed bl k li ) d h i i i f i i i l l i d 0 0.5 1 1.5 Temperature error propagation [%] 10 15 20 25 30 35 40 Altitude [km] HDO 0 25 50 75 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0’ 0’ 1’ 3’ 5’ 10’ -50 -25 0 25 50 xγ - Axtrue [%] 10 15 20 25 30 35 40 Altitude [km] 0 1 2 3 FWHM A [km] Fig. 6. The retrieval error, the FWHM of the averaging kernels, and xγ −Axtrue for HDO retrieval with a systematic pointing error. For reference, the retrieval results with no offset is illustrated as well for the case that pointing information is not retrieved (dashed black line) and that pointing information is simultaneously retrieved (solid black line). The pointing offset covers 1–10 arc min, which corresponds to a shift of the tangent height of 200 m to 2 km for the lowest tangent points. In the right panel the solid black line refers to the retrieval error in case of no pointing offset. 0 0.5 1 1.5 Temperature error propagation [%] 10 15 20 25 30 35 40 Altitude [km] H2 18O 0 25 50 75 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0’ 0’ 1’ 3’ 5’ 10’ -50 -25 0 25 50 xγ - Axtrue [%] 10 15 20 25 30 35 40 Altitude [km] 0 1 2 3 FWHM A [km] Fig. 6. 6.1 Pointing The uncertainty in the pointing of the telescope contributes generally signiﬁcantly to the error budget in limb retrieval. It is therefore mandatory to inspect its inﬂuence on the re- trieval. Currently, TELIS receives its pointing information from the MIPAS instrument, which is equipped with a dedi- cated attitude and heading reference system (AHRS) (Friedl- Vallon et al., 2004). The stability of this system is much better than 1 arc minute. However, both TELIS and MIPAS are connected by several springs to the gondola frame and it remains untested how these springs will inﬂuence the actual pointing with respect to the received pointing information. It is estimated that this pointing mismatch is up to 10 arc min. Sx = DKT ST KT T DT, (14) www.atmos-meas-tech.net/2/423/2009/ Atmos. Meas. Tech., 2, 423–435, 2009 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS 430 30 . de a ge et a .: St atos 0 0.5 1 1.5 Temperature error propagation [%] 10 15 20 25 30 35 40 Altitude [km] H2 18O 0 0.5 1 1.5 Temperature error propagation [%] 10 15 20 25 30 35 40 Altitude [km] HDO Fig. 5. The error propagation of the limited accuracy on the temperature on the retrieved HDO and H2 18 roﬁles is depicted. The error is given in % relative to the true state vector and can thus directly be compar gainst the retrieval error. Fig. 5. The error propagation of the limited accuracy on the tem- perature on the retrieved HDO and H218O proﬁles is depicted. The error is given in % relative to the true state vector and can thus di- rectly be compared against the retrieval error. 0 0.5 1 1.5 Temperature error propagation [%] 10 15 20 25 30 35 40 Altitude [km] H2 18O 0 0.5 1 1.5 Temperature error propagation [%] 10 15 20 25 30 35 40 Altitude [km] HDO Fig. 5. The error propagation of the limited accuracy on the temperature on the retrieved HDO and H2 18O proﬁles is depicted. The error is given in % relative to the true state vector and can thus directly be compared against the retrieval error. Fig. 5. 6.1 Pointing -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] 50 75 rror [%] 0’ 0’ 1’ 3’ 5’ 10’ 0 1 2 3 FWHM A [km] 0 25 50 75 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0’ 0’ 1’ 3’ 5’ 10’ 0 1 2 3 FWHM A [km] 0.4 A Fig. 7. The retrieval error, the widths of the averaging kernels, and the averaging kernels for H218O retrieval with a systematic point- ing error. For reference, the retrieval results with no offset is illus- trated as well for the case that pointing information is not retrieved (dashed black line) and that pointing information is simultaneously retrieved (solid black line). The pointing offset covers 1–10 arc min, which corresponds to a shift of the tangent height of 200 m to 2 km for the lowest tangent points. In the above, only a systematic pointing offset was investi- gated. The results for a random pointing error for each spec- trum in the limb sequence, however, are virtually the same. This is due to the implementation of the pointing retrieval. A pointing offset is not retrieved as a single parameter, but is retrieved separately for each spectrum in the limb sequence. The rationale behind this is that it is anticipated that the ac- tual pointing angles of real measurements will show a gen- eral offset with a random component superimposed due to the springs by which TELIS is attached to the gondola, and hence, it must be retrieved for every spectrum separately. The retrieval results are therefore very comparable for a system- atic offset or an offset with a random nature. which is of similar form as Eq. (1) albeit with an extra scal- ing factor for both sidebands. From these equations it can be seen that an error in the knowledge of the sideband ratio will lead to erroneous contributions of the spectra of the two side- bands. To compensate for this effect, the retrieval algorithm will alter the state vector, and thus the proﬁle, effectively nul- lifying these scaling factors. In conclusion, if there is an uncertainty in the pointing in- formation, both HDO and H218O can still be retrieved above 20 km, up to a pointing error of about 5 arc min. 6.1 Pointing For HDO the retrieval is also possible below 20 km, albeit that the re- trieval error peaks to ≈35%. In this range H218O retrieval is hampered due to H2O interference, as is explained in Sect. 5 The HDO line occurs in this study in the lower sideband and from Eq. (16) it is then expected that an overestimation of the sideband ratio by a factor ϵ will lead to an underestima- tion of the retrieved proﬁle by a factor of ϵ 2, for the assumed sideband ratio s=1.0. The retrieval results on this systematic effect are shown in Fig. 8. Indeed, for small values of ϵ the systematic underestimation of the HDO proﬁle is ϵ 2. Above 20 km the systematic error in the HDO proﬁle is smaller than the retrieval error if ϵ<0.06, and below 20 km if ϵ<0.12. 6.1 Pointing When a pointing offset is applied, the retrieval error and averaging kernel below 20 km are virtually unaffected with www.atmos-meas-tech.net/2/423/2009/ Atmos. Meas. Tech., 2, 423–435, 2009 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS 431 0 25 50 75 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0’ 0’ 1’ 3’ 5’ 10’ -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] 0 1 2 3 FWHM A [km] Fig. 7. The retrieval error, the widths of the averaging kernels, and the averaging kernels for H218O retrieval with a systematic point- ing error. For reference, the retrieval results with no offset is illus- trated as well for the case that pointing information is not retrieved (dashed black line) and that pointing information is simultaneously retrieved (solid black line). The pointing offset covers 1–10 arc min, which corresponds to a shift of the tangent height of 200 m to 2 km for the lowest tangent points. The retrieval results for H218O for a systematic pointing offset are depicted in Fig. 7. Above 20 km the averaging kernel is unaffected by a pointing error, as was also the case with HDO. For H218O, however, the retrieval error seems to be slightly higher than for HDO, with the exception of the largest offset of 10 arc min. When no pointing offset is ap- plied, the averaging kernels of the retrievals with or without pointing information, are very similar. This is in contrast to HDO where a narrowing is observed below 20 km. When a pointing offset is applied, the averaging kernels show some variation below 20 km, which is governed by noise rather than systematic effects, as is the case with HDO. The re- trieval errors are comparable to the retrieval errors of HDO. In contrast to HDO, however, is the observation of large sys- tematic errors in the H218O proﬁle below 20 km (not shown). This is, however, of minor importance as the retrievals in this altitude range are ambiguous even for the case of perfect in- strument knowledge as is explained in Sect. 5. 6.2 Sideband ratio Moreover, a ripple with a frequency of ≈600 MHz and an amplitude of 0.03 is super-imposed on this slope. Even in this case the retrieval results for H218O are virtually identical to the retrieval with perfect knowledge on the sideband ratio. For all altitudes and over the whole range of ratios xγ −Axtrue does not show signiﬁcant deviations from zero for H218O (not shown), i.e. an uncertainty in the sideband ratio causes no systematic effect on the H218O proﬁle. This indicates that the sideband ratio can be retrieved to such an extent that the retrieval of H218O is virtually unaffected. A ﬁrst measurement of the sideband ratio at 625 GHz showed a variation in the sideband ratio of 1.2–1.3 over 2 GHz. To see how robust the retrieval is for a spectrally non- constant sideband ratio, the observed variation in the side- band ratio is modelled. This is done as follows; ϵ increases from 0.2 to 0.3 over the IF frequency range νIF=5–7 GHz. Moreover, a ripple with a frequency of ≈600 MHz and an amplitude of 0.03 is super-imposed on this slope. Even in this case the retrieval results for H218O are virtually identical to the retrieval with perfect knowledge on the sideband ratio. It is noted that for water dynamic studies, the ratio rare isotopologue/main isotopologue is of importance. If the wa- ter proﬁle is taken from the rare isotopologue measurement windows, then the effect of an error in the sideband ratio may inﬂuence this ratio. In case of H218O both isotopologues re- side in the same sideband and systematic effects of an erro- neous sideband ratio on the isotope ratio will cancel, even if the sideband ratio is not retrieved. In case of HDO, however, both isotopologues are in different sidebands and an error in the sideband ratio will cause a systematic error in the isotope ratio. For a systematic error of 2.5% in the HDO proﬁle, the systematic error is 5% in the HDO/H2O ratio. For all altitudes and over the whole range of ratios xγ −Axtrue does not show signiﬁcant deviations from zero for H218O (not shown), i.e. an uncertainty in the sideband ratio causes no systematic effect on the H218O proﬁle. This indicates that the sideband ratio can be retrieved to such an extent that the retrieval of H218O is virtually unaffected. 6.2 Sideband ratio -20 -10 0 10 20 xγ - Axtrue [%] 10 15 20 25 30 35 40 Altitude [km] 0.02 0.04 0.11 0.22 0 0.1 0.2 0.3 0.4 ε -0.2 -0.15 -0.1 -0.05 0 Systematic error Fig. 8. The systematic error on the retrieved HDO proﬁle induced by an error ϵ in the sideband ratio. In the right panel the average values of the altitude range 20–35 km are plotted (solid line). The dotted line corresponds to −ϵ/2. 0 5 10 15 20 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0.00 0.00 0.11 0.35 0.86 0.25 ripple 0 1 2 3 FWHM A [km] -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] Fig. 9. Retrieval results for H218O with an error ϵ in the sideband ratio. For reference the results for a retrieval with a known sideband ratio is illustrated as well in case this ratio is not retrieved (dashed line) and in case the ratio is retrieved (solid black line). Note that i th l tt th li i b d b th th lt i di t 0 5 10 15 20 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0.00 0.00 0.11 0.35 0.86 0.25 ripple 0 1 2 3 FWHM A [km] -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] Fig. 9. Retrieval results for H218O with an error ϵ in the sideband ratio. For reference the results for a retrieval with a known sideband ratio is illustrated as well in case this ratio is not retrieved (dashed line) and in case the ratio is retrieved (solid black line). Note that in the latter case the line is obscured by the other results, indicat- ing that an uncertainty in the knowledge of the sideband ratio does not inﬂuence the retrieval. In addition to the sideband ratio is the ozone proﬁle retrieved. The grey line refers to a situation where the sideband ratio varies over the simulated spectrum over a range of 1.2–1.3 with an average value of 1.25. 6.2 Sideband ratio An error in the knowledge of the sideband ratio (SBR) leads to a systematic shift in the contributions of the two sideband spectra, and may lead to a systematic over- or underestima- tion of the retrieved species. This can be seen as follows. Suppose that the relative error in the knowledge of this ratio is ϵ, then Eq. (1) becomes If one or more lines of a single species is present in both sidebands, the sideband ratio may be retrieved assuming a stable SBR over the frequency interval. For H218O retrieval this is the case; O3 shows spectral features from both side- bands in the retrieval window. The retrieval of the sideband ratio is of no consequence for the H218O retrieval (not de- picted). If the error ϵ is set to zero, the retrieval errors and the averaging kernels are almost identical whether the SBR is retrieved or not. In fact, the retrieval results do not show signiﬁcant change over the wide range of uncertainties in the sideband ratio of ϵ=0.0–0.86. This, however, assumes per- fect knowledge of O3. I = s(1 + ϵ) s(1 + ϵ) + 1IUSB + 1 s(1 + ϵ) + 1ILSB, (15) (15) which, by neglecting higher order terms in ϵ, can be rewritten as I ≈ s s + 1 1 + ϵ s + 1 IUSB + 1 s + 1 1 − ϵ s + 1 ILSB, (16) In Fig. 9 it is shown how the retrieval of the sideband ratio inﬂuences the retrieval of the H218O proﬁle retrieval if ozone (16) www.atmos-meas-tech.net/2/423/2009/ Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ Atmos. Meas. Tech., 2, 423–435, 2009 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS 432 -20 -10 0 10 20 xγ - Axtrue [%] 10 15 20 25 30 35 40 Altitude [km] 0.02 0.04 0.11 0.22 0 0.1 0.2 0.3 0.4 ε -0.2 -0.15 -0.1 -0.05 0 Systematic error Fig. 8. The systematic error on the retrieved HDO proﬁle induced by an error ϵ in the sideband ratio. In the right panel the average values of the altitude range 20–35 km are plotted (solid line). The dotted line corresponds to −ϵ/2. 6.2 Sideband ratio 0 0.1 0.2 0.3 0.4 ε -0.2 -0.15 -0.1 -0.05 0 Systematic error -20 -10 0 10 20 xγ - Axtrue [%] 10 15 20 25 30 35 40 Altitude [km] 0.02 0.04 0.11 0.22 -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] 0 5 10 15 20 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0.00 0.00 0.11 0.35 0.86 0.25 ripple 0 1 2 3 FWHM A [km] 0 A Fig. 8. The systematic error on the retrieved HDO proﬁle induced by an error ϵ in the sideband ratio. In the right panel the average values of the altitude range 20–35 km are plotted (solid line). The dotted line corresponds to −ϵ/2. Fig. 9. Retrieval results for H218O with an error ϵ in the sideband ratio. For reference the results for a retrieval with a known sideband ratio is illustrated as well in case this ratio is not retrieved (dashed line) and in case the ratio is retrieved (solid black line). Note that in the latter case the line is obscured by the other results, indicat- ing that an uncertainty in the knowledge of the sideband ratio does not inﬂuence the retrieval. In addition to the sideband ratio is the ozone proﬁle retrieved. The grey line refers to a situation where the sideband ratio varies over the simulated spectrum over a range of 1.2–1.3 with an average value of 1.25. is retrieved as well. Between 20 and 35 km, the retrieval of the sideband ratio in conjunction with ozone, leaves the re- trieved proﬁle unaffected. Only below 20 km the averaging kernel broadens and correspondingly the retrieval error de- creases, but as explained in Sect. 5 this range is of limited interest only, due to the water interference. retrieved HDO proﬁle. The actual sideband ratio in all mea- surement windows are to be determined. A ﬁrst measurement of the sideband ratio at 625 GHz showed a variation in the sideband ratio of 1.2–1.3 over 2 GHz. To see how robust the retrieval is for a spectrally non- constant sideband ratio, the observed variation in the side- band ratio is modelled. This is done as follows; ϵ increases from 0.2 to 0.3 over the IF frequency range νIF=5–7 GHz. 6.2 Sideband ratio y In conclusion, above 20 km an error in the sideband ratio does not affect the proﬁle retrieval of H218O as the sideband ratio can be retrieved. For HDO, matters are somewhat more complicated as the sideband ratio may not be retrieved di- rectly from spectral features in the retrieval window. It re- mains to be seen as to what accuracy the sideband ratio may be determined in this measurement window, but uncertain- ties of a few percent may already cause biases in the re- trieved HDO proﬁle. Currently, no variations in the side- band ratio larger than 5% are observed over a bandwidth of 2 GHz, which would imply a systematic error of 2.5% in the retrieved HDO proﬁle. The retrieval error for HDO and the averaging kernel are not inﬂuenced by an error in the side- band ratio. Unfortunately, in the HDO retrieval window no lines of a single species are available in both sidebands. Ozone lines, however, do occur in the 2 GHz HDO measurement window. These lines may be used to retrieve a sideband ratio, albeit at a slightly different frequency. The only measurement of the sideband ratio at 625 GHz showed a variation in the side- band ratio of less than 5% over 2 GHz. This would imply a systematic error in the retrieved proﬁle of less than 2.5%. Another option is to take the sideband ratio of the H218O measurement window as the LO frequencies of the measure- ment windows of the two water isotopologues differ by less than 2 GHz, giving rise to a similar systematic error in the Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS 433 0 5 10 15 20 Retrieval error [%] 10 15 20 25 30 35 40 Altitude [km] 0 MHz 0 MHz 1 MHz 2 MHz 3 MHz 4 MHz 5 MHz 0 1 2 3 FWHM A [km] -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] Fig. 10. Retrieval results for H218O with an error in the LO fre- quency. For reference the results are also depicted for a retrieval without a shift in LO frequency for the case that the LO frequency is not retrieved (dashed black line) and that this frequency is re- trieved (solid black line). 6.4 Field of view and instrumental lineshape The width of the ﬁeld of view is known within several per- cent. Simulations show that the importance of this uncer- tainty becomes only relevant if this value is larger than 10%, and then only in a slight systematic bias below 15 km of less than 1%. This is true even for the case where the ﬁeld of view width is not retrieved, and hence this error is of no relevance for isotopic water retrieval with TELIS. An asymmetry in the ﬁeld of view, may in ﬁrst order be interpreted as a pointing offset which is discussed in Sect. 6.1. expected in the stratosphere and consequently the retrieval error will then be ≈4%. Expected uncertainties in the local oscillator signal (central frequency, shape, and width), the width of the ﬁeld of view, and in case of H218O, also the side- band ratio have only limited impact on these retrievals. If the pointing error does not exceed ≈5 arc min, this uncertainty is also of minor relevance. In fact, only the sideband ratio may induce a systematic shift in the HDO proﬁle. For the estimated upper limit of 5% in the knowledge of this ratio, the bias in the HDO proﬁle is estimated to be less than 2.5%, which is slightly smaller than the retrieval error. This means that isotopic water can be measured from a single limb scan down to the lower stratosphere with an accuracy and reso- lution that is sufﬁcient to address the origin of stratospheric water and stratospheric water dynamics. The width of the instrumental lineshape depends on the locking mechanism of the LO frequency, but is in both cases much narrower than the pressure broadened atmospheric lines. It is therefore expected that the width of this lineshape is only of minor importance. Indeed, an uncertainty of even 50% does not signiﬁcantly inﬂuence the retrievals, also if this width is kept ﬁxed and is not retrieved. An asymmetry in the instrumental lineshape, may in ﬁrst order be interpreted as an LO frequency shift which is discussed in Sect. 6.3. HDO can even be retrieved between 10–20 km probing the upper troposphere as well. With perfect knowledge of the instrument this can be done with a retrieval error of 1–6% and a FWHM of the averaging kernel of 1.5–2.5 km. However, this retrieval is deteriorated when pointing information has to be retrieved as well. 6.4 Field of view and instrumental lineshape In that case the retrieval error increases up to ≈35%. 6.3 LO frequency -0.2 0 0.2 0.4 0.6 0.8 1 A 10 15 20 25 30 35 40 Altitude [km] The LO frequency has an intrinsic instability of 1 MHz due to signal processing. In frequency locking mode the central frequency shows an additional uncertainty of ≈2 MHz. In Fig. 10 it can be seen that shifts up to 3 MHz do not inﬂuence the retrieval of the H218O proﬁle, when the LO frequency is retrieved as well. If the uncertainty in LO frequency in- creases to 5 MHz, the averaging kernel broadens somewhat below 20 km, and in conjunction the retrieval error decreases. No systematic errors are observed at altitudes up to ﬂight al- titude. At 40 km, however, a systematic error of +25% can be observed (not depicted), which is more than the retrieval error of 15% at that altitude. This altitude lies in the range where no tangent points are possible, and is only of minor importance. A Fig. 10. Retrieval results for H218O with an error in the LO fre- quency. For reference the results are also depicted for a retrieval without a shift in LO frequency for the case that the LO frequency is not retrieved (dashed black line) and that this frequency is re- trieved (solid black line). The latter is obscured by the other re- trieval results, indicating that the retrieval is almost not inﬂuenced by an uncertainty in this frequency. p For HDO similar retrieval results are obtained. Hence, uncertainties in the exact LO frequency are not rel- evant for isotopic water retrievals with TELIS. 6.2 Sideband ratio The latter is obscured by the other re- trieval results, indicating that the retrieval is almost not inﬂuenced by an uncertainty in this frequency. Atmos. Meas. Tech., 2, 423–435, 2009 References Rothman, L., Jacquemart, D., Barbe, A., Benner, D. C., Birk, M., Brown, L., Carleer, M., Jr., C. C., Chance, K., Coudert, L., Dana, V., Devi, V., Flaud, J.-M., Gamache, R., Goldman, A., Hartmann, J.-M., Jucks, K., Maki, A., Mandin, J.-Y., Massie, S., Orphal, J., Perrin, A., Rinsland, C., Smith, M., Tennyson, J., Tolchenov, R., Toth, R., Auwera, J. V., Varanasi, P., and Wagner, G.: The HI- TRAN 2004 molecular spectroscopic database, J. Quant. Spec- trosc. Radiat. Transfer, 96, 139–204, 2005. Anderson, G., Chetwynd, J., Clough, S., Shettle, E., and Kneizys, F.: AFGL atmospheric constituent proﬁles (0-120km), Tech. Rep. AFGL-TR-86-0110, Air Force Geophysics Laboratory, Hanscom Air Force Base, Bedford, Mass., 1986. V., Devi, V., Flaud, J.-M., Gamache, R., Goldman, A., Hartmann, J.-M., Jucks, K., Maki, A., Mandin, J.-Y., Massie, S., Orphal, J., Perrin A Rinsland C Smith M Tennyson J Tolchenov R Brewer, A.: Evidence for a world circulation provided by the mea- surements of helium and water vapor distribution in the strato- sphere, Q. J. Roy. Meteorol. Soc., 75, 351–363, 1949. Danielsen, E.: A dehydration mechanism for the stratosphere, Geo- phys. Res. Lett., 9, 605–608, 1982. Sherwood, S. and Dessler, A.: On the control of stratospheric hu- midity, Geophys. Res. Lett., 27, 2513–2516, 2000. Franz, P. and R¨ockmann, T.: High-precision isotope measure- ments of H2-16O, H2-17O, H2-18O, and the Delta 17O-anomaly of water vapor in the southern lowermost stratosphere, Atmos. Chem. Phys., 5, 2949–2959, 2005, http://www.atmos-chem-phys.net/5/2949/2005/. Smith, J., Ackerman, A., Jensen, E., and Toon, O.: Role of deep convection in establishing the isotopic composition of water va- por in the tropical transition layer, Geophys. Res. Lett., 33, L06812, doi:10.1029/2005GL024078, 2006. http://www.atmos-chem-phys.net/5/2949/2005/. Steinwagner, J., Milz, M., von Clarmann, T., Glatthor, N., Grabowski, U., H¨opfner, M., Stiller, G. P., , and R¨ockmann, T.: HDO measurements with MIPAS, Atmos. Chem. Phys., 7, 2601– 2615, 2007, http://www.atmos-chem-phys.net/7/2601/2007/. Friedl-Vallon, F., Maucher, G., Seefeldner, M., Trieschmann, O., Kleinert, A., Lengel, A., Keim, C., Oelhaf, H., and Fischer, H.: Design and characterization of the balloon-borne michelson interferometer for passive atmospheric sounding (MIPAS-B2), Appl. Opt., 43, 3335–3355, 2004. http://www.atmos-chem-phys.net/7/2601/2007/. Tikhonov, A.: On the solution of incorrectly stated problems and a method of regularization, Dokl. Akad. Nauk SSSR, 151, 501– 504, 1963. Hansen, P.: Analysis of discrete ill posed problems by means of the L-curve, SIAM Rev., 34, 561–580, 1992. Holton, J. and Gettelman, A.: Horizontal transport and the dehy- dration of the stratosphere, Geophys. Res. References Lett., 28, 2799–2802, 2001. Twomey, S.: On the numerical solution of Fredholm integral equa- tions of the ﬁrst kind by inversion of the linear system produced by quadrature, J. Assoc. Comput. Mach., 10, 97–101, 1963. Hoogeveen, R., Yagoubov, P., de Lange, A., Selig, A., Koshelets, V., Ellison, B., and Birk, M.: Superconducting integrated receiver development for TELIS, Proc. SPIE, 5978, 440–450, 2005. Urban, J., Lauti´e, N., Murtagh, D., Eriksson, P., Kasai, Y., Loßow, S., Dupuy, E., de La No¨e, J., Frisk, U., Olberg, M., Flochmo¨en, E. L., and Ricaud, P.: Global observations of middle atmospheric water vapour by the Odin satellite, Planet. Space Sc., 55, 1093– 1102, 2007. Johnson, D., Jucks, K., Traub, W., and Chance, K.: Isotopic com- position of stratospheric water vapor: Implications for transport, J. Geophys. Res., 106, 12219–12226, 2001. Vleck, J. V. and Weisskopf, V.: On the Shape of Collision- Broadened Lines, Rev. Mod. Phys., 17, 227–236, 1945. Koshelets, V., Ermakov, A., Filippenko, L., Lyudmila, V., Khud- chenko, A., Kiselev, O., Sobolev, A., Alexander, S., Torgashin, M., Yagoubov, P., Hoogeveen, R., and Wild, W.: Superconduct- ing integrated submillimeter receiver for TELIS, IEEE T. Appl. Supercon., 17, 336–342, doi:10.1109/TASC.2007.898622, 2007. Webster, C. and Heymsﬁeld, A.: Water Isotope Ratios D/H, 18O/16O, 17O/16O in and out of Cloud Map Dehydration Path- ways, Science, 302, 1742–1745, 2003. Weidner, F., B¨osch, H., Bovensmann, H., Burrows, J., Butz, A., Camy-Peyret, C., Dorf, M., Gerilowski, K., Gurlit, W., Platt, U., von Friedeburg, C., Wagner, T., and Pfeilsticker, K.: Balloon- borne limb proﬁling of UV/vis skylight radiances, O3, NO2, and BrO: technical set-up and validation of the method, Atmos. Chem. Phys., 5, 1409–1422, 2005, http://www.atmos-chem-phys.net/5/1409/2005/. Liebe, H.: MPM-An atmospheric millimeter-wave propagation model, Int. J. Infrared and Millimeter Waves, 10, 631–650, 1989. Moyer, E., Irion, F., Yung, Y., and Gunson, M.: ATMOS stratospheric deuterated water and implications for troposphere- stratosphere transport, Geophys. Res. Lett., 23, 2385–2388, 1996. Nassar, R., Bernath, P. F., Boone, C. D., Gettelman, A., McLeod, S. D., and Rinsland, C. P.: Variability in HDO/H2O abundance ratios in the tropical tropopause layer, J. Geophys. Res., 112, D21305, doi:10.1029/2007JD008417, 2007. Worden, J., Bowman, K., Noone, D., Beer, R., Clough, S., Elder- ing, A., Fisher, B., Goldman, A., Gunson, M., Herman, R., Ku- lawik, S. 7 Conclusions This paper demonstrates the capability of the 480–650 GHz channel of the TELIS instrument to retrieve proﬁles for H218O and HDO from one single limb scan with an inte- gration time of only 1.5 s per spectrum. This enables water isotopologue proﬁle retrieval at a high spatial/temporal reso- lution in contrast to current operational platforms. In that altitude range, the proﬁle of H218O becomes in- termingled with the proﬁle of the main water isotopologue hampering straightforward interpretation. In conclusion HDO and H218O can both be retrieved in the altitude range 20–37 km from a single limb scan. In addition HDO can also be retrieved between 10 and 20 km, albeit with an enhance retrieval error. It is noted that the sideband ratio may cause a systematic error up to 2.5% in the case of HDO. TELIS can therefore address scientiﬁc issues pertaining to water dynamics in the upper troposphere and lower strato- sphere. For both isotopologues the retrieval error is typically 3% in the altitude range of 20–37 km and the averaging kernel width is ≈1.5 km, corresponding to the altitude step between two consecutive tangent heights in the limb scan and to the extension of the ﬁeld of view at tangent point. This is for the undepleted case. For HDO depletions of 30–50% can be Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ 434 A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS Acknowledgements. The authors would like to acknowledge Thomas R¨ockmann for fruitful discussions on isotopic water and its relevance to stratospheric research. ing, and in-situ freeze-drying in the Tropical Tropopause Layer, Atmos. Chem. Phys., 8, 6051–6067, 2008. ing, and in-situ freeze-drying in the Tropical Tropopause Layer, Atmos. Chem. Phys., 8, 6051–6067, 2008. Ridal, M., Jonsson, A., Werner, M., and Murtagh, D. P.: A one- dimensional simulation of the water vapor isotope HDO in the tropical stratosphere, J. Geophys. Res., 106, 32283–32294, 2001. Edited by: G. Stiller Rodgers, C. D.: Inverse Methods for Atmospheric Sounding: The- ory and Practice, World Scientiﬁc, River Edge, N. J., 2000. Rosenlof, K. H.: How Water Enters the Stratosphere, Science, 302, 1691–1692, 2003. Zahn, A., Franz, P., Bechtel, C., Grooss, J.-U., and R¨ockmann, T.: Modelling the budget of middle atmospheric water vapour iso- topes, Atmos. Chem. Phys., 6, 2073–2090, 2006, http://www.atmos-chem-phys.net/6/2073/2006/. A. de Lange et al.: Stratospheric isotopic water proﬁles from a s A., van Kuik, B., de Vries, E., Dercksen, J., Hoogeveen, R., Filippenko, L., Ermakov, A., and Koshelets, V.: Superconduct- ing Integrated Receiver on Board TELIS, in: Proceedings of the 19th International Symposium on Space Terahertz Technology, SRON Netherlands Institute for Space Research, Groningen, the Netherlands, 2008. Zelinger, Z., Barret, B., Kub´at, P., Ricaud, P., Attie, J.-L., Urban, E. L. F. J., Murtagh, D., and Stˇriˇz´ık, M.: Observation of HD-18O, CH3OH and vibrationally-excited N2O from Odin/SMR mea- surements, Molec. Phys., 104, 2815–2820, 2006. A. de Lange et al.: Stratospheric isotopic water proﬁles from a single limb scan by TELIS www.atmos-meas-tech.net/2/423/2009/ Atmos. Meas. Tech., 2, 423–435, 2009 References S., Lampel, M., Luo, M., Osterman, G., Rinsland, C., Rodgers, C., Sander, S., Shephard, M., , and Worden, H.: Tropo- spheric Emission Spectrometer observations of the tropospheric HDO/H2O ratio: Estimation approach and characterization, J. Geophys. Res., 111, D16309, doi:10.1029/2005JD006606, 2006. Yagoubov, P., de Lange, G., Golstein, H., de Jong, L., de Lange, Phillips, P.: A technique for the numerical solution of certain in- tegral equations of the ﬁrst kind, J. Assoc. Comput. Mach., 9, 84–97, 1962. Geophys. Res., 111, D16309, doi:10.1029/2005JD006606, 2006. Yagoubov, P., de Lange, G., Golstein, H., de Jong, L., de Lange, Read, W., Schwartz, M., Lambert, A., Su, H., Livesey, N., Daffer, W., and Boone, C.: The roles of convection, extratropical mix- Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/ 435 Zelinger, Z., Barret, B., Kub´at, P., Ricaud, P., Attie, J.-L., Urban, E. L. F. J., Murtagh, D., and Stˇriˇz´ık, M.: Observation of HD-18O, CH3OH and vibrationally-excited N2O from Odin/SMR mea- surements, Molec. Phys., 104, 2815–2820, 2006. Zahn, A., Franz, P., Bechtel, C., Grooss, J.-U., and R¨ockmann, T.: Modelling the budget of middle atmospheric water vapour iso- topes, Atmos. Chem. Phys., 6, 2073–2090, 2006, http://www.atmos-chem-phys.net/6/2073/2006/. Atmos. Meas. Tech., 2, 423–435, 2009 www.atmos-meas-tech.net/2/423/2009/
https://openalex.org/W3041517651	https://journal.iainkudus.ac.id/index.php/tawazun/article/download/6068/pdf	Indonesian	null	Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah dan Ibnu Khaldun	Tawazun	2,019	cc-by-sa	6,539	Tawazun: Journal of Sharia Economic Law P-ISSN: 2655-9021, E-ISSN: 2502-8316 Volume 2, Nomor 2, September 2019 http://journal.stainkudus.ac.id/index.php/tawazun/index Tawazun: Journal of Sharia Economic Law P-ISSN: 2655-9021, E-ISSN: 2502-8316 Volume 2, Nomor 2, September 2019 http://journal.stainkudus.ac.id/index.php/tawazun/index Tawazun: Journal of Sharia Economic Law P-ISSN: 2655-9021, E-ISSN: 2502-8316 Volume 2, Nomor 2, September 2019 http://journal.stainkudus.ac.id/index.php/tawazun/index Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun Muzakkir S. Sekolah Tinggi Ilmu Syariah Darul Falah Mataram NTB ibnusesela@gmail.com Abstract Modern business is a very complex reality. Many factors also influence and determine business activities, including organizational-managerial factors, scientific-technological factors and political-social-cultural factors. Business complexity cannot be understood separately from the community in which the community itself has a complex structure. In the Islamic concept, price determination is done by the strength of demand and the power of supply. The second meeting must be based and occur on the principle of being willing to be willing, no party is forced, deceived or there is an error in the transaction object in making certain goods transactions. If such things happen, the structure and attributes of the market will become unstable and have a vatal effect on the sustainability of the economic system. The course of the market mechanism greatly influences the normal pricing conditions. Increased demand tends to lead to efforts to raise prices and encourage producers to increase production, but if the ability of producers to supply goods increases and demand decreases, prices will go down. Balanced prices become the best conditions in the sustainability of the market mechanism, but the realization of normal and balanced prices becomes an important study in the literacy of the thoughts of figures such as Ibn Taymiyah and Ibn Khaldun. This study seeks to explain and describe the ideas of two major figures in the field of Islamic economics so that they can become standards and references in realizing Al-Tas'īr Al-lAdl which are in accordance with the concept of Islamic economics. Keywords: Construction, Al-Tas'īr Al-‘Adl, Ibn Taimiyah, and Ibn Khaldun. Keywords: Construction, Al-Tas'īr Al-‘Adl, Ibn Taimiyah, and Ibn Khaldun Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 Abstrak Bisnis modern merupakan realitas yang amat kompleks. Banyak faktor turut mempengaruhi dan menentukan kegiatan bisnis, antara lain faktor organisatoris- manajerial, faktor ilmiah-teknologis dan faktor politik-sosial-kultural. Kompeleksitas bisnis tidak bisa dipahami secara terpisah dari masyarakat yang dalam diri masyarakat itu sendiri terdapat struktur yang kompleks. Dalam konsep Islam, penentuan harga dilakukan oleh kekuatan permintaan dan kekuatan penawaran. Pertemuan keduanya harus didasarkan dan terjadi atas asas rela sama rela, tidak ada pihak yang terpaksa, tertipu atau adanya kekeliruan objek transaksi dalam melakukan transaksi barang tertentu. Bila terjadi hal-hal demikian, maka struktur dan atribut pasar akan menjadi tidak stabil dan berakibat vatal bagi keberlangsungan sistem ekonomi. Jalannya mekanisme pasar sangat mempengaruhi ketentuan harga normal. Peningkatan permintaan cenderung menimbulkan upaya menaikkan harga dan mendorong produsen memperbanyak produksi, namun jika kemampuan produsen dalam menyediakan barang meningkat dan permintaan menurun, maka harga akan turun. Harga seimbang menjadi kondisi terbaik dalam keberlangsungan Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 147 Muzakkir S. mekanisme pasar, namun realisasi dari harga normal dan seimbang menjadi kajian penting dalam literasi pemikiran para tokoh seperti Ibnu Taimiyah dan Ibnu Khaldun. Kajian ini berupaya mengekplanasi dan mendeskripsikan gagasan dua tokoh besar pada bidang ekonomi Islam agar bisa menjadi standar dan acuan dalam merealisasikan Al-Tas’īr Al-‘Adl yang sesuai dengan konsep ekonomi Islam. Kata kunci: Konstruksi, Al-Tas’īr Al-‘Adl, Ibnu Taimiyah, dan Ibnu Khaldun. Kata kunci: Konstruksi, Al-Tas’īr Al-‘Adl, Ibnu Taimiyah, dan Ibnu Khaldun PENDAHULUAN Bisnis merupakan komponen yang tidak bisa dipisahkan dari ruang gerak hidup manusia, baik dalam rangka memenuhi kebutuhan maupun dalam upaya melayani keinginan. Begitu kuat dorongan al- Qur’an tentang ekonomi dan bisnis dapat dicermati dalam berbagai terminologi dan keyword yang terkait dengan dunia ekonomi dan bisnis itu sendiri (Faurani, 2006: 91) seperti term al-bai’, al-tijārah, dan isytara. (Abdul Baqi, 2002). Hal itu mengindikasikan bahwa bisnis merupakan elemen penting dalam siklus kehidupan manusia yang menentukan arah kebutuhan dan keinginan manusia dapat atau tidak dapat terpenuhi. Sehingga bisnis merupakan keniscayaan yang mesti ada bahkan para nabi menjadikan bisnis sebagai salah satu media penyebaran idiologi dan ajaran-ajaran tentang misi visi kenabian. Maka dengan tegas al-Qur’an mendeklarasikan bisnis pada dasarnya merupakan hal yang halal. p y p y g Meningkatnya frekuensi perkembangan bisnis yang semakin kompleks, sudah banyak mempengaruhi dimensi ruang bisnis yang menuntut sikap responsif solutif dari para pelaku bisnis dengan tetap memperhatikan etika dalam bisnis itu sendiri. Kontestasi persaingan bisnis dalam pasar kian tidak terbendung yang secara tidak langsung berpengaruh pada struktur dan mekanisme pasar itu sendiri. Dalam perekonomian Islam pasar mendapat kedudukan yang amat urgen. Islam menekankan adanya moralitas di pasar seperti persaingan sehat, kejujuran, keadilan, dan keterbukaan yang harus diimplementasikan oleh semua pihak sebagai bentuk tanggung jawab kepada Allah Swt (Rivai dkk, 2012). A. Epistimologi Al-Tas’īr Al-‘Adl Al-Tas’īr merupakan bentuk maṣdar dari derivasi akar kata sa’ara, yusaa’iru, tas’īran yang artinya proses penentuan harga (Manzur, 2015). Sa’ara itu sendiri merupakan bentuk fi’il ṡulaṡi Māzīd biharfin atau fi’il maḍi yang mendapat tambahan satu huruf di antara ‘ain fi’il dan lām fi’il dengan menambahkan huruf yang sama dengan ‘ain fi’il-nya berasal dari akar kata sa’ara yang artinya menyalakan api. Hubungan makna “harga” dengan “menyalakan” adalah karena saat api menyala kobarannya akan naik ke atas sementara harga selalu identik dengan dua sifatnya yaitu naik dan turun. Dalam fiqh dikenal dua istilah berbeda mengenai harga suatu Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 148 Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun barang, yaitu ṡaman dan si’r. Ṡaman merupakan patokan harga suatu barang, sedangkan si’r adalah harga yang berlaku secara aktual di dalam pasar. Fluktuasi harga sebuah komoditas erat kaitannya dengan terminologi si’r bukan ṡaman (Budi Utomo, 2003). Sedangkan term tas’īr merupakan kajian tentang proses terhadap penentuan harga suatu komoditas yang beredar di kalangan masyarakat atau sering disebut dengan kebijakan harga yang diperankan oleh pemegang otoritas dalam hal ini pemerintah. barang, yaitu ṡaman dan si’r. Ṡaman merupakan patokan harga suatu barang, sedangkan si’r adalah harga yang berlaku secara aktual di dalam pasar. Fluktuasi harga sebuah komoditas erat kaitannya dengan terminologi si’r bukan ṡaman (Budi Utomo, 2003). Sedangkan term tas’īr merupakan kajian tentang proses terhadap penentuan harga suatu komoditas yang beredar di kalangan masyarakat atau sering disebut dengan kebijakan harga yang diperankan oleh pemegang otoritas dalam hal ini pemerintah. Adapun pengertian al-Tas’īr menurut ulama fiqh bisa dianalisis dari beberapa pendapat berikut; 1. Menurut Syekh Zakaria Al-Anshari salah seorang ulama mazhab Syafi’i menyatakan ُبَيِيْعُوْا ِل ُأِن ِ لسُّاُقِة ْ لُاِ لْي ِبَيِأُمْر ُأِن ِكِذ بْسْوُر َإْل ُأِمُتْوِتِهْم Al-Tas’īr adalah perintah seorang pemimpin kepada warganya untuk tidak menjual barang-barang mereka kecuali dengan harga yang sudah pasti (Al-Anshari, 1995). 1. Menurut Syekh Zakaria Al-Anshari salah seorang ulama mazhab Syafi’i menyatakan َبَُب yafi i menyatakan ُبَيِيْعُوْا ِل ُأِن ِ لسُّاُقِة ْ لُاِ لْي ِبَيِأُمْر ُأِن ِكِذ بْسْوُر َإْل ُأِمُتْوِتِهْم م إْ Al-Tas’īr adalah perintah seorang pemimpin kepada warganya untuk tidak menjual barang-barang mereka kecuali dengan harga yang sudah pasti (Al-Anshari, 1995). y g p ( ) 2. A. Epistimologi Al-Tas’īr Al-‘Adl Imam as-Syaukani berpendapat ِهْا ُأِن ِبَيِأُمْر ْ لسُّلُطِان ُأِو ْنِاِ بْه ُأِو ُّكْل ُمِن وِلَى ُمْن ْأْمْاُر ِ لمْسُلْمْعُن أِمُر ِأِهُل ْ لسُّاُق َأِل ُبَيِيْعُوْا ُأِمُتْوِتِهْم َإْل بْسْوُر ِكِذ ْفِعِمُنِع ِمْن ْ لز ْبَيِادِة ْعِلِعُه ْأِو ْ لنُّقُصِان لْمِصُلِحِة Al-Tas’īr merupakan keterlibatan dan instruksi pemerintah kepada warganya untuk menjual barang-barang mereka dengan standar harga yang baik demi kemaslahatan bersama (Muhammad, 2004). y g p ( 2. Imam as-Syaukani berpendapat ِ ُِ 2. Imam as-Syaukani berpendapat ِهْا ُأِن ِبَيِأُمْر ْ لسُّلُطِان ُأِو ْنِاِ بْه ُأِو ُّكْل ُمِن وِلَى ُمْن ْأْمْاُر ِ لمْسُلْمْعُن أِمُر ِأِهُل ْ لسُّاُق َأِل ُبَيِيْعُوْا ُأِمُتْوِتِهْم َإْل بْسْوُر ِكِذ ْفِعِمُنِع ِمْن ْ لز ْبَيِادِة ْعِلِعُه ْأِو ْ لنُّقُصِان لْمِصُلِحِة Al-Tas’īr merupakan keterlibatan dan instruksi pemerintah kepada y p p ِهْا ُأِن ِبَيِأُمْر ْ لسُّلُطِان ُأِو ْنِاِ بْه ُأِو ُّكْل ُمِن وِلَى ُمْن ْأْمْاُر ِ لمْسُلْمْعُن أِمُر ِأِهُل ْ لسُّاُق َأِل ُبَيِيْعُوْا ُأِمُتْوِتِهْم َإْل بْسْوُر ِكِذ ْفِعِمُنِع ِمْن ْ لز ْبَيِادِة ْعِلِعُه ْأِو ْ لنُّقُصِان لْمِصُلِحِة ْبَي عْ ُْ Al-Tas’īr merupakan keterlibatan dan instruksi pemerintah kepada warganya untuk menjual barang-barang mereka dengan standar harga yang baik demi kemaslahatan bersama (Muhammad, 2004). g y g 3. Menurut Imam Taqiyuddin An-Nabhani ُُ ِ ُِّ 3. Menurut Imam Taqiyuddin An-Nabhani ِهْا ُأِن ِبَيِأُمْر ْ لسُّلُطِان ُأِو ْنِاِ بْه ُأِو ُّكْل ُمِن وِلَى ُمْن ْأْمْاُر ِ لمْسُلْمْعُن أِمُر ِأِهُل ْ لسُّاُق َأِل ُبَيِيْعُوْا ِ لس ْلُع َإْل بْسْوُر ِكِذ ُفِعِمُنِوْا ِمْن ْ لز ْبَيِادِة ْعِلِعُه حِتَى ِل ُبَيْغْلًّا ِ ألِسُوِار ْأِو ْ لنُّقُصِان ْعِنُه حِتَى ِل ُبَيْضِارْبْا ،ُغِعُرِهْم أي ِبَيِمُنِوْاُن ِمْن ْ لز ْبَيِادِة ْأِوْ لنَقُص ْعِن ْ لس ْوُر ْلْمِصُلِحِة ْ لنَاس ْس Al-Tas’īr adalah perintah penguasa atau para wakilnya atau siapa saja yang mengatur urusan kaum muslimin kepada pelaku pasar agar mereka tidak menjual barang dagangan mereka kecuali dengan harga tertentu, dan mereka dilarang menambah atas harga itu agar mereka tidak melonjakkan harga, atau mengurangi dari harga itu agar mereka tidak merugikan lainnya. Artinya, mereka 149 Muzakkir S. dilarang menambah atau mengurangi dari harga itu demi kemaslahatan masyakarat (An-Nabhani, 2010). y 4. Sedangkan Qadhi ‘Iyadh menyatakan bahwa al-Tas’īr merupakan harga maklum yang ditetapkan berdasarkan mekanisme pasar (Hammad, 2008). 4. Sedangkan Qadhi ‘Iyadh menyatakan bahwa al-Tas’īr merupakan harga maklum yang ditetapkan berdasarkan mekanisme pasar (Hammad, 2008). Dari beberapa definisi di atas, secara umum dapat disederhanakan bahwa al-Tas’īr merupakan otoritas atau kebijakan yang dimiliki pemerintah dalam upaya menentukan harga demi kemaslahatan bersama. B. Dialektika Al-Tas’īr Menurut Para Ulama Perlu diingat bahwa sebagai sebuah konsep, al-tas’īr banyak mengalami diskursus dan dialektika antara ulama-ulama fiqh. Berdasarkan peta sejarah dan generasi, paling tidak ada lima tokoh yang bisa dikemukakan dalam kajian ini, yaitu; A. Epistimologi Al-Tas’īr Al-‘Adl Menarik bila meneliti batasan yang diberikan empat tokoh di atas yang secara umum semuanya bermuara pada satu fokus yaitu al-Tas’īr merupakan proses penentuan harga. Ulama fiqh mengklasifikasikan al-tas’īr menjadi dua macam. Pertama, harga yang berlaku secara natural tanpa intervensi pemerintah. Dalam hal ini, pedagang bebas menjual barang dagangannya dengan harga yang wajar, dengan mempertimbangkan keuntungan yang ingin diperoleh. Artinya para pedagang dan produsen diberikan kebebasan dalam menentukan harga sesuai dengan ekspektasi keuntungan atau disebut dengan al-tas’īr al-ṭabi’i. Kedua, al-tas’īr al-jabbari yaitu harga suatu komoditas yang ditetapkan pemerintah atau pemegang otoritas kekuasaan setelah mempertimbangkan modal dan keuntungan wajar bagi pedagang maupun produsen serta melihat keadaan ekonomi yang nyata dan daya beli masyarakat (Budi Utomo, 2003). j y 1. Abu Yusuf (112-182 H/ 731-798 M) ( ) Abu Yusuf menyatakan bahwa mekanisme pasar terbentuk dari pemberian kebebasan yang optimal kepada para produsen dan konsumen. Pemerintah tidak diperbolehkan intervensi dalam harga, penentuan harga sepenuhnya diperankan oleh kekuatan permintaan dan penawaran karena suatu hal selain monopoli atau aksi sepihak yang tidak wajar (Nur Chamid, 2010). Abu Yusuf menentang pemerintah yang menetapkan harga dan masalah pengendalian harga (price control) inilah yang menjadi poin kontroversial dari pemikiran Abu Yusuf. Namun perlu dicatat bahwa yang mempengaruhi pemikiran Abu Yusuf tentang penentangan tidak bolehnya penetapan harga oleh pemerintah disebabkan karena fenomena dan sosial politik yang dihadapi oleh AbuYusuf saat itu. Abu Yusuf adalah seorang mufti pada kekhalifahan Harun Al-Rasyid yang pernah meminta Abu Yusuf untuk menulis kitab pertama yang berjudul Al-Kharaj yang menjelaskan Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 150 Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun tentang sistem perpajakan yang dijadikan sebagai paduan perpajakan dalam Islam saat itu. Fenomena yang terjadi saat itu adalah ketika terjadi kelangkaan barang maka harga cenderung naik, sedangkan pada saat persedian barang melimpah harga cenderung turun. Kondisi seperti ini dikritisi dan dibantah oleh Abu Yusuf karena faktanya tidak selalu bila persediaan barang sedikit barang akan mahal dan bila persediaan melimpah barang akan murah. Namun hal itu bisa terbalik yang terkadang komoditas berlimpah tetapi harga tetap mahal dan terkadang persediaan barang sedikit namun harga tetap murah. Menurut Abu Yusuf, peningkatan atau penurunan harga tidak selamanya berhubungan dengan peningkatan atau penurunan permintaan dan produksi, karena harga tidak bergantung pada permintaan saja namun juga tergantung pada kekuatan penawaran. Abu Yusuf menegaskan bahwa ada variabel lain yang mempengaruhi, tetapi beliau tidak menjelaskan lebih rinci. Namun para pemikir ekonom muslim sering mengasumsikan bahwa bisa jadi variabel peningkatan atau penurunan harga adalah akibat dari pergeseran dalam permintaan atau jumlah uang yang beredar di suatu negara, atau penimbunan dan penahanan barang, atau keterkaitan masing-masing variabel tersebut (Rivai Zainal dkk, 2014). Dalam kasus yang dihadapi saat itu bahwa hasil panen yang banyak bukan alasan untuk menurunkan harga panen dan kelangkaan tidak selamanya mengakibatkan harga makin melambung karena fakta menunjukkan bahwa ada kemungkinan kelebihan hasil panen berbanding lurus dengan harga yang tinggi dan kelangkaan suatu komoditas tidak sebanding lurus dengan harga yang murah (Nur Chamid, 163). Pada dasarnya, tidak ada batasan tertentu yang dapat dipastikan sebagai standar penentuan harga. Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 2. Yahya Ibn ‘Umar (213-289 H) 2. Yahya Ibn ‘Umar (213-289 H) Sebagai seorang fuqaha mazhab Maliki, Yahya ibn ‘Umar menyatakan bahwa eksistensi harga merupakan hal yang sangat penting dalam sebuah transaksi dan al-tas’īr atau penetapan harga tidak boleh dilakukan dan ditetapkan oleh siapa pun berdasarkan hadits Anas bin Malik. Penetapan harga merupakan tema sentral dalam kitab Yahya Ibn ‘Umar yang berjudul al-Ahkam al-Sūq (Amalia, 2010). Menurut Yahya Ibn ‘Umar, harga ditentukan oleh kekuatan permintan dan kekuatan penawaran serta mekanisme harga harus tunduk pada kaidah-kaidah. Pandangan Yahya Ibn ‘Umar tentang ketidakbolehan penetapan harga mengindikasikan bahwa ia mendukung kebebasan ekonomi dan kebebasana kepemilikan, sikap dan pendapat itu didasarkan pada sikap Rasul Saw saat menolak melakukan penetapan harga sebagaimana hadits Anas bin Malik. Beliau menambahkan bahwa pemerintah tidak diperbolehkan melakukan intervensi pasar kecuali dalam dua hal, yaitu; a. Para pedagang tidak akan menjual barangnya yang sangat dibutuhkan oleh masyarakat dan sikap itu akan dapat menimbulkan kemudaratan dan merusak mekanisme pasar; dan b. Para pedagang melakukan praktik siyasah al-Ighraq atau banting harga (dumping) yang dapat menimbulkan persaingan yang tidak sehat dan dapat mengacaukan stabilitas harga pasar (Amalia, 160). j y 1. Abu Yusuf (112-182 H/ 731-798 M) Murah bukan karena melimpahnya komoditi dan mahal tidak juga disebabkan kelangkaan komoditi karena murah dan mahal merupakan ketentuan Allah Swt. Artinya harga mahal tidak selalu ketika persediaan barang sedikit dan harga murah ketika persediaan barang melimpah, karena bisa saja pada saat persediaan barang melimpah harga barang tetap mahal dan pada saat persediaan barang sedikit harga malah rendah (Nur Chamid, 163). Untuk kasus kekinian, fenomena seperti itu bisa terlihat pada saat menjelang bulan Ramadhan, hari Raya, dan hari-hari besar keagamaan lainnya yang banyak terjadi saat-saat ini, di mana persediaan barang yang begitu melimpah tidak menjadikan harga menjadi murah justru yang terjadi adalah sebaliknya, seperti daging sapi, kurma, pakaian dan kebutuhan di bulan Ramadhan dan menjelang ‘Idul Fithri lainnya. Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 151 Muzakkir S. 2. Yahya Ibn ‘Umar (213-289 H) Sebagai seorang fuqaha mazhab Maliki, Yahya ibn ‘Umar menyatakan bahwa eksistensi harga merupakan hal yang sangat penting dalam sebuah transaksi dan al-tas’īr atau penetapan harga tidak boleh dilakukan dan ditetapkan oleh siapa pun berdasarkan hadits Anas bin Malik. Penetapan harga merupakan tema sentral dalam kitab Yahya Ibn ‘Umar yang berjudul al-Ahkam al-Sūq (Amalia, 2010). Menurut Yahya Ibn ‘Umar, harga ditentukan oleh kekuatan permintan dan kekuatan penawaran serta mekanisme harga harus tunduk pada kaidah-kaidah. Pandangan Yahya Ibn ‘Umar tentang ketidakbolehan penetapan harga mengindikasikan bahwa ia mendukung kebebasan ekonomi dan kebebasana kepemilikan, sikap dan pendapat itu didasarkan pada sikap Rasul Saw saat menolak melakukan penetapan harga sebagaimana hadits Anas bin Malik. Beliau menambahkan bahwa pemerintah tidak diperbolehkan melakukan intervensi pasar kecuali dalam dua hal, yaitu; a. Para pedagang tidak akan menjual barangnya yang sangat dibutuhkan oleh masyarakat dan sikap itu akan dapat menimbulkan kemudaratan dan merusak mekanisme pasar; dan b. Para pedagang melakukan praktik siyasah al-Ighraq atau banting harga (dumping) yang dapat menimbulkan persaingan yang tidak sehat dan dapat mengacaukan stabilitas harga pasar (Amalia, 160). 3. Al-Ghazali (450-505 H/1058-1111 M) 3. Al-Ghazali (450-505 H/1058-1111 M) Secara mengejutkan, Al-Ghazali menjabarkan secara rinci peran aktivitas perdagangan dan timbulnya pasar yang harganya bergerak sesuai dengan kekuatan permintaan dan penawaran, menurutnya pasar merupakan bagian dari keteraturan alami dan itu merupakan mekanisme pasar (Rivai Zainal, 233). Teori permintaan dan penawaran Al-Ghazali tersirat dalam sebuah ilustrasi yang dijabarkan bahwa jika petani tidak mendapatkan pembeli ia akan menjualnya pada harga yang lebih murah, dan harga dapat diturunkan dengan menambah jumlah barang di pasar (Muhammad, 2012), dari kasus ini kemudian lahir teori permintaan dan penawaran Al-Ghazali. Sepanjang tulisan- tulisannya, Al-Ghazali sering menggunakan istilah”harga yang berlaku seperti yang ditentukan oleh praktik-praktik pasar”. Dari konsep tersebut kemudian lahir konsep al-ṣaman al-‘adl (harga yang adil) atau equilibrium price (harga seimbang). (Abdullah, 2010) Di samping itu, Al-Ghazali juga memperkenalkan teori elastisitas permintaan dengan menyebutkan bahwa mengurangi margin keuntungan dengan menjual pada harga lebih murah akan Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 152 Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun meningkatkan volume penjualan dan pada gilirannya akan meningkatkan keuntungan. Imam Al-Ghazali mengidentifikasi permintaan produk makanan bagian dari in-elastis, karena makanan adalah kebutuhan pokok yang harus seminimal mungkin didorong oleh motif mencari keuntungan untuk menghindari ekploitasi melalui pengenaan harga yang tinggi dan keuntungan yang besar karena jika ingin mencari keuntungan tinggi dari perdagangan selayaknya dicari dari barang-barang yang bukan merupakan kebutuhan pokok. (Rivai Zainal dkk, 234) 4. Ibn Taimiyah (661-728 H/1263-1328 M) Ibn Taimiyah dengan tegas menyatakan bahwa harga ditentukan oleh kekuatan permintaan dan penawaran karena naik dan turunnya harga tidak selalu disebabkan oleh tindakan tidak adil dari sebagian orang yang terlibat dalam transaksi, namun bisa jadi karena permintaan menurun akibat dari in-efesiensi produksi, penurunan jumlah impor barang yang diminta, intensitas dan besarnya permintaan, kelangkaan atau melimpahnya barang, kondisi kepercayaan, diskonto dari pembayaran tunai atau karena adanya tekanan pasar. Besar kecilnya kenaikan harga tergantung pada besarnya perubahan permintaan atau penawaran, dan bila seluruh transaksi sesuai dengan aturan maka kenaikan harga yang terjadi merupakan kehendak Allah Swt. Permintaan terhadap barang sering kali mengalami perubahan yang disebabkan karena ketergantungan pada jumlah penawaran, jumlah orang yang menginginkannya, lemah kuatnya dan besar kecilnya kebutuhan terhadap barang tersebut. Pandangan Ibn Taimiyah mengenai mekanisme pasar sebenarnya terfokus pada masalah pergerakan harga yang terjadi pada waktu itu, akan tetapi ia letakkan dalam kerangka mekanisme pasar dan secara umum ia menunjukkan the beauty of market (kaidah-kaidah mekanisme pasar) sebagai mekanisme ekonomi. Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 3. Al-Ghazali (450-505 H/1058-1111 M) (Rianto dan Amalia, 2010: 270) Regulasi harga merupakan pengaturan terhadap harga-harga barang yang dilakukan oleh pemerintah yang bertujuan untuk memelihara kejujuran dan kemungkinan penduduk untuk bisa memenuhi kebutuhan pokok (Amalia, 2010). Kemudian Ibn Taimiyah membedakan dua tipe regulasi harga, yaitu regulasi harga yang adil dan regulasi yang tidak adil. Jika regulasi yang dilakukan terdapat unsur kezaliman terhadap manusia dan bersifat memaksa tanpa hak untuk menjual barang yang diinginkan atau melarang mereka dari barang yang dilarang oleh Allah Swt maka regulasi tersebut hukumnya haram. Tapi jika regulasi harga memiliki unsur keadilan bagi masyarakat dengan harga standar dan normal, melarang mereka dari Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 153 Muzakkir S. hal-hal yang diharamkan oleh Allah Swt untuk mengambil tambahan di atas harga normal maka hal ini diperbolehkan (Amalia, 2010). 1. Perspektif Ibnu Taimiyah 1. Perspektif Ibnu Taimiyah Dalam kitab al-Hisbah penetapan harga diperlukan untuk mencegah seorang menjual makanan dan barang lainnya kepada kelompok tertentu dengan harga yang ditetapkan sesuai dengan keinginan mereka. Oleh karena itu, regulasi harga sangat mempermudah usaha mikro dalam menghadapi maipulasi pasar yang umumnya dilakukan oleh penguasa besar (Taqiyuddin Ahmad, 35). Di samping tentang regulasi harga, dalam kitab al-Hisbah fi al-Islam Ibn Taimiyah menyebutkan tiga term penting yang mengarah pada mekanisme penentuan harga dan ketiganya menjadi indikasi penentuan harga yang menjadi barometer terhadap fenomena transaksi ekonomi bisnis yang bervariasi dan selalu dinamis saat ini. Tiga term tersebut adalah ṣaman al-Miṡl, ‘iwaḍ al-Miṡl, dan qīmah al-Miṡl. 5. Ibn Khaldun (732-808 H/1332-1406 M) ( / ) Menurut Ibn Khaldun harga merupakan hasil dari hukum permintaan dan penawaran dengan asumsi bahwa apabila suatu barang langka dan banyak diminta maka harganya akan tinggi, namun jika suatu barang berlimpah maka harganya turun (Abdullah, 2010). Hampir berbeda dengan Abu Yusuf, terbentuknya harga menurut Ibn Khaldun tidak semata-mata melalui mekanisme pasar akan tetapi pada kondisi dan situasi tertentu diperlukan peran pemerintah dalam mengintervensi harga dengan cara melakukan regulasi harga. Faktor yang mendorong perlunya regulasi harga apabila ketentuan harga berjalan tidak stabil dalam mekanisme pasar. Oleh sebab itu, kombinasi antara mekanisme pasar dan intervensi pemerintah menunjukkan adanya interaksi antara kondisi sosial ekonomi dan hukum dalam upaya menentukan harga. Penurunan harga yang sangat drastis akan merugikan pengerajin dan pedagang serta mendorong mereka keluar dari pasar, sedangkan kenaikan harga yang drastis akan menyusahkan konsumen. Harga yang rendah dibutuhkan karena memberikan kelapangan bagi kaum miskin, sedangkan harga yang wajar menjadikan pedagang mendapatkan tingkat pengembalian yang baik dan dapat menciptakan kegairahan pasar dengan meningkatkan penjualan untuk mendapatkan tingkat keuntungan dan kemakmuran tertentu (Chamid, 257). g g ( ) Menurut Ibn Khaldun faktor yang mempengaruhi penetapan penawaran adalah permintaan, tingkat keuntungan relatif, tingkat usaha manusia, besarnya tenaga buruh termasuk ilmu pengetahuan dan keterampilan yang dimiliki, ketenangan dan keamanan, kemampuan teknik serta perkembangan masyarakat secara keseluruhan (Chamid, 251). Sementara itu, faktor yang menentukan penetapan permintaan adalah pendapatan, jumlah penduduk, kebiasaan dan adat istiadat masyarakat serta pembangunan dan kemakmuran masyarakat secara umum. Inti dari faktor tersebut adalah kenaikan penawaran atau penurunan permintaan menyebabkan kanaikan harga. Dari lima tokoh di atas, tercermin skeam konsturksi al-tas’īr yang bervariasi dengan asumsi dasarnya adalah “tergantung pada kekuatan pasar”. Terbentuknya harga banyak dipengaruhi oleh permintaan dan penawaran dalam mekenisme pasar dan ketentuan harga yang normal sangat dipengaruhi oleh stabilitas jalannya mekanisme pasar yang diukur melalui efektifitas tingkat kelangkaan dan pengadaan barang. Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 154 Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun a. Ṡaman al-Miṡl Ṡ a. Ṡaman al Miṡl Ṡaman al-Miṡl adalah harga normal yang beredar di pasar atau dalam istilah ekonomi disebut dengan just price atau price of the equivalent. Ṡaman al-Miṡl merupakan harga dasar standar atau normal yang diciptakan oleh para pelaku pasar dalam transaksi baik penjual, pembeli maupun pemilik sumber. Di samping itu ṣaman al-Miṡl merupakan nilai harga yang dijual oleh seorang dan diterima secara umum oleh masyarakat luas sebagai hal yang sepadan dengan barang yang dijual atau barang yang sejenis lainnya di tempat dan waktu tertentu (Ahmad, 41). Sederhananya, ṣaman al-Miṡl merupakan harga yang terbentuk dari kekuatan permintaan dan penawaran, dan beliau mengilustrasikannya bahwa jika seorang menjual barangnya dengan harga yang normal (wajh al-maʻrūf), maka harga bisa meningkat karena kekurangan pasokan komoditas dan karena tingginya permintaan (Ahmad, 22). p gg y p ( ) Formulasi harga dilandaskan pada prinsip kesepakatan masing-masing pihak atau sama-sama ridha yaitu kerelaan dari masing-masing pihak dalam melakukan transaksi. Dalam bisnis Islam prinsip ini merupakan prinsip fundamental dalam upaya stabilisasi mekanisme pasar dengan kompetisi sehat, karena terakomodirnya kebutuhan masing-masing pihak, di mana penjual dapat menjual komoditi yang ditawarkan dan pembeli dapat membeli komoditi yang diinginkan tanpa ada paksaan, penipuan, perilaku monopoli, intervensi, ekploitasi, intimidasi dan praktik korupsi lainnya dengan harga yang telah disepakati. Setiap harga lainnya yang ada karena ketidaksempurnaan pasar akan sangat mempengaruhi tingkat kesejahteraan manusia, dan kondisi tersebut membutuhkan intervensi pemerintah untuk mengontrol harga (Rozalinda, 2014: 161). Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 155 Muzakkir S. Indikasi harga sangat ditentukan oleh unsur kerelaan dalam kemampuan penjual untuk menyediakan barang dan kemampuan pembeli untuk mendapatkan barang tersebut. Keseimbangan dua elemen (supply dan demand) menjadi penentu dalam terbentuknya harga yang berasaskan kerelaan. Naik dan turunnya harga bisa saja disebabkan karena kekurangan produksi atau penurunan impor barang yang dibutuhkan. Sehingga bila permintaan naik dan penawaran turun harga akan naik, dan bila persediaan barang meningkat sementara permintaan menurun maka harga akan ikut turun, maka menurut Ibn Taimiyah inilah yang dikatakan mekanisme pasar yang Islami (Rozalinda, 2014: 160). Dalam al-Hisbah Ibn Taimiyah menjelaskan apa yang dimaksud dengan Ṡaman al-Miṡl, yaitu; g , y ; “Apabila orang-orang memperjualbelikan barang dagangannya dengan cara-cara yang biasa dilakukan, tanpa ada pihak yang dizalimi kemudian harga mengalami kenaikan karena berkurangnya persediaan barang ataupun karena bertambahnya jumlah permintaan, maka itu semata-mata karena Allah Swt. a. Ṡaman al-Miṡl Ṡ Dalam hal demikian, memaksa para pedagang untuk menjual barang dagangannya pada harga tertentu merupakan tindakan pemaksaan yang tidak dapat dibenarkan”. b. ‘Iwaḍ al-Miṡl Kata ‘iwaḍ merupakan bentuk singuler dan pluralnya adalah a’wāḍ yang berarti pengganti atau kompensasi. Dari asal kata ini lahir derivasi dalam bentuk bina musyārakah yaitu al-mu’āwaḍah yang berarti pertukaran. al-mu’āwaḍah tersebut mengandung dua pengertian, pertama, pertukaran barang dengan alat pembayaran, dan kedua, pertukaran barang dengan barang lain yang senilai dan sesuai dengan kesepakatan para pihak dan disebut dengan istilah barter (Ibn Manzhur 299). Awalnya term ‘iwaḍ al-Miṡl berorientasi pada tiga hal, pertama, kompensasi terkait dengan perusakan jiwa, harta, dan barang yang mengandung nilai dan manfaat, kedua, pertukaran dengan orang lain, dan ketiga, istilah keseimbangan balasan yang sepadan seperti orang yang berbuat baik akan mendapat kebaikan dan sebagainya. ‘Iwaḍ al-Miṡl adalah penggantian sama yang merupakan nilai harga yang sepadan dari sebuah benda menurut adat istiadat setempat. Kompensasi yang adil diukur dan ditaksir oleh hal-hal yang setara tanpa ada tambahan dan pengurangan dan itu merupakan esensi dari sebuah keadilan (Amalia, 210). Sehingga ‘iwaḍ al-Miṡl mengarah pada dua hal yaitu “nilai harga” dan “adat kebiasaan”. Menurut Ibn Taimiyah nilai terhadap penggantian yang sepadan didasarkan pada taksiran suatu benda menurut adat istiadat b. ‘Iwaḍ al-Miṡl Kata ‘iwaḍ merupakan bentuk singuler dan pluralnya adalah a’wāḍ yang berarti pengganti atau kompensasi. Dari asal kata ini lahir derivasi dalam bentuk bina musyārakah yaitu al-mu’āwaḍah yang berarti pertukaran. al-mu’āwaḍah tersebut mengandung dua pengertian, pertama, pertukaran barang dengan alat pembayaran, dan kedua, pertukaran barang dengan barang lain yang senilai dan sesuai dengan kesepakatan para pihak dan disebut dengan istilah barter (Ibn Manzhur 299). b. ‘Iwaḍ al-Miṡl Kata ‘iwaḍ merupakan bentuk singuler dan pluralnya adalah a’wāḍ yang berarti pengganti atau kompensasi. Dari asal kata ini lahir derivasi dalam bentuk bina musyārakah yaitu al-mu’āwaḍah yang berarti pertukaran. al-mu’āwaḍah tersebut mengandung dua pengertian, pertama, pertukaran barang dengan alat pembayaran, dan kedua, pertukaran barang dengan barang lain yang senilai dan sesuai dengan kesepakatan para pihak dan disebut dengan istilah barter (Ibn Manzhur 299). Awalnya term ‘iwaḍ al-Miṡl berorientasi pada tiga hal, pertama, kompensasi terkait dengan perusakan jiwa, harta, dan barang yang mengandung nilai dan manfaat, kedua, pertukaran dengan orang lain, dan ketiga, istilah keseimbangan balasan yang sepadan seperti orang yang berbuat baik akan mendapat kebaikan dan sebagainya. ‘Iwaḍ al-Miṡl adalah penggantian sama yang merupakan nilai harga yang sepadan dari sebuah benda menurut adat istiadat setempat. a. Ṡaman al-Miṡl Ṡ Kompensasi yang adil diukur dan ditaksir oleh hal-hal yang setara tanpa ada tambahan dan pengurangan dan itu merupakan esensi dari sebuah keadilan (Amalia, 210). Sehingga ‘iwaḍ al-Miṡl mengarah pada dua hal yaitu “nilai harga” dan “adat kebiasaan”. Menurut Ibn Taimiyah nilai terhadap penggantian yang sepadan didasarkan pada taksiran suatu benda menurut adat istiadat ( ) Awalnya term ‘iwaḍ al-Miṡl berorientasi pada tiga hal, pertama, kompensasi terkait dengan perusakan jiwa, harta, dan barang yang mengandung nilai dan manfaat, kedua, pertukaran dengan orang lain, dan ketiga, istilah keseimbangan balasan yang sepadan seperti orang yang berbuat baik akan mendapat kebaikan dan sebagainya. ‘Iwaḍ al-Miṡl adalah penggantian sama yang merupakan nilai harga yang sepadan dari sebuah benda menurut adat istiadat setempat. Kompensasi yang adil diukur dan ditaksir oleh hal-hal yang setara tanpa ada tambahan dan pengurangan dan itu merupakan esensi dari sebuah keadilan (Amalia, 210). Sehingga ‘iwaḍ al-Miṡl mengarah pada dua hal yaitu “nilai harga” dan “adat kebiasaan”. Menurut Ibn Taimiyah nilai terhadap penggantian yang sepadan didasarkan pada taksiran suatu benda menurut adat istiadat Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 156 Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun setempat, dan hal ini yang dianggap sebagai keadilan dan adat kebiasaan masyarakat yang sebenarnya (Ahmad, 35). Dengan demikian dapat dikatakan bahwa ‘iwaḍ al-Miṡl adalah harga pasar yang sedang beredar dan disesuaikan dengan adat kebiasaan masyarakat dan dalam istilah ekonomi dikenal dengan just compensation atau compensation of the equivalent. Kompensasi yang adil adalah penggantian sepadan yang merupakan nilai harga setara yang diukur dan ditaksir oleh hal-hal yang setara pula tanpa ada tambahan dan pengurangan (Rivai Zainal dkk, 299). Lebih lanjut, dalam mendefinisikan kompensasi yang adil Ibn Taimiyah berkata “yang dimaksud dengan kesetaraan adalah kuantitas dari objek khusus dalam penggunaan secara umum dan berkaitan dengan nilai dasar (rate/si’r) serta kebiasaan”. Di samping itu, beliau menambahkan “evaluasi yang benar terhadap konpensasi yang adil didasarkan atas analogi dan taksiran dari barang tersebut dengan barang lain yang setara (ekuivalen). Inilah yang benar-benar adil dan benar-benar diterima dalam penggunaannya” (Amalia, 211). p gg y ( ) Tentang harga dan kompensasi yang adil, Ibn Taimiyah menguraikan ada dua macam jumlah kuantitas yang tercatat dalam kontrak. Pertama, jumlah kuantitas yang sangat akrab dengan masyarakat dan biasa mereka gunakan. Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 a. Ṡaman al-Miṡl Ṡ Kedua, jenis kuantitas yang tidak lazim (nadir) sebagai akibat dari meningkat dan menurunnya kemauan (raghbah) atau faktor lain (Ahmad, 31). Menurutnya harga yang adil atau setara lebih bervariasi yang dipengaruhi oleh kekuatan pasar dan kebutuhan masyarakat, sedangkan kompensasi yang adil merupakan fenomena yang lebih bertahan lama akibat terbentuknya kebiasaan dan itu bersifat relatif. Kompensasi yang adil merupakan sebuah pedoman bagi masyarakat yang adil dan para hakim, karena tujuan dari harga yang adil adalah memberikan panduan bagi para penguasa dalam mengembangkan kehidupan ekonomi. g g p c. Qīmah al-Miṡl Dalam kamus ekonomi Islam kata qīmah berarti harga, bentuk kata qīmah adalah singuler dan pluralnya adalah qiyam. Awalnya term qīmah al-Miṡl digunakan untuk suatu yang menyamai nilai sesuatu yang lain menurut keinginan masyarakat. Konsep ini merupakan pengembangan dari konsep ‘iwaḍ al-Miṡl karena masih erat kaitannya dengan faktor adat kebiasaan masyarakat yang kemungkinan ada dua macam yaitu, pertama, kemungkinan terjadinya kesepakatan antara para pihak yang didasarkan kerelaan dalam menentukan harga, kedua, kemungkinan adanya penyimpangan yang dilakukan oleh salah satu pihak dalam Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 157 Muzakkir S. berinteraksi untuk mentransaksikan barang dagangan sehingga pertukaran barang dan harga yang harus dibayar tidak seimbang sahingga salah satu pihak menjadi dirugikan. Dengan demikian, rumusan qīmah al-Miṡl merupakan nilai harga yang ditetapkan oleh pemerintah untuk mengantisipasi distorsi pasar yang dilakukan oleh para pelaku pasar terutama produsen (Ahmad, 21). Qīmah al-Miṡl merupakan harga pasar yang sedang beredar setelah melalui proses normalisasi atau dalam ekonomi dikenal dengan istilah fixing price. Term qīmah al-Miṡl pernah digunakan oleh Rasulullah Saw pada saat mengawasi harga budak yang hendak dibebaskan, di mana budak yang akan dimerdekakan dengan cara dijual dan majikannya tetap mendapatkan kompensasi dengan harga yang adil atau qīmah al-Miṡl atau qīmah al-‘adl. q Istilah qīmah al-Miṡl banyak digunakan oleh para hakim yang telah mengkodifikasikan hukum Islam tentang transaksi bisnis dalam objek barang cacat yang dijual, perubatan kekuasaan, memaksan penimbun barang untuk menjual barang timbunan, membuang jaminan atas hak milik dan lain sebagainya (Boedi Abdullah, 388). 2. Perspektif Ibnu Khaldun Menurut Ibn Khaldun harga merupakan hasil dari hukum permintaan dan penawaran dengan asumsi bahwa apabila suatu barang langka dan banyak diminta maka harganya akan tinggi, namun jika suatu barang berlimpah maka harganya turun (Boedi Abdullah, 2010: 292). Hampir berbeda dengan Abu Yusuf, terbentuknya harga menurut Ibn Khaldun tidak semata-mata melalui mekanisme pasar akan tetapi pada kondisi dan situasi tertentu diperlukan peran pemerintah dalam mengintervensi harga dengan cara melakukan regulasi harga. Faktor yang mendorong perlunya regulasi harga apabila ketentuan harga berjalan tidak stabil dalam mekanisme pasar. Oleh sebab itu, kombinasi antara mekanisme pasar dan intervensi pemerintah menunjukkan adanya interaksi antara kondisi sosial ekonomi dan hukum dalam upaya menentukan harga. Penurunan harga yang sangat drastis akan merugikan pengerajin dan pedagang serta mendorong mereka keluar dari pasar, sedangkan kenaikan harga yang drastis akan menyusahkan konsumen. Harga yang rendah dibutuhkan karena memberikan kelapangan bagi kaum miskin, sedangkan harga yang wajar menjadikan pedagang mendapatkan tingkat pengembalian yang baik dan dapat menciptakan kegairahan pasar dengan meningkatkan penjualan untuk mendapatkan tingkat keuntungan dan kemakmuran tertentu (Nur Chamid, 251). Menurut Ibn Khaldun faktor yang mempengaruhi penetapan penawaran adalah permintaan, tingkat keuntungan relatif, tingkat usaha Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 158 Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun manusia, besarnya tenaga buruh termasuk ilmu pengetahuan dan keterampilan yang dimiliki, ketenangan dan keamanan, kemampuan teknik serta perkembangan masyarakat secara keseluruhan. Sementara itu, faktor yang menentukan penetapan permintaan adalah pendapatan, jumlah penduduk, kebiasaan dan adat istiadat masyarakat serta pembangunan dan kemakmuran masyarakat secara umum. Inti dari faktor tersebut adalah kenaikan penawaran atau penurunan permintaan menyebabkan kanaikan harga. Formulasi harga yang digagas oleh Ibn Khaldun di atas erat kaitannya dengan teori nilai. Karena nilai suatu barang ditentukan oleh banyakanya aktifitas kerja dan akumulasi biaya yang dikeluarkan untuk memproduksi suatu barang, artinya aktivitas kuantitas kerja dan hasil produksi memiliki hubungan timbal balik yaitu bila kualitas dan kuantitas nilai kerja menurun, maka nilai produksi akan menurun dan begitu sebaliknya (Amalia, 241). Hubungan teori nilai dengan terbentuknya harga terformulasikan dalam hukum permintaan dan penawaran yaitu nilai kemanfaatan suatu barang. Nilai kemanfaatan suatu barang menentukan tingkat permintaan dan penawaran terhadap barang tersebut. Barang yang memiliki nilai kemanfaatan yang tinggi bagi pemenuhan kebutuhan seseorang akan mempengaruhi tingkat permintaan terhadap barang tersebut dan begitu sebaliknya. 2. Perspektif Ibnu Khaldun Dengan demikian, Ibn Khaldun menguraikan teori nilai yang berdasarkan tenaga kerja, teori uang yang kuantitatif, dan teori harga yang ditentukan oleh kekuatan hukum permintaan dan penawaran. Teori harga ini kemudian mengantarkannya untuk menganalisis fenomena distribusi (Boedi Abdullah, 2010: 292). Menurut A. Mannan, secara toeritis bahwa ketentuan harga dalam kontek Islam dapat dinyatakan dengan munculnya sebuah keadilan yang datang dari dalam masyarakat itu sendiri, pengawasan dan regulasi yang dapat dipengaruhi dari nilai-nilai Islam yang berlaku bagi mereka. Karena prinsip dasarnya adalah kerja sama dan kompetisi yang sehat bukan kontestasi monopolis tetapi suatu persaingan yang bebas dari spekulasi, penimbunan, penyelundupan dan lain-lain. Dalam konteks ini, ketentuan harga yang adil perlu diatur di bawah pengawasan hukum, namun perlu ditegaskan bahwa untuk menciptakan kondisi kerja sama yang baik dalam jangka panjang antara produsen dan konsumen diperlukan semangat Islam, nilai-nilai, serta aturan bisnis Islam yang berlaku sebagai proses edukatif bagi semua elemen yang terkait. Sementara dalam jangka pendek diperlukan intervensi pemerintah untuk memastikan terbentuknya harga yang adil dan normal bagi masyarakat (Manna, 1995: 150). Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 159 Muzakkir S. yang diberikan, sehingga dengan dalih keterdesakan itu nilai kerelaan pun sudah hilang. yang diberikan, sehingga dengan dalih keterdesakan itu nilai kerelaan pun sudah hilang. KESIMPULAN Terbentuknya harga secara operasional lahir dari kesepakatan para pihak karena apabila saat negosiasi harga dalam transaksi para pihak telah sepakat maka keduanya terikat oleh wajibnya melangsungkan transaksi dan sekaligus menandai sahnya transaksi yang dilakukan. Terbentuknya harga melalui kesepakatan para pihak mencerminkan keadilan bagi keduanya. Menarik bila melihat aspek suka sama suka masing-masing pihak menjadi standar yang sering kali muncul dalam sebuah transaksi atau kontrak. Namun, karena kerelaan merupakan suatu yang abstrak maka susah untuk merealisasikan dan mengaktualisasikan dalam konteks bisnis modern saat ini. Oleh sebab itu, “kerelaan” menurut penulis harus memiliki kriteria dan standar yang jelas dengan mempertimbangkan beberapa hal di antaranya; 1. Aspek akad, yaitu dengan melihat aspek isi akad, unsur-unsur akad, dan prinsip-prinsip akad dapat dijadikan sebagai acuan dalam mengakutalkan konsep “suka sama suka” dalam sebuah bisnis,; 2. Aspek pressure (tekanan), yaitu melihat faktor tekanan yang ada pada masing-masing pihak, baik tekanan dari diri sendiri atau tekanan dari orang lain saat melakukan transaksi, yang dalam istilah Ibn Khaldun disebut dengan faktor psikografi,; 3. Aspek sosiologis, yaitu keadaan masing-masing pihak secara sosilogi terdesak atau terpaksa untuk melakukan transaksi. Transaksi yang lahir karena faktor keterdesakan dan keterpaksaan dalam transaksi belum memenuhi standar “suka sama suka” dari masing-masing pihak. Bila seseorang melakukan transaksi dalam keadaan terdesak atau terpaksa maka secara zahir “kerelaan” sulit untuk dimasukkan. Kerelaan merupakan suatu yang ada dalam hati, begitu juga keterpaksaan dan keterdesakan bagian dari sesuatu yang ada dalam hati. Sehingga kondisi terpaksa sering kali dijadikan celah untuk mewujudkan kerelaan. Tiga standar tersebut paling tidak bisa menjadi standar untuk mengakutalisasikan konsep “kerelaan” dalam bisnis terutama pergerakan bisnis modern saat ini. Dalam soal akad, kerelaan tidak cukup hanya dengan adanya tanda tangan, karena tanda tangan belum bisa menjadi standar absolut adanya kerelaan. Di samping adanya tanda tangan, aspek tidak ada tekanan dari salah satu pihak dan tidak ada keterpaksaan atau keterdesakan patut menjadi pertimbangan dalam melanjutkan suatu akad atau transaksi. Kondisi yang sulit untuk dicermati adalah kondisi psikografi dalam hal ini konsumen, konsumen yang terpaksa dan terdesak sering kali tidak menyadari bahkan tidak peduli dengan tingginya harga Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 160 Konstruksi At-Tas’īr Al-‘Adl dalam Dialektika Pemikiran Ibnu Taimiyah Dan Ibnu Khaldun Veithzal Rivai Zainal, dkk., Islamic Business Management; Praktik Manajemen Bisnis yang Sesuai Syari’ah Islam, (Yogyakarta: BPFE, 2014). j y g y ( gy ) Veithzal Rivai Zainal, dkk.,., Islamic Business and Economic Ethics, (Jakarta; PT. Bumi Aksara, 2012) Zakariya Al-Anshari, Asnal Mathalib Syarah Raudhah Ath-Thalib, Jilid. II., (Bairut: Dār al-Kutub al-‘Ilmiyah, 1995) Veithzal Rivai Zainal, dkk., Islamic Business Management; Praktik Manajemen Bisnis yang Sesuai Syari’ah Islam, (Yogyakarta: BPFE, 2014). Veithzal Rivai Zainal, dkk.,., Islamic Business and Economic Ethics, (Jakarta; PT. Bumi Aksara, 2012) Zakariya Al-Anshari, Asnal Mathalib Syarah Raudhah Ath-Thalib, Jilid. II., (Bairut: Dār al-Kutub al-‘Ilmiyah, 1995) Veithzal Rivai Zainal, dkk., Islamic Business Management; Praktik Manajemen Bisnis yang Sesuai Syari’ah Islam, (Yogyakarta: BPFE, 2014). Veithzal Rivai Zainal, dkk.,., Islamic Business and Economic Ethics, (Jakarta; PT. Bumi Aksara, 2012) Z k i Al A h i A l M th lib S h R dh h Ath Th lib Jilid II DAFTAR PUSTAKA Abu Hamid Muhammad bin Muhammad Al-Ghazali, Ihyā’Ulum ad-Dīn, Jilid.II., (Bairut: Dār al-Kutub al-‘Ilmiyah, 2012) Abu Isa Muhammad bin Isa bin Saurah At-Turmuzi, Al-Jāmi’ al-Shahih wahua Sunan Al-Turmuzi, Jilid, III., (Mesir: Mushtafa Albāni Alhalabī, tt) Ahmad bin Abdu al-Halim bin Taimiyah, Al-Hisbah Fi Al-Islām Au Wazīfah Al-Hukuman Al-Islamiyah, (Bairut: Dār Al-Kutub Al-Ilmiyah, tt) Boedi Abdullah, Peradaban Pemikiran Ekonomi Islam, (Bandung: Pustaka Setia, 2010) Euis Amalia, Sejarah Pemikiran Ekonomi Islam, (Depok: Gramata Publishing, 2010) Ibn Manzhur, Lisān al-‘Arab, jilid, III., ( Dār al-Ma’arif, t.t) Lukman Faurani, Arah dan Strategi Ekonomi Islam (Yogyakarta: Magistra Insani Press, 2006) Muhammad A. Mannan, Teori dan Praktek Ekonomi Islam, (Yogyakarta: PT. Dana Bhakti Wakaf, 1995) Muhammad bin Ali bin Muhammad As-Syaukani, Nail al-Authar min Ahādits Sayyid al-Akhbar, jilid. 5, (Bairut: Dār al-Kutub al-‘Ilmiyah, 2004) Muhammad Fuad Abdul Baqi, Mu’jam Mufahras Li Alfādzi al-Qur’an (Bairut: Dārul Marifah, 2002) M. Nur Rianto Al-Arif dan Euis Amalia, Teori Mikroekonomi; suatu Perbandingan Ekonomi Islam dan Ekonomi konvensional, (Jakarta: Kencana, 2010) Naziyah Hammad, Mu’jam al-Muṣtalahāt al-Māliyah wa al-Iqtiṣādiyah fī Lugat al-Fuqaha (Dimsyak: Dār al-Qalam, 2008) Nur Chamid, Jejak Langkah dan Sejarah Pemikiran Ekonomi Islam, (Yogyakarta: Pustaka Pelajar, 2010) Rozalinda, Ekonomi Islam; Teori dan Aplikasinya pada Aktivitas Ekonomi, (Jakarta: Rajawali Pers, 2014) Setiawan Budi Utomo, Fiqh Aktual: Jawaban Tuntas Masalah Kontemporer, (Jakarta: Gema Insani, 2003), Taqiyuddin An-Nabhani , An-Niẓam Al-Iqtiṣadi fil Islam, Terj. Redaksi Al- Azhar Press, (Bogor: Al-Azhar Press, 2010) Taqiyuddin Ahmad bin Abdu al-Halim bin Taimiyah, Al-Hisbah Fi Al- Islām Au Wazīfah Al-Hukuman Al-Islamiyah, (Bairut: Dār Al-Kutub Al- Ilmiyah, tt) Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019 161 Muzakkir S. 162 Tawazun: Journal of Sharia Economic Law Vol. 2 No. 2 2019
https://openalex.org/W2024173091	https://europepmc.org/articles/pmc4335406?pdf=render	English	null	Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae	BMC Microbiology	2,015	cc-by	13,255	Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae Endres et al. Endres et al. BMC Microbiology (2015) 15:30 DOI 10.1186/s12866-015-0365-0 * Correspondence: karl-erich.jaeger@fz-juelich.de; r.batra-safferling@fz-juelich.de †Equal contributors 1Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany 2Institute of Complex Systems, ICS-6: Structural Biochemistry, Forschungszentrum Jülich, D-52425 Jülich, Germany Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae Endres et al Endres et al. BMC Microbiology (2015) 15:30 DOI 10.1186/s12866-015-0365-0 Endres et al. BMC Microbiology (2015) 15:30 DOI 10.1186/s12866-015-0365-0 Open Access Structure and function of a short LOV protein from the marine phototrophic bacterium Dinoroseobacter shibae Stephan Endres1†, Joachim Granzin2†, Franco Circolone1†, Andreas Stadler3, Ulrich Krauss1, Thomas Drepper1, Vera Svensson1, Esther Knieps-Grünhagen1, Astrid Wirtz1, Anneliese Cousin2, Petra Tielen4, Dieter Willbold2,5, Karl-Erich Jaeger1,6* and Renu Batra-Safferling2* Background fused effector domain, short LOV proteins can act as genuine blue-light receptors, probably transducing the signal via interactions with a partner protein. g Signal perception in LOV domains is achieved via a blue-light (λ = 440–482 nm) absorbing flavin mononu- cleotide (FMN) or, in some cases, via a flavin adenine dinucleotide (FAD) chromophore, that in the dark is non-covalently bound within the LOV domain [1,2]. Blue-light absorption triggers the formation of a transi- ent adduct between the FMN-C4a atom and the sulfur atom of a strictly conserved cysteine residue in the LOV domain active site [1]. Once illumination has ceased, this covalent bond is broken thus concluding the photocycle. While the initial photochemical reaction and the adduct formation occurs on a timescale of microseconds [3], adduct decay can take seconds to hours depending on the LOV protein [3-7] (Additional file 1: Table S1). The longest living intermediate, the FMN-cysteinyl-thiol adduct, has been proven to represent the structural signaling state of LOV photoreceptors [8,9]. The major- ity of LOV photoreceptors are multi-domain sensory systems, where light-signal perception by the LOV do- main influences the “activity” of fused effector domains such as kinases, anti-sigma factors, helix-turn-helix DNA binding domains, phosphodiesterases and cyclases [10]. All short LOV proteins structurally characterized so far possess N- and/or C-terminal helical extensions (N- terminal N-cap or A´α-helix, and C-terminal Jα-helix) outside the conserved LOV core domain suggested to be involved in signaling [7,24,26]. In multi-domain LOV photoreceptors, these extensions often link the sensor LOV and associated effector domains and are suggested to act as signal-transducing linker elements or spacers providing flexibility [13,27]. Another feature common to most LOV domains is their dimerization propensity. In particular, a light- dependent alteration of the monomer-dimer equilibrium represents a feasible mode of signal-relay for short LOV proteins, where one of the two quaternary structural states selectively mediates the interaction with down- stream signaling partners [25,28,29]. For short LOV pro- teins crystallized as full-length constructs, dimerization is mostly mediated by either the N-terminal A´α-helix (N-cap) and/or, if present, the respective C-terminal Jα-helices [24,25,28]. The presence and structure of N- and C-terminal extensions largely determine the mode of dimerization and hence the corresponding light-dependent conformational change. Thus, the study of short LOV proteins with unusual LOV-core exten- sions might provide further insights into the diversity of LOV-protein dimerization and signaling. Abstract Background: Light, oxygen, voltage (LOV) domains are widely distributed in plants, algae, fungi, bacteria, and represent the photo-responsive domains of various blue-light photoreceptor proteins. Their photocycle involves the blue-light triggered adduct formation between the C(4a) atom of a non-covalently bound flavin chromophore and the sulfur atom of a conserved cysteine in the LOV sensor domain. LOV proteins show considerable variation in the structure of N- and C-terminal elements which flank the LOV core domain, as well as in the lifetime of the adduct state. Results: Here, we report the photochemical, structural and functional characterization of DsLOV, a LOV protein from the photoheterotrophic marine α-proteobacterium Dinoroseobacter shibae which exhibits an average adduct state lifetime of 9.6 s at 20°C, and thus represents the fastest reverting bacterial LOV protein reported so far. Mutational analysis in D. shibae revealed a unique role of DsLOV in controlling the induction of photopigment synthesis in the absence of blue-light. The dark state crystal structure of DsLOV determined at 1.5 Å resolution reveals a conserved core domain with an extended N-terminal cap. The dimer interface in the crystal structure forms a unique network of hydrogen bonds involving residues of the N-terminus and the β-scaffold of the core domain. The structure of photoexcited DsLOV suggests increased flexibility in the N-cap region and a significant shift in the Cα backbone of β strands in the N- and C-terminal ends of the LOV core domain. Conclusions: The results presented here cover the characterization of the unusual short LOV protein DsLOV from Dinoroseobacter shibae including its regulatory function, extremely fast dark recovery and an N-terminus mediated dimer interface. Due to its unique photophysical, structural and regulatory properties, DsLOV might thus serve as an alternative model system for studying light perception by LOV proteins and physiological responses in bacteria. Keywords: Aerobic anoxygenic photosynthesis, Blue-light photoreceptor, Crystal structure, Dimerization, Dinoroseobacter shibae, LOV domain, PAS domain, Photocycle © 2015 Endres et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Endres et al. BMC Microbiology (2015) 15:30 Page 2 of 15 Background g p p y Based on this modularity, LOV photoreceptors in turn control or influence a multitude of cellular light re- sponses in plants, algae, fungi and bacteria [11]. In plants, the biological function of LOV photoreceptors is well-established, including plant phototropism, chloro- plast relocation, light-induced leaf movement and regu- lation of stomatal opening [12,13]. In contrast, the understanding of regulatory functions of their counter- parts in bacteria is still limited, some examples were de- scribed recently (comprehensively reviewed in [14]). The best characterized bacterial LOV photoreceptor is YtvA from the soil bacterium Bacillus subtilis [15], which is part of the stressosome, involved in the σB-mediated general stress response of this organism [9,16-18]. Re- cently, additional regulatory functions of bacterial LOV proteins were described such as control of cell-adhesion in Caulobacter cresentus [19], virulence in Brucella abortus [20], motility of Pseudomonas syringae [19,21,22] and in the counteraction of plant immune response dur- ing citrus canker in Xanthomonas citri [23]. Here, we report on the identification of an unusual short LOV protein DsLOV from the marine phototrophic α-proteobacterium Dinoroseobacter shibae DFL12T. Combining crystallography and biochemical methods, we investigate the molecular mechanism of photoexci- tation in DsLOV, which displays a recovery on the timescale of seconds. Furthermore, we demonstrate that the fast recovering DsLOV is a key regulator for light mediated triggering of photopigments bacteriochlo- rophyll a and spheroidenone formation in D. shibae. Dinoroseobacter shibae harbors three putative LOV photoreceptor genes Remarkably, in addition to the multi-domain LOV photoreceptors, a large subset of LOV photoreceptors exist in bacteria and fungi that do not possess fused ef- fector domains and are thus called “short” LOV proteins. Functionally and structurally characterized examples in- clude the fungal photoreceptor VVD [7], the LOV pro- teins PpSB1-LOV and PpSB2-LOV of Pseudomonas putida KT2440 [4,5,24] and the recently characterized RsLOV protein of Rhodobacter sphaeroides 2.4.1 [25,26]. Physiological studies on Neurospora crassa VVD and re- cent data for RsLOV indicate that despite the lack of a The Gram-negative α-proteobacterium Dinoroseobacter shibae is a member of the Roseobacter clade, a bacterial lineage that is globally abundant in different marine habitats. Light is an important environmental factor for D. shibae because it can produce ATP via aerobic anoxygenic photosynthesis [30,31]. Sequence similarity analysis of the published D. shibae DFL12T genome re- vealed the presence of three genes encoding putative blue-light photoreceptors of the LOV family (Additional file 1: Figure S1), possessing all the sequence features necessary for FMN binding and photocycling [1]. The Endres et al. BMC Microbiology (2015) 15:30 Page 3 of 15 blue-light illumination, reflected by the loss of its absorp- tion within the blue-region of the spectrum could only be observed when the sample temperature was reduced to 4°C (Figure 1B, dashed lines) suggesting a rapid dark recovery which was determined as τREC = 9.6 ± 0.1 s at 20°C (Figure 1C). predicted LOV proteins encoded by the genes Dshi_1135 and Dshi_1893 show either one single histidine kinase domain or four domains with homologies to PAS, histidine kinase and response regulator protein domains, which are fused to the respective C-termini of the pre- dicted LOV domains (Figure 1A). In contrast, the gene Dshi_2006 codes for a short LOV protein that lacks fused effector domains. Among bacterial short LOV pro- teins, DsLOV appears exceptional as it harbors an N- terminal cap domain but lacks a C-terminal extension and is thus architecturally very similar to fungal VVD [7]. DsLOV exhibits fast recovery kinetics The soluble photoreceptor protein DsLOV was expressed in E. coli BL21 (DE3) as C-terminal His6-tagged protein and was purified via immobilized-metal affinity chroma- tography (IMAC). In the dark, DsLOV exhibits the characteristic absorption bands of the LOV dark state with λmax = 449 nm (Figure 1B, solid lines). Remarkably, forma- tion of the DsLOV photoadduct (λmax = 390 nm) after Figure 1 DsLOV domain architecture and photochemistry. (A) Domain architecture of the three D. shibae LOV proteins. Locus tags and accession numbers (in brackets) are given in front of each protein. The length of each protein and the type and relative location of all predicted Pfam domains are specified. LOV: light, oxygen voltage; HK: histidine kinase; PAS: PER-ARNT-SIM; RR: response regulator. (B) Absorbance spectra of DsLOV in the dark and light state. In the dark, DsLOV displays the characteristic UV/vis absorbance bands with a λmax of about 449 nm (solid line). To sufficiently populate the light state, the DsLOV protein was exposed to blue-light for 30 seconds at 4°C (dashed lines), resulting in a decreased absorbance at 447 nm and the concomitant formation of a new absorbance maximum at 390 nm. The inset shows the light–dark difference spectrum of DsLOV. (C) Dark recovery of DsLOV measured at 20°C. LOV protein dark recovery was monitored by recording time traces for the absorbance recovery at 485 nm after blue-light illumination. The data (squares) was fitted using a mono-exponential decay function. Figure 1 DsLOV domain architecture and photochemistry. (A) Domain architecture of the three D. shibae LOV proteins. Locus tags and accession numbers (in brackets) are given in front of each protein. The length of each protein and the type and relative location of all predicted Pfam domains are specified. LOV: light, oxygen voltage; HK: histidine kinase; PAS: PER-ARNT-SIM; RR: response regulator. (B) Absorbance spectra of DsLOV in the dark and light state. In the dark, DsLOV displays the characteristic UV/vis absorbance bands with a λmax of about 449 nm (solid line). To sufficiently populate the light state, the DsLOV protein was exposed to blue-light for 30 seconds at 4°C (dashed lines), resulting in a decreased absorbance at 447 nm and the concomitant formation of a new absorbance maximum at 390 nm. The inset shows the light–dark difference spectrum of DsLOV. (C) Dark recovery of DsLOV measured at 20°C. Crystal structure of DsLOV reveals a unique N-cap region Overall structure Monoclinic crystals of the DsLOV protein were obtained in the dark, which diffracted to a resolution of 1.5 Å. The statistics of X-ray data collection and refinement are documented in Table 1. The final monomer model (one molecule per asymmetric unit) comprises residues 20–139 of the protein and the chromophore riboflavin (RBF) (Figure 2; the secondary structure elements are colored with helices in light blue; β-strands in light green, and loops in yellow; the chromophore is shown as stick model and is colored by element: carbon in yellow, nitrogen in blue and oxygen in red). Residues 1–19 DsLOV exhibits fast recovery kinetics The chromophore riboflavin (RBF) is shown as stick model and is colored by element: carbon, yellow; nitrogen, blue; oxygen, red. β-sheet comprising Aβ (residue 36–40), Bβ (residue 49–52), Gβ (residue 98–105), Hβ (residue 111–122) and Iβ (residue 128–137), and four α-helices Cα (residue 54–60), Dα (residue 64–67), Eα (residue 72–75) and Fα (residue 82–94). In addition, a noncanonical structural element designated as the N-cap is present that includes a N-terminal turn (residues 20–23), helix (A´α, residues 24–27) and a connecting loop (28–35) (Figure 2). Dimer interface Th D LOV The DsLOV crystal structure has one molecule in the asymmetric unit even though the protein exists as a dimer in solution as revealed by SEC and small angle X- ray scattering experiments described below. The most probable higher-order assembly proposed by PISA ana- lysis [33] is a dimer, where the N-terminal A´α helix is part of the dimer interface that is unique amongst LOV protein structures (Figure 3A). The buried surface area of ~950 Å2 involves a set of interchain interactions be- tween charged residues (D20-OD2…NH1-R119´, 3.4 Å) (residues in italics with prime refer to those from the symmetry equivalent molecule), hydrogen bonds (D20- O…N-Y122´, 2.7 Å, A22-N…O-Y122´, 2.8 Å) and several hydrophobic interactions between residues in the N- terminal cap (including helix A´α) and the β-scaffold of the opposite subunit (Hβ and Iβ) (Figure 3A). Residues constituting the dimer interface are I21, L24, I50, L68, G69, I121, D123, P124, E125, M129 and F130 from the two-fold related monomers. aStatistics for the highest-resolution shell are shown in parentheses. could not be traced in the electron density map and are likely to be disordered. Also, no electron density was observed for the C-terminal residues 139–146 that comprise the His6-tag. According to Ramachan- dran plot generated with Molprobity [32], the model exhibited good geometry with none of the residues in disallowed regions. DsLOV exhibits fast recovery kinetics LOV protein dark recovery was monitored by recording time traces for the absorbance recovery at 485 nm after blue-light illumination. The data (squares) was fitted using a mono-exponential decay function. Endres et al. BMC Microbiology (2015) 15:30 Page 4 of 15 Table 1 Data collection and refinement statistics DsLOV “dark” DsLOV “photoexcited” Wavelength (Å) 0.93340 (Beamline: ESRF, ID14-1) 0.95372 (Beamline: ESRF, ID23-1) Detector type ADSC Quantum Q210 ADSC Quantum Q315r Resolution range (Å) 45.4 - 1.5 (1.53 - 1.5)a 26.1 - 2.0 (2.07 - 2.0)a Space group C 1 2 1 C 1 2 1 Unit cell a = 89.98 (Å) a = 90.78 (Å) b = 30.60 (Å) b = 30.94 (Å) c = 49.33 (Å) c = 49.54(Å) β = 113.03° β = 113.09° Total reflections 65088 30165 Unique reflections 19912 8206 Multiplicity 3.3 (2.5) 3.7 (4.0) Completeness (%) 98.98 (91.59) 93.48 (98.15) Mean I/σ (I) 19.7 (4.3) 16.3 (10.4) Wilson B-factor (Å2) 14.08 10.62 R-sym 0.037 (0.203) 0.061 (0.085) R-work 0.1298 (0.1115) 0.1633 (0.1595) R-free 0.1789 (0.2031) 0.1824 (0.1586) Coordinate error (maximum- likelihood based) (Å) 0.13 0.16 Number of atoms 1125 1015 Macromolecules 981 904 Ligands 32 27 Water molecules 112 84 Protein residues 120 115 RMS (bonds) (Å) 0.014 0.004 RMS (angles) (°) 1.56 0.77 Ramachandran favored (%) 99 99 Ramachandran outliers (%) 0 0 Clash score 2.99 4.92 Average B-factor (Å2) 19.9 14.7 PDB entry 4KUK 4KUO aStatistics for the highest-resolution shell are shown in parentheses. Table 1 Data collection and refinement statistics Figure 2 Crystal structure of DsLOV. Ribbon representation of the dark state of DsLOV (residues 20–139) colored according to secondary structure elements with helices in light blue; β-strands in light green, and loops in yellow). The chromophore riboflavin (RBF) is shown as stick model and is colored by element: carbon, yellow; nitrogen, blue; oxygen, red. Figure 2 Crystal structure of DsLOV. Ribbon representation of the dark state of DsLOV (residues 20–139) colored according to secondary structure elements with helices in light blue; β-strands in light green, and loops in yellow). The chromophore riboflavin (RBF) is shown as stick model and is colored by element: carbon, yellow; nitrogen, blue; oxygen, red. Figure 2 Crystal structure of DsLOV. Ribbon representation of the dark state of DsLOV (residues 20–139) colored according to secondary structure elements with helices in light blue; β-strands in light green, and loops in yellow). Figure 3 Dimer interface and photoexcited state of DsLOV. Figure 3 Dimer interface and photoexcited state of DsLOV. g p (A) The dimer of DsLOV (dark state) showing the N-terminus (A´α) mediated dimer interface, based on PISA analysis [33]. The secondary structure elements are colored with helices in light blue; β strands in light green, and loops in yellow. The chromophore riboflavin (RBF) is shown as stick model and is colored by element: carbon, yellow; nitrogen, blue; oxygen, red. The intersubunit hydrogen bonds are shown as broken lines between the residues shown as stick models and colored by element. Specifications on the hydrogen bond distance and the atoms involved are provided in the text. Side chain conformations of R117 and R119 in photoexcited state are shown superimposed in coral. Position of L27 and E32 are indicated by arrowheads. (B) Superposition of dark and photoexcited crystal structures. Side chains of residues shown as stick models are colored by element for dark state as in A, and in coral for photoexcited state. A shift seen in the respective Iβ backbone (dark state- light blue and photoexcited- coral) is highlighted in the oval area. For clarity, rest of the region is shown as ribbon representation in transparent grey. Note the additional H-bond between Q135-NE2 and N5 of RBF molecule in the photoexcited state. The view in this figure is of the monomer (right) of the dimer shown in panel A, which is rotated ~ 180° around the horizontal axis. The mode of protein-chromophore interaction is conserved amongst the LOV protein family, where rigid coordination of the chromophore molecule in the binding pocket is mediated by a network of hydro- gen bonds, van der Waals and electrostatic interac- tions. Mainly, residues in the 310 Eα-helix (N71, R73, Q76), Hβ-strand (N104, N114), and Iβ strand (Q135) constitute most of the hydrogen bonding interactions with the RBF molecule. Residues forming hydrophobic contacts with the isoalloxazine ring are V38, C72, I88, R89, L92, L116, I118, F131, A132 and G133. In all LOV proteins, two conserved arginine residues make contact with the phosphate group of the chromo- phore. In DsLOV, the corresponding arginines are R73 and R89, where only R73 forms a hydrogen bond (R73- NH2…O5´-RBF, 3.0 Å) with the ribityl chain. Structural comparison with other LOV domains The residues 36–137 comprise the conserved LOV core domain with a typical α/β-fold PAS topology. The core domain is made up of a five-stranded antiparallel Although the LOV core domain is structurally well con- served, diversity is seen for the noncanonical N- and C-terminal elements [7,24,26,34,35]. These regions adopt Page 5 of 15 Endres et al. BMC Microbiology (2015) 15:30 VVD: 71 aa long) and secondary structure, superposition of the VVD dark state (PDB ID 2PD7, orange in Additional file 1: Figure S2, panel B) shows the highest similarity, where residues 43–50 and 67–72 follow the trace corresponding to the N-cap residues of DsLOV. Moreover, N-cap mediated dimerization is globally similar in both structures. Functional implications of this similarity are discussed below in the section ‘light-induced conformational changes’. VVD: 71 aa long) and secondary structure, superposition of the VVD dark state (PDB ID 2PD7, orange in Additional file 1: Figure S2, panel B) shows the highest similarity, where residues 43–50 and 67–72 follow the trace corresponding to the N-cap residues of DsLOV. Moreover, N-cap mediated dimerization is globally similar in both structures. Functional implications of this similarity are discussed below in the section ‘light-induced conformational changes’. Figure 3 Dimer interface and photoexcited state of DsLOV. Please note that the interaction between the terminal end of chromophore ribityl chain and the protein is most likely disturbed due to presence of RBF (and not FMN) in the DsLOV crystal structure, hence preventing any conclu- sions with regard to the FMN-phosphate coordination in DsLOV in solution, where FMN is the predominant chromophore (see above results of HPLC analysis). the helical secondary structure in the previously pub- lished LOV protein structures (Additional file 1: Figure S2). An N-terminal turn-helix-turn motif has been pre- viously reported [34] in the crystal structure of AsLOV2 (PDB ID 2V0U, yellow in Additional file 1: Figure S2). In DsLOV, the N-terminal extension shows similar second- ary structure elements with a turn, helix and a long connecting loop (Figure 2). However, superposition of the core domains (Additional file 1: Figure S2) shows different positioning of the corresponding N-terminal elements. In contrast to monomeric AsLOV2, the non- canonical N-terminal element of DsLOV is involved in dimer formation. We next compared DsLOV to fungal VVD [28] as both proteins consist of the conserved core domain with an N-terminal extension. Surprisingly, des- pite the differences in N-cap length (DsLOV: 35 and the helical secondary structure in the previously pub- lished LOV protein structures (Additional file 1: Figure S2). An N-terminal turn-helix-turn motif has been pre- viously reported [34] in the crystal structure of AsLOV2 (PDB ID 2V0U, yellow in Additional file 1: Figure S2). In DsLOV, the N-terminal extension shows similar second- ary structure elements with a turn, helix and a long connecting loop (Figure 2). However, superposition of the core domains (Additional file 1: Figure S2) shows different positioning of the corresponding N-terminal elements. In contrast to monomeric AsLOV2, the non- canonical N-terminal element of DsLOV is involved in dimer formation. We next compared DsLOV to fungal VVD [28] as both proteins consist of the conserved core domain with an N-terminal extension. Surprisingly, des- pite the differences in N-cap length (DsLOV: 35 and Flavin binding pocket l As seen in previously reported dark state LOV protein structures, a flavin chromophore molecule is noncova- lently bound within a tight pocket formed by the β- scaffold and the surrounding Eα- and Fα-helices (Figure 2). Surprisingly, the chromophore bound in the DsLOV struc- ture is RBF and not FMN; where both are identical except for the terminal phosphate group that is absent in RBF. Prior to crystallization setups, purified DsLOV protein was denatured and the released chromophores were ana- lyzed by HPLC demonstrating that it predominantly binds FMN (74.3 ± 0.2%) and FAD (25.7 ± 0.2%) whereas no riboflavin could be detected (data not shown). We suspect that the FMN bound to the DsLOV protein in solution got hydrolyzed to RBF during the crystallization time period of several weeks in acidic conditions [36]. Light-induced conformational changes Additionally, comparison of the spectra obtained from single crystal microspectrometry confirmed typical dark state (λmax = 447 nm) and light state (λmax ≈390 nm) absorbance spectra for the DsLOV crystals grown in dark, and after blue-light illumination, respectively (Additional file 1: Figure S4, panel A). In order to verify that spectral changes observed in the crystal are not the result of irre- versible photobleaching, dark grown DsLOV crystals were transferred to the microspectrometer under con- stant blue-light illumination in the cryo stream (100 K) and an absorbance spectrum was recorded. Subse- quently, the cryo stream was blocked and the light was switched off to allow dark recovery of the protein at room temperature. The corresponding spectra shows full recovery of the flavin specific absorption band in the blue- region of the spectrum within 5 minutes (Additional file 1: Figure S4, panel B). In contrast, no dark recovery occured within 1 hour at cryogenic temperatures (Additional file 1: Figure S4, panel C). Taken together, the data demonstrates that the protein in dark grown DsLOV crystals is capable of reversible photocycling, suggesting that the structural changes observed in photoexcited crystals are most likely the result of illumination and are not related to irrevers- ible photobleaching. Despite the increased flexibility in the N-terminus (absence of electron density for residues 29–33 located in the A´α-Aβ loop) in the photoexcited state, residues 20–28 were still traceable (Additional file 1: Figure S3, panel C). The position of the N-cap, in particular, is stabilized by hydrogen bonding interactions between dimer-interface residues D20-Y122´, and A22-Y122´, that exist in both the dark- and photoexcited-states. It should be noted that the photoexcited state structure was obtained upon illumination of dark-grown crystals, which is not sufficient to cause large conformational changes of functional relevance that would require breaking of the crystal lattice constraints. Interestingly, the side chain conformation of R119, involved in the dimer interface differs in both the states, facing towards and away from the dimer interface in the dark and pho- toexcited state, respectively (Figure 3A). As a result, in- teractions between R119 and D20´, R119 and L27, and R119 and E32 are absent. Energetically, this might in- crease the flexibility of the N-terminus as seen in the photoexcited state (disordering of the A´α-Aβ loop). An- other major structural change is observed for the side chain conformation of R117 in the photoexcited state. Light-induced conformational changes The photochemistry of LOV domains is reversible in so- lution as well as in crystals [7,34,35,37]. We thus solved the structure of DsLOV in the photoexcited state after Endres et al. BMC Microbiology (2015) 15:30 Page 6 of 15 Aβ strand (~0.6 Å), and the connecting loop (A´α-Aβ loop in the N-terminus) (Additional file 1: Figure S3, panels C-D); (iv) the hydrophobic pocket around the dimethyl-benzene ring of the chromophore is also rear- ranged; for example, A132 and G133 no longer make hydrophobic contacts as in the dark state. An interesting observation is that the side chain conformation of F74 which is aligned parallel to the flavin ring system in the dark rotates towards the flavin ring in the photoexcited state (Figure 3B). Notably, we observed similar side chain orientations for this structurally conserved phenyl- alanine residue in previously published crystal structures of C. reinhardtii Phot LOV1 (PDB ID 1N9L, 1N9N, 1N9O), B. subtilis YtvA LOV (PDB ID 2PR5, 2PR6), R. sphaeroides RsLOV (PDB ID HJ6, 4HNB) and P. putida PpSB1-LOV (PDB ID 3SW1). Mutation of the corre- sponding phenylalanine residue in YtvA-LOV to histi- dine accelerated the dark recovery of the protein 25-fold [38]. This hints a possible involvement of F74 in photo- activation, signaling and adduct state stability in LOV proteins and further suggests that the structural differ- ences observed between dark and photoexcited structures of DsLOV are a consequence of photoactivation. illuminating crystals grown in the dark (Table 1). The difference electron density map (Fdark-Flight) does not show the presence of a covalent bond between residue C72 and flavin C4a atom (Additional file 1: Figure S3, panels A-B). The interatomic distance between the C72- Sγ and RBF-C4a is 3.44 Å, marginally smaller than 3.54 Å observed in the DsLOV dark state structure. The presence or absence of a covalent adduct in LOV protein crystals has been used in previous studies to define ei- ther the ‘light’ or ‘dark’ state, respectively. The absence of a covalent adduct in the photoexcited crystal structure of DsLOV can be due to two reasons: (i) fast dark recovery of the protein (τREC = 9.6 s) prevents adduct stabilization, and/or (ii) radiation damage with radiolysis of the Cys72-RBF thioether bond caused by the high- energy X-ray beam. The latter has been previously ob- served for other LOV proteins [7,34,37]. Light-induced conformational changes Additionally, comparison of the spectra obtained from single crystal microspectrometry confirmed typical dark state (λmax = 447 nm) and light state (λmax ≈390 nm) absorbance spectra for the DsLOV crystals grown in dark, and after blue-light illumination, respectively (Additional file 1: Figure S4, panel A). In order to verify that spectral changes observed in the crystal are not the result of irre- versible photobleaching, dark grown DsLOV crystals were transferred to the microspectrometer under con- stant blue-light illumination in the cryo stream (100 K) and an absorbance spectrum was recorded. Subse- quently, the cryo stream was blocked and the light was switched off to allow dark recovery of the protein at room temperature. The corresponding spectra shows full recovery of the flavin specific absorption band in the blue- region of the spectrum within 5 minutes (Additional file 1: Figure S4, panel B). In contrast, no dark recovery occured within 1 hour at cryogenic temperatures (Additional file 1: Figure S4, panel C). Taken together, the data demonstrates that the protein in dark grown DsLOV crystals is capable of reversible photocycling, suggesting that the structural changes observed in photoexcited crystals are most likely the result of illumination and are not related to irrevers- ible photobleaching. Several distinct conformational differences between dark and photoexcited DsLOV structures were observed illuminating crystals grown in the dark (Table 1). The difference electron density map (Fdark-Flight) does not show the presence of a covalent bond between residue C72 and flavin C4a atom (Additional file 1: Figure S3, panels A-B). The interatomic distance between the C72- Sγ and RBF-C4a is 3.44 Å, marginally smaller than 3.54 Å observed in the DsLOV dark state structure. The presence or absence of a covalent adduct in LOV protein crystals has been used in previous studies to define ei- ther the ‘light’ or ‘dark’ state, respectively. The absence of a covalent adduct in the photoexcited crystal structure of DsLOV can be due to two reasons: (i) fast dark recovery of the protein (τREC = 9.6 s) prevents adduct stabilization, and/or (ii) radiation damage with radiolysis of the Cys72-RBF thioether bond caused by the high- energy X-ray beam. The latter has been previously ob- served for other LOV proteins [7,34,37]. Light-induced conformational changes Here, the mutations I427V (AsLOV2), I39V (YtvA) and I85V (VVD) accelerated the recovery of the respective proteins 8-, 5- and 23-fold [40,45]. Several structural interpretations have been proposed for the observed mutation-induced kinetic effects. For AsLOV2, I427V was proposed to be in van der Waals contact with the sulfhydryl group of the photoactive cysteine (C450) [45]. The authors suggested that structurally, I427 pro- vides steric support to the active site region near the photoactive cysteine and that removal of this support by introducing a valine at the respective position destabilizes the adduct state and hence results in a faster dark re- covery. Moreover, different imidazole-base dependent adduct-decay rate accelerating effects were observed for VVD and the corresponding I85V mutant. In par- ticular, the adduct-decay rate acceleration was more pronounced for VVD-I85V than for the wild-type protein. This suggests better access of imidazole to the active site of VVD-I85V where it acts as a base to ab- stract the proton from N5 proton in the light state, hence accelerating the photocycle dark recovery [40]. Taken together, residue-alterations at the respective position on Bβ seem to influence the dark recovery of LOV proteins by both steric effects acting on the photoactive cysteine and by controlling solvent/base access to the active site, which drives adduct scission by N5 deprotonation. β p In VVD, structures of dark-grown crystals are mono- meric [7]. Both, solution studies and light state crystal structure (obtained from crystals grown under light conditions) show homodimer formation [28]. Structural rearrangements in the latter include release of the N- terminal ‘latch’, which then binds to the symmetry equivalent molecule allowing dimer formation. In con- trast, the N-terminus is packed against the β-sheet of the LOV core domain in structures obtained from dark- grown crystals. An important role in the process is played by the hinge region that connects the N-terminus and the core domain, and shows structural changes as a result of FMN-cysteinyl-thiol adduct formation. Interest- ingly, the corresponding region in DsLOV (residues 28–35 in A´α-Aβ loop) shows no electron density upon photoexcitation. Light-induced conformational changes Here, R117 forms a salt bridge with N136 on Iβ (R117- NH1…OD1-N136, 2.49 Å and R117-NH2…OD1-N136, 2.71 Å). In the corresponding dark state structure, the side chain of R117 is flipped towards the N-terminal cap (Figure 3A) breaking the interaction with N136. Residue Q135, that forms a single hydrogen bond with the RBF isoalloxazine ring – O4…NE2-Q135, 3.07 Å, shows two possible hydrogen bonds O4…NE2-Q135, 2.93 Å, and Several distinct conformational differences between dark and photoexcited DsLOV structures were observed, even though direct evidence supporting an adduct for- mation in the electron density map is absent in the latter (Figure 3B): (i) several residues primarily localized at the boundary of the chromophore binding pocket show dif- ferent side chain conformations in the photoexcited state; these include V37, V38, M49, E65, V67, R70, R73, F74, R97, R117, R119, I121 and E125; (ii) due to absence of electron density, residues 29–33 located in the A´α- Aβ loop could not be traced in the photoexcited state; (iii) a significant shift is observed in the Cα backbone of the Iβ strand (~0.7 Å Cα - Cα distance) (Figure 3B), the Page 7 of 15 Endres et al. BMC Microbiology (2015) 15:30 Page 7 of 15 N5…NE2-Q135, 3.02 Å in the photoexcited state (Figure 3B) (Additional file 1: Figure S3, panel B). Hydrogen bonding between the conserved glutamine and the newly protonated N5 atom of the flavin ring has been observed in other light state crystal structures [24,35,37,39]. This light-induced structural change of Q135 is one of the initial steps in LOV photoactivation that probably contributes to the observed displacement of the Iβ strand in the photoexcited state of DsLOV. NE2…C4a-RBF, 18.5 Å), no direct or indirect proton relay network can be identified in the structure, ruling out a significant impact of the respective residue on the fast recovery of DsLOV. One major structural difference in the vicinity of the flavin chromophore between DsLOV and other plant and bacterial LOV domains is the presence of a methio- nine residue (M49) on Bβ at the dimethylbenzene side of the flavin isoalloxazine ring. In other LOV domains, the corresponding position is either occupied by an isoleu- cine (YtvA, AsLOV2, VVD) [40] or leucine (PpSB1-LOV) [24]. For AsLOV2, YtvA and VVD, mutation of the re- spective isoleucine residue strongly influences the adduct- state lifetime. Light-induced conformational changes Considering the analogy between DsLOV and VVD that show N-cap mediated dimer interface, it is possible that the mode of interaction as well as signal transfer are globally similar in both proteins where an illumination-triggered relay of structural changes occurs from the chromophore binding pocket towards the N- and C-terminus, gated via residues on the central β-scaffold (Iβ and Hβ). These might involve change in conformation of the A´α-Aβ loop in the N-cap region, which could consequently enable signal-transduction to an as yet unknown downstream partner protein of DsLOV within the cellular context. Structural basis of the fast dark recovery of DsLOV Most positions in the vicinity of the flavin chromophore known to influence the adduct decay such as V38 (V416 on Aβ in AsLOV2 [40,41]), N104 and N114 (N94 and N104; YtvA numbering, [42]) which form hydrogen- bonds to the isoalloxazine C(4) = O and C(2) = O atoms, as well as N47 on the Aβ-Bβ loop (N37, YtvA number- ing, [43]) are conserved between DsLOV and other LOV proteins, and are thus less likely to be involved in accel- erating the dark recovery of DsLOV compared to other LOV proteins. Additionally, it has been previously sug- gested that adduct decay can be influenced by surface exposed histidine residues by base-catalysis via a pro- posed hydrogen-bonding network of the base, the chromophore and intraprotein water molecules [3,44]. While DsLOV possesses one surface exposed histidine (H83) about 18 Å away from the RBF-C4a atom (H83- In the DsLOV dark-state crystal structure, M49 pos- sesses two conformations (Additional file 1: Figure S3, panel A), one where the δ-sulfur atom of the methionine side-chain is facing towards C72-Sγ (C72-SG…SD-M49, 3.43 Å) and a second mirrored conformation where the δ-sulfur is facing away from the photoactive cysteine (C72-SG…SD-M49, 3.89 Å). The double occupancy of M49 is indicative of structural flexibility of the respective residue in the dark. Even though we do not directly ob- serve C72-Sγ - FMN-C4a bond formation in photoex- cited crystals of DsLOV, adduct formation would result in a movement of C72-Sγ towards the RBF-C4a atom and hence away from M49. For reversal of the reaction, the C72-Sγ – RBF-C4a bond has to be broken and C72 Page 8 of 15 Endres et al. BMC Microbiology (2015) 15:30 scattering curves were calculated for the monomer and the dimer of the crystal structure, and fitted against the experimental SAXS data. As shown in Figure 4A, the theoretical curve of the monomer is a poor fit that yields a large χ2-value of 47. A better fit with dimer results in a reduction of χ2-value to 11.9, however, this is still signifi- cantly large. Notably, the dimer fits well in the small scattering vector range up to 0.1 Å−1 whereas deviations become visible at larger q-values. Scattering in the small angle region is sensitive to the molecular mass and to the global shape, whereas the large q-region is more sensitive to the tertiary structure of the protein. Small angle X-ray scattering reveals dimeric DsLOV in solution We performed SAXS measurements to determine the oligomeric state and envelope of the molecule in solu- tion. The molecular mass determined from the Porod volume and ab initio modelling was 33.4 kDa and 33.7 kDa, respectively. The calculated molecular mass from the amino acid sequence of monomeric DsLOV is 16.85 kDa. Analytical size-exclusion chromatography (SEC) revealed an apparent molecular mass of 35.6 ± 0.5 kDa for DsLOV, corresponding to the calculated mass of the dimer (33.7 kDa) (data not shown). There- fore, both SEC and SAXS results demonstrate that the protein forms a dimer in solution. Next, theoretical The envelope of DsLOV was determined by ab initio modelling. The distance distribution function P(r) is shown in Figure 4B. The ab initio model obtained using Figure 4 Solution behavior of DsLOV measured by small angle X-ray scattering. (A) Experimental data (open circles), calculated curves of the crystal structure of the monomer (blue line), the crystal structure of the dimer (red line), and the crystal structure of the dimer including flexible ends (green line). The inset shows the zoom in on the low q-range, where a small fraction of aggregated particles cause the slight uptick at q < 0.024 Å−1. The lower panel shows the same experimental data and the fits in a Kratky-plot. (B) The distance distribution function P(r) of DsLOV as calculated for the range of q > 0.024 Å−1, where effects of protein aggregation are negligible. (C) Ab initio model of DsLOV (cyan, shown in mesh) determined by SAXS and aligned crystal structure of the dimer (dimer orientation as in Figure 3A, two protein molecules are shown in coral and cyan with bound RBF molecules as stick model). Figure 4 Solution behavior of DsLOV measured by small angle X-ray scattering. (A) Experimental data (open circles), calculated curves of the crystal structure of the monomer (blue line), the crystal structure of the dimer (red line), and the crystal structure of the dimer including flexible ends (green line). The inset shows the zoom in on the low q-range, where a small fraction of aggregated particles cause the slight uptick at q < 0.024 Å−1. The lower panel shows the same experimental data and the fits in a Kratky-plot. (B) The distance distribution function P(r) of DsLOV as calculated for the range of q > 0.024 Å−1, where effects of protein aggregation are negligible. Structural basis of the fast dark recovery of DsLOV The positions of the first 19 N-terminal residues and the C-terminal His6-tag are not resolved in the crystal struc- ture. Disorder and flexibility of the missing regions was thus accounted for by modelling these regions in the crystal structure of DsLOV dimer using the EOM program [46]. The resulting theoretical curve pro- duces the best fit with a reduced χ2-value of 5.8 (Figure 4A). The discrepancy of the theoretical curve calculated from the dimer structure is thus related to the missing terminal regions. has to move back towards M49. Consequently, this back movement might be eased by the flexibility of M49 in the dark state of DsLOV, whereas it would be more restricted in the presence of a branched amino acid such as isoleucine in AsLOV2, VVD and YtvA, thus resulting in a slower dark recovery of the latter proteins compared to DsLOV. Likewise, M49 might act as a “gate-keeper” restricting/enabling the solvent/base access to FMN-N5 hence influencing adduct decay by N5 deprotonation as suggested for VVD [40]. Small angle X-ray scattering reveals dimeric DsLOV in solution (C) Ab initio model of DsLOV (cyan, shown in mesh) determined by SAXS and aligned crystal structure of the dimer (dimer orientation as in Figure 3A, two protein molecules are shown in coral and cyan with bound RBF molecules as stick model). Page 9 of 15 Endres et al. BMC Microbiology (2015) 15:30 Page 9 of 15 program GASBOR combined with the aligned crystal structure of the dimer is shown in Figure 4C. The gen- eral shape of the envelope confirms the dimeric state of the protein and overlaps the dimer structure. In fact, the non-filled parts of the envelope correspond to the loca- tions of the crystallographically non-resolved residues in the N- and C-terminus. namely (i) daylight emulating neon tubes emitting broad spectrum light of wavelengths from 400 nm to 720 nm; (ii) infrared-light (IR) LEDs exhibiting a maximal emis- sion at 855 nm (half-maximal width 837–864 nm) and (iii) blue-light LEDs maximally emitting at 462 nm (half-maximal width 455–477 nm). These specific emission wavelengths were chosen because they are suitable for the excitation of BChl a (in vivo absorption in the blue and infrared range of the light spectrum) and spheroidenone (absorption of blue-light). After cultivation, differences in photopigment accumulation were analyzed (Figure 5A). DsLOV is involved in the upregulation of photopigment synthesis in the absence of blue-light The facultative phototrophic bacterium D. shibae can grow either heterotrophically in the dark or photoheter- otrophically in the light by using a photosystem that contains the photopigments bacteriochlorophyll (BChl) a and the carotenoid spheroidenone [47]. In contrast to anoxygenic photosynthetic α-proteobacteria, D. shibae synthesizes its photopigments in the presence of oxygen [30,48]. However, since photoexcited BChl a efficiently transfers energy to molecular oxygen which in turn leads to the formation of toxic reactive oxygen species (ROS), D. shibae down regulates the synthesis of photopigments upon illumination [30,31,49]. As expected, significant amounts of photopigments accumulated in D. shibae wild-type strain in the dark in- dicated by a deep red coloration of the corresponding cell pellet. In contrast, illumination with broad-spectrum light led to an almost complete loss of coloration (Figure 5A). Interestingly, blue-light illumination of D. shibae wild-type cells resulted in a similar down regu- lation of pigment synthesis, whereas no inhibitory effect was observed upon infrared-light irradiation. In this case, cells showed a red coloration that had so far only been observed for dark grown cells. We first analyzed the effect of blue-light on the accu- mulation of BChl a and spheroidene in D. shibae to determine if the photoreceptor DsLOV is involved in light-dependent control of photopigment formation. For this, we comparatively cultivated D. shibae cells hetero- trophically in the dark as well as photoheterotrophically using three different light sources for illumination, To quantitatively analyze the pigment composition of dark-grown and illuminated cells, photopigments were extracted and the absorption of spheroidenone and BChl a (λmax of isolated photopigments: 482 nm for spheroi- denone and 772 nm for BChl a) were determined as described in the methods section (Figure 5B). Dark Figure 5 Characterization of a DsLOV-deficient D. shibae mutant strain. (A) Influence of different light regimes on pigment composition of D. shibae wild-type (WT) and the DsLOV-deficient mutant strain (Δdslov). Strains were grown in the dark, under white-, infrared-, and blue-light conditions until the cells reached the late logarithmic growth phase. After harvesting 1 ml of cell culture by centrifugation, cell pellets were photographed to document the individual pigmentation. (B) Spectroscopic determination of light-dependent photopigment composition in D. shibae wild-type and mutant Δdslov. Figure 5 Characterization of a DsLOV-deficient D. shibae mutant strain. (A) Influence of different light regimes on pigment composition of D. shibae wild-type (WT) and the DsLOV-deficient mutant strain (Δdslov). DsLOV is involved in the upregulation of photopigment synthesis in the absence of blue-light Strains were grown in the dark, under white-, infrared-, and blue-light conditions until the cells reached the late logarithmic growth phase. After harvesting 1 ml of cell culture by centrifugation, cell pellets were photographed to document the individual pigmentation. (B) Spectroscopic determination of light-dependent photopigment composition in D. shibae wild-type and mutant Δdslov. Figure 5 Characterization of a DsLOV-deficient D. shibae mutant strain. (A) Influence of different light regimes on pigment composition of D. shibae wild-type (WT) and the DsLOV-deficient mutant strain (Δdslov). Strains were grown in the dark, under white-, infrared-, and blue-light conditions until the cells reached the late logarithmic growth phase. After harvesting 1 ml of cell culture by centrifugation, cell pellets were photographed to document the individual pigmentation. (B) Spectroscopic determination of light-dependent photopigment composition in D. shibae wild-type and mutant Δdslov. Endres et al. BMC Microbiology (2015) 15:30 Page 10 of 15 Page 10 of 15 30 m. Because D. shibae is a marine bacterium which lives in symbiosis with dinoflagellates in coast-near aquatic habitats [47], blue light might be the most sensitive envir- onmental factor for the proposed adaptation process. grown and infrared illuminated cells showed comparable absorption values for both photopigments. In contrast, the absorption values of photopigments extracted from white or blue-light irradiated D. shibae cells were negli- gible indicating that neither BChl a nor spheroidenone accumulated under these conditions. These observations indicate that photopigment synthesis in D. shibae is pre- dominantly controlled by a blue-light signal provided here by the blue-light LEDs or by the predominant blue part of the white-light spectrum. In contrast to the light regulated photopigment synthe- sis in D. shibae, expression of photosynthesis genes in the facultative photosynthetic bacterium R. sphaeroides is pre- dominantly regulated by oxygen tension. High oxygen concentration leads to activation of the repressor molecule PpsR which strictly prevents the expression of photosyn- thesis genes [51,52]. However, a second blue-light trig- gered regulatory mechanism comes into play under micro-aerobic conditions where the blue-light photo- receptors AppA and CryB bind to the repressor PpsR in the dark to inactivate its suppressing function [53-56]. Also, the short LOV blue-light receptor RsLOV is in- volved in the light-mediated control of photosynthesis related genes [25,26,56]. An RsLOV mutant strain exhib- ited a stronger pigmentation and a higher BChl a content during the late exponential phase. DsLOV is involved in the upregulation of photopigment synthesis in the absence of blue-light Furthermore, RsLOV effects the gene expression of photooxidative stress re- sponse genes as well as genes for chemotaxis and carbohy- drate metabolism [26]. Very recently, it was published that the regulator AerR acts as an antirepressor of the PpsR homolog CrtJ in the closely related phototrophic bac- terium R. capsulatus, which inhibits the CrtJ-mediated repression of photosynthesis genes in a vitamin B12- dependent and light-responsive manner [57,58]. Despite missing information about the detailed mode of intracellu- lar signal transduction, our functional data suggests that photosynthesis pigment formation is apparently controlled very differently in D. shibae than in other closely related photosynthetic microbes, even though similar blue-light responsive photoreceptors are employed. We next analyzed if the blue-light photoreceptor DsLOV is involved in this regulatory process. We con- structed a D. shibae DsLOV deletion strain (D. shibae Δdslov) and subsequently analyzed pigment accumula- tion under the illumination conditions described above. The results clearly demonstrate that the Δdslov strain did not show any coloration, regardless of the applied il- lumination conditions (Figure 5A, B) demonstrating that induction of pigment formation in the DsLOV-deficient D. shibae strain was almost completely abolished under all tested light regimes. Our data suggest that photopigment synthesis in D. shibae is primarily controlled by blue-light with the short LOV photoreceptor DsLOV acting as a key regula- tor. Based on the presented observations, we propose a unique regulatory function where the dark state of DsLOV seems to be the regulatory relevant state which is directly or indirectly involved in the activation of photopigment synthesis. As shown for other aerobic anoxygenic phototrophic bacteria (AAPB), D. shibae performs anoxygenic photo- synthesis in the presence of light and oxygen to gain additional ATP. However, since light-excitation of BChl a in the presence of molecular oxygen can result in the formation of cytotoxic reactive oxygen species (ROS) including singlet oxygen (1O2) [50], it might be of vital importance for D. shibae to control photopigment synthe- sis in response to changing light regimes. Accordingly, Tomasch and coworkers could demonstrate by transcrip- tome analysis that the transition from dark to light condi- tions resulted in an induction of ROS-mediated stress response accompanied by a down-regulation of photosyn- thesis genes [30]. Remarkably, based on our finding, the photoreceptor DsLOV might act as a ROS-independent dark-sensor which is essential for induction of pigment formation under non-hazardous illumination conditions. DsLOV is involved in the upregulation of photopigment synthesis in the absence of blue-light In accordance with this assumption, light-mediated inhib- ition of photopigment synthesis was only provoked by blue light whereas infrared light that is particularly absorbed by Bchl a is irrelevant for the observed regula- tory process. In this context, it is worth mentioning that infrared light is rapidly attenuated in water and thus does not penetrate a water column further than ca. 5 m, whereas blue light can reach a water depth of more than Cloning of DsLOV conformational changes at a larger scale than observed here as permitted by the crystal lattice. Additionally, we have demonstrated that DsLOV is essential for the in- duction of photopigment formation in the dark whereas blue-light specifically leads to almost complete downreg- ulation of Bchl a and spheroidenone accumulation. First, a 423-bp DNA fragment encompassing the DsLOV gene without (i) the first ATG codon at the 5′-end encod- ing one of two annotated N-terminal methionine residues and (ii) its stop codon, was PCR-amplified by using the primers DsLOV + NdeI-up (5′-GAGTCGCATATGCG CAGACATTATCGCGACCTGAT-3′) and DsLOV + XhoI- Stop-dn (5′-AATAATCTCGAGGACCGGCTTCTGGGC GCCTGCGAAGAA-3′) as well as the genomic DNA of Dinoroseobacter shibae DFL12T as template DNA. The PCR fragment was ligated into the SmaI-site of pBluescript KS (+). Next, the recombinant plasmid pBluescript-DsLOV was hydrolyzed with NdeI and XhoI to isolate the 419-bp DsLOV DNA fragment which was subsequently cloned into the respective sites of the T7 expression vector pRhotHi-2 [60]. The final DsLOV expression vector was designated as pRhotHi-DsLOV. The correct se- quences for all vectors described in the manuscript were verified by DNA sequencing. The results of our functional studies show that photo- pigment synthesis in D. shibae takes place only in the dark when DsLOV is likely to be in a dimeric state. Compared to the crystal structure of the dark state dimer, the DsLOV photoexcited state shows several structural rearrangements including an increase in flexi- bility in the N-cap region involved in dimer formation. It is thus tempting to speculate that these blue-light mediated structural changes (that might eventually be more pronounced in solution) could lead to a quater- nary structural change of the protein. This in turn could result in altered protein-protein interactions in- side the cell, which would enable the reported physio- logical response. Construction of a D. shibae Δdslov strain The D. shibae dslov (dshi_2006) deletion strain was constructed using the suicide plasmid pEX18-Ap [61]. To generate the gene deletion vector, the PCR-primer pair 5′-DsLOV + Ext (GAGCTCTCGGTCAGGTCCG GATATG-3′) and 3′-DsLOV + Ext (5′-AAGCTTGGAA GAGCACCGTGAACTC-3′) was used to amplify a 1853- bp PCR product which contains the dslov (dshi_2006) encoding sequence together with up- and downstream non-coding sequences to enable homologous recombin- ation. The PCR product which contained recognition sites for restriction nucleases SacI at the 5′-end and HindIII at the 3′-end was cloned into the corresponding sites of plasmid pEX18Ap. Conclusions h h h The photochemical and structural characterization of the ‘short’ DsLOV protein highlights some unique properties including a very fast recovery, a flexible N- terminal cap region as well as a dimeric structure in solution stabilized mainly via an unusual set of interac- tions at the molecule interface. Based on the differences between the crystal structures of the dark state and the photoexcited state, we identified several key residues in the chromophore binding pocket which play an import- ant role in signaling and are located in the hinge region connecting the N-cap to the LOV core domain. These results lead us to postulate that in DsLOV subtle light- induced structural changes in the chromophore binding pocket are subsequently propagated, via several residues located in the Iβ and Hβ strands of the central β- scaffold, towards the peripheral regions and manifested as partially disordered loop in the N-cap region. How- ever, transfer of the light signal in DsLOV might require Page 11 of 15 Endres et al. BMC Microbiology (2015) 15:30 Cloning of DsLOV To interrupt the DsLOV encoding se- quence, the gentamicin interposon cassette was amplified from plasmid pWKR202 [62] in a second PCR. At the same time, XagI-recognition sequences (CCTNNNNNAGG) were introduced at both ends of the PCR product using primers 5′GmR XagI: CCTCTTCGAGGTATCCATCAG GCAACGACG and 3′GmR XagI: CCTTGCACAGG TACGGCCACAGTAACCAAC. The modified cassette was ligated into the dslov XagI site of the recombinant pEX18- Ap plasmid. The successful insertion of the resistance cas- sette and its correct orientation (i.e. the same orientation as the DsLOV gene to avoid putative polar effects due to the interposon insertion) was further analyzed by restriction analysis and corroborated by DNA sequencing. The result- ing pEX18ApΔdslov::Gmr plasmid was used for inactivation of the chromosomal dslov gene of D. shibae DFL12T. E. coli ST18 [63] cells were transformed with the suicide plasmid and used as donor strain for conjugative gene transfer to D. shibae DFL12T wild-type cells [64]. Homologous re- combination by a double-crossover event was confirmed by antibiotic resistance screenings and analytic PCR. Its unique photophysical properties and regulatory functions, as well as the distinct and quantifiable pheno- type of a mutant, make DsLOV a new and promising model system to study the intramolecular and regulatory processes of LOV photoreceptors. Bacterial strains and growth conditions Bacterial strains were cultivated as batch cultures in ster- ilized Erlenmeyer flasks with a culture to vessel volume ratio of at least 1:5. E. coli strains were grown in LB- medium (Carl Roth, Karlsruhe, Germany) at 37°C under continuous shaking at 130 rpm. D. shibae strains were cultivated under equal shaking conditions in marine bouillon medium (Carl Roth, Karlsruhe, Germany) at 30°C. Depending on the plasmid mediated resistance, culture media were supplemented with kanamycin (50 μg/ml), ampicillin (100 μg/ml) or gentamycin (100 μg/ml). Aminolevulinic acid (ALA, 50 μg/ml) was added to E. coli ST18 cultures. Light-conditioned experi- ments were performed in darkness, infrared, blue and white-light. For the blue and infrared illumination, a set of previously described light emitting diodes (LED)-arrays were used [59]. Each of the two LED panels contain 120 blue LEDs (NSSB100BT, λmax = 462 nm, Nichia, Japan) and 120 high power infrared LEDs (SFH 4257, λmax = 855 nm, Osram, Munich, Germany). The average photon flux at a distance of approximately 12 cm from the LED panel was as follows: blue-light (462 nm): 55 μmol m−2 s−1, Infra-red light (855 nm): 43 μmol m−2 s−1. White-light illumination was carried out with daylight emulating neon tubes ‘865 Lumilux Cool Daylight’ (Osram, Munich, Germany). Cell cultures were placed between the light sources at a constant distance of 12.5 cm on each side. Page 12 of 15 Endres et al. BMC Microbiology (2015) 15:30 SAXS measurements SAXS d SAXS data was measured at beamline BM29 at the ESRF using a X-ray wavelength of 1 Å at 10°C. Two samples of DsLOV with concentrations of 7.2 and 14.8 mg/ml were measured in phosphate buffer (10 mM NaH2PO4, 300 mM NaCl, pH 8.0). The buffer was measured before and after each protein sample. The samples were purged through a quartz capillary during X-ray exposure. For each sample ten frames with an exposure time of 2 sec each were recorded, and the frames without radiation damage were merged. Data analysis was done using the ATSAS software package [66]. Measured data were scaled by the concen- tration, where the lower concentration was used for the smaller q-range, while the data at higher concentration was taken for the high q-range. The excluded volume was calculated with the program DATPOROD and the molecular mass was estimated by applying a division factor of 1.7 [66]. Theoretical scattering curves of the crystallographic structures of the monomer and the dimer were calculated and fitted to the experimental SAXS data using the program CRYSOL. The program EOM was used to generate 10000 native-like conforma- tions of N-terminal residues 1–19 and the C-terminal His6-tag (LEHHHHHH), while the crystal structure of the dimer was held fixed. Consecutively, representative ensembles were selected, which best describe the mea- sured data. The distance distribution function P(r) was determined using the program DATGNOM. Ab initio models were generated using the programs GASBOR and DAMMIF applying two-fold symmetry. In both cases 20 ab initio models were generated, averaged and the filtered model was used. The molecular mass was Protein expression and purification p p DsLOV was isolated from E. coli BL21(DE3) harboring pRhotHi-DsLOV E. coli cultivated in auto induction medium (TB, 0.2% lactose, 0.05% glucose, 50 μg/ml kanamycin) [4] and purified as described previously [24]. Briefly, the cell pellet (about 5 g cells, wet weight) was dissolved in 30 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Cells were lysed by passing the cell suspension four times through a french pressure cell (Thermo Scientific, Waltham, MA). The soluble fraction and pellet were separated by centrifugation (9200 × g, 30 min). The His6-tagged DsLOV proteins were subsequently puri- fied by metal affinity chromatography using a super- flow Ni-NTA resin (QIAGEN, Hilden, Germany). Elution was performed with a gradient between wash- ing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). All purification steps were carried out at 4°C. The pur- ity of the eluted fractions was anaylzed by SDS-PAGE. Pure fractions were pooled, and the elution buffer was exchanged to 10 mM Tris–HCl, pH 7.0 supplemented with 10 mM NaCl. Desalting was achieved using Vivaspins with a cut-off of 10 kDa. The maximal con- centration of imidazole in all samples is estimated to be <10 μM. Hence, the presence of imidazol in the re- spective DsLOV preparations is expected to contribute only minimally to the observed adduct decay rates. Spectroscopic techniques All spectroscopic analyses were carried out under red light at 20°C, using either a Beckmann UV/Vis spectro- photometer DU-650 for absorbance and recovery kinetic measurements or by employing a Perkin-Elmer Luminescence Spectrometer LS 50B for fluorescence measurements as described previously [24]. For all measurements, the samples were diluted to an OD450 of 0.1 with 10 mM phosphate buffer, pH 8.0 supple- mented with 300 mM NaCl. In order to generate the LOV proteins light state, the samples were illuminated for 30 s using a high-power blue-light (450 nm)-emitting LED (Luxeon Lumileds, Phillips, Aachen Germany). Dark state recovery was measured from illuminated samples by recording the absorption recovery at 485 nm. Reported adduct state lifetimes are derived from three independent measurements and were obtained by fitting experimental data to a mono-exponential decay function using soft- ware Origin (OriginLab corporation, Northhampton, MA, USA). Photopigments were detected by their specific absorb- ance at 772 nm ±2 nm for bacteriochlorophyll a and at 482 nm ±2 nm for spheroidenone. D. shibae cultures were cultivated as described above for 44 hours. After- wards, cells corresponding to an optical density of 1 at 660 nm were harvested by centrifugation (5000 × g, 10 min, 20°C). The photopigments were extracted from cell pellets by addition of 1 ml ethanol and sub- sequent incubation for 10 minutes at 50°C in the dark. Afterwards, the cell-debris was separated by centrifu- gation (9000 × g, 10 min, 20°C) and the supernatant was transferred into silica glass cuvettes. For spectral analysis, the absorbance was detected using the stand- ard UV/Vis spectrometer Genesys6 (ThermoFischer Scientific, Madison, WI USA) with a resolution of 1 nm. Acknowledgements We are grateful to the beamline scientists at the European Synchrotron Radiation Facility (Grenoble, France) for providing assistance with the use of beamlines ID14-1, ID23-1 and BM-29. This work was partially supported by grants from the Federal Ministry of Education and Research (Project OptoSys, FKZ 031A16). References 1. Salomon M, Christie JM, Knieb E, Lempert U, Briggs WR. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry-Us. 2000;39(31):9401–10. 1. Salomon M, Christie JM, Knieb E, Lempert U, Briggs WR. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry-Us. 2000;39(31):9401–10. 2. Christie JM, Salomon M, Nozue K, Wada M, Briggs WR. LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc Natl Acad Sci U S A. 1999;96(15):8779–83. Unless otherwise indicated, figures were generated with MOLSCRIPT [73] and RASTER3D [74] using secondary structure assignments as given by the DSSP program [75]. 3. Swartz TE, Corchnoy SB, Christie JM, Lewis JW, Szundi I, Briggs WR, et al. The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J Biol Chem. 2001;276(39):36493–500. 4. Jentzsch K, Wirtz A, Circolone F, Drepper T, Losi A, Gärtner W, et al. Mutual exchange of kinetic properties by extended mutagenesis in two short LOV domain proteins from Pseudomonas putida. Biochemistry-Us. 2009;48(43):10321–33. Competing interests p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors’ contributions JG, AS, UK, TD, PT, DW, KEJ and RBS conceived and designed the experiments. SE, FC, JG, AS, UK, VS, EKG, AW and AC performed the experiments. JG, SE, FC, AS, UK, TD, PT, DW, KEJ and RBS analyzed the data. SE, JG, FC, AS, UK, TD, KEJ and RBS wrote the paper. All authors read and approved the final manuscript. Additional file The purified protein was concentrated to 25 mg/ml and crystallization set-ups were performed using the vapor- diffusion method. Crystals were grown in the dark using 1.8 μl sitting drops (0.9 μl purified protein + 0.9 μl reser- voir solution) against 0.3 M MgCl2, 8% PGA-LM and 0.1 M sodium acetate, pH 5.0 at 19°C. Typically, crystals appeared after three weeks. No crystals could be grown in continuous blue-light conditions. Additional file 1: Table S1. Lifetimes for the dark recovery reaction in LOV proteins. Figure S1. Identification of three putative LOV genes in the genome of D. shibae DFL12T. Figure S2. Superposition of LOV domains. Figure S3. Differences between dark and photoexcited crystal structures of DsLOV. Figure S4. Single crystal microspectrometry of dark-grown DsLOV crystals. Author details 1 1Institute of Molecular Enzyme Technology, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany. 2Institute of Complex Systems, ICS-6: Structural Biochemistry, Forschungszentrum Jülich, D-52425 Jülich, Germany. 3Juelich Centre for Neutron Science JCNS (JCNS-1) & Institute for Complex Systems (ICS-1), Forschungszentrum Jülich, D-52425 Jülich, Germany. 4Institute for Microbiology, Technische Universität Braunschweig, D-38106 Braunschweig, Germany. 5Institute of Physical Biology, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany. 6Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany. Received: 4 July 2014 Accepted: 29 January 2015 Received: 4 July 2014 Accepted: 29 January 2015 Chromatographic techniques Detection and quantification of the putative chromo- phores, i.e. FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and riboflavin, respectively, were conducted as described previously [65]. Page 13 of 15 Endres et al. BMC Microbiology (2015) 15:30 Data Bank (http://www.pdb.org/pdb/) under accession codes 4KUK and 4KUO. Data Bank (http://www.pdb.org/pdb/) under accession codes 4KUK and 4KUO. estimated from the excluded volume of the filtered DAMMIF ab initio model using a division factor of 2 [66]. The crystal structure of the dimer was aligned in the filtered model. The envelope function was deter- mined using the SITUS package. Ethics statement This research did not involve human subjects, human material, or human data. Data collection and structure determination Prior to cryo cooling, the crystals were soaked stepwise in reservoir solution containing up to 20% (v/v) glycerol. Both X-ray diffraction data were collected at 100 K, one under constant dark and one under constant light condi- tions. In the light condition, the crystals were exposed to blue-light prior to cryo cooling and in the subsequent steps. Please note that the crystals used for the datasets reported here were from the same drop and were stored in the dark at 100 K (in cryo tanks) until data collection. The X-ray diffraction data were recorded at beamline ID14-1 and ID23-1 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The data collection strategies taking radiation damage into account were based on calculations using the program BEST [67]. Data processing was carried out using MOSFLM and SCALA, which is part of the CCP4 software package [68]. The dark state structure was determined by molecular replacement using PHASER [part of the PHENIX software suite [69]]. The search model was built by homology mod- eling via the ExPASy web server [70,71], based on the template of the PDB ID 2V0U (phototropin 1 from Avena sativa). Phase determination was followed by several cycles of automated model building and refinement using the PHENIX package. The model was further improved by manual rebuilding using the program COOT [72]. The refinement and rebuilding of the photoexcited structure was carried out with the finished dark state structure. For statistics on data collection and refinement refer to Table 1. Abbreviations ESRF E ESRF: European Synchrotron Radiation Facility; FMN: Flavin mononucleotide; LOV: Light–oxygen–voltage; PAS: Per-ARNT-Sim; PCR: Polymerase chain reaction; PDB: Protein Data Bank; SAXS: Small angle X-ray scattering. References 1. Salomon M, Christie JM, Knieb E, Lempert U, Briggs WR. Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry-Us. 2000;39(31):9401–10. 2. Christie JM, Salomon M, Nozue K, Wada M, Briggs WR. LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc Natl Acad Sci U S A. 1999;96(15):8779–83. 3. Swartz TE, Corchnoy SB, Christie JM, Lewis JW, Szundi I, Briggs WR, et al. The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J Biol Chem. 2001;276(39):36493–500. 4. Jentzsch K, Wirtz A, Circolone F, Drepper T, Losi A, Gärtner W, et al. Mutual exchange of kinetic properties by extended mutagenesis in two short LOV domain proteins from Pseudomonas putida. Biochemistry-Us. 2009;48(43):10321–33. PDB accession codes Coordinates and structure factors for DsLOV (dark) and DsLOV (illuminated) have been deposited in the Protein Endres et al. BMC Microbiology (2015) 15:30 Page 14 of 15 5. Krauss U, Losi A, Gärtner W, Jaeger K-E, Eggert T. Initial characterization of a blue-light sensing, phototropin-related protein from Pseudomonas putida: a paradigm for an extended LOV construct. Phys Chem Chem Phys. 2005;7(14):2804–11. 28. Vaidya AT, Chen CH, Dunlap JC, Loros JJ, Crane BR. Structure of a light-activated LOV protein dimer that regulates transcription. Sci Signal. 2011;4(184):ra50. 29. Zoltowski BD, Crane BR. Light activation of the LOV protein vivid generates a rapidly exchanging dimer. Biochemistry-Us. 2008;47(27):7012–9. 6. Losi A, Quest B, Gärtner W. Listening to the blue: the time-resolved thermodynamics of the bacterial blue-light receptor YtvA and its isolated LOV domain. Photochem Photobiol Sci. 2003;2(7):759–66. 30. Tomasch J, Gohl R, Bunk B, Diez MS, Wagner-Döbler I. Transcriptional response of the photoheterotrophic marine bacterium Dinoroseobacter shibae to changing light regimes. ISME J. 2011;5(12):1957–68. 7. Zoltowski BD, Schwerdtfeger C, Widom J, Loros JJ, Bilwes AM, Dunlap JC, et al. Conformational switching in the fungal light sensor Vivid. Science. 2007;316(5827):1054–7. shibae to changing light regimes. ISME J. 2011;5(12):1957–68. 31. Wagner-Döbler I, Biebl H. Environmental biology of the marine Roseobacter lineage. Annu Rev Microbiol. 2006;60:255–80. 32. Chen VB, Arendall 3rd WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 1):12–21. 8. Christie JM, Swartz TE, Bogomolni RA, Briggs WR. Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function. Plant J. 2002;32(2):205–19. 33. Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372(3):774–97. 9. Avila-Perez M, Vreede J, Tang Y, Bende O, Losi A, Gartner W, et al. In vivo mutational analysis of YtvA from Bacillus subtilis: mechanism of light activation of the general stress response. J Biol Chem. 2009;284(37):24958–64. 34. Halavaty AS, Moffat K. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry-Us. 2007;46(49):14001–9. 10. Losi A, Gärtner W. Bacterial bilin- and flavin-binding photoreceptors. Photochem Photobiol Sci. 2008;7(10):1168–78. 35. Möglich A, Moffat K. Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA. J Mol Biol. 2007;373(1):112–26. 11. Losi A, Gartner W. The evolution of flavin-binding photoreceptors: an ancient chromophore serving trendy blue-light sensors. PDB accession codes Annu Rev Plant Biol. 2012;63:49–72. 36. Florian J, Warshel A. Phosphate ester hydrolysis in aqueous solution: Associative versus dissociative mechanisms. J Phys Chem B. 1998;102(4):719–34. 12. Demarsy E, Fankhauser C. Higher plants use LOV to perceive blue light. Curr Opin Plant Biol. 2009;12(1):69–74. 37. Fedorov R, Schlichting I, Hartmann E, Domratcheva T, Fuhrmann M, Hegemann P. Crystal structures and molecular mechanism of a light-induced signaling switch: The Phot-LOV1 domain from Chlamydomonas reinhardtii. Biophys J. 2003;84(4):2474–82. 13. Herrou J, Crosson S. Function, structure and mechanism of bacterial photosensory LOV proteins. Nat Rev Microbiol. 2011;9(10):713–23. 14. Losi A, Mandalari C, Gärtner W. From Plant Infectivity to Growth Patterns: The Role of Blue-Light Sensing in the Prokaryotic World. Plants. 2014;3(1):70–94. 38. Möglich A, Moffat K. Engineered photoreceptors as novel optogenetic tools. Photochem Photobiol Sci. 2010;9(10):1286–300. 15. Losi A, Polverini E, Quest B, Gartner W. First evidence for phototropin- related blue-light receptors in prokaryotes. Biophys J. 2002;82(5):2627–34. 39. Crosson S, Moffat K. Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch. Plant Cell. 2002;14(5):1067–75. 16. Avila-Perez M, Hellingwerf KJ, Kort R. Blue light activates the sigmaB- dependent stress response of Bacillus subtilis via YtvA. J Bacteriol. 2006;188(17):6411–4. 40. Zoltowski BD, Vaccaro B, Crane BR. Mechanism-based tuning of a LOV domain photoreceptor. Nat Chem Biol. 2009;5(11):827–34. 17. Jurk M, Schramm P, Schmieder P. The blue-light receptor YtvA from Bacillus subtilis is permanently incorporated into the stressosome independent of the illumination state. Biochem Biophys Res Commun. 2013;432(3):499–503. 41. Kawano F, Aono Y, Suzuki H, Sato M. Fluorescence Imaging-Based High-Throughput Screening of Fast- and Slow-Cycling LOV Proteins. Plos One. 2013;8(12):e82693. 42. Raffelberg S, Mansurova M, Gärtner W, Losi A. Modulation of the photocycle of a LOV domain photoreceptor by the hydrogen-bonding network. J Am Chem Soc. 2011;133(14):5346–56. 18. van der Steen JB, Nakasone Y, Hendriks J, Hellingwerf KJ. Modeling the functioning of YtvA in the general stress response in Bacillus subtilis. Mol Biosyst. 2013;9(9):2331–43. 43. Raffelberg S, Gutt A, Gartner W, Mandalari C, Abbruzzetti S, Viappiani C, et al. The amino acids surrounding the flavin 7a-methyl group determine the UVA spectral features of a LOV protein. Biol Chem. 2013;394(11):1517–28. 19. Purcell EB, Siegal-Gaskins D, Rawling DC, Fiebig A, Crosson S. A photosensory two-component system regulates bacterial cell attachment. Proc Natl Acad Sci U S A. 2007;104(46):18241–6. 44. Alexandre MTA, Arents JC, van Grondelle R, Hellingwerf KJ, Kennis JTM. PDB accession codes A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain. Biochemistry-Us. 2007;46(11):3129–37. 20. Swartz TE, Tseng TS, Frederickson MA, Paris G, Comerci DJ, Rajashekara G, et al. Blue-light-activated histidine kinases: two-component sensors in bacteria. Science. 2007;317(5841):1090–3. 45. Christie JM, Corchnoy SB, Swartz TE, Hokenson M, Han IS, Briggs WR, et al. Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1. Biochemistry. 2007;46(32):9310–9. 21. Rio-Alvarez I, Rodriguez-Herva JJ, Martinez PM, Gonzalez-Melendi P, Garcia-Casado G, Rodriguez-Palenzuela P, et al. Light regulates motility, attachment and virulence in the plant pathogen Pseudomonas syringae pv tomato DC3000. Environ Microbiol. 2014;16(7):2072–85. 46. Bernado P, Mylonas E, Petoukhov MV, Blackledge M, Svergun DI. Structural characterization of flexible proteins using small-angle X-ray scattering. J Am Chem Soc. 2007;129(17):5656–64. 46. Bernado P, Mylonas E, Petoukhov MV, Blackledge M, Svergun DI. Structural characterization of flexible proteins using small-angle X-ray scattering. J Am Chem Soc. 2007;129(17):5656–64. 22. Wu L, McGrane RS, Beattie GA. Light regulation of swarming motility in Pseudomonas syringae integrates signaling pathways mediated by a bacteriophytochrome and a LOV protein. MBio. 2013;4(3):e00334–00313. 47. Biebl H, Allgaier M, Tindall BJ, Koblizek M, Lunsdorf H, Pukall R, et al. Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated from dinoflagellates. Int J Syst Evol Microbiol. 2005;55(Pt 3):1089–96. 23. Kraiselburd I, Daurelio LD, Tondo ML, Merelo P, Cortadi AA, Talon M, et al. The LOV Protein of Xanthomonas citri subsp citri Plays a Significant Role in the Counteraction of Plant Immune Responses during Citrus Canker. Plos One. 2013;8(11):e80930. 48. Yurkov V, Beatty JT. Isolation of aerobic anoxygenic photosynthetic bacteria from black smoker plume waters of the juan de fuca ridge in the pacific ocean. Appl Environ Microbiol. 1998;64(1):337–41. 49. Yurkov V, Gemerden H. Impact of light/dark regimen on growth rate, biomass formation and bacteriochlorophyll synthesis in Erythromicrobium hydrolyticum. Arch Microbiol. 1993;159(1):84–9. 24. Circolone F, Granzin J, Jentzsch K, Drepper T, Jaeger KE, Willbold D, et al. Structural basis for the slow dark recovery of a full-length LOV protein from Pseudomonas putida. J Mol Biol. 2012;417(4):362–74. 25. Conrad KS, Bilwes AM, Crane BR. Light-induced subunit dissociation by a light-oxygen-voltage domain photoreceptor from Rhodobacter sphaeroides. Biochemistry-Us. 2013;52(2):378–91. 50. Borland CF, Cogdell RJ, Land EJ, Truscott TG. Bacteriochlorophyll Alpha-Triplet State and Its Interactions with Bacterial Carotenoids and Oxygen. J Photoch Photobio B. 1989;3(2):237–45. 51. Gomelsky M, Kaplan S. PDB accession codes Molecular genetic analysis suggesting interactions between AppA and PpsR in regulation of photosynthesis gene expression in Rhodobacter sphaeroides 2.4.1. J Bacteriol. 1997;179(1):128–34. 26. Metz S, Jager A, Klug G. Role of a short light, oxygen, voltage (LOV) domain protein in blue light- and singlet oxygen-dependent gene regulation in Rhodobacter sphaeroides. Microbiology. 2012;158(Pt 2):368–79. 27. Nash AI, McNulty R, Shillito ME, Swartz TE, Bogomolni RA, Luecke H, et al. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/ helix-turn-helix (LOV-HTH) DNA-binding protein. Proc Natl Acad Sci U S A. 2011;108(23):9449–54. 52. Gomelsky M, Horne IM, Lee HJ, Pemberton JM, McEwan AG, Kaplan S. Domain structure, oligomeric state, and mutational analysis of PpsR, the Rhodobacter sphaeroides repressor of photosystem gene expression. J Bacteriol. 2000;182(8):2253–61. Page 15 of 15 Page 15 of 15 Endres et al. BMC Microbiology (2015) 15:30 53. Braatsch S, Gomelsky M, Kuphal S, Klug G. A single flavoprotein, AppA, integrates both redox and light signals in Rhodobacter sphaeroides. Mol Microbiol. 2002;45(3):827–36. 54. Han Y, Meyer MHF, Keusgen M, Klug G. A haem cofactor is required for redox and light signalling by the AppA protein of Rhodobacter sphaeroides Mol Microbiol. 2007;64(4):1090–104. 55. Metz S, Haberzettl K, Fruhwirth S, Teich K, Hasewinkel C, Klug G. Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes. Nucleic Acids Res. 2012;40(13):5901–9. 56. Hendrischk AK, Fruhwirth SW, Moldt J, Pokorny R, Metz S, Kaiser G, et al. A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides. Mol Microbiol. 2009;74(4):990–1003. 57. Cheng Z, Li K, Hammad LA, Karty JA, Bauer CE. Vitamin B12 regulates photosystem gene expression via the CrtJ antirepressor AerR in Rhodobacter capsulatus. Mol Microbiol. 2014;91(4):649–64. 58. Klug G. Beyond catalysis: vitamin B12 as a cofactor in gene regulation. Mol Microbiol. 2014;91(4):635–40. 58. Klug G. Beyond catalysis: vitamin B12 as a cofactor in gene regulation. Mol Microbiol. 2014;91(4):635–40. 59. Kaschner M, Loeschcke A, Krause J, Minh BQ, Heck A, Endres S, et al. Discovery of the first light-dependent protochlorophyllide oxidoreductase in anoxygenic phototrophic bacteria. Mol Microbiol. 2014;93(5):1066–78. 60. Katzke N, Arvani S, Bergmann R, Circolone F, Markert A, Svensson V, et al. A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus. Protein Expr Purif. 2010;69(2):137–46. 61. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. PDB accession codes A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene. 1998;212(1):77–86. 62. Drepper T, Gross S, Yakunin AF, Hallenbeck PC, Masepohl B, Klipp W. Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus. Microbiology. 2003;149(Pt 8):2203–12. 63. Thoma S, Schobert M. An improved Escherichia coli donor strain for diparental mating. FEMS Microbiol Lett. 2009;294(2):127–32. 64. Piekarski T, Buchholz I, Drepper T, Schobert M, Wagner-Doebler I, Tielen P, et al. Genetic tools for the investigation of Roseobacter clade bacteria. BMC Microbiol. 2009;9:265. 65. Cao Z, Buttani V, Losi A, Gärtner W. A blue light inducible two-component signal transduction system in the plant pathogen Pseudomonas syringae pv. tomato. Biophys J. 2008;94(3):897–905. 66. Petoukhov MV, Franke D, Shkumatov AV, Tria G, Kikhney AG, Gajda M, et al. New developments in the ATSAS program package for small-angle scattering data analysis. J Appl Crystallogr. 2012;45:342–50. 67. Bourenkov GP, Popov AN. Optimization of data collection taking radiation damage into account. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):409–19. 68. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):235–42. 69. Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr. 2002;58(Pt 11):1948–54. 70. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 2006;22(2):195–201. 71. Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T. The SWISS-MODEL Repository and associated resources. Nucleic Acids Res. 2009;37(Database issue):D387–92. PDB accession codes Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 72. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 12 Pt 1):2126–32. 73. Kraulis PJ. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J Appl Crystallogr. 1991;24:946–50. • Convenient online submission plots of protein structures. J Appl Crystallogr. 1991;24:946–50. • Thorough peer review 74. Merritt EA, Bacon DJ. Raster3D: photorealistic molecular graphics. Methods Enzymol. 1997;277:505–24. 75. Kabsch W, Sander C. Dictionary of Protein Secondary Structure - Pattern- Recognition of Hydrogen-Bonded and Geometrical Features. Biopolymers. 1983;22(12):2577–637.
https://openalex.org/W2138250996	https://open.library.ubc.ca/media/download/pdf/52383/1.0132619/2	English	null	Incidental littoral cell angioma of the spleen	World journal of surgical oncology	2,008	cc-by	4,092	Published: 19 August 2008 Published: 19 August 2008 World Journal of Surgical Oncology 2008, 6:87 doi:10.1186/1477-7819-6-87 This article is available from: http://www.wjso.com/content/6/1/87 © 2008 Tee et al; licensee BioMed Central Ltd. © 2008 Tee et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Incidental littoral cell angioma of the spleen May Tee1, Patrick Vos2, Peter Zetler3 and Sam M Wiseman4 Address: 1Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada, 2Department of Radiology, St. Paul's Hospital & University of British Columbia, Vancouver, British Columbia, Canada, 3Department of Pathology & Laboratory Medicine, St. Paul's Hospital & University of British Columbia, Vancouver, British Columbia, Canada and 4Department of Surgery, St. Paul's Hospital & University of British Columbia, Vancouver, British Columbia, Canada Email: May Tee - mctee@interchange.ubc.ca; Patrick Vos - pvos@providencehealth.bc.ca; Peter Zetler - PZetler@providencehealth.bc.ca; Sam M Wiseman - smwiseman@providencehealth.bc.ca mail: May Tee - mctee@interchange.ubc.ca; Patrick Vos - pvos@providencehealth.bc.ca; Peter Zetler - PZetler@prov am M Wiseman* - smwiseman@providencehealth.bc.ca * Corresponding author Abstract Background: Littoral cell angioma (LCA) is a recently described primary vascular neoplasm of the spleen that may be associated with other malignancies and may itself also have malignant potential. Case presentation: We present a case of LCA that was discovered incidentally in a 52-year-old woman who presented with biliary colic at the time of consultation for cholecystectomy. This vascular neoplasm was evaluated by ultrasound, CT, MRI, Tc-99m labelled red blood cell scintigraphy, and core biopsy. A splenectomy revealed LCA by pathological evaluation. Post- operative outcome was favourable with no evidence of complication or recurrent disease. Following this case presentation, clinical, radiographic, and pathological features of LCA will be reviewed as well as recent advances in our understanding of this uncommon splenic lesion. Conclusion: LCA is a rare, generally benign, primary vascular tumour of the spleen that typically is discovered incidentally. Individuals diagnosed with this tumour must be carefully evaluated to exclude primary, secondary, and synchronous malignancies. presentation of LCA ranges from being completely asymp- tomatic and discovered incidentally, to presenting with a constellation of signs and symptoms such as abdominal pain, vague constitutional symptoms, splenomegaly, and hypersplenism [2-6]. Although first described as benign, LCA has recently been shown to exhibit malignant poten- tial [11,12] and may also be associated with other visceral malignancies [13]. In this report, we present a case of LCA including its diagnostic work-up, surgical treatment, path- ological evaluation, and post-operative outcome. A dis- cussion regarding the clinical, radiological, and pathological features of LCA, as well recent advances in our understanding of this uncommon splenic tumour are also presented. World Journal of Surgical Oncology Open Access Case presentation nondiagnostic as histological evaluation showed skeletal muscle. Overall, it was felt that the splenic lesion had a benign appearance and would be followed up with imag- ing. p A 52-year-old woman was evaluated for symptoms of bil- iary colic for possible cholecystectomy. She described intermittent episodes of right upper quadrant pain with no history of jaundice, nausea, vomiting, or changes in bowel habits. Laboratory tests revealed elevated liver enzymes of a cholestatic nature but total bilirubin within normal limits (ALP = 139 U/L, GGT = 67 U/L, total bilirubin = 6 μmol/L). Haemoglobin, white blood cells, and platelet counts were within normal limits. Ultra- sound (US) revealed cholelithiasis, a normal appearing biliary tree, and fatty infiltration of the liver. Notably, US also identified a hyperechoic, well-circumscribed, 3 cm lesion located at the inferior aspect of the spleen (figure 1). No significant vascularity was noted on the color Dop- pler images. A laparoscopic cholecystectomy was performed. No splenic lesion was grossly evident at the time of laparos- copy. The postoperative course was uneventful and epi- sodes of abdominal pain resolved post-operatively. A colonoscopy was also performed, which was normal. A repeat CT scan 6 months postoperatively showed interval enlargement of the splenic lesion as well as the develop- ment of an adjacent satellite lesion with a similar radio- logical appearance. As a result of the enlargement of the lesion, and development of a new lesion, a decision was made to carry out a splenectomy. The splenic lesion was further evaluated with CT, MRI. A hypodense well defined lesion with some internal enhancement in the arterial and venous phases was dem- onstrated on the contrast enhanced CT scan (Fig. 2A, 2B). The lesion was isodense compared to the surrounding splenic parenchyma on the 5 minutes delayed images (Fig. 2C). On MR, the lesion was hypointense on the T1, and hyperintense on the T2-weighted sequences (Fig. 3A). After the administration of IV gadolinium the lesion dem- onstrated some internal linear enhancement in the portal venous phase on the T1-weighted fat suppressed sequence (Fig. 3B) and became isointense on the delayed images. A Tc-99m labelled red blood cell scan showed the splenic lesion to be 'cold'. Background Littoral Cell Angioma (LCA) of the spleen was recently described by Falk et al. in 1991 [1]. This group reviewed 200 surgical specimens of benign vascular splenic tumours and found 17 similar tumours that appeared related to the cells lining the red pulp splenic sinuses [1]. These tumours were unique in that they displayed both epithelial and histiocytic properties based on their cell of origin, the splenic littoral cells [1]. From these observa- tions, this group designated this new vascular tumour of the spleen LCA [1]. Since this initial description, there have been scattered case reports and few case series of LCA [2-10]. The clinical Page 1 of 5 (page number not for citation purposes) Page 1 of 5 (page number not for citation purposes) World Journal of Surgical Oncology 2008, 6:87 http://www.wjso.com/content/6/1/87 Case presentation A percutaneous ultrasound guided core biopsy of the lesion was subsequently carried out but was At the time of open splenectomy, the spleen was unre- markable in terms of size and appearance, and was removed in its entirety for pathological evaluation. This patient had an uneventful post-operative recovery and has remained well at fifteen months of post-operative follow- up. Pathologic evaluation identified a non-encapsulated but well-circumscribed reddish nodule that was 4 × 3 × 3 cm in size and located at the anterior-inferior pole of the spleen. Histologically, this lesion was described as a vas- cular neoplasm forming anastomosing vascular channels lined by histiocytes with occasional papillary structure, consistent with Littoral Cell Angioma (Fig. 4). Page 2 of 5 (page number not for citation purposes) Discussion i Primary vascular tumours of the spleen are uncommon but represent the majority of non-hematolymphoid splenic tumours [14]. The differential diagnosis of splenic vascular tumours is broad and may represent benign (hae- mangioma, haemartoma, lymphangioma), indeterminate (littoral cell angioma, haemangioendothelioma, haeman- giopericytoma), or malignant neoplasms (angiosarcoma) [14]. LCA is a recently described vascular tumour of the spleen that is now classified as having uncertain biological behaviour, given several case reports which have identi- fied malignant potential [14]. Ultrasound of the spleen demonstrates a well-defined hyper- echoic lesion Figure 1 Ultrasound of the spleen demonstrates a well-defined hyper- echoic lesion. The exact incidence of LCA is unknown although the inci- dence of splenic haemangioma varies from 0.03% to as high as 14% in one reported autopsy series [15]. LCA does not have any particular gender or age predilection although the median age in Falk et al.,'s original study of LCA was 49 years [1,14]. As was the case for our patient, LCA may be completely asymptomatic and represent an incidental finding by imaging [13,16]. LCA may also present with a myriad of possible signs and symptoms, such as: splenomegaly with or without abdominal pain, hypersplenism with ensuing anaemia and/or thrombocy- topenia, and constitutional symptoms such as intermit- Ultrasound of the spleen demonstrates a well-defined hyper- echoic lesion Figure 1 Ultrasound of the spleen demonstrates a well-defined hyper- echoic lesion. Page 2 of 5 (page number not for citation purposes) Page 2 of 5 (page number not for citation purposes) World Journal of Surgical Oncology 2008, 6:87 http://www.wjso.com/content/6/1/87 CT scan after oral and iv-contrast in the arterial phase A and portal venous phase B demonstrates a hypodense well defined round lesion in the posterior portion of the spleen with some linear internal enhancement Figure 2 CT scan after oral and iv-contrast in the arterial phase A and portal venous phase B demonstrates a hypodense well defined round lesion in the posterior portion of the spleen with some linear internal enhancement. C. The lesion is isodense compared to the normal spleen on the 5 minutes delayed image. p p p yp p p p g CT scan after oral and iv-contrast in the arterial phase A and portal venous phase B demonstrates a hypodense well defined round lesion in the posterior portion of the spleen with some linear internal enhancement. C. Page 3 of 5 (page number not for citation purposes) Discussion i Anastamosing vascular channels lined by plump cells with the appearance of sinus lining (arrow: 'littoral' cells) (H&E 100×) Figure 4 Anastamosing vascular channels lined by plump cells with the appearance of sinus lining (arrow: 'littoral' cells) (H&E 100×). Gross pathology of LCA is characterized by single, or more commonly, multiple pigmented focal nodules well-delin- eated from normal spleen parenchyma [28,30]. The col- our of these nodules may be dark red, brown, or black, consistent with blood or blood products of varying chro- nicity [28]. Rarely, LCA appears white on gross pathology [1,30]. The size of these lesions varies and may range from 0.1 cm to 11 cm in diameter [1,30,31]. The spleen itself may appear grossly enlarged or, as was true for our case, look otherwise unremarkable [31,32]. Anastamosing vascular channels lined by plump cells with the appearance of sinus lining (arrow: 'littoral' cells) (H&E 100×) Figure 4 Anastamosing vascular channels lined by plump cells with the appearance of sinus lining (arrow: 'littoral' cells) (H&E 100×). Anastamosing vascular channels lined by plump cells with the appearance of sinus lining (arrow: littoral cells) (H&E 100×) Figure 4 Anastamosing vascular channels lined by plump cells with the appearance of sinus lining (arrow: 'littoral' cells) (H&E 100×). Microscopically, there are several distinguishing histolog- ical and molecular features of LCA. Histologically, LCA has specific features that differentiate it from other pri- mary vascular tumours, including angiosarcoma [1]. LCA are composed of anastomosing vascular channels resem- bling splenic sinusoids and have irregular lumina featur- ing papillary projections and cyst-like spaces (Figure 4) [1]. Tall endothelial cells with histiocytic properties that slough off into the vascular lumen are common, as is the absence of atypical cells and presence of low mitotic activ- ity [1]. By immunohistochemical staining, these tumour cells will express endothelial and histiocyte antigens, a reflection of the distinct dual differentiation potential of LCA [1]. Such expression includes endothelial markers (factor VIII Ag and CD 31/BMA 120) as well as histiocytic markers (CD 68/KP 1 and lysozyme) [1,4,30,31,33]. The expression of these molecular markers has also been dem- onstrated in fine-needle aspiration biopsies of LCA [33]. have prompted recommendations to closely evaluate and provide surveillance to patients with LCA for the develop- ment of other malignancies [20]. Discussion i The lesion is isodense compared to the normal spleen on the 5 minutes delayed image. tent fevers. More dramatically, LCA has been reported to present as splenic rupture and haemoperitoneum [13-15]. The pathogenesis of LCA remains unclear, but given its association with autoimmune disorders such as Crohn's disease and inborn metabolic diseases such as Gaucher's disease, immune system dysfunction has been postulated as a possible important pathogenic mechanism [10,19]. Supporting this hypothesis, other reports have suggested that chronic infection and systemic immunosuppression may contribute to LCA development [13,18]. LCAs are believed to originate from the littoral cells, the cells that line red pulp sinuses of the spleen [1,17]. From studies performed as far back as the 1930s, endothelial cells lining the vascular sinuses of the spleen were consid- ered unique in that they exhibited both phagocytic and hematopoietic properties [10]. Neoplasia of these cells results in the formation of LCA, which exhibit histologic and molecular features consistent with both these epithe- lial and histiocytic cell types [17,18]. Indeed, immune system dysregulation may explain the association of LCA with other cancer types. The other can- cer types associated with LCA include: thyroid, colorectal, renal, pancreatic, hematologic (lymphoma), ovarian, and testicular cancer [10,13,18,20,21]. These observations A. The lesion is hyperintense on the T2 weighted fast recovery fast spin echo (FRFSE) image Figure 3 A. The lesion is hyperintense on the T2 weighted fast recovery fast spin echo (FRFSE) image. B. Internal linear enhancement is noted on the T1 weighted fat saturated image after iv-gadolinium in the portal venous phase. A. The lesion is hyperintense on the T2 weighted fast recovery fast spin echo (FRFSE) image Figure 3 A. The lesion is hyperintense on the T2 weighted fast recovery fast spin echo (FRFSE) image. B. Internal linear enhancement is noted on the T1 weighted fat saturated image after iv-gadolinium in the portal venous phase. Page 3 of 5 (page number not for citation purposes) World Journal of Surgical Oncology 2008, 6:87 http://www.wjso.com/content/6/1/87 studies with Tc-99m labelled RBC scintigraphy can be use- ful to differentiate splenic lesions from splenic haemangi- omas [27,28]. However, the radiologic features of LCA are rarely diagnostic since many other splenic neoplasms such as haemartomas, haemangiomas, lymphomas, met- astatic disease and infectious processes exhibit similar imaging characteristics [29]. Discussion i Conversely, the associa- tion of LCA with other cancer types may also be a result of making an incidental diagnosis of LCA during extensive radiological imaging for other diseases, given the largely asymptomatic presentation of these tumours [22]. However, close follow-up of LCA may be warranted due to the potential for their malignant transformation. The two subtypes of LCA with malignant potential have been described as "littoral cell angiosarcoma" [18,23,24] and "littoral cell haemangioendothelioma" [11,12,18]. These LCA variants may present with distant metastasis several months after splenectomy; histologic evaluation reveals features consistent with LCA histopathology as well as abnormal architecture, nuclear atypia, and necrosis [11,12,23,24]. Symptomatic LCA are often relieved by splenectomy, and given the association of LCA with other malignancies and reported cases of metastasizing LCA, splenectomy is both diagnostic and therapeutic. While there have been reports of medical therapy with glucocorticoids and angioembol- ization of splenic haemangiomas [34], splenectomy is still considered the gold standard for treatment of vascular splenic tumours [15]. Radiologically, LCA may be evaluated by several imaging modalities such as US, CT, MRI, or nuclear medicine stud- ies (Tc-99m labelled RBC scintigraphy). US may reveal lobular splenomegaly with heterogeneous nodules (either hypo- or hyper-echoic) that may be solitary or multiple (Figure 1) [25]. On non-contrast CT, LCA appear as hypo- attenuating masses; given the vascular nature of these neo- plasms, they tend to enhance homogeneously. On MRI, a minority of cases may be hypointense on both T1- weighted and T2-weighted scans because of hemosiderin content of the tumour [14]. However, as significant siderosis is seen in less than 50% of LCA cases [1], lesions tend to be hyperintense on the T2 weighted images, as was the case in our patient (Figure 3) [26]. Nuclear medicine References 1. Falk S, Stutte HJ, Frizzera G: Littoral cell angioma – a novel splenic vascular lesion demonstrating histiocytic differentia- tion. Am J Surg Pathol 1991, 15(11):1023-1033. 29. Bhatt S, Huang J, Dogra V: Littoral cell angioma of the spleen. AJR American J Roentgenol 2007, 188:1365-1366. J J g 30. Musgrave NJ, Williamson RM, O'Rourke NA, Searle JW: Inciden- tally discovered splenic vascular lesion. Pathology 2002, 34:579-581. J g , ( ) 2. Dascalescu CM, Wendum D, Gorin NC: Littoral cell angioma as a cause of splenomegaly. New Engl J Med 2001, 345(10):772-773. J g ( ) 2. Dascalescu CM, Wendum D, Gorin NC: Littoral cell angioma as a cause of splenomegaly. New Engl J Med 2001, 345(10):772-773. J g ( ) 2. Dascalescu CM, Wendum D, Gorin NC: Littoral cell angioma as a cause of splenomegaly. New Engl J Med 2001, 345(10):772-773. 3. Collins PJ, Ettler H, Amann J, Rajgopal C: Soft-tissue images: splenic littoral cell angioma. Can J Surg 2003, 46(3):204-205. 4. Ziske C, Meybehn M, Sauerbruch T, Schmidt-Wolf IGH: Littoral cell angioma as a rare cause of splenomegaly. Ann Hematol 2001, 80:45-48. 2. Dascalescu CM, Wendum D, Gorin NC: Littoral cell angioma as a cause of splenomegaly. New Engl J Med 2001, 345(10):772-773. 3. Collins PJ, Ettler H, Amann J, Rajgopal C: Soft-tissue images: splenic littoral cell angioma. Can J Surg 2003, 46(3):204-205. p g y e g J ed , ( ) 3. Collins PJ, Ettler H, Amann J, Rajgopal C: Soft-tissue images: splenic littoral cell angioma Can J Surg 2003 46(3):204-205 p g y g J , ( ) 3. Collins PJ, Ettler H, Amann J, Rajgopal C: Soft-tissue images: splenic littoral cell angioma. Can J Surg 2003, 46(3):204-205. 31. Veillon DM, Williams RB, Sardenga LJ, Harrison GK, Cotelingam JD: "Little" littoral cell angioma of the spleen. Am J Surg Pathol 2000, 24(2):306-327. splenic littoral cell angioma. Can J Surg 2003, 46(3):204 205. 4. Ziske C, Meybehn M, Sauerbruch T, Schmidt-Wolf IGH: Littoral cell angioma as a rare cause of splenomegaly. Ann Hematol 2001, 80:45-48. p g J g , ( ) 4. Ziske C, Meybehn M, Sauerbruch T, Schmidt-Wolf IGH: Littoral cell angioma as a rare cause of splenomegaly. Ann Hematol 2001, 80:45-48. ( ) 32. Cheng SP, Yang TL, Chen BF, Liu CL: Image of the month: littoral cell angioma. Arch Surg 2005, 140:1127. 5. Authors' contributions g 21. Akyildiz H, Akcan A, Soyuer I, Ibrahim Karahan O, Sozuer E: Littoral cell angioma mimicking pancreatic tumour. Surgery 2006, 141(5):690-691. MT performed the literature review and drafted the man- uscript. PV reviewed and revised the manuscript and pro- vided radiographic images. PZ reviewed and revised the manuscript and provided pathologic images. SW origi- nated the idea and assisted with drafting and revising the manuscript. All authors read and approved the final man- uscript. ( ) 22. Kutok JL, Fletcher CDM: Splenic vascular tumours. Semin Diag Pathol 2003, 20:128-139. 23. Rosso R, Paulli M, Gianelli U, Boveri E, Stella G, Magrini U: Littoral cell angiosarcoma of the spleen. Am J Surg Pathol 1995, 19(10):1203-1208. ( ) 24. Rosso R, Paulli M: Littoral cell angiosarcoma: a truly malignant tumour. Am J Surg Pathol 2004, 28:1255. J g 25. Giovagnoni A, Giorgi C, Goteri G: Tumours of the spleen. Cancer Imaging 2005, 5:73-77. Conclusion LCA is a recently described primary vascular neoplasm of the spleen that may be associated with other malignancies and may itself also have malignant potential. Several radi- ological studies may suggest LCA, although a pathological diagnosis, either by core biopsy or diagnostic splenectomy is imperative. This rare case illustrates the importance of thoroughly evaluating incidental vascular splenic Page 4 of 5 (page number not for citation purposes) Page 4 of 5 (page number not for citation purposes) World Journal of Surgical Oncology 2008, 6:87 http://www.wjso.com/content/6/1/87 15. Wilcox TM, Speer RW, Schlinkert RT, Sarr MG: Hemangioma of the spleen: presentation, diagnosis, and management. J Gas- trointest Surg 2000, 4:611-613. tumours. Although the vast majority of LCAs are benign, their differential diagnosis must include both primary and secondary malignancy, given LCA's association with other cancer types as well as their uncertain malignant poten- tial. With this in mind, gold standard management remains splenectomy and long-term follow-up for the development of synchronous tumours or metastatic lesions is advised. g 16. Chen LW, Chien RN, Yen CL, Chang LC: Splenic tumour: a clin- icopathological study. Int J Clin Pract 2004, 58:924-927. 17. Arber DA, Strickler JG, Chen YY, Weiss LM: Splenic vascular tumors: a histologic, immunophenotypic, and virologic study. Am J Surg Pathol 1997, 21(7):827-835. y J g ( ) 18. Fadare O, Hileeto D, Mariappan MR: Multiple splenic lesions in a bacteremic patient. Arch Pathol Lab Med 2004, 128:1183-1185. p 19. Suvajdzic N, Cemerikic-Martinovic V, Saranovic D, Petrovic M, Popo- vic M, Artiko V, Cupic M, Elezovic I: Littoral-cell angioma as a rare cause of splenomegaly. Clin Lab Haem 2006, 28:317-320. Acknowledgements 26. Levy AD, Abbott RM, Abbondanzo SL: Littoral cell angioma of the spleen: CT features with clinicopathologic comparison. Radiology 2004, 230:485-490. Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written con- sent is available for review by the Editor-in-Chief of this journal. gy 27. Wijaya J, Kapoor R, Roach P: Tc-99m-labelled RBC scintigraphy and splenic hemangioma. Clin Nuc Med 2001, 26(12):1022-1023. 28 J h C G l M Ki B W d hl D N i i k K R di l 27. Wijaya J, Kapoor R, Roach P: Tc 99m labelled RBC scintigraphy and splenic hemangioma. Clin Nuc Med 2001, 26(12):1022-1023. 28. Johnson C, Goyal M, Kim B, Wasdahl D, Nazinitsky K: Radiology- Pathology Conference: Littoral cell angioma. Clin Imaging 2007, 31:27-28. 28. Johnson C, Goyal M, Kim B, Wasdahl D, Nazinitsky K: Radiology- Pathology Conference: Littoral cell angioma. Clin Imaging 2007, 31:27-28. Pathology Conference: Littoral cell angioma. Clin Imaging 2007, 31:27-28. Competing interests p g y 20. Bisceglia M, Sickel JZ, Giangaspero F, Gomes V, Amini M, Michal M: Littoral cell angioma of the spleen: an additional report of four cases with emphasis on the association with visceral organ cancers. Tumori 1998, 84:595-599. The authors declare that they have no competing interests. The authors declare that they have no competing interests. References Espanol I, Lerma E, Fumanal V, Palmer J, Roca M, Domingo-Albos A, Pujol-Moix N: Littoral cell angioma with severe thrombocyto- penia. Ann Hematol 2000, 79:46-49. g g 33. Hesse J, Bocklage T: Specimen fine-needle aspiration cytology of littoral cell angioma with histologic and immunohisto- chemical confirmation. Diagn Cytopathol 2000, 22:39-44. p 6. Goldfeld M, Cohen I, Loberant N, Mugrabi A, Katz I, Papura S, Noi I: Littoral cell angioma of the spleen: appearance on sonogra- phy and CT. J Clin Ultrasound 2002, 30:510-513. g y p 34. Islam S, Newman EA, Strousse PJ, Geiger JD: Antiangiogenic ther- apy for a large splenic hemangioma. Pediatr Surg Int 2005, 21:1007-1010. p y J 7. Ercin C, Gurbuz Y, Hac-Hanefioglu A, Karakaya AT: Multiple litto- ral cell angioma of the spleen in a case of myelodysplastic syndrome. Hematology 2005, 10(2):141-144. y e to ogy , ( ) 8. Grantham M, Einstein D, McCarron K, Lichtin A, Vogt D: Littoral cell angioma of the spleen. Abdom Imaging 1998, 23:633-635. g p g g 9. Qu ZB, Liu LX, Wu LF, Zhao S, Jiang HC: Multiple littoral cell angioma of the spleen: a case report and review of literature. Onkologie 2007, 30(5):256-258. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 5 of 5 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Publish with BioMed Central and every scientist can read your work free of charge g ( ) 10. Gupta MK, Aguilera NS, Pastores GM: Littoral cell angioma of the spleen in a patient with Gaucher disease. Am J Hematol 2001, 68:61-62. 11. Fernandez A, Cook GW, Arber DA: Metastasizing splenic littoral cell hemangioendothelioma. Am J Surg Pathol 2006, 30(8):1036-1040. ( ) 12. Ben-Izhak O, Bejar J, Ben-Eliezer S, Vlodavsky E: Splenic littoral cell hemangioendothelioma: a new low-grade variant of malig- nant littoral cell tumour. Histopathology 2001, 39:469-475. p gy 13. Harmon RL, Cerruto CA, Scheckner A: Littoral cell angioma: a case report and review. Current Surgery 2006, 63(5):345-350. p g y ( ) 14. Abbott RM, Levy AD, Aguilera NS, Gorospe L, Thompson WM: Pri- mary vascular neoplasms of the spleen: radiologic-patho- logic correlation. Radiographics 2004, 24(4):1137-1163.
https://openalex.org/W4206725276	https://jurnal.sttsetia.ac.id/index.php/pkm/article/download/181/133	Indonesian	null	PERTUMBUHAN IMAN KAUM TUNANETRA KEPADA KRISTUS DI MASA PANDEMI COVID-19 DI JAWA BARAT, JAKARTA DAN MEDAN	Jurnal PkM Setiadharma	2,021	cc-by	5,879	Abstract During the Covid-19 pandemic, the church was limited by government regulations not to hold on-site service but was replaced with online service via YouTube, Zoom Meetings or other media. Not everyone can use this technology, especially those who have limitations as experienced by the blind. As believers they still have to grow in faith. The problem in the research is how blind people continue to grow in faith in Christ even during the Covid-19 pandemic? This study uses a qualitative method with a literature study approach together with interviews with blind people about the need for their growth in faith. The growth in faith can be obtained through worship, Bible reading. By stopping the weekly service as usual, it is through reading the Bible that they gain faith growth. But they are constrained by funding problems to have a special Bible for them. The result of this research is that the blind need Braille Bible. The author lists the needs of the Braille Bible and distributes it for free as a form of community service. The author hopes that this research can raise the concern of Christians for the needs of the blind and those who need help. PERTUMBUHAN IMAN KAUM TUNANETRA KEPADA KRISTUS DI MASA PANDEMI COVID-19 DI JAWA BARAT, JAKARTA DAN MEDAN Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Sekolah Tinggi Teologi Kharisma Bandung tjutjun.setiawan65@gmail.com, andre.kaizenskinexpert@gmail.com, ronald.rachmat@gmail.com, iwansetiyawan0508@gmail.com, Diterima: 24-07-2021 Direview: 28-07-2021 24-09-2021 Direvisi: 20-08-2021 07-10-2021 Diterbitkan: 31-12-2021 Keywords: Bible, braille, blind, devotion, covid-19 Kata Kunci: Alkitab, braille, tunanetra, pengabdian, covid- 19 Diterima: 24-07-2021 Direview: 28-07-2021 24-09-2021 Direvisi: 20-08-2021 07-10-2021 Diterbitkan: 31-12-2021 Keywords: Bible, braille, blind, devotion, covid-19 Kata Kunci: Alkitab, braille, tunanetra, pengabdian, covid- 19 Diterima: 24-07-2021 Direview: 28-07-2021 24-09-2021 Direvisi: 20-08-2021 07-10-2021 Diterbitkan: 31-12-2021 Keywords: Bible, braille, blind, devotion, covid-19 Kata Kunci: Alkitab, braille, tunanetra, pengabdian, covid- 19 PERTUMBUHAN IMAN KAUM TUNANETRA KEPADA KRISTUS DI MASA PANDEMI COVID-19 DI JAWA BARAT, JAKARTA DAN MEDAN Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Sekolah Tinggi Teologi Kharisma Bandung tjutjun.setiawan65@gmail.com, andre.kaizenskinexpert@gmail.com, ronald.rachmat@gmail.com, iwansetiyawan0508@gmail.com, 140 1 Desca Lidya Natalia and Hanni Sofia, “Presiden Umumkan Kasus Infeksi Corona Pertama Di Indonesia,” Antaranews.Com. 2 Aldi Ahmad Rifai and Sahadi Humaedi, “INKLUSI PENYANDANG DISABILITAS DALAM SITUASI PANDEMI COVID-19 DALAM PERSPEKTIF SUSTAINABLE DEVELOPMENT GOALS (SDGs),” Prosiding Penelitian dan Pengabdian kepada Masyarakat (2020). 3 Ibid. 4 Ibid. 5 Dani Prabowo, “Ada Yang Lebih Susah Dari Kita, Yaitu Penyandang Disabilitas,” Kompas.Com. 6 Arie Nugraha, “Rumah Ibadah Di Bandung Belum Ramah Bagi Disabilitas,” Liputan6.Com. 7 Sigit Triyono, “Alkitab Dan Disabilitas - Lembaga Alkitab Indonesia,” Https://Indonesia.Bible. Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 g g g p 7 Sigit Triyono, “Alkitab Dan Disabilitas - Lembaga Alkitab Indonesia,” Https://Indonesia.Bible. Abstrak Pada masa pandemi Covid-19, gereja dibatasi aturan pemerintah untuk tidak mengadakan ibadah secara onsite tetapi diganti dengan ibadah online melalui YouTube, Zoom Meeting atau media yang lain. Tidak semua orang dapat memanfaatkan teknologi ini terutama mereka yang mempunyai keterbatasan sebagaimana dialami kaum tunanetra. Sebagai orang percaya mereka tetap harus bertumbuh dalam iman. Rumusan masalah dalam penelitian adalah bagaimana kaum tunanetra tetap bertumbuh dalam iman kepada Kristus meskipun di masa pandemi Covid-19? Penelitian ini menggunakan metode kualitatif dengan pendekatan studi kepustakaan dibarengi dengan wawancara terhadap kaum tunanetra tentang kebutuhan akan pertumbuhan iman mereka. Pertumbuhan iman bisa didapat melalui ibadah, pembacaan Alkitab. Dengan dihentikan ibadah minggu seperti biasa maka melalui pembacaan Alkitablah mereka mendapatkan pertumbuhan iman. Tetapi mereka terkendala masalah dana untuk memiliki Alkitab khusus bagi mereka. Hasil dari penelitian ini adalah kaum tunanetra membutuhkan Alkitab Braille. Penulis mendata kebutuhan Alkitab Braille dan membagikannya secara gratis sebagai bentuk pengabdian kepada masyarakat. Penulis berharap penelitian ini dapat membangkitkan kepedulian umat Kristen akan kebutuhan kaum tunanetra maupun mereka yang membutuhkan pertolongan. 5 Dani Prabowo, “Ada Yang Lebih Susah Dari Kita, Yaitu Penyandang Disabilitas,” Kompas.Com. 6 Arie Nugraha, “Rumah Ibadah Di Bandung Belum Ramah Bagi Disabilitas,” Liputan6.Com. 7 , g , y g , p 6 Arie Nugraha, “Rumah Ibadah Di Bandung Belum Ramah Bagi Disabilitas,” Liputan6.Com. 7 5 Dani Prabowo, “Ada Yang Lebih Susah Dari Kita, Yaitu Penyandang Disabilitas,” Kompas.Com. 6 141 8 Sugiyono, Metode Penelitian Kuantitatif Kualitatif Dan R&D (Bandung: Alfabeta, 2011). 9 Ferry Simanjuntak, Metode Penelitian (Bandung: Sekolah Tinggi Teologi Kharisma, 2016). 10 Samuel Santoso, “Wawancara” (Solo, 2020). 11 Dini Widinarsih, “Penyandang Disabilitas Di Indonesia: Perkembangan Istilah Dan Definisi,” Jurnal Ilmu Kesejahteraan Sosial (2019). 12 Ibid. 13 Ibid. METODE PELAKSANAAN Metode yang digunakan dalam penelitian ini adalah kualitatif dengan pendekatan studi kepustakaan (library research).8 Penulis mengumpulkan data dari informasi internet, buku-buku, jurnal, dan juga Alkitab, yang berkaitan dengan masalah yang diteliti,9 menggali dan mendalami apa yang menjadi kebutuhan kelompok tunanetra di Jawa Barat, Jakarta dan Medan supaya iman mereka tetap bertumbuh di tengah keterbatasan mereka dan di masa pandemi Covid-19 ini di mana ada pembatasan dalam beribadah secara langsung. Penulis juga melakukan wawancara dengan kaum tunanetra ini,10 sehingga dengan demikian dapat dihasilkan suatu pembahasan yang objektif dan menyeluruh, dapat dipertanggungjawabkan, yang pada akhirnya didapat suatu kesimpulan yang menjawab rumusan masalah dalam penelitian ini dan dapat membantu kaum tunanetra akan pertumbuhan imannya. Pengabdian kepada masyarakat ini persiapannya dimulai pada bulan Desember 2020. PENDAHULUAN Meskipun mereka memiliki keterbatasan tetapi kelompok disabilitas Kristen mempunyai kebutuhan yang sama seperti orang Kristen pada umumnya, mereka membutuhkan eksistensi, relasi dan bertumbuh di dalam Kristus.7 Juga mempunyai 140 PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) kebutuhan untuk dapat mengakses dan membaca Alkitab, sehingga mereka dapat bertumbuh dalam iman. kebutuhan untuk dapat mengakses dan membaca Alkitab, sehingga mereka dapat bertumbuh dalam iman. Dari paparan di atas didapat rumusan masalah yang akan diteliti yaitu bagaimana kaum tunanetra tetap bertumbuh dalam iman kepada Kristus meskipun di masa pandemi Covid-19? apa yang menjadi kebutuhan kaum tunanetra supaya iman mereka dapat tetap bertumbuh meskipun di tengah pandemi Covid-19? dan bagaimana membantu mereka dalam hal pertumbuhan iman tersebut? Adapun tujuan dari penelitian ini adalah supaya kaum tunanetra yang sangat rentan terdampak dalam pandemi yang terjadi di Indonesia ini tetap dapat bertumbuh dalam iman dan pengharapan dalam Kristus. Penulis juga berharap dengan penelitian ini dapat menggerakkan dan membangkitkan kepedulian dari setiap orang Kristen yang mempunyai berkat lebih terhadap kaum penyandang disabilitas pada umumnya dan tunanetra pada khususnya. PENDAHULUAN Wabah virus corona (Covid-19) yang berkepanjangan telah mengubah tatanan kehidupan masyarakat sehari-hari. Pada bulan Maret tahun 2020. Presiden Joko Widodo mengumumkan bahwa Indonesia sudah mulai dijangkiti oleh virus corona yang dikenal dengan sebutan Covid-19,1 dan pada akhirnya Covid-19 telah dinyatakan sebagai pandemi global dan telah menyebar ke banyak negara di seluruh dunia. Covid-19 menyebar dengan cepat melalui kontak langsung maupun melalui droplet (tetesan pernapasan) dari orang yang telah terpapar atau terinfeksi.2 Dari sejak saat itu tatanan kehidupan masyarakat berubah, baik dalam pekerjaan, kehidupan sehari-hari di mana masyarakat sekarang ini harus terbiasa dengan pola hidup bersih, selalu mencuci tangan, selalu memakai masker dan juga dalam pergaulan sosial mengalami perubahan, tidak lagi bisa seperti sebelum pandemi terjadi. Bahkan dalam tatanan kehidupan beribadah juga mengalami perubahan, di mana masyarakat kristiani yang biasa selalu beribadah pada hari Minggu dan bahkan mengadakan acara- acara rohani di gereja dengan datang secara langsung, kini semua itu diatur oleh pemerintah. Jika kasus-kasus baru melandai maka jemaat bisa hadir secara fisik di gereja dengan aturan yang ketat di mana protokol kesehatan dilakukan secara ketat dan juga kehadiran jemaat dibatasi jumlah dalam kehadiran tiap-tiap ibadahnya. Jika keadaan kasus baru yang terjangkit Covid-19 semakin meningkat maka ibadah langsung di gereja ditiadakan dan diganti dengan ibadah secara online. Pemberlakuan Pembatasan Sosial Berskala Besar (PSBB) adalah suatu kebijakan protokol pencegahan Covid-19 di Indonesia yang diterapkan di beberapa wilayah yang memiliki kasus positif Covid-19 terbanyak. Dengan berlakunya PSBB memiliki dampak kepada seluruh lapisan masyarakat dalam aspek sosial, ekonomi, maupun lingkungan.3 Hal ini dikarenakan dibatasinya kegiatan masyarakat di luar rumah dan penutupan fasilitas umum seperti sekolah diliburkan dan tempat kerja, penutupan tempat ibadah dan objek wisata, termasuk pembatasan transportasi umum.4 Dampak tersebut pun dirasakan oleh kelompok rentan seperti penyandang disabilitas dengan beragam disabilitasnya yang salah satunya adalah komunitas tunanetra yang banyak dari komunitas ini kehilangan mata pencaharian.5 Selain berdampak pada kehidupan ekonomi mereka, tetapi juga berdampak kepada kehidupan rohani mereka, di mana mereka tidak bisa lagi beribadah sebagaimana biasanya. Di masa sebelum pandemi saja tidak mudah bagi mereka untuk beribadah karena keterbatasan gereja menyediakan sarana untuk penyandang disabilitas.6 Mereka harus berdiam diri di rumah dan mengikuti secara online yang tentu saja tidak mudah bagi mereka untuk melakukan seperti itu. 142 14 “Arti Kata Tunanetra - Kamus Besar Bahasa Indonesia (KBBI) Online” (KBBI Online, 2021). 15 Halahan D.P and Kauffman J.M, Exceptional Learners: Introduction to Special Education, 10th ed. (USA: Pearson, 2006). 16 Vanaja Syifa Radissa et al., “PEMENUHAN KEBUTUHAN DASAR PENYANDANG DISABILITAS PADA MASA PANDEMI COVID-19,” Focus : Jurnal Pekerjaan Sosial (2020). 17 Ibid. 18 Ibid. 19 Nadia Khairina Budiman, Penyebab Dan Pencegahan Kebutaan (Bandung, 2019). 20 Kirk. A Samuel, Klasifikasi Kebutaan (USA, 1991). Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 Tunanetra menurut Kamus Besar Bahasa Indonesia adalah tidak dapat melihat; buta.14 Tunanetra termasuk ke dalam kelompok difabel atau penyandang cacat, yang memiliki hambatan dalam penglihatan. Tunanetra dapat diklasifikasikan kedalam dua golongan yaitu: buta total (blind) dan low vision. Definisi tunanetra menurut Kaufman & Hallahan adalah individu yang memiliki lemah penglihatan atau akurasi penglihatan kurang dari 6/60 setelah dikoreksi atau tidak lagi memiliki penglihatan.15 Karena tunanetra memiliki keterbatasan dalam indera penglihatan maka proses pembelajaran menekankan pada alat indera yang lain yaitu indera peraba dan indera pendengaran. Tunanetra adalah mereka yang tidak memiliki penglihatan sama sekali (buta total) hingga mereka yang masih memiliki sisa penglihatan tetapi tidak mampu menggunakan penglihatannya. g Menurut Undang-Undang Nomor 8 Tahun 2016 tentang Penyandang Disabilitas, setiap orang yang mengalami keterbatasan fisik, intelektual, mental dan/atau sensorik dalam jangka waktu lama yang dalam berinteraksi dengan lingkungan dapat mengalami hambatan dan kesulitan untuk berpartisipasi secara penuh dan efektif dengan warga negara lainnya berdasarkan kesamaan hak.16 Lebih lanjut dinyatakan bahwa jenis disabilitas dikaregorikan menjadi empat yaitu disabilitas fisik, intelektual, mental, dan/atau sensorik.17 Tunanetra termasuk ke dalam kategori disabilitas sensorik yaitu penyandang disabilitas yang fungsi panca indera yaitu penglihatan terganggu.18 19 Nadia Khairina Budiman, Penyebab Dan Pencegahan Kebutaan (Bandung, 2019). 20 Kirk. A Samuel, Klasifikasi Kebutaan (USA, 1991). Definisi Tunanetra Kata Tuna mengandung beberapa arti harfiah: luka; rusak; kurang; tidak memiliki.11 Berasal dari Bahasa Jawa kuno yang berarti rusak atau rugi, tetapi kata ini tidak biasa digunakan untuk mengacu kepada barang yang rusak, seperti halnya kata cacat. Pada awalnya istilah tuna ini digunakan untuk memperhalus kata cacat sebagai bentuk tetap menghormati penyandangnya.12 Dapat dihubungkan dengan kekurangan fungsi tubuh dalam diri seseorang seperti tunadaksa (cacat tubuh); tunagrahita (cacat pikiran, keterbelakangan mental; tunanetra (tidak dapat melihat, buta).13 141 143 21 Ade Nasihudin Al Ansori, “4 Klasifikasi Tunanetra Berdasarkan Jenis Kelainan Hingga Waktu Terjadinya,” Http://Liputan6.Com. 22 Fetty Ismandari, Situasi Gangguan Penglihatan, Infodatin Kemenkes RI (Jakarta, 2018). 23 Zaid Haron Musa Jawasreh, Noraidah Sahari Ashaari, and Dahlila Putri Dahnil, “The Acceptance of Braille Self-Learning Device,” International Journal on Advanced Science, Engineering and Information Technology (2020). 24 Farid Afandi, Mari Mengenal Huruf Braille (Sidoarjo, 2017). 25 Ibid. Klasifikasi Tunanetra Sebagai organisasi Kesehatan dunia, World Health Organization (WHO) berdasarkan International Statistical Classification of Diaseases and Related Health Problems, 10th revision (ICD-10) telah memberikan definisi tentang kebutaan sebagai tajam penglihatan kurang dari 3/60 atau lapang pandang kurang dari 10 derajat pada mata terbaik dengan koreksi refraksi yang tersedia. Adapun gangguan penglihatan sedang dikategorikan jika seseorang memiliki tajam penglihatan diatas 6/60 namun kurang dari 6/18. Sedangkan gangguan penglihatan berat dikategorikan jika seseorang memiliki tajam penglihatan di bawah 6/60 namun lebih dari 3/60. Kategori 1 dan 2 disebut dengan low vision dan kategori 3 – 5 disebut dengan kebutaan.19 Tunanetra ringan (defective vision/low vision) adalah seseorang yang memiliki gangguan dalam penglihatan namun mereka masih bisa mengikuti kegiatan pendidikan atau proses pembelajaran dan mampu melakukan pekerjaan yang menggunakan fungsi penglihatan. Adapun tunanetra dengan gangguan sedang (partially sighted) adalah seseorang yang kehilangan sebagian daya penglihatan, hanya dengan menggunakan kaca pembesar mampu mengikuti pendidikan biasa dan atau mampu membaca tulisan berhuruf tebal. Tunanetra dengan gangguan berat (totally blind) yakni mereka yang sama sekali tidak dapat melihat, dan mengalami kebutaan total.20 Tunanetra dapat dikelompokkan sebagai berikut: tunanetra sejak lahir terdiri dari orang yang mengalami kebutaan pada saat dalam kandungan; tunanetra setelah lahir atau pada usia kecil; tunanetra yang mengalami pada usia sekolah anak atau 142 PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) remaja; tunanetra dewasa adalah orang dewasa yang mengalami kebutaan pada usia dewasa; dan tunanetra yang mengalami kebutaan pada usia lanjut.21 remaja; tunanetra dewasa adalah orang dewasa yang mengalami kebutaan pada usia dewasa; dan tunanetra yang mengalami kebutaan pada usia lanjut.21 Faktor yang menyebabkan gangguan pada penglihatan terbanyak di seluruh dunia adalah gangguan refraksi yang tidak terkoreksi (48,99%), katarak (25,81%) dan Age related Macular Degeneration (AMD, 4,1%). Sedangkan faktor terbanyak yang menyebabkan kebutaan adalah katarak (34,47%), lalu gangguan refraksi yang tidak terkoreksi (20,26%), dan glukoma (8,30%). Lebih dari 75% gangguan penglihatan adalah gangguan penglihatan yang dapat dicegah. Katarak atau kekeruhan lensa mata adalah penyebab utama kebutaan di lndonesia, 77,7% kebutaan dikarenakan katarak. Sedangkan prevalensi kebutaan akibat katarak pada penduduk umur 50 tahun ke atas di Indonesia sebesar 1,9%. Katarak adalah merupakan proses degeneratif yang sangat dipengaruhi umur. Dengan meningkatnya umur harapan hidup maka proporsi penduduk umur ≥50 tahun akan meningkat sehingga jumlah penderita katarak juga akan makin meningkat.22 144 26 Suhento Liauw, Doktrin Alkitab Alkitabiah (Jakarta: Gereja Baptis Independen Alkitabiah GRAPHE, 2001). 27 Budisatyo Taniharjo, Integritas Seorang Pemimpin Rohani, ke 5. (Yogyakarta, 2019). 28 Jonar T.H Situmorang, Bibliologi, Ke 5. (Yogyakarta: Penerbit Andi, 2017). 29 Ibid. 30 French L Arrington, Doktrin Kristen Perspektif Pentakosta, Ke 6. (Yogyakarta: Penerbit Andi, 2020). 31 Liauw, Doktrin Alkitab Alkitabiah. 32 Arrington, Doktrin Kristen Perspektif Pentakosta. 33 Ibid. 34 Ibid. 35 Triyono, “Alkitab Dan Disabilitas - Lembaga Alkitab Indonesia.” Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 Bertumbuh dalam Iman Dalam Roma 10:17, “Jadi, iman timbul dari pendengaran, dan pendengaran oleh firman Kristus.” Seseorang yang ingin bertumbuh imannya tidak bisa dilepaskan dari firman Yesus Kristus, dan itu hanya akan bisa didapat dengan membaca Alkitab dan mendengarkan Alkitab yang adalah firman Tuhan. Rasul Paulus menasihati Timotius untuk selalu mengingat dan memberi perhatian pada Alkitab, yang pada zaman itu hanya ada perjanjian Lama, “Ingatlah juga bahwa dari kecil engkau sudah mengenal Kitab Suci yang dapat memberi hikmat kepadamu dan menuntun engkau kepada keselamatan oleh iman kepada Kristus Yesus.” (2Tim. 3:15).26 Bahkan dituliskan lebih lanjut mengenai manfaat yang bisa diperoleh dengan mendalami Alkitab sebagaimana tertulis dalam 2 Timotius 3:16, “Segala tulisan yang diilhamkan Allah memang bermanfaat untuk mengajar, untuk menyatakan kesalahan, untuk memperbaiki kelakuan dan untuk mendidik orang dalam kebenaran.” Jadi di dalam Alkitab ada banyak pengajaran-pengajaran, bermanfaat untuk menyatakan kesalahan, dan untuk memperbaiki kelakuan dan juga membawa orang ke dalam kebenaran yang berpusat pada Kristus. Dengan bertekun dalam firman Tuhan akan terjadi pertumbuhan baik pribadi maupun dalam jemaat sebagaimana jemaat mula- mula bertekun dalam pengajaran rasul-rasul dan mengalami pertumbuhan (Kis. 2:42).27 Huruf Braille Braille adalah suatu sistem membaca dan menulis yang digunakan oleh tunanetra sehingga ia mendapatkan informasi pengetahuan. Menguasai Kode braille membawa penglihatan bagi orang-orang tunanetra dan memainkan peran penting dalam kehidupan mereka. Di era digital seperti sekarang ini, individu tunanetra memiliki banyak pilihan yang sangat efektif untuk komunikasi. Terbaru teknologi telah memungkinkan mereka untuk menjadi bagian dari masyarakat berpengetahuan dengan memungkinkan mereka untuk memahami isi situs web atau buku apa pun.23 Hambatan di dalam penglihatan mengakibatkan kaum tunanetra mengalami kesulitan dalam menerima informasi dalam bentuk visual, juga termasuk dalam bentuk tulisan. Dalam keterbatasannya mereka lebih mengoptimalkan kepekaan indera lain seperti indera pendengaran, indera penciuman, dan indera raba. Karena itu, bagi mereka, informasi yang sebelumnya dalam bentuk visual harus diubah menjadi bentuk lain seperti bunyi suara, bau-bauan, atau sentuhan untuk memudahkan penyandang tunanetra menerima informasi.24 Huruf braille adalah huruf dengan sistem tulisan sentuh yang digunakan oleh penyandang disabilitas tunanetra untuk membaca dan menulis. Sistem ini pertama kali digunakan di L’Institution Nationale des Jeunes Aveugles, Paris, dalam rangka mengajar siswa-siswa tunanetra. Dengan ditemukannya sistem ini membawa harapan bagi kaum tunanetra untuk mengakses dan mendapatkan informasi dalam bentuk tulisan sekaligus memudahkan dalam proses pendidikan. Penemu dari sistem sentuh huruf braille ini diciptakan oleh seorang tunanetra asal Perancis, yang bernama Louis Braille, itulah mengapa huruf tersebut disebut huruf Braille mengacu kepada nama penemunya.25 143 Ibid. 35 Triyono, “Alkitab Dan Disabilitas - Lembaga Alkitab Indonesia.” 26 Suhento Liauw, Doktrin Alkitab Alkitabiah (Jakarta: Gereja Baptis Independen Alkitabiah GRAPHE, 01) Alkitab Standar dan Alkitab Braille Kata Alkitab berasal dari kata dalam bahasa Yunani yaitu biblion atau biblos yang artinya adalah gulungan atau buku.28 Alkitab seringkali disebut Kitab Suci yang dalam bahasa Inggris disebut Scripture yang dapat diatikan firman, dan dalam bahasa Latin, Alkitab disebut Scriptura atau dalam bahasa Yunani dengan sebutan Graphe.29 Alkitab adalah firman Allah, kebenaran dari Allah,30 firman yang tertulis, wahyu yang sempurna yang diadakan Allah melalui proses yang panjang.31 Dalam menyatakan diri-Nya kepada manusia, Allah menggunakan berbagai sarana dan Alkitab adalah salah satunya, sebuah sarana yang istimewa sebab Alkitab adalah firman Allah.32 Alkitab terdiri dari dua kumpulan tulisan kudus yang disebut Perjanjian Lama yang berisi 39 kitab dan Perjanjian Baru yang berisi 27 kitab sehingga dengan demikian jumlahnya adalah 66 kitab.33 Kitab-kitab itu memuat tentang sejarah, hukum, nubuat, hikmat, puisi, surat-surat, dan apokaliptis.34 Dan inti dari keseluruhan kitab adalah berbicara tentang Yesus yang dinubuatkan dalam Perjanjian Lama, tentang Yesus yang datang sebagai penggenapan Mesias yang dijanjikan itu dan Yesus yang akan datang kembali untuk menjemput umat-Nya. Kaum tunanetra mempunyai kebutuhan untuk dapat mengakses dan membaca Alkitab, sehingga mereka dapat bertumbuh dalam iman.35 Kaum disabilitas fisik terutama para tunanetra, tunarungu, dan tunawicara pastilah memiliki keterbatasan Ibid. 35 Triyono, “Alkitab Dan Disabilitas - Lembaga Alkitab Indonesia.” 144 PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) berinteraksi dengan Alkitab bila menggunakan Alkitab cetak yang standar. Perlu ada Alkitab khusus yang memudahkan mereka berinteraksi dengan Alkitab.36 berinteraksi dengan Alkitab bila menggunakan Alkitab cetak yang standar. Perlu ada Alkitab khusus yang memudahkan mereka berinteraksi dengan Alkitab.36 Kesaksian suami isteri Brikjon Purba, bersama istrinya, Laoli, berdomisili di kota Siantar, mereka sama – sama tunanetra, mengungkapkan kegembiraannya dapat membaca Alkitab Braille yang mereka pinjam dari Rumah Baca Mutiara Bangsa.37 Mereka sudah lama memiliki kerinduan untuk membaca Alkitab dengan huruf Braille karena dengan keterbatasan mereka tentunya mereka tidak bisa membaca Alkitab cetak standar. Mereka pernah memesan Alkitab Braille tersebut dari rekan-rekan yang lain dan ternyata harganya cukup mahal dan barangnya-pun sulit ditemukan.38 y g y g y Alkitab Braille memang berbeda dengan Alkitab cetak yang standar yang hanya satu buku tebal saja. Sedangkan satu Alkitab Braille Perjanjian Lama dan Perjanjian baru berjumlah 39 buku atau jilid yang jika ditumpukkan menjadi satu maka 1 Alkitab Braille tumpukannya cukup tinggi dengan bobot lebih dari 20 kilogram, adapun rinciannya adalah sebagai berikut: 1. Kejadian jilid 1 2. Kejadian jilid 2 3. Keluaran jilid 1 4. Alkitab Standar dan Alkitab Braille Keluaran jilid 2 5. Imamat-Bilangan 6. Bilangan-Ulangan 7. Yosua - Hakim2 8. Rut-1 Samuel 9. 2 Samuel 10. 1 Raja-raja 11. 2 Raja-raja 12. Ezra-Nehemia 13. Ayub-Pengkotbah 14. Mazmur jilid 1 15. Mazmur jilid 2 16. Yesaya jilid 1 17. Yesaya jilid 2 18. Yeremia 19. Yehezkiel 20. Daniel-Hosea 21. Yoel-Mikha 22. Habakuk-Maleakhi 23. Tawarikh 24. Ester 25. Kidung Agung 26. Matius Jilid 1 27. Matius Jilid 2 28. Markus 29. Lukas jilid 1 30. Lukas jilid 2 31. Yohanes 32. Kisah pararasul jilid1 33. Kisah pararasul jilid2 34. Roma 35. 1&2 Korintus 36. Galatia-Kolose 37. 1 Tesalonika-Filemon 38. Ibrani - 2 Petrus 39. Yohanes-Wahyu Dengan terbagi menjadi 39 jilid maka meskipun sebuah Yayasan tunanetra tidak memiliki Alkitab dalam jumlah yang banyak tetapi bagian-bagian Alkitab itu bisa dibaca oleh banyak orang secara bergantian dan saling tukar menukar. 36 Ibid. 37 Tim, “Bisa Baca Buku Gratis Dan Alkitab Tulisan Braille, Pasangan Suami Istri Tunanetra Apresiasi Rumah Baca Mutiara Bangsa,” Https://Hetanews.Com. 38 Ibid. Kehidupan yang Berdampak Alkitab mengajarkan kepada setiap orang percaya untuk membantu yang lemah sebagaimana tertulis dalam Roma 15:1, “Kita, yang kuat, wajib menanggung kelemahan orang yang tidak kuat dan jangan kita mencari kesenangan kita sendiri.” Tuhan Yesus sendiri mengatakan bahwa hendaknya semua orang percaya menjadi garam dan terang dunia sehingga orang-orang melihat perbuatan baik dari setiap orang percaya dan memuliakan Bapa di sorga (Mat. 5:13-16), karena hanya manusia saja yang bisa memuliakan Allah, sedangkan ciptaan yang lain hanya bisa memancarkan kemuliaan 145 Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 Allah.39 Ada dampak yang baik dan positif yang diberikan oleh orang percaya pada kehidupan orang lain.40 Hendaklah kamu murah hati sama seperti Bapamu adalah murah hati (Luk. 6:36), firman Tuhan yang mengingatkan setiap orang percaya untuk bermurah hati kepada sesama, menolong yang lemah dan berkurangan sebagai wujud ucapan syukur kepada Tuhan sebab Ia sudah menebus dan menyelamatkan setiap orang yang percaya dari dosa. Siapa menaruh belas kasihan kepada orang yang lemah, memiutangi Tuhan, yang akan membalas perbuatannya itu (Ams. 19:17) Allah.39 Ada dampak yang baik dan positif yang diberikan oleh orang percaya pada kehidupan orang lain.40 Hendaklah kamu murah hati sama seperti Bapamu adalah murah hati (Luk. 6:36), firman Tuhan yang mengingatkan setiap orang percaya untuk bermurah hati kepada sesama, menolong yang lemah dan berkurangan sebagai wujud ucapan syukur kepada Tuhan sebab Ia sudah menebus dan menyelamatkan setiap orang yang percaya dari dosa. Siapa menaruh belas kasihan kepada orang yang lemah, memiutangi Tuhan, yang akan membalas perbuatannya itu (Ams. 19:17) Perencanaan Berawal dari adanya permintaan Alkitab Braille dari seorang yang bernama Andreas, pendeta GPdI Margatiga, Kabupaten Lampung Timur, yang bersangkutan sebenarnya sudah mempunyai Alkitab Braille tetapi karena sering dipergunakan maka titik timbul dari huruf braille tersebut sudah mulai menipis sehingga menimbulkan kesulitan dalam membacanya. Meskipun sekarang sudah ada Alkitab dalam bentuk audio, tetapi pendeta ini tetap lebih menyukai membaca Alkitab Braille dan untuk itulah ia menyampaikan permintaan kepada penulis. Penulis mengadakan komunikasi satu dan lainnya melalui media WhatsApp untuk membahas ini, mencari informasi kemana harus mencari Alkitab Braille. Pada akhirnya penulis berkordinasi dengan Lembaga Alkitab Indonesia (LAI), dan setelah melalui proses administrasi, LAI mendukung dan memberikan 1 set Alkitab Braille seperti yang diminta secara gratis dan penulis hanya membayar ongkos kirim saja ke Bandung di mana penulis berdomisili. p Setelah Alkitab Braille tersebut sudah diterima dari LAI Jakarta dan penulis menghubungi pendeta yang mengajukan permintaan itu, ternyata ia sudah mendapatkan kiriman dari temannya yang lain. Dengan adanya kejadian seperti ini lalu penulis kembali berkomunikasi membahas masalah ini. Lalu diputuskan untuk mengadakan aksi pembagian Alkitab yang lebih banyak lagi dengan mendata siapa saja dari kaum tunanetra Kristen yang membutuhkan Alkitab Braille tetapi terkendala dengan dana, baik perorangan maupun yayasan yang menaungi para tunanetra akan penulis bagikan secara gratis tanpa dipungut biaya sedikitpun. Ini dilakukan sebagai bentuk pengabdian kepada masyarakat, menjadi berkat bagi sesama yang membutuhkan. Dan setelah melakukan pendataan penulis berkordinasi kembali dengan LAI untuk meminta Alkitab Braille lebih banyak lagi sebagai tambahan. Keberagaman Etika Dalam Kekristenan,” Jurnal Teologi Berita Hidup (2021). g , g p ( ) 40 Rubin Adi Abraham, Menjadi Berkat Berbuah Lebat, ke 5. (Yogyakarta: Penerbit Andi, 2015). 39 Tony Salurante, Aprianus Moimau, and Filmon Berek, “Tujuan Penciptaan Sebagai Cara Memaham Keberagaman Etika Dalam Kekristenan,” Jurnal Teologi Berita Hidup (2021). 40 Rubin Adi Abraham, Menjadi Berkat Berbuah Lebat, ke 5. (Yogyakarta: Penerbit Andi, 2015). 39 Tony Salurante, Aprianus Moimau, and Filmon Berek, “Tujuan Penciptaan Sebagai Cara Memahami Keberagaman Etika Dalam Kekristenan,” Jurnal Teologi Berita Hidup (2021). 40 Rubin Adi Abraham, Menjadi Berkat Berbuah Lebat, ke 5. (Yogyakarta: Penerbit Andi, 2015). 39 Tony Salurante, Aprianus Moimau, and Filmon Berek, “Tujuan Penciptaan Seb Keberagaman Etika Dalam Kekristenan,” Jurnal Teologi Berita Hidup (2021). 40 Pelaksanaan Dari pendataan yang dilakukan, diperoleh data pribadi, Yayasan yang menaungi para tunanetra dan gereja di mana ada anggota jemaatnya yang masuk dalam kelompok tunanetra yang membutuhkan Alkitab Braille, yaitu sebagai berikut:  Yayasan Elsafan, Jl. Belitung Raya no. 49 RT/RW 5, Pondok Bambu, Duret Sawit, Jakarta Timur, Daerah khusus Ibu Kota Jakarta -13430. Pelaksanaan: dikirim melalui kurir ekspedisi sebanyak 3 set Alkitab.  Yayasan Pendidikan Tunanetra, Sumatera, KM 21.5 Dusun III. Desa Tanjung Baru, Kecamatan Tanjung Morawa 20362. Kab Deli Serdang, Sumatera Utara. Attn: Bapak Yabes Silaban. Pelaksanaan: dikirim melalui kurir ekspedisi sebanyak 5 set Alkitab. 146 PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak)  GBI Lippo Cikarang, Perumahan Puri Cijambe blok C12 no, 2e, Sukadami Cikarang Selatan, Kab Bekasi, Attn: Deddy Silalahi. Pelaksanaan: dikirim melalui kurir ekspedisi sebanyak 3 set Alkitab.  GBI Lippo Cikarang, Perumahan Puri Cijambe blok C12 no, 2e, Sukadami Cikarang Selatan, Kab Bekasi, Attn: Deddy Silalahi. Pelaksanaan: dikirim melalui kurir ekspedisi sebanyak 3 set Alkitab.  Pdt. Simon. Toko Fatigon. Jl. Mandalagiri 17, Garut. Pelaksanaan: dikirim melalui kurir ekspedisi sebanyak 1 set Alkitab.  Pdt. Simon. Toko Fatigon. Jl. Mandalagiri 17, Garut. Pelaksanaan: dikirim melalui kurir ekspedisi sebanyak 1 set Alkitab.  Yayasan Rumah Orang Tua Tunanetra Eben Haizer, Jl. Maribaya no. 24, Lembang, Kab Bandung Barat. Jawa Barat-40391, Attn: Bapak Sitompul. Pelaksanaan: Diberikan langsung kepada pengurus Yayasan sebanyak 4 set.  Pdt. Dicky Dauhan, Cimahi. Pelaksanaan: Dikirim melalui kurir ekspedisi sebanyak 1 set Alkitab.  Pdt. Dicky Dauhan, Cimahi. Pelaksanaan: Dikirim melalui kurir ekspedisi sebanyak 1 set Alkitab.  Solo Disability Ministry, Jl. Embarkasi H. Banaran, Gagaksipat, Kec. Ngemplak, Kab Boyolali, Jawa Tengah – 57375. Attn: Samuel Santoso Telepon: +628122311381, +628586134355, Pelaksanaan: Diserahkan langsung kepada yang bersangkutan sebanyak 1 Set Alkitab. Pelaksanaan penyerahan Alkitab Braille dilakukan dengan 2 metode yaitu metode pengiriman melalui kurir/ekspedisi dan penyerahan langsung di mana Akitab tersebut diserahkan langsung oleh para penulis kepada yang membutuhkan dengan menggunakan protokol kesehatan yang ketat karena masih berada dalam pandemi Covid-19. Evaluasi dan Tindak Lanjut Dari 39 jilid yang seharusnya ada dalam 1 set Alkitab tersebut, yang penulis kirim baru 29 jilid saja dikarenakan 10 jilid yang lain stoknya sedang kosong di LAI. Jika yang 10 jilid itu sudah tersedia, maka penulis merencanakan akan melakukan pengiriman kembali untuk kekurangan tersebut. Tidak tertutup kemungkinan jika di kemudian hari ada permintaan yang baru dari kaum tunanetra yang lain, sebagai bentuk pelayanan dan pengabdian kepada masyarakat maka penulis akan memproses permintaan itu sebagaimana yang telah dilakukan sebelumnya. Respons dari mereka yang menerima Alkitab Braille tersebut sungguh baik dan mereka sungguh bersukacita menerimanya sebab sudah lama mereka mendambakan memiliki Alkitab Braille untuk memenuhi kerinduan mereka akan pembacaan firman Tuhan. Penulis menghubungi yayasan yang menjadi penerima seperti Yayasan Pendidikan Tunanetra Medan untuk memantau sejauh mana manfaat dari pengabdian yang penulis lakukan. Di yayasan tersebut ada 64 orang tunanetra dari berbagai kelompok usia dan setiap hari dari senin sampai saptu diadakan ibadah sebanyak dua kali, yaitu pagi dan sore, di mana peserta ibadah secara bergantian membaca Alkitab Braille tersebut dan merenungkannya. Pelaksanaan Pelaksanaan penyerahan Alkitab Braille dilakukan dengan 2 metode yaitu metode pengiriman melalui kurir/ekspedisi dan penyerahan langsung di mana Akitab tersebut diserahkan langsung oleh para penulis kepada yang membutuhkan dengan menggunakan protokol kesehatan yang ketat karena masih berada dalam pandemi Covid-19. Sehubungan dengan pandemi covid-19 ini di mana ada pembatasan ruang gerak sebagaimana di atur oleh keputusan pemerintah, maka tidak semua Yayasan atau penerima dari pembagian Alkitab Braille ini dikunjungi oleh penulis, hanya beberapa saja yang dapat dikunjungi dan itupun dengan protokol kesehatan yang ketat. Pembagian Alkitab Braille ini merupakan kegiatan tanpa dipungut biaya sedikitpun, penulis memberikan kepada para penerima sebagaimana dituliskan di atas secara gratis. Biaya pengiriman Alkitab tersebut baik dari LAI ke Bandung ke tempat di mana penulis berdomisili maupun biaya pengiriman ke masing-masing penerima Alkitab tersebut, semua biaya menjadi tanggung jawab dari para penulis secara kolektif sebagai bentuk berbagi kasih dan bentuk kepedulian kepada sesama anak Tuhan yang membutuhkan. Mengingat bahwa kaum tunanetra ini adalah kelompok yang sudah dapat membaca huruf braille, maka penulis tidak melakukan kegiatan mengajar huruf tetapi penulis memberikan motivasi dan mendorong penerima baik yang dikunjungi langsung maupun yang dikunjungi melalui sarana komunikasi yang tersedia untuk memanfaatkan Alkitab Braille tersebut dengan membaca serta merenungkannya karena itu akan 147 Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 berdampak bagi pertumbuhan iman mereka sebagaimana tertulis dalam Roma 10:17, “Jadi iman timbul dari pendengaran, dan pendengaran oleh firman Kristus.” berdampak bagi pertumbuhan iman mereka sebagaimana tertulis dalam Roma 10:17, “Jadi iman timbul dari pendengaran, dan pendengaran oleh firman Kristus.” Pengawasan Terhadap Alkitab yang diserahkan langsung tentunya tidak dibutuhkan pengawasan karena Alkitab-Alkitab tersebut diserahkan dan diterima secara langsung baik yang mengatasnamakan pribadi maupun Yayasan dan serah terimanya didokumentasikan. Tetapi untuk Alkitab-Alkitab yang pengirimannya dilakukan melalui jasa kurir/ekspedisi, penulis melakukan pemantauan atau pengawasan sampai kiriman tersebut diterima oleh pribadi maupun Yayasan yang membutuhkan, dan penulis melakukan komunikasi dengan pihak penerima dalam hal ini dan juga penulis meminta kepada mereka untuk dibuatkan photo penerimaan jika sudah diterima sehingga terdokumentasi dengan baik dan lebih daripada itu apa yang mereka butuhkan yaitu Alkitab Braille di mana mereka dapat membacanya, merenungkan firman Tuhan sehingga iman mereka bertumbuh dapat terpenuhi dengan baik. UCAPAN TERIMA KASIH Pengabdian kepada masyarakat dalam hal membagikan Alkitab Braille kepada kaum tunanetra sehingga mereka dapat membaca Alkitab yang adalah firman Tuhan dengan tulisan yang dapat mereka pahami sehubungan dengan adanya hambatan yang mereka miliki dalam hal melihat, sehingga dengan demikian mereka dapat bertumbuh dalam iman kepada Kristus. Penulis mengucap syukur kepada Tuhan atas pelaksanaan ini dan juga penulis berterimakasih kepada Lembaga Alkitab Indonesia (LAI) yang telah menjalin kerjasama dengan penulis sehingga pengabdian kepada masyarakat ini dapat terlaksana. 148 PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) KESIMPULAN Alkitab adalah firman Allah. Di mana di dalamnya nyata kebenaran Allah, bermanfaat bagi setiap orang, ada banyak pengajaran-pengajaran, untuk menyatakan kesalahan, untuk memperbaiki kesalahan dan pada akhirnya membawa orang pada kebenaran, mendidik orang pada kebenaran. Bagi orang yang tidak termasuk kategori penyandang disabilitas fisik seperti tunanetra tentu saja tidak mempunyai hambatan dalam membaca Alkitab cetak standar, tetapi bagi kaum tunanetra ini menjadi masalah. Mereka membutuhkan Alkitab khusus yang ditulis dalam huruf Braille di mana mereka membacanya dengan memakai indera peraba. Faktor kesulitan untuk mendapatkan Alkitab berhuruf Braille dan juga ketiadaan dana yang menyebabkan tidak semua tunanetra memiliki Alkitab Braille ini. Dengan melakukan kegiatan pengabdian kepada masyarakat yang penulis lakukan untuk memenuhi kebutuhan kaum tunanetra untuk tetap bertumbuh dalam iman apalagi dalam masa pandemi Covid-19 ini di mana gereja di dalam melakukan ibadahnya berganti dengan cara online melalui media YouTube, Zoom Meeting atau aplikasi yang lain yang tentu saja tidak mudah juga bagi kaum tunanetra ini untuk mengaksesnya, beribadah dan bertumbuh dalam iman. Adapun kegiatan yang dilakukan adalah dengan membagikan Alkitab Braille itu kepada mereka yang sebelumnya sudah penulis data baik itu perorangan maupun Yayasan yang menaungi beberapa tunanetra dan juga gereja yang memiliki anggota jemaat yang mengalami keterbatasan dalam melihat. Semua diberikan secara gratis dan tidak dipungut biaya sedikitpun. Melalui kegiatan pengabdian masyarakat ini penulis menggugah setiap orang PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) PERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald Rachmat, Linutama Setiyawan, Ferry Simanjuntak) ERTUMBUHAN IMAN KAUM TUNANETRA … (Tjutjun Setiawan, Andreas Kurniawan, Ronald achmat, Linutama Setiyawan, Ferry Simanjuntak) KESIMPULAN Alkitab adalah firman Allah. Di mana di dalamnya nyata kebenaran Allah, bermanfaat bagi setiap orang, ada banyak pengajaran-pengajaran, untuk menyatakan kesalahan, untuk memperbaiki kesalahan dan pada akhirnya membawa orang pada kebenaran, mendidik orang pada kebenaran. Bagi orang yang tidak termasuk kategori penyandang disabilitas fisik seperti tunanetra tentu saja tidak mempunyai hambatan dalam membaca Alkitab cetak standar, tetapi bagi kaum tunanetra ini menjadi masalah. Mereka membutuhkan Alkitab khusus yang ditulis dalam huruf Braille di mana mereka membacanya dengan memakai indera peraba. Faktor kesulitan untuk mendapatkan Alkitab berhuruf Braille dan juga ketiadaan dana yang menyebabkan tidak semua tunanetra memiliki Alkitab Braille ini. Dengan melakukan kegiatan pengabdian kepada masyarakat yang penulis lakukan untuk memenuhi kebutuhan kaum tunanetra untuk tetap bertumbuh dalam iman apalagi dalam masa pandemi Covid-19 ini di mana gereja di dalam melakukan ibadahnya berganti dengan cara online melalui media YouTube, Zoom Meeting atau aplikasi yang lain yang tentu saja tidak mudah juga bagi kaum tunanetra ini untuk mengaksesnya, beribadah dan bertumbuh dalam iman. Adapun kegiatan yang dilakukan adalah dengan membagikan Alkitab Braille itu kepada mereka yang sebelumnya sudah penulis data baik itu perorangan maupun Yayasan yang menaungi beberapa tunanetra dan juga gereja yang memiliki anggota jemaat yang mengalami keterbatasan dalam melihat. Semua diberikan secara gratis dan tidak dipungut biaya sedikitpun. Melalui kegiatan pengabdian masyarakat ini penulis menggugah setiap orang Kristen yang mempunyai kemampuan keuangan atau berkat yang lebih untuk tergerak, bangkit, dan mulai peduli akan kebutuhan kaum tunanetra ini maupun juga kepada mereka yang membutuhkan pertolongan. 149 Jurnal PkM Setiadharma Volume 2, Nomor 3, Desember 2021 DAFTAR PUSTAKA Abraham, Rubin Adi. Menjadi Berkat Berbuah Lebat. Ke 5. Yogyakarta: Penerbit Andi, 2015. Afandi, Farid. Mari Mengenal Huruf Braille. Sidoarjo, 2017. Afandi, Farid. Mari Mengenal Huruf Braille. Sidoarjo, 2017. Al Ansori, Ade Nasihudin. “4 Klasifikasi Tunanetra Berdasarkan Jenis Kelainan Hingga Waktu Terjadinya.” Http://Liputan6.Com. Arrington, French L. Doktrin Kristen Perspektif Pentakosta. Ke 6. Yogyakarta: Penerbit Andi, 2020. Bebeng, Agus. “Sejarah Hari Braille Sedunia Di Tengah Ancaman Teknologi.” Http://Www.Kumparan.Com. p // p Budiman, Nadia Khairina. Penyebab Dan Pencegahan Kebutaan. Bandung, 2019. D.P, Halahan, and Kauffman J.M. Exceptional Learners: Introduction to Special Education. 10th ed. USA: Pearson, 2006. Hsu, Bi Min. “Braille Recognition for Reducing Asymmetric Communication between the Blind and Non-Blind.” Symmetry (2020). y y ( ) andari, Fetty. Situasi Gangguan Penglihatan. Infodatin Kemenkes RI. Jakarta, 2018. Jawasreh, Zaid Haron Musa, Noraidah Sahari Ashaari, and Dahlila Putri Dahnil. “The Acceptance of Braille Self-Learning Device.” International Journal on Advanced Science, Engineering and Information Technology (2020). Liauw, Suhento. Doktrin Alkitab Alkitabiah. Jakarta: Gereja Baptis Independen Alkitabiah GRAPHE, 2001. Natalia, Desca Lidya, and Hanni Sofia. “Presiden Umumkan Kasus Infeksi Corona Pertama Di Indonesia.” Antaranews.Com. Nugraha, Arie. “Rumah Ibadah Di Bandung Belum Ramah Bagi Disabilitas.” Liputan6.Com. Prabowo, Dani. “Ada Yang Lebih Susah Dari Kita, Yaitu Penyandang Disabilitas.” Kompas.Com. Radissa, Vanaja Syifa, Hery Wibowo, Sahadi Humaedi, and Maulana Irfan. “PEMENUHAN KEBUTUHAN DASAR PENYANDANG DISABILITAS PADA MASA PANDEMI COVID- 19.” Focus : Jurnal Pekerjaan Sosial (2020). Rifai, Aldi Ahmad, and Sahadi Humaedi. “INKLUSI PENYANDANG DISABILITAS DALAM SITUASI PANDEMI COVID-19 DALAM PERSPEKTIF SUSTAINABLE DEVELOPMENT GOALS (SDGs).” Prosiding Penelitian dan Pengabdian kepada Masyarakat (2020). Salurante, Tony, Aprianus Moimau, and Filmon Berek. “Tujuan Penciptaan Sebagai Cara Memahami Keberagaman Etika Dalam Kekristenan.” Jurnal Teologi Berita Hidup (2021). Samuel, Kirk. A. Klasifikasi Kebutaan. USA, 1991. Santoso, Samuel. “Wawancara,” 2020. Simanjuntak, Ferry. Metode Penelitian. Bandung: Sekolah Tinggi Teologi Kharisma j y g gg g Situmorang, Jonar T.H. Bibliologi. Ke 5. Yogyakarta: Penerbit Andi, 2017. Situmorang, Jonar T.H. Bibliologi. Ke 5. Yogyakarta: Penerbit Andi, 2017. Sugiyono. Metode Penelitian Kuantitatif Kualitatif Dan R&D. Bandung: Alfabeta, 2011. Taniharjo, Budisatyo. Integritas Seorang Pemimpin Rohani. Ke 5. Yogyakarta, 2019. g y f f g aniharjo, Budisatyo. Integritas Seorang Pemimpin Rohani. Ke 5. Yogyakarta, 2019. j y g g p gy Tim. “Bisa Baca Buku Gratis Dan Alkitab Tulisan Braille, Pasangan Suami Istri Tunanetra Apresiasi Rumah Baca Mutiara Bangsa.” Https://Hetanews.Com. p g p // Triyono, Sigit. “Alkitab Dan Disabilitas - Lembaga Alkitab Indonesia.” Https://Indonesia.Bible. p // Widinarsih, Dini. DAFTAR PUSTAKA “Penyandang Disabilitas Di Indonesia: Perkembangan Istilah Dan Definisi.” Jurnal Ilmu Kesejahteraan Sosial (2019). “Arti Kata Tunanetra-Kamus Besar Bahasa Indonesia (KBBI) Online.” KBBI Online, 2021. 150
https://openalex.org/W2951591995	https://europepmc.org/articles/pmc6924977?pdf=render	English	null	Preimplantation genetic testing for Huntington disease: the perspective of one Portuguese center	Porto biomedical journal/Porto Biomedical Journal	2,019	cc-by	3,533	a Serviço de Genética, Departamento de Patologia, Faculdade de Medicina, University of Porto, b Instituto de Investigação e Inovação em Saude, i3s, Universidade do Porto, c Centro de Genética da Reprodução A. Barros, d Departamento de Ginecologia-Obstetrícia e Pediatria, Faculdade de Medicina, Serviço de Ginecologia e Obstétricia, Centro Hospitalar Universitário S. João, Universidade do Porto, Porto, Portugal. ∗Corresponding author. Serviço de Genética, Departamento de Patologia, Faculdade de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal. E-mail address: ﬁlipac@med.up.pt (Filipa Carvalho). Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of PBJ-Associação Porto Biomedical/Porto Biomedical Society. All rights reserved. This is an open-access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Porto Biomed. J. (2019) 4:5(e48) Received: 12 March 2019 / Accepted: 4 July 2019 http://dx.doi.org/10.1097/j.pbj.0000000000000048 Original Article Original Article OPEN Abstract Background: Huntington disease (HD) is an autosomal dominant late-onset neurodegenerative disease caused by an unstable cytosine-adenine-guanine trinucleotide repeat expansion in the huntingtin (HTT) gene. Preimplantation genetic testing (PGT) is a diagnostic procedure available for these individuals, because they carry a high risk of transmitting this genetic condition to their offspring. Methods: Information about 15 HD couples referred for PGT and 21 cycles performed from 2009 to 2018 was collected retrospectively. PGT provide direct testing of embryos obtained after intracytoplasmic sperm injection, using polymerase chain reaction multiplex as the genetic testing protocol. Results:PGT for HD was performed in 15 couples, with no history of previous attempts, in a total of 21 cycles. The mean number of biopsied embryos per cycle was 4.9. The ampliﬁcation efﬁciency in blastomeres was 87.4%. From the 90 ampliﬁed embryos, 32 were normal and suitable for transfer. The mean number of transferred embryos per couple was 1.2. Overall, 3 positive human chorionic gonadotropin tests were obtained in 3 couples, resulting in 2 clinical pregnancies. The 2 ongoing clinical pregnancies had normal evolution, and culminated in 2 deliveries, resulting in the birth of 2 healthy children. Conclusions: PGT for HD is considered an effective and safe reproductive option for couples who are at risk of transmitting HD, when proper genetic and reproductive counseling is warranted. D is considered an effective and safe reproductive option for couples who are at risk of transmitting HD, eproductive counseling is warranted. Keywords: assisted reproductive technologies, embryo, Huntington, preimplantation genetic testing Preimplantation genetic testing for Huntington disease: the perspective of one Portuguese center Diogo Ferreira, MDa, Berta Carvalho, MSca,b, Ana P. Neto, MSca,b, Joaquina Silva, MDc, Ana M. Póvoa, MD, PhDb,d, Alberto Barros, MD, PhDa,b,c, Filipa Carvalho, PhDa,b,∗ ∗Corresponding author. Serviço de Genética, Departamento de Patologia, Faculdade de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal. E-mail address: ﬁlipac@med.up.pt (Filipa Carvalho). Porto Biomed. J. (2019) 4:5(e48) Methods All therapeutic procedures were done in accordance with the National Ethical Committee and National Council for Assisted Medical Reproduction. Informed consent was obtained from both partners after careful explanation of the treatment technique. This study was approved by the Ethical Committee from Centro Hospitalar S. João (Protocol n° 357/18). The mean number of biopsied embryos per cycle was 4.9. Laser drilling was the preferred method for zona breaching, during embryo biopsy. Embryos were biopsied at cleavage-stage, on day 3, and 1 or 2 blastomeres were retrieved for genetic analysis. Multiplex PCR was the genetic testing method used for DNA ampliﬁcation of each biopsied embryo. In average, 4.3 embryos per PGT-M cycle obtained a positive signal in the PCR reaction (Fig. 1). The ampliﬁcation efﬁciency in blastomeres was 87.4%. From the 90 ampliﬁed embryos, 32 were genetically transferable, whereas 58 were genetically not transferable (including mutated, haploid, and inconclusive embryos) (Table 1). ADO was detected in 3 embryos, from 3 different cycles. Couples obtained genetic and reproductive counseling by a clinical geneticist before being referred for PGT-M. All patients had normal karyotypes and were considered suitable candidates for this procedure. Controlled ovarian hyperstimulation was done by a gonadotro- pin releasing hormone agonist or antagonist protocol on female patients. After this treatment, oocytes were collected by ultraso- nography-guided follicular aspiration. Oocytes were fertilized by intracytoplasmic sperm injection (ICSI), which is preferred to conventional in vitro fertilization, because it prevents DNA contamination with sperm and/or cumulus cells during embryo biopsy. Embryo’s development was carefully evaluated every day. Embryos of type A (no anucleated fragmentation), type B (1%– 20% fragmentation), and type C (21%–50% fragmentation) were biopsied in day 3 after ICSI. One (embryos with 6 cells) or 2 (embryos with 7 or more cells) blastomeres were removed from each embryo. Controlled ovarian hyperstimulation was done by a gonadotro- pin releasing hormone agonist or antagonist protocol on female patients. After this treatment, oocytes were collected by ultraso- nography-guided follicular aspiration. Oocytes were fertilized by intracytoplasmic sperm injection (ICSI), which is preferred to conventional in vitro fertilization, because it prevents DNA contamination with sperm and/or cumulus cells during embryo biopsy. Embryo’s development was carefully evaluated every day. Results From 2009 to 2018, ﬁfteen HD couples were referred for PGT-M for HD. Twenty-one cycles were performed and 6 couples repeated the cycle once corresponding to a mean of 1.4 cycles per couple. Mutation was paternally derived in 6 couples, whereas in the other nine couples it was maternally derived corresponding to a male:female ratio of carriers/at risk persons of 1:1.5. None of these couples had a previous PGT-M attempt. The mean female age at beginning of each cycle was 34.5 years (Table 1). The Department of Genetics in the Faculty of Medicine/Centro Hospitalar Universitário São João has been the only Portuguese public center, since 1998, performingthis technique. Since then, the range of chromosomal disorders and monogenetic disorders for which PGT is available has expanded worldwide. In 2009, for the ﬁrst time, it was performed PGT-M in an HD couple in Portugal. The main goal of this work was to provide an overview about the uptake and outcome of PGT-M technique in HD couples, in the perspective of a Portuguese public center. The Department of Genetics in the Faculty of Medicine/Centro Hospitalar Universitário São João has been the only Portuguese public center, since 1998, performingthis technique. Since then, the range of chromosomal disorders and monogenetic disorders for which PGT is available has expanded worldwide. In 2009, for the ﬁrst time, it was performed PGT-M in an HD couple in Portugal. One couple had a reported male infertility history and so the sperm was obtained by Testicular Sperm Extraction. The main goal of this work was to provide an overview about the uptake and outcome of PGT-M technique in HD couples, in the perspective of a Portuguese public center. Following ovarian stimulation, the mean number of cumulus- oocyte complexes retrieved per cycle was 9.5 (ranging from 3 to 19). Overall, a mean of 7.6 oocytes per cycle were considered mature (metaphase II oocytes) and a mean of 5.6 oocytes were successfully fertilized (Table 1). Methods In 13 cycles it was possible to perform embryo transfer, with 17 embryos being transferred (including a frozen-thawed embryo transfer cycle from couple 8), corresponding to 14 transfers and a mean number of transferred embryos per couple of 1.2 (in the range 1–2). In some couples, although genetically transferable embryos were available, the transfer was not performed due to poor morphological quality of the embryos. Embryos of type A (no anucleated fragmentation), type B (1%– 20% fragmentation), and type C (21%–50% fragmentation) were biopsied in day 3 after ICSI. One (embryos with 6 cells) or 2 (embryos with 7 or more cells) blastomeres were removed from each embryo. Overall, 3 positive human chorionic gonadotropin (hCG) tests were obtained in 3 couples, resulting in 2 clinical pregnancies. One of the positive hCG ended as an ectopic pregnancy and termination of pregnancy (couple 13). The clinical pregnancy rate was 14.3% per transfer. Amniocentesis conﬁrmed PGT-M result in 1 couple, whereas the other couple decided not to perform prenatal diagnosis. There are different strategies to perform genetic analysis on single cells, but the most widely procedure is a multiplex polymerase chain reaction (PCR). In this protocol, ampliﬁcation of the disease-associated locus along with different informative polymorphic markers, known as “short tandem repeats,” which ﬂank the mutated HTT gene is done (D4S1614, D4S412, D4S127, and IVS-1 intronic marker). This strategy overcomes the potential threat of ADO and allows the detection of contamination. The 2 ongoing clinical pregnancies had normal evolution, and culminated in 2 deliveries (delivery rate/embryo transfer of 14.3%). The 2 cesarean sections deliveries resulted in the birth of 2 healthy children, 1 girl and 1 boy (Table 2). Before PCR ampliﬁcation, blastomeres were lysed with proteinase K by incubation at 45°C for 15minutes and then 98°C for 10minutes. Five sets of primers labelled with different ﬂuorochromes were used in multiplex PCR (Platinum Multiplex PCR Master Mix, Applied Biosystems, Foster City, Califórnia, USA) with the following PCR conditions: initial denaturation at 95°C for 2minutes and then 45 cycles with 95°C for 30seconds, 60°C for 90seconds, and 72°C for 60seconds. Final extension was done at 60°C for 30minutes. The ﬂuorescent PCR products were separated based on their size and ﬂuorochrome in an ABI PRISM 3500 Genetic Analyzer and analyzed using the appropriate software (Genemapper, Applied Biosystems). Introduction 1 Ferreira et al Porto Biomed. J. (2019) 4:5 Porto Biomedical Journal Porto Biomedical Journal Ferreira et al Porto Biomed. J. (2019) 4:5 After genetic diagnosis, 1 or 2 unaffected embryos (without expansion)andwithgoodmorphologicalqualitywereselectedand transferred into the uterus, on blastocyst stage (day 5 post-ICSI). Despite its numerous advantages, this procedure presents some risks and ethical and legal issues. It is technically complex and misdiagnosis may occur due to allele drop-out (ADO), an event in which one of the alleles is not properly ampliﬁed, and mosaicism, in case the biopsied blastomere is not representative of the total embryo. Main ethical problems relate to the moral status of the human embryo, embryo manipulation via assisted reproductive techniques, and eugenics.5 Introduction larger dimensions, due to a polyglutamine repeat in its structure. The elongated protein is fragmented in smaller toxic portions, which attach to each other and accumulate in different tissues, mainly in neurons, inducing their dysfunction and, ultimately, their death. Huntington disease (HD) is an autosomal dominantly inherited, late-onset neurodegenerative disease caused by a dynamic mutation in the huntingtin (HTT) gene: an expanded cytosine- adenine-guanine (CAG) triplet repeat.1HTT gene is responsible for the synthesis of the huntingtin protein. Normally, the CAG segment is repeated 10 to 35 times within the gene. In patients with HDtheCAGsegmentisrepeated36to>120times.HDisobserved at reduced penetrance for repeat ranging between 36 and 39 and at full penetrance for repeat counts >40.2 Repeat length is not stable during meiosis, and it can expand in the subsequent generations, particularly when mutation is paternally derived. HD prevalence in Western Europe varies between 5 and 10 per 100,000, similar to what is observed in Portugal.3,4 Clinically, symptoms begin at age of 35 to 44 years and rapidly progress, signiﬁcantly affecting patients’ quality of life. CAG repeat length is inversely correlated to age of onset. In fact, juvenile HD, a variant form of HD in young adults, is characterized by a large number of repeats, usually >60. Overall survival stands at 15 to 18 years and there is no current effective treatment.3 Normal HTT plays a vital role in brain development, being mainly found in striatum and cerebral cortex. Mutated HTT has Preimplantation genetic testing (PGT) is performed for couples at a high risk of transmitting a known genetic condition to their offspring and allows the diagnosis of chromosomal structural rearrangements (PGT-SR) and monogenic diseases (PGT-M). PGT-M is one of the reproductive options available for these individuals,becausethere isa 50% risk of a carriertransmitting the mutation to the offspring.4 It requires a multidisciplinary approach by a team of experts in gynecology/obstetrics, embryology, and medical genetics, which will follow the couple from the adequate genetic counseling until the birth of a healthy child. PGT-M is an alternative to prenatal diagnosis, involving the biopsy and genetic testing of single or few cells from preimplantation embryos and transfer of unaffected embryos for the genetic condition being tested to the patient’s uterus. PGT- M avoids the risk of induced abortion, the psychological burden associated to termination of pregnancy, and it is the most suitable option for couples with an increased genetic risk combined with infertility. Discussion Reproductive options available for couples with genetic disorders should be analyzed in each individual case, concerning their advantages and disadvantages. Although prenatal diagnosis has higher success rates, the possibility of termination of pregnancy in case of an unfavorable result represents one of the major concerns. Whether the introduction of PGT for HD has reduced or not the use of prenatal diagnosis in these patients is an interesting question, and should be studied in further inves- tigations in our population. 2 Ferreira et al Porto Biomed. J. Discussion (2019) 4:5 www.portobiomedicaljournal.com Table 1 Preimplantation Genetic Testing-M cycles performed for Huntington disease couples Table 1 Preimplantation Genetic Testing-M cycles performed for Huntington disease couples Couple Clinical cycle Female age, (yr) No oocytes No MII oocytes No fertilized oocytes No biopsied embryos No ampliﬁed embryos No genetically transferable embryos No genetically not transferable embryos ∗ No embryos transferred† hCG result Clinical pregnancy Delivery 1 1 29 10 9 8 8 8 4 4 0 2 1 36 7 5 4 4 4 1 3 1 – – No 2 37 8 6 4 4 4 1 3 1 – – No 3 1 34 7 5 5 5 5 3 2 1 – – No 2 35 12 9 3 2 2 1 1 0 4 1 35 5 4 3 3 3 1 2 0 5 1 35 4 3 3 2 2 2 0 2 + + Yes 6 1 33 5 3 3 3 3 0 3 0 7 1 37 14 12 4 3 2 1 1 0 8 1 38 13 12 9 8 0 0 0 0 2 39 11 10 9 7 7 4 3 1/1 –/–‡ –/– No 9 1 36 13 11 8 8 6 2 4 1 – – No 2 37 10 7 6 6 5 1 4 1 – – No 10 1 32 19 12 11 10 10 4 6 2 + + Yes 11 1 33 13 11 4 3 3 0 3 0 12 1 36 9 8 5 5 5 2 3 2 – – No 2 37 3 3 1 1 1 1 0 1 – – No 13 1 29 12 10 7 7 7 2 5 1 + –x No 2 30 6 5 2 2 2 1 1 1 – – No 14 1 28 14 11 11 11 10 0 10 0 15 1 39 5 4 2 1 1 1 0 1 – – No ∗Mutated, haploid, and inconclusive embryos. † In some cycles, although genetically transferable embryos were available, the transfer was not performed due to poor morphological quality of the embryos. ‡ Frozen-thawed embryo transfer cycle. x Ectopic pregnancy. Discussion Table 1 Preimplantation Genetic Testing-M cycles performed for Huntington disease couples tion Genetic Testing-M cycles performed for Huntington disease couples PGT has speciﬁc indications and its implementation is regulated by law and subject to the National Council for Medically Assisted Procreation approval. One of the indications accepted by law is HD. PGT is one of the available reproductive options for these couples, acknowledging the possibility of analysis for the presence of the triplet expansion, and/or genetically linked markers associated with the dynamic mutation, in human embryos. Figure 1. Electropherogram of the multiplex PCR amplifying simultaneously the cytosine-adenine-guanine (CAG) repeat (red) and the polymorphic markers D4S1614 (blue), D4S412 (blue), D4S127 (green), and IVS1-intronic marker (green). Figure 1. Electropherogram of the multiplex PCR amplifying simultaneously the cytosine-adenine-guanine (CAG) repeat (red) and the polymorphic markers D4S1614 (blue), D4S412 (blue), D4S127 (green), and IVS1-intronic marker (green). 3 Ferreira et al Porto Biomed. J. (2019) 4:5 Porto Biomedical Journal Table 2 Clinical information of the 2 babies at birth Baby 1 Baby 2 Couple 5 10 Sex Male Female Delivery mode Caesarian Caesarian Weight (g) 3045 3290 Height (cm) 48 50 Head circumference (cm) 34.5 35.5 Apgar score (1 min/5 min) 9/10 9/10 cycles. Increasing number of linked informative polymorphic markers are being used, to reduce the risk of misdiagnosis. The mean number of transferred embryos per couple was 1.2 (ranging from 1 to 2), comparable with that found in the literature.8 The clinical pregnancy rate and the delivery rate were both 14.3% per embryo transfer, which signiﬁcantly differs from what is observed in our center for other pathologies and also depicted in literature.7 One potential reason, which may explain this difference, is the small sized PGT-M sample for HD in this study. The pregnancy rates of the Department of Genetics in the Faculty of Medicine/Centro Hospitalar Universitário São João are in accordance with international data on PGT pregnancy rates for other pathologies. This analysis can be performed in 2 different modalities: direct testing of embryos or exclusion testing. References The mean age of woman at beginning of the ﬁrst cycle (34.5 years) was a bit higher than that reported in literature (32 years).7 This may be caused by the delay of Portuguese couples on searching for these treatments, waiting list, or by the lack of available information about PGT. [1] Sun YM, Zhang YB, Wu ZY. Huntington’s disease: relationship between phenotype and genotype. Mol Neurobiol. 2017;54:342–348. [2] Novak MJ, Tabrizi SJ. Huntington’s disease: clinical presentation and treatment. Int Rev Neurobiol. 2011;98:297–321. [3] Van Rij MC, De Rademaeker M, Moutou C, et al. Preimplantation genetic diagnosis (PGD) for Huntington’s disease: the experience of three European centres. Eur J Hum Genet. 2012;20:368–375. In our study, male:female ratio of HD carriers or at-risk persons was 2:3, matching the 40:60 ratio reported for couples opting for presymptomatic testing.3 [4] Associação Portuguesa de Doentes de Huntington. Hereditariedade [Web page] Alvor: APDH; 2008. Available from: http://www.huntington- portugal.com/hereditariedade. Accessed February 10, 2019. Although all couples were selected for ICSI procedure, most parents who undergo PGT do not have fertility problems, except 1 couple with a reported male infertility history. None of the 15 couples have beneﬁted on this technique in the past. [5] Teles NO. Diagnóstico genético pré-implantação – aspectos técnicos e considerações éticas. Acta Med Port. 2011;24:987–996. [6] De Die-Smulders CE, De Wert GM, Liebaers I, et al. Reproductive options for prospective parents in families with Huntington’s disease: clinical, psychological and ethical reﬂections. Hum Reprod Update. 2013;19: 304–315. According to literature, day 3 cleavage stage embryo biopsy remains the preferable biopsy method for PGT-M cycles, although it may switch to day 5 biopsy in the very near future.7 In our study, 100% of embryos were biopsied in day 3 after ICSI. From the embryos successfully biopsed, 87.4% gave a diagnostic result (vs 91%, from literature).7 Multiplex PCR may be subjected to several problems, including sample contamination, total PCR failure, and ADO. The latter phenomenon was detected in 3 (3.3%) embryos, from 3 different According to literature, day 3 cleavage stage embryo biopsy remains the preferable biopsy method for PGT-M cycles, although it may switch to day 5 biopsy in the very near future.7 In our study, 100% of embryos were biopsied in day 3 after ICSI. [7] De Rycke M, Goossens V, Kokkali G, et al. ESHRE PGD Consortium data collection XIV-XV: cycles from January 2011 to December 2012 with pregnancy follow-up to October 2013. Acknowledgements None. The authors report no conﬂicts of interest. The authors report no conﬂicts of interest. Conﬂicts of interest Our main results on PGT cycles for HD patients were compared to those internationally published, although we were aware on implications of working on different sample sizes. Discussion The latter is not approved in Portugal, but it is performed in other European centers, when couple decided not to be informed about their HD carrier status, and do not want to be subjected to presymptomatic testing.6 Despite the reported differences, we concluded that PGT-M for HD is considered an effective and safe reproductive option for couples who are at risk of transmitting HD, when proper genetic and reproductive counseling is warranted. European Society of Human Reproduction and Embryology PGT Consortium, established in 1997, collects, retrospectively and prospectively, data on PGT cycles, pregnancies, deliveries, and children. The later published report, covering monogenetic diseases, HLA typing, and chromosome abnormalities, docu- mented cycles performed from 2011 to 2012. References Hum Reprod. 2017;32: 1974–1994. y y p y From the embryos successfully biopsed, 87.4% gave a diagnostic result (vs 91%, from literature).7 Multiplex PCR may be subjected to several problems, including sample contamination, total PCR failure, and ADO. The latter phenomenon was detected in 3 (3.3%) embryos, from 3 different From the embryos successfully biopsed, 87.4% gave a diagnostic result (vs 91%, from literature).7 Multiplex PCR may be subjected to several problems, including sample contamination, total PCR failure, and ADO. The latter phenomenon was detected in 3 (3.3%) embryos, from 3 different [8] Van Rij MC, De Koning Gans PA, Van Belzen MJ, et al. The uptake and outcome of prenatal and pre-implantation genetic diagnosis for Huntington’s disease in the Netherlands (1998–2008). Clin Genet. 2014;85:87–95.
https://openalex.org/W2508045367	https://europepmc.org/articles/pmc4981397?pdf=render	English	null	Action Sport Cameras as an Instrument to Perform a 3D Underwater Motion Analysis	PloS one	2,016	cc-by	8,731	Gustavo R. D. Bernardina1, Pietro Cerveri2, Ricardo M. L. Barros3, João C. B. Marins1, Amanda P. Silvatti1 1 Department of Physical Education, Universidade Federal de Viçosa, Minas Gerais, Brasil, 2 Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Itália, 3 Faculty of Physical Education, Universidade Estadual de Campinas, São Paulo, Brasil a1111 gustavordalla@gmail.com * gustavordalla@gmail.com Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: RMLB received funding from Fundação de Amparo à Pesquisa do Estado de São Paulo (São Paulo Research Foundation) (www.fapesp.br) through Award Numbers 00/1293-1, 2006/02403-1, and 2009/09359-6. RMLB and APS received funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico (National Counsel of Technological and Scientific Development) (www. cnpq.br) through Award Numbers 473729/2008-3, 304975/2009-5, 478120/2011-7, 234088/2014-1, and 481391/2013-4. GRDB and this study received RESEARCH ARTICLE Abstract Action sport cameras (ASC) are currently adopted mainly for entertainment purposes but their uninterrupted technical improvements, in correspondence of cost decreases, are going to disclose them for three-dimensional (3D) motion analysis in sport gesture study and athletic performance evaluation quantitatively. Extending this technology to sport analy- sis however still requires a methodologic step-forward to making ASC a metric system, encompassing ad-hoc camera setup, image processing, feature tracking, calibration and 3D reconstruction. Despite traditional laboratory analysis, such requirements become an issue when coping with both indoor and outdoor motion acquisitions of athletes. In swim- ming analysis for example, the camera setup and the calibration protocol are particularly demanding since land and underwater cameras are mandatory. In particular, the underwa- ter camera calibration can be an issue affecting the reconstruction accuracy. In this paper, the aim is to evaluate the feasibility of ASC for 3D underwater analysis by focusing on cam- era setup and data acquisition protocols. Two GoPro Hero3+ Black (frequency: 60Hz; image resolutions: 1280×720/1920×1080 pixels) were located underwater into a swimming pool, surveying a working volume of about 6m3. A two-step custom calibration procedure, consisting in the acquisition of one static triad and one moving wand, carrying nine and one spherical passive markers, respectively, was implemented. After assessing camera param- eters, a rigid bar, carrying two markers at known distance, was acquired in several positions within the working volume. The average error upon the reconstructed inter-marker dis- tances was less than 2.5mm (1280×720) and 1.5mm (1920×1080). The results of this study demonstrate that the calibration of underwater ASC is feasible enabling quantitative kine- matic measurements with accuracy comparable to traditional motion capture systems. Editor: Roi Gurka, Coastal Carolina University, UNITED STATES Editor: Roi Gurka, Coastal Carolina University, UNITED STATES Received: March 4, 2016 Accepted: July 20, 2016 Published: August 11, 2016 Copyright: © 2016 Bernardina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. OPEN ACCESS Citation: Bernardina GRD, Cerveri P, Barros RML, Marins JCB, Silvatti AP (2016) Action Sport Cameras as an Instrument to Perform a 3D Underwater Motion Analysis. PLoS ONE 11(8): e0160490. doi:10.1371/ journal.pone.0160490 Action Sport Cameras as an Instrument to Perform a 3D Underwater Motion Analysis Gustavo R. D. Bernardina1, Pietro Cerveri2, Ricardo M. L. Barros3, João C. B. Marins1, Amanda P. Silvatti1 ASC to Perform a 3D Underwater Motion Analysis rehabilitation [8–9] and clinics [10–11]. However, optoelectronics and electromagnetic devices, mainly devoted to laboratory analysis, feature high costs and are not designed for both outdoor and underwater usage. Qualisys company distributes a video-based commercial system [12], specifically designed for underwater measurements using devoted illumination to enhance image quality. However, cameras still demand cables and the system is very expensive. funding from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazilian Federal Agency for Support and Evaluation of Graduation Education) (www.capes.gov.br) through Award Numbers 2011/ 10-7 and 08/2014. APS received funding from Fundação de Amparo à Pesquisa de Minas Gerais (Minas Gerais Research Foundation) (www.fapemig. br) through Award Number PEE-00596-14. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. An alternative video-based technology is represented by action sport cameras (ASC), which are currently used mainly for recreational purposes. Their uninterrupted technical improve- ments, in terms of image resolution and capture frequency, in correspondence of a cost decrease, are enabling them to sport gesture study and athletic performance evaluation [13– 17]. Recent works in the literature described the application of ASC for two-dimensional (2D) analysis [18–21]. Extending this technology for three-dimensional (3D) sport analysis using multiple cameras, however, still requires a methodologic step-forward to making ASC a metric system, encompassing ad-hoc camera setup, synchronization of the acquisitions, and devoted calibration protocols. Competing Interests: The authors have declared that no competing interests exist. In swimming motion analysis for example, the camera setup is particularly demanding as the athletic gesture develops both in air and underwater concurrently. ASC manufacturers par- tially addressed this issue by developing different accessories, especially designed for underwa- ter usage, as waterproof housings and support with suction cups to secure the cameras to the wall of the swimming pool. From an operational point of view, camera calibration represents a bottleneck to the development of video-based underwater motion analysis systems because of two main issues. First, the 3D reconstruction of the complete athletic gesture requires the cali- bration of both air and underwater cameras and a coordinate system registration in between them. At present time however, there are no standardized protocols available. Second, under- water calibration can require specific solutions addressing water disturbance of the image qual- ity to ensure high reconstruction accuracy. In order to achieve high accurate 3D underwater movement analysis, our group already addressed some critical points related mainly to underwater camera calibration. Using indus- trial cameras, we showed that the accuracy results of the wand-based and 2D plate-based cali- bration methods were less associated to the testing tool position in the working volume and provided better accuracy than the graduated rod-based calibration with nonlinear DLT [22]. The main advantage of using wand-based calibration was the equalization of the reconstruction error across the working volume, ensured by the bundle adjustment of the camera parameters. In contrast, 2D plate-based calibration led to an unregistered camera network as each camera was calibrated separately. However, 2D plate-based calibration was less sensitive to water qual- ity than wand-based. In the [23], we applied wand-based underwater calibration to reconstruct with high accuracy the hand trajectory of four swimmers during front-crawl, breaststroke and butterfly styles. In this paper, the underwater wand-based calibration procedure was applied to the ASC. Experimentally, two cameras were submerged into a swimming pool and located steady at the corners of a working volume of about 6m3. The measurement protocol encompassed calibra- tion and testing acquisitions to compute camera parameters and evaluate the reconstruction accuracy, respectively, with two different image resolutions. The effect of a set of calibration data acquisition strategies, on the 3D accuracy, was investigated. Introduction Motion capture systems are traditionally adopted to reconstruct the movements of animals and humans in different applications such as biomechanics [1–2], sport gesture analysis [3–7], PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 1 / 14 NE \| DOI:10.1371/journal.pone.0160490 August 11, 201 Materials and Methods Instruments The data acquisition was performed in a swimming pool. Two action sport cameras with water- proof housings (GoPro, Hero3+, Black Edition1 / USA), were fixed on the wall of the swimming 2 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 ASC to Perform a 3D Underwater Motion Analysis pool (see Fig 1). The view angle and acquisition frequency were 127° and 60Hz, respectively. Two different image resolutions were investigated, namely 1280×720 (LOWRES) and 1920×1080 (HIGHRES) pixels. In order to synchronize the cameras, we used the Wi-Fi remote GoPro control (see Fig 1B). After acquisition, the videos were converted to AVI movie format in the GoPro studio software. Calibration quality The calibration quality was evaluated by the accuracy of 3D reconstruction in both image reso- lutions (HIGHRES and LOWRES). We used five acquisitions of the rigid bar, carrying two markers at known distance (dn: 250 mm), moved within the working volume during 15 seconds (Fig 1C). The wand was manufactured by CNC machine ensuring a marker positioning accu- racy of about 10μm. In order to describe the 3D accuracy of ASC the following quantities were calculated for each trial: a) mean value of the marker distance; b) the standard deviation of the distance distribution; c) the mean absolute error (difference between the nominal and the mea- sured marker distances); d) the percentage accuracy (the ratio between the absolute accuracy and the maximum diagonal of the working volume) [27]. The error distributions for HIGHRES and LOWRES were compared using a non-parametric test (Wilcoxon rank sum) with a signifi- cance of 1%. The calibration quality was also evaluated in laboratory and were compared to the underwater condition, using same camera setup (two cameras, HIGHRES and LOWRES, 60Hz, camera position) and calibration protocol (triad and wand tools). Camera calibration procedure The wand-based calibration method consists in the acquisition of one static (a triad structure) and one moving (a wand structure) tool, carrying nine and one spherical passive markers, respectively. One waterproof orthogonal triad structure (1×1×1m) was built by a computer numerical control machine (CNC) screwing onto it nine spherical black markers (;: 35 mm) in known positions (10μm accuracy). The triad was located at the floor of the swimming pool, in the center of the working volume (1×4×1.5m3) and acquired for 5 seconds (see Fig 1A). 2D marker segmentation in videos and centroid computation were performed using “Dvideo” soft- ware [24]. 2D data of triad markers were used to assess the initial intrinsic and extrinsic param- eters of the cameras, using DLT method disregarding optical distortions [22], and define the origin and orientation of the working volume. In order to refine the camera parameters, also ensuring nonlinear optical distortion correction [25], a wand, carrying one spherical marker located at its extremity, was moved in the working volume, during about 20 seconds. “Dvideo” software was used again to track the marker in the image sequence of the two cameras. Four hundred useful video frames were used into a bundle adjustment nonlinear optimization, using control points with both known (triad markers) and unknown (wand marker) 3D coordinates [26]. The bundle adjustment iteratively estimates the parameters of all the cameras along with the unknown 3D marker coordinates by minimizing the 2D projection error (measured vs pre- dicted by the camera model) on the images. The optical distortion was taken into account by adding one radial parameter into the camera model (Fig 2). Calibration dependability Since the calibration quality can be affected by the performed wand acquisition movement [26], we evaluated the camera dependability testing three different acquisitions, namely zig-zag (M1), circular (M2) and up and down (M3). For this test, we used the high image resolution. As far as M1 is concerned, the operator was instructed to well cover all the camera field of view. In the M2 wand movement, the operator was instructed to perform circular movements 3 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 ASC to Perform a 3D Underwater Motion Analysis Fig 1. A) Calibration tools. B) Action sport cameras (GoPro, Hero3+, Black). C) Rigid bar used to 3D reconstruction accuracy evaluation. D) Camera position and acquisition volume. doi:10.1371/journal.pone.0160490.g001 Fig 1. A) Calibration tools. B) Action sport cameras (GoPro, Hero3+, Black). C) Rigid bar used to 3D reconstruction accuracy evaluation. D) Camera position and acquisition volume. doi:10.1371/journal.pone.0160490.g001 doi:10.1371/journal.pone.0160490.g001 within the working volume. In M3, the operator was instructed to move up-down the wand within the working volume. In addition, we evaluated whether to add an inter-marker distance constraint into the bundle adjustment (two-marker in the wand tool) could result in an improvement of the 3D reconstruction accuracy. Operatively, five different calibration tests (see Fig 3), explicitly M1-1 (one marker with zig-zig movement), M2-1 (one marker with circu- lar movement), M3-1 (one marker with up-down), M1-2 (two markers with zig-zig movement) and M2-2 (two markers with circular movement), were performed. The quality of all the five calibrations was assessed again by reconstructing the distance between the markers of the test rigid bar moved within the working volume during about 20s. within the working volume. In M3, the operator was instructed to move up-down the wand within the working volume. In addition, we evaluated whether to add an inter-marker distance constraint into the bundle adjustment (two-marker in the wand tool) could result in an improvement of the 3D reconstruction accuracy. Operatively, five different calibration tests (see Fig 3), explicitly M1-1 (one marker with zig-zig movement), M2-1 (one marker with circu- lar movement), M3-1 (one marker with up-down), M1-2 (two markers with zig-zig movement) and M2-2 (two markers with circular movement), were performed. The quality of all the five calibrations was assessed again by reconstructing the distance between the markers of the test rigid bar moved within the working volume during about 20s. Calibration quality In the five repeated calibrations, the reconstruction error was below 2.6mm for both image res- olutions. In Table 1, we listed mean of the distance between the markers, standard deviation and mean absolute errors (bias) of the five trials of the dynamic rigid bar test (2 markers). As expected, the bias of HIGHRES was lower (1.28mm) than that of LOWRES (2.41mm). The two error distributions were statistically (p-value<0.0001) different (see Fig 4). For com- parison, the 3D reconstruction in laboratory using HIGHRES and LOWRES led to a mean absolute error of 1.56mm and 1.67mm, respectively. In the five repeated calibrations, the reconstruction error was below 2.6mm for both image res- olutions. In Table 1, we listed mean of the distance between the markers, standard deviation and mean absolute errors (bias) of the five trials of the dynamic rigid bar test (2 markers). As expected, the bias of HIGHRES was lower (1.28mm) than that of LOWRES (2.41mm). The two error distributions were statistically (p-value<0.0001) different (see Fig 4). For com- parison, the 3D reconstruction in laboratory using HIGHRES and LOWRES led to a mean absolute error of 1.56mm and 1.67mm, respectively. Calibration dependability In order to analyze how the wand movement type and the number of markers affected the calibration dependability, we calculated the minimum, mean and maximum value of the dis- tance between the markers, the standard deviation and the mean absolute error, in all the five acquisition protocols (M1-1, M2-1, M3-1, M1-2 and M2-2). The five distance error distribu- tions were statistically analyzed by using a non-parametric test (Kruskal-Wallis) with a post- hoc (Tukey: p-value<0.05) (Matlab1 2012). PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 Calibration dependability There was a significant difference (p<0.001) among the five different calibrations (Table 2). Since the movement M1 spread more the working volume, we found the best accuracy results in M1-1 (1.17 mm) and M1-2 (1.28 mm). PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 4 / 14 ASC to Perform a 3D Underwater Motion Analysis Fig 2. Schematic workflow of the two-stage camera calibration for a generic number of cameras. doi:10.1371/journal.pone.0160490.g002 Fig 2. Schematic workflow of the two-stage camera calibration for a generic number of cameras. doi:10 1371/journal pone 0160490 g002 Fig 2. Schematic workflow of the two-stage camera calibration for a generic number of cameras. doi:10.1371/journal.pone.0160490.g002 doi:10.1371/journal.pone.0160490.g002 PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 5 / 14 ASC to Perform a 3D Underwater Motion Analysis Fig 3. Three different calibration wand movements to evaluate the calibration dependability. doi:10.1371/journal.pone.0160490.g003 Fig 3. Three different calibration wand movements to evaluate the calibration dependability. doi:10.1371/journal.pone.0160490.g003 doi:10.1371/journal.pone.0160490.g003 No significant difference was found when we compared M1 movement using or not the dis- tance constraints (Table 2). When the movement did not spread systematically the working volume (M2), the usage of the distance constraint in the bundle calibration improved signifi- cantly the accuracy of results (M2-1 = 1.80mm, M2-2 = 1.47mm, p<0.001). M3-1 acquisition protocol (non-systematic movement and one marker wand) yielded the worst reconstruction error (2.63mm). As noticed, when considering M1-2 and M2-2 (different movements but with bar length constraint), no statistical difference was found. Discussion Performing 3D kinematic analysis in sports, as in the case of swimming, requires high recon- struction accuracy. The usage of nonlinear camera calibration was reported to improve the accuracy results found in laboratory conditions [25, 28, 29]. Reconstruction errors, ranging from 0.58 to about 1mm, were obtained when using optoelectronic systems and industrial cam- eras [15, 30, 23]. The reconstruction accuracy of ASC (wide-angle lenses—GoPro) was evalu- ated out of water conditions obtaining 10mm error in correspondence of a linear camera model [31]. In the same paper, the authors reported that the accuracy increased by five times (2mm) when adopting a nonlinear camera model. This last result is in agreement with our GoPro laboratory test (cfr. Table 1). namic rigid bar test (HIGHRES and LOWRES). Nominal distance dn between the two markers: 250mm. Table 1. Results of the 5 trials of dynamic rigid bar test (HIGHRES and LOWRES). Nominal distance dn between the two markers: 250mm. Trial Mean ± SD (mm) Mean Absolute Error (mm) Error related to volume size HIGHRES LOWRES HIGHRES LOWRES HIGHRES LOWRES 1 249.95±1.81 248.0±1.70 1.36 2.22 1:3000 1:5200 2 250.27±1.67 247.8±1.30 1.29 2.28 1:3000 1:5300 3 249.91±1.55 247.86±1.78 1.22 2.37 1:3000 1:5500 4 250.11±1.71 247.51±1.78 1.35 2.63 1:3000 1:6100 5 250.04±1.48 247.60±1.65 1.19 2.55 1:3000 1:6000 Land 248.53±1.08 248.56±1.40 1.56 1.67 1:3600 1:3800 doi:10 1371/journal pone 0160490 t001 Table 1. Results of the 5 trials of dynamic rigid bar test (HIGHRES and LOWRES). Nominal distance dn betwe Table 1. Results of the 5 trials of dynamic rigid bar test (HIGHRES and LOWRE PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 6 / 14 ASC to Perform a 3D Underwater Motion Analysis Fig 4. The histograms of the residual error distribution (cumulated over the five trials) for HIGHRES (1920–1080) and LOWRES (1280–720). The average values were 1.28 and 2.41mm, respectively. doi:10.1371/journal.pone.0160490.g004 Fig 4. The histograms of the residual error distribution (cumulated over the five trials) for HIGHRES (1920–1080) and LOWRES (1280–720). The average values were 1.28 and 2.41mm, respectively. doi:10.1371/journal.pone.0160490.g004 doi:10.1371/journal.pone.0160490.g004 Underwater 3D analysis, based on cumbersome calibration structure and linear camera models, using traditional video-based systems, provided a reconstruction error higher than 5mm [32–35]. In [22], it was shown that underwater camera calibration using industrial cam- eras and nonlinear camera model, improves on average the reconstruction accuracy up to Table 2. ASC to Perform a 3D Underwater Motion Analysis 1mm across a working volume of about 7m3. This result was comparable with the values (2mm at 10m distance) reported by commercial systems devoted to 3D underwater analysis [12]. In the light of such prior results, the present study evaluated the reconstruction accuracy of underwater ASC calibrated using the wand method with a nonlinear camera model encom- passing optical distortions [22]. We found that the average error upon the reconstructed inter- marker distances was less than 1.5mm (HIGHRES underwater and Land) on average across the whole working volume of about 6m3, comparable with the values reported in our previous work [22], with reconstruction errors reported in [36] using GoPro cameras and in [12] using the Oqus-Underwater system. As far as the calibration dependability is concerned, the wand calibration movement, as expected, affected the reconstruction accuracy results. As shown (cfr. Table 2), spreading the wand systematically across the whole working volume (M1), led to the best accuracy results. The M3 protocol, featuring up-down wand movements without any systematic control, led in contrast to the poorest results. Circular movement (M2), while not systematically covering the entire working volume, ensured accuracy results less than 2mm, nonetheless worse than the results obtained with M1. The analysis of the calibration dependability related to the number of markers upon the calibration wand showed that one marker is sufficient when it is acquired well spread within the whole working volume. We point out that the use of two markers (dis- tance constraint) can improve the reconstruction accuracy making the result less dependent on the wand movement performed by the operator (see Table 2: M1-2 vs M2-2). Based on these considerations, we can argue that the wand-based calibration makes ASC suitable competitive to industrial cameras for underwater motion analysis. Some limits of the present study must be however discussed. First, we performed an evalua- tion of the potentiality of ASC in terms of calibration setup and reconstruction accuracy, disre- garding the environmental issues relative to underwater conditions. For instance, the image contrast, which can be affected by the illumination of swimming pool, and the water distur- bance, which is directly related to the speed of the swimmer, deteriorate the marker detection quality on the images (see Fig 5). Such environmental issues demand therefore specific testing to evaluate their effects on the 3D reconstruction accuracy. PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 Discussion Minimum, mean and maximum inter-marker distance, averaged across 5 trials and the corresponding mean absolute error (dn: 250mm). M1-1 (one marker with zig-zig movement); M2-1 (one marker with circular movement); M3-1 (one marker with up-down); M1-2 (two markers with zig-zig movement); M2-2 (two markers with circular movement). The post-hoc comparison results were reported (p<0.05). Table 2. Minimum, mean and maximum inter-marker distance, averaged across 5 trials and the corresponding mean absolute error (dn: 250mm). M1-1 (one marker with zig-zig movement); M2-1 (one marker with circular movement); M3-1 (one marker with up-down); M1-2 (two markers with zig-zig movement); M2-2 (two markers with circular movement). The post-hoc comparison results were reported (p<0.05). Calibration Distance (mm) Mean Absolute Error (mm) Post-hoc comparison p-value Minimum Mean Maximum M1-1 249.31 249.43 249.55 1.17 M1-2 0.08 M2-1 0.000 M2-2 0.01* M3-1 0.000* M1-2 249.07 249.15 249.23 1.28 M2-1 0.000* M2-2 0.06 M3-1 0.000* M2-1 248.57 248.60 248.67 1.80 M2-2 0.000* M3-1 0.000* M2-2 248.89 248.95 249.08 1.47 M3-1 0.000* M3-1 247.92 248.13 248.29 2.63 doi:10.1371/journal.pone.0160490.t002 Table 2. Minimum, mean and maximum inter-marker distance, averaged across 5 trials and the correspond M1-1 (one marker with zig-zig movement); M2-1 (one marker with circular movement); M3-1 (one marker with up-do movement); M2-2 (two markers with circular movement). The post-hoc comparison results were reported (p<0.05) er-marker distance, averaged across 5 trials and the corresponding mean absolute error (dn: 250mm). 1 (one marker with circular movement); M3-1 (one marker with up-down); M1-2 (two markers with zig-zig ovement). The post-hoc comparison results were reported (p<0.05). PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 7 / 14 We plan to perform a systematic analysis of this effect in future works. Second, the issue relative to the 3D kinematic analysis underwater is concerning the marker protocol utilized to compute the absolute and angular kinematics. While increasing the num- ber of markers ensures a better body segment definition, marker labeling and tracking proce- dures complicate and the swimmer performance would be greater affected by water drag increase [37]. In order to avoid it, the use of crosses or circles drawn on the swimmer skin was proposed [38]. Alternatively, a complete markerless approach could be applied as described in [39–40]. However, the segmentation of the complete swimmer silhouette on the images demand a complex network of camera with an enhanced underwater illumination to increase swimmer-to-water contrast [40]. Third, we did not perform an extensive evaluation of the role of the GoPro acquisition setup (image resolution, acquisition frequency and view angle of the camera). In this work, we aimed at studying the calibration accuracy for a typical size of the working volume ensuring at least one complete front crawl cycle. Arranging a 6m working volume wide and camera-to-working volume distance of about 3m required a camera view angle of about 130°. The only option from GoPro setup to cope with such demands was “medium”, featuring a view angle of 127°. With this camera setup we had available 24Hz, 30Hz, 48Hz and 60Hz frequencies. In order to cope with typical swimmer speed we choose 60Hz. A more systematic comparison among dif- ferent setup, allowed by GoPro (resolution, view angle and acquisition frequency), will be the subject of future evaluations. 8 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 ASC to Perform a 3D Underwater Motion Analysis Fig 5. Two instants of the front-crawl swim cycle surveyed by two cameras. The swimmer is equipped with surface markers attached to the right arm. This real condition elucidates how poor image contrast and water disturbance can complicate the automatic marker detection on the image and tracking analysis, affecting the accuracy of the 3D kinematic analysis. Fig 5. Two instants of the front-crawl swim cycle surveyed by two cameras. The swimmer is equipped with surface markers attached to the right arm. This real condition elucidates how poor image contrast and water disturbance can complicate the automatic marker detection on the image and tracking analysis, affecting the accuracy of the 3D kinematic analysis. Conclusions This article was conceived to mainly demonstrate the feasibility of the quantitative 3D mea- surements underwater using action sport cameras. We showed that, by endowing action sport cameras with an opportune calibration methodology (handy tools and bundle adjustment), they can be made an accurate metric system. Compared to optoelectronic devices, especially designed for 3D motion analysis, this technology features low cost, reduced size, high portabil- ity, wireless facility and waterproof housings. Swimming, underwater gait, water aerobics, water polo are relevant potential applications for such an emerging technology. doi:10.1371/journal.pone.0160490.g005 doi:10.1371/journal.pone.0160490.g005 Fourth, we did not consider wearable technology for benchmark comparison. Especially, inertial-magnetic measurements units (IMMUs) have been recently proposed in the literature for underwater kinematic analysis [41]. While being in principle plug & play as they are wire- less, these sensors can affect the swimmer performance due to the drag effect, which is aug- mented as the swimmer speed increases. It has to be pointed out that the drag causes for instance vibrations of the sensors, affecting the quality of the kinematic measure. In addition, to ensure underwater wireless data transmission, high capacity batteries are mandatory to cope with power consumption, increasing the size of the wearable devices. Supporting Information This is the 2D coordinates of the second rigid bar test acquisition, carrying two markers at known distance. File S. This is the Trial_3. This is the 2D coordinates of the third rigid bar test acqui- sition, carrying two markers at known distance. File T. This is the Trial_4. This is the 2D coor- dinates of the fourth rigid bar test acquisition, carrying two markers at known distance. File U. This is the Trial_5. This is the 2D coordinates of the fifth rigid bar test acquisition, carrying two markers at known distance. File V. This is the MotionSequence3D_Trial_1. This is the 3D coordinates of the first rigid bar test acquisition. File W. This is the MotionSequence3D_ Trial_2. This is the 3D coordinates of the second rigid bar test acquisition. File X. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File Y. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File Z. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.1.2_Land > S1.1.2.1_Highres > S1.1.2.1.1_Calibration > File AA. This is the BUNDLE_CameraParameters. This is the camera calibration parameters. File AB. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File AC. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.2.1.2_Accuracy > File AD. This is the Trial_Land. This is the 2D coordi- nates of the first rigid bar test acquisition, carrying two markers at known distance. File AE. This is the MotionSequence3D_Land. This is the 3D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. S1.1.2.2_Lowres > S1.1.2.2.1_Calibra- tion File AF. This is the BUNDLE_CameraParameters. This is the camera calibration parameters. File AG. This is the Triad. This is the 2D coordinates of the waterproof orthogo- nal triad structure carrying nine spherical black markers. File AH. This is the WandCalibra- tion. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.2.2.2_Accuracy > File AI. This is the Trial_Land. This is the 2D coordinates of the first rigid bar test acquisitions, carrying two markers at known distance. File AJ. This is the is the camera calibration parameters. File B. This is the Triad. Supporting Information This is the 2D coordinates of the second rigid bar test acquisition, carrying two mark- ers at known distance. File F. This is the Trial_3. This is the 2D coordinates of the third rigid bar test acquisition, carrying two markers at known distance. File G. This is the Trial_4. This is the 2D coordinates of the fourth rigid bar test acquisition, carrying two markers at known distance. File H. This is the Trial_5. This is the 2D coordinates of the fifth rigid bar test acqui- sition, carrying two markers at known distance. File I. This is the MotionSequence3D_Trial_ 1. This is the 3D coordinates of the first rigid bar test acquisition. File J. This is the MotionSe- quence3D_Trial_2. This is the 3D coordinates of the second rigid bar test acquisition. File K. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File L. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File M. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.1.1.2_Lowres > S1.1.1.2.1_Calibra- tion > File N. This is the BUNDLE_CameraParameters. This is the camera calibration parameters. File O. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File P. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.1.2.2_Accuracy > File Q. This is the Trial_1. This is the 2D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. File R. This is the Trial_2. This is the 2D coordinates of the second rigid bar test acquisition, carrying two markers at known distance. File S. This is the Trial_3. This is the 2D coordinates of the third rigid bar test acqui- sition, carrying two markers at known distance. File T. This is the Trial_4. This is the 2D coor- dinates of the fourth rigid bar test acquisition, carrying two markers at known distance. File U. This is the Trial_5. This is the 2D coordinates of the fifth rigid bar test acquisition, carrying two markers at known distance. File V. This is the MotionSequence3D_Trial_1. This is the 3D coordinates of the first rigid bar test acquisition. File W. This is the MotionSequence3D_ Trial_2. Supporting Information S1 Supporting Information. S1.1_Calibration Quality > S1.1.1_Underwater > S1.1.1.1_ Highres > S1.1.1.1.1_Calibration > File A. This is the BUNDLE_CameraParameters. This PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 9 / 14 ASC to Perform a 3D Underwater Motion Analysis is the camera calibration parameters. File B. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File C. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.1.1.2_Accuracy > File D. This is the Trial_1. This is the 2D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. File E. This is the Trial_2. This is the 2D coordinates of the second rigid bar test acquisition, carrying two mark- ers at known distance. File F. This is the Trial_3. This is the 2D coordinates of the third rigid bar test acquisition, carrying two markers at known distance. File G. This is the Trial_4. This is the 2D coordinates of the fourth rigid bar test acquisition, carrying two markers at known distance. File H. This is the Trial_5. This is the 2D coordinates of the fifth rigid bar test acqui- sition, carrying two markers at known distance. File I. This is the MotionSequence3D_Trial_ 1. This is the 3D coordinates of the first rigid bar test acquisition. File J. This is the MotionSe- quence3D_Trial_2. This is the 3D coordinates of the second rigid bar test acquisition. File K. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File L. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File M. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.1.1.2_Lowres > S1.1.1.2.1_Calibra- tion > File N. This is the BUNDLE_CameraParameters. This is the camera calibration parameters. File O. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File P. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.1.2.2_Accuracy > File Q. This is the Trial_1. This is the 2D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. File R. This is the Trial_2. Supporting Information This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File C. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.1.1.2_Accuracy > File D. This is the Trial_1. This is the 2D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. File E. This is the Trial_2. This is the 2D coordinates of the second rigid bar test acquisition, carrying two mark- ers at known distance. File F. This is the Trial_3. This is the 2D coordinates of the third rigid bar test acquisition, carrying two markers at known distance. File G. This is the Trial_4. This is the 2D coordinates of the fourth rigid bar test acquisition, carrying two markers at known distance. File H. This is the Trial_5. This is the 2D coordinates of the fifth rigid bar test acqui- sition, carrying two markers at known distance. File I. This is the MotionSequence3D_Trial_ 1. This is the 3D coordinates of the first rigid bar test acquisition. File J. This is the MotionSe- quence3D_Trial_2. This is the 3D coordinates of the second rigid bar test acquisition. File K. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File L. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File M. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.1.1.2_Lowres > S1.1.1.2.1_Calibra- tion > File N. This is the BUNDLE_CameraParameters. This is the camera calibration parameters. File O. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File P. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. is the camera calibration parameters. File B. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File C. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.1.1.2_Accuracy > File D. This is the Trial_1. This is the 2D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. File E. This is the Trial_2. PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 Supporting Information This is the 3D coordinates of the second rigid bar test acquisition. File X. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File Y. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File Z. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.1.2_Land > S1.1.2.1_Highres > S1.1.2.1.1_Calibration > File AA. This is the BUNDLE_CameraParameters. This is the camera calibration parameters. File AB. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File AC. This is the WandCalibration. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.2.1.2_Accuracy > File AD. This is the Trial_Land. This is the 2D coordi- nates of the first rigid bar test acquisition, carrying two markers at known distance. File AE. This is the MotionSequence3D_Land. This is the 3D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. S1.1.2.2_Lowres > S1.1.2.2.1_Calibra- tion File AF. This is the BUNDLE_CameraParameters. This is the camera calibration parameters. File AG. This is the Triad. This is the 2D coordinates of the waterproof orthogo- nal triad structure carrying nine spherical black markers. File AH. This is the WandCalibra- tion. This is the 2D coordinates of the wand structure carrying one spherical black marker. S1.1.2.2.2_Accuracy > File AI. This is the Trial_Land. This is the 2D coordinates of the first rigid bar test acquisitions, carrying two markers at known distance. File AJ. This is the PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 10 / 14 ASC to Perform a 3D Underwater Motion Analysis MotionSequence3D_Land. This is the 3D coordinates of the first rigid bar test acquisitions. S1.2_Calibration Dependability > S1.2.1_Calibration > File AK. This is the BUNDLE_Ca- meraParameters_ Cal_Mov1_1p. This is the camera calibration parameters of the first move- ment (M1) with one marker. File AL. This is the BUNDLE_CameraParameters_Cal_Mov1_ 2p. This is the camera calibration parameters of the first movement (M1) with two markers. File AM. This is the BUNDLE_CameraParameters_Cal_Mov2_1p. This is the camera cali- bration parameters of the second movement (M2) with one marker. File AN. This is the BUN- DLE_CameraParameters_Cal_Mov2_2p. This is the camera calibration parameters of the second movement (M2) with two markers. File AO. This is the BUNDLE_CameraParame- ters_Cal_Mov3_1p. Supporting Information This is the camera calibration parameters of the third movement (M3) with one marker. File AP. This is the Triad. This is the 2D coordinates of the waterproof orthogonal triad structure carrying nine spherical black markers. File AQ. This is the Cal_Mo v1_1p. This is the 2D coordinates of the wand structure carrying one spherical black marker using the first movement (M1). File AR. This is the Cal_ Mov1_2p. This is the 2D coordinates of the wand structure carrying two spherical black markers using the first movement (M1). File AS. This is the Cal_Mov2_1p. This is the 2D coordinates of the wand structure carrying one spherical black marker using the second movement (M2). File AT. This is the Cal_Mov2_ 2p. This is the 2D coordinates of the wand structure carrying two spherical black markers using the second movement (M2). File AU. This is the Cal_ Mov3_1p. This is the 2D coordi- nates of the wand structure carrying one spherical black marker using the third movement (M3). S1.2.2_Accuracy > File AV. This is the Trial_1. This is the 2D coordinates of the first rigid bar test acquisition, carrying two markers at known distance. File AW. This is the Trial_2. This is the 2D coordinates of the second rigid bar test acquisition, carrying two mark- ers at known distance. File AX. This is the Trial_3. This is the 2D coordinates of the third rigid bar test acquisition, carrying two markers at known distance. File AY. This is the Trial_4. This is the 2D coordinates of the fourth rigid bar test acquisition, carrying two mark- ers at known distance. File AZ. This is the Trial_5. This is the 2D coordinates of the fifth rigid bar test acquisition, carrying two markers at known distance. S1.2.2.1_Mov1_1p > File BA. This is the MotionSequence3D_Trial_1. This is the 3D coordinates of the first rigid bar test acquisition. File BB. This is the MotionSequence3D_ Trial_2. This is the 3D coordinates of the second rigid bar test acquisition. File BC. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File BD. This is the MotionSequen- ce3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File BE. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acqui- sition. S1.2.2.2_Mov1_2p > File BF. This is the MotionSequence3D_Trial_1. PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 Writing - review & editing: GRDB APS PC RMLB JCBM. Writing - review & editing: GRDB APS PC RMLB JCBM. Supporting Information This is the 3D coordinates of the first rigid bar test acquisition. File BV. This is the MotionSequence3D_Trial_2. This is the 3D coordinates of the second rigid bar test acqui- sition. File BW. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File BX. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File BY. This is the MotionSequen- ce3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. (ZIP) Supporting Information This is the 3D coordinates of the first rigid bar test acquisition. File BG. This is the MotionSequence3D_ Trial_2. This is the 3D coordinate of the second rigid bar test acquisition. File BH. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File BI. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File BJ. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.2.2.3_Mov2_1p > File BK. This is the MotionSequence3D_Trial_1. This is the 3D coordinates of the first rigid bar test acquisition. File BL. This is the MotionSequence3D_Trial_2. This is the 3D coordinates of the second rigid bar test acquisition. File BM. This is the MotionSequence3D_Trial_3. This is the 3D PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 11 / 14 ASC to Perform a 3D Underwater Motion Analysis This is the 3D coordinates of the second rigid bar test acquisition. File BR. This is the Motion- Sequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File BS. This is the MotionSequence3D_ Trial_4. This is the 3D coordinate of the fourth rigid bar test acquisition. File BT. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.2.2.5_Mov3_1p > File BU. This is the MotionSequen- ce3D_Trial_1. This is the 3D coordinates of the first rigid bar test acquisition. File BV. This is the MotionSequence3D_Trial_2. This is the 3D coordinates of the second rigid bar test acqui- sition. File BW. This is the MotionSequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File BX. This is the MotionSequence3D_Trial_4. This is the 3D coordinates of the fourth rigid bar test acquisition. File BY. This is the MotionSequen- ce3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. (ZIP) This is the 3D coordinates of the second rigid bar test acquisition. File BR. This is the Motion- Sequence3D_Trial_3. This is the 3D coordinates of the third rigid bar test acquisition. File BS. This is the MotionSequence3D_ Trial_4. This is the 3D coordinate of the fourth rigid bar test acquisition. File BT. This is the MotionSequence3D_Trial_5. This is the 3D coordinates of the fifth rigid bar test acquisition. S1.2.2.5_Mov3_1p > File BU. This is the MotionSequen- ce3D_Trial_1. Conceptualization: GRDB APS PC JCBM. Conceptualization: GRDB APS PC JCBM. Funding acquisition: APS RMLB. Investigation: GRDB APS. Resources: APS RMLB. Resources: APS RMLB. Software: GRDB APS PC RMLB. Supervision: APS. Visualization: APS PC. Visualization: APS PC. Writing - original draft: GRDB APS. Writing - review & editing: GRDB APS PC RMLB JCBM. PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 5. Joyce C, Burnett A, Cochrane J, Ball K. Three-dimensional trunk kinematics in golf: between-club differ- ences and relationships to clubhead speed. Sport Biomech. 2013; 12(2): 108–120. References Wahid F, Begg R, McClelland JA, Webster KE, Halgamuge S, Ackland DC. A multiple regression nor- malization approach to evaluation of gait in total knee arthroplasty patients. Clin Biomech. 2016; 32:92–101. doi: 10.1016/j.clinbiomech.2015.12.012 12. Qualysis Oqus Underwater. 2014. Available: http://www.qualisys.com/wp-content/uploads/2014/01/ PI_Oqus_Underwater.pdf 13. Chong AK. Low-cost compact cameras: a medical application in CMT disease monitoring. Photogramm Rec. 2011; 26(134): 263–273. doi: 10.1111/j.1477-9730.2011.00638.x 14. Silvatti AP, Sarro KJ, Cerveri P, Baroni G, Barros RML. A 3D kinematic analysis of breathing patterns in competitive swimmers. J Sports Sci. 2012; 30(14): 1551–1560. doi: 10.1080/02640414.2012.713976 PMID: 22897476 15. Bernardina, GRD., Silva, RG, Cerveri, P., Barros RML and Silvatti AP, Accuracy of sport action cam- eras for 3d underwater motion analysis. Proceedings of the 32 International Conference of Biomechan- ics in Sports 2014. pp. 505–508 ISSN 1999-4168. 16. Monnet T, Samson M, Bernard A, David L, Lacouture P. Measurement of three-dimensional hand kine- matics during swimming with a motion capture system: a feasibility study. Sports Eng. 2014; 17(3): 171–181. doi: 10.1007/s12283-014-0152-4 17. Fujii K, Shinya M, Yamashita D, Kouzaki M, Oda S. Anticipation by basketball defenders: An explana- tion based on the three-dimensional inverted pendulum model. Eur J Sport Sci. 2014; 14(6): 538–546. doi: 10.1080/17461391.2013.876104 PMID: 24397711 18. García-Ramos A, Feriche B, de la Fuente B, Argüelles-Cienfuegos J, Strojnik V, Strumbelj B, et al. Relationship between different push-off variables and start performance in experienced swimmers. Eur J Sport Sci. 2015; 15(8): 687–695. doi: 10.1080/17461391.2015.1063699 PMID: 26305175 19. McDonnell L, Hume P, Nolte V. Sprint kayaking stroke rate reliability, variability and validity of the Digi- trainer accelerometer compared to GoPro video measurement. In Proceedings of the XXX International Symposium on Biomechanics in Sports, Melbourne, Australia. 2012. pp. 316–319. ISSN 1999-4168. 20. Timmis MA, Turner K, van Paridon KN. Visual Search Strategies of Soccer Players Executing a Power vs. Placement Penalty Kick. PLoS ONE. 2014; 9(12): 115–179. doi: 10.1371/journal.pone.0115179 21. Vieira LHP, Pagnoca EA, Milioni F, Barbieri RA, Menezes RP, Alvarez L, et al. Tracking futsal players with a wide-angle lens camera: accuracy analysis of the radial distortion correction based on an improved Hough transform algorithm. Comput Methods Biomech Biomed Eng Imaging Vis. 2015. doi: 10.1080/21681163.2015.1072055 22. Silvatti AP, Dias FAS, Cerveri P, Barros RML. Comparison of different camera calibration approaches for underwater applications. J Biomech. 2012; 45(6): 1112–1116. doi: 10.1016/j.jbiomech.2012.01.004 PMID: 22284990 23. Silvatti AP, Cerveri P, Telles T, Dias FAZ, Baroni G, Barros RML. References 1. Olsen E, Andersen PH, Pfau T. Accuracy and Precision of Equine Gait Event Detection during Walking with Limb and Trunk Mounted Inertial Sensors. Sensors. 2012; 12(6): 8145–8156. doi: 10.3390/ s120608145 PMID: 22969392 2. Safayi S, Jeffery ND, Shivapour SK, Zamanighomi M, Zylstra TJ, Bratsch-Prince J, et al. Kinematic analysis of the gait of adult sheep during treadmill locomotion: Parameter values, allowable total error, and potential for use in evaluating spinal cord injury. J Neurol Sci. 2015; 358: 107–112. doi: 10.1016/j. jns.2015.08.031 PMID: 26341152 3. Sinclair J, Richards J, Taylor PJ, Edmundson CJ, Brooks D, Hobbs SJ. Three-dimensional kinematic comparison of treadmill and overground running. Sport Biomech. 2013; 12(3): 272–282. doi: 10.1080/ 14763141.2012.759614 4. Bates NA, Ford KR, Myer GD, Hewett TE. Impact differences in ground reaction force and center of mass between the first and second landing phases of a drop vertical jump and their implications for injury risk assessment. J Biomech. 2013; 46(7):1237–1241. doi: 10.1016/j.jbiomech.2013.02.024 PMID: 23538000 5. Joyce C, Burnett A, Cochrane J, Ball K. Three-dimensional trunk kinematics in golf: between-club differ- ences and relationships to clubhead speed. Sport Biomech. 2013; 12(2): 108–120. PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 12 / 14 ASC to Perform a 3D Underwater Motion Analysis 6. Seminati E, Marzari A, Vacondio O, Minetti AE. Shoulder 3D range of motion and humerus rotation in two volleyball spike techniques: injury prevention and performance. Sport Biomech. 2015; 14(2):216– 231. doi: 10.1080/14763141.2015.1052747 7. Begon M, Leardini A, Belvedere C, Farahpourc N, Allard P. Effects of front a land sagittal thorax atti- tudes in gait on trunk and pelvis three-dimensional kinematics. Med Eng Phys. 2015; 37(10):1032–6. doi: 10.1016/j.medengphy.2015.08.001 PMID: 26337553 8. Klotz MCM, Kost L, Braatz F, Ewerbeck V, Heitzmann D, Gantz S, et al. Motion capture of the upper extremity during activities of daily living in patients with spastic hemiplegic cerebral palsy. Gait Posture. 2013; 38(1):148–152. doi: 10.1016/j.gaitpost.2012.11.005 PMID: 23218727 9. Maier MW, Niklasch M, Dreher T, Zeifang F, Rettig O, Klotz MC, et al. Motion patterns in activities of daily living: 3- year longitudinal follow-up after total shoulder arthroplasty using an optical 3D motion analysis system. BMC Musculoskelet Disord. 2014; 15(244). doi: 10.1186/1471-2474-15-244 10. Gefen A, Megido-Ravid M, Itzchak Y, Arcan M. Biomechanical Analysis of the Three-Dimensional Foot Structure During Gait: A Basic Tool for Clinical Applications. J Biomech Eng. 2000; 122(6): 630–639. doi: 10.1115/1.1318904 PMID: 11192385 11. PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 References Quantitative underwater 3D motion analysis using submerged video cameras: accuracy analysis and trajectory reconstruction. Comput Methods Biomech Biomed Eng. 2013; 16(11): 1240–1248. doi: 10.1080/10255842.2012.664637 24. Figueroa PJ, Leite NJ, Barros RML. A flexible software for tracking of markers used in human motion analysis. Comput Methods Programs Biomed. 2003; 72(2): 155–165. doi: 10.1016/S0169-2607(02) 00122-0 PMID: 12941519 25. Cerveri P, Borghese NA, Pedotti A. Complete calibration of a stereo photogrammetric system through control points of unknown coordinates. J Biomech. 1998; 31(10): 935–940. doi: 10.1016/S0021-9290 (98)00104-3 PMID: 9840759 13 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 ASC to Perform a 3D Underwater Motion Analysis 26. Cerveri P, Pedotti A, Borghese NA. Combined evolution strategies for dynamic calibration of video- based measurement systems. IEEE Trans Evolut Comput. 2001; 5(3): 271–282. doi: 10.1109/4235. 930315 27. Chiari L, Della Croce U, Leardini A, Cappozzo A., Human movement analysis using stereophotogram- metry. Part 2: instrumental errors. Gait Posture. 2005, 21(2):197–211. doi: 10.1016/j.gaitpost.2004.04. 004 PMID: 15639399 28. Zhang Z. A flexible new technique for camera calibration. IEEE Trans Pattern Anal. 2000; 22(11): 1330–1334. doi: 10.1109/34.888718 29. Wang J, Shi F, Zhang J, Liu Y. A new calibration model of camera lens distortion. Pattern Recognit. 2008; 41(2): 607–615. doi: 10.1016/j.patcog.2007.06.012 30. Pribanić T, Sturm P, Cifrek M. Calibration of 3D kinematic systems using 2D calibration plate. In Pro- ceedings of the XXVI International Symposium on Biomechanics in Sports, Seoul, Korea. 2008. pp. 77–80. ISSN 1999-4168. 31. Balletti C, Guerra F, Tsioukas V, Vernier P. Calibration of action cameras for photogrammetric pur- poses. Sensors. 2014; 14(9): 17471–17490. doi: 10.3390/s140917471 PMID: 25237898 32. Yanai T, Hay JG, Gerot JT. Three-dimensional videography of swimming with panning periscopes. J Biomech. 1996; 29(5): 673–678. doi: 10.1016/0021-9290(95)00123-9 PMID: 8707797 33. Kwon YH, Lindley SL. Applicability of four localized-calibration methods in underwater motion analysis. In Proceedings of the XVIII International Symposium on Biomechanics in Sports, Hong Kong, China. 2000. pp. 48–55. ISSN 1999-4168. 34. Gourgoulis V, Aggeloussis N, Kasimatis P, Vezos N, Boli A, Mavromatis G. Reconstruction accuracy in underwater three-dimensional kinematic analysis. J Sci Med Sport. 2008; 11(2): 90–95. doi: 10.1016/j. jsams.2007.02.010 PMID: 17544326 35. Jesus K, Jesus K, Figueiredo P, Vilas-Boas JP, Fernandes RJ, Machado LJ. Reconstruction accuracy assessment of surface and underwater 3D motion analysis: a new approach. Comput Math Methods Med. 2015. Article ID 269264. doi: 10.1155/2015/269264 36. Shortis M. Calibration Techniques for accurate measurements by underwater camera systems. PLOS ONE \| DOI:10.1371/journal.pone.0160490 August 11, 2016 41. Fantozzi S, Giovanardi A, Borra D, Gatta G. Gait kinematic analysis in water using wearable inertial magnetic sensors. PLos ONE. 2015; 10(9): e0138105. doi: 10.1371/journal.pone.0138105 PMID: 26368131 References Sen- sors 2015, 15(12), 30810–30826; doi: 10.3390/s151229831 PMID: 26690172 37. Magalhaes FA, Sawacha Z, Di Michele R, Cortesi M, Gatta G, Fantozzi S., Effectiveness of an auto- matic tracking software in underwater motion analysis. J Sports Sci Med. 2013; 12(4):660–7. PMID: 24421725 38. Ceccon S, Ceseracciu E, Sawacha Z, Gatta G, Cortesi M, Cobelli C, Fantozzi S. Motion analysis of front crawl swimming applying CAST technique by means of automatic tracking. J Sports Sci., 2013; 31(3) 276–287. doi: 10.1080/02640414.2012.729134 PMID: 23057667 39. Ceseracciu E, Sawacha Z, Fantozzi S, Cortesi M, Gatta G, Corazza S, et al. Markerless analysis of front crawl swimming. J Biomech. 2011; 44(12): 2236–2242. doi: 10.1016/j.jbiomech.2011.06.003 PMID: 21719017 40. Ceseracciu E, Sawacha Z, Cobelli C. Comparison of markerless and marker-based motion capture technologies through simultaneous data collection during gait: proof of concept. PLoS ONE. 2014; 9 (3): e87640. doi: 10.1371/journal.pone.0087640 PMID: 24595273 41. Fantozzi S, Giovanardi A, Borra D, Gatta G. Gait kinematic analysis in water using wearable inertial magnetic sensors. PLos ONE. 2015; 10(9): e0138105. doi: 10.1371/journal.pone.0138105 PMID: 26368131 14 / 14
https://openalex.org/W3042811906	https://europepmc.org/articles/pmc7363091?pdf=render	English	null	Clarifying the structure of serious head and spine injury in youth Rugby Union players	PloS one	2,020	cc-by	7,602	PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Clarifying the structure of serious head and spine injury in youth Rugby Union players Koh SasakiID1, Haruhiko Sato2, Akihiko Nakamura3, Takumi Yamamoto4, Ichiro Watanabe5, Takashi Katsuta6, Ichiro Kono7 1 Research Center of Health, Physical Fitness, and Sports, Nagoya University, Nagoya, Japan, 2 Department of Neurosurgery, Seirei Mikatahara General Hospital, Shizuoka, Japan, 3 Department of Pediatric Surgery, Nakamura Hospital, Tokyo, Japan, 4 Faculty of Education, National Defense Academy, Yokosuka, Japan, 5 Faculty of Liberal Arts, Tokyo City University, Tokyo, Japan, 6 High Performance Sport Center, Japan Sport Council, Tokyo, Japan, 7 2019 Rugby World Cup Organizing Committee, Tokyo, Japan sasakikoh@htc.nagoya-u.ac.jp a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS OPEN ACCESS Citation: Sasaki K, Sato H, Nakamura A, Yamamoto T, Watanabe I, Katsuta T, et al. (2020) Clarifying the structure of serious head and spine injury in youth Rugby Union players. PLoS ONE 15 (7): e0235035. https://doi.org/10.1371/journal. pone.0235035 Editor: Filipe Manuel Clemente, Instituto Politecnico de Viana do Castelo, PORTUGAL Received: September 30, 2019 Accepted: June 8, 2020 Published: July 15, 2020 OPEN ACCESS Citation: Sasaki K, Sato H, Nakamura A, Yamamoto T, Watanabe I, Katsuta T, et al. (2020) Clarifying the structure of serious head and spine injury in youth Rugby Union players. PLoS ONE 15 (7): e0235035. https://doi.org/10.1371/journal. pone.0235035 Editor: Filipe Manuel Clemente, Instituto Politecnico de Viana do Castelo, PORTUGAL Received: September 30, 2019 Accepted: June 8, 2020 Published: July 15, 2020 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0235035 Abstract This study aimed to clarify the cause of rugby head and spinal cord injuries through a net- work centrality analysis of 14-year (2004–2018) longitudinal data in Japan. The study hypothesis is that understanding the causal relationship among the occurrence of serious injuries, the quality of player experience and play situation as a network structure could be possible to obtain practical knowledge on injury prevention. In this study, bipartite graphs are used to make it easier to understand the situation of players and injuries. This would also help to elucidate more characteristic subgroup. A network bipartite graph and subgroup (cluster) analyses were performed to clarify the injured players’ experience and the cause of injury. We used the algorithm of R program, IGRAPH, clustering edge betweenness. For subgroup extraction, the modularity Q value was used to determine which step to cut. The Japanese rugby population was 93,873 (2014–2018 average), and 27% were high school students. The data showed that careful attention would be particularly needed for groups of inexperienced Japanese high school players. Our study suggests that we should consider introducing rules that prohibit “head-on collisions” in youth rugby. * sasakikoh@htc.nagoya-u.ac.jp PLOS ONE PLOS ONE Introduction Some traditional studies focusing on network structures were introduced in the period from 1960 to 1970, including the “small world phenomenon”, “the strength of weak ties”, and “scale free network”. In addition, the specification of the “cluster community” or “sub-group network” were also developed to determine with social network robustness. Sociological approaches are widely used for predictive models of big data in the field of information tech- nology and cerebral functions in biology [8,9,10]. Network analysis methodologies have been applied in social and natural science approaches used in sport sciences. Within the social-science approaches, there are social psychological debates on how sports assist the development of cooperation values within a specific society [6,11]. In addition, political and economic studies have been reported that declining trends in sports and health promotion activities have a negative impact on the civil economy network [12,13,14,15]. Natural science approaches include discussions on the causes of sports injuries [4,13]. The factors that play a central role in various physiological parameters during exercise-induced fatigue were clarified by network analysis, and its application to risk management was discussed [13]. The direct relationship among some performance indicators in sports had also been investigated [14,15,16,17,18,19], which involves strategies and tactics in competitive dimensions. It was con- sidered that there was a central player called a hub in the passing behavior research of a soccer game, and there was a power law there. Furthermore, the hub dynamically switched throughout the game. The difference between dynamic networks and static networks is that the former focuses on the variable and diverse play structures occurring in sports games [18]. How is the importance of network dynamism discussed? There exist important hub func- tions, sub-group communities, and actors playing some central roles within a network. The important role of network analysis would be to reveal the decisive and pragmatic structures required to obtain specific goals among complex networks as well as to identify individuals who are fulfilling the principal roles. This could be discussed from the perspective of network centrality [20,21]. Network centrality clarifies an organization’s temporal and bipartite or mul- tilayered structures, which would help us understand the driving force of the network dyna- mism [22]. This study aimed to investigate the network relationship of injury cause structure in a sports field by clarifying the situation of serious injury by network centrality analysis. Introduction In Japan, approximately 20 cases of serious injuries occurred annually since 2011 [1]. Data uti- lizing long-term data would contribute to its prevention. In rugby, tackle plays to stop the opponent’s attack by physical contact are allowed by rules. This could lead to more serious injuries in adolescents, where the growth gap is large. A longitudinal study of adolescent inju- ries may be an important issue. However, studies on serious rugby injuries are few, which are mostly short-term or conceptual ones [2,3,4], and longitudinal studies on these cases are scarce. The reason why there are few longitudinal studies would be that the social rules for eth- ical use of medical diagnosis and for protecting personal information were not sufficiently established. Copyright: © 2020 Sasaki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. The occurrence of injury will have a relationship structure, such as experience differences and growth differences, as well as physical contact situations. The approach of grasping the PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 1 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby Funding: Initials of the authors who received JSPS KAKENHI grant Number 16K01658(2016-2018) and 19K11549(2019-2021). Funding: Initials of the authors who received JSPS KAKENHI grant Number 16K01658(2016-2018) and 19K11549(2019-2021). network structure of its occurrence might be useful for safety management of tackle situations in Rugby. Network analysis has applications in many disciplines including physics, computer science, biology, economics, ecology, and sociology. From a broad perspective, networks can be viewed as integrative structures of the physical society or lifeforce. Additionally, the relationship that links structural entities is always dynamic. Network analysis was found to be a valuable new tool for exploring, depicting, and explaining the individual relationships that impact team dynamics [5]. Studying these collaborative structures might allow discovery of the possibilities of new survival abilities in a society. Competing interests: The authors have declared that no competing interests exist. Network analysis has been developed in the field of communication network studies [6,7]. Introduction The study hypothesis is that understanding the causal relationship among the occurrence of serious injuries, the quality of player experience and the play situation as a network structure, it could be possible to obtain more practical knowledge on injury prevention. Data collection The data were obtained from rugby serious injury reports for the past 14 years [1]. Registered teams of the Japan Rugby Football Union are obligated to report within 3 days, 2 months, and PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 2 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby 6 months after a serious injury. Multiple specialist doctors determine the classification of the injury: 1) death, 2) loss of consciousness over 24 hours, 3) spinal cord injury with quadriplegia, 4) craniotomy and spinal surgery, 5) visceral injury surgery, and 6) seriously injured from medical certificate. The referring data of the Japan Rugby Union serious injury report were fully anonymized. The anonymized data were locked in multiple stages in a hard disk drive that was not connected to the internet. All procedures used in this study were approved by the Ethics Committee of the Research Center for Health, Physical Fitness, and Sports, Nagoya University. In this study, high school players were divided into the following groups according to their age and years of experience: 16E1 (players aged 16 years with 1-year rugby experience), 17E2 (players aged 17 years with 1-2-year experience), 18E3 (players aged 18 years with 1-3-year experience), 16EM (players aged 16 years with many years of experience), and 17EM (players aged 17 years with many years of experience), 18EM (players aged 18 years with many years of experience). Caused of injuries were categorized into 1) own tackle, 2) oppose tackle, 3) rack, a one-on- one situation; 4) collision not categorized as above, 5) saving, hold the ball on the ground, or 6) others or unknown. In addition, the causes of injury were further classified as mauls (3 or more players battling the ball) and scrum (8 players battling the ball). Moreover, seasons were categorized as summer training camp (August) and the main official season (November, Feb- ruary). These classification processes were performed by the safety management committee of the Japanese Rugby Football Union. This committee works closely with the medical commit- tee, coaching committee, referee committee, and technical committee in Japanese Rugby Union, cooperates in classification of the cause of injury based on the detailed play situation reported by the team manager and doctor. PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 Network analysis and statistical analysis Nodes of network in this study are player groups (age and years of experience), types of inju- ries, and play styles that cause injuries (tackle, tackled, scrum, etc.). It clarifies the continuity of what kind of players’ experience years, what kind of play, and what part of body collision caused the injury. These are the dynamic networks which focus on the diverse play structures occurring throughout rugby game. Although this study would clarify a kind of local properties of networks, it might be considered as global properties by promoting international joint study in the future. A bipartite graph divides a network into two subsets and has no edges between nodes in each set [23]. In this study, bipartite graphs are used to make it easier to understand the situa- tion of players and injuries. Map layout was calculated by Fruchterman-Reingold Algorithm which is a force-directed layout algorithm for centralization of the injury factors [24]. This would also help to elucidate more characteristic subgroup. We used the algorithm of R pro- gram, IGRAPH, clustering edge betweenness. The idea of the edge betweenness based commu- nity structure detection is that edges connecting separate modules have high edge betweenness as all the shortest paths from one module to another must traverse through them [25,26]. Edge-betweenness centrality represents the degree of being located in the shortest path bridge between nodes. It was adopted because it would indicate a close relationship among players, injuries, and the causes. The limitation of this metric is that its approximate value may become unstable in case of a large-scale network graph [27,28]. In the bipartite graphs, players’ experiences, injury details, and caused plays were plotted in the adjacent matrix. For drawing the graph, centrality, obtained by identifying which items occupy the critical positions in injury condition, is a major focus. Subgroup that was central in PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 3 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby the network area are extracted from the relatively dense area. If the adjacent matrix of the graph is A = (αij), the number of nodes of the entire graph is n, the number of nodes belonging the subgroup is ns, and the cohesion S of the subgroup is next [10]. Network analysis and statistical analysis S ¼ P i2S P j2Saij nsðns 1Þ = P i2S P j=2Saij nsðn nsÞ There were some clustering methods that extract subgroup such as random walks [29], greedy algorithm [30], overlapping [31], or spin glass [32]. The present study repeated the cal- culation of edge-betweenness centrality and the removal of the edge with the maximum betweenness centrality to detect a highly cohesive subgroup. The use of edge-mediated central- ity makes it possible to extract a subgroup relatively easily [25,26] For subgroup extraction, the modularity Q value was used to determine which step to cut. If the extracted subgroup set is C, the number of edges connected from community i to com- munity j is ???, and the total number of edges included in the entire graph structure is m, Mod- ularity is defined as follows [33]: Q ¼ X i2C feij=2m ð X j2C eij=2mÞ ^2Þg The closer the modularity Q value is to 1, the more appropriate the cluster is divided. There were few graphs close to 1 in this study. Results In the entire domestic rugby population of 93,873 (2004–2018 average), there is a higher per- centage (27%) of high school students (~18 years old). Among all high school players, 57% (24,918–10,693 = 14,225) started from high school, indicating that they have a relatively low experience. The serious injury ratio of the high school players was high (head: 41%, spine: 41%, chest abdominal: 50%, circulation: 38%, average: 43%. High school students suffered a relatively high frequency of head injuries during the summer camp in August. This season would be a first time for high school first graders (16 years old) to be suffered of high load practices and games of rugby. Many injuries occurred during the summer camp (August) and the game season (February). (Table 1) Table 1. Japanese rugby population, number (ration) of serious injuries, and number of head injuries (HI) and all serious injuries (ASI) by month in High school players (nc; not clear). Japanese Rugby Population Age grade -12 -15 -18 -22 23- (women) Total Number (%) 19,975 (21) 10,510(11) 24.918(27) 10,693(12) 26,204(28) 570 (1) 93,873(100) Number (Ration) of serious injuries in Japan (nc; not clear) Head nc nc 48 (41) nc nc nc 117 Spine nc nc 54 (41) nc nc nc 133 Chest Abdominal nc nc 4 (50) nc nc nc 8 Circulation nc nc 10 (38) nc nc nc 26 Number of head injuries (HI) and all serious injuries (ASI) by month in High school players (nc; not clear) Month 4 5 6 7 8 9 10 11 12 1 2 3 nc N of HI 2 0 5 4 14 3 6 3 2 5 0 3 0 N of ASI 6 3 2 4 7 4 7 3 2 5 7 3 2 ation) of serious injuries, and number of head injuries (HI) and all serious injuries (ASI) by month in High school Table 1. Japanese rugby population, number (ration) of serious injuries, and number of head injuries (HI) and all serious injuries (ASI) by month in High school players (nc; not clear). panese rugby population, number (ration) of serious injuries, and number of head injuries (HI) and all serious injuries (A not clear). Table 1. Japanese rugby population, number (ration) of serious injuries, and number of head injuries (HI) and all seriou players (nc; not clear). Head injury Table 2 shows that there is a higher occurrence of acute subdural hematoma (ASH) in the high school players. Thirty-five cases (76%) out of the 48 head injuries were occurred in inexperi- enced players. The causes of the injuries were own tackle and oppose tackle, accounting for 83% of cases. There were also other collision situations. Table 3 shows the cause of play and the body area of collision. Injury reports suggested that the head position of the injured player tended to be lower than that the opposite’s knee height. Many injured parts were at frontal depressions and many situations were in tackles (Table 3). The ratio of inexperienced players with ASH was high (79%; 22 out of the 28), and the cause play was still due to tackle (Table 2). The number of players who recovered were 13, The ratio of inexperienced players with ASH was high (79%; 22 out of the 28), and the cause play was still due to tackle (Table 2). The number of players who recovered were 13, those with aftereffects were 6, those who died were 2, and those with unknown injuries were 7. The 16E1 group showed unfortunate results. Table 2. Results PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 4 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby Spinal cord injury A total of 54 spinal cord injuries occurred in high school students (Table 1). Forty-three play- ers (80%) with spinal cord injuries were inexperienced (Table 2). Causes of spinal cord injuries in the inexperienced players were OT (oppose tackle), T (own tackle), Ruck (a one-on-one sit- uation), Maul (3 or more players battling the ball), C (collision not categorized as above), Sav- ing, and SC (8 players battling with the ball), (Table 3). The cause of the load occurred not only in one-on-one tackles, but also under heavy weights pressure by multiple players, espe- cially in the head and spine, such as scrum or maul (Table 3). Head injury High school players’ head injuries, spinal cord injuries, the age and experiences of playing years (ASH: acute subdural hematoma, FR: fracture, AEH; acute epidural hematoma, ICH/CC: intra cerebral hemorrhage, cerebral contusion, CI/CA: cerebral infarction, cerebrovascular accident O: others or not clear, OTHG: oppose tackle and head to ground, THG; tackle and head to ground, THB: tackle and head to oppose body, C: collision not categorized as above, R: rack, S: saving, VF: vertebral fractureDS: dislocation of spine, FD: fracture dislocation, CC: central cord, S: Spinal cord, CSC: Cervical spinal cord, OT: oppose tackle, T: own tackle, Rack: a one-to-one situation, M: maul (3 or more players battling the ball), C: collision not categorized as above, Saving; hold the ball on the ground, SC: scrum (8 players battling with the ball)) p y g )) Players 16all 16E1 17all 17E2 18all 18E3 Total head Injuries ASH 15 12 8 6 5 4 28 FR 2 1 3 1 0 0 5 AEH 2 1 0 0 3 3 5 ICH/CC 0 0 1 1 4 2 5 CI/CA 1 0 1 1 0 0 2 O 2 2 1 1 0 0 3 Total 22 16 14 10 12 9 48 cause play of ASH OTHG 7 7 3 1 1 1 11 THG 3 2 4 4 2 2 9 THB 3 2 0 0 0 0 3 C 2 1 0 0 0 0 2 R 0 0 1 0 1 S 0 0 1 1 1 O 1 1 0 0 1 Total 15 12 8 6 5 4 28 Spinal cord injuries VF 1 1 6 6 6 3 13 DS 1 1 7 5 5 5 13 FD 2 0 6 6 3 3 11 CC 0 0 2 1 4 1 6 S 1 1 3 3 0 0 4 CSC 3 2 0 0 2 2 5 O 1 0 1 1 0 0 2 Total 9 7 25 22 20 14 54 cause play of spinal cord injuries OT 2 1 3 2 2 1 7 T 2 2 10 10 9 7 21 R 2 1 5 4 3 1 10 M 0 0 2 2 3 3 5 C 0 0 2 1 0 0 2 S 1 1 0 0 0 0 1 SC 3 2 3 3 2 2 8 Total 10 6 25 22 19 14 54 https //doi org/10 1371/jo rnal pone 0235035 t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 5 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby Table 3. Head injury Causes of injury and the body area of collision (H-G; head to ground, H-H; head to head, H-L; head to leg, H-B; head to the other part of opponent’s body part, LH; low head (head under the opponent’s body), O; others, T; own tackle, OT; oppose tackle, R; ruck (a one-on-one situation), C; collision not categorized as above, S; saving (hold the ball on the ground), O; other or unknown, SC; scrum (8 players battling with the ball), M; maul (3 or more players battling the ball), and cause of the load (MW; multi players weights, LH; low head (head under oppose’ body), H-B; head to the other part of oppose body, N-B: Neck to other part of oppose body, O: Others). Table 3. Causes of injury and the body area of collision (H-G; head to ground, H-H; head to head, H-L; head to leg, H-B; head to the other part of opponent’s body part, LH; low head (head under the opponent’s body), O; others, T; own tackle, OT; oppose tackle, R; ruck (a one-on-one situation), C; collision not categorized as above, S; saving (hold the ball on the ground), O; other or unknown, SC; scrum (8 players battling with the ball), M; maul (3 or more players battling the ball), and cause of the load (MW; multi players weights, LH; low head (head under oppose’ body), H-B; head to the other part of oppose body, N-B: Neck to other part of oppose body O: Others) causes of injury and the body area of collision cause of the load H-G H-H H-L H-B LH O Total MW LH H-B N-B O Total T 12 1 0 0 0 0 13 4 7 4 2 4 21 OT 6 4 5 7 3 2 27 2 0 0 3 2 7 R 0 0 2 0 0 0 2 8 0 0 0 2 10 C 1 2 0 0 0 1 4 0 0 2 0 0 2 S 0 0 1 0 0 0 1 0 0 1 0 0 1 O 0 0 0 0 0 0 1 SC 8 0 0 0 0 8 M 5 0 0 0 0 5 https://doi org/10 1371/journal pone 0235035 t003 https://doi.org/10.1371/journal.pone.0235035.t003 Junior high and elementary school players A total of 20 serious injuries were found in elementary and junior high school players (Table 4). The injuries in these age groups should also be prevented the expansion. In terms of body parts and caused of injuries, there were many cases of head and spinal cord injuries caused by a tackle. There were also many cases of the head hitting the ground. A total of 20 serious injuries were found in elementary and junior high school players (Table 4). The injuries in these age groups should also be prevented the expansion. In terms of body parts and caused of injuries, there were many cases of head and spinal cord injuries caused by a tackle. There were also many cases of the head hitting the ground. Table 4. Serious injuries, injured body parts and caused plays, and the possible sequelae and the collision situations (ASH: acute subdural hematoma, ICH/CC: intra cerebral hemorrhage, cerebral contusion, TSH: traumatic subarachnoid hemorrhage, FR: Fracture, H-G: head to ground, H-H/B: head to head or/and the other part of opposes’ body, nc: not clear). Serious injury Injured body parts and caused plays year 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Age (years old) Caused plays -12 13 14 15 T OT R M O Head 2 0 0 0 1 3 1 0 1 0 1 1 4 4 3 3 5 1 2 Spine 0 0 1 0 0 2 0 0 1 0 0 1 4 1 2 1 2 chest abdominal 0 0 0 0 0 0 0 0 0 0 2 0 1 1 1 heatstroke 0 0 1 1 0 0 0 0 0 0 0 0 Injuries, the possible sequelae and the collision situations. possible sequelae collision situations. possible sequelae Total H-G H-H/B nc ASH 2 8 3 3 ICH/CC 1 2 1 TSH 1 1 FR 3 3 https://doi.org/10.1371/journal.pone.0235035.t004 Table 4. Serious injuries, injured body parts and caused plays, and the possible sequelae and the collision situations (ASH: acute subdural hematoma, ICH/CC: intra cerebral hemorrhage, cerebral contusion, TSH: traumatic subarachnoid hemorrhage, FR: Fracture, H-G: head to ground, H-H/B: head to head or/and the other part of opposes’ body, nc: not clear). Junior high and elementary school players parts and caused plays, and the possible sequelae and the collision situations (ASH: acute subdural hematoma, ICH/CC: tusion, TSH: traumatic subarachnoid hemorrhage, FR: Fracture, H-G: head to ground, H-H/B: head to head or/and the ) Table 4. Serious injuries, injured body parts and caused plays, and the possible sequelae and the collision situations (ASH: acute subdural hematoma, ICH/CC: intra cerebral hemorrhage, cerebral contusion, TSH: traumatic subarachnoid hemorrhage, FR: Fracture, H-G: head to ground, H-H/B: head to head or/and the other part of opposes’ body, nc: not clear). PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 6 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby Clustering by edge betweenness centrality Given that the number of serious injury data were not large enough (48 head injuries, 54 spinal cord injuries), we tried to extract the central subgroup in the network. The ratio of the rela- tionship density between the nodes in the subgroup and the nodes outside the subgroup indi- cates the cohesion of the subgroup. It is a community detection approach based on edge betweenness centrality. The graph is divided from the calculation of the edge-betweenness cen- trality and the removal of the edge having the maximum betweenness centrality, and a highly cohesive community is detected. Head injuries We made a bipartite graph of age, years of experience, and head injury symptoms (Fig 1). From the edge-betweenness centrality analysis in the network, the central subgroup comprised young and inexperienced players [16E1 (players aged 16 years with 1-year rugby experience), 17E2 (players aged 17 years with 2-year experience), 18E3 (players aged 18 years with 3-year experience), 16EM (players aged 16 years with many years of experience)] suffered serious injuries such as ASH, skull fracture, cerebral infarction/cerebrovascular accident, and others (Fig 1, clustering edge-betweenness, Modularity Q value = 0.5). A subgroup analysis of causes of serious injuries include own tackle and opponents’ tackle situations. The collisions parts were “head to ground”, “head to head”, ‘head to opponent’s body”, “head under opponent’s body” and others. The collision position of the head during Fig 1. Age, grade, years of rugby playing experience, presence of head injury [inexperienced players (16 years old and with only 1 year of experience, 16 years old and with many years of experience, 17 years old and with 2 years of experience, 17 years old and with many years of experience), and presence of serious injuries like such as ASH: acute subdural hematoma, FR: skull fracture, CI/CA: cerebral infarction/cerebrovascular accident others (the circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.5)]. htt //d i /10 1371/j l 0235035 001 Fig 1. Age, grade, years of rugby playing experience, presence of head injury [inexperienced players (16 years old and with only 1 year of experience, 16 years old and with many years of experience, 17 years old and with 2 years of experience, 17 years old and with many years of experience), and presence of serious injuries like such as ASH: acute subdural hematoma, FR: skull fracture, CI/CA: cerebral infarction/cerebrovascular accident others (the circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.5)]. https://doi.org/10.1371/journal.pone.0235035.g001 Fig 1. Head injuries Age, grade, years of rugby playing experience, presence of head injury [inexperienced players (16 years old and with only 1 year of experience, 16 years old and with many years of experience, 17 years old and with 2 years of experience, 17 years old and with many years of experience), and presence of serious injuries like such as ASH: acute subdural hematoma, FR: skull fracture, CI/CA: cerebral infarction/cerebrovascular accident others (the circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.5)]. https://doi.org/10.1371/journal.pone.0235035.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 7 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby Fig 2. Causes of serious injuries (T: own tackle, OT: oppose tackle, S: saving, R: rack, C: other collisions, H-H: head to head, H-B: head to opponent’s body, RH: head under opponent’s body, O: other; the circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.23). https://doi org/10 1371/journal pone 0235035 g002 T: own tackle, OT: oppose tackle, S: saving, R: rack, C: other collisions, H-H: head to head, H-B: head to opponent’s body, O: other; the circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.23). Fig 2. Causes of serious injuries (T: own tackle, OT: oppose tackle, S: saving, R: rack, C: other collisions, H-H: head to head, H-B: head to opponent’s body, RH: head under opponent’s body, O: other; the circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.23). https://doi.org/10.1371/journal.pone.0235035.g002 tackle and the manner of falling after tackle could be related to serious injuries (Fig 2, Modu- larity Q value = 0.23). Focusing on the injury and the cause of injury for ASH only, it became clear that a young and inexperienced player tackled and collided his head with the ground or the opponent’s body part, or was involved in other collisions. The data on whether the player left the place as soon as possible after the tackle was also extracted (Fig 3, Modularity Q value = 0.37). Spinal cord injuries The spinal injury graphs and the result of the cluster analysis showed that players with spinal cord injuries were older than those with head injuries, but both patient groups had short play- ing experience. The injuries including VF: vertebral fracture, DS: dislocation of spine, FD: frac- ture dislocation, S: spinal cord occurred in inexperienced players with 3 years of experience (16E1, 17E2, 18 E3) (Fig 4, Modularity Q value = 0.14) The causes of the spinal cord injuries include plays under multiple weight pressure, such as scrums, in addition to the one-on-one situation of tackles. It would be necessary to strictly manage the scrum and maul plays in high school players (Fig 5, Modularity Q value = 0.1). Network analysis revealed that high school players with one or two years of experience tended to suffer from serious head and spinal injuries caused by tackles. Careful consideration for this age would be desired. Discussion The study hypothesis was to clarify the fact that the causes of serious rugby injuries such as head injury and spinal cord injury are due to the years of player experience. That knowledge could help to propose practical rules for injury prevention. A playing experience of 1 year was defined as the period ranging from 1 to 365 days. First year high school students (16 years old) experience first summer camp in approximately 100 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 8 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby Fig 3. Injury and cause of injury for acute subdural hematoma only (THG: tackle and head to ground, THB: tackle and head to the opponent’s body, OTHG: oppose tackle and head to ground, C: other collision, S: saving, R: rack, O: other; circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.37). Fig 3. Injury and cause of injury for acute subdural hematoma only (THG: tackle and head to ground, THB: tackle and head to the opponent’s body, OTHG: oppose tackle and head to ground, C: other collision, S: saving, R: rack, O: other; circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0 37) https://doi.org/10.1371/journal.pone.0235035.g003 days (3–4 months) after their starting day. The inexperienced players are forced to face severe physical contacts with opponents with large differences in physique and experience. Many team players who participate in national competitions have more than 10 years of experience. These teams and teams including inexperienced players play against each other during sum- mer camps or national competition qualifiers in the Autumn. There is no doubt that the so- called mismatch situation would be one of the causes of serious injuries [34,35]. Based on the findings of this study, it is an urgent issue to construct special guidelines that would consider the mismatch in cross-age and cross-body size of players. Previous studies on rugby injury also mentioned the difference in physique concerning serious injury [1,2]. Our study suggests that we should consider introducing rules that prohibit head-on collisions in youth inex- perienced rugby players. Such specific rules could be developed in collaboration with medical, refereeing, technical and coaching experts. PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 https://doi.org/10.1371/journal.pone.0235035.g003 Discussion The injury prevention guidelines would also signalize the attention of governments (Japan Sports Agency, Japan Sports Council) and elite rugby organi- zations (World Rugby) in terms of sports policies that can prevent these serious injuries. The numbers of female rugby players are also increasing. The empirical study to prevent serious injuries to female players is also urgent. Our study of female rugby players suggested that serious injuries at the lower leg, especially the knee joint that is unique to female athletes, as well as head injuries including concussion [3]. With the aim of acquiring valuable knowledge from data with a graph structure, we per- formed graph mining [33]. The approaches include frequent pattern detection [36], structure prediction [37], extraction of subgraphs that extract dense nodes (such as subgroup extrac- tion), and calculation of node importance. Among them, it is important to identify closely related communities from network graphs by subgroup extraction and to find highly influen- tial nodes [33]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 9 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby Fig 4. Spinal injuries and years of playing experience (VF: vertebral fracture, DS: dislocation of spine, FD: fracture dislocation, S: spinal cord, CC: central cord, CSC: cervical spinal cord, O: others; Circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.14). https://doi org/10 1371/journal pone 0235035 g004 Fig 4. Spinal injuries and years of playing experience (VF: vertebral fracture, DS: dislocation of spine, FD: fracture dislocation, S: spinal cord, CC: central cord, CSC: cervical spinal cord, O: others; Circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.14). Fig 4. Spinal injuries and years of playing experience (VF: vertebral fracture, DS: dislocation of spine, FD: fracture dislocation, S: spinal cord, CC: central cord, CSC: cervical spinal cord, O: others; Circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.14). https://doi.org/10.1371/journal.pone.0235035.g004 https://doi.org/10.1371/journal.pone.0235035.g004 The closer the modularity Q value is to 1, the more appropriate the cluster is divided. There were few graphs close to 1 in this study. Whether this is due to the number of data or different factors would be a future task. The value of 0.5 (Fig 1) was a relatively good clustering, which leads to some persuasive considerations. However, even if the modularity values were less than Fig 5. Discussion Years of playing experience and cause of spinal injuries (T: tackle, OT: oppose tackle, SC: scrum, R: rack, M: maul, SV: saving, C: other collision; circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.1). https://doi org/10 1371/journal pone 0235035 g005 Fig 5. Years of playing experience and cause of spinal injuries (T: tackle, OT: oppose tackle, SC: scrum, R: rack, M: maul, SV: saving, C: other collision; circle was the extracted subgroup by clustering edge betweenness, Modularity Q value = 0.1). https://doi.org/10.1371/journal.pone.0235035.g005 10 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby 0.5, those graphs might be useful findings, suggesting structures of serious injury processes in young rugby players. This study examined the cause of serious injuries with a bipartite graph. However, in order to consider the relational structure more deeply, multi-stage analysis would be required in the future. In addition, discussion on injury reporting methods in each country seems to be an urgent issue. Serious injury occurs in other ball games and martial arts sports. Cross-sectional study, including specific injury prevention methods, would also be important. The aim of this study was not to scare young individuals of the possible injuries that could occur when playing rugby because it may be a dangerous sports activity, but it was our goal to design a positive system by sharing information networks structure that for a safer playing environment. This study has several limitations. First, the small number of data would be a limitation of a one-country study. In the future, collaborative study with foreign countries would be neces- sary. To unify the data collection methods, it would be necessary to resolve the differences in sports insurance systems in each country. Second, there were causes of injuries that were diffi- cult to determine [34,35], and specifying which situation caused the injury is also difficult to determine. Third, it is also deeply involved with the insurance system. Despite these limita- tions, our data using more than 14 years of longitudinal data could contribute in preventing serious injuries in rugby players. Conclusion Younger and inexperienced rugby players tended to suffer from serious injuries. Our 14-years (2004–2018) longitudinal data showed that approximately 20 cases of serious injuries occurred in one year. 48 head injuries in 14 years divided 28 subdural hematoma, 5 fracture, 5 acute epi- dural hematoma, 5 intra cerebral hemorrhage/cerebral contusion, 2 cerebral infarction/cere- brovascular accident, and 3 others. 35 of the 48 (76%) were inexperienced players. 54 Spine cord injuries in 14 years divided 13 fracture dislocation, 13 central cord, 12 spinal cord, 6 cer- vical spinal cord, 4 vertebral fracture, 4 dislocation of spine, and 2 others. 43 of the 54 (80%) were inexperienced players. Causes of injury were “head to ground”, “head to head”, “head to leg”, “head to the opponent’s body part”, “head under the opponent’s body”, and others. The play styles were own tackle, oppose tackle, ruck (a one-on-one situation), other collision not categorized as above, saving (hold the ball on the ground), and other or unknown. In the bipartite graph of age, years of experience and injury symptoms obtained from 14 years’ data, the edge-betweenness centrality network analysis could be an effective method for understanding occurrence structure. Our study suggests that we should consider introducing rules that prohibit head-on collisions in youth rugby. References 1. Japan Rugby Football Union. Rugby serious injury report. 2004–2018. 2. Fuller CW, Brooks JH, Kemp SP. Spinal injuries in professional rugby union: a prospective cohort study. Clinical Journal of Sport Medicine. 2007: 17(1): 10–16. https://doi.org/10.1097/JSM. 0b013e31802e9c28 PMID: 17304000 3. Silver JR. The impact of the 21st century on rugby injuries. Spinal Cord. 2002: 40(11): 552–559. https:// doi.org/10.1038/sj.sc.3101349 PMID: 12411962 4. Sasaki K, Watanabe I, Yamamoto T, Yamashita S, Tanaka A, Okuwaki T. An empirical study of Japa- nese women’s rugby injury 2016. Japanese Journal of Rugby Science. 2017: 28(1): 56–60. 5. Warner S, Bowers MT, Dixon M. Team dynamics: a social network perspective. Journal of Sport Man- agement. 2012: 26(1): 53–66. 6. Sasaki K, Yamamoto T, Komatsu K, Ueno Y, Katsuta T, Kono I. Cognitive societal human values of sports: after the 2011 disaster of Japan. Social Sciences. 2013: 2(1): 1–6. 7. Sasaki K, Yamamoto T, Murakami J, Ueno Y. Defense performance analysis of rugby union in Rugby World Cup 2011: network analysis of the turnover contributors. Performance Analysis of Sport. 2013: IX: 94–99. 8. Junker BH, Schreiber F. Analysis of Biological Networks. Hoboken (NJ): John & Wiley & Sons, Inc. 2008. 9. Sanz-Leon P, Knock SA, Spiegler A, Jirsa VK. Mathematical framework for large-scale brain network modeling in The Virtual Brain. NeuroImage. 2015: 111: 385–430. https://doi.org/10.1016/j.neuroimage. 2015.01.002 PMID: 25592995 10. Suzuki T. Network Analysis. Tokyo (JPN): Kyouritsu Shuppan. 2009. 11. Sasaki K, Yamamoto T, Watanabe I, Katsuta T, Kono I. Athletes’ pride bridge: network centrality analy- sis to clarify the societal values of sports after the 2011 disaster in Japan. Advances in Social Sciences Research Journal. 2019: 6(2): 440–450. 12. Newell P, Timmons R. The Globalization and Environment Reader. Hoboken (NJ): Blackwell Publish- ing. 2016. 13. Pereira VH, Gama MCT, Sousa FAB, Lewis TG, Gobatto CA, Manchado-Gobatto FB. Complex network models reveal correlations among network metrics, exercise intensity and role of body changes in the fatigue process. Scientific Report. 2015: 5: 10489. 14. Hambrick ME. Social Network Analysis in Sport Research. Cambridge Scholars Publishing. 2019. 15. Putnam RD. Bowling alone. Simon and Schuster. 2001. 15. Putnam RD. Bowling alone. Simon and Schuster. 2001. 16. Duch J, Waitzman JS, Nunes Amaral LA. Quantifying the performance of individual players in a team activity. PLoS One. 2010: 5(6): e10937. https://doi.org/10.1371/journal.pone.0010937 PMID: 20585387 17. Passos P, Davis K, Araujo D, Paz N, Minguens J, Mendes J. Supervision: Ichiro Watanabe. Validation: Koh Sasaki, Akihiko Nakamura, Takumi Yamamoto, Ichiro Kono. Visualization: Koh Sasaki. Supervision: Ichiro Watanabe. Validation: Koh Sasaki, Akihiko Nakamura, Takumi Yamamoto, Ichiro Kono. Visualization: Koh Sasaki. Writing – original draft: Koh Sasaki, Ichiro Watanabe, Takashi Katsuta, Ichiro Kono. Author Contributions Conceptualization: Haruhiko Sato, Akihiko Nakamura, Ichiro Watanabe, Takashi Katsuta, Ichiro Kono. Data curation: Haruhiko Sato, Akihiko Nakamura, Takumi Yamamoto, Ichiro Watanabe. Formal analysis: Takumi Yamamoto. Funding acquisition: Koh Sasaki. Investigation: Takashi Katsuta. Methodology: Koh Sasaki, Akihiko Nakamura, Takumi Yamamoto, Takashi Katsuta. Methodology: Koh Sasaki, Akihiko Nakamura, Takumi Yamamoto, Takashi Katsuta Methodology: Koh Sasaki, Akihiko Nakamura, Takumi Yamamoto, Takashi Katsuta. Project administration: Ichiro Kono. 11 / 13 PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby References Network as a novel tool for studying team ball sports as complex social systems. Journal of Science and Medicine in Sport. 2011: 14: 170–176. https://doi.org/10.1016/j.jsams.2010.10.459 PMID: 21145787 18. Yamamoto Y, Yokoyama K. Common and unique network dynamics in football games. PLoS One. 2011: 6(12): e29638. https://doi.org/10.1371/journal.pone.0029638 PMID: 22216336 19. Sasaki K, Yamamoto T, Miyao M, Katsuta T, Kono I. Network centrality analysis to determine the tacti- cal leader of a sports team. International Journal of Performance Analysis in Sport. 2017: 17(6): 822– 831. 20. Shalley CE, Perry-Smith JE. The emergence of team creative cognition: the role of diverse outside ties, sociocognitive network centrality, and team evolution. Strategic Entrepreneurship Journal. 2008: 2(1): 23–41. 21. Zuo XN, Ehmke R, Mennes M, Imperati D, Castellanos FX, Sporns O, et al. Network centrality in the human functional connections. Cereb Cortex. 2011: 22(8): 18621875. PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 12 / 13 PLOS ONE A network edge-betweenness centrality analysis revealed the cause of serious injury in Rugby 22. Ramos J, Lopes RJ, Arau´jo D. What’s next in complex networks? Capturing the concept of attacking play in invasive team sports. Sports Medicine. 2018: 48(1): 17–28. https://doi.org/10.1007/s40279-017- 0786-z PMID: 28918464 23. Faust K. Centrality in affiliation network. Social networks. 1997: 19(29: 157–191. 24. Fruchterman TMJ and Reingold EM. Graph drawing by force-directed placement. Software-practice and Experience. 1991: 42(11): 149–160. 25. Newman M and Girvan M. Finding and evaluating community structure in networks, Physical Review. 2004: E 69, 026113. 26. Girvan M and Newman M. Community structure in social and biological networks. Proceedings of the National Academy of Sciences of United States of America. 2002: 99 (12) 7821–7826. 27. Nakaminami T, Nakayama S, Kobayashi S and Yamaguchi H. Ulnerability assessment of emergency transportation road network based on eigenvalue analysis. Civil engineering. 2018: 74(5) I_1141– I_1148. 28. Hayashi T, Akiba T, Yoshida Y. A fast metrics for computing edge-betweenness centrality in dynamic network. DEIM (Data Engineering and Information Management) Forum. 2015: E3–4. 29. Pons P, Latapy M. Computing communities in large networks using random walks. Journal of Graph Algorithms and Applications. 2006: 10(2): 191–218. 30. Clauset A, Newman MEJ, Moore C. Finding community structure in very large networks. Physical Review E. 2005: 70(6): 066111. 31. Ahn YY, Bagrow JP, Lehmann S. Link communities reveal multiscale complexity in networks. Nature. 2010: 466(7307): 761–764. https://doi.org/10.1038/nature09182 PMID: 20562860 32. Reichardt J, Bornholdt S. Statistical mechanics of community detection. PLOS ONE \| https://doi.org/10.1371/journal.pone.0235035 July 15, 2020 References Physical Review E. 2006: 74 (1): 016110. 33. Iida Y, Kishimoto Y, Fujiwara Y, Shiokawa H, Onizuka M. Finding communities and ranking for large- scale graphs—fast algorithms and applications. AI. 2014: 29(5): 472479. 34. Bleakley C, Tully M, O’Connor S. Epidemiology of Adolescent Rugby Injuries: A Systematic Review. Journal of Athletic Training. 2011: 46(5): 555–565. https://doi.org/10.4085/1062-6050-46.5.555 PMID: 22488143 35. McIntosh AS, McCrory P, Finch CF, Wolfe R. Head, face and neck injury in youth rugby: incidence and risk factors. British Journal of Sports Medicine. 2010: 44(3): 188–193. https://doi.org/10.1136/bjsm. 2007.041400 PMID: 18385188 36. Inokuchi A, Washio T, Motoda H. An Apriori-Based Algorithm for Mining Frequent Substructures from Graph Data. Heidelberg (Berlin): Springer. 2000. 37. Kashima H. Prediction of network structure. AI. 2007: 22(3): 344–351. 13 / 13
https://openalex.org/W2807380917	https://europepmc.org/articles/pmc5909242?pdf=render	English	null	Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease	BMC structural biology	2,018	cc-by	5,400	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Due to dengue virus disease, half of the world population is at severe health risk. Viral encoded NS2B-NS3 protease complex causes cleavage in the nonstructural region of the viral polyprotein. The cleavage is essentially required for fully functional viral protein. It has already been reported that if function of NS2B-NS3 complex is disrupted, viral replication is inhibited. Therefore, the NS2B-NS3 is a well-characterized target for designing antiviral drug. Results: In this study docking analysis was performed with active site of dengue NS2B-NS3 protein with selected plant flavonoids. More than 100 flavonoids were used for docking analysis. On the basis of docking results 10 flavonoids might be considered as the best inhibitors of NS2B-NS3 protein. The interaction studies showed resilient interactions between ligand and receptor atoms. Furthermore, QSAR and SAR studies were conducted on the basis of NS2B-NS3 protease complex docking results. The value of correlation coefficient (r) 0.95 shows that there was a good correlation between flavonoid structures and selected properties. Conclusion: We hereby suggest that plant flavonoids could be used as potent inhibitors of dengue NS2B-NS3 protein and can be used as antiviral agents against dengue virus. Out of more than hundred plant flavonoids, ten flavonoid structures are presented in this study. On the basis of best docking results, QSAR and SAR studies were performed. These flavonoids can directly work as anti-dengue drug or with little modifications in their structures. Keywords: Flavonoids, QSAR, SAR, Molecular docking, Dengue virus, NS2B-NS3 Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease Muhammad Waseem Sarwar1, Adeel Riaz1, Syed Muhammad Raihan Dilshad5, Ahmed Al-Qahtani2,3,4, Muhammad Shah Nawaz-Ul-Rehman1 and Muhammad Mubin1* Background encoded NS2B/NS3 protease and the host proteases into structural proteins C, M, and E as well as nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 to initiate the replication of dengue virus [2, 3]. Dengue virus belonging to family Flaviviridae is the most prevalent arthropod transmitted virus in humans. It can cause symptoms ranging from self-limiting dengue fever to sometimes-fatal dengue hemorrhagic fever [1]. The NS2B-NS3 protease contains two functional re- gions i.e., a C-terminal region acting as RNA helicase and a N-terminal 180-residue is a trypsin like serine pro- tease (Fig. 1). NS3 protease requires the central hydro- philic region of NS2B (NS2B; residues 49 to 95) to perform proteolytic activity and to stabilize folding. Thus, hydrophilic domain of NS2B interacts with NS3 protease and forms full active site [4]. The activity of Dengue virus is a positive sense single stranded ssRNA virus with 10.7 kb genome. Viral RNA is translated into a single polyprotein. The poly protein is cleaved by virus * Correspondence: mmubin@uaf.edu.pk 1Virology Lab, Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Jail road, Faisalabad 38000, Pakistan Full list of author information is available at the end of the article Sarwar et al. BMC Structural Biology (2018) 18:6 https://doi.org/10.1186/s12900-018-0084-5 Sarwar et al. BMC Structural Biology (2018) 18:6 https://doi.org/10.1186/s12900-018-0084-5 Open Access Methods All analyses presented here were performed using 64-bit Operating System and Intel(R) Core(TM) i5-5200 U processor with 2.2 GHz processing speed. MOE (Mo- lecular Operating Environment) software was used for computational analysis, provided by chemical computing Page 2 of 10 Sarwar et al. BMC Structural Biology (2018) 18:6 Fig. 1 Structure of dengue NS2B-NS3 (2FOM); Catalytic site is shown in ball stick model group Inc. and Chimera software was used for protein structure manipulation. Ligand preparation More than 100 chemical structures of ligand flavonoid molecules were downloaded online from chebi (http:// www.ebi.ac.uk/chebi/) in .mol format. These structures were prepared for docking in LigX module of MOE pro- gram with parameters (gradient: 0.05, Force Field: MMFF94X). Fig. 1 Structure of dengue NS2B-NS3 (2FOM); Catalytic site is shown in ball stick model NS2B/NS3 is critical for viral replication [5] as the dis- ruption of NS2B-NS3 function inhibits viral replication [6–8]. So NS2B/NS3 protease could be targeted for the development of anti-DENV inhibitors. Docking setup and run The binding sites for the target protein were calculated, for docking analysis, by MOE site finder and then con- firmed with the binding site reported in literature. Dur- ing docking setup, only this binding site (His51, Asp75 and Ser135) was used (Fig. 1) to find the correct con- formation of the ligand. To bind the selected ligands with receptor protein, MOE docking program with de- fault parameters was used. MOE London dG scoring function was used to estimate free energy of binding for each ligand from a given pose [22]. The functional form of London dG scoring function is a sum of terms: Plants had served as a source of medicinal com- pounds for a long time and are basis of many phar- maceuticals now days [9]. Flavonoids are plant based phenolic compounds [10] having various biological properties like antiviral [11, 12], antioxidant, antifun- gal [13], anti-cancerous [14, 15], anti-angiogenic [16] and anti-inflammatory properties [17, 18]. Henceforth, flavonoids may act as inhibitors of dengue NS2B-NS3. In this study, in-silico screening using auto- mated docking method was performed and binding models of dengue NS2B-NS3 protease with selected plant flavonoids are proposed. Finally, ten plant flavo- noids were suggested as potential inhibitors of dengue virus NS2B-NS3 complex. Furthermore extensive studies of binding modes were performed using SAR model i.e., (Structure Activity Relationship) and QSAR model i.e., (Quantity Structure Activity Relationship) [19]. This study provides the novel insights in the development of anti-viral drugs against dengue virus. ΔG ¼ c þ Eflex þ X h−bonds cHBf HB þ X m−lig cM f M þ X atoms i ΔDi The difference in desolvation energies is calculated ac- cording to the formula [23]. ΔDi ¼ ciRi 3 ∭ u∉A∪B u j j−6du−∭ u∉B u j j−6du The final docking output was saved for further investigations. Preparation of receptor structure Crystal structure of NS3-NS2B protease was obtained from Protein Data Bank (http://www.rcsb.org) with PDB ID 2FOM [20]. The protein consists of two chains and 185 residues length with resolution 1.5 Å. The ribbon diagram of target structure with catalytic site is shown in Fig. 1. This structure was subjected to 3D protonation and energy minimization using parameters like (gradi- ent: 0.05, Force Field: MMFF94X + Solvation) using MOE Program. For docking the minimized structure was used as the receptor protein [21]. Calculation of ligand interaction Ligand interactions were obtained by MOE program. The Ligand Interactions studies are used to visualize an active site of a complex in diagrammatic form. Depicted view of 3D interaction is shown in Fig. 3. Flavonoid 1 formed an interaction with three amino acids of viral protein (Gly 87, Val 146 and Asn 167) with in the active site (Fig. 4a). Flavonid 2 formed the interaction with two amino acids of viral protein (Lys 74 and Ile 165) with in active site (Fig. 4b). Flavonoid 3 formed an arene-arene link with amino acid Trp 83 (Fig. 4c). Flavonoid 4 formed interaction with three amino acids (Trp 83 Gly 87 and Val 146) (Fig. 5a). Flavonoid 5 formed an arene cationic link with amino acid Lys 74 (Fig. 5b). Flavonoid 6 formed the interaction with Lys 74 (Fig. 5c). Flavonoid 7 interacted as a cationic arene interaction with Lys 74 and a hydrogen bond donation with Trp 83 (Fig. 6a). Fla- vonoid 8 formed interaction with Asn 167, Val 147 and Trp 89 (Fig. 6b). Flavonoid 9 has shown an interaction with amino acid Gly 87 and Trp 83 (Fig. 6c). Flavonoid 10 formed a cationic arene link with Lys 74 and an hydrogen bond with Trp 83 (Fig. 6d). Ligand interactions studies The ligand interactions were studied by Ligand Interaction module in MOE Program. It includes 2D and 3D repre- sentations of ligand and receptor protein interactions and Sarwar et al. BMC Structural Biology (2018) 18:6 Page 3 of 10 Page 3 of 10 calculated distances among ligand and protein interacting atoms. descriptors, with the variation in the biological activity of protein. To test the reliability of results, regression analysis was performed using inhibitory activity as dependent variable and the descriptor as predictor vari- ables. After making sure the reasonable correlation of inhibitory activity with the individual descriptor, QSAR models were derived The predictor variables with p value greater than 0.05 were eliminated while obtaining the QSAR models, to assure their statistical reliability. Docking analysis Docking of all flavonoid structures (Fig. 2) was done against the active site of dengue NS2B-NS3 protein. Docking analysis provided a number of configurations that were scored to determine favorable binding modes. The flavonoid structures with high docking scores with molecular data are summarized in Table 1. Calculation of descriptors p Descriptor refers to the two-dimensional (2-D) or three- dimensional (3-D) physiochemical property of a mol- ecule [23]. The descriptors that were calculated in this study are as follows. logP(o/w) is the logarithm of the octanol and water partition coefficient. b_count is the number of bonds (including implicit hydrogens). This is calculated by the addition of (di/2 + hi) over all nontriv- ial atoms i. b-rotN is the number of rotatable bonds. With order 1, having at least two heavy neighbors and provided it is not in a ring, a bond is said to be rotatable. chi1 is the atomic connectivity index. a_acc is the num- ber of hydrogen bonds of acceptor atoms (not counting acidic atoms but counting atoms that are both hydrogen bond donors and acceptors such as -OH). Q_pc + is the total positive partial charge i.e., the sum of the positive qi. Q_PC+ is identical to PC+ that has been reserved for compatibility. Q_pc-PC is the total negative partial charge and it is the sum of the all negative qi. SlogP is the logarithm of the octanol and water partition coeffi- cient (including hydrogens). Log S is the logarithm of the aqueous solubility (mol/L). apol is the sum of the atomic polarizabilities. a_don is the number of hydrogen bond acceptor atoms. vsurf_g is the surface globularity. vsurf_wpu is the Hydrophilic volume. E is the value of the potential energy. E_ele is the electrostatic compo- nent of the potential energy. E_nb is the value of the potential energy with all bonded positions that have been disabled. Studies about structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) The Structure-Activity Relationship (SAR) report was generated by MOE application to find common scaffolds in flavonoid structures used in this study. The input data was flavonoid structures in .mol file format. Structure- Activity Report involves gathering of input data (mole- cules, activity, predefined scaffolds, etc.) and detection of common scaffolds. After that, Alignment of the scaffolds was done to produce a common numbering system by using ligand alignment module of MOE program. Structure activity report Structure activity report The Structure-Activity Report is a MOE application, which generates a web page housing an interactive view onto a collection of molecules, which are related by a small number of related common scaffolds. The applica- tion is designed to operate on small databases of drug- like molecules, which are in the process of being refined for improved binding affinity and other pharmacological properties. Report was generated using a panel from an interactive session of MOE, using a MOE molecular database. The default browser was launched to show the results, when the generation was completed. The Quantitative Structure Activity Relationship (QSAR) was done in MOE. All docked molecules were used for training set. In a test set, QSAR model corre- lates the activities with properties inherent to each mol- ecule. Different molecular descriptors were used to evaluate these properties. QSAR studies involve two steps. In first step, descriptors were generated that en- code chemical structure information. During second step, a statistical regression technique is employed to correlate the structural variation, as encoded by the Sarwar et al. BMC Structural Biology (2018) 18:6 Page 4 of 10 Fig. 2 Flavonoid structures that shown best results of docking analysis; 1. quercetin 3-O-(2″,3″-digalloyl)-β-D-galactopyranoside 2. quercetin 3-O-α- (6″‘-caffeoylglucosyl-β-1,2-rhamnoside) 3. schaftoside 4. myricetin 5. quercetin 3-sulfate 6. eriocitrin 7. catiguanin B 8. 4′,5,7-trihydroxy-3-methoxyflavone-7-O- α-L-arabinofuranosyl(1 →6)-β-D-glucopyranoside 9. wogonin 7-O-β-D-glucuronide 10. silychristin Fig. 2 Flavonoid structures that shown best results of docking analysis; 1. quercetin 3-O-(2″,3″-digalloyl)-β-D-galactopyranoside 2. quercetin 3-O-α- (6″‘-caffeoylglucosyl-β-1,2-rhamnoside) 3. schaftoside 4. myricetin 5. quercetin 3-sulfate 6. eriocitrin 7. catiguanin B 8. 4′,5,7-trihydroxy-3-methoxyflavone-7-O- α-L-arabinofuranosyl(1 →6)-β-D-glucopyranoside 9. wogonin 7-O-β-D-glucuronide 10. silychristin Fig. 2 Flavonoid structures that shown best results of docking analysis; 1. quercetin 3-O-(2″,3″-digalloyl)-β-D-galactopyranoside 2. quercetin 3-O-α- (6″‘-caffeoylglucosyl-β-1,2-rhamnoside) 3. schaftoside 4. myricetin 5. quercetin 3-sulfate 6. eriocitrin 7. catiguanin B 8. 4′,5,7-trihydroxy-3-methoxyflavone-7-O- α-L-arabinofuranosyl(1 →6)-β-D-glucopyranoside 9. wogonin 7-O-β-D-glucuronide 10. silychristin Sarwar et al. BMC Structural Biology (2018) 18:6 Page 5 of 10 Table 1 Selected flavonoid molecules downloaded from chebi (http://www.ebi.ac.uk/chebi/), shown higher docking score Flavonoid No. Structure activity report Chebi ID Name Plant Sources Docking Score (Kcal/mol) 1 66,287 quercetin 3-O-(2″,3″-digalloyl)-β-D-galactopyranoside Euphorbia lunulata −26.101 2 66,284 quercetin 3-O-α-(6″‘-caffeoylglucosyl-β-1,2-rhamnoside) Sedum sarmentosum −24.987 3 9047 schaftoside Passiflora tripartita −23.399 4 18,152 myricetin Myrica rubra −21.987 5 17,730 quercetin 3-sulfate Anethum graveolens −20.989 6 28,709 eriocitrin Citrus lumia −20.693 Cyclopia subternata 7 65,602 catiguanin B Trichilia catigua −20.414 8 68,348 4′,5,7-trihydroxy-3-methoxyflavone-7-O-α-L-arabinofuranosyl (1 →6)-β-D-glucopyranoside Lepisorus contortus −20.378 9 61,282 wogonin 7-O-β-D-glucuronide Scutellaria baicalensis −20.102 10 9143 silychristin Silybum marianum −20.085 Scaffold categorization needs a small number of common scaffolds for best results. Alignment of the scaffolds was done to produce a common numbering system by using ligand alignment module of MOE program. One common scaffold was obtained from the alignment of all ten flavonoid structures (Fig. 7). These results show that there is a prevalence of simi- larity among the structural formulas of different fla- vonoid compounds performing the same function i.e. binding with active site of NS3-NSB protein of den- gue virus. observed parameters with a correlation coefficient (r) of value 0.956. So to clarify conceivable QSARs for each compound with its biological activity, atomic models of flavonoids were constructed. The structures of flavonoid compounds were shown in Fig. 2 and data was presented in Table 1. These flavonoids mod- eled keeping in view the patterns of hydrogen bond- ing and hydrophobic interactions and the induced fit phenomena that best express the binding affinity of each structure as accessed from logP values. These logP were separated as training set and test set values. Since there was no pervious information of flavonoid inhibition of dengue NS2B-NS3 complex available, 10 flavonoids showing best docking results were used for both training set as well as test set. The values of test set were approximately the same as that for training set (Fig. 8) clearly showing that there is a positive correlation between the values of logP and activities. The parameters used in QSAR model QSAR model The biochemical activities in this work were analyzed for cross correlation of activities and chemical de- scriptors to find out whether for observed inhibitory effect against NS2B-NS3 complex is there any com- mon structure activity relationship. It was discovered that there was a high level of correlation between the Fig. 3 3D interaction diagram; Dotted lines showing the interactions between ligand and receptor protein atoms with calculated distances Fig. 3 3D interaction diagram; Dotted lines showing the interactions between ligand and receptor protein atoms with calculated distances Fig. 3 3D interaction diagram; Dotted lines showing the interactions between ligand and receptor protein atom Page 6 of 10 Sarwar et al. BMC Structural Biology (2018) 18:6 Fig. 4 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No.1 b. Flavonoid No. 2 c. Flavonoid No. 3 Fig. 5 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No. 4 b. Flavonoid No. 5 c. Flavonoid No. 6 Fig. 4 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No.1 b. Flavonoid No. 2 c. Flavonoid No. 3 Fig. 5 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein Dotted lines are showing specific Fig. 5 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No. 4 b. Flavonoid No. 5 c. Flavonoid No. 6 Fig. 4 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No.1 b. Flavonoid No. 2 c. Flavonoid No. 3 Fig. 4 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No.1 b. Flavonoid No. 2 c. Flavonoid No. 3 Fig. 5 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No. 4 b. Flavonoid No. 5 c. Flavonoid No. 6 Fig. 5 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No. 4 b. Flavonoid No. QSAR model 5 c. Flavonoid No. 6 Page 7 of 10 Sarwar et al. BMC Structural Biology (2018) 18:6 Fig. 6 2D interaction diagram; showing the interacting ligand atoms with specific residues in receptor protein. Dotted lines are showing specific interaction. a. Flavonoid No.7 b. Flavonoid No. 8 c. Flavonoid No. 9 d. Flavonoid No. 10 and the value of logP Correlation Co-efficient ob- tained are shown below and the value of logP Correlation Co-efficient ob- tained are shown below Activity field ¼ logP o=w ð Þ; Descriptor ¼ SlogP; PLS ¼ RMSE ¼ 0:24071; R2 ¼ 0:97662 logP Correlation Coeffient ¼ þ0:77652; Normalization ¼ þ0:95672 Accordingly partial least square (PLS) experiment was performed to achieve three dimensional-QSAR (Fig. 9). Leave one out statistical procedure was used to get ideal number of components. It was done to build a regression model that is statistically significant. Cross- validated coefficient q2 was used to measure the model quality. Conventional correlation coefficient r2 was utilized to obtain the external predictivity. Discussion Dengue is a mosquito-borne viral hemorrhagic illness that is very dangerous to human wellbeing in tropical and subtropical areas. The virus-encoded protease activ- ity, important for dengue virus infection, requires two viral proteins, NS2B and NS3 [24]. It has been already established that the disruption of NS2B-NS3 protein complex has negative effect on viral replication [25]. In the present investigation, plant flavonoids have been tested as potential inhibitor of NS2B-NS3 complex of viral protease. Previously, Plant flavonoids were known for anti-inflammatory, anticancer and antiviral activities [12, 14]. In present work we used structure based com- putational docking analysis tool to find the anti-dengue Fig. 7 Common Scaffolds obtained from Structure Activity Report; a. Scaffold 1 common in sevenall flavonoid structures Fig. 7 Common Scaffolds obtained from Structure Activity Report; a. Scaffold 1 common in sevenall flavonoid structures Fig. 7 Common Scaffolds obtained from Structure Activity Report; a. Scaffold 1 common in sevenall flavonoid structures Sarwar et al. BMC Structural Biology (2018) 18:6 Page 8 of 10 potential of flavonoids. More than 100 flavonoid struc- tures were downloaded from chemebi. Docking of all fla- id d i h i i f ligand and protein. In our study we found more than one flavonoid as potential inhibitor of dengue NS2B- NS3 i fi d h Fig. 8 QSAR model; a. Training set b. Test set Fig. 8 QSAR model; a. Training set b. Test set Fig. 8 QSAR model; a. Training set b. Test set potential of flavonoids. More than 100 flavonoid struc- tures were downloaded from chemebi. Docking of all fla- vonoid structures was done against the active site of dengue NS2B-NS3 protein. Docking analysis provided various designs that were scored to decide ideal restrict- ing modes. We found very high scores of docking than previously reported data [26]. Docking analysis contrib- uted towards basic idea and binding energy values for each ligand. After this, ligand interaction studies were done for the further confirmation of docking results and to have an insight into each interaction of atoms of ligand and protein. In our study we found more than one flavonoid as potential inhibitor of dengue NS2B- NS3 so it was necessary to find out the common struc- ture among all those flavonoids that showed high bind- ing score. Conclusions In present work, different plant flavonoids were docked and structure based SAR and QSAR analysis was done for dengue NS2B-NS3 protein. The results of statistical analysis of both models i.e., SAR and QSAR were con- vincing and comparable. The generated model was used to predict the effect of important structural characteris- tics of the potent inhibitor. The generated 3D-QSAR models were further verified by the docking studies of most potent and least active inhibitors so can be used for better drug development. Abbreviations a_acc: Number of hydrogen bond acceptor atoms; apol: Sum of the atomic polarizabilities; Asp: Aspartic acid; b rotN: Number of rotatable bond; Chi1: Atomic connectivity index; DENV: Dengue virus; E_ele: Electrostatic component of the potential energy; E_nb: Value of the potential energy with all bonded term disabled; His: Histidine; Log P: Partition coefficient; MOE: Molecular operating environment; NS2B: Non structural protein 2B; NS3: Non structural protein 3; OPLS-AA: Optimized potentials for liquid simulations for amino acids; PLS: Partial least square analysis; Q_pc+: Total positive partial charge; QSAR: Quantitative structure activity relationship; R2: Conventional correlation coefficient; RMSE: Root mean square error; SAR: Structure activity relationship; Ser: Serine; SlogP: Log of the octanol/ water partition coefficient; vsurf_g: Surface globularity; s rf p H drophilic ol me Availability of data and materials The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Discussion As the compounds that have common or correlated structures have similar physicochemical prop- erties and thus have similar binding modes and subse- quently comparable biological activities. To find out a common structural feature among flavonoids, structure activity report was generated and a common conform- ation was obtained. We are reporting this conformation Sarwar et al. BMC Structural Biology (2018) 18:6 Page 9 of 10 Fig. 9 3D Correlation Plot for the activities of QSAR; X-axis logP(o/w), Y-axis SlogP, Z-axis logS Fig. 9 3D Correlation Plot for the activities of QSAR; X-axis logP(o/w), Y-axis SlogP, Z-axis logS for very first time and it is an important step towards development of novel drugs against dengue virus. Quan- titative structure activity relationships (QSAR) have been used for the development of relationships between phys- icochemical properties of compounds and their bio- logical activities to get a statistical model, which prove their binding assays. The basic principle involved in this process is that the difference in the structure of chemical compounds is accountable for the variation in their bio- logical activities [27]. In the present study the value of correlation obtained from QSAR model showed that there is linear relationship between selected calculated descriptor values and activities of compounds. We are reporting ten naturally occurring flavonoids as potential inhibitors of dengue NS2B-NS3 protein complex, which can inhibit viral replication. These flavonoids were not known previously for their anti-dengue activity. Funding This work was funded by the IFS Research Grant No. C/5260-1 to Dr. Muhammad Mubin and IFS Research grant No. C/5434-1 to Dr. Muhammad Shah Nawaz ul Rehman. Funding bodies have no role in the design of the study and collection, analysis, and interpretation of data and in writing this manuscript. Acknowledgements k l d ll We acknowledge all members of virology lab for their help and support for this work. Authors’ contributions MM and MSN conceived the idea and designed the experiments. MWS and AR did all experiments. MM, SMRD and MAAQ analyze and interpret the data. MM, MAAQ, SMRD and MSN wrote and critically reviewed the manuscript. All authors have read and approved the manuscript. Ethics approval and consent to participate N/A Ethics approval and consent to participate This study is an effort to explore novel inhibitors of dengue NS2B-NS3 and to through light on the structural and inhibitory activity of plant flavonoids. Consequently, ten flavonoids were found as inhibitors of NS2B-NS3. Henceforth, QSAR and SAR data provided in this study will serve as basis for a better drug development. Competing interests Competing interests The authors declare that they have no competing interests. References Rothan HA, Han HC, Ramasamy TS, Othman S, Rahman NA, Yusof R. Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1. BMC Infect Dis. 2012;12:314. 9. Cragg GM, Grothaus PG, Newman DJ. Impact of natural products on developing new anti-cancer agents. Chem Rev. 2009;109(7):3012–43. developing new anti-cancer agents. Chem Rev. 2009;109(7):3012 10. Beecher GR. Overview of dietary flavonoids: nomenclature, occurrence and intake. J Nutri. 2003;133(10):3248S–54S. 11. Giovannini C, Filesi C, D'Archivio M, Scazzocchio B, Santangelo C, Masella R. Polyphenols and endogenous antioxidant defences: effects on glutathione and glutathione related enzymes. Ann Ist Super Sanita. 2006;42(3):336–47. 12. Sang S, Hou Z, Lambert JD, Yang CS. Redox properties of tea polyphenols and related biological activities. Antioxid Redox Signal. 2005;7(11-12):1704–14. Sang S, Hou Z, Lambert JD, Yang CS. Redox properties of tea polypheno 13. Friedman M. Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas. Mol Nutri Food Res. 2007;51(1):116–34. 14. Li Y, Fang H, Xu W. Recent advance in the research of flavonoids as anticancer agents. Mini-Rev Med Chem. 2007;7(7):663–78. 15. Fresco P, Borges F, Diniz C, Marques MP. New insights on the anticancer properties of dietary polyphenols. Med Res Rev. 2006;26(6):747–66. 16. Oak MH, El Bedoui J, Schini-Kerth VB. Antiangiogenic properties of natural polyphenols from red wine and green tea. J Nutri Biochem. 2005;16(1):1–8. 16. Oak MH, El Bedoui J, Schini-Kerth VB. Antiangiogenic properties of natura polyphenols from red wine and green tea. J Nutri Biochem. 2005;16(1):1– 17. Middleton E Jr. Effect of plant flavonoids on immune and inflammatory c function. Adv Exp Med Biol. 1998;439:175–82. 17. Middleton E Jr. Effect of plant flavonoids on immune and inflammatory cell function. Adv Exp Med Biol. 1998;439:175–82. 17. Middleton E Jr. Effect of plant flavonoids on immune and inflammatory cell function. Adv Exp Med Biol. 1998;439:175–82. 18. Lu Y, Yeap FL. The polyphenol constituents of grape pomace. Food Chem. 1999;65(1):1–8. 18. Lu Y, Yeap FL. The polyphenol constituents of grape pomace. Food Chem. 1999;65(1):1–8. 19. Su BH, Shen MY, Esposito EX, Hopfinger AJ, Tseng YJ. In silico binary classification QSAR models based on 4D-fingerprints and MOE descriptors for prediction of hERG blockage. J Chem Inf Model. 2010;50(7):1304–18. 20. Erbel P, Schiering N, D'Arcy A, Renatus M, Kroemer M, Lim SP, Yin Z, Keller TH, Vasudevan SG, Hommel U. Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. receptor: iterative selection and rescoring of docked poses systematically improves the correlation. J Comput Aided Mol Des. 2013;27(8):707–21. 23. Santos CB, Vieira JB, Lobato CC, Hage-Melim LI, Souto RN, Lima CS, Costa EV, Brasil DS, Macedo WJ, Carvalho JC. A SAR and QSAR study of new artemisinin compounds with antimalarial activity. Molecules. 2013;19(1):367–99. 24. Noble CG, Seh CC, Chao AT, Shi PY. Ligand-bound structures of the dengue virus protease reveal the active conformation. J Virol. 2012;86(1):438–46. 25. Robin G, Chappell K, Stoermer MJ, Hu SH, Young PR, Fairlie DP, Martin JL. Structure of West Nile virus NS3 protease: ligand stabilization of the catalytic conformation. J Mol Biol. 2009;385(5):1568–77. 26. Qamar TU, Mumtaz A, Ashfaq UA, Azhar S, Fatima T, Hassan M, Hussain SS, Akram W, Idrees S. Computer aided screening of phytochemicals from Garcinia against the dengue NS2B/NS3 protease. Bioinformation. 2014;10(3):115–8. 27. Verma J, Khedkar VM, Coutinho EC. 3D-QSAR in drug design-a review. Curr Top Med Chem. 2010;10(1):95–115. References 1. Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev. 1998;11(3):480–96. 1. Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev. 1998;11(3):480–96. 1. Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev. 1998;11(3):480–96. 2. Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Ann Rev Microbiol. 1990;44:649–88. 2. Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Ann Rev Microbiol. 1990;44:649–88. 3. Falgout B, Pethel M, Zhang YM, Lai CJ. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991;65(5):2467–75. 3. Falgout B, Pethel M, Zhang YM, Lai CJ. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol. 1991;65(5):2467–75. 4. Yang CC, Hsieh YC, Lee SJ, Wu SH, Liao CL, Tsao CH, Chao YS, Chern JH, Wu CP, Yueh A. Novel dengue virus-specific NS2B/NS3 protease inhibitor, BP2109, discovered by a high-throughput screening assay. Antimicrob Agents Chemother. 2011;55(1):229–38. 4. Yang CC, Hsieh YC, Lee SJ, Wu SH, Liao CL, Tsao CH, Chao YS, Chern JH, Wu CP, Yueh A. Novel dengue virus-specific NS2B/NS3 protease inhibitor, BP2109, discovered by a high-throughput screening assay. Antimicrob Agents Chemother. 2011;55(1):229–38. 5. Lescar J, Luo D, Xu T, Sampath A, Lim SP, Canard B, Vasudevan SG. Towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional NS3 protein from dengue virus as a target. Antivir Res. 2008; 80(2):94–101. 5. Lescar J, Luo D, Xu T, Sampath A, Lim SP, Canard B, Vasudevan SG. Towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional NS3 protein from dengue virus as a target. Antivir Res. 2008; 80(2):94–101. 6. Geiss BJ, Stahla H, Hannah AM, Gari AM, Keenan SM. Focus on flaviviruses: current and future drug targets. Future Med Chem. 2009;1(2):327–44. 6. Geiss BJ, Stahla H, Hannah AM, Gari AM, Keenan SM. Focus on flaviviruses: current and future drug targets. Future Med Chem. 2009;1(2):327–44. 7. Rothan HA, Abdulrahman AY, Sasikumer PG, Othman S, Rahman NA, Yusof R. Protegrin-1 inhibits dengue NS2B-NS3 serine protease and viral replication in MK2 cells. J Biomed Biotechnol. 2012;2012:251482. 7. Rothan HA, Abdulrahman AY, Sasikumer PG, Othman S, Rahman NA, Yusof R. Protegrin-1 inhibits dengue NS2B-NS3 serine protease and viral replication in MK2 cells. J Biomed Biotechnol. 2012;2012:251482. 8. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Page 10 of 10 Page 10 of 10 Page 10 of 10 Sarwar et al. BMC Structural Biology (2018) 18:6 26. Qamar TU, Mumtaz A, Ashfaq UA, Azhar S, Fatima T, Hassan M, Hussain SS, Akram W, Idrees S. Computer aided screening of phytochemicals from Garcinia against the dengue NS2B/NS3 protease. Bioinformation. 2014;10(3):115–8. 27. Verma J, Khedkar VM, Coutinho EC. 3D-QSAR in drug design-a review. Curr Top Med Chem. 2010;10(1):95–115. 25. Robin G, Chappell K, Stoermer MJ, Hu SH, Young PR, Fairlie DP, Martin JL. Structure of West Nile virus NS3 protease: ligand stabilization of the catalytic conformation. J Mol Biol. 2009;385(5):1568–77. Author details 1 l b C 1Virology Lab, Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Jail road, Faisalabad 38000, Pakistan. 2Department of Infection and Immunity, Research Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. 3Department of Microbiology and Immunology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia. 4Liver Disease Research Center, King Saud University, Riyadh, Saudi Arabia. 5Department of Theriogenology, Faculty of Veterinary and Animal Sciences, Gomal University, Dera Ismail Khan, Pakistan. Received: 10 July 2017 Accepted: 11 April 2018 Received: 10 July 2017 Accepted: 11 April 2018 24. Noble CG, Seh CC, Chao AT, Shi PY. Ligand-bound structures of the dengue virus protease reveal the active conformation. J Virol. 2012;86(1):438–46. References Nat Struct Mol Biol. 2006; 13(4):372–3. 21. Sarwar MW, Saleem IB, Ali A, Abbas F. Insilico characterization and homology modeling of Arabitol dehydrogenase (ArDH) from Candida albican. Bioinformation. 2013;9(19):952–7. 22. Wright JS, Anderson JM, Shadnia H, Durst T, Katzenellenbogen JA. Experimental versus predicted affinities for ligand binding to estrogen 22. Wright JS, Anderson JM, Shadnia H, Durst T, Katzenellenbogen JA. Experimental versus predicted affinities for ligand binding to estrogen
https://openalex.org/W2950334163	https://link.springer.com/content/pdf/10.1007/s40037-019-0519-0.pdf	English	null	Seeking coherence between ‘mobile learning’ applications and the everyday lives of medical residents	Perspectives on medical education	2,019	cc-by	5,886	Abstract Introduction The role of technology in health professions education has received increased research attention. Research has examined the interaction between humans and technology, focusing on the mutual inﬂuence between people and technology. Little attention has been given to the role of motivation and incentives in how learning technologies are used in relation to daily activities. This research aims to understand the relationship between medical-learning technology and its users. Methods A mixed-method case study of a new medical-learning mobile application (app) for family medicine residents was undertaken at a Canadian university hospital. The Information Assessment Method is a custom-made app to help residents prepare for the College of Family Physicians of Canada licensing examination. Residents’ use of the app was tracked over a 7-month period and individual, semi-structured interviews were conducted with users. Data were thematically analyzed and correlated with app use data. Results Factors identiﬁed as shaping residents’ mobile app use for learning, included: efﬁciency, mobility and resonance with life context; credibility of information retrieved; and relevance of content. Most inﬂuential was stage of residency. Second-year residents were more selective and strategic than ﬁrst-year residents in their app use. Discussion An emphasis on coherence between self-directed learning and externally dictated learning provides a framework for understanding the relationship between users and mobile-learning technology. This framework can guide the design, implementation and evaluation of learning interventions for healthcare professionals and learners. Keywords Mobile learning technology · Educational technology · Medical residency · Spaced lear https://doi.org/10.1007/s40037-019-0519-0 Perspect Med Educ (2019) 8:152–159 https://doi.org/10.1007/s40037-019-0519-0 Perspect Med Educ (2019) 8:152–159 ORIGINAL ARTICLE What this paper adds nation (agency), provides a framework for understanding and assessing technological innovations in HPE. Since sec- ond-year residents already make strategic use of such apps, special efforts must be made to inform ﬁrst-year residents of how they can use such technology to support their learn- ing. After all, they tend, relative to later-year residents, to be externally directed by learning technology, either using it rigidly, or abandoning it entirely. There is little research conducted on mobile applications in health professions education (HPE), despite proliferation of their use in healthcare. There is even less guidance on how their use aligns with the everyday lives of the trainees who use them. This qualitative study shows that creative and strategic information use is a learning skill increas- ingly developed by medical trainees. ‘Coherence’, deﬁned in terms of external inﬂuence (structure) and self-determi- Diana Ramos1 · Roland Grad1 · Alenoush Saroyan2 · Peter Nugus1,3 Published online: 7 June 2019 © The Author(s) 2019 Published online: 7 June 2019 © The Author(s) 2019 Seeking coherence between ‘mobile learning’ applications and the everyday lives of medical residents Diana Ramos1 · Roland Grad1 · Alenoush Saroyan2 · Peter Nugus1,3 Diana Ramos diana.ramos@mail.mcgill.ca Diana Ramos diana.ramos@mail.mcgill.ca 1 Department of Family Medicine, McGill University, Montreal, Canada 2 Department of Counseling and Educational Psychology, McGill University, Montreal, Canada 3 Institute of Health Sciences Education and Department of Family Medicine, McGill University, Montreal, Canada 1 Department of Family Medicine, McGill University, Montreal, Canada Methods This paper focuses on the introduction of a new medical learning mobile application for family medicine residents at a Canadian university teaching hospital—the ‘Informa- tion Assessment Method (IAM)’ app—to optimize exam preparation. The IAM app was custom-made speciﬁcally based on the curriculum and licensing requirements of the College of Family Physicians of Canada (CFPC), to guide residents in preparation for the licensing examination. Sam- pled purposively, then, among other apps that are clinically based only, the app has links that speciﬁcally align with the deﬁnitions and details of topics and their corresponding objectives required for the CFPC physician licensing exam. Family medicine residents from a different institution chose the speciﬁc content. The content was reviewed and mod- erated by the second author, a family physician. The ﬁrst author of the paper subsequently uploaded the content to the app. Social scientiﬁc research on how technology is used has generally focused on how humans interact with technology. The discipline of sociology, in particular, has drawn on its traditional distinction between ‘structure’ and ‘agency’ [3, 4, 7, 8]. This involves a focus on the extent to which human beings exercise freewill (agency), as opposed to being rel- atively bound by external (structural) constraints. More re- cently, social scientiﬁc studies have focused on the mutually inﬂuential relationship between humans and technology [3, 4, 7–10], where both human and non-human elements act and are acted upon. Our study draws on a framework of ‘sociomateriality’—a notion that assumes the mutual inﬂu- ence, or ‘entanglement’ of humans and technology [11, 12]. The idea is that a human being is part of a network in which they are inﬂuenced by what others do, in ways often un- knowable to them [7–10]. The effects of this sociomaterial network on behaviour depend on the nature of the overlap between interests among individuals and their mutual rela- tionships, how attracted particular individuals feel towards technology, and individuals’ shared cultural beliefs about technology [9–12]. Today, researchers tend to favour a view of ‘hybridity’ across humans and technology—that is, the co-identiﬁcation of people with technologies and the ‘en- meshment’ of technologies with human bodies themselves [12, 13]. For instance, the response of a mobile-phone user to the ‘beep’ of a text message shows the potential hybridity between people and technology. Introduction A dramatic increase in the use of mobile phone applica- tions (apps) among healthcare professionals has come about through evolving medical knowledge bases, the develop- ment and popularity of mobile communication devices, and the continually increasing specialization of medicine [1]. Currently, more than 7,000 health-related apps are avail- able and used by healthcare professionals around the world [2]. Educational and social scientists have paid increased 153 Mobile applications and medical resident learning attention to the role of mobile technology in work and ed- ucation [3]. Several studies have identiﬁed advantages of using mobile apps in clinical practice, including facilita- tion of communication, portability, and efﬁcient time use [4]. In a recent study, most physicians reported using apps in their clinical practice [5]. Apps cover diverse topics and functions. The most commonly used are drug guides (79%), medical calculators (18%), coding and billing apps (4%), and calculator apps (e.g. pregnancy wheels) (4%). The most frequently downloaded apps are textbooks or ref- erence materials (55%), classiﬁcation and treatment algo- rithms (46%), and general medical knowledge compilations (43%) [6]. That clinicians are evidently downloading apps to assemble information to aid their practice suggests a bur- geoning ﬁeld of human-technology interaction for health professional education researchers to examine. everyday action. Such research is needed if HPE research is to keep pace with innovations in learning technology. Moreover, few studies have examined the inﬂuence of mo- bile technology use on learning patterns in the context of formal exam preparation. This study aimed to understand the role of a learner’s everyday ‘mobile’ life in terms of the relationship between mobile medical learning technologies and users’ priorities. Methods The IAM app periodically alerts users to one of 99 clin- ical topics delineated by the College of Family Physicians of Canada as learning priorities for the ﬁnal examination, which residents take to obtain a license. A weekly ‘prompt’ of one priority topic (also referred to as an ‘alert’ or ‘push notiﬁcation’), reminds residents to study at regular inter- vals, in a ‘spaced’ fashion. The app’s design is based on ‘s- paced learning theory,’ suggesting that distribution of learn- ing spreads out study activities over time, improving the long-term acquisition of information [16]. We characterize the app’s ‘prompt’ to the user as an incursion by the exter- nal environment into the everyday, mobile life of the user, placing a demand on users to re-order their learning [16]. The residents’ responses to the app’s prompts were tracked throughout the study and their views elicited to understand the nature of its inﬂuence on their study pat- terns. This case study employed a mixed-methods approach and was conducted over a 7-month period (October–May 2015). Following approval by a university Institutional Review Board (otherwise known as a Human Research Ethics Committee), 20 family medicine residents (of a co- hort of 46 invitees) consented to participate in the study. Eight participants were in their ﬁrst year of residence, that is, postgraduate year 1 (PGY1) and 12 in their second However, little research has focused on the relationship between mobile technology and users’ wider everyday con- texts, and the extent to which apps match the situated ac- tions that people perform in their everyday lives [13–15]. In general, the need for efﬁcient mobility and time manage- ment are central, but under-explored, dimensions of tech- nology use [13, 14]. Few studies have examined the role of motivation and incentives in the way learning technolo- gies are used in relation to daily activities, including the relationship between intentional planning and spontaneous 154 D. Ramos et al. Fig. 1 IAM app levels of information Fig. 1 IAM app levels of information year (PGY2). Inclusion criteria were limited to owning a smartphone with an iPhone (iOS 5.0+) or Android (2.3+) platform. Participants received instructions to download the IAM app onto their smartphones. of use. Two PGY2s were interviewed twice, based on the detail of their insights and the speciﬁcity of their feedback on the app’s technical functioning in the ﬁrst interview. Methods The interviews were evenly spaced over three stages of the data collection process to address the change in usage patterns of learning technology over time. Topics covered in the in- terviews included: perceptions of the app’s functionality, at- titudes towards the app, perceptions of personal behaviours around the prompts, and perceptions of the relationship be- tween the app and the participants’ learning preferences. Data sources included log ﬁles, which enabled us to track app use, and individual, open-ended, semi-structured inter- views, which helped us to understand users’ experiences and perceptions of using an app to prepare for an exam that is an important milestone in their professional careers. To create the log ﬁles, a ‘hit’ was recorded whenever a user opened the app at the level of pages of clinical information (see Fig. 1). Every hit was logged as a separate entry in a password-protected database at www.99prioritytopics.ca. Log-ﬁle data included each participant’s name as well as the frequency, date, and time of page access. The interviews, featuring a combination of open ques- tions (designed to elicit relatively unguided perceptions) and prompt questions (designed to seek more information about speciﬁc points raised) [17], were audio-recorded and transcribed verbatim. The software MaxQDA was used to organize the codes into which qualitative data were orga- nized. The transcripts were analyzed independently by three coders who met to discuss and resolve a limited number (ap- proximately 10%) of categorization differences. Thematic analysis was used to generate initial codes, compare and contrast data segments and organize segments into codes and then into categories, in order to conceptualize the re- lationships between the categories and develop conceptual themes [18]. Microsoft excel was used to calculate the de- scriptive statistics. The app use log ﬁles enabled triangula- tion with the data ﬁndings from interview responses. The cut-off point of app use for inclusion in the study was 2 months (the length of two clinical rotations). This was because some clinical rotations have a greater work- load than others, and 4 weeks (one clinical rotation) would not have been enough time to show the residents’ app use. All 20 participants installed the app. Methods Based on the app use log ﬁle, participants were classiﬁed into three usage pat- terns: 3 were ‘non-users’ who installed the app but had no recorded hits; 9 were ‘discontinued users’, who had page hits but had stopped using the app ≥2 months before the study ended; and 8 were ‘persistent users,’ who had page hits and continued to use the app until the end of the study. Alerts were sent at the start of the weekly mandatory aca- demic half-day, during which residents were released from clinical duties. The intention was to try and neutralize as a variable any possible patterns of difference in the busy- ness of the clinical work of either R1s or R2s. The inﬂuence of learning level on app use Several factors were identiﬁed as inﬂuential in determin- ing the character of app use among participants. The most A total of 15 semi-structured interviews were conducted with 13 participants representing each of the three levels Mobile applications and medical resident learning 155 Table 1 Usage duration by year of residency and number of participants Year of residency Usage frequency (based on number of participants) Total number of participants 2 months 3≥months PGY 1 4 3 7 PGY 2 5 5 10 Total 9 8 17 PGY postgraduate year inﬂuential was learning level, measured across PGY1s and PGY2s. Clear differences were discernible in the degree of agency (i.e., freewill) residents demonstrated in using the app. Despite a baseline level of agency among all partici- pants, app use by PGY1s could be characterized as ‘struc- tured’ (i.e., guided by the app). PGY1s allowed the app itself to play a greater role in dictating how it would be used. When the app failed to align with their daily routine (i.e., when the prompt was unwelcome), they ceased using it entirely. A typical comment by PGY1s was: Table 1 Usage duration by year of residency and number of participants alerted for that week by PGY2s, compared with PGY1s. Tab. 1 illustrates the analysis of the tracked data. I [would like] to base my preparation on this app, cause it’s always nice to have one structure ... just to follow [its] information. (PGY1, Interview 1) These ﬁndings demonstrate the app’s perceived relevance and its strategic use by PGY2s. This signals a degree of command over the sociomaterial engagement between the app and its user. The app was designed to deliver regular prompts on a particular topic. However, the app’s use in- creased dramatically 3 months prior to the examination as illustrated in Fig. 2. This pattern points to a self-mandated use, especially by PGY2s, who might have been more mo- tivated by a high-stakes professional examination. Thus, app use among PGY1s appeared to be relatively ‘ex- ternally/other-directed.’ Conversely, use by PGY2s could be characterized as more ‘agential’ or ‘self-directed.’ PGY2s took initiative in determining how they would use the app. Although there is evidence of sociomaterial inﬂuence of and on all app users, PGY2s appeared less troubled by the sometimes-unwelcomed interruptions of the prompt com- pared with PGY2s, simply ignoring the app on such occa- sions and opening it later without being prompted. The inﬂuence of learning level on app use They evinced more autonomy and strategy than the PGY1s in usage patterns: The inﬂuence of credibility and relevance on app use The relevance and credibility of the app, which were per- ceived to be high, reportedly increased use. Thus, these fac- tors were persuasive in promoting mobile app use, while not strongly inﬂuential in determining the particular patterns of app use. The initial participant–app interaction evolved into an ongoing relationship for those users who perceived the app to be reliable and credible. Thus, reliability and cred- ibility were factors in shaping the extent and nature of the sociomaterial entanglement of user and app. Although the sustainability of this trusting relationship was predicated, ﬁrst and foremost, on relevance, users did not question the app’s relevance. The app was designed to help prepare res- idents for their certiﬁcation examination, and as such their conﬁdence in its relevance led to their participation in this study. Moreover, we found that establishing credibility re- lied, at least partly, on the users’ perceptions of the content’s trustworthiness and its currency. Both factors had an impact on app use: Efﬁciency, which complements free physical mobility, was a criterion of use in the context of various other knowledge resources competing for the resident user’s attention: Usually, if I have time to prepare, I look at ‘UpToDate’ or ‘Essential Evidence,’ but as I get more used to [the IAM app], and if I ﬁnd it helpful and it’s brief and to the point, then I would use that. (PGY1, Interview 6) Usually, if I have time to prepare, I look at ‘UpToDate’ or ‘Essential Evidence,’ but as I get more used to [the IAM app], and if I ﬁnd it helpful and it’s brief and to the point, then I would use that. (PGY1, Interview 6) Closely aligned to the need for efﬁcient time use and user mobility, ease of use (i.e., conﬁdence in using the technol- ogy) and technological threshold (i.e., tolerance for perse- vering in the face of the user’s technical uncertainty or the software’s technical faults) also aligned with the extent of app use. In general, the shared perception of ease of use and high technological threshold among the participants ensured that these variables did not strongly inﬂuence if, or how, residents used the app in this study. However, in some cases, technical hitches did impact app use. Techni- cal hitches included the failure of the app to open in some iPhone operating systems, only opening reliably in Android form. The inﬂuence of eﬃciency, mobility and resonance on app use Underlying all factors affecting mobile app use in this study were mobility and efﬁciency. Users were more willing to ini- tiate interactions with the app (i.e., respond to its prompt- ing ‘beep’) if the app supported efﬁcient use of their time, in building knowledge for the exam, and aligned with their physical mobility in accomplishing daily tasks. Framing use in general was the resonance of the app and its prompts with the routines and moment-to-moment priorities of each user. Reported app use was more likely to increase if its prompts aligned with the user’s daily life. Speciﬁcally, consistent app use was partially tied to residents’ need to efﬁciently prepare for a high-stakes exam within the 2 years of resi- dency, while juggling competing personal and professional priorities. At a micro level, this also involved the need to Underlying all factors affecting mobile app use in this study were mobility and efﬁciency. Users were more willing to ini- tiate interactions with the app (i.e., respond to its prompt- ing ‘beep’) if the app supported efﬁcient use of their time, in building knowledge for the exam, and aligned with their physical mobility in accomplishing daily tasks. Framing use in general was the resonance of the app and its prompts with the routines and moment-to-moment priorities of each user. I would click on [the app] and I’ll read the topic of the week ... for my own study purposes. [Only one] topic [per] week is nice now, but the closer I get to the exam, it’s [going to] be a topic [per] day. (PGY2, Interview 9) I would click on [the app] and I’ll read the topic of the week ... for my own study purposes. [Only one] topic [per] week is nice now, but the closer I get to the exam, it’s [going to] be a topic [per] day. (PGY2, Interview 9) When asked whether they would follow the weekly prompts, one PGY2 said: Probably not. [I wouldn’t follow the prompts just be- cause] an app is telling me that this week it’s ‘this topic’, which is arbitrary. [It] doesn’t make much sense to me. (PGY2, Interview 7) Log-ﬁle data seemed to match this concern, showing a higher rate of searching for topics other than the topic Fig. 2 Monthly app use 156 D. Ramos et al. The inﬂuence of eﬃciency, mobility and resonance on app use be able to quickly manage (by reading or ignoring) the in- terruption the prompt posed in the ﬂow of their daily lives: exam and use the app to help them do so, but only when it met the functional needs of their everyday life, expressed in notion of mobility and reliability. I mean, I don’t have a lot of time every night to do something, [but I aim to read] a little bit about it, like what the full history would be one night and exam and treatment, and kind of go through it like that. (PGY1, Interview 6) I mean, I don’t have a lot of time every night to do something, [but I aim to read] a little bit about it, like what the full history would be one night and exam and treatment, and kind of go through it like that. (PGY1, Interview 6) I mean, I don’t have a lot of time every night to do something, [but I aim to read] a little bit about it, like what the full history would be one night and exam and treatment, and kind of go through it like that. (PGY1, Interview 6) None of the participants reported problems to the re- search team prior to being asked speciﬁcally in the in- terviews. In the interviews, most participants reported mi- nor technical hitches or questions. When asked how they sought to resolve technical issues, no one, including persis- tent users, reported approaching another resident to discuss it. The enmeshment between user and device appeared to be a solitary relationship, the residents reporting to work and solve problems in relative isolation from each other. To be usable and used, then, the app needed to accommo- date time-efﬁcient use, complementing rather than imped- ing the user’s free mobility in their daily life. Free physical mobility was key to its use: [The app is] much more available [than other re- sources]. I have the phone with me all the time and it’s a good way to keep that there, at (my) ﬁngertips. It’s great. (PGY2, Interview 9) [The app is] much more available [than other re- sources]. I have the phone with me all the time and it’s a good way to keep that there, at (my) ﬁngertips. It’s great. (PGY2, Interview 9) The inﬂuence of credibility and relevance on app use The other hitch was that the app required internet use, and that internet coverage was often poor in hospitals. The impact, and potential impact, of technical faults underlines the importance of the app resonating with the users by aligning well with their daily routines: Will these topics be updated? [That’s important to me]. (PGY1, Interview 1) If these could be updated regularly throughout the year, it would be in my best interest to go back and visit these more than once. (PGY2, Interview 4) When participants were asked what they thought about par- ticipating in shaping the content of the mobile app, contra- dictory comments emerged: I was using [the app] at ﬁrst. It was really, for me, the alarms and push notiﬁcations to guide me .... So when I stopped getting those push notiﬁcations ... it wasn’t as useful to me. (PGY2, Interview 13) Ideally, a resident, who has ﬁnished studying for his or her exam, could go through all the topics with their other resources ... and see which topics were incom- plete on the app. (PGY2, Interview 15) The failure of the app to open on some iPhone operating systems was ﬁxed within a week of the app’s introduction. Indeed, only one person actually withdrew from the study on account of technical problems. In general, then, residents were apparently prepared to work hard to prepare for the I’m always scared with resident notes, because you’re always worried that something is ... wrong. (PGY2, Interview 11). 157 Mobile applications and medical resident learning In terms of learning approaches, the following resident uniquely reﬂected on the app’s potential to facilitate col- lective learning among residents: terns paralleled the difference in learning phases between PGY1s and PGY2s and the associated differences in their incentives for learning. Undoubtedly, PGY1s are more able to build knowledge cumulatively for its own sake, whereas PGY2s have the added pressure (or incentive) to pass a high-stakes physician-qualifying exam to be admitted as family medicine physicians. [The app could] even encourage [us] to study in groups early on. [Because] that’s what I found to be the most helpful thing in preparing for the exam. ... We could sit as a group with the app to make sure we hit all points. (PGY2, Interview 11) The distinction between structure and agency is not ﬁxed, but is mutual and recursive among both human and non-hu- man elements [22]. The inﬂuence of credibility and relevance on app use It functions within a complex system of sociomaterial networks, in which a collection of ele- ments deliver effects, rather than easily discernible causal relationships [8]. Accordingly, this study demonstrates both users’ inﬂuence on technology and technology’s inﬂuence on users, as part of an increasingly sociomaterial reality. This system of inﬂuence can be characterized as part of a search for coherence between individual will and external inﬂuences [23, 24]. Thus, at least one participant considered the possibility that groups could delegate the organization of their collective learning to the app. However, this was the only resident who referred to group work or the use of the app with oth- ers, when asked how he or she would use the app to prepare for the exam. Despite the residents’ desire for group learn- ing, for most participants the app seemed to support self- learning, thus, to some extent, exacerbating the tendencies of those already inclined to prepare, and deal with learning issues, alone: As technology progressively permeates our lives, indi- viduals try, within the constraints and opportunities of daily living and learning environments, to ﬁnd a coherent, self- determined path [25, 26]. The success of the mutual entan- glement between technology and its users depends on the extent to which the goals of technology designers and users intertwine. The study ﬁndings demonstrate the signiﬁcance of the resonance of technology’s content and prompts with users’ priorities, technological thresholds, learning levels, perceptions of credibility and currency, and needs for ef- ﬁcient time use and mobility. Rather than relying strictly on the distinction between structure agency, accounting for structural elements through a lens of coherence can help us create a conceptual framework to evaluate the extent to which technology serves human needs. Such an assessment is vital in a ﬁeld like healthcare, which aims to improve people’s lives through interventions, increasingly of a tech- nological nature, and in which technology could easily be- come an end in itself. I think there were some people studying in groups. But I was more on my own. (PGY2, Interview 12). Conclusions 1. Aungst TD, Clauson KA, Misra S, et al. How to identify, assess and utilise mobile medical applications in clinical practice. Int J Clin Prac. 2014;68:155–62. The framework of coherence emerging from this study can help clinician educators to shape exam preparation strate- gies, select mobile resources that resonate with students’ learning environments and needs, and guide the use and evaluation of mobile learning technologies according to learning levels. Such targeted interventions will promote agency within the mobile reality in which learners increas- ingly ﬁnd themselves. 2. Kailas A, Chong C-C, Watanabe F. From mobile phones to personal wellness dashboards. Pulse IEEE. 2010;1:57–63. 3. Gourlay L. Posthuman texts: nonhuman actors, mediators and the digital university. Soc Sem. 2015;25:484–500. 4. McBride N. Actor-network theory and the adoption of mobile com- munications. Geography. 2003;88:266–76. 5. Wallace S, Clark M, White J. ‘It’s on my iPhone’: attitudes to the use of mobile computing devices in medical education, a mixed- methods study. BMJ Open 2012;2:e001099. https://doi.org/10. 1136/bmjopen-2012-001099. New clinical practice apps frequently appear on the mar- ket including, for example, calculators for risk stratiﬁcation and treatment options. It is rare to ﬁnd institutionally sanc- tioned apps devoted to preparation for licensing examina- tions. A rare exception is an app similar to the IAM app that was later developed by Western University in Ontario, Canada. Since this study, the IAM app has been integrated with the Western University’s app, which had a similar focus—training for the CFPC licensing examination. The IAM’s technical ﬂaws have been remedied as part of this integration, and some of the content has been updated or otherwise revised. The app is not yet available for wider use. The efﬁcacy of the combined apps awaits further study. Al- though residents sometimes study in groups, neither this nor other similar apps have acquired an inter-dependent online functionality—supporting sociomaterial collaboration—but this would be a valuable innovation. 6. Franko OI, Tirrell TF. Smartphone app use among medical providers in ACGME training programs. J Med Sys. 2012;36:3135–9. 7. Latour B. Reassembling the social: an introduction to actor-net- work-theory. Oxford: Oxford University Press; 2005. p. 316. 8. Law J. Actor network theory and material semiotics. The new Blackwell companion to social theory. Oxford: Blackwell; 2008. pp. 141–58. 9. Colbjørnsen T. Digital divergence: analysing strategy, interpretation and controversy in the case of the introduction of an ebook reader technology. Inf Commun Soc. 2015;18:32–47. 10. Thompson L, Cupples J. Discussion This paper has expanded previous literature featuring lo- cal experiments in design technology for medical trainees’ learning, and perceptions of usefulness. As a core assump- tion, the notion of sociomateriality accounts for the mutual and increasing entanglement of human beings with tech- nology [11]. Very little was known about the way medi- cal trainees actually use learning technology in their daily lives. This paper offers an account of the tension between structure (other-directed) and agency (user-directed) in nav- igating mobile-learning tools as a function of learning in- centives and the developmental stages of learning. While it is increasingly less common to characterize human-technol- ogy interaction using the traditional sociological distinction between structure and agency [3, 4, 7, 8], its persistence is evident in the ﬁndings. We found that mobile learning applications may exacer- bate learners’ isolation from one another. Medical students have been found to be particularly individualistic and com- petitive in the way they study and learn [27]. This is not necessarily undesirable. Yet, policy-makers and educators should consider incentives for learning, with a particularly critical eye on exam-driven behaviour and the culture that drives it. Such considerations are particularly salient given that the potential of mobile learning applications is appar- ently not being fully used to support collaborative learning among medical trainees [28]. Understanding the way learn- ers interact with technology enables policymakers and edu- cators to intervene, to foster learning beyond sociomaterial individualism, to learning through sociomaterial collabora- tion. The app’s weekly prompt was intended to instil a habit of regular, structured learning—traditionally called ‘spaced learning’ or ‘spaced education’ [19, 20]—in its users, thus re-ordering their learning. Spaced learning contrasts with a mode of study colloquially known as ‘cramming’ or massed practice before an exam. In terms of the structure- agency distinction, PGY1s’ app use was externally gov- erned by the learning mode and organization proposed by the app’s designer [21]. App use by PGY2s, on the other hand, was internally governed; and these users exhibited more agency and willingness to use the app if, when and how they desired. The observed differences in use pat- 158 D. Ramos et al. most appropriate time during a training program to intro- duce particular educational interventions. Study limitations most appropriate time during a training program to intro- duce particular educational interventions. A limitation of the study is the relatively small sample size and the collection of data among one specialty at one in- stitution. Nevertheless, the study focused on the relatively generic subject of mobile learning among residents, sug- gesting broader resonance of its ﬁndings about how app use interacts with one’s personal life with residency trainees worldwide. After all, the topics discussed in interviews were not of a personal nature or idiosyncratic to the institution or region. Further research among other specialties and coun- tries, and involving direct observation of mobile app use for learning, rather than reliance on second-hand reporting, would be illuminating. Acknowledgements The authors wish to thank the participants of this study, and the anonymous reviewers for Perspectives on Medical Edu- cation for their suggestions which improved this article. Conﬂict of interest D. Ramos, R. Grad, A. Saroyan and P. Nugus de- clare that they have no competing interests. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 20. Kerfoot BP, Baker H. An online spaced-education game for global continuing medical education: a randomized trial. Ann Surg. 2012;256:33–8. 23. Beck U. Risk society: towards a new modernity. Translated by Mark Ritter. London: SAGE; 1992. 24. Beck U. World at risk: translated by Ciaran Cronin. Cambridge: Polity; 2009. Conclusions Seen and not heard? Text messaging and digital sociality. Soc Cult Geogr. 2008;9:95–108. 11. Orlikowski WJ. Using technology and constituting structures: a practice lens for studying technology in organizations. Organ Sci. 2000;11:404–28. 12. Suchman L. Human-machine reconﬁgurations: plans and situated actions. Cambridge: Cambridge University Press; 2007. 13. Söderström S. Staying safe while on the move. Nord J Youth Res. 2011;19:91–109. 14. Peters P, Kloppenburg S, Wyatt S. Co-ordinating passages: under- standing the resources needed for everyday mobility. Mobilities. 2010;5:349–68. Technology designers interested in mobile learning tech- nology can use the factors identiﬁed in this study as inﬂu- encing technology adoption to guide and evaluate the bal- ance between end users’ needs with educators’ interests. We suggest that alerts from an app be provided to res- idents from their ﬁrst year onwards. Further research into the culture of (and behaviours relating to) exam preparation can shed light on learning strategies and perceived learning gaps. Such research ought to provide guidance as to the 15. Kailas A, Chong C-C, Watanabe F. From mobile phones to personal wellness dashboards. IEEE Pulse. 2010;1:57–63. 16. Xue G, Mei L, Chen C, et al. Spaced learning enhances subse- quent recognition memory by reducing neural repetition suppres- sion. J Cogn Neurosci. 2011;23:1624–33. 17. Maxwell JA. Qualitative research design: an interactive approach. London: SAGE; 2012. 18. Patton M. Qualitative research. New York: Wiley; 2005. 19. Cain LF, Willey RDV. The effect of spaced learning on the curve of retention. J Exp Psychol. 1939;25:209. Mobile applications and medical resident learning 159 24. Beck U. World at risk: translated by Ciaran Cronin. Cambridge: Polity; 2009. y 25. Lemke T. Foucault, governmentality, and critique. Rethink Marx. 2002;14:49–64. 21. Grad RM, Pluye P, Wong E, et al. Do weekly alerts from a mo- bile application inﬂuence reading during residency? PRiMER. 2017;1:8. https://doi.org/10.22454/PRiMER.2017.243866. 26. Drake F. Protesting mobile phone masts: risk, neoliberalism, and governmentality. Sci Technol Hum Values. 2011;36:522–48. 22. Nugus P. The Interactionist self and grounded research: reﬂexivity in a study of emergency department clinicians. Qual Sociol Rev. 2008;4:189–204. 27. Lempp H, Seale C. The hidden curriculum in undergraduate medi- cal education: qualitative study of medical students’ perceptions of teaching. BMJ. 2004;329:770–3. 28. Bleakley A. Broadening conceptions of learning in medical educa- tion: the message from teamworking. Med Educ. 2006;40:150–7. 23. Beck U. Risk society: towards a new modernity. Translated by Mark Ritter. London: SAGE; 1992.
https://openalex.org/W2007570291	https://qmro.qmul.ac.uk/xmlui/bitstream/123456789/15830/1/Wills%20et%20al.%202011%20Report%20from%20the%20second%20cytomegalovirus%20and%20immunosenescence%20workshop.pdf	English	null	Report from the second cytomegalovirus and immunosenescence workshop	Immunity & ageing	2,011	cc-by	8,031	© 2011 Wills et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Report from the second cytomegalovirus and immunosenescence workshop Mark Wills1, Arne Akbar2, Mark Beswick3, Jos A Bosch4,5, Calogero Caruso6, Giuseppina Colonna-Romano6, Ambarish Dutta7, Claudio Franceschi8, Tamas Fulop9, Effrossyni Gkrania-Klotsas10, Joerg Goronzy11, Stephen J Griffiths2, Sian M Henson2, Dietmar Herndler-Brandstetter12, Ann Hill13, Florian Kern14, Paul Klenerman15, Derek Macallan16, Richard Macaulay2, Andrea B Maier17, Gavin Mason1, David Melzer7, Matthew Morgan12, Paul Moss3, Janko Nikolich-Zugich18, Annette Pachnio3, Natalie Riddell2, Ryan Roberts1, Paolo Sansoni19, Delphine Sauce20, John Sinclair1, Rafael Solana21, Jan Strindhall22, Piotr Trzonkowski23, Rene van Lier24, Rosanna Vescovini19, George Wang25, Rudi Westendorp26 and Graham Pawelec27 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 IMMUNITY & AGEING Open Access Open Access Abstract The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion. duly held in Cambridge, UK, 2-4th December, 2010. The second meeting format was modified to include a day of formal seminar presentations with some emphasis on the presentation of data that addressed open questions highlighted in the first meeting. This was then followed by a half-day round-table discussion. Background The first International Workshop on CMV & Immuno- senescence was held in Tubingen, Germany, December 2009. The discussions focused on trying to define what immunosenescence is exactly, the phenotypes of the cells associated with it and what evidence was available that long term carriage of HCMV into old age was det- rimental to health as assessed either by measuring all- cause mortality or degradation of the immune system such that older HCMV-seropositive individuals respond less well to neo-antigens such as seasonal influenza vac- cination. The outcome of the first workshop was sum- marized in this Journal by Pawelec et al. [1]. The attendees at that workshop decided that it would be extremely valuable to meet again in a year’s time with a remit to update participants on progress on questions left open from the first meeting’s discussion, and to extend participation to other scientists omitted from or unable to attend the first workshop. The Second Inter- national Workshop on CMV & Immunosenescence was The seminar presentations covered four separate ses- sions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This com- mentary summarizes the major findings of these presen- tations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion. Correspondence: mrw1004@cam.ac.uk 1Department of Medicine, University of Cambridge, Cambridge, UK Full list of author information is available at the end of the article Cytomegalovirus and T cell phenotypes The impression left on the immune system of the majority of the population (as characterized by changes in T cell phenotype) following primary CMV infection * Correspondence: mrw1004@cam.ac.uk 1Department of Medicine, University of Cambridge, Cambridge, UK Full list of author information is available at the end of the article Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Page 2 of 8 Page 2 of 8 and its subsequent life-long carriage is well-established and uncontroversial. Nonetheless, certain special popu- lations may resist the effects of CMV infection on their immune signatures [2]. As these data stem from obser- vations in exceptionally long-lived families, and because such longevity is likely influenced by multiple genes, sharing of more or less of the latter in the general popu- lation is likely to make the impact of CMV a continuous rather than a discrete variable. This complicates inter- pretation of data treating the presence of CMV infection as simply present or absent (ie. seropositive or seronega- tive). Speiser (Lausanne) presented evidence that the late-differentiated HCMV-specific CD8+ T cell clones in the periphery had a restricted T cell receptor usage and particular clonotypes segregated with particular states of differentiation. Interestingly, once established, these pat- terns were stable over many years. This may again sug- gest a subtle genetic control of anti-CMV surveillance. duration of HCMV replication after transplantation and the percentage of EMRA HCMV-specific CD8+ T cells [3,4]. All these data are consistent with the potential of some (CMV, HIV) but not all (eg. HSV, HCV) [5] chronic viral infections to drive the acquisition of immune signatures associated with “immunosenes- cence”. However, the relationship, if any, between such immune signatures and mortality in any populations except the very old (> 85 yr) Swedes investigated in the OCTO/NONA studies [6] still remain to be established, as does the question of whether the contribution of CMV infection is causative or merely reactive. y Kern (Brighton) presented a study to establish the most relevant target proteins recognized by ageing CMV-positive individuals so that a knowledge base can be established to inform researchers which proteins to use for monitoring and analyzing T-cell responses. Cur- rently the focus is very much on pp65 and IE-1; how- ever, many other proteins are recognized by T cells and there is good reason to believe that studies on pp65 and IE-1 alone are not representative of the entire immune response to CMV. Cytomegalovirus and T cell phenotypes The reported study investigates T- cell responses to 19 ORFs (previously identified by Syl- wester et al. [7] to be the most important target pro- teins) in approx. 100 older people (including 50 people over 75+). The study addresses a spectrum of different T-cell functions with respect to each target protein and it will facilitate a wider, more comprehensive view of the T-cell response to CMV in older age. The study will be concluded in 2011 and results are expected to be published in 2012. Vescovini and Sansoni (Parma) made available their unpublished data on an immune phenotype and fre- quency study in a cohort of 125 HCMV-seropositive elderly subjects aged between 60-100 years. The study concluded that there was a significant correlation between the magnitude of CMV-specific CD4+ T-cell responses and accumulation of effector T-cells within the CD4+ T cell compartment. There was also a signifi- cant correlation in the magnitude of CMV-specific CD8 + T-cell responses and number of total CD8+ T-cells as well as the number of both memory and effector CD8+ T-cells. The study did not find significant correlations between the magnitude of CMV-specific T-cell responses and a shortage of either CD4+ or CD8+ naïve T-cells. In this instance, the presence or absence of CMV infection did seem to behave as a discrete variable in its effects on naïve T cells. These findings emphasize the over-riding impact of CMV infection per se on com- monly-measured immune parameters, and those com- monly attributed to immunosenescence (ie. loss of naïve T cells). Solana (Cordóba) presented further evidence that there is an accumulation of CD8+ CD28- T cells with short telomeres that tend to also acquire inhibitory NK receptors in healthy elderly people. He noted that similar changes can be seen in chronic stimulation situa- tions such as patients with melanoma, HIV infection and RA. The HCMV pp65-specific T cells from elderly individuals were found to be heavily enriched in CD45RA+ CD27- CD28- CCR7- cells and evidence was presented that these had a low proliferative capacity fol- lowing specific stimulation with pp65 peptides [3]. He noted that similar phenotype changes can be seen in HCMV-pp65 specific CD8 T cells in solid organ trans- plantation that were associated with age and with epi- sodes of CMV replication after transplantation. In addition a correlation was observed between the T cell memory frequency, inflation and immunosenescence Using two sepa- rate models of replication and spread-deficient virus that still establishes viral latency, memory T cell infla- tion nonetheless occurred and was maintained. Inhibi- tion of viral replication by inhibiting viral DNA replication also had no effect on the T cell response over time [9]. exhaustion of the naïve T cell pool [12]. Moreover, in a separate study in HAART-treated HIV patients, mount- ing strong CMV-specific responses appeared to impact on both naïve CD4+ T-cell counts and their recovery upon antiretroviral therapy [13]. In these two clinical settings, T cell responses to HCMV have a profound impact of the immune system as a whole. In the special cases of thymectomy and HIV infection, the impact of CMV may therefore be more pronounced and easier to determine unequivocally. Further discussion considering the impact of CMV on immunosenescence led to revisiting the question of what is the definition of T cell senescence? Senescence is a term generally used to describe replication incompe- tence of fibroblast cells after a certain finite number of population doublings. Replicative senescent cells have been defined in the brain and in cardiology but how should it be done for the immune system? Are there any such cells in the constantly-renewing T cell com- partment? The use of the term in describing CMV-spe- cific T cell populations is more controversial as it is acknowledged that many of the late-differentiated T cells do retain replication competence. However, it should be noted that their definition by CD45RA expression and lack of either CD27 or CD28 or both does not define a homogeneous cell population, as many more markers can be added (eg CD57, KLRG1, PD1) to further sub-divide late-stage T cell populations which may yet include an antigen-specific population that is indeed unable to divide further. Another compli- cation is that the term “senescent” is also being used to imply non-functionality, whereas data from many laboratories in human, rhesus and murine systems have shown that late-differentiated cells maintain function. In vivo, the work of Macallan (London) suggests that these cells turn over only very slowly. There was a feeling that we might have become too fixated on the replication capacity of these cells. Of more importance could be their direct effector function. Indeed, the suggestion was made (Pawelec, Tübingen) that some of the defining characteristics of senescent cells which T cells share with the “classic” fibroblast model (ie. T cell memory frequency, inflation and immunosenescence Setting aside the difficulties in proving that accumula- tion of cytomegalovirus-specific T cells with the distinct phenotypic and functional characteristics described above are responsible for an increase in all-cause mor- tality or a decreased ability to mount good quality responses to new antigens, and whether their specificity is important in this context, both Hill (Portland) and Klenerman (Oxford) addressed the biological phenom- enon of “memory T cell inflation” using the murine CMV model. Klenerman has investigated the mechan- ism that leads to memory inflation while Hill has asked what happens to the CMV-specific T cell response if cytomegalovirus replication is inhibited. This latter investigation is of particular importance as it has been suggested that elderly HCMV-seropositive people may benefit from anti-herpes virus chemotherapy. The ratio- nale would be that anti-viral therapy would decrease HCMV replication and thus chronic antigenic stimula- tion which might reduce the frequency of the late-stage differentiated T cell pool and thus allow an increase in Page 3 of 8 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 the naïve pool with the hope that T cell responses to neo-antigens would improve. Klenerman addressed the issue of why memory inflation is triggered by some anti- gens but not others. Memory inflation is defined as the inordinate increase in frequency of CD8+ T cells speci- fic for certain antigens with time post-exposure to anti- genic stimulation. The phenomenon has been seen with CMV, HIV and parvovirus B19 infections; however, cer- tain antigens from these viruses but not to others within the same virus are found to cause this effect, and other viruses do not cause it at all. The “inflating” cells have a distinct transcriptional profile as compared to non- inflating T cells from the same animal. It is not clear if persistence of viral antigen is required regardless of viral reactivation or why some epitopes cause inflation while others do not. The Klenerman study concluded that viral replication was not required for memory inflation and that epitopes that showed inflation had a reduced dependence on the immunoproteasome [8]. Hill also addressed memory inflation in MCMV specifically to understand what happens to the T cell response if cyto- megalovirus replication was inhibited? Discussion of CMV and Immunosenescence - Is it associated at all? If chronic challenge with CMV contributes to causing immunosenescence, it might be expected that more fre- quent CMV reactivation would accelerate this process. However, it is not known whether CMV does reactivate more frequently in the elderly. It was felt that higher HCMV IgG levels might reflect this, but in many sick people they stay the same, so did this mean that HCMV was not reactivating? A study of elderly people suffering from acute bacterial infections (e.g. S. pneumonia, E. coli, S. aureus, K. pneumonia) did not show any reacti- vation of CMV infections measured by IgM antibodies (n = 20). However, serology does not measure viral levels so this is a mute point. Nonetheless, it was strongly felt by a number of clinicians involved with the care of older adults that “It is very unlikely that CMV reactivation and viral replication is a problem in the very elderly.” Wang (Baltimore) provided some evidence for this, indicating that a small-scale study of older adults (n = 40) categorised as either frail or not frail showed no HCMV DNA in serum in either group, with the exception of one frail individual who had 8 copies/ mL (this would be on the very edge of detection). Peo- ple were reminded that VZV reactivation occurs in the elderly and that it had been correlated with decrease in CD4 cells and decrease in IFN gamma responses [15]. Data on CMV are lacking. y It should be noted that not all studies do show such an association, and that when they do, this does not necessarily imply causality. Maier (Leiden) presented results from four cohorts comprising the Leiden 85-plus study, Danish Twin study, Leiden Longevity Study and the PROSPER study with a mortality follow-up of 6 to 12 years. The results did not support a relationship with CMV serostatus or antibody levels in all-cause mortality or inflammation markers in any of the 4 cohorts. Con- sistent with this, a recent study presented at the 2011 Cytomegalovirus Workshop in Nuremberg, Germany by Stuart Adler concerned a prospective longitudinal study with an 18 year follow-up of 915 patients undergoing coronary angiography. It was concluded that neither CMV seropositivity nor antibody levels were associated with longevity irrespective of the presence or absence of coronary heart disease (personal communication). T cell memory frequency, inflation and immunosenescence lack of prolifera- tive capacity, apoptosis resistance, inflammatory media- tor secretion) are not markers of senescence in the sense that they are detrimental (which by definition of the term “senescence” has to be shown to justify its use). Instead, one could view these properties as cellular protection mechanisms responsible for maintaining an active population of cells required for immunosurveil- lance, ie. required for prevention of peripheral clonal deletion of necessary cells. In fact, there is some evi- dence from the longitudinal NONA studies that at the terminal phase of life in the very elderly, the number of CD8+ clonal T cell expansions, most of which are Taking these two studies together, the data from the murine model would suggest that treating humans with anti-herpesvirus chemotherapy may not reverse the effects of HCMV on the memory T cell compartment. Most studies of this type have been carried out in humans or mice, but Nikolich-Zugich (Tucson) pre- sented data looking at the maintenance of T cell mem- ory in the face of CMV and ageing using the Rhesus CMV (RhCMV) model. The study examined the RhCMV CD4+ and CD8+ T cell responses in adult ani- mals aged 7-10 years and aged animals 19-26 years old. The study concluded that there was no difference in the phenotype, numbers, proliferative capacity or functional- ity of the T cells between the two groups of animals and that there was no evidence for either a functional decline or exhaustion in aged animals [10]. This was further corroborated by their study using systemic HSV- 1 infection, which closely models MCMV infection in mice [11]. However, given that CMV and HSV have very different effects in humans [5], this result in mon- keys is perhaps not unexpected. Further, in the context of the impact of HCMV on immune signatures, Sauce (Paris) presented her studies on the impact of HCMV infection in young adults thy- mectomized during early childhood. The data presented suggested that HCMV infection tended to cause the Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Page 4 of 8 specific for CMV, is inversely related to remaining survi- val time [14]. HCMV in aging, mortality and disease states HCMV in aging, mortality and disease states In the studies of Swedish octogenarians and nonagenar- ians (OCTO and NONA) an immune risk profile (IRP) associated with increased mortality on 2, 4 and 6-year follow-up was defined, comprising reduced B cell num- bers, an inverted CD4:CD8 T cell ratio, increased fre- quency of CD8+ CD28- T cell populations, a poor proliferative response to mitogen stimulation and HCMV seropositivity. Strindhall (Jönköping) outlined the results from a new study examining whether this immune profile is already established in a cohort of much younger older people in their sixties, the HEXA study. The initial results show that this profile does exist in hexagenarians. Almost 15% of the cohort had an inverted CD4:CD8 ratio, a frequency very similar to that seen in 85-year-olds, and these individuals also showed characteristics previously identified in IRP individuals of T cell memory frequency, inflation and immunosenescence Work on data taken from the National Health and Nutrition Examination study (NHANES) III concluded that CMV-seropositivity was associated with an increased risk of all-cause mortality in > 14,000 adults at least 25 years of age followed for > 10 years; especially individuals with a raised inflamma- tory marker (C-reactive protein CRP) were at a higher risk of both all-cause mortality and cardiovascular-asso- ciated mortality [19]. Discussion of CMV and Immunosenescence - Is it associated at all? These data are therefore different from a smaller, shorter, study from Finland on one-third the number of patients with stable cardiovascular disease which did show a positive relationship between highest quartile IgG anti- body titer and mortality [16]. This crucial question therefore remains to be resolved, as discussed intensely at the Workshop. T cell memory frequency, inflation and immunosenescence More recently, data from a 7-year follow- up of subjects 88 years old at baseline in the Leiden 85+ study has shown that the degree of accumulation of late-differentiated CD8 cells, putatively mostly specific for CMV, is positively associated with longer survival (Derhovanessian et al., 2011 submitted). the very old. The study now has the opportunity to examine morbidity and mortality to determine if this truly defines a risk profile in people only in their sixties. It is clearly of great importance to establish if long- term carriage of HCMV is associated with an increased likelihood of all-cause mortality. A number of studies addressing this issue have already been published [16,17] and the results from further studies were pre- sented at this meeting. Wang spoke about a study of 635 70-79 year-old women living in the community. The results demonstrated that women in the highest HCMV IgG quartile had a significantly higher incidence of frailty as compared to HCMV-seronegative women at baseline. Furthermore, CMV IgG concentration in the highest quartile also increased the risk of five-year mor- tality, independent of potential confounders including cardiovascular disease [18]. Work on data taken from the National Health and Nutrition Examination study (NHANES) III concluded that CMV-seropositivity was associated with an increased risk of all-cause mortality in > 14,000 adults at least 25 years of age followed for > 10 years; especially individuals with a raised inflamma- tory marker (C-reactive protein CRP) were at a higher risk of both all-cause mortality and cardiovascular-asso- ciated mortality [19]. the very old. The study now has the opportunity to examine morbidity and mortality to determine if this truly defines a risk profile in people only in their sixties. It is clearly of great importance to establish if long- term carriage of HCMV is associated with an increased likelihood of all-cause mortality. A number of studies addressing this issue have already been published [16,17] and the results from further studies were pre- sented at this meeting. Wang spoke about a study of 635 70-79 year-old women living in the community. The results demonstrated that women in the highest HCMV IgG quartile had a significantly higher incidence of frailty as compared to HCMV-seronegative women at baseline. Furthermore, CMV IgG concentration in the highest quartile also increased the risk of five-year mor- tality, independent of potential confounders including cardiovascular disease [18]. Discussions on the Immune risk profile, IRP The idea that CD8+ memory T cells compete for space in a T cell compartment that is constrained in its maximum size has led to the suggestion that over-abun- dant memory T cells such as those seen in people with latent HCMV carriage could have a detrimental effect by displacing naïve or other memory T cells. Could the occupation of the “immunological space” by large and inflating HCMV-specific T cells contribute to immuno- senesence? Van Lier (Amsterdam) presented data from a study comparing the frequency of EBV- and HCMV- specific T cells in the blood and lymph node (LN) of donors showing that while the percentages of EBV-spe- cific T cells were comparable between blood and LN, HCMV had a much lower LN frequency which included the late-differentiated cells as determined by CD45RA+ and CD27- phenotyping. The study concluded that strong immune responses to HCMV were unlikely to restrict space for naïve or other memory T cells in LN. Although these results are intriguing, the impact of aging was not addressed in that study. In particular, as CMV infection has been shown to impair the response to a co-resident EBV infection in middle-aged and elderly persons, but not in young adults [21], the study raises a very important question. If CMV infection does not impair the survival of naive T cells in the LN, the major reservoir of naive T cells, how is the decline of naive T cells in the peripheral blood of CMV-seroposi- tive versus CMV-seronegative persons explained? [22,23]. The ability of CMV to significantly decrease peripheral naive T cells in young persons with poten- tially intact or at least more residual thymic T cell gen- eration indicates that CMV infection may not only drive peripheral naive T cell exhaustion but also affect T cell generation in the thymus. Accordingly, there is a need to delineate the mechanism of naive T cell generation/ exhaustion and CMV infection as well as to analyze the impact of CMV infection not only in the peripheral blood, but also in lymphoid and extra-lymphoid organs, to improve our understanding of HCMV-specific T cell migration, differentiation and expansion throughout life. Westendorp (Leiden) presented a study on the effi- The IRP was defined in the Swedish OCTO and NONO studies, but it was left as an open question if the IRP actually applies generally to human populations? Discussions on the Immune risk profile, IRP It was suggested that a cohort in Spain does show the IRP (Solana) and Moss has cohorts in the UK which also show the IRP; however, not all cohorts do, as an aged community in the mountain region in Sicily do not show the IRP (Caruso, Palermo). However it was felt that this population was not old enough as so far we only know that the IRP is relevant from age 85. The IRP does not take into account the absolute num- ber of naïve T cells, nor the absolute numbers of mem- ory cells: the IRP was based on percentages. The percentage of CD8+CD28- cells was part of the IRP, but it is also clear that there is an increase in the absolute number of these cells per ml of blood in the risk group. This raises the question at what levels of naïve CD4 or CD8 T cells do people start to experience problems? Sauce suggested from her work that a level of < 50 naive CD8+ T cells per μl and < 100 naïve CD4+ T cells is crucial, although different specific cut-offs might be relevant at different age ranges. A further refinement of the accumulation of CD8+ CD28- T cells defined in the IRP was suggested to take into account lymphope- nia, as it was felt that those individuals who were both lymphopenic and had large CD8+ CD28- expansions had a poor outcome. Discussion on Cohort studies of all-cause mortality CMV seropositive vs negative It was felt that defining the confounding factors and how to correct for them is crucial in these analyses, Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Page 5 of 8 Henson (London) reported that late-stage memory cells also express inhibitory receptors such as PD-1 and KLRG1 and this tends to be higher on T cells from indi- viduals who are HCMV-seropositive. This has been observed by many others, including several Workshop participants and seems to be a general finding. She showed that blockade of PD-1 increased proliferation of these cells and no additive effect was seen with dual PD-1 and KLRG-1 blockade, nor did it increase telomer- ase activity. although it is not clear what best practice is. The dis- similar populations in these cohort studies likely also contribute to the divergent findings on the association between HCMV carriage and mortality. However, the requirement for standardization in the quantitation of HCMV IgG levels would also be an important step and was discussed further along with other metrics that require standardization. These are outlined in the new questions section of this report. although it is not clear what best practice is. The dis- similar populations in these cohort studies likely also contribute to the divergent findings on the association between HCMV carriage and mortality. However, the requirement for standardization in the quantitation of HCMV IgG levels would also be an important step and was discussed further along with other metrics that require standardization. These are outlined in the new questions section of this report. The immunobiology of cytomegalovirus-specific T cells and effects on vaccination Macallan presented a study to determine why highly differentiated HCMV-specific CD8+ T cells accumulate and in particular to distinguish accumulation due to rapid proliferation versus long-term persistence due to impaired apoptosis. They demonstrated that these expansions are often oligoclonal and these clones are stable over many years. In addition, comparison of CD8 cells from young and old donors showed no difference in either proliferative activity or cytotoxic function. Assessment of in vivo turnover rate by deuterated glu- cose experiments suggest that the cells have a low pro- liferative rate in vivo and as such probably accumulate with time rather than by proliferation [20]. Westendorp (Leiden) presented a study on the effi- cacy of influenza vaccination in a cohort of elderly indi- viduals living in care facilities. The study design encompassed a number of different dosing regimes and Page 6 of 8 Page 6 of 8 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 was able to examine influenza seroconversion in relation to HCMV seropositivity. The study concluded that in this particular cohort previous CMV infection does not explain poor responsiveness to influenza vaccination and as such does not support CMV infection as being the origin of this dysfunction in the immune system of these older people [24]. These results contrast with an earlier study [25], in which elderly HCMV-seropositive individuals where shown to respond less well to influ- enza vaccination. It should be noted that only the domi- nant H3N2 strain of influenza strain was used in the den Elzen study as opposed to all three seasonal strains in the Trzonkowski study, and that the weakest correla- tion was between HCMV seropositivity and seroconver- sion to H3N2 in the latter. It is clear that this important area deserves further study. If HCMV does drive immu- nosenescence, a tangible outcome such as poor vaccina- tion responses would be predicted and importantly this evidence could support a rationale for a modified vacci- nation protocol in elderly HCMV seropositive individuals. specific immune responses (MASIR). A goal for presen- tation at the next meeting is to determine a set of recommendations for standardization along the lines outlined in [26,27]. Where are the CMV-specific T cells, where is the reservoir, is it the spleen? Or perhaps marginal pools? The immunobiology of cytomegalovirus-specific T cells and effects on vaccination This reservoir, and there may be more than one, seems dynamic and provides rapid access to the blood, as per- ipheral blood cell numbers show rapid fluctuations. The stress hormone adrenaline can regulate this exchange with the blood. Bosch (Birmingham) presented data showing that physiological stressors (exercise, psycholo- gical stress) cause swift (< 10 minutes) and robust (up to +300%) release of differentiated (CD27-CD28-CCR7-) memory CD8+ T cells as well as CMV-specific CD8+ T cells into peripheral blood. Using pharmacological infu- sion and blockade studies, he provided evidence that this release is regulated by the stress hormone adrena- line and dependent on activation of b-adrenergic recep- tors. This finding coincides with reports of upregulated expression of b2-adrenergic receptors on differentiated T cells [28]. Significantly, 1 to 2 hours post-stress these cells cleared from the blood, with cell numbers dropping to 70% below resting levels [29,30]. Bosch suggested that this may reflect a rapid egress into tissue. New Questions raised by the discussions Standardization - HCMV serotesting the means of detection, the measurement of viremia and T cell assays? It is not at all clear what antibody responses are actually being measured when HCMV serotesting is reported. There is a requirement for standardization in the CMV antigens that are used on the plate coatings. This is of particular importance as studies of the effect of HCMV on mortality or measures of health often group patients based on the level of antibody response (into four quar- tile groups). Comparison of different studies is not helped if what is being measured in one study/labora- tory is not the same as another study/laboratory. It was felt that there is a need for standardization such that results can be compared, and for further sophistication in dissecting out antibody repertoires. The subclass of IgG is never considered; IgM titer rarely, the avidity of antibody never. All these factors could be informative. In accordance with their effector-memory phenotype, CMV-specific T cells should preferentially migrate to and reside in extra-lymphoid organs and the bone mar- row. Yet the frequency of CMV-specific CD8+ and CD4 + T cells in the human bone marrow is similar [31,32] or even lower compared to the peripheral blood [33]. Nonetheless, Letch et al. did show that CMV-specific CD8 cells with a central memory phenotype, while pre- sent in low frequencies in the peripheral blood, were present at higher levels in the BM. The immunobiology of cytomegalovirus-specific T cells and effects on vaccination Moreover, CMV-spe- cific T cells from the BM underwent greater clonal expansion in vitro than PB cells. This might also be of practical importance in transplantation (independently of any ageing considerations). Although the impact of aging on the frequency of CMV-specific T cells in the bone marrow has not yet been addressed, the currently available data may support a role of the bone marrow as a reservoir for highly functional CMV-specific central- memory T cells (Herndler-Brandstetter, Birmingham). In a similar fashion, concerns about the comparability of T cell assays between groups in the field was felt to be an issue. Very little standardization was evident between different flow cytometry-based T cell assays. Parameters that were of particular concern were: the use of fresh vs frozen PBMC samples, the anti-coagu- lants used to collect blood samples, the combination of cell surface phenotypes used to determine T cell subpo- pulations (variations on the use of CD27 or CD28 with CD45RA eg). These concerns are not unique to this field. Kern noted that there was a move to adopt a stan- dardization provision of minimal information on T cell assays (MIATA). Similar concerns were raised from a series of conferences on the measurement of antigen- Is it how someone deals with CMV over a long period of time which is actually important? y p The Leiden Longevity Study examines familial longevity in an F2 generation with a 30% decreased standardised mortality rate between the ages of 40 and 80. This is proposed to be due to a genetic influence - an enrich- ment of “longevity-assurance” genes. The F2 subjects studied here showed some indications of resistance to becoming infected with CMV even when their partner (sometimes over decades) was CMV-seropositive. More- over, when they were infected, they did not show the characteristically lower frequencies of naïve CD8+ T cells and higher frequencies of late-differentiated CD8+ CD28- T cells seen in the general population [2]. They also showed lower levels of markers of inflammation in the plasma and less potentially inflammatory-mediated disease (eg. type 2 diabetes). The link between CMV, inflammation, disease, longevity and genetics is being studied in these remarkable people. Would treatment of CMV with antivirals have a beneficial effect on the immune system? Evidence from Hill’s studies suggest not! However, is the MCMV model sufficiently close to HCMV that we can extrapolate? In the face of the not yet sufficiently documented (and to some extent controversial) health risks of persistent CMV infection, it is highly speculative whether long-term treatment with antiviral drugs will result in any measurable health benefits. In particular, long-term treatment with antiviral drugs may not be a good option, as the treatment is likely to have side effects and to facilitate viral resistance. Some people felt that this argued in favour of the ongoing efforts to develop an efficient CMV vaccine able to protect from primary infection. Such a vaccine would most likely be cost-effective in preventing from any detrimental CMV- related impairments of the immune system. It was felt that currently-available antivirals also have too much toxicity and adverse-effect burden to tip the benefit- risk balance toward warranting administration to other- wise healthy humans. The administration of these antivir- als to immunosuppressed transplant recipients is warranted because of the significant morbidity and mortal- ity caused by CMV in this patient population. If further evidence suggests that halting HCMV replication and reactivation confers a beneficial effect on the immune sys- tem, the development of next-generation antivirals with significantly less toxicity would be a high priority. 27Department of Internal Medicine II, University of Tübingen, Tübingen, Germany. Author details 1 1Department of Medicine, University of Cambridge, Cambridge, UK. 2Division of Infection and Immunity, University College London, London, UK. 3School of Cancer Sciences, University of Birmingham, Birmingham, UK. 4College of Life and Environmental Sciences, University of Birmingham, Birmingham, UK. 5Mannheim Institute of Public Health, Social and Preventive Medicine, University of Heidelberg, Mannheim, Germany. 6Department of Pathobiology and Medical and Forensic Biotechnologies, University of Palermo, Palermo, Italy. 7Epidemiology and Public Health Group, Peninsula Medical School, University of Exeter, Exeter, UK. 8Dept. Experimental Pathology, University of Bologna, Bologna, Italy. 9Research Centre on Aging, Immunology Program, Geriatric Division, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Canada. 10Addenbrooke’s Hospital, Cambridge, UK. 11Stanford University School of Medicine, Stanford, CA, USA. 12School of Immunity and Infection, University of Birmingham, Birmingham, UK. 13Dept Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA. 14BSMS, University of Sussex Campus, Falmer, UK. 15NIHR Biomedical Research Centre, Nuffield Dept of Clinical Medicine, University of Oxford, Oxford, UK. 16Centre for Infection, St George’s, University of London, Cranmer Terrace, London, UK. 17Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, The Netherlands. 18Department of Immunobiology and the Arizona Center on Aging, University of Arizona, Tucson, AZ, USA. 19Department of Internal Medicine and Biomedical Sciences, University of Parma, Parma, Italy. 20INSERM, Infections and Immunity, Universite Pierre et Marie Curie-Paris, Paris, France. 21Instituto Maimonides de Investigación Biomédica de Cordoba (IMIBIC), Hospital Universitario Reina Sofía, Universidad de Cordoba, Cordoba, Spain. 22Department of Natural Science and Biomedicine, School of Health Sciences, Jönköping University, Jönköping, Sweden. 23Department of Clinical Immunology and Transplantology, Medical University of Gdańsk, Gdańsk, Poland. 24Sanquin Blood Supply Foundation and Department of Experimental Immunology, Amsterdam, The Netherlands. 25Division of Geriatric Medicine and Gerontology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 26Department of Public Health and Primary Care, Leiden University Medical Center, Leiden, The Netherlands. 27Department of Internal Medicine II, University of Tübingen, Tübingen, Germany. Are large expansions of HCMV specific T cells detrimental? Moss asked us to directly address the issue that if CMV has any association with immunosenescence, old people without CMV must have better immune responses to neo-antigens. It was further suggested that CMV-sero- positive vs negative showed no correlation with inflam- matory markers - and it would be expected that it should. However, could there be a subset or group of Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Page 7 of 8 Page 7 of 8 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Page 7 of 8 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 Acknowledgements Th W k h The Workshop was supported by the EU Network of Excellence “LifeSpan” (FP6-036894) Conclusions d certain individuals that are affected and this is not seen at a population level? Moss suggested this was the case in a cohort of CML patients where those with large CD4+ CD28- T cell expansions with a loss of naïve cells showed an increase in infections. This 2nd Workshop was attended by twice the number of people than the first original discussion group. The interaction of so many additional scientists with exper- tise in basic cytomegalovirus immunology, virology and epidemiology was invaluable. As a group, a large num- ber of the most important questions are being addressed and importantly new questions are being raised and dis- cussed which is vital for the field to move forwards. It was proposed that a 3rd meeting should be organised and this had overwhelming support. We are grateful to Rafael Solana (rsolana@uco.es) for offering to do this in Cordoba, Spain, 8-9th March, 2012. Authors’ contributions All authors contributed to the writing of this Commentary and all have approved the final version. References Derhovanessian E, Maier AB, Hahnel K, Beck R, de Craen AJ, Slagboom EP, Westendorp RG, Pawelec G: Infection with cytomegalovirus but not herpes simplex virus induces the accumulation of late differentiated CD4+ and CD8+ T-cells in humans. J Gen Virol 2011. 24. den Elzen WP, Vossen AC, Cools HJ, Westendorp RG, Kroes AC, Gussekloo J: Cytomegalovirus infection and responsiveness to influenza vaccination in elderly residents of long-term care facilities. Vaccine 2011, 29:4869. 6. Derhovanessian E, Larbi A, Pawelec G: Biomarkers of human immunosenescence: impact of Cytomegalovirus infection. Curr Opin Immunol 2009, 21:440. 25. Trzonkowski P, Mysliwska J, Szmit E, Wieckiewicz J, Lukaszuk K, Brydak LB, Machala M, Mysliwski A: Association between cytomegalovirus infection, enhanced proinflammatory response and low level of anti- hemagglutinins during the anti-influenza vaccination–an impact of immunosenescence. Vaccine 2003, 21:3826. 7. Sylwester AW, Mitchell BL, Edgar JB, Taormina C, Pelte C, Ruchti F, Sleath PR, Grabstein KH, Hosken NA, Kern F, Nelson JA, Picker LJ: Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects. J Exp Med 2005, 202:673. 26. Maecker HT, McCoy JP, Amos M, Elliott J, Gaigalas A, Wang L, Aranda R, Banchereau J, Boshoff C, Braun J, Korin Y, Reed E, Cho J, Hafler D, Davis M, Fathman CG, Robinson W, Denny T, Weinhold K, Desai B, Diamond B, Gregersen P, Di Meglio P, Nestle FO, Peakman M, Villanova F, Ferbas J, Field E, Kantor A, Kawabata T, Komocsar W, Lotze M, Nepom J, Ochs H, O’Lone R, Phippard D, Plevy S, Rich S, Roederer M, Rotrosen D, Yeh JH: A model for harmonizing flow cytometry in clinical trials. Nat Immunol 2010, 11:975. 8. Hutchinson S, Sims S, O’Hara G, Silk J, Gileadi U, Cerundolo V, Klenerman P: A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus. PLoS One 2011, 6:e14646. 9. Snyder CM, Allan JE, Bonnett EL, Doom CM, Hill AB: Cross-presentation of a spread-defective MCMV is sufficient to prime the majority of virus- specific CD8+ T cells. PLoS One 2010, 5:e9681. 10. Cicin-Sain L, Sylwester AW, Hagen SI, Siess DC, Currier N, Legasse AW, Fischer MB, Koudelka CW, Axthelm MK, Nikolich-Zugich J, Picker LJ: Cytomegalovirus-specific T cell immunity is maintained in immunosenescent rhesus macaques. J Immunol 2010, 187:1722. 27. References Britten CM, Janetzki S, van der Burg SH, Huber C, Kalos M, Levitsky HI, Maecker HT, Melief CJ, O’Donnell-Tormey J, Odunsi K, Old LJ, Pawelec G, Roep BO, Romero P, Hoos A, Davis MM: Minimal information about T cell assays: the process of reaching the community of T cell immunologists in cancer and beyond. Cancer Immunol Immunother 2011, 60:15. 11. Lang A, Nikolich-Zugich J: Functional CD8 T cell memory responding to persistent latent infection is maintained for life. J Immunol 2011, 187:3759. 28. Dimitrov S, Benedict C, Heutling D, Westermann J, Born J, Lange T: Cortisol and epinephrine control opposing circadian rhythms in T cell subsets. Blood 2009, 113:5134. 12. Sauce D, Larsen M, Fastenackels S, Duperrier A, Keller M, Grubeck- Loebenstein B, Ferrand C, Debre P, Sidi D, Appay V: Evidence of premature immune aging in patients thymectomized during early childhood. J Clin Invest 2009, 119:3070. 29. Turner JE, Aldred S, Witard OC, Drayson MT, Moss PM, Bosch JA: Latent cytomegalovirus infection amplifies CD8 T-lymphocyte mobilisation and egress in response to exercise. Brain Behav Immun 2009, 24:1362. 13. Sauce D, Larsen M, Fastenackels S, Pauchard M, Ait-Mohand H, Schneider L, Guihot A, Boufassa F, Zaunders J, Iguertsira M, Bailey M, Gorochov G, Duvivier C, Carcelain G, Kelleher AD, Simon A, Meyer L, Costagliola D, Deeks SG, Lambotte O, Autran B, Hunt PW, Katlama C, Appay V: HIV disease progression despite suppression of viral replication is associated with exhaustion of lymphopoiesis. Blood 2011, 117:5142. 30. Campbell JP, Riddell NE, Burns VE, Turner M, van Zanten JJ, Drayson MT, Bosch JA: Acute exercise mobilises CD8+ T lymphocytes exhibiting an effector-memory phenotype. Brain Behav Immun 2009, 23:767. 31. Letsch A, Knoedler M, Na IK, Kern F, Asemissen AM, Keilholz U, Loesch M, Thiel E, Volk HD, Scheibenbogen C: CMV-specific central memory T cells reside in bone marrow. Eur J Immunol 2007, 37:3063. 14. Hadrup SR, Strindhall J, Kollgaard T, Seremet T, Johansson B, Pawelec G, thor Straten P, Wikby A: Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly. J Immunol 2006, 176:2645. 32. References 19. Simanek AM, Dowd JB, Pawelec G, Melzer D, Dutta A, Aiello AE: Seropositivity to cytomegalovirus, inflammation, all-cause and cardiovascular disease-related mortality in the United States. PLoS One 2011, 6:e16103. 1. Pawelec G, Akbar A, Beverley P, Caruso C, Derhovanessian E, Fulop T, Griffiths P, Grubeck-Loebenstein B, Hamprecht K, Jahn G, Kern F, Koch SD, Larbi A, Maier AB, Macallan D, Moss P, Samson S, Strindhall J, Trannoy E, Wills M: Immunosenescence and Cytomegalovirus: where do we stand after a decade? Immun Ageing 2010, 7:13. 20. Wallace DL, Masters JE, De Lara CM, Henson SM, Worth A, Zhang Y, Kumar SR, Beverley PC, Akbar AN, Macallan DC: Human cytomegalovirus- specific CD8(+) T-cell expansions contain long-lived cells that retain functional capacity in both young and elderly subjects. Immunology 2011, 132:27. 2. Derhovanessian E, Maier AB, Beck R, Jahn G, Hahnel K, Slagboom PE, de Craen AJ, Westendorp RG, Pawelec G: Hallmark features of immunosenescence are absent in familial longevity. J Immunol 2010, 185:4618. 21. Khan N, Hislop A, Gudgeon N, Cobbold M, Khanna R, Nayak L, Rickinson AB, Moss PA: Herpesvirus-specific CD8 T cell immunity in old age: cytomegalovirus impairs the response to a coresident EBV infection. J Immunol 2004, 173:7481. 3. Cantisan S, Solana R, Lara R, Rodriguez-Benot A, Vaquero JM, Gutierrez- Aroca J, Gayoso I, Montejo M, Rivero A, Torre-Cisneros J: CD45RA expression on HCMV-specific effector memory CD8+ T cells is associated with the duration and intensity of HCMV replication after transplantation. Clin Immunol 2010, 137:81. 22. Almanzar G, Schwaiger S, Jenewein B, Keller M, Herndler-Brandstetter D, Wurzner R, Schonitzer D, Grubeck-Loebenstein B: Long-term cytomegalovirus infection leads to significant changes in the composition of the CD8+ T-cell repertoire, which may be the basis for an imbalance in the cytokine production profile in elderly persons. J Virol 2005, 79:3675. 4. Cantisan S, Torre-Cisneros J, Lara R, Rodriguez-Benot A, Santos F, Gutierrez- Aroca J, Gayoso I, Gonzalez-Padilla M, Casal M, Rivero A, Solana R: Age- dependent association between low frequency of CD27/CD28 expression on pp65 CD8+ T cells and cytomegalovirus replication after transplantation. Clin Vaccine Immunol 2009, 16:1429. 23. Weinberger B, Lazuardi L, Weiskirchner I, Keller M, Neuner C, Fischer KH, Neuman B, Wurzner R, Grubeck-Loebenstein B: Healthy aging and latent infection with CMV lead to distinct changes in CD8+ and CD4+ T-cell subsets in the elderly. Hum Immunol 2007, 68:86. 5. Conflicts of interests Conflicts of interests The authors declare that they have no competing interests. Received: 12 October 2011 Accepted: 28 October 2011 Published: 28 October 2011 Received: 12 October 2011 Accepted: 28 October 2011 Published: 28 October 2011 Received: 12 October 2011 Accepted: 28 October 2011 Published: 28 October 2011 Page 8 of 8 Page 8 of 8 Wills et al. Immunity & Ageing 2011, 8:10 http://www.immunityageing.com/content/8/1/10 doi:10.1186/1742-4933-8-10 Cite this article as: Wills et al.: Report from the second cytomegalovirus and immunosenescence workshop. Immunity & Ageing 2011 8:10. References Libri V, Azevedo RI, Jackson SE, Di Mitri D, Lachmann R, Fuhrmann S, Vukmanovic-Stejic M, Yong K, Battistini L, Kern F, Soares MV, Akbar AN: Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4+CD45RA+CD27+ T cells: the potential involvement of interleukin-7 in this process. Immunology 2011, 132:326. y 15. Arvin A, Abendroth A: VZV: immunobiology and host response. 2007. 16. Strandberg TE, Pitkala KH, Tilvis RS: Cytomegalovirus antibody level and mortality among community-dwelling older adults with stable cardiovascular disease. Jama 2009, 301:380. 33. Palendira U, Chinn R, Raza W, Piper K, Pratt G, Machado L, Bell A, Khan N, Hislop AD, Steyn R, Rickinson AB, Buckley CD, Moss P: Selective accumulation of virus-specific CD8+ T cells with unique homing phenotype within the human bone marrow. Blood 2008, 112:3293. 17. Roberts ET, Haan MN, Dowd JB, Aiello AE: Cytomegalovirus antibody levels, inflammation, and mortality among elderly Latinos over 9 years of follow-up. Am J Epidemiol 2010, 172:363. doi:10.1186/1742-4933-8-10 Cite this article as: Wills et al.: Report from the second cytomegalovirus and immunosenescence workshop. Immunity & Ageing 2011 8:10. 18. Wang GC, Kao WH, Murakami P, Xue QL, Chiou RB, Detrick B, McDyer JF, Semba RD, Casolaro V, Walston JD, Fried LP: Cytomegalovirus infection and the risk of mortality and frailty in older women: a prospective observational cohort study. Am J Epidemiol 2010, 171:1144. 18. Wang GC, Kao WH, Murakami P, Xue QL, Chiou RB, Detrick B, McDyer JF, Semba RD, Casolaro V, Walston JD, Fried LP: Cytomegalovirus infection and the risk of mortality and frailty in older women: a prospective observational cohort study. Am J Epidemiol 2010, 171:1144.
https://openalex.org/W2027962422	https://hal.science/hal-01189739/document	English	null	Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources	BMC genomics	2,010	cc-by	11,299	To cite this version: Camille Rustenholz, Pete E. Hedley, Jenny Morris, Frédéric Choulet, Catherine Feuillet, et al.. Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources. BMC Genomics, 2010, 11, 10.1186/1471-2164-11-714. hal-01189739 Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources Camille Rustenholz, Pete E. Hedley, Jenny Morris, Frédéric Choulet, Catherine Feuillet, Robbie Waugh, Etienne Paux Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources Camille Rustenholz, Pete E. Hedley, Jenny Morris, Frédéric Choulet, Catherine Feuillet, Robbie Waugh, Etienne Paux RESEARCH ARTICLE Open Access © 2010 Rustenholz et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources Camille Rustenholz1†, Pete E Hedley2†, Jenny Morris2, Frédéric Choulet1, Catherine Feuillet1, Robbie Waugh2, Etienne Paux1* * Correspondence: etienne.paux@clermont.inra.fr † Contributed equally 1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100 Clermont-Ferrand, France Full list of author information is available at the end of the article Abstract Background: Because of its size, allohexaploid nature and high repeat content, the wheat genome has always been perceived as too complex for efficient molecular studies. We recently constructed the first physical map of a wheat chromosome (3B). However gene mapping is still laborious in wheat because of high redundancy between the three homoeologous genomes. In contrast, in the closely related diploid species, barley, numerous gene-based markers have been developed. This study aims at combining the unique genomic resources developed in wheat and barley to decipher the organisation of gene space on wheat chromosome 3B. Results: Three dimensional pools of the minimal tiling path of wheat chromosome 3B physical map were hybridised to a barley Agilent 15K expression microarray. This led to the fine mapping of 738 barley orthologous genes on wheat chromosome 3B. In addition, comparative analyses revealed that 68% of the genes identified were syntenic between the wheat chromosome 3B and barley chromosome 3 H and 59% between wheat chromosome 3B and rice chromosome 1, together with some wheat-specific rearrangements. Finally, it indicated an increasing gradient of gene density from the centromere to the telomeres positively correlated with the number of genes clustered in islands on wheat chromosome 3B. Conclusion: Our study shows that novel structural genomics resources now available in wheat and barley can be combined efficiently to overcome specific problems of genetic anchoring of physical contigs in wheat and to perform high-resolution comparative analyses with rice for deciphering the organisation of the wheat gene space. HAL Id: hal-01189739 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 A high throughput anchoring method g t oug put a c o g et od To assess the efficiency of wheat-barley cross-species hybridisation for gene-based physical map anchoring, a barley Agilent 15K unigene microarray was hybridised with 60 three-dimensional (plate, row, column) BAC pools from the MTP of the wheat chromosome 3B [17]. After signal quantification and normalisation, hybridisa- tion data were evaluated with four complementary scor- ing methods to reliably locate as many barley gene homologs as possible on the wheat BACs (see Methods). Using the most stringent “automated scoring” method, 3355, 3401 and 3286 probes were identified as positive with the plate, row and column pools, respectively. Deconvolution of the pool data led to the identification of 571 unambiguous BAC addresses for 566 unigenes, defining 561 unique genomic loci and 5 duplicated loci. The less stringent “boxplot scoring” method led to the identification of 6205, 5103 and 6761 positive probes for the plate, row and column pools, respectively. With this method, 770 probes having unambiguous BAC addresses were identified, including 481 that were already identi- fied with the “automated” method. Out of the 289 newly identified probes, we selected 86 probes (100 loci) that correspond to the most robust data (i.e. located on two to three overlapping BACs). Finally the “semi- automated” and the “manual scoring” methods added additional BAC addresses for 13 and 78 probes respec- tively that showed missing coordinates with the two other methods due to technical limitations (detailed below). We recently constructed a physical map of chromo- some 3B, the largest wheat chromosome (1 Gb, 2.5 times the whole rice genome) [17]. The map consists of 1036 contigs spanning 811 Mb, of which 611 Mb are anchored with 1443 molecular markers. However, very few contigs are anchored by gene-derived markers. Indeed despite the development of genomic resources, such as extensive marker collections and saturated genetic maps [18-20], genetic mapping of genes in wheat is still hampered by the lack of polymorphism and the presence of the three homoeologous copies of each gene. As a result, no high density transcript genetic map is available. In contrast, several gene maps have been constructed for barley [21-25] (Hordeum vulgare L.) that diverged from wheat ~10-12 MYA [26,27] and belongs to the same tribe (Tri- ticeae). Background Arabidopsis thaliana (125 Mb), Brachypodium distach- yon (272 Mb) and Oryza sativa (389 Mb) exhibit fairly homogenous gene distribution along their chromosomes [3-5]. The transition from a homogenous to a non- homogenous gene distribution seems correlated to the genome size. Indeed, in intermediate size genome, such as Populus trichocarpa (485 Mb) and Vitis vinifera (487 Mb), large regions alternating between high and low gene density were observed [6,7], whereas larger genomes, such as Glycine max (1115 Mb) and Zea mays (2300 Mb), display an increasing gradient of gene den- sity from the centromere to the telomeres [8,9]. The term “gene space” refers to the fraction of the gen- ome corresponding to protein coding genes and, by extension, to the distribution of these genes [1]. In large genomes that contain abundant repetitive DNA, it encompasses also the notion of regions containing genes, the so-called gene-rich regions, surrounded by gene-poor regions composed of repeats [2]. With the growing number of sequenced plant gen- omes, it becomes obvious that the distribution pattern of genes is far from random and not universal across the plant kingdom. Small plant genomes, such as Because of its size (17000 Mb), allohexaploid nature (A, B and D-genomes) and high repeat content (>80%) [10], the bread wheat (Triticum aestivum L.) genome is among the largest and most complex plant genomes and has always been considered too complex for * Correspondence: etienne.paux@clermont.inra.fr † Contributed equally 1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100 Clermont-Ferrand, France Full list of author information is available at the end of the article Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 2 of 12 molecular analyses. As a result, no genome sequence is available yet and very little is known about the organisa- tion of the wheat gene space. The first insights were obtained from the mapping of wheat gene-based mar- kers in wheat aneuploid genotypes called deletion lines where fragments of chromosomes or deletion bins are missing [11]. Based on EST and Pst1 genomic clone mapping, Erayman et al. [12] suggested a very heteroge- neous distribution of the genes along the wheat chro- mosomes, with 94% of the genes being located in only 29% of the entire wheat chromosomes and mostly at their telomeric parts. In contrast, by EST mapping on chromosome group 3 deletion bins, Munkvold et al. Background [13] observed a slight gradient of the gene density along the chromosomes as well as a significant number of genes in the most proximal bins thereby suggesting a more homogeneous distribution. More recently, indivi- dual BAC sequencing [14,15] confirmed a rather homo- geneous gene distribution in wheat with an average of one gene per BAC. Finally, Choulet et al. [16] investi- gated megabase-sized regions from various parts of chromosome 3B and indicated that the gene-free regions are much smaller than expected by Erayman et al. [12], i.e. not larger than 1 Mb. Moreover, they found evidence for a slight gradient (twofold) of the gene density distri- bution from the centromere to the telomeres. Thus, additional whole genome or whole chromosome ana- lyses are needed to better characterize the gene space organisation in wheat. microarrays to identify the location of genes along the wheat 3B physical map. The results show that such barley-wheat cross-hybridisations represent high- throughput cost-efficient approaches for anchoring genes on wheat physical maps and for performing com- parative genomics studies between wheat and other grass genomes. In addition, the possibility to locate genes precisely within BAC contigs that were anchored by other markers onto the chromosome 3B enabled us to gain new insights into the distribution of genes along a wheat chromosome. A high throughput anchoring method With a size of 4.9 Gb [10] and a repeat content of over 80% [28], the diploid barley genome (2n = 14) is very similar to the wheat subgenomes and several map- ping studies have demonstrated a high collinearity between barley and wheat [26,27,29-32]. In total, the combination of four methods enabled us to identify 762 unambiguous wheat BAC addresses for 743 barley probes. A BLASTN search [33] against the Triticeae repeat database TREP [34], indicated that five probes had high sequence identity (>86%) with TEs and were removed from further analysis. Each of the remain- ing 738 non TE-related genes was assigned to one to three wheat BACs resulting in 757 gene loci identified on the wheat chromosome 3B physical map [17]. These barley unigenes were located on 624 wheat BACs that corresponded to 388 individual contigs of 187 kb to 3.8 Mb and 86 singletons. Here, we wanted to explore the possibility of using barley transcript genetic maps as a surrogate to anchor and order the wheat physical contigs. BAC pools repre- senting the minimal tiling path (MTP) of wheat chro- mosome 3B were hybridised onto barley expression Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 3 of 12 Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 We tested the reliability of the 757 genes using the sequenced contigs available on chromosome 3B [16]. We found that 74% (23/31) of the genes located on the sequenced contigs through hybridisation gave a hit on the sequenced contigs after a BLASTN analysis. Out of these genes, 91% (21/23) matched a gene on the sequenced contigs at their expected location. Eight uni- genes (26%) were assigned to these contigs but their position was not supported by sequence information. Several hypotheses can be proposed to explain such dis- crepancy between hybridization and sequencing data, including false positives, misassembled MTP BACs or gaps in the sequence. In addition, 30 out of the 15208 barley Agilent microarray unigenes matched a gene after a BLASTN analysis against the sequenced contigs but were not located on a BAC through hybridisation. How- ever the sequence identity of these 30 unigenes (84%) was lower than the sequence identity of the 21 unigenes located on the sequenced contigs through hybridisation (90%). These unigenes would therefore hardly be located on a BAC through hybridisation. A high throughput anchoring method These data validated this cross-species hybridisation approach as a powerful and reliable method to map genes to BAC contigs. sequence conservation played a key role in the detection of hybridisation signals and that more than half of the potentially positive barley probes generated a near unde- tectable hybridisation signal with the wheat BACs. A second origin of the discrepancy likely originates from the presence of gene families located at multiple loci. The wheat genome is allohexaploid (three subgenomes: A, B and D) and at least one copy of each wheat gene is expected to be present on the three homoeologous chro- mosomes. In addition, there is increasing evidence for high level of tandem and interchromosomal duplication events in wheat and perhaps barley genomes since their divergence from the other grasses [16,39]. Thus there is a good probability that some genes are found in multiple copies on chromosome 3B. Such genes can result in mul- tiple non-overlapping BAC addresses that cannot be resolved without ambiguity and are therefore excluded from our analysis. Another critical point affecting the efficiency of the approach lies in the putative heterogene- ity of the BAC pools. Indeed, each three-dimensional MTP pool contains more than 300 BACs (see Methods), making it difficult to guarantee equimolarity for all BACs. In some extreme cases, this heterogeneity in indi- vidual BAC quantity may lead to weak signal intensity for positive probes resulting in missing coordinates. These two limitations could be circumvented by the use of six- dimension pools of the complete chromosome 3B BAC library [40]. However, such pools would have required almost 3 times more hybridisations than the three- dimensional MTP pools (177 vs. 60) thereby reducing the cost-efficiency of the approach. g g The 738 probes correspond to roughly 40% of the bar- ley unigenes that were expected to be present on the wheat chromosome 3B physical map. Indeed, chromo- some 3 H accounts for approximately 14.8% of the bar- ley genome [35,36]. Assuming a comparable gene density for all barley chromosomes, 2250 probes out of the 15208 unigenes are expected to be located on chro- mosome 3 H. As the MTP covers 82% of the whole wheat chromosome 3B, about 1845 probes should in theory be present on the wheat chromosome 3B physi- cal map assuming that all barley genes are conserved in wheat. A high throughput anchoring method The difference of 60% between expected and observed results could be explained by both biological and technical limitations of our experiment. First, sequence divergence between wheat and barley genes may have significantly impacted the efficiency of this approach. Letowski et al. [37] estimated that hybridising a probe and a DNA target sharing 90% of sequence identity results in 73% to 99% decrease in hybridisation signal intensity compared to a probe and a DNA sharing 100% of sequence identity. Blasting the barley 60-mer probes against the 6162 wheat cv. Chinese Spring full- length cDNA dataset [38] revealed that 56% of the hits show more than 10% nucleotide divergence (86% iden- tity on average). Moreover we found that the unigenes located on the sequenced contigs of the wheat chromo- some 3B [16] through BLASTN and hybridisation showed a significantly higher sequence identity (90%) compared to the ones that were located on the sequenced contigs through BLASTN only (83%) (T-test, P-value = 5E-6). Therefore, one can estimate that Despite these limitations, this single experiment per- mitted the localisation of 738 genes on the wheat chro- mosome 3B contigs and allowed us to get novel information for the order of the BAC contigs along the chromosome based on the barley EST genetic maps. So far, genetic mapping of genes in wheat has been ham- pered by the lack of polymorphism in the genic sequences and the presence of several homoeologous copies. As a consequence, only a third of the 680 mar- kers located on the chromosome 3B genetic map con- structed using the ‘neighbours’ approach correspond to ESTs [17]. Here, we established a barley whole genome neighbour map using the same criteria as the IBM neighbour map of maize [41] and used it to assess the order of wheat contigs based on the EST order found on the genetic map. Out of the 738 probes assigned to BAC contigs of wheat chromosome 3B, 308 (42%) were mapped to the barley neighbour map including 209 on chromosome 3 H and 99 on other chromosomes. Using the barley 3 H mapping data, 151 BAC contigs and 20 singletons from the wheat chromosome 3B physical map were genetically ordered. Only 30% of these contigs were previously ordered genetically using the wheat Rustenholz et al. A high throughput anchoring method BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 4 of 12 probes located on the contigs of wheat chromosome 3B, 209 were mapped on barley chromosome 3 H and 99 on other barley chromosomes. Rice orthologous genes were identified unambiguously for 659 of the 738 probes located on wheat chromosome 3B, of which 389 are located on rice chromosome 1and 270 on other rice chromosomes (Table 1). These results suggest that, at the whole chromosome scale, 68% and 59% of wheat chromosome 3B genes are syntenic with genes located on the orthologous barley chromosome 3 H and rice chromosome 1, respectively. These results are consistent with previous studies that estimated between 59% and 74% of the genes were in conserved positions between wheat chromosome group 3 and rice chromosome 1 and 75% between wheat chromosome group 3 and bar- ley chromosome 3 H [13,25,43-50]. The genes that are not syntenic between wheat chromosome 3B and barley chromosome 3 H mapped on the other barley chromo- somes with no significant bias towards any other single chromosome (Chi2 test, P-value = 0.16). In contrast, the non-syntenic genes between wheat chromosome 3B and rice chromosome 1 are biased in favour of genes mapped on the rice chromosomes carrying the highest number of genes (chromosomes 3 and 5) and against the rice chromosome carrying the lowest number of genes (chromosome 9) (Chi2 test, P-value < 10-5). Further analyses confirmed that the distribution of the non-syntenic genes between wheat chromosome 3B and rice chromosome 1 on the other rice chromosomes is correlated with the number of genes per rice chromo- some (Pearson’s correlation coefficient r = 0.735; P-value = 0.01) [3]. Thus, no real mapping bias was identified towards any of the non-syntenic barley or rice chromosomes. chromosome 3B neighbour map, whereas 44% were mapped to the wheat chromosome 3B deletion bin map, but not ordered in bins, and 26% were not anchored at all [17]. In addition, it is worth noting that 36% of the 151 contigs are only anchored by gene-based markers. This is consistent with the results of Paux et al. [17] who showed that some regions of the genome can only be anchored by specific types of markers (ESTs, SSRs, ISBPs) and that 35% of the contigs were anchored by ESTs only. A high throughput anchoring method Therefore, we conclude that the cross-hybri- disations of wheat BAC pools with barley expression microarrays is a straight forward approach to order wheat contigs with gene-based markers without the dif- ficulty of EST genetic mapping in wheat. Moreover, the total cost for these 60 pool hybridisa- tions on 15 microarrays was approximately 8800 USD. For the same price, PCR screening of individual EST markers on the same BAC pools (including primers and amplification) would only have allowed testing of 500 markers. Thus, the method is a cost-efficient alternative to PCR-based physical map anchoring. However, despite its convenience and its cost-efficiency, this technique is still limited in the number of contigs anchored and ordered but it would be greatly improved by technologi- cal developments in the near future. First, use of the barley 44K Agilent expression microarray will signifi- cantly increase the number of positive probes, regardless of the experiment efficiency. Second, as large amounts of barley SNPs are becoming available [22], the number of genetically mapped genes will increase in the coming years, therefore improving the efficiency of the anchor- ing strategy. Finally, wheat-barley cross-species hybridisation is a convenient, cost-efficient and relatively high-throughput approach for gene-based physical map anchoring and ordering of wheat BAC contigs. However, even if the use of barley genomic resources circumvents the limita- tions caused by the complexity of the wheat genome, the divergence between the two species is large enough to observe synteny breaks. Thus, we performed a com- parative study between wheat, barley and rice to assess the extent to which the barley gene order is transferable to wheat. Interestingly, a number of genes located on wheat chromosome 3B were not syntenic with barley chromo- some 3 H but their homologs were syntenic between barley and rice. For example, 11 wheat chromosome 3B genes mapped on barley chromosome 2 H and on its ortholog in rice (chromosome 4). We found another example with 9 wheat chromosome 3B genes mapping on barley chromosome 6 H and on the orthologous rice chromosome 2 [44]. This result indicates that these genes have undergone rearrangements specifically in wheat and supports the recent finding of Choulet et al. [16] for extensive interchromosomal duplications in wheat. Comparative genomics between wheat, barley and rice We calculated the per- centage of probes that are syntenic to barley chromo- some 3 H genes for each deletion bin of chromosome 3B and found that the conservation of genes is signifi- cantly uniform along chromosome 3B (Chi2 test, P-value = 0.84) with 73% of syntenic genes per bin on average (Table 1). We performed the same calculation with the 285 genes assigned to wheat chromosome 3B deletion bins and syntenic to genes on rice chromosome 1. In this case, the distribution of syntenic genes was nega- tively correlated with the distance to the centromere (Pearson’s correlation coefficient r = -0.742; P-value = 0.04). In other words, the level of synteny between wheat chromosome 3B and rice chromosome 1 decreases from the centromere to the telomeres. This is in complete agreement with the results of Akhunov et al. [39] who correlated this with the recombination rate along wheat chromosomes. However, using the data from Saintenac et al. [51] who performed an analysis of the distribution of the recombination rate among chro- mosome 3B, we did not find any correlation between the synteny level and crossing-over frequency (Pearson’s correlation coefficient r = -0.378; P-value = 0.36). Com- parisons between the sequences of 18 Mb sized contigs of chromosome 3B with the rice and Brachypodium genomes led to the same conclusions [16]. Moreover, the authors found a positive correlation between trans- posable element activity and the number of non syntenic genes. Thus, it is likely that the synteny level between wheat chromosome 3B and rice chromosome 1 that decreases from the centromere to the telomeres results from a combination of factors that have still to be identified. The links between the barley chromosome 3 H genetic map, the rice chromosome 1 sequence and the wheat chromosome 3B deletion bin map were used to analyse the collinearity, i.e. the order of the genes [42], between the genes on wheat chromosome 3B and on barley chro- mosome 3 H and between the genes on wheat chromo- some 3B and on rice chromosome 1. As the relative order of wheat genes in a given deletion bin is not known, we inferred this order from the barley chromo- some 3 H and rice chromosome 1 data. Comparative genomics between wheat, barley and rice Comparative genomics between wheat, barley and rice In addition to its interest for anchoring physical maps, cross-species hybridisation also provides valuable data for comparative genomics as it allows the mapping of barley (and to some extent rice) orthologous genes on wheat chromosomes. We studied synteny, i.e. the con- servation of the genes on the orthologous chromosomes of wheat chromosome 3B in barley (chromosome 3H) and rice (chromosome 1) without the assumption of the conservation of the gene order [42]. Out of the 738 Out of the 219 gene loci orthologous to barley chro- mosome 3 H genes, 153 have been located on wheat BACs assigned to one of the eight deletion bins of wheat chromosome 3B using the physical map data [17]. Their approximate location on the chromosome arms was thus inferred from the mapping data of the BAC. This enabled us to study the synteny between wheat, Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 5 of 12 Page 5 of 12 Table 1 Mapping data in wheat chromosome 3B deletion bins Wheat Barley Rice 3B Deletion Bin Bin size (Mb) Number of loci Density (locus/ Mb) Gene loci mapped on 3H Gene loci mapped on the other chromosomes Collinear gene loci between 3B and 3H Gene loci mapped on Os01 Gene loci mapped on the other chromosomes Collinear gene loci between 3B and Os01 3BS8- 0.78-1.00 44.2 37 0.84 10 9 7 15 14 10 3BS9- 0.57-0.78 43.2 43 1.00 12 4 9 22 16 16 3BS1- 0.33-0.57 94.3 86 0.91 14 11 8 40 33 20 C-3BS1- 0.33 58.3 34 0.58 9 1 4 23 7 16 C-3BL2- 0.22 45.7 36 0.79 15 3 8 27 6 18 3BL2- 0.22-0.50 74.9 78 1.04 25 10 21 47 26 38 3BL10- 0.50-0.63 40.1 40 1.00 13 5 8 18 17 8 3BL7- 0.63-1.00 155.5 165 1.06 55 14 37 93 57 59 Total assigned 556.2 519 0.93 153 57 102 285 176 185 Not assigned 438.8 238 / 66 42 / 111 104 / Total 995 757 / 219 99 / 396 280 / 318 676 Table 1 Mapping data in wheat chromosome 3B deletion bins Wheat Barley Table 1 Mapping data in wheat chromosome 3B deletion bins barley and rice at a finer scale. Comparative genomics between wheat, barley and rice This virtual order was used to define eight synteny blocks on barley chromosome 3 H and rice chromosome 1 corresponding to the eight deletion bins of wheat chromosome 3B (Figure 1). Genes being assigned to a given deletion bin of wheat and to the corresponding synteny block of bar- ley or rice were considered as collinear. Those not being conserved at syntenic positions were considered as non collinear. In total we found that 102 (67%) genes were collinear between wheat 3B and barley 3 H and 185 (65%) between wheat 3B and rice 1 (Table 1 & Figure 1). We calculated the percentage of collinear genes between wheat chromosome 3B and barley chromosome 3 H and between wheat chromosome 3B and rice chromosome 1 Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 6 of 12 Figure 1 Collinearity between wheat chromosome 3B, barley chromosome 3 H and rice chromosome 1. Each colour on wheat chromosome 3B corresponds to a deletion bin. Yellow: 3BS8-0.78- 1.00; light green: 3BS9-0.57-0.78; pink: 3BS1-0.33-0.57; red: C-3BS1- 0.33; orange: C-3BL2-0.22; purple: 3BL2-0.22-0.50; dark green: 3BL10- 0.50-0.63 and brown: 3BL7-0.63-1.00. The black segments correspond to the heterochromatic regions identified by C-banding, the coloured segments to the euchromatic regions and the circle to the centromere. The coloured blocks represent regions where the genes are collinear between two chromosomes. The lines represent the genes that are not collinear between two chromosomes. As the relative order of wheat genes in a given deletion bin is not known, we inferred this order from the barley chromosome 3 H and from rice chromosome 1 data. we had to use a barley neighbour map combining these maps to optimize the anchoring experiment. However, the gene order is not fully reliable in such maps especially in the pericentromeric and centromeric parts of the chro- mosomes where recombination is reduced or totally sup- pressed [25]. Finally, the use of wheat neighbour genetic mapping data suggests additional rearrangements in bins (data not shown) as suggested by Liu et al. [53] and Chantret et al. [54] and this may also lead to some discrepancies. Altogether, our results regarding conservation between wheat chromosome 3B, barley chromosome 3 H and rice chromosome 1 at the whole chromosome and at the deletion bins scales are in agreement with previous studies. Wheat gene space organisation For the first time, we were able to assign precisely a large number of genes to individual BACs and BAC contigs whose order is known on wheat chromosome 3B. This led us to analyse the pattern of gene distribu- tion along the chromosome. Out of the 757 loci mapped on the wheat chromosome 3B physical map, 519 loci were assigned to the eight deletion bins (Table 1). The density of loci per deletion bin was calculated by divid- ing the number of loci assigned to each deletion bin by the cumulative length of contigs in the bin [17]. The density of loci showed a slight increasing gradient from the centromere to the telomeres (Figure 2) with a posi- tive correlation with the distance to the centromere (Pearson’s correlation coefficient r = 0.664), even though this correlation was not statistically significant using a 5% threshold (P-value = 0.07). The highest density was found on the most distal 3BL7-0.63-1.00 deletion bin of the long arm (1.06 probes per megabase), whereas the lowest density was observed on the most proximal C-3BS1-0.33 bin of the short arm (0.58 probes per megabase). Such a gradient could be artificially created by a difference in gene sequence conservation from the centromere to the telomeres between wheat and barley. To test this hypothesis, we calculated the coefficient of correlation between the gene density and the percentage of sequence identity per bin. No significant correlation was found (Pearson’s correlation coefficient r = -0.478; P-value = 0.23), demonstrating that the gene density gradient is not biased by the differences in the similarity Figure 1 Collinearity between wheat chromosome 3B, barley chromosome 3 H and rice chromosome 1. Each colour on wheat Figure 1 Collinearity between wheat chromosome 3B, barley chromosome 3 H and rice chromosome 1. Each colour on wheat for each bin. We found that the distribution of collinear genes is significantly uniform along chromosome 3B (Chi2 test, P-value = 0.89 and 0.69 for barley and rice respectively). Some translocations of genes can be observed between wheat chromosome 3B and barley chromosome 3 H and between wheat chromosome 3B and rice chromosome 1 (Figure 1) that are similar to pre- vious studies [13,52]. However, we expected a higher col- linearity between the wheat and barley group 3 chromosomes with regard to previous results [29-32]. Comparative genomics between wheat, barley and rice However, we also noticed some wheat-specific rearrangements of the genes that disrupt the collinearity between wheat and barley and between wheat and rice. Thus, globally we expected the genes to be in the same order between wheat and barley but rearrangements are likely to be observed locally. So the results of anchoring and ordering of the wheat BAC contigs along chromo- some 3B using the barley mapping data should be con- sidered with caution as they may not be perfectly exact. Wheat gene space organisation We think that this apparent discrepancy may originate from the construction of the barley neighbour map. None of the five individual barley genetic maps available to date holds a sufficient number of genes, and therefore Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 7 of 12 Figure 2 Gene density in the eight deletion bins of wheat chromosome 3B. The density of isolated genes is represented by blue bars. The density of the genes organized in island is represented by red bars. The proportions of genes organized in island per deletion bins are shown as percentages within the red bars. Figure 2 Gene density in the eight deletion bins of wheat chromosome 3B. The density of isolated genes is represented by blue bars. The density of the genes organized in island is represented by red bars. The proportions of genes organized in island per deletion bins are shown as percentages within the red bars. 3B. This resulted in an estimate of 904 loci assigned to the eight deletion bins compared to the 519 loci identi- fied by hybridisation in this study. Recently Choulet et al. [16] estimated that chromosome 3B carries 8400 genes. We then extrapolated the gene density by consid- ering 8400 loci assigned to the eight deletion bins. We found that the distal bin 3BL7-0.63-1.00 and the proxi- mal bin C-3BS1-0.33 would have a gene density of 1 gene per 101 kb and 1 per 185 kb, respectively. There- fore, even in the least gene-dense regions of the chro- mosome, our results indicate that there may be one gene on average every 185 kb and therefore no mega- base-sized regions devoid of genes. This is consistent with RNA hybridisations on chromosome 3B MTP arrays that showed that the largest region without genes is about 800 kb long and that genes are distributed across the entire chromosome 3B [16]. between wheat and barley gene sequences. Thus, the gene density distribution observed here provides a reli- able snapshot of the gene space organisation along wheat chromosome 3B and led to a hypothesis where the gene density is higher in the distal parts than in the proximal parts of the chromosome (Figure 2). Conclusions Our study demonstrates that hybridisations of the barley Agilent 15K expression microarray with wheat chromo- some 3B MTP pools is a convenient and cost-efficient technique to perform physical map anchoring with gene-based markers. Our comparative genomics analysis between wheat, barley and rice confirms good global collinearity between these species, with a few wheat-spe- cific rearrangements that could lead to local mis-order- ing of wheat contigs using the barley gene order. Using this technique, we also confirmed previous studies that the gene space organisation follows a gradient of gene density along chromosome 3B from centromere to telo- meres without large “gene-free” regions. We also demonstrated that this gradient was generated by a dif- ferential accumulation of gene islands between the cen- tromere and the telomeres with more genes in islands in the distal parts of the chromosome. Such results have far-reaching implications in terms of strategies to sequence the wheat genome. Indeed, our results confirm that to access the whole wheat gene set, the entire wheat genome needs to be sequenced. A wheat expres- sion microarray is currently being utilised to increase the density of genes at the BAC scale located along wheat chromosome 3B and to improve our understand- ing of the wheat gene space organisation. To further study the gene space and especially the genes clustered in islands in more detail, we considered gene islands as multiple genes located on the same BAC or overlapping BACs, i.e. separated by less than 150 kb. Out of the 757 loci mapped on wheat chromosome 3B physical map, 303 loci, i.e. 40%, were considered part of gene islands, whereas the 454 remaining genes (60%) were considered as isolated genes. In contrast to the dis- tribution of isolated genes that we found significantly uniform along chromosome 3B (Chi2 test, P-value = 0.97), the distribution of genes organised in islands was significantly non-uniform along chromosome 3B (Chi2 test, P-value < 10-5) with a positive correlation between the density of genes in islands and the distance to the centromere (Pearson’s correlation coefficient r = 0.762; P-value = 0.03) (Figure 2). We also found a correlation between the density of genes in islands and the overall gene density (Pearson’s correlation coefficient r = 0.956, P-value < 10-3). Wheat gene space organisation As we found that 87% of the genes were mapped on the 81% most distal parts of chromosome 3B, our result is in agreement with the moderate gradient of gene density along wheat chromosomes suggested by Munkvold et al. [13], Devos et al. [15], Charles et al. [14] and more recently, Choulet et al. [16]. The discrepancy between these results and the first suggestions of Erayman et al. [12] that most of the genes are located in the distal parts of the wheat chromosomes may originate from their use of a consensus deletion bin map from the A, B and D chromosomes, where markers were non-systema- tically assigned to the deletion bins [51]. This likely led to approximations of the relative positions of the mar- kers and the postulation that genes are mostly found in gene-rich regions in wheat. Here, the possibility to assign genes on physical contigs that cover 82% of a specific chromosome 3B [17] enabled us to derive more precise information about the gene distribution. However, our approach suffers from a major limitation to estimate the gene density precisely. Here, the gene densities estimated for the 3BS8-0.78-1.00 and 3BL7- 0.63-1.00 telomeric deletion bins were lower than the ones found through RFLP hybridisation with ESTs by Munkvold et al. [13] (normalized gene densities: 0.928 versus 1.190 and 1.176 versus 1.421, respectively). One of the characteristics of telomeric parts of wheat chro- mosomes is that they accumulate tandemly duplicated genes at a high rate [16,39]. Thus, it is likely that the differences in gene density observed between the two experiments reflect the inability of gene mapping based We then extrapolated the expected gene density per deletion bin to the whole set of chromosome 3B genes. We first estimated the number of genes per bin by con- sidering the bins fully covered by contigs and by keeping the same gene density distribution along chromosome Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 8 of 12 on BAC hybridisation to detect tandemly duplicated genes. This method is only qualitative and detects the presence or absence of a gene on a BAC but it does not indicate whether a gene located on a BAC is present in single or multiple copies. Wheat gene space organisation Thus, the gene density estab- lished through gene mapping based on BAC hybridisa- tion is likely underestimated in the distal regions and therefore one can expect an even higher gene density gra- dient. If we consider that the difference in gene density between the two studies is only due to tandemly dupli- cated genes, we could estimate that we missed 28% and 21% of genes for 3BS8-0.78-1.00 and 3BL7-0.63-1.00 deletion bins, respectively. This also explains why our estimation of gene density at telomeres was lower com- pared to the gene density of sequenced contigs located in distal regions of chromosome 3B (1 gene per 101 kb ver- sus 1 gene per 86 kb) whereas our estimation at the cen- tromere precisely fits the gene density of sequenced contigs located in proximal regions (1 gene per 185 kb versus 1 gene per 184 kb). Assuming that we missed 21% of genes due to tandem duplications in 3BL7-0.63-1.00 deletion bin, the gene density would be 1 gene per 90 kb. This demonstrates that all these studies can give an indi- cation of the general gene space organisation along wheat chromosomes but are unable to precisely estimate the local gene density of specific regions. organisation. We confirmed that the gene density distri- bution along the chromosome 3B follows a slight gradi- ent from the centromere to the telomeres and we suggest that the presence of more gene islands in the distal part of the chromosome explains this gradient. However, the ultimate experiment to access the whole set of genes and confirm the gene density distribution at a high resolution along a wheat chromosome will be high-quality sequen- cing and annotation. This is currently underway for chro- mosome 3B (C. Feuillet, personal communication). Conclusions This strongly suggests that the gradient of gene density between centromeric and telomeric regions is due to the differential distribution of genes organised in islands across the chromosome with pro- portionately more genes in islands in the distal parts compared to the proximal parts. Methods l Golden Promise RNAs (equal amounts of leaf, root and inflorescence) labelled with Cy5. RNA (5 μg) was labelled as described by Ducreux et al. [55]. Amplified BAC pool DNA (200 ng) was labelled using a modified BioPrime Genomic DNA Labelling System (Invitrogen, Carlsbad, California USA): BAC pool DNA in 11 μl was added to 10 μl Random Primer Reaction Buffer mix and denatured at 95°C for 5 min prior to cooling on ice and to this was added 2.5 μl modified 10× dNTPs buffer (1.2 mM each of dATP, dGTP, dTTP; 0.6 mM dCTP; 10 mM Tris pH8.0; 1 mM EDTA), Cy3 dCTP (1 μl of 1 nM) and 0.5 μl Kle- now enzyme (20U) followed by incubation for 16 h at 37°C. Labelled samples (BAC DNA & reference RNA) for each array were combined and unincorporated dyes removed using the Qiaquick PCR Purification Kit (Qia- gen, Hilden, Germany) as recommended, eluting with 20 μl EB buffer (Qiagen, Hilden, Germany). Hybridisa- tions and washing were carried out as recommended (Agilent Protocol v5.5). Scanning was performed with an Agilent G2505B scanner using default settings and data extracted using Agilent FE software (v 9.5.3). All data has been submitted to ArrayExpress [56] (accession # E-TABM-1011) under MIAME guidelines [57]. a barley probe. For each barley probe, we identified the positive pools to determine the original MTP BAC address on which it is located. Each type of pool does not contain the same number of BACs (plate: 384 BACs/pool; row: 480 BACs/pool; column: 320 BACs/ pool). The first normalisation step undertaken addressed the fact that the 60 samples had different hybridisation sig- nal averages. Medians were calculated for each pool independently and each value was divided by the median corresponding to the pool type. This led to comparable hybridisation values for each pool. A second normalisa- tion step was undertaken for each probe, based on the same method. After this second normalisation step, probe hybridisation values were all comparable, while pool hybridisation values were not significantly changed. p y g y g To identify pools with positive signal, we first used an automated classical outlier detection method, that we called the “automated scoring” method. The mean and the standard deviation were calculated for each probe and used to define a different threshold for each probe. Blast analyses y A BLASTN analysis [33] was performed with the 60-mer barley probes against the TREP database [34] to identify probes that could hybridise with TEs of the wheat BACs. We considered that a probe could generate a false positive due to TEs if we found 80% identity on a minimum 45 nucleotides. Then a BLASTN analysis [33] was performed with the 15208 60-mer barley probes against the sequenced contigs of wheat chromosome 3B [16]. The annotation of the best hit on the sequenced contigs was viewed using Artemis [58]. The best hit and the query barley probe were then aligned using Clus- talW2 [59] and the sequence identity was calculated using the entire barley probe length. In addition, a BLASTN analysis [33] was performed with the 60-mer barley probes against 6162 wheat cv Chinese Spring full- length cDNAs developed by Kawaura et al. [38]. The sequence identity between the best hit and the query barley probe was calculated on the entire barley probe length as previously described. The most significant rice homologues to the unigenes used to design the barley microarray probes were identified by BLASTN searches of the gene models from the Rice Genome Annotation Project from Michigan State University (Rice Pseudomo- lecules v5 database [60]). Following this “automated scoring” method, a “semi- automated” one was performed to identify missing coor- dinates from probes having five positive pools (e.g. two plate coordinates, two row coordinates and one column coordinate). Here, a combination of all possible coordi- nates was used to try to identify two overlapping BACs. A “manual scoring” analysis was also performed to iden- tify the missing coordinate from probes having two posi- tive pools. The final analysis that we called “boxplot scoring” method was performed whereby a boxplot was drawn using R software [61] for each probe for each of the three pool types and the upper outlier values were con- sidered as positive pools. This analysis was less stringent than the “automated scoring” so we kept only the probes that were located on two overlapping BACs. To rebuild the BAC addresses, we collated the positive pools for each probe. Some probes gave one positive pool per pool type which enables us to identify an unambiguous BAC address. However, some probes gave more positive pools per pool type. We therefore looked at every combination and used the chromosome 3B Methods l Calculation of the thresholds was different for each pool type (plate: Mean + 2.8 × Standard Deviation; row: Mean + 2.5 × Standard Deviation; column: Mean + 3 × Standard Deviation). All the pools with probe signal above this threshold were considered positive. We repeated this step twice by deleting positive signals pre- viously detected, calculating the mean, standard devia- tion and the threshold again for each probe and selecting the new positive signals above the new thresh- olds. The calculation of the thresholds for the three pool types remained the same. Methods l Barley expression microarray and hybridisations The barley Agilent 15K expression microarray contains 15208 barley 60-mer probes derived from unigenes of HarvEST assembly #25 used to originally design probe sets for the 22K Barley1 Affymetrix GeneChip [21]. BACs (7440 in total) arranged in twenty 384-well plates were selected to build a wheat chromosome 3B Minimal Tiling Path covering 82% of the whole chromosome with ~30% overlap as described by Paux et al. [17]. These twenty plates were pooled in three dimensions (20 plate pools, 16 row pools and 24 column pools) to generate 60 samples by CNRGV (Toulouse, France) and the BAC pools were amplified as described by Paux et al. [17]. Two channels processing of the microarrays was used, with BAC pool DNA labelled with Cy3 and a In conclusion, our cross-species hybridisation techni- que allowed us to assign a large number of genes onto wheat chromosome 3B at the BAC resolution and to obtain original results on the wheat gene space Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 9 of 12 mixed reference set of barley cv. Golden Promise RNAs (equal amounts of leaf, root and inflorescence) labelled with Cy5. RNA (5 μg) was labelled as described by Ducreux et al. [55]. Amplified BAC pool DNA (200 ng) was labelled using a modified BioPrime Genomic DNA Labelling System (Invitrogen, Carlsbad, California USA): BAC pool DNA in 11 μl was added to 10 μl Random Primer Reaction Buffer mix and denatured at 95°C for 5 min prior to cooling on ice and to this was added 2.5 μl modified 10× dNTPs buffer (1.2 mM each of dATP, dGTP, dTTP; 0.6 mM dCTP; 10 mM Tris pH8.0; 1 mM EDTA), Cy3 dCTP (1 μl of 1 nM) and 0.5 μl Kle- now enzyme (20U) followed by incubation for 16 h at 37°C. Labelled samples (BAC DNA & reference RNA) for each array were combined and unincorporated dyes removed using the Qiaquick PCR Purification Kit (Qia- gen, Hilden, Germany) as recommended, eluting with 20 μl EB buffer (Qiagen, Hilden, Germany). Hybridisa- tions and washing were carried out as recommended (Agilent Protocol v5.5). Scanning was performed with an Agilent G2505B scanner using default settings and data extracted using Agilent FE software (v 9.5.3). All data has been submitted to ArrayExpress [56] (accession # E-TABM-1011) under MIAME guidelines [57]. mixed reference set of barley cv. Synteny and collinearity analyses In total, five barley genetic maps [21-25] were used to establish a barley neighbour map using the same criteria as the IBM neighbour map of maize [41]. For two maps [24,25], we had to perform a BLASTN analysis to link the markers to the barley unigenes mapped on wheat chro- mosome 3B. The best hits with at least 85% identity over 100 nucleotides were selected for each unigene. The order of the rice genes along the chromosomes was established using the rice gene numbering annotation. As the relative order of wheat genes in a given deletion bin is not known, we inferred this order from the barley chromosome 3 H and rice chromosome 1 data. The soft- ware GenomePixelizer [62] was used for the graphical display of the collinearity between wheat chromosome 3B, barley chromosome 3 H and rice chromosome 1. manuscript. The final version of the manuscript was approved by all the authors. Authors’ contributions PH and JM carried out the hybridisations; CR, PH and FC performed data analysis; RW, CF and EP helped with the interpretation of the results; CR drafted the manuscript; FC, RW, CF and EP were involved in improving the Author details 1 1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100 Clermont-Ferrand, France. 2Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK. After identification of BACs carrying barley orthologs, we used the physical map [17] to locate them in their respective contig and possibly in one of the eight chro- mosome 3B deletion bins used for this study. 1INRA UMR 1095, Génétique Diversité et Ecophysiologie des Céréales, 63100 Clermont-Ferrand, France. 2Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK. References 1 J k Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 2007, 449:463-467. Data deconvolution Following hybridisation, signals were analysed to rebuild the MTP addresses of the BACs carrying an ortholog of Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Page 10 of 12 (FP7/2007-2013) under the grant agreement n°FP7-212019. CR was financially supported by Région Auvergne. (FP7/2007-2013) under the grant agreement n°FP7-212019. CR was financially supported by Région Auvergne. physical map data [17] to assist finding addresses of overlapping BACs where the probe is located on the overlap itself. 11. Endo TR, Gill BS: The deletion stocks of common wheat. J Hered 1996, 87:295-307. 10. Zonneveld BJ, Leitch IJ, Bennett MD: First nuclear DNA amounts in more than 300 angiosperms. Ann Bot (Lond) 2005, 96:229-244. 12. Erayman M, Sandhu D, Sidhu D, Dilbirligi M, Baenziger PS, Gill KS: Demarcating the gene-rich regions of the wheat genome. Nucleic Acids Res 2004, 32:3546-3565. References 1 J k 1. Jackson S, Hass Jacobus B, Pagel J: The Gene Space of the Soybean Genome. In Legume Crop Genomics. Edited by: Wilson RF, Stalker HT, Brummer EC. Champaign: AOCS Press; 2004:187-193. Brummer EC. Champaign: AOCS Press; 2004:187 193. 2. Varshney RK, Hoisington DA, Tyagi AK: Advances in cereal genomics and applications in crop breeding. Trends Biotechnol 2006, 24:490-499. 3. International Rice Genome Sequencing Project: The map-based sequence of the rice genome. Nature 2005, 436:793-800. g 2. Varshney RK, Hoisington DA, Tyagi AK: Advances in cereal genomics and applications in crop breeding. Trends Biotechnol 2006, 24:490-499. 3. International Rice Genome Sequencing Project: The map-based sequence of the rice genome. Nature 2005, 436:793-800. 2. Varshney RK, Hoisington DA, Tyagi AK: Advances in cereal genomics and applications in crop breeding. Trends Biotechnol 2006, 24:490-499. . a s ey , o s gto , yag : d a ces ce ea ge o cs a d applications in crop breeding. Trends Biotechnol 2006, 24:490-499. 3. International Rice Genome Sequencing Project: The map-based sequence of the rice genome. Nature 2005, 436:793-800. The Arabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 2000, 408:796-814. The International Brachypodium Initiative: Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 2010, 463:763-768. The Arabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 2000, 408:796-814. The International Brachypodium Initiative: Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 2010, 463:763-768. 6. 6. Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyere C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Cattonaro F, Anthouard V, Vico V, Del Fabbro C, Alaux M, Di Gaspero G, Dumas V, et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 2007, 449:463-467. . Statistical analyses The statistical analyses including the T-test, Chi2 and Pearson’s correlation coefficient tests were performed using R software [61] at a 5% threshold. The distance to centromere was estimated from the centromere to the middle of deletion bins. We calculated the gene density per deletion bins by dividing the number of genes assigned to the bin by the length of the contigs assigned to the same bin. The gene density per bin of Munkvold et al. [13] was estimated by dividing the number of genes they assigned to the bin by the total size of the bin. The normalized gene densities per deletion bin were calcu- lated by dividing the density for each bin by the mean of the densities along chromosome 3B. The Rice Genome Annotation Project from Michigan State University (Rice Pseudomolecules v6.1 database [60]) was used to esti- mate the number of annotated genes on the rice chromo- somes. For the Chi2 tests, to test the uniformity of the percentages and the densities along chromosome 3B, we estimated the number of genes per deletion bins that would have generated a uniform distribution and we used these numbers as theoretical values. 7. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A, Schein J, Sterck L, Aerts A, Bhalerao RR, Bhalerao RP, Blaudez D, Boerjan W, Brun A, Brunner A, Busov V, Campbell M, Carlson J, Chalot M, Chapman J, Chen GL, Cooper D, Coutinho PM, Couturier J, Covert S, Cronk Q, et al: The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 2006, 313:1596-1604. mpbell M, Carlson J, Chalot M, Chapman J, Chen GL, Cooper D, 8. Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, et al: Genome sequence of the palaeopolyploid soybean. Nature 2010, 463:178-183. 9. Abbreviations MTP: minimal tiling path; TE: transposable element. 9. Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, Minx P, Reily AD, Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Courtney B, Rock SM, Belter E, Du F, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, et al: The B73 maize genome: complexity, diversity, and dynamics. Science 2009, 326:1112-1115. Statistical analyses Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, Minx P, Reily AD, Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Courtney B, Rock SM, Belter E, Du F, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, et al: The B73 maize genome: complexity, diversity, and dynamics. Science 2009, 326:1112-1115. 10. Zonneveld BJ, Leitch IJ, Bennett MD: First nuclear DNA amounts in more than 300 angiosperms. Ann Bot (Lond) 2005, 96:229-244. 11. Endo TR, Gill BS: The deletion stocks of common wheat. J Hered 1996, 87:295-307. 12. Erayman M, Sandhu D, Sidhu D, Dilbirligi M, Baenziger PS, Gill KS: 12. Erayman M, Sandhu D, Sidhu D, Dilbirligi M, Baenziger PS, Gill KS: Demarcating the gene-rich regions of the wheat genome. Nucleic Acids Res 2004, 32:3546-3565. Demarcating the gene-rich regions of the wheat genome. Nucleic Acids Res 2004, 32:3546-3565. 13. Munkvold JD, Greene RA, Bermudez-Kandianis CE, La Rota CM, Edwards H, Sorrells SF, Dake T, Benscher D, Kantety R, Linkiewicz AM, Dubcovsky J, Akhunov ED, Dvorak J, Gustafson JP, Pathan MS, Nguyen HT, Matthews DE, Chao S, Lazo GR, Hummel DD, Anderson OD, Anderson JA, Gonzalez- Hernandez JL, Peng JH, Lapitan N, Qi LL, Echalier B, Gill BS, Hossain KG, et al: Group 3 Chromosome Bin Maps of Wheat and Their Relationship to Rice Chromosome 1. Genetics 2004, 168:639-650. Abbreviations MTP: minimal tiling path; TE: transposable element. 13. Munkvold JD, Greene RA, Bermudez-Kandianis CE, La Rota CM, Edwards H, Sorrells SF, Dake T, Benscher D, Kantety R, Linkiewicz AM, Dubcovsky J, Akhunov ED, Dvorak J, Gustafson JP, Pathan MS, Nguyen HT, Matthews DE, Chao S, Lazo GR, Hummel DD, Anderson OD, Anderson JA, Gonzalez- Hernandez JL, Peng JH, Lapitan N, Qi LL, Echalier B, Gill BS, Hossain KG, et al: Group 3 Chromosome Bin Maps of Wheat and Their Relationship to Rice Chromosome 1. Genetics 2004, 168:639-650. g The authors would like to thank Christel Laugier for interesting discussions about data analysis. The research leading to these results has received funding from the European Community’s Seventh Framework Programme Acknowledgements Paux E, Sourdille P, Salse J, Saintenac C, Choulet F, Leroy P, Korol A, Michalak M, Kianian S, Spielmeyer W, Lagudah E, Somers D, Kilian A, Alaux M, Vautrin S, Bergès H, Eversole K, Appels R, Safar J, Simkova H, Dolezel J, Bernard M, Feuillet C: A physical map of the 1Gb bread wheat chromosome 3B. Science 2008, 322:101-104. 39. Akhunov ED, Goodyear AW, Geng S, Qi LL, Echalier B, Gill BS, Gustafson JP, Lazo G, Chao S, Anderson OD, Linkiewicz AM, Dubcovsky J, La Rota M, Sorrells ME, Zhang D, Nguyen HT, Kalavacharla V, Hossain K, Kianian SF, Peng J, Lapitan NL, Gonzalez-Hernandez JL, Anderson JA, Choi DW, Close TJ, Dilbirligi M, Gill KS, Walker-Simmons MK, Steber C, et al: The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms. Genome Res 2003, 13:753-763. 18. Lehmensiek A, Bovill W, Wenzl P, Langridge P, Appels R: Genetic Mapping in the Triticeae. In Genetics and Genomics of the Triticeae. Edited by: Feuillet C, Muelhlbauer G. Berlin: Springer; 2009:201-235. 19. Paux E, Sourdille P: A Toolbox for Triticeae Genomics. In Genetics and Genomics of the Triticeae. Edited by: Feuillet C, Muelhlbauer G. Berlin: Springer; 2009:255-283. 40. Klein PE, Klein RR, Cartinhour SW, Ulanch PE, Dong J, Obert JA, Morishige DT, Schlueter SD, Childs KL, Ale M, Mullet JE: A high-throughput AFLP-based method for constructing integrated genetic and physical maps: progress toward a sorghum genome map. Genome Res 2000, 10:789-807. 20. GrainGenes 2.0. [http://wheat.pw.usda.gov/GG2]. 21. Chen X, Hackett CA, Niks RE, Hedley PE, Booth C, Druka A, Marcel TC, Vels A, Bayer M, Milne I, Morris J, Ramsay L, Marshall D, Cardle L, Waugh R: An eQTL analysis of partial resistance to Puccinia hordei in barley. PLoS One 2010, 5:e8598. 41. Cone KC, McMullen MD, Bi IV, Davis GL, Yim YS, Gardiner JM, Polacco ML, Sanchez-Villeda H, Fang Z, Schroeder SG, Havermann SA, Bowers JE, Paterson AH, Soderlund CA, Engler FW, Wing RA, Coe EH Jr: Genetic, physical, and informatics resources for maize. On the road to an integrated map. Plant Physiol 2002, 130:1598-1605. 22. Acknowledgements The authors would like to thank Christel Laugier for interesting discussions about data analysis. The research leading to these results has received funding from the European Community’s Seventh Framework Programme Page 11 of 12 Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 14. Charles M, Belcram H, Just J, Huneau C, Viollet A, Couloux A, Segurens B, Carter M, Huteau V, Coriton O, Appels R, Samain S, Chalhoub B: Dynamics and differential proliferation of transposable elements during the evolution of the B and A genomes of wheat. Genetics 2008, 180:1071-1086. 34. Wicker T, Matthews DE, Keller B: TREP: a database for Triticeae repetitive elements. Trends Plant Sci 2002, 7:561-562. 35. Mayer KF, Taudien S, Martis M, Simkova H, Suchankova P, Gundlach H, Wicker T, Petzold A, Felder M, Steuernagel B, Scholz U, Graner A, Platzer M, Dolezel J, Stein N: Gene content and virtual gene order of barley chromosome 1H. Plant Physiol 2009, 151:496-505. 15. Devos KM, Ma J, Pontaroli AC, Pratt LH, Bennetzen JL: Analysis and mapping of randomly chosen bacterial artificial chromosome clones from hexaploid bread wheat. Proc Natl Acad Sci USA 2005, 102:19243-19248. 36. Suchánková P, Kubaláková M, KováŐová P, Bartoš J, Číhalíková J, Molnár- Láng M, Endo T, Doležel J: Dissection of the nuclear genome of barley by chromosome flow sorting. Theor Appl Genet 2006, 113:651-659. 37. Letowski J, Brousseau R, Masson L: Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays. J Microbiol Methods 2004, 57:269-278. 16. Choulet F, Wicker T, Rustenholz C, Paux E, Salse J, Leroy P, Schlub S, Le Paslier MC, Magdelenat G, Gonthier C, Couloux A, Budak H, Breen J, Pumphrey M, Liu S, Kong X, Jia J, Gut M, Brunel D, Anderson JA, Gill BS, Appels R, Keller B, Feuillet C: Megabase Level Sequencing Reveals Contrasted Organization and Evolution Patterns of the Wheat Gene and Transposable Element Spaces. Plant Cell 2010, 22:1686-1701. 38. Kawaura K, Mochida K, Enju A, Totoki Y, Toyoda A, Sakaki Y, Kai C, Kawai J, Hayashizaki Y, Seki M, Shinozaki K, Ogihara Y: Assessment of adaptive evolution between wheat and rice as deduced from full-length common wheat cDNA sequence data and expression patterns. BMC Genomics 2009, 10:271. 17. Acknowledgements Close TJ, Bhat PR, Lonardi S, Wu Y, Rostoks N, Ramsay L, Druka A, Stein N, Svensson JT, Wanamaker S, Bozdag S, Roose ML, Moscou MJ, Chao S, Varshney RK, Szucs P, Sato K, Hayes PM, Matthews DE, Kleinhofs A, Muehlbauer GJ, DeYoung J, Marshall DF, Madishetty K, Fenton RD, Condamine P, Graner A, Waugh R: Development and implementation of high-throughput SNP genotyping in barley. BMC Genomics 2009, 10:582. 42. Keller B, Feuillet C: Colinearity and gene density in grass genomes. Trends Plant Sci 2000, 5:246-251. 43. Bilgic H, Cho S, Garvin DF, Muehlbauer GJ: Mapping barley genes to chromosome arms by transcript profiling of wheat-barley ditelosomic chromosome addition lines. Genome 2007, 50:898-906. 23. Potokina E, Druka A, Luo Z, Wise R, Waugh R, Kearsey M: Gene expression quantitative trait locus analysis of 16000 barley genes reveals a complex pattern of genome-wide transcriptional regulation. The Plant Journal 2008, 53:90-101. 44. Bolot S, Abrouk M, Masood-Quraishi U, Stein N, Messing J, Feuillet C, Salse J: The ‘inner circle’ of the cereal genomes. Curr Opin Plant Biol 2009, 12:119-125. 24. Sato K, Nankaku N, Takeda K: A high-density transcript linkage map of barley derived from a single population. Heredity 2009, 103:110-117. 45. Cho S, Garvin DF, Muehlbauer GJ: Transcriptome analysis and physical mapping of barley genes in wheat-barley chromosome addition lines. Genetics 2006, 172:1277-1285. 25. Stein N, Prasad M, Scholz U, Thiel T, Zhang HN, Wolf M, Kota R, Varshney RK, Perovic D, Grosse I, Graner A: A 1,000-loci transcript map of the barley genome: new anchoring points for integrative grass genomics. Theor Appl Genet 2007, 114:823-839. 46. Devos KM: Updating the ‘crop circle’. Curr Opin Plant Biol 2005, 8:155-162. 26. Chalupska D, Lee HY, Faris JD, Evrard A, Chalhoub B, Haselkorn R, Gornicki P: Acc homoeoloci and the evolution of wheat genomes. Proc Natl Acad Sci USA 2008, 105:9691-9696. 47. Gaut BS: Evolutionnary dynamics of grass genomes. New Phytol 2002, 154:15-28. 48. La Rota M, Sorrells ME: Comparative DNA sequence analysis of mapped wheat ESTs reveals the complexity of genome relationships between rice and wheat. Funct Integr Genomics 2004, 4:34-46. 27. Dvorak J, Akhunov ED: Tempos of gene locus deletions and duplications and their relationship to recombination rate during diploid and polyploid evolution in the Aegilops-Triticum alliance. Genetics 2005, 171:323-332. 49. Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 Acknowledgements Thiel T, Graner A, Waugh R, Grosse I, Close TJ, Stein N: Evidence and evolutionary analysis of ancient whole-genome duplication in barley predating the divergence from rice. BMC Evol Biol 2009, 9:209. 28. Wicker T, Narechania A, Sabot F, Stein J, Vu GT, Graner A, Ware D, Stein N: Low-pass shotgun sequencing of the barley genome facilitates rapid identification of genes, conserved non-coding sequences and novel repeats. BMC Genomics 2008, 9:518. 50. Varshney RK, Sigmund R, Börner A, Korzun V, Stein N, Sorrells ME, Langridge P, Graner A: Interspecific transferability and comparative mapping of barley EST-SSR markers in wheat, rye and rice. Plant Science 2005, 168:195-202. 29. Bennetzen JL, Ramakrishna W: Numerous small rearrangements of gene content, order and orientation differentiate grass genomes. Plant Mol Biol 2002, 48:821-827. 51. Saintenac C, Falque M, Martin OC, Paux E, Feuillet C, Sourdille P: Detailed Recombination Studies along Chromosome 3B Provide New Insights on Crossover Distribution in Wheat (Triticum aestivum L). Genetics 2009, 181:393-403. 30. Devos KM, Gale MD: Genome relationships: The grass model in current research. Plant Cell 2000, 12:637-646. 31. Dubcovsky J, Luo MC, Zhong GY, Bransteitter R, Desai A, Kilian A, Kleinhofs A, Dvorak J: Genetic map of diploid wheat, Triticum monococcum L, and its comparison with maps of Hordeum vulgare L. Genetics 1996, 143:983-999. 52. Salse J, Bolot S, Throude M, Jouffe V, Piegu B, Quraishi UM, Calcagno T, Cooke R, Delseny M, Feuillet C: Identification and characterization of shared duplications between rice and wheat provide new insight into grass genome evolution. Plant Cell 2008, 20:11-24. 32. Moore G, Devos KM, Wang Z, Gale MD: Cereal genome evolution. Grasses, line up and form a circle. Curr Biol 1995, 5:737-739. 53. Liu S, Zhang X, Pumphrey MO, Stack RW, Gill BS, Anderson JA: Complex microcolinearity among wheat, rice, and barley revealed by fine mapping of the genomic region harboring a major QTL for resistance to Fusarium head blight in wheat. Funct Integr Genomics 2005, 1-7. 33. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402. Page 12 of 12 Page 12 of 12 Rustenholz et al. BMC Genomics 2010, 11:714 http://www.biomedcentral.com/1471-2164/11/714 54. Acknowledgements Chantret N, Salse J, Sabot F, Bellec A, Laubin B, Dubois I, Dossat C, Sourdille P, Joudrier P, Gautier MF, Cattolico L, Beckert M, Aubourg S, Weissenbach J, Caboche M, Leroy P, Bernard M, Chalhoub B: Contrasted microcolinearity and gene evolution within a homoeologous region of wheat and barley species. J Mol Evol 2008, 66:138-150. 55. Ducreux LJ, Morris WL, Prosser IM, Morris JA, Beale MH, Wright F, Shepherd T, Bryan GJ, Hedley PE, Taylor MA: Expression profiling of potato germplasm differentiated in quality traits leads to the identification of candidate flavour and texture genes. J Exp Bot 2008, 59:4219-4231. 56. ArrayExpress. [http://www.ebi.ac.uk/microarray-as/ae/]. 57. Minimum Information About a Microarray Experiment - MIAME. [http:// www.mged.org/Workgroups/MIAME/miame.html]. 57. Minimum Information About a Microarray Experiment - MIAME. [http:// www.mged.org/Workgroups/MIAME/miame.html]. 58. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation. Bioinformatics 2000, 16:944-945. 58. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation. Bioinformatics 2000, 16:944-945. 59. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal × version 2.0. Bioinformatics 2007, 23:2947-2948. 59. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal × version 2.0. Bioinformatics 2007, 23:2947-2948. 60. Rice Genome Annotation from Michigan State University. [http://rice. plantbiology.msu.edu/]. 61. R software. [http://www.r-project.org]. 61. R software. [http://www.r-project.org]. 62. GenomePixelizer. [http://www.atgc.org/Ge GenomePixelizer_Welcome.html]. 62. GenomePixelizer. [http://www.atgc.org/GenomePixelizer/ l l h l 62. GenomePixelizer. [http://www.atgc GenomePixelizer_Welcome.html]. GenomePixelizer_Welcome.html]. doi:10.1186/1471-2164-11-714 Cite this article as: Rustenholz et al.: Specific patterns of gene space organisation revealed in wheat by using the combination of barley and wheat genomic resources. BMC Genomics 2010 11:714. 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https://openalex.org/W3152524270	https://digital.library.adelaide.edu.au/dspace/bitstream/2440/130433/2/hdl_130433.pdf	English	null	Applying a framework to assess the impact of cardiovascular outcomes improvement research	Health research policy and systems	2,021	cc-by	9,348	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Applying a framework to assess the impact of cardiovascular outcomes improvement research Mitchell N. Sarkies1,2* , Suzanne Robinson2, Tom Briffa3, Stephen J. Duffy4,5, Mark Nelson6, John Beltrame7,8,9, Louise Cullen10,11,12, Derek Chew13, Julian Smith14,15, David Brieger16, Peter Macdonald17, Danny Liew5 and Chris Reid5,18 Abstract Background: Health and medical research funding agencies are increasingly interested in measuring the impact of funded research. We present a research impact case study for the first four years of an Australian National Health and Medical Research Council funded Centre of Research Excellence in Cardiovascular Outcomes Improvement (2016– 2020). The primary aim of this paper was to explore the application of a research impact matrix to assess the impact of cardiovascular outcomes improvement research. Methods: We applied a research impact matrix developed from a systematic review of existing methodological frameworks used to measure research impact. This impact matrix was used as a bespoke tool to identify and under- stand various research impacts over different time frames. Data sources included a review of existing internal docu- mentation from the research centre and publicly available information sources, informal iterative discussions with 10 centre investigators, and confirmation of information from centre grant and scholarship recipients. Results: By July 2019, the impact on the short-term research domain category included over 41 direct publications, which were cited over 87 times (median journal impact factor of 2.84). There were over 61 conference presentations, seven PhD candidacies, five new academic collaborations, and six new database linkages conducted. The impact on the mid-term research domain category involved contributions towards the development of a national cardiac regis- try, cardiovascular guidelines, application for a Medicare Benefits Schedule reimbursement item number, introduction of patient-reported outcome measures into several databases, and the establishment of nine new industry collabora- tions. Evidence of long-term impacts were described as the development and use of contemporary management for aortic stenosis, a cardiovascular risk prediction model and prevention targets in several data registries, and the establishment of cost-effectiveness for stenting compared to surgery. Conclusions: We considered the research impact matrix a feasible tool to identify evidence of academic and policy impact in the short- to midterm; however, we experienced challenges in capturing long-term impacts. Cost contain- ment and broader economic impacts represented another difficult area of impact to measure. *Correspondence: mitchell.sarkies@mq.edu.au 1 Centre for Healthcare Resilience and Implementation Science, Australian Institute of Health Innovation, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW 2109, Australia Full list of author information is available at the end of the article Sarkies et al. Health Res Policy Sys (2021) 19:67 https://doi.org/10.1186/s12961-021-00710-4 Sarkies et al. Health Res Policy Sys (2021) 19:67 https://doi.org/10.1186/s12961-021-00710-4 Open Access © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 12 Sarkies et al. Health Res Policy Sys (2021) 19:67 Keywords: Research impact, Impact matrix, Research output, Implementation science, Cardiovascular outcomes, Evaluation, Health research, Research translation, Knowledge translation, Dissemination Page 2 of 12 Sarkies et al. Health Res Policy Sys (2021) 19:67 Sarkies et al. Health Res Policy Sys Keywords: Research impact, Impact matrix, Research output, Implementation science, Cardiovascular outcomes, Evaluation, Health research, Research translation, Knowledge translation, Dissemination Keywords: Research impact, Impact matrix, Research output, Implementation science, Cardiovascular outcomes, Evaluation, Health research, Research translation, Knowledge translation, Dissemination utilization to knowledge mobilization [12]. Early tools for the assessment of impact almost solely captured aca- demic productivity, rather than broader impacts to soci- ety [13]. In recent years, research impact is increasingly conceptualized in terms of promoting national prosper- ity, as the focus of research policy has turned toward impacts on the economy, society, environment, and cul- ture; for example, the introduction of a “National Inter- est Test” in 2018 for Australian research grants allows the Commonwealth Minister for Education veto power to reject applications recommended for funding by the Australian Research Council [14]. Given the conceptual ambiguity of “research impact”, it can be difficult for aca- demics to articulate the patterns and pathways by which transformation of their research into “impact” occurs. Background Here, we explore the application of a research impact matrix to assess the impact of car- diovascular outcomes improvement research, including both “hard” impacts that can be directly attributed and “soft” impacts indirectly attributed to an Australian col- laborative research centre. We present a programme impact evaluation case study for the first four years of a Centre of Research Excellence in Cardiovascular Out- comes Improvement (2016–2020) initiative, funded for AUD$ 2,583,231 by the Australian National Health and Medical Research Council. The Centre of Research Excel- lence funding scheme provides support for teams of researchers to pursue collaborative research and develop capacity in clinical, health services, public health, and dementia research. The objective of the scheme is to improve health outcomes and promote or improve trans- lation of research outcomes into policy and practice, in addition to supporting researchers in capacity-building activities [23]. ultimately improve healthcare delivery, human health and quality of life, and generate benefits for the economy, society, culture, public policy or the environment.” [10, 16]. The importance of measuring this impact is clear. Large amounts of public funding are directed towards health research, so it is important that recipients of those funds show accountability and demonstrate relevance to the needs of clinicians, patients, and the health systems within which they operate [17]. Furthermore, the alloca- tion of research funding increasingly relies on informa- tion regarding the impact of previous projects. A range of approaches to measuring research impact have been adopted internationally [18], perhaps most prominently the “Payback” method [19]. Payback meas- ures both academic outputs and wider societal ben- efits to assess the impact of health research [20]. There are two components to the Payback, one is a model that studies specific components of research and dissemina- tion, and the second is a classification scheme for out- puts, outcomes, and impacts. However, the Payback and similar frameworks are generally used to measure research benefit at the macro-scale (e.g. nationally) over long time periods (often decades) and may be cumber- some to apply to smaller, time-constrained programmes of research or individual projects, where specific funding for this additional layer of evaluation may not be avail- able. More recently, Searles et al. developed the Frame- work to Assess the Impact from Translational (FAIT) health research, which can be feasibly applied to individ- ual research projects. Background g Health and medical research funding agencies are increasingly interested in measuring return on invest- ment through improved health outcomes. This focus reflects the ultimate goal of research to generate new knowledge that can be applied to improve health and reduce the burden of illness. Historical investment in health research has not always translated into healthcare policy and practice [1–3], leading to an equivocal use of scarce resources in both the generation of research and functioning of health systems [4, 5]. Consequently, meas- uring the impact of research including and beyond aca- demic outputs, such as peer-reviewed journal articles, is crucial [6–9]. However, there is a paucity of evidence suggesting an increase in the number of health research projects that measure impact, particularly beyond aca- demic criteria [10]. There are numerous ways to define research impact [16]. The International School on Research Impact Assessment, which was set up to facilitate the application of robust and repeatable evaluation methods for research impact, recommends analysis of the research con- text prior to conducting a research impact assessment. This paper centres on a case study for the evaluation of research impact in cardiovascular outcomes research; therefore, we selected a research impact definition that was considered contextually applicable, defined as “the demonstrable effect from basic, health systems, patient and population-oriented research, and clinical trials that Measuring the impact of health research is an evolv- ing and contested activity. The pathway of research impact is often conceptualized and represented as a sim- ple linear process (Fig. 1). However, impacts are rarely manifested through linear pathways in complex systems such as healthcare [11]. This leaves the rigidity of many frameworks for measuring research impact as potentially unfit for this purpose, if they misrepresent the complex research—practice ecosystem. The focus of research policy has also shifted over the years from research Fig. 1 Linear research impact pathways (Source: Fryirs et al. [12], modified from Morgan et al. [15]. Use of this image is supported by the Creative Commons Attribution Non-Commercial (CC BY-NC 4.0) license. See: http://creativecommons.org/licenses/by-nc/4.0/) Sarkies et al. Health Res Policy Sys (2021) 19:67 Page 3 of 12 Page 3 of 12 documenting the wider impact of specific cardiovascu- lar research projects. Background This framework incorporates three approaches: (1) modifications to the Payback Framework to help quantify research impact; (2) an economic meas- ure of social return on investment; and (3) narrative case studies of how the research generated impact, particu- larly for those that are difficult to quantify [21]. The FAIT relies on two assumptions: (1) that research translation activity has occurred within the life cycle of the project and (2) measurement has been applied prospectively. For projects where these assumptions cannot be met, a prag- matic approach could be considered. Bespoke tools that consider a suite of potentially appropriate metrics drawn from multiple frameworks may allow more consideration of the complex, contextually dependent nature of trans- lating research into impact. We document the feasibility, lessons learned, and chal- lenges encountered when applying a research impact matrix to explore how research output from this centre has influenced healthcare policy and practice. The pri- mary aim of this paper was to explore the application of a research impact matrix to assess the impact of cardiovas- cular outcomes improvement research. Selecting a research impact frameworkh In light of these challenges, we have outlined how we allocated each measurement and metric to the research impact matrix categories. We did not take a deductive approach of pre-specifying metrics; rather an iterative stance was taken to ensure metrics were contextually relevant to the specific cardiovascular health research undertaken. In the early stages of identifying potentially relevant metrics and measurements of impact, it became clear that mapping and attributing impacts across various programmes of research within an umbrella consortium would be particularly difficult. There were a wide range of both direct and indirect impacts, and uncoupling the centre’s activities from those of its component members or determining the counterfactual of impact that would have occurred without the existence of the centre pre- sented unique challenges. To address these challenges, we attempted to articulate what we considered both “direct” and “indirect” impacts, which were aligned to the notion of “soft” and “hard” impacts used in the River Styles Framework, which is another research impact framework used in the environment sciences [12]. There is currently little consensus on the most appro- priate tool to measure research impact. We conducted a literature review to identify a health-focused research impact framework that would meet the specific needs of the key stakeholders, primarily (1) without a requirement for prospectively applied measures and (2) a simple and feasible approach to include different health impact cate- gories and metrics within a single tool. However, we were unable to identify a unique research impact assessment framework meeting the full set of requirements. Instead, we took a pragmatic approach and considered the application of a research impact matrix developed by Rivera et al. [24]. This research impact matrix was developed from a systematic review of existing meth- odological frameworks used to measure research impact, summarizing common themes and metrics. It allows the selection of components from existing methodological frameworks, generating a bespoke tool to understand and maximize various impact pathways over different time frames. Rivera et al. identified five major impact cat- egories, 16 impact subgroups, and 80 different metrics across 24 existing methodological frameworks [24]. The five impact categories are (1) primary research-related impact, (2) influence on policy-making, (3) health and health systems impact, (4) health-related and societal impact, and (5) broader economic impact. Case study contexth The process of applying a framework to assess the impact of cardiovascular outcomes improvement research is explored through a nationally funded research centre. This centre involved a collaborative team of 10 investi- gators from multiple academic institutions and health service organizations across Australia. The consortium of research-focused clinicians, academics, consumers, and other stakeholders fostered development of national capability in cardiovascular comparative effectiveness and quality of outcomes research, by utilizing established and developing the footprint of cardiovascular registries. A key aim of the centre was to develop a national plat- form for encouraging and facilitating excellence in car- diovascular trials, achieved through the establishment of the Australian and New Zealand Alliance for Cardiovas- cular Trials network. Themes of research for the centre were risk factors, acute cardiovascular disease, coronary artery revascularization procedures (cardiac percutane- ous coronary intervention and cardiac surgery), and the management of heart failure. Details of the seed projects and scholarships directly funded by the centre are pro- vided in Additional file 1. Cardiovascular research provides a good case study for the evaluation of impact, due to the large volume of allo- cated funding and academic outputs produced. Broadly, the long-term return from cardiovascular research has seen substantial improvements in survival of people affected by cardiovascular disease [22]. Whether new research findings can continue the success in translat- ing findings into improved health outcomes remains an open question. To date, there is a paucity of information Sarkies et al. Health Res Policy Sys (2021) 19:67 Page 4 of 12 Sarkies et al. Health Res Policy Sys (2021) 19:67 Sarkies et al. Health Res Policy Sys Selecting a research impact frameworkh Researchers do not necessarily need to address every aspect of the methodological framework, as every project is expected to impact the categories and subgroups differently. The original authors suggest a multidimensional approach that adopts narratives, quantitative metrics, and elements from other frameworks, arguably representing a novel and more comprehensive method of impact assessment than reliance on a single tool. Research‑related impact A d i Academic measurements capturing metrics such as knowledge generation, dissemination, and the develop- ment of capacity and networks were considered short- term impacts, as the first evidence of return on research investment. Direct research-related impacts were those metrics (e.g. publications) arising from projects funded by the centre’s grants and scholarships. Indirect impacts were metrics associated with the centre, for example, supporting the establishment of a clinical trial network, which was a joint effort of cardiovascular researchers both internal and external to the centre. Case study: Centre for Research Excellence in Cardiovascular Outcomes Improvementi Case study: Centre for Research Excellence in Cardiovascular Outcomes Improvementi Stage 1: Involved preliminary mapping of the centre impacts according to the matrix of impact categories and subgroups. Review of existing internal centre documen- tation and publicly available information sources (e.g. Google Scholar) was used to allocate known impacts according to short-, mid- and long-term timelines. Stage 1 was conducted by an independent researcher (MS) who was not involved in any of the projects or running of the centre. The initial research impact matrix was then sent to the centre investigators for their review and amend- ments, completing the first draft. Over the first four years of the research centre, there were 13 seed grant projects, seven scholarships, and three investigator-led projects. Seed grant projects typi- cally funded early career researcher-led investigations under the supervision of one or more of the centre’s investigators. The scholarships were directed towards PhD candidates who were considered to have high poten- tial to attract additional sources of funding for their work. Three investigator-led projects encompassed larger initiatives, which were part of broader efforts outside the centre. Scholarship recipients were expected to obtain further funding from national and other sources to free up scholarship funding for more researchers, which was achieved throughout the life of the centre. The research impact matrix results for the centre’s projects are pre- sented in Table 1.h , p gi Stage 2: Informal semi-structured telephone discus- sions were then conducted between April and June 2019 by the independent researcher (MS) with each of the centre investigators for every project, grant, and scholar- ship. These telephone discussions were used to identify the relevant impacts achieved and which impact category and subgroup they best aligned with (approximately 20–60 min). The conversation was structured using the research impact matrix developed by Rivera et al. [24], and questions were asked about each of the impact cate- gories and subgroups. Further prompts were provided for specific example metrics when the centre investigators were uncertain of how to interpret the impact categories and subgroups. Notes were taken by the independent researcher throughout the discussion, which were used to summarize the research impact matrix results for the centre’s projects. Iterative discussions were decided as the best way to elicit different forms of impact, as it allowed the investigators to explore their projects and clarify whether certain measures and metrics could be consid- ered “impact”. Processh The research impact assessment was conducted in two stages, as described below. Health and health systems impact, health‑related and societal impact, and broader economic impact Health and health systems impact, health‑related and societal impact, and broader economic impacth Health and health systems impact, health‑related and societal impact, and broader economic impacth Health and health systems impact, health‑related and societal impact, and broader economic impacth discussions with each investigator, additional impacts were sought by emailing the completed impact matrix to each of the centre’s grant and scholarship recipients to confirm the information obtained and add anything missed during the discussions with centre investiga- tors. These grant and scholarship recipients were mostly early career researchers who were able to provide addi- tional contextual information regarding the projects and research impact. A manual search of academic outputs was then performed to obtain citations and impact fac- tors for the identified publications. These impacts represented a time frame beyond five years according to the research impact matrix, requir- ing a mix of ex-post and ex-ante reporting, as the cen- tre had only been running for four years at the time of research impact evaluation. However, some of the impact subgroups had been captured within the four-year time frame. Influence on policy‑making h d h Choosing the measurement approach and metrics Impact does not occur at single time points. It can be sporadic or sustained. Rarely will linear pathways ade- quately capture the complex and contested nature of this process. Therefore, we must acknowledge both the limi- tation of categorizing certain types of impact based on time frames as well as the pragmatic necessity of doing so. How we represent and measure the impact of cardio- vascular outcomes research according to short-, mid- and long-term timelines poses a particular challenge, as in some instances research has taken 25–50 years to deliver impact [25–27]. Impactful research can lead to paradigm shifts that reshape the field and context of application, framing further phases of research, policy and practice. Capturing the entirety of these processes can be chal- lenging, if not impossible. Metrics that captured the interaction between academ- ics and policy-makers to develop and implement pol- icy were considered mid-term impacts (approximately 1–3 years). These outcomes represented interim impacts that are reliant upon building an evidence base and pro- vide a pathway to translating the benefits of this evidence into practice. Direct influence on policy-making impacts was articulated as metrics with clear attributable links to centre-funded activity, such as publications from centre- funded projects being cited in clinical practice guide- lines or centre-funded PhD candidates transitioning to industry, clinical, or academic roles. Indirect impacts were framed as the involvement of centre investigators in policy-making (e.g. contributing to the development of a national cardiovascular registry). Page 5 of 12 Sarkies et al. Health Res Policy Sys (2021) 19:67 Sarkies et al. Health Res Policy Sys (2021) 19:67 Sarkies et al. Health Res Policy Sys Case study: Centre for Research Excellence in Cardiovascular Outcomes Improvementi This predominantly synchronous exchange was considered a more appropriate approach, as asyn- chronous communication over email or via a survey was thought to potentially risk constraining the process and lead to missing key impacts. Most of the investigators were located in different states or internationally at the time of the evaluation. One of the investigators could not be interviewed, as they passed away shortly after the cen- tre’s commencement. Upon completion of the telephone The centre covered short-term impacts across research and innovation outcomes, dissemination and knowledge transfer, academic collaborations, research networks, and data sharing. The use of prompts by the independ- ent researcher elicited previously unreported impact metrics, allowing quantitative measurement. Mid-term impacts were captured under one category of influencing and involvement in policy-making, with three subgroups: level of policy-making, type and nature of policy impact, and policy networks. While these impacts were known to the investigators, they had not been explicitly meas- ured as research impacts elsewhere. Descriptions of these impacts were difficult to quantify, such as contributions to establishment of registries, guideline development, and Medicare Benefits Scheme reimbursement item number applications. Long-term impacts were reported under three categories, across nine subgroups. Impact for some subgroups were unable to be identified due to the formative nature and preliminary timing of this impact evaluation. A mix of descriptive and quantitative meas- urements were reported for evidence of change in clinical Page 6 of 12 Sarkies et al. Health Res Policy Sys (2021) 19:67 Table 1 The research impact matrix for the Centre for Research Excellence in Cardiovascular Outcomes Improvement projects Time frame Impact categories Impact subgroups Outputs/outcomes Short-term 1. Case study: Centre for Research Excellence in Cardiovascular Outcomes Improvementi Research-related impact Research and innovation outcomes Direct Peer-reviewed publications (n = 41) Number of citations (n = 87) Journal impact factor (median 2.843; range 1.248 to 23.239) Manuscripts under review or development (n = 26) Indirect Associated publications (n = 156) Dissemination and knowledge transfer Direct Conference presentations (n = 61) Total PhD candidacies commenced (n = 7): PhD candidacies with cross-institutional collabora- tion (n = 1) Academic collaborations, research networks and data sharing Direct Total new academic collaborations (n = 5): International collaborations (n = 3) New database linkages (n = 6): Admitted episodes & emergency episode National Death Index Ambulance Victoria Australian and New Zealand Society of Cardiac and Thoracic Surgeons National database Combined Australian acute coronary syndrome registries (n = 16,500) Development of a multistate linked data platform for analysis of coronary heart disease in younger adults Indirect Utilization and reinforcement of a national general practice network to conduct research (> 2000 members) Establishment of the Australian and New Zealand Alliance for Cardiovascular Trials network (> 200 members) Mid-term 2. Policy networks Case study: Centre for Research Excellence in Cardiovascular Outcomes Improvementi Influencing and involvement in policy-making Level of policy-making Indirect Centre contribution to the development of a national cardiac data registry Centre contribution to the development of the National Heart Foundation guidelines [35-37] Type and nature of policy impact Direct Contribution to Medicare Benefits Schedule reim- bursement item number application: Ambulatory blood pressure monitoring Implementation of patient reported outcome measures in several data registries: Coronary Angiogram Database of South Australia Victorian Cardiac Outcomes Registry Policy networks Direct Establishment of new industry collaborations (n = 9) Embedded academic and statistician roles in industry Western Australian Department of Health PhD candidate transition to industry role (n = 1) x for the Centre for Research Excellence in Cardiovascular Outcomes Improvement projects Impact subgroups Outputs/outcomes Research and innovation outcomes Direct Peer-reviewed publications (n = 41) Number of citations (n = 87) Journal impact factor (median 2.843; range 1.248 to 23.239) Manuscripts under review or development (n = 26) Indirect Associated publications (n = 156) Dissemination and knowledge transfer Direct Conference presentations (n = 61) Total PhD candidacies commenced (n = 7): PhD candidacies with cross-institutional collabora- tion (n = 1) Academic collaborations, research networks and data sharing Direct Total new academic collaborations (n = 5): International collaborations (n = 3) New database linkages (n = 6): Admitted episodes & emergency episode National Death Index Ambulance Victoria Australian and New Zealand Society of Cardiac and Thoracic Surgeons National database Combined Australian acute coronary syndrome registries (n = 16,500) Development of a multistate linked data platform for analysis of coronary heart disease in younger adults Indirect Utilization and reinforcement of a national general practice network to conduct research (> 2000 members) Establishment of the Australian and New Zealand Alliance for Cardiovascular Trials network (> 200 members) t in policy-making Level of policy-making Indirect Centre contribution to the development of a national cardiac data registry Centre contribution to the development of the National Heart Foundation guidelines [35-37] Type and nature of policy impact Direct Contribution to Medicare Benefits Schedule reim- bursement item number application: Ambulatory blood pressure monitoring Implementation of patient reported outcome measures in several data registries: Coronary Angiogram Database of South Australia Victorian Cardiac Outcomes Registry Policy networks Direct Establishment of new industry collaborations (n = 9) Embedded academic and statistician roles in industry Western Australian Department of Health PhD candidate transition to industry role (n = 1) Table 1 The research impact matrix for the Centre for Research Excellence in Cardiovascular Outcomes Improvement projects Time frame Impact categories Impact subgroups Outputs/outcomes Short-term 1. Case study: Centre for Research Excellence in Cardiovascular Outcomes Improvementi Research-related impact Research and innovation outcomes Direct Peer reviewed publications (n 41) Mid-term 2. Influencing and involvement in policy-making Level of policy-making Type and nature of policy impact Page 7 of 12 Sarkies et al. Health Res Policy Sys (2021) 19:67 Table 1 (continued) Time frame Impact categories Impact subgroups Long-term 3. Health and health systems impact Evidence-based practice Outputs/outcomes Quality of care and service delivery 4. Health-related and societal impacts 4. Health-related and societal impacts Improved social equity, inclusion or cohesion 5. Broader economic impacts The term industry refers to all non-academic organizations, including governmental, non-governmental, and private practice, patient outcomes, consumer involvement, and further research funding. Cost containment and effectiveness Cost containment and effectiveness Quality of care and service delivery Lessons learned from the application of a research impact matrix In applying a research impact matrix for a case study in cardiovascular outcomes improvement it became clear that there is no one tool (or set of tools) that can cover the diversity of circumstances in health research. The complexity of capturing all relevant measures of impact not only presented itself when trying to choose a frame- work, but became more apparent when identifying and capturing potential metrics as well as categorizing these according to timeframes and domains of impact. On reflection, it was important to tailor the choice of frame- work and measures of impact iteratively throughout the process, particularly given the cyclical nature of inputs, activities, outputs, and outcomes in research which is striving to generate new knowledge. Feasibility of the research impact matrix We considered the research impact matrix a feasible tool to identify evidence of academic and policy impact across short- and mid-term timeframes, otherwise not Sarkies et al. Health Res Policy Sys (2021) 19:67 Page 8 of 12 documented through traditional research criterion and project evaluations. We did not identify any types of impact that could not be categorized into the research impact matrix domains. For the centre’s projects, the matrix assisted the investigators in contemplating out- puts across the range of categories and subgroups that otherwise would have gone undocumented. Support and prompting from the independent researcher were crucial to the identification of these outputs and impact, as well as describing and quantifying metrics for each domain. Given the importance of this synchronous exchange, we remain convinced that relying on the centre documenta- tion and publicly available information alone would not have been sufficient to capture the full range of impacts. Further, an asynchronous approach using survey or email exchange would likely not have resulted in the same depth of exploration with the centre’s investigators. Most of the reported research impact was captured under the short-term research domain category, such as scientific journal publications, citations of published work, dis- semination of information and knowledge transfer (e.g. conference presentations), and academic collaboration activity. The most challenging areas of impact to meas- ure were related to long-term health and health sys- tems, health-related and societal, and broader economic outcomes. of the centre’s work. Capturing broader societal changes in health literacy, knowledge, attitudes, and behaviours proved beyond much of the data captured by clinical research translation activities. The paucity of available data in these areas of research impact indicates that pro- spective planning and data collection may be warranted for other projects wishing to capture these categories of research impact. Cost containment and broader economic impacts rep- resented another difficult area of impact to measure. The data needed to model economic returns was not always adequately captured. Further, input from a health econo- mist, while available for our work, is not always resourced in project teams. Strengths and limitations approach exploring the research impact on cardiovascu- lar outcomes improvement ex-post from the first four years of the collaborative centre. This was done to ensure that findings could be used by the investigators to inform ongoing decisions regarding the investment of research funding towards implementation and translation activi- ties, before the end of the funding period. Four years is a relatively short time period in cardiovascular outcomes research [16]. However, anecdotal feedback from the cen- tre’s investigators indicated that exploring their research impact formatively encouraged prospective investment of resources in research translation projects and activi- ties. The centre investigators often articulated how their research might achieve impact ex-ante, through the discussions when applying the research impact matrix to their projects. The formative application of impact frameworks to encourage research translation has been proposed by Ramanathan et al., which provides the addi- tional benefit of facilitating collection of process, output, and interim outcomes related to research impact [10]. One of the main strengths of this study was the prag- matic approach to apply a research impact matrix that summarized common themes and metrics used across several existing methodological frameworks. This approach had two main benefits; firstly, it allowed the sufficient tailoring to the context of application where specific measures of impact were not collected prospec- tively, and secondly, provided a simple, contemporane- ous, and resource-efficient process to identify different impact categories across the programme of research. The time and cost involved in applying many research impact assessment frameworks is an important consideration for research teams and funding agencies, given the potential opportunity costs associated with their implementation. Opportunity costs occur because resources spent on conducting a research impact assessment are the same resources that can no longer be used to conduct other- wise valuable research [33]. Therefore, it is important to ensure the benefits of research impact assessment out- weigh the costs of engaging in this activity [34]. Given the pragmatic nature of our approach, it was important to ensure robustness and trustworthiness. Multiple data sources, including documentation review, bibliometric searches, and discussions with the centre investigators, were used to triangulate information ideally ensuring multiple accounts pointed to the same result. An inde- pendent researcher collated the information and engaged in the discussions with centre investigators, providing a validating check to ensure suggested outputs and impact were objective, administratively efficient to apply metrics, transparent, and comparable. Strengths and limitations Further, claims made by the centre investigators were confirmed with scholarship and grant recipients as an additional source of validation.h Challenges encountered when applying the research impact matrixh The impact categories considered within the long-term time frame were the most challenging aspects to measure and report. Measuring health and health system impact through changes in evidence-based practice would require fundamentally different study designs to those typically employed in clinical research projects. Most of the funded work within the centre was engaged in effec- tiveness research, focused on translating the benefits of therapies from efficacy trials to real-world settings. How- ever, an entirely different set of study designs is required to enable changes in evidence-based practice, namely encompassed within the field of implementation science. Implementation studies are positioned at the end of the research translation pipeline, and typically require the evidence base for an intervention, programme, or policy to be established prior to exploring how it may be imple- mented into practice [28, 29]. The time frames for this process are typically beyond what would be considered feasible for an ex post research impact evaluation of this nature, given it reportedly takes, on average, approxi- mately 17 years for only 14% of research into practice [30]. An important lesson to arise from our application of a research impact framework is the predominantly con- text-dependent nature of research impact [16, 31]. The presented case study is situated in a mixed public and private funded health system from a high-income coun- try, where impact is typically conceptualized as a direct and linear process from quantitative findings to relatively short-term changes one step removed from patient out- comes [18]. Our case study unsurprisingly favoured a similar narrative, focusing on potentially high economic returns without in-depth exploration of the interactions by which indirect impacts might occur. Methods that incorporate co-design principles, participatory designs, and rigorous qualitative approaches might have allowed us to capture the nature and mechanisms of research impact, potentially creating a more complete under- standing of this multidimensional process within com- plex health systems. Another important learning from this work pertains to the respective value and benefit of a formative or sum- mative approach to research impact. We took a formative Changes in health-related societal impacts were also difficult to measure given the first four-year time frame Sarkies et al. Health Res Policy Sys (2021) 19:67 Sarkies et al. Health Res Policy Sys (2021) 19:67 Page 9 of 12 Conclusion Th l The application of a bespoke research impact matrix uncovered the impacts of cardiovascular outcomes improvement research across and range of domains and timeframes. We did not identify any types of impact that could not be categorized into the research impact matrix domains. Using both synchronous and asynchronous means of capturing data from multiple sources required support and prompting from the independent researcher but led to identification of impact that had not otherwise been captured via traditional criterion and evaluations. We considered the research impact matrix a feasible tool to identify evidence of academic and policy impact in the short- to mid-term; however, we experienced chal- lenges in capturing long-term impacts of cardiovascular outcomes improvement research. Cost containment and broader economic impacts represented another difficult area of impact to measure. Availability of data and materials Availability of data and materials y Data are available from the corresponding author on reasonable request. Authors’ contributions MS, SR, SD, and CR contributed to the initial conceptualization and overall study design. Refinement of project data collection approach, definition of research impact, and selection of research impact matrix was conducted by MS, SR, TB, SD, MN, JB, LC, DC, JS, DB, PM, DL, and CR. MS was responsible for the data collection and descriptive analysis. MS drafted the initial manuscript, which was modified by SR, TB, SD, MN, JB, LC, DC, JS, DB, PM, DL, and CR. All authors read and approved the final manuscript. Suggestions for future research impact assessment in cardiovascular outcomes research Health Res Policy Sys (2021) 19:67 Sarkies et al. Health Res Policy Sys (2021) 19:67 Page 10 of 12 Page 10 of 12 as part of our evaluation due to its formative nature and limited prospectively collected data regarding cost- effectiveness. Our limited exploration of the activities and pathways to research impact constrained the abil- ity to trace a linear logic from “the bench to bedside”. This was perhaps a result of the research impact matrix selected and focus of the relevant stakeholders’ needs. Arguably, a more in-depth exploration of the pathways to impact may have elucidated where to best direct research translation investment, but this was beyond the scope of our project. While some efforts were taken to verify the claimed impacts from the centre’s research, much of the data collection was reliant on self-reported measures. This reliance brought about a tension between attribu- tion and contribution, making it difficult to attribute cer- tain impacts to particular studies, especially when much of the centre’s work was incremental and collaborative. However, unlike the scientific approach which aims to produce valid, reliable, and generalizable findings, the exploration of research impact seeks to understand how research has led to impact from the perspectives of spe- cific stakeholders [34]. Acknowledgements The authorship team would also like to acknowledge contributions by Julia Fallon-Ferguson, who helped coordinate the project. Funding This work was supported by the Australian National Health and Medical Research Council (NHMRC) Centres of Research Excellence (CRE) commencing in 2015, grant number 1111170. Declarations Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval and consent to participate is not applicable, as the project is considered a quality assurance exercise, not research conducted on humans. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. Author details 1 Centre for Healthcare Resilience and Implementation Science, Australian Institute of Health Innovation, Faculty of Medicine, Health and Human Sci- ences, Macquarie University, 75 Talavera Road, Sydney, NSW 2109, Australia. 2 Health Systems and Health Economics Group, Health Research and Data Analytics Hub, School of Public Health, Faculty of Health Sciences, Curtin University, Perth, Australia. 3 Faculty of Health and Medical Sciences, Popula- tion and Public Health, The University of Western Australia, Perth, Australia. 4 Department of General Cardiology, Alfred Health, Melbourne, Australia. 5 Department of Epidemiology and Preventative Medicine, Monash University, Melbourne, Australia. 6 Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia. 7 Discipline of Medicine, University of Adelaide, Adelaide, Australia. 8 Cardiology Department, Central Adelaide Local Health Network, Adelaide, Australia. 9 Cardiology Department, Lyell McEwin Hospital, Adelaide, Australia. 10 Institute of Health and Biomedical Innovation and School of Public Health and Social Work, Faculty of Health, Queensland University of Technology, Brisbane, Australia. 11 Emergency and Trauma Centre, Royal Bris- bane and Women’s Hospital, Brisbane, Australia. 12 School of Medicine, Faculty of Health and Behavioural Sciences, The University of Queensland, Brisbane, Australia. 13 Department of Cardiovascular Medicine, Flinders University, Ade- laide, Adelaide, Australia. 14 Department of Surgery (School of Clinical Sciences At Monash Health), Monash University, Melbourne, Australia. 15 Department of Cardiothoracic Surgery, Monash Health, Melbourne, Australia. 16 Division of Cardiology, Concord Hospital and University of Sydney, Sydney, Australia. 17 St Vincent’s Hospital, Victor Chang Cardiac Research Institute, University of New South Wales, Sydney, Australia. 18 NHMRC Centre for Research Excel- lence in Cardiovascular Outcomes Improvement, Health Research and Data Analytics Hub, School of Public Health, Faculty of Health Sciences, Curtin University, Perth, Australia. Abbreviations FAIT: Framework to Assess the Impact from Translational health research. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12961-021-00710-4. Additional file 1: Appendix S1. Seed grants and scholarships directly funded by the CRE in cardiovascular outcomes improvement. Abbreviations FAIT: Framework to Assess the Impact from Translational health research. Received: 10 May 2020 Accepted: 21 March 2021 Suggestions for future research impact assessment in cardiovascular outcomes research In the presented case study, we applied the research impact matrix retrospectively to the first four years of the collaborative centre, identifying mostly the ex- post impacts of the centre. We propose that a prospec- tive approach from the beginning of the funding period (including prospective data collection) may have yielded more insightful findings, as relevant measurements could have been preplanned to capture the expected research impacts over the lifetime of the funded centre. We were unable to capture many important metrics or the requi- site data to conduct an economic impact evaluation. Fur- ther, articulating ex-ante pathways for research impact at the beginning of the case study projects may have promoted additional translation- and implementation- focused designs to be incorporated with the studies on the effectiveness of different treatment modalities for cardiovascular outcomes improvement. Future evalu- ations of the impact of research may provide a more accurate, nuanced, and useful evaluation if prospective planning can take place prior to awarding of research funding. If appropriate measures can be identified and collected for both formative and summative evaluation, research activity may be able to be more targeted towards translation and impact, particularly at the later periods of funded projects. Research funding agencies may be well served by requiring reporting of the impact and transla- tion of funded research projects to ensure future award- ing decisions are made to groups with a track record of translating and implementing research into practice [32]. g There were limitations to conducting the research impact evaluation before the completion of the cen- tre’s funding term. The impact of research usually takes a long time to occur, often well beyond the completion and dissemination of the findings. The relatively short time frame of four years since research programme com- mencement meant that it was difficult to capture meas- ures of impact beyond short-term academic outputs and mid-term influence on health policy. Reliance on ret- rospective, self-reported recall from investigators also limited the ability to apply quantitative measures and metrics for several of the reported impacts. This limita- tion resulted in the reliance on descriptions, which are difficult to verify. Further, a more robust qualitative approach could have been used to obtain more trustwor- thy responses during the semi-structured discussions with centre investigators. Some research impact evalu- ations include an exploration of the economic benefits of the research conducted, but this was not completed Sarkies et al. Abbreviations FAIT Framework Supplementary Information References Osteoarthritis of the Knee Clinical Care Standard. Australian Commission on Safety and Quality in Health Care Sydney, Australia; 2017. 31. Spaapen J, Van Drooge L. Introducing ‘productive interactions’ in social impact assessment. Res Eval. 2011;20(3):211–8. 6. Rosenberg G. Research excellence framework 2014: manager’s report. London; 2015. 32. Kamenetzky A, Hinrichs-Krapels S. How do organisations implement research impact assessment (RIA) principles and good practice? A narra- tive review and exploratory study of four international research funding and administrative organisations. Health Res Policy Syst. 2020;18(1):6. 7. Kalucy EC, Jackson-Bowers E, McIntyre E, Reed R. The feasibility of deter- mining the impact of primary health care research projects using the Payback Framework. Health Res Policy Syst. 2009;7(1):11. 8. Cohen G, Schroeder J, Newson R, King L, Rychetnik L, Milat AJ, et al. Does health intervention research have real world policy and practice impacts: testing a new impact assessment tool. Health Res Policy Syst. 2015;13(1):3. 33. Haines TP, Bowles K-A, Mitchell D, O’Brien L, Markham D, Plumb S, et al. Impact of disinvestment from weekend allied health services across acute medical and surgical wards: 2 stepped-wedge cluster randomised controlled trials. PLOS Med. 2017;14(10):e1002412. 9. Donovan C, Butler L, Butt AJ, Jones TH, Hanney SR. Evaluation of the impact of National Breast Cancer Foundation-funded research. Med J Austr. 2014;200(4):214–8. 34. Adam P, Ovseiko PV, Grant J, Graham KEA, Boukhris OF, Dowd A-M, et al. ISRIA statement: ten-point guidelines for an effective process of research impact assessment. Health Res Policy Syst. 2018;16(1):8. 10. Ramanathan S, Reeves P, Deeming S, Bailie RS, Bailie J, Bainbridge R, et al. Encouraging translation and assessing impact of the Centre for Research Excellence in Integrated Quality Improvement: rationale and protocol for a research impact assessment. BMJ Open. 2017;7(12):e018572. 35. Chew DP, Scott IA, Cullen L, French JK, Briffa TG, Tideman PA, et al. National Heart Foundation of Australia and Cardiac Society of Australia and New Zealand: Australian clinical guidelines for the management of acute coronary syndromes 2016. Med J Aust. 2016;205(3):128–33. 11. Braithwaite J, Churruca K, Long JC, Ellis LA, Herkes J. When complexity sci- ence meets implementation science: a theoretical and empirical analysis of systems change. BMC Med. 2018;16(1):63. 36. Brieger D, Amerena J, Attia J, Bajorek B, Chan KH, Connell C, et al. References National Heart Foundation of Australia and the Cardiac Society of Australia and New Zealand: Australian Clinical Guidelines for the Diagnosis and Man- agement of Atrial Fibrillation 2018. Heart Lung Circ. 2018;27(10):1209–66. 12. Fryirs KA, Brierley GJ, Dixon T. Engaging with research impact assessment for an environmental science case study. Nat Commun. 2019;10(1):4542. 37. Atherton JJ, Sindone A, De Pasquale CG, Driscoll A, MacDonald PS, Hop- per I, et al. National Heart Foundation of Australia and Cardiac Society of Australia and New Zealand: Guidelines for the Prevention, Detection, and Management of Heart Failure in Australia 2018. Heart Lung Circ. 2018;27(10):1123–208. 13. Hicks D. Performance-based university research funding systems. Res Policy. 2012;41(2):251–61. y 14. Moodie G. National interest test for research grants could further erode pure research: The Conversation; 2018 https://theconversation.com/ national-interest-test-for-research-grants-could-further-erode-pure-resea rch-106061. 38. International Consortium for Health Outcome Measurement. Case Study: Implementing ICHOM’s Standard Sets of Outcomes – (CADOSA) 2017. https://www.ichom.org/news/case-study-implementing-ichoms-stand ard-sets-of-outcomes-cadosa/. 15. Morgan B. Income for outcome. Nature. 2014;511(7510):S72 16. Penfield T, Baker MJ, Scoble R, Wykes MC. Assessment, evaluations, and definitions of research impact: a review. Res Eval. 2014;23(1):21–32. 39. Lim HS, Andrianopoulos N, Sugumar H, Stub D, Brennan AL, Lim CC, et al. Long-term survival of elderly patients undergoing percutaneous coro- nary intervention for myocardial infarction complicated by cardiogenic shock. Int J Cardiol. 2015;195:259–64. 17. Dodd R, Ramanathan S, Angell B, Peiris D, Joshi R, Searles A, et al. Strengthening and measuring research impact in global health: lessons from applying the FAIT framework. Health Res Policy Syst. 2019;17(1):48. 40. Yudi MB, Ramchand J, Farouque O, Andrianopoulos N, Chan W, Duffy SJ, et al. Impact of door-to-balloon time on long-term mortality in high-and low-risk patients with ST-elevation myocardial infarction. Int J Cardiol. 2016;224:72–8. 18. Greenhalgh T, Raftery J, Hanney S, Glover M. Research impact: a narrative review. BMC Med. 2016;14(1):78. 19. Buxton M, Hanney S. How can payback from health services research be assessed? J Health Serv Res Policy. 1996;1(1):35–43. 20. Donovan C, Hanney S. The ‘payback framework’explained. Res Eval. 2011;20(3):181–3. 41. Karim MN, Reid CM, Huq M, Brilleman SL, Cochrane A, Tran L, et al. Predicting long-term survival after coronary artery bypass graft surgery. Interactive CardioVasc Thor Surg. 2018;26(2):257–63. 21. Searles A, Doran C, Attia J, Knight D, Wiggers J, Deeming S, et al. An approach to measuring and encouraging research translation and research impact. Health Res Policy Syst. 2016;14(1):60. 42. References 1. Sarkies MN, Bowles K-A, Skinner EH, Haas R, Lane H, Haines TP. The effec- tiveness of research implementation strategies for promoting evidence- informed policy and management decisions in healthcare: a systematic review. Implement Sci. 2017;12(1):132. 1. Sarkies MN, Bowles K-A, Skinner EH, Haas R, Lane H, Haines TP. The effec- tiveness of research implementation strategies for promoting evidence- informed policy and management decisions in healthcare: a systematic review. Implement Sci. 2017;12(1):132. nto products, policy, and practice. Health Res Policy Syst. 2015;13(1 26. Sarkies M. Effectiveness of research implementation strategies for evi- dence informed healthcare policy and practice [Doctorate]. Melbourne, Australia: Monash University; 2019. 2. Sarkies MN, White J, Morris ME, Taylor NF, Williams C, O’Brien L, et al. Implementation of evidence-based weekend service recommendations for allied health managers: a cluster randomised controlled trial protocol Implement Sci. 2018;13(1):60. 2. Sarkies MN, White J, Morris ME, Taylor NF, Williams C, O’Brien L, et al. Implementation of evidence-based weekend service recommendations for allied health managers: a cluster randomised controlled trial protocol Implement Sci. 2018;13(1):60. y 27. G. R. Response to second HEFCE consultation on the Research Excellence Framework 2009. 3. Sarkies MN, White J, Henderson K, Haas R, Bowles J. Additional weekend allied health services reduce length of stay in subacute rehabilitation wards but their effectiveness and cost-effectiveness are unclear in acute general medical and surgical hospital wards: a systematic review. J Physi- other. 2018;64(3):142–58. 28. Sarkies MN, Skinner EH, Bowles K-A, Morris ME, Williams C, O’Brien L, et al. A novel counterbalanced implementation study design: methodological description and application to implementation research. Implement Sci. 2019;14(1):45. 3. Sarkies MN, White J, Henderson K, Haas R, Bowles J. Additional weekend allied health services reduce length of stay in subacute rehabilitation wards but their effectiveness and cost-effectiveness are unclear in acute general medical and surgical hospital wards: a systematic review. J Physi- other. 2018;64(3):142–58. 29. Sarkies MN, Maloney S, Symmons M, Haines TP. Video strategies improved health professional knowledge across different contexts: a helix counter- balanced randomized controlled study. J Clin Epidemiol. 2019;112:1–11. 4. Glasziou P, Straus S, Brownlee S, Trevena L, Dans L, Guyatt G, et al. Evidence for underuse of effective medical services around the world. Lancet. 2017;390(10090):169–77. 30. Morris ZS, Wooding S, Grant J. The answer is 17 years, what is the ques- tion: understanding time lags in translational research. J R Soc Med. 2011;104(12):510–20. 5. Safety ACo, Care QiH. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12961-021-00710-4. Received: 10 May 2020 Accepted: 21 March 2021 Received: 10 May 2020 Accepted: 21 March 2021 Additional file 1: Appendix S1. Seed grants and scholarships directly funded by the CRE in cardiovascular outcomes improvement. Page 11 of 12 Page 11 of 12 Sarkies et al. Health Res Policy Sys (2021) 19:67 Sarkies et al. Health Res Policy Sys 25. Hanney SR, Castle-Clarke S, Grant J, Guthrie S, Henshall C, Mestre- Ferrandiz J, et al. How long does biomedical research take? Studying the time taken between biomedical and health research and its translation into products, policy, and practice. Health Res Policy Syst. 2015;13(1):1.f References Si S, Hillis GS, Sanfilippo FM, Smith J, Tran L, Reid CM, et al. Surgical aortic valve replacement in Australia, 2002–2015: temporal changes in clinical practice, patient profiles and outcomes. ANZ J Surg. 2019;89(9):1061–7. 22. Permanyer-Miralda G, Hinrichs-Krapels S, Adam P. The social impact of cardiology research: beyond management. Revista Española de Cardi- ología (English Edition). 2016;69(7):639–43. 43. Dhurandhar V, Parikh R, Saxena A, Vallely MP, Wilson MK, Black DA, et al. Early and late outcomes following valve sparing aortic root reconstruc- tion: the ANZSCTS database. Heart Lung Circ. 2016;25(5):505–11. g g 23. National Health and Medical Research Council. Centres of Research Excel- lence: NHMRC; 2021. https://www.nhmrc.gov.au/funding/find-funding/ centres-research-excellence. 44. Karim MN, Reid CM, Cochrane A, Tran L, Alramadan M, Hossain MN, et al. Mortality risk prediction models for coronary artery bypass graft surgery: current scenario and future direction. J Cardiovasc Surg (Torino). 2017;58(6):931–42. 24. Rivera SC, Kyte DG, Aiyegbusi OL, Keeley TJ, Calvert MJ. Assessing the impact of healthcare research: a systematic review of methodological frameworks. PLoS Med. 2017;14(8):e1002370. Sarkies et al. Health Res Policy Sys (2021) 19:67 Page 12 of 12 surgery versus stenting in high-risk patients: propensity score-matched analysis of a single-centre experience. Appl Health Econ Health Pol. 2018;16(5):661–74. surgery versus stenting in high-risk patients: propensity score-matched analysis of a single-centre experience. Appl Health Econ Health Pol. 2018;16(5):661–74. 49. Ariyaratne TV, Ademi Z, Ofori-Asenso R, Huq MM, Duffy SJ, Yan BP, et al. The cost-effectiveness of guideline-driven use of drug-eluting stents: propensity-score matched analysis of a seven-year multicentre experi- ence. Curr Med Res Opin. 2020;36(3):419–26. 49. Ariyaratne TV, Ademi Z, Ofori-Asenso R, Huq MM, Duffy SJ, Yan BP, et al. The cost-effectiveness of guideline-driven use of drug-eluting stents: propensity-score matched analysis of a seven-year multicentre experi- ence. Curr Med Res Opin. 2020;36(3):419–26. 45. Karim MN, Reid CM, Tran L, Cochrane A, Billah B. Missing Value Imputation improves mortality risk prediction following cardiac surgery: an investiga- tion of an australian patient Cohort. Heart Lung Circ. 2017;26(3):301–8. 46. Andrianopoulos N, Dinh D, Duffy SJ, Clark DJ, Brennan AL, Chan W, et al. Quality control activities associated with registries in interventional cardiology and surgery. Heart Lung Circ. 2011;20(3):180–6. 47. Ariyaratne TV, Yap C-H, Ademi Z, Rosenfeldt F, Duffy SJ, Billah B, et al. A systematic review of cost-effectiveness of percutaneous coronary intervention vs. surgery for the treatment of multivessel coronary artery disease in the drug-eluting stent era. Eur Heart J. 2016;2(4):261-70. 48. Ariyaratne TV, Ademi Z, Huq M, Rosenfeldt F, Duffy SJ, Parkinson B, et al. The real-world cost-effectiveness of coronary artery bypass surgery versus stenting in high-risk patients: propensity score-matched analysis of a single-centre experience. Appl Health Econ Health Pol. 2018;16(5):661–74. 47. Ariyaratne TV, Yap C-H, Ademi Z, Rosenfeldt F, Duffy SJ, Billah B, et al. A systematic review of cost-effectiveness of percutaneous coronary intervention vs. surgery for the treatment of multivessel coronary artery disease in the drug-eluting stent era. Eur Heart J. 2016;2(4):261-70. 45. Karim MN, Reid CM, Tran L, Cochrane A, Billah B. Missing Value Imputation improves mortality risk prediction following cardiac surgery: an investiga- tion of an australian patient Cohort. Heart Lung Circ. 2017;26(3):301–8.f 46. Andrianopoulos N, Dinh D, Duffy SJ, Clark DJ, Brennan AL, Chan W, et al. Quality control activities associated with registries in interventional cardiology and surgery. Heart Lung Circ. 2011;20(3):180–6.f Publisher’s Note S Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 48. Ariyaratne TV, Ademi Z, Huq M, Rosenfeldt F, Duffy SJ, Parkinson B, et al. The real-world cost-effectiveness of coronary artery bypass • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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https://openalex.org/W4388630737	https://www.nature.com/articles/s41598-023-47022-x.pdf	English	null	Adaptive walking control for quadruped robot by using oscillation patterns	Scientific reports	2,023	cc-by	7,164	Adaptive walking control for quadruped robot by using oscillation patterns OPEN Yong Zhang 1, Yijia Qian 1, Yi Ding 1, Beiping Hou 1 & Rongyang Wang 2 To improve the adaptability of quadruped robot in multiple scenarios, an adaptive locomotive system based on the double-layered central pattern generator (CPG) is proposed. The novel CPG network consists of double master units and subsets of slave units based on gyroscope signals including yaw and pitch angle. The response of master units provides the ability to control the 1st joins of quadruped robot, while slave units can generate the symmetry signals to control the 2nd and 3rd joints. The CPG network enables the seamless switching of locomotion gaits to stops and starts by using an ultrasonic sensor. Through adjusting the mutually dependent parameters, joints can generate the joint angles to achieve steering behavior. For adaptive movement on an irregular surface, stable ranges of the robot body yaw and pitch angles are proposed by using gyroscope signals. The experimental results verify that the quadruped robot with the proposed double-layered CPG network can perform stable trot pattern in a complex environment. Quadruped robots have a more remarkable capability to move with diverse movement patterns than wheeled robots especially in unstructured terrain1. Numerous outstanding control methods have been developed to control quadruped robot movements such as walking, running and steering2. These control methods include model-based control, compliant control and CPG control3. Model-based control is the most widely used method in the motion control of legged robots4. The calculation of the mechanical characteristics of the robot is an important task5. The compliant control method can improve the environmental adaptability based on feedback information6. The virtual model controller was proposed to control the quadruped robot as a PID controller7. However, researchers have consistently focused on the center of gravity using dynamics equations8. When the road conditions change, it is necessary to recalculate the rigid dynamics. With the rapid development of the neural control field, quadruped robots have been studied to utilize neural oscillator techniques to achieve the bionic movement patterns9. p In biology, the CPG (central pattern generators) unit located in the spinal cord of vertebrates is able to generate high-dimensional signals for coordinated symmetry movements10. The biological CPG network has proved to be a multi-layered structure11. Inspired by this method, researchers have developed many important mathematical models to control the quadruped robot12. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2023) 13:19756 Quadruped robot model p Figure 1a,b present the appearance of the quadruped robot named Standford Pupper, which controlled by 12 degrees of freedom. Each leg has three active joints that provide a total of three degree of freedom: first pitch joint, second yaw joint and third yaw joint. The second and third joints mostly provide the primary power to the forward movement of the robot. These joints are driven by a steering actuator. The servomotor of the robot is Servo CL S6336HV. The communication among the servo motors is serial communication bus. To recognize obstacles during robot movement, an ultrasonic sensor is installed on the body of the robot. To measure the body angle, a gyroscope sensor is equipped inside of the body to feedback the yaw, pitch and roll angles. The robot con- troller installed inside the robot body can generate the signal of each joint. The power is supplied from a battery. The overall dimensions are 20 cm height, 33 cm length, and 1.65 kg total weight. Due to the small size of robot, the dynamic characteristics will affect the motion stability. The constraint condition of stability is the distances from the projection point of the COG (center of gravity) to the sides of the supporting polygon. The stability margin of the trot gaits is always positive in the moving processes. To design the phase of the trot pattern, the sequence of the leg is defined as Hind-Left (HL) leg → Front-Right (FR) leg → Hind-Right (HR) leg → Front- Left (FL) leg during movement in the trot pattern, as shown in Fig. 1c. Through the joints control with the trot walking pattern, the quadruped robot can suitable for complex environments. This mechanical setup is based on our previous work on the model of Featherstone rigid dynamics28. By using this mechanical structure, the quad- ruped robot has the ability to move with a trot pattern. The physical parameters of the robot are listed in Table 1. Figure 1a,b present the appearance of the quadruped robot named Standford Pupper, which controlled by 12 degrees of freedom. Each leg has three active joints that provide a total of three degree of freedom: first pitch joint, second yaw joint and third yaw joint. The second and third joints mostly provide the primary power to the forward movement of the robot. These joints are driven by a steering actuator. www.nature.com/scientificreports/ under the trot pattern. This kind of controller has the ability to achieve adaptive motion in the quadruped robot in multiple scenarios27. In this paper, an adaptive locomotive system based on the double-layered central pattern generator (CPG) is proposed, to improve the adaptability of the quadruped robot in multiple scenarios. The novel CPG network consists of double master units and subsets of slave units. The response of master units renders them the ability to control the 1st joins of quadruped robot, while slave units can generate the symmetry signals to control the 2nd and 3rd joints. By implementing this concept, the CPG networks of central pattern generators feature the capability to generate the symmetry signals for stable movement and steering under the trot pattern. For adap- tive motion on an irregular surface, a stable range of the robot body yaw and pitch angles are proposed based on gyroscope signals. The CPG network enables the seamless switching of locomotion gaits to stops and starts based on an ultrasonic sensor. The experimental results verify that the quadruped robot with the proposed double-layered CPG network can perform stable trotting in a complex environment.h y p g p The rest of the paper is organized as follows. In “Quadruped robot model” section, the model of quadruped robot is presented and the contribution of different body parameters are discussed. “Model of CPG unit” sec- tion discusses the central pattern generator and formulates a trajectory generator based on different mutually dependent parameters. In “Model of CPG network” section, the network of this control system is discussed and verified. In “Experiment” section, the results are further evaluated and compared with the experimental results. In “Conclusions” section the conclusions are presented. Adaptive walking control for quadruped robot by using oscillation patterns OPEN A two-layered CPG network including self-learning function was proposed for the walking motion control of legged robots on irregular terrain13. The multi-layered structure allows the biological CPG network to hierarchically process sensory information to control the body parts to improve environmental adaptability14.f p p p y As for a multi-layered CPG network, researchers have used the properties of higher-order differential equa- tions to generate the periodic signals15. Through adjusting the parameters of oscillator units, the CPG network has the ability to generate a stable rhythm signal16. However, when the robot moves in a diverse environment, the gait planning becomes single17. Thus, getting the adaptive values of mutually dependent parameters is an important work in CPG networks18,19. Researchers have proposed to use feedback information to adjust the joints angles for a diversity of environments20–22.ifh g y Diversified movement patterns are the most effective in unstructured environments23. The trot movement pattern is a gait plan in which the diagonal legs pair up and hit down at separate intervals in animals such as the horse24. Quadruped robots use the trot pattern to control the center of gravity easily25. Because the double-layered CPG network consists of higher order differential equations, it is easy to integrate abundant sensing signals26. Through information from the environment, CPG networks have appropriate characteristics for motion control 1School of Automation and Electrical Engineering, Zhejiang University of Science and Technology, Hangzhou, China. 2Key Laboratory of Robot System Integration and Intelligent Equipment of Huzhou City, Huzhou, China. email: zhangy@zust.edu.cn \| https://doi.org/10.1038/s41598-023-47022-x www.nature.com/scientificreports/ Model of CPG unit In order to achieve movement in the trot pattern, the model of CPG unit is proposed. There is an excitatory and an inhibitory unit to generate the periodic signals depending on the coupling parameters. The internal state of the excitatory unit is ue and the inhibitory unit is ui . The joint angles are generated by mutually dependent parameters in real time. Figure 2 shows the block diagram of the proposed CPG model. A function of the dynamic parameters is output as a series of periodic signals for controlling the joint motor, where a represents the intrinsic excitatory coupling parameter, d represents the intrinsic inhibitory coupling parameter, b represents the inhibitory coupling factor and c is the excitatory coupling factor. To control the joint angle directly, the motor dynamic parameters are used. Km represents the gain constant of the motor, τm repre- sents the time constant, Kg represents the gear ratio, and Ka represents the gain of the motor amplifier. Based on the dynamic parameters of the motor, the model of CPG unit can generate the periodic signals in the robot joints directly. The parameter sets are shown in Table 2. The dynamic equations of the proposed CPG model are formulated as follows: (1) τm d2ue dt2 = −due dt −oﬀsetg −KgKmKaa tan−1 ue + KgKmdP −KgKmKac tan−1 ui + d tan−1 ue τm d2ui dt2 = −dui dt −oﬀsetg + KgKmKab tan−1 ue + KgKmdP + d tan−1 ui oﬀsetg = Kr(θ −θo) dp=        1 Ui ≥10 −   1 n i=1 Ui   2 otherwise (1) The gyroscope can detect the body angle to the CPG model in real time, where oﬀsetg represents the param- eters to feedback this information, and Kr represents the feedback gain. For the first joint angle of the robot, θ represents the yaw angle measured in real time, and θo represents the stable body yaw angle of the robot. For the other joint angles of the robot, θ represents the pitch angle measured in real time, and θo represents the stable body pitch angle of the robot. If the robot approaches an upward or downward inclination road, its yaw and pitch angle changes quickly. The gyroscope will detect this information and feed it back to the CPG model immedi- ately. Quadruped robot model The servomotor of the robot is Servo CL S6336HV. The communication among the servo motors is serial communication bus. To recognize obstacles during robot movement, an ultrasonic sensor is installed on the body of the robot. To measure the body angle, a gyroscope sensor is equipped inside of the body to feedback the yaw, pitch and roll angles. The robot con- troller installed inside the robot body can generate the signal of each joint. The power is supplied from a battery. Th ll d h h l h d k l h h ll f b y g g jh p pp y The overall dimensions are 20 cm height, 33 cm length, and 1.65 kg total weight. Due to the small size of robot, the dynamic characteristics will affect the motion stability. The constraint condition of stability is the distances from the projection point of the COG (center of gravity) to the sides of the supporting polygon. The stability margin of the trot gaits is always positive in the moving processes. To design the phase of the trot pattern, the sequence of the leg is defined as Hind-Left (HL) leg → Front-Right (FR) leg → Hind-Right (HR) leg → Front- Left (FL) leg during movement in the trot pattern, as shown in Fig. 1c. Through the joints control with the trot walking pattern, the quadruped robot can suitable for complex environments. This mechanical setup is based on our previous work on the model of Featherstone rigid dynamics28. By using this mechanical structure, the quad- ruped robot has the ability to move with a trot pattern. The physical parameters of the robot are listed in Table 1. Figure 1. The quadruped robot platform: (a) mechanical structure of the quadruped robot; (b) structural sketch of robot; (c) the trot gait. Figure 1. The quadruped robot platform: (a) mechanical structure of the quadruped robot; (b) structural sketch of robot; (c) the trot gait. Table 1. The dimensions of robot. 1st joint 2nd joint 3th joint Length (cm) 5 12 15 Width (cm) 7 4 1.5 Table 1. The dimensions of robot. https://doi.org/10.1038/s41598-023-47022-x Scientific Reports \| (2023) 13:19756 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ original value again. By means of this concept, the robot can overcome the terrain condition through adaptive rhythmic joint motion. Here, dP represents feedback parameters for the ultrasonic sensor, Ui represents the value of ultrasonic sensor in real time, and Ku is the gain constant. Depending on the value of dP , the CPG model will be changed to adjust the first joints of robot. If there is an obstacle detected by the ultrasonic sensor, the value of dp will change. The oscillation network can output the signals with higher amplification. The robot will turn and leave the obstacle until no obstacles are ahead. At this time, dp switches to 1 again. The CPG model can continue to generate peri- odic signals for the forward movement of the robot. Model of CPG network In order to generate the fundamental gait signals for the quadruped robot, the CPG models are mounted on each leg, as depicted in Fig. 3. Here, FL, FR, HL, and HR denote the Front-Left leg, Front-Right leg, Hind-Left leg and Hind-Right leg, respectively. In this research, the CPG network consists of double master units and subsets of slave units. The response of master units Me1 , Mi1 , Me2 and Mi2 have the ability to control all the 1st joins. The models are connected to each other by coupling parameters k12 and k21 . The parameter dP feedbacks the signals of ultrasonic sensor. Moﬀsetg represents the yaw angles of the robot body. ﬀ g y g y Slave units can generate the symmetry signals to control all of the 2nd and 3rd joints of the quadruped robot. The mutually dependent parameters, including kMj and kSj(j = 1 −4) , can be adjusted. Soﬀsetg represents the pitch angles of the robot body. Through the mutually dependent parameters, the models of CPG units are con- nected to control the quadruped robot. The dynamic equations of the CPG network in the Front-Left leg are expressed as follows: (2)                        τm d2uMe1 dt2 = −duMe1 dt −Moﬀsetg −KgKmKaaM1 tan−1 uMe1 + dM1 tan−1 uSe1 −KgKmKacM1 tan−1 uMi1 + KgKmdp + k12 tan−1 uMe2 τm d2uMi1 dt2 = −duMi1 dt −Moﬀsetg + KgKmKabM1 tan−1 uMe1 + KgKmdi1dp + kM4 tan−1 uSe4 Moﬀsetg = Kyθyaw (2) (3)                  τm d2uSe1 dt2 = −duSe1 dt −Soﬀsetg −KgKmKaaS1 tan−1 uSe1 + dS1 tan−1 uMe1 −KgKmKacS1 tan−1 uSi1 + KgKm τm d2uSi1 dt2 = −duSi1 dt −Soﬀsetg + KgKmKabS1 tan−1 uSe1 + KgKmdi1 Soﬀsetg = Kpθpitch (3) Once the CPG network reaches a stable oscillation state, it generates periodic signals whose shapes depend on the internal parameters, the network topology and interconnection weights, as shown in Fig. 4. Model of CPG unit If the body angle deviates from the stable range, oﬀsetg will rise to a high value to control the CPG model to generate a high cyclic period until the body angle returns to the stable range. At this time, oﬀsetg becomes the 3 Vol.:(01234 13:19756 \| https://doi.org/10.1038/s41598-023-47022-x Figure 2. The block diagram of CPG unit. Table 2. CPG parameter sets. Parameters a b c d Kg Ka Km Kr Ku Value 2.1 17.1 18.1 − 18.1 1.6 4.9 120 14 18 h Figure 2. The block diagram of CPG unit. https://doi.org/10.1038/s41598-023-47022-x Scientific Reports \| (2023) 13:19756 \| www.nature.com/scientificreports/ Model of CPG network The coefficients and limits of each polynomial are found experimentally to yield a good fitting with low-order polynomials.i i In order to create the stable periodic signal of the CPG model, a limit cycle is designed that defines a series of exact repetitions with a closed trajectory for the trotting motion of the robot. The trajectory of the limit cycle 4 6 \| htt //d i /10 1038/ 41598 023 47022 Figure 3. The framework of CPG network. Figure 3. The framework of CPG network. Figure 3. The framework of CPG network. Scientific Reports \| (2023) 13:19756 \| https://doi.org/10.1038/s41598-023-47022-x https://doi.org/10.1038/s41598-023-47022-x www.nature.com/scientificreports/ Figure 4. Examples of the phase-coordinated CPG network; the relevant parameters are provided in Table 2. (a) The response of CPG network under dP switching; (b) Evolution of the limit cycle during the gait switch. Figure 4. Examples of the phase-coordinated CPG network; the relevant parameters are provided in Table 2 (a) The response of CPG network under dP switching; (b) Evolution of the limit cycle during the gait switch. starts with a fixed point and continues around a nominal orbit swift. Depending on resetting the phase, oscillation network can generate a series of signals to spin in circles.hih The amplifier and frequency are used to control the quadruped robot. The CPG control system generates the joint angles with the trot pattern. Through the ultrasonic sensor, the response of uMe1 and uMi1 is based on the adjustment of dp . At 5.5s, the value of dP become 10, the CPG network can be updated to adjust gait patterns of output, and the joints stop immediately using the internal feedback signals. By using this way, the CPG network has ability to generate suitable motions.h y g Through setting the mutually dependent parameters, k12 , k21 , kMj and kSj(j = 1 −4) , the joints can generate the joint angles to achieve steering behavior. The angle of steering behavior is π/2 . Because the robot is steering right with the trot pattern, the amplitudes for the right swing joints are smaller than those for the left swing joints. The diagonal limbs move out of phase, and the generated trajectories must be in phase. The responses are shown in Fig. 5. The first joints, second joints and third joints are depicted in blue, yellow and red lines, respectively. Model of CPG network All of the generated trajectories respect the coordination constraints imposed by the couplings.hf The phase difference between the output signals is determined by the internal parameters of the neurons, the CPG network topology and the inter-connection weights. These parameters have been experimentally chosen to yield output signals with a shape resembling a periodic wave as much as possible, given the weights and internal parameters. By adjusting the value of feedback parameters, the response of uMe1 and uMi1 can reset the amplifier and frequency of signals. By adjusting the value of feedback parameter, the output of oscillation network can generate periodic signals to respond to the external stimuli. The parameters uMe1 and uMi1 generate the periodic signals stably by using mutually dependent parameters. The coefficients of the low-order polynomials and the corresponding coefficients of determination are given in Table 3. Experimenti p In order to confirm the validity of the CPG network, two sets of experiments are conducted for the application of adaptive movement system for quadruped robot control. Firstly, the robot trots forward and steers by using the ultrasonic sensor. Secondly, the robot trots on an irregular surface by using its gyroscope sensor. The controller of the robot is Raspberry Pi. To record the data, wireless modules are installed in the robot body. By using the XBee modules, the controller can send the joint angles to a computer. Scientific Reports \| (2023) 13:19756 \| https://doi.org/10.1038/s41598-023-47022-x www.nature.com/scientificreports/ Figure 5. The response of CPG network for steering motion under mutually parameters control. The joints angle of FL, FR, HL, HR legs of robot. Figure 5. The response of CPG network for steering motion under mutually parameters control. The joints angle of FL, FR, HL, HR legs of robot. k12 k21 kM1 kS1 kM2 kS2 kM3 kS3 kM4 kS4 Walk forward − 1.8 1.8 1.1 1.15 1.8 − 1.6 − 1.8 1.8 − 1.6 1.6 Steering motion 2.1 2.3 0.8 2 − 1.8 1.6 1.8 − 1 1.6 − 2 Table 3. The mutually parameters sets. In the first experiment, the experimental environment is shown in Fig. 6. The distance of region A is set to 10 cm. Such information is unknown to the quadruped robot. The mutually dependent parameters of CPG networks are shown in Table 2. The response of each neuron can generate the robot joint based on the stimuli of feedback parameters. The experimental results demonstrate that the oscillation network can autonomously generate the periodic signals for the quadruped robot. Figure 7 illustrates the body angle along the X axis, Y axis and Z axis during the whole process. Before 6s, the robot moves along the road in a trot pattern. Depending on the CPG network, the speed is stable, and the ampli- fier and frequency of body angle is regular. The quadruped robot is able to trot stably. However, at 6s, the robot trots into the A area, and the ultrasonic sensor detects the obstacle. The body angles change sharply because of the parameters dp adjusting. At this time, the master and slave oscillation units adjust the mutually dependent parameters. The oscillation networks change the amplifier of robot joints to steer for leaving the narrow space. Experimenti At 7.5 s, the robot finishes the steering behavior, the ultrasonic sensor detects the distance of obstacle, and robot needs to continue to steer. At 9.8 s, there is no obstacle in front of the quadruped robot and it stops steering. The mutually dependent parameters switch to moving forward. At last, the robot leaves the narrow space in a trot pattern by using CPG network control. The experiment demonstrates that the robot body angles are stable due to the adjustment of feedback parameter including the feedback of ultrasonic sensor. Th f d d b h h l h d h f db k f l The scenes of quadruped robot in the whole experiment are shown in Fig. 8. Based on the feedback of ultra- sonic sensor, the quadruped robot can move forward and then stop in front of the obstacle. Through adjusting the mutually dependent parameters, the double-layered CPG network can set the response for steering behavior. The quadruped robot can move stably in a trot pattern. Figure 6. The map of the first experiment. Figure 6. The map of the first experiment. Figure 6. The map of the first experiment. Scientific Reports \| (2023) 13:19756 \| https://doi.org/10.1038/s41598-023-47022-x www.nature.com/scientificreports/ Figure 7. The yaw, roll and pitch angles of robot in the first experiment. Figure 7. The yaw, roll and pitch angles of robot in the first experiment. Figure 7. The yaw, roll and pitch angles of robot in the first experiment. In the second experiment, the irregular surface includes an up-down slope, as shown in Fig. 9, with the angle of slope at 15 degrees. On the up and downward surface, there is a slope with a 10-degree angle and the robot is unaware of this irregular road condition. Using the gyroscope signals can provide feedback in real time. The range of the pitch and yaw angle are within ± 8 degrees. If the pitch and yaw angle deviates from this range, the CPG network will change the parameters for the slope. Figure 10 shows the body angle in the X axis, Y axis and Z axis during the whole process. Before 5 s, the quadruped robot moves on the flat surface. Because the pitch and yaw angle do not deviate from the stable range, the robot moves using the parameter set for the flat surface. Experimenti At 6 s, the robot moves on the upward slope, where the pitch angle deviates from the stable range. The yaw angle also increases suddenly, and the gyroscope detects Figure 8. The frame of first experiment. Figure 8. The frame of first experiment. Figure 8. The frame of first experiment. In the second experiment, the irregular surface includes an up-down slope, as shown in Fig. 9, with the angle of slope at 15 degrees. On the up and downward surface, there is a slope with a 10-degree angle and the robot is unaware of this irregular road condition. Using the gyroscope signals can provide feedback in real time. The range of the pitch and yaw angle are within ± 8 degrees. If the pitch and yaw angle deviates from this range, the CPG network will change the parameters for the slope. In the second experiment, the irregular surface includes an up-down slope, as shown in Fig. 9, with the angle of slope at 15 degrees. On the up and downward surface, there is a slope with a 10-degree angle and the robot is unaware of this irregular road condition. Using the gyroscope signals can provide feedback in real time. The range of the pitch and yaw angle are within ± 8 degrees. If the pitch and yaw angle deviates from this range, the CPG network will change the parameters for the slope. Figure 10 shows the body angle in the X axis Y axis and Z axis during the whole process Before 5 s the In the second experiment, the irregular surface includes an up-down slope, as shown in Fig. 9, with the angle of slope at 15 degrees. On the up and downward surface, there is a slope with a 10-degree angle and the robot is unaware of this irregular road condition. Using the gyroscope signals can provide feedback in real time. The range of the pitch and yaw angle are within ± 8 degrees. If the pitch and yaw angle deviates from this range, the CPG network will change the parameters for the slope. g p p Figure 10 shows the body angle in the X axis, Y axis and Z axis during the whole process. Before 5 s, the quadruped robot moves on the flat surface. Experimenti Because the pitch and yaw angle do not deviate from the stable range, the robot moves using the parameter set for the flat surface. At 6 s, the robot moves on the upward slope, where the pitch angle deviates from the stable range. The yaw angle also increases suddenly, and the gyroscope detects g p p Figure 10 shows the body angle in the X axis, Y axis and Z axis during the whole process. Before 5 s, the quadruped robot moves on the flat surface. Because the pitch and yaw angle do not deviate from the stable range, the robot moves using the parameter set for the flat surface. At 6 s, the robot moves on the upward slope, where the pitch angle deviates from the stable range. The yaw angle also increases suddenly, and the gyroscope detects Scientific Reports \| (2023) 13:19756 \| https://doi.org/10.1038/s41598-023-47022-x www.nature.com/scientificreports/ Figure 9. Up-down slope used in experiment. Figure 10. The yaw, roll and pitch angles of robot in the second experiment. Figure 9. Up-down slope used in experiment. Figure 9. Up-down slope used in experiment. Figure 9. Up-down slope used in experiment. Figure 10. The yaw, roll and pitch angles of robot in the second experiment. this information. By changing the parameters of feedback, the first joint angles adjust to keep the pitch angle in a stable range. The second and third joint angles can generate the appropriate periodic joint motions. At 18 s, the robot approaches the down-slope, and the yaw and pitch angles change acutely. At this time, the CPG network generates high cyclic period signals. Figures 11 show the responses of the joint angles. When the robot enters the up-down slope, it detects the change in the road surface and the internal feedback parameters are adjusted automatically. The results for the quadruped robot are shown in Fig. 12.h The above experiments show that the proposed CPG control system can allow the robot to move in a complex environment to avoid abrupt changes, demonstrating improved adaptability in multiple scenarios. This result is crucial for extending the practical applications. The double-layered CPG network can generate the periodic joint angle to control the quadruped robot on the irregular terrain. For adaptive movement on an irregular surface, a stable range of the robot body yaw and pitch angles are proposed based on gyroscope signals. Conclusions The response of each neuron can generate the robot joint based on the stimuli of feedback parameters. Through adjusting the mutually dependent parameters of CPG networks, the joint angle can be controlled based on feedback information. For adaptive movement on an irregular surface, a stable range of the robot body pitch and yaw angle are proposed by using gyroscope signals. To detect the obstacle, the ultrasonic signals are utilized for adjusting the parameters. The double-layered CPG networks have a capability to generate the symmetry signals for stable movement on a complex surface. output of each neuron is directly connected to the slave neuron. The response of master units is able to control the 1st joins of the quadruped robot, while the slave units can generate the symmetry signals to control the 2nd and 3rd joints. The response of each neuron can generate the robot joint based on the stimuli of feedback parameters. Through adjusting the mutually dependent parameters of CPG networks, the joint angle can be controlled based on feedback information. For adaptive movement on an irregular surface, a stable range of the robot body pitch and yaw angle are proposed by using gyroscope signals. To detect the obstacle, the ultrasonic signals are utilized for adjusting the parameters. The double-layered CPG networks have a capability to generate the symmetry signals for stable movement on a complex surface. y y g p By using our proposed concept, the quadruped robot can be controlled by double-layered CPG network, while generating the symmetry signals periodically depend on feedback from the information of sensors. This realizes the effective implementation of adaptive motion in the quadruped robot. Two set of experiments confirm the validity of the proposed control system. Data availability y All data generated during this study are included in this published article. The datasets used and analyzed du the current study available from the corresponding author on reasonable request. Received: 3 July 2023; Accepted: 8 November 2023 Received: 3 July 2023; Accepted: 8 November 2023 4. Lewis, M. A., Hartmann, M. J., Cummings, R. E. & Cohen, A. H. Control of a robot leg with an adaptive aVLSI CPG chip. Neuro- computing 38, 1409–1421 (2001). 3. Zhang, Y., Qiao, G. F., Qi, W., Tian, L. & Liu, D. A novel double-layered central pattern generator-based motion controller for the hexapod robot. Mathematics 11(617), 1–15 (2023). p g ( ) 5. Nessler, J. A., Minakata, K., Sharp, K. & Reinkensmeyer, D. J. Robot-assisted hindlimb extension increases the probability of swing initiation during treadmill walking by spinal cord contused rats. J. Neurosci. Methods 159(1), 66–77 (2007). g gi 2. Inagaki, S., Yuasa, H. & Arai, K. CPG model for autonomous decentralized multi-legged robot system generation and tran of oscillation patterns and dynamics of oscillators. Robot. Auton. Syst. 44(3–4), 171–179 (2003). Conclusions In this research, the model of CPG unit including motor dynamics parameters for quadruped robot has been proposed. An adaptive locomotive system based on the double-layered central pattern generator (CPG) intro- duced to improve the adaptability of the quadruped robot in multiple scenarios. The novel CPG network consists of double master units and subsets of slave units based on gyroscope signals, including yaw and pitch angle. To generate the regular oscillatory signals for the quadruped robot, the master and slave CPG models are mutually connected to each other to control the joints of each leg. In the master unit, by using coupling parameters, the Scientific Reports \| (2023) 13:19756 \| https://doi.org/10.1038/s41598-023-47022-x www.nature.com/scientificreports/ output of each neuron is directly connected to the slave neuron. The response of master units is able to control the 1st joins of the quadruped robot, while the slave units can generate the symmetry signals to control the 2nd and 3rd joints. The response of each neuron can generate the robot joint based on the stimuli of feedback parameters. Through adjusting the mutually dependent parameters of CPG networks, the joint angle can be controlled based on feedback information. For adaptive movement on an irregular surface, a stable range of the robot body pitch and yaw angle are proposed by using gyroscope signals. To detect the obstacle, the ultrasonic signals are utilized for adjusting the parameters. The double-layered CPG networks have a capability to generate the symmetry signals for stable movement on a complex surface. By using our proposed concept, the quadruped robot can be controlled by double-layered CPG network, while Figure 11. The response of FL, FR, HL, HR in the second experiment. Figure 12. The frame of second experiment. Figure 11. The response of FL, FR, HL, HR in the second experiment. Figure 11. The response of FL, FR, HL, HR in the second experiment. Figure 11. The response of FL, FR, HL, HR in the second experiment. Figure 12. The frame of second experiment. Figure 12. The frame of second experiment. Figure 12. The frame of second experiment. output of each neuron is directly connected to the slave neuron. The response of master units is able to control the 1st joins of the quadruped robot, while the slave units can generate the symmetry signals to control the 2nd and 3rd joints. www.nature.com/scientificreports/ Learning diverse motor patterns with a single multi-layered multi-pattern cpg for a humanoid obot in 2014 IEEE RAS International Conference on Humanoid Robots 1016 1021 (2014) 20. Khan, M. R., Billah, M. M. CPG-based control system for flexible snake robot locomotion. in 2014 IEEE International Symposium on Robotics and Manufacturing Automation (ROMA) 254–257 (2014).i f g 21. Yang, Y. Q., Wang, J. A. & Wu, Z. X. Fault-tolerant control of a CPG-governed robotic fish. Engineering 6, 861–868 (2018). 22. Asadi, F., Khorram, M., Moosavian, S. A. A. CPG-based gait transition of a quadruped robot. in 2015 3rd RSI International Confer- ence on Robotics and Mechatronics (ICROM) 210–215 (2015). 23. Teng, L., Wu, X., Chen, W., Wang, J. Center of gravity balance approach based on CPG algorithm for locomotion control of a quadruped robot. in 2013 IEEE/ASME International Conference on Advanced Intelligent Mechatronics 325–329 2013 (. q p f g 4. Gui, B., Wang, H., Chen, W. Stability analysis for a hexapod robot walking on slopes. in 2015 IEEE International Conference on Robotics and Biomimetics (ROBIO) 1888–1893 (2015). 5. Rostro, H., Cerna, P. A., Trejo, G., Capulin, C. & Huitzil, C. T. A CPG system based on spiking neurons for hexapod robot locomo tion. Neurocomputing 170, 47–54 (2015). p g 6. Qin, G., Wang, Z. L., Hu, W. J. & Zhao, L. Y. Gait simulation of snake robot based on CPG method. J Simul 27(6), 1374 (2015). 7. Yu, H. T. et al. Gait generation with smooth transition using CPG-based locomotion control for hexapod walking robot. IEEE Trans. Ind. Electron. 63(9), 5488–5500 (2016). 27. Yu, H. T. et al. Gait generation with smooth transition using CPG-based locomotion control for hexapod walking robot. IEEE Trans. Ind. Electron. 63(9), 5488–5500 (2016). 8. Yong, Z. & Yasuno, T. Adaptive walking control for quadruped robot on irregular terrain based on CPG network. J. Signal Process 15(4), 315–318 (2011). 28. Yong, Z. & Yasuno, T. Adaptive walking control for quadruped robot on irregular terrain based on CPG network. J. Signal Process. 15(4), 315–318 (2011). Competing interests h p g The authors declare no competing interests. Funding d Funding Funding was provided by Public Welfare Technology Application Research Project of Zhejiang Province grant number LGF22F030005 and “Pioneer” and “Leading Goose” R&D Program of Zhejiang Province grant number 2022C04012. Author contributions Author contributions Y.Z. and Y.Q. wrote the main manuscript text; Y.D. prepared the software of programming; B.H. and R.W. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Additional information Correspondence and requests for materials should be addressed to Y.Z. Correspondence and requests for materials should be addressed to Y.Z. References References 1. Zhao, W., Hu, Y. & Zhang, L. Design and CPG-based control of biomimetic robotic fish. IET 3(3), 281–293 (2009). y y 3. Zhang, Y., Qiao, G. F., Qi, W., Tian, L. & Liu, D. A novel double-layered central pattern generator-based motion controller for the hexapod robot. Mathematics 11(617), 1–15 (2023). 3. Zhang, Y., Qiao, G. F., Qi, W., Tian, L. & Liu, D. A novel double-layered central pattern generator-based motion controller for the hexapod robot. Mathematics 11(617), 1–15 (2023). p 4. Lewis, M. A., Hartmann, M. J., Cummings, R. E. & Cohen, A. H. Control of a robot leg with an adaptive aVLSI CPG chip. Neuro- computing 38, 1409–1421 (2001). 4. Lewis, M. A., Hartmann, M. J., Cummings, R. E. & Cohen, A. H. Control of a robot leg with an adaptive aVLSI CPG chip. Neuro- computing 38, 1409–1421 (2001). https://doi.org/10.1038/s41598-023-47022-x Scientific Reports \| (2023) 13:19756 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ 6. Huang, H. J. & Ferris, D. P. Computer simulations of neural mechanisms explaining upper and lower limb excitatory neural cou- pling. J. Neuroeng. Rehabil. 7(1), 1–13 (2010). p g g 7. Heliot, R. & Espiau, B. Multisensor input for CPG-based sensory motor coordination. IEEE Trans. Robot. 24(1), 195–191 (2008) . Tutsoy, O. CPG based RL algorithm learns to control of a huma ed RL algorithm learns to control of a humanoid robot leg. Int. J. R y g g 9. Spaeth, A., Tebyani, M., Haussler, D., Teodorescu, M. Neuromorphic closed-loop control of a flexible modular robot by a simulatett 9. Spaeth, A., Tebyani, M., Haussler, D., Teodorescu, M. Neuromorphic closed-loop control of a flexible modular robot by a simu spiking central pattern generator. in 2020 3rd IEEE International Conference on Soft Robotics (RoboSoft) 46-51 (2020).f 9. Spaeth, A., Tebyani, M., Haussler, D., Teodorescu, M. Neuromorphic closed-loop control of a flexible modular robot by a simulated spiking central pattern generator in 2020 3rd IEEE International Conference on Soft Robotics (RoboSoft) 46 51 (2020) 9. Spaeth, A., Tebyani, M., Haussler, D., Teodorescu, M. Neuromorphic closed loop control of a flexible modular robot by a simulated spiking central pattern generator. in 2020 3rd IEEE International Conference on Soft Robotics (RoboSoft) 46-51 (2020).f y p pl y ng central pattern generator. in 2020 3rd IEEE International Conference on Soft Robotics (RoboSoft) 46-51 (2020).f p g p g f ft 10. Jouaiti, M., Henaff, P. CPG-based controllers can trigger the emergence of social synchrony in human IEEE Workshop on Advanced Robotics and its Social Impacts (ARSO) 71–76 (2018). 0. Jouaiti, M., Henaff, P. CPG-based controllers can trigger the emergence of social synchrony in human-robot interactions. in 2018 IEEE Workshop on Advanced Robotics and its Social Impacts (ARSO) 71–76 (2018). p p 1. Wang, X. J., Zhang, Q., Zhang, Y. X., Wang, S. P. Rhythmic control method of a worm robot based on neural CPG. in 2018 13th IEEE Conference on Industrial Electronics and Applications (ICIEA) 1108–1112 (2018). g g g y e on Industrial Electronics and Applications (ICIEA) 1108–1112 (2 f pp 2. Shi, R. D., Zhang, X. L., Tian, Y. B., Dong, S. Y., Yao, Y. A. A CPG-based control method for the rolling locomotion of a desert spider. in 2016 IEEE Workshop on Advanced Robotics and its Social Impacts (ARSO) 243–248 (2016). 12. Shi, R. D., Zhang, X. L., Tian, Y. www.nature.com/scientificreports/ B., Dong, S. Y., Yao, Y. A. A CPG-based control method for the rolling loc spider. in 2016 IEEE Workshop on Advanced Robotics and its Social Impacts (ARSO) 243–248 (2016). 2. Shi, R. D., Zhang, X. L., Tian, Y. B., Dong, S. Y., Yao, Y. A. A CPG based control method for the rolling locomotion of a dese spider. in 2016 IEEE Workshop on Advanced Robotics and its Social Impacts (ARSO) 243–248 (2016). 3. Peng, L., Wang, C., Luo, L., Chen, S., Hou, Z. G., Wang, W. Q. A CPG-inspired assist-as-needed controller for an upp rehabilitation robot. in 2018 IEEE Symposium Series on Computational Intelligence (SSCI) 2200–2206 (2018). 14. Wang, Y. X., Zhu, A. B., Wu, H. L., Zhu, P. C., Zhang, X. D., Cao, G. Z. Control of lower limb rehabilitation exoskeleton robot based on CPG neural network in 2019 16th International Conference on Ubiquitous Robots (UR) 678 682 (2019) 14. Wang, Y. X., Zhu, A. B., Wu, H. L., Zhu, P. C., Zhang, X. D., Cao, G. Z. Control of lower limb rehabilitation exoskeleton robot 14. Wang, Y. X., Zhu, A. B., Wu, H. L., Zhu, P. C., Zhang, X. D., Cao, G. Z. Control of lower limb rehabilitation exos on CPG neural network. in 2019 16th International Conference on Ubiquitous Robots (UR) 678–682 (2019). 5. Donati, E., Indiveri, G., Stefanini, C. A novel spiking CPG-based implementation system to control a lamprey robot. in 2016 6th IEEE International Conference on Biomedical Robotics and Biomechatronics (BioRob) 1364–1364 (2016). 6. Oliveira, M., Matos, V., Santos, C. P., Costa, L. Multi-objective parameter CPG optimization for gait generation of a biped robot in 2013 IEEE International Conference on Robotics and Automation 3130–3135 (2013).i os, V., Santos, C. P., Costa, L. Multi-objective parameter CPG opti 7. Wang, W. & Xie, G. CPG-based locomotion controller design for a boxfish-like robot. Int. J. Adv. Robot. Syst. 11(87), 147–169 (2014).f 8. Zhang, X. L., Gong, J. Q. & Yao, Y. A. Effects of head and tail as swinging appendages on the dynamic walking performance of a quadruped robot. Robot 34(12), 2878–2891 (2016). q p 19. Debnath, S., Nassour, J., Cheng, G. Learning diverse motor patterns with a single multi-layered multi-pattern cpg for a hum robot. in 2014 IEEE-RAS International Conference on Humanoid Robots 1016–1021 (2014).l p Debnath, S., Nassour, J., Cheng, G. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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https://openalex.org/W4386027197	https://www.researchsquare.com/article/rs-2234670/latest.pdf	English	null	An efficient implementation of novel Polar encoder with Wavelet pipelining Architecture for 5G Networks	Research Square (Research Square)	2,023	cc-by	4,383	Albert Raj A (  albert@skcet.ac.in ) Sri Krishna College of Engineering and Technology https://orcid.org/0000-0002-4507-3524 vijila ebenezer Abstract The polar code is one of the error correcting codes which was proposed by Dr. Arıkan It attracts the attention of researchers due to the capacity achieving algorithms over a wide range of channels. However polar codes sacrifices encoding complexity and latency to improve error performance. To address this issue, wave pipelining is proposed in this article. The main advantage of pipelining is parallelism which leads to the improvement in latency. In addition, the system gets the benefit of the increased computation rate, or throughput. The absence of registers at the intermediate stages in the wave pipeline makes the polar code more power efficient and efficient area overheads. It provides a method for significantly reducing clock by keeping the external functionality and timing of any circuit. This architecture is coded in VHDL language and the design is synthesized in SYNOPSYS tool and implemented in the Virtex-7 VC 709 FPGA device. The synthesis report proved that 50 % of efficiency in area and timing in the wave pipelining compared to the conventional methods. The polar code is one of the error correcting codes which was pro attention of researchers due to the capacity achieving algorithms polar codes sacrifices encoding complexity and latency to improv issue, wave pipelining is proposed in this article. The main advant leads to the improvement in latency. In addition, the system gets t rate, or throughput. The absence of registers at the intermediate s polar code more power efficient and efficient area overheads. It pr reducing clock by keeping the external functionality and timing of in VHDL language and the design is synthesized in SYNOPSYS to 709 FPGA device. The synthesis report proved that 50 % of efficie pipelining compared to the conventional methods. Research Article Keywords: Polar codes, Encoder, Wave pipelining, VHDL, FPGA Posted Date: August 21st, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2234670/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/15 Page 1/15 1 INTRODUCTION Binary Memoryless Symmetric (BMS) channel otherwise known as Polar Codes [1] has a low-end complexity in terms of encoding and decoding. Perfect bit or noisy bit channels in a N channel polar code approaching infinity is based on the polarization phenomena of merging and splitting the channels thus assembling perfect bit channels of capacity I symmetric to the channels W. The channel W being an independent channel which is also a Binary Memoryless Symmetric channel, polarization happens on merging and splitting thereby transmitting data in the form of polar codes. This transmission is made possible through perfect bit channels that also promotes in working towards fixing frozen bits in channels that are completely noisy. According to Shannon’s coding theorem, several studies have worked together in achieving a noiseless channel transmission. Lately, on comparison with existing coding techniques, an advanced method to construct codes namely channel polarization was proposed [1–4], which aided to attain a symmetrical capacity of BDMC (Binary to Discrete Memoryless Channel) specifically BSC(p). Another study [5] has deconstructed the polar codes in BSC. [6–7] has characterized bounds and exponents in the construction of BSC for polar codes [8] has proposed a decoder based on LP (Linear Programming) that simulates differentiated decoders to hand out a SC (Successive Cancellation) Decoder and BEC at the same time. Bakshi et.al [9] has proposed a study to merge a concatenated RS(Reed – Solomon) code that uses inner codes and outer codes simultaneously. Polar codes are built to pick particular bit channels from N bits in [1] to [9] studies. Monte –Carlo Algorithms simulate complex channels [1] iteratively. [2] and [3] studies proposed that Polar codes work towards convoluting function but its metrics are not precise enough on complex calculations. Telatar et.al [4] has put forward approximation of bit channel with Nµ log 2 µ, where the user defined parameter, µ controls the output at an approximation stage. GA (Gaussian Approximation) uses Additive White Gaussian Noise (AWGN) to build polar codes in those channels proposed in [5–7]. Building Polar Codes on the basis of Polarization Weight (PW) does approximation of bit channels independently suggested by [8] and [9]. Code rates and code lengths need to be arbitrary which is achieved by the codes that are shortened and Page 2/15 Page 2/15 punctured are put forward in [10] to [17]. 1 INTRODUCTION Puncturing procedure is performed channel independently [10] to set a stopping point with the minimum bit value. These punctured bits are also known as decoders.[11] has proposed another puncturing algorithm which known as QUP (Quasi Uniform Puncturing) to puncture the bits that are in the reverse order from the initial value to P (P is the total number which indicates the number of bits to be punctured). Re-ordering mechanism followed by puncturing is suggested in [12] which makes use of GA methodology that punctures bits from positions that are frozen. Tal et.al [13] has used RCPP which optimizes the code family with unalike code rates. These conventional codes use the frozen bits that are all zero thus increasing the performance of decoders. Methodology to shorten the polar codes are suggested by [14] and [15] which generates the weights along the column of the matrices. A unique method that shortens the polar code is suggested in [16] which also punctured by a less complex construction method. An optimized algorithm with a pattern to shorten polar codes with a set of frozen codes is put forward in [17]. Nevertheless the pattern of the channels on being punctured and shortened, it is mandatory for re-arranging the channel bits based on the operations performed. Re-ordering mechanisms through GA is performed in AWGN channels so as to puncture the bits that are coded or shorten the bits that are coded. Selected codes that are nevermore in the underlying channel W undergoes puncturing and shortening that are parallel to the underlying channel. Original channel differ from the bit channel built from the underlying channel without undergoing the process of being punctured and shortened. Re- arranging of these bit channels helps to avoid performance distortion. Anyhow, GA is only pertinent to be used in AWGN channels. Advanced measures need to be performed to shorten the polar codes or to puncture the polar codes which is one inspiration that this article provides. Punctured or shortened coded bits aid in the building of polar codes, by considering the underlying channels to generalize the issue in which the BMS channels need to be independent regardless of the identical ones. The symmetric nature of polar codes proven in [1] vary the underlying channel in the new environment provided. The relationship based on Tal–Vardy’s algorithm is found to degrade when operated in such environment. 1 INTRODUCTION By analyzing a modified version Tal–Vardy’s algorithm [4] theoretically, the new environment becomes applicable for BMS channel that rearranges the bits in the underlying channels that could be unconstrained or different. Efficient FPGA Implementation of Chaotic based Communication System with Polar Encoding is proposed in [18]. In this article the parallel processing is discussed and the author claimed the latency reduction. Based on the theoretical analysis, a modification to the Tal–Vardy algorithm [4] that is applicable to any BMS channel is proposed to reorder the bit channels when the underlying channels are independent BMS channels (which, again, could be different channels). shortens the polar code is suggested in [16] which also punctured by a less complex construction method. An optimized algorithm with a pattern to shorten polar codes with a set of frozen codes is put forward in [17]. Nevertheless the pattern of the channels on being punctured and shortened, it is mandatory for re-arranging the channel bits based on the operations performed. Re-ordering mechanisms through GA is performed in AWGN channels so as to puncture the bits that are coded or shorten the bits that are coded. Selected codes that are nevermore in the underlying channel W undergoes puncturing and shortening that are parallel to the underlying channel. Original channel differ from the bit channel built from the underlying channel without undergoing the process of being punctured and shortened. Re- arranging of these bit channels helps to avoid performance distortion. Anyhow, GA is only pertinent to be used in AWGN channels. Advanced measures need to be performed to shorten the polar codes or to puncture the polar codes which is one inspiration that this article provides. Punctured or shortened coded bits aid in the building of polar codes, by considering the underlying channels to generalize the issue in which the BMS channels need to be independent regardless of the identical ones. The symmetric nature of polar codes proven in [1] vary the underlying channel in the new environment provided. The relationship based on Tal–Vardy’s algorithm is found to degrade when operated in such environment. By analyzing a modified version Tal–Vardy’s algorithm [4] theoretically, the new environment becomes applicable for BMS channel that rearranges the bits in the underlying channels that could be unconstrained or different. Efficient FPGA Implementation of Chaotic based Communication System with Polar Encoding is proposed in [18]. 1 INTRODUCTION In this article the parallel processing is discussed and the author claimed the latency reduction. Based on the theoretical analysis, a modification to the Tal–Vardy algorithm [4] that is applicable to any BMS channel is proposed to reorder the bit channels when the underlying channels are independent BMS channels (which, again, could be different channels). The rest of this paper is organized as follows. In Section 2, we briefly introduce the basics Encoding of polar codes. The general construction of Wave pipelining encoder procedure is presented in Section 3. The coding is done in VHDL language and the discussion of result is presented in Section 4 and this article ends with concluding remarks which is given in Section 5. 2 GENERAL ENCODER IMPLEMENTATION Page 3/15 2.1 Encoding of Polar code Polar codes are unique in the way they split the channel as good and bad channels, (during the encoding the polar code make the channel as reliable and unreliable) and the message bits are transmitted only through the induced good(reliable) channels, while the information to the unreliable channels are almost frozen. Let the length of the codeword is N, the messages are transmitted into k reliable channels then and (N − k) channels are unreliable and they are set to 0 as they are not useful for message transmission. This N-k bits are called frozen bits. Let W be a binary discrete memoryless channel (B-DMC) with u and x are the input and output respectively. When two bits (u1, u2) are transmitted, two values (x1, x2) are received (as shown in Fig. 1). The general equation for encoder for 2-bit value is given in (1). Let W be a binary discrete memoryless channel (B-DMC) with u and x are the input and output respectively. When two bits (u1, u2) are transmitted, two values (x1, x2) are received (as shown in Fig. 1). The general equation for encoder for 2-bit value is given in (1). . -----------------------------------------------------------------------(1) x2 1 = u2 1G2 . x2 1 = u2 1G2 Where G2 is the generator matrix and its value is Where G2 is the generator matrix and its value is ( ) 10 11 ( ) 10 11 For encoding 2-bit value (Fig. 1)using the above notation we obtain(2). For encoding 2-bit value (Fig. 1)using the above notation we obtain(2). For encoding 2-bit value (Fig. 1)using the above notation we obtain(2). x1 = (u1 ⊕ u2) and x2 = u2 ---------------------------------------------------(2) Similarly for encoding N = 4 bits (Fig. 2) we need to use the matrix G4. We obtain (3) Similarly for encoding N = 4 bits (Fig. 2) we need to use the matrix G4. We obtain (3) -----------------------------------------------------------------(3) x4 1 = u4 1G4 - x4 1 = u4 1G4 --(3) In this case we have x1 = (u1⊕u2⊕u3⊕u4), x2 = (u3⊕u4), x3 = (u2⊕u4) and x4 = u4. In the same way we can obtain ‘n’ bit value encoder. To encode more bits, this kind of process may sacrifice complexity and latency. That may not be desirable for a high-speed data network like 5G. 3.2 Wave Pipeline Design for Polar Code A maximum-rate pipeline is not achievable using conventional pipelining so only wave pipleining is proposed in this article. However, by removing the intermediate registers within a pipeline, a wave pipeline becomes a close realization of a maximum-rate pipeline. Wave pipelining in Polar encoder can reduce the clock load of a circuit as well as the associated area and power while maintaining the timing of the circuit. Another difference between conventional and wave pipelines is the relationship between clock rate and path delay. In the conventional 4 bit encoding method the system uses 10 intermediate registers. Obviously 10 clock cycle were required to move the inputs to the output. Where as in the wave pipelining method the number of tasks (xor operation) are executed in parallel (computed concurrently). Thus, minimum number of clock cycles are expected here for a wave to propagate through the wave pipeline before being latched by the output register. In addition, by removing intermediate registers in this process wave pipelining reduced the clock load of a circuit as well as the associated area and power while maintaining the timing of the circuit. 3.1 Wave Pipelining A fundamental and well explored concept used in the design of digital systems is pipelining. Pipelining is a popular hardware design technique that takes advantage of parallelism. The key benefit of utilizing a pipeline design is the increased computation rate, or throughput, available. For a computational task, throughput is typically increased by reducing the time at which it takes to complete said task. Pipelining Page 4/15 increases throughput by increasing the number of tasks that can be executed in parallel and therefore increasing the number of tasks that can be computed concurrently, otherwise known as increasing bandwidth. increases throughput by increasing the number of tasks that can be executed in parallel and therefore increasing the number of tasks that can be computed concurrently, otherwise known as increasing bandwidth. The throughput of a pipelined system is determined by the maximum rate at which the pipeline can operate. In this model, the maximum clock rate is limited by the maximum path delay difference across a combinational logic rather than the maximum path delay. Six stage pipeline is explained in Fig. 4 there are 6, tasks processing concurrently in this design. The latency for each task is 6 clock cycles, but as previously explained, the latency between outputs is only 1 clock cycle. 4. Implementation Results In this proposed research work, the architectural design of Wave pipeline Polar codes is implemented using Xilinx 9.2i ISE and Model Sim 6.3. The program was written in VHDL coding and the encoded output is shown Fig 5. The WP – Polar architecture is also synthesized in both Xilinx and Synopsys tools. The above implementation provides us a detailed analysis on area and power consumptions. WP – Polar reduces the area overheads and reduced power dissipation due to the absence of intermediate registers. In the conventional 4 bit encoding method (Fig 6) the system uses 10 intermediate registers. Obviously 10 clock cycle were required to move the inputs to the output. In the wave pipelining method the number of tasks (xor operation) were executed in parallel (computed concurrently). Thus, minimum number of clock cycles were expected here for a wave to propagate through the wave pipeline before being latched by the output register(fig 7). Due to absence of the intermediate register and pipeline stages the reduce in the static and dynamic power hence SoC power gets reduced. Here latency is calculated by the time taken by any encoder design to generate its output after the clock signal is applied. Table 1 provides the device utilization summary of Page 5/15 two, three stage and Wave pipelining is given. As the stages of pipe-line increases, the incremental gate count and number of flip flops also increases due to the intermediate registers. Gate count and flip flops have increased from 794 in 2-stage pipeline to 1702 in 3-stage pipeline. where as it has been observed that there is a significant reduction in number of flip flops and gate count used for wave pipelining. As the table showcase, efficient utilization of equivalent gate count and the number of slices is achieved by wave pipelined architecture with a count of 302 and 12 respectively. The gate count increases with the increase in the number of pipeline stages because of the insertion of additional registers. Table 1: Device Utilization summary(area) The bar chart (Fig. 8) is plotted considering device utilization without pipeline and with different pipeline stages. Table 1: Device Utilization summary(area) The bar chart (Fig. 8) is plotted considering device utilization without pipeline and with different pipeline stages. The bar chart (Fig. 8) is plotted considering device utilization without pipeline and with different pipeline stages. The summary of timing analysis is given in Table 2. 4. Implementation Results Here the timing analysis is based on the maximum time required by the output calculated in nanoseconds (ns). The time required by the two stages and three stage encoders is 4.89 ns and 4.04 ns respectively. The time required by the wave pipelined architecture is 3.939 ns. In normal pipelining stages, intermediate data are stored in registers, whereas in wave pipelining the data are handled by delays. Table 2: Summary of Timing Analysis Table 2: Summary of Timing Analysis Table 2: Summary of Timing Analysis Table 2: Summary of Timing Analysis The architecture of wave pipelining was synthesized using Synopsys where utilization of area and power was obtained. Fig 9 indicates the cell area utilization of pipe-lined and wave pipelined architecture. Due to the absence of intermediate registers in the pipelined stages, Cell area utilized by wave pipelined Polar encoder is much lesser compared to other pipelined architectures. Fig 10 shows the cell area utilization of (8,4) Polar Encoder, where the area is measured in μm2 . The total cell area occupied by normal design, two stage, three stage and wave pipelining are 43,660.715 μm2 The below chart showcases the power Utilization of different pipelined architecture of Polar encoder. Analysis is made using Synopsys tool which is measured in milliwatt (mW) (Fig. 10). Page 6/15 The power utilized by normal design, two stage and three stage pipelines are 4.125 mW, 5.05 mW and 5.9 mW respectively, which is effectively reduced by using wave pipelined structure and the power utilized is 3.8 mW. Field-Programmable Gate Array (FPGAs) is explicitly intended to address the issues of high volume, cost-delicate purchaser electronics applications. The five-part family offers densities going from 100,000 to 1.6 million framework gates. The Spartan-3E family. expands on the accomplishment of the prior spartan 3E family by expanding the measure of rationale per I/O, essentially reducing the expense per logic cell. New highlights improve framework execution and diminish the expense of arrangement. These Spartan-3E FPGA improvements, joined with cutting edge 90 nm measure innovation, convey more usefulness and transfer speed per dollar than already conceivable, setting new guidelines in the programmable logic industry. The Spartan 3E family is a better option than mask modified ASICs. This result is obtained from the synthesis report from Synopsys. Declarations Conflicts of interest The first author of the article, on his behalf and that of all the authors, declares that there is no potential conflict of interest related to the article. Funding: No funding was received Data Availability Statement All data generated or analyzed during this study are included in this published article Data Availability Statement All data generated or analyzed during this study are included in this published article 5 CONCLUSIONS In the article, wave pipelined architecture for Polar encoder is introduced. The same is simulated using Modelsim tool and synthesized using both Synopsys and Xilinx. After the synthesis, the module is targeted to Virtex-7 VC 709 FPGA device. The test reports of the Synopsys, proved that the power dissipation area for wave pipelined architecture is more efficient compared to Conventional and other wave pipelining architectures. The absence of intermediate registers in the wave pipeline made the polar code more power efficient and efficient area overheads. It provides a method for significantly reducing clock by keeping the external functionality and timing of any circuit. The synthesis report proved that 50% of efficiency in area and Latency in the wave pipelining compared to the conventional methods. References 1. Arıkan E. Channel polarization: a method for constructing capacity achieving codes for symmetric binary-input memory less channels. IEEE Transactions on Information Theory, 2009, 55(7): 3051−3073 2. Arıkan E. A performance comparison of polar codes and Reed-Muller codes.IEEE communications Letters, 2008, 12(6): 447−449 Page 7/15 Page 7/15 3. Şaşoğlu E, Telatar E, Arikan E. Polarization for arbitrary discrete memoryless channels. Proceedings of the 2009 IEEE Information Theory Workshop on Networking and Information Theory (ITW’09), Jun 10−12, 2009, Volos, Greece. Piscataway, NJ, USA: IEEE, 2009: 144−148 4. Telatar E. Polar coding: a brief tour. Proceedings of the 2010 International Conference on Signal Processing and Communications (SPCOM’10), Jul 18−21, 2010, Lausanne, Switzerland. Piscataway, NJ, USA: IEEE, 2010: 2p 5. Mari R, Tanaka T. Performance and construction of polar codes on symmetric binary-input memoryless channels. Proceedings of the IEEE International Symposium on Information Theory (ISIT’09), Jun 28−Jul 3, 2009, Seoul, Republic of Korea. Piscataway, NJ, USA: IEEE, 2009:−1500 6. Korada S B, Şaşoğlu E, Urbanke R. Polar codes: characterization of exponent, bounds, and constructions. IEEE Transactions on Information Theory, 2010, 56(12): 6253−6264 7. Korada S B. Polar codes for channel and source coding. doctoral dissertation.Ph D Thesis Lausanne, Switzerland: Ecole Polytechnique Fédérale de Lausanne (EPFL), 2009 8. Goela N, Korada S B, Gastpar M. On LP decoding of polar codes. Proceedings of the 2010 IEEE Information Theory Workshop on Networking and Information Theory (ITW’10), Aug 30−Sept 3, 2010, Dublin, Ireland. Piscataway, NJ, USA: IEEE, 2010: 5p 9. Bakshi M, Jaggi S, Effros M. Concatenated polar codes. Proceedings of the IEEE International Symposium on Information Theory (ISIT’10), Jun 13−18, 2010, Pasadena, CA, USA. Piscataway, NJ, USA: IEEE, 2010:918−922. 10. E. Arikan, “Channel polarization: A method for constructing capacityachieving codes for symmetric binary-input memoryless channels,” IEEE Trans. Inf. Theory, vol. 55, no. 7, pp. 3051–3073, Jul. 2009. 11. R. Mori and T. Tanaka, “Performance and construction of polar codes on symmetric binary-input memoryless channels,” in Proc. IEEE Int. Symp. Inf. Theory, Jun. 2009, pp. 1496–1500. 12. R. Mori and T. Tanaka, “Performance of polar codes with the construction using density evolution,” IEEE Commun. Lett., vol. 13, no. 7, pp. 519–521, Jul. 2009. 13. I. Tal and A. Vardy, “How to construct polar codes,” IEEE Trans. Inf. Theory, vol. 59, no. 10, pp. 6562– 6582, Oct. 2013. 14. P. Trifonov, “Efficient design and decoding of polar codes,” IEEE Trans. Commun., vol. 60, no. 11, pp. Figures Figures Page 9/15 g Figure 1 Polar encoding for 2-bit data 18. Y. Zhou, R. Li, H. Zhang, H. Luo, and J. Wang, “Polarization weight family methods for polar code construction,” in Proc. IEEE 87th Veh. Technol. Conf. (VTC Spring), Jun. 2018, pp. 1–5. 19. A. Eslami and H. Pishro-Nik, “A practical approach to polar codes,” in Proc. IEEE Int. Symp. Inf. Theory Proc., Jul. 2011, pp. 16–20. 20. K. Niu, K. Chen, and J.-R. Lin, “Beyond turbo codes: Rate-compatible punctured polar codes,” in Proc. IEEE Int. Conf. Commun. (ICC), Jun. 2013, pp. 3423–3427. [12] L. Zhang, Z. Zhang, X. Wang, Q. Yu, and Y. Chen, “On the puncturing patterns for punctured polar codes,” in Proc. IEEE Int. Symp. Inf. Theory, Jun. 2014, pp. 121–125. References 3221–3227, Nov. 2012. 15. J. Dai, K. Niu, Z. Si, C. Dong, and J. Lin, “Does Gaussian approximation work well for the long-length polar code construction?” IEEE Access, vol. 5, pp. 7950–7963, Apr. 2017. 16. D. Wu, Y. Li, and Y. Sun, “Construction and block error rate analysis of polar codes over AWGN channel based on Gaussian approximation,” IEEE Commun. Lett., vol. 18, no. 7, pp. 1099–1102, Jul. 2014. 17. G. He et al., “Beta-expansion: A theoretical framework for fast and recursive construction of polar codes,” in Proc. GLOBECOM-IEEE Global Commun. Conf., Dec. 2017, pp. 1–6. Page 8/15 Page 8/15 18. Y. Zhou, R. Li, H. Zhang, H. Luo, and J. Wang, “Polarization weight family methods for polar code construction,” in Proc. IEEE 87th Veh. Technol. Conf. (VTC Spring), Jun. 2018, pp. 1–5. 19. A. Eslami and H. Pishro-Nik, “A practical approach to polar codes,” in Proc. IEEE Int. Symp. Inf. Theory Proc., Jul. 2011, pp. 16–20. 20. K. Niu, K. Chen, and J.-R. Lin, “Beyond turbo codes: Rate-compatible punctured polar codes,” in Proc. IEEE Int. Conf. Commun. (ICC), Jun. 2013, pp. 3423–3427. [12] L. Zhang, Z. Zhang, X. Wang, Q. Yu, and Y. Chen, “On the puncturing patterns for punctured polar codes,” in Proc. IEEE Int. Symp. Inf. Theory, Jun. 2014, pp. 121–125. Figure 1 Polar encoding for 2-bit data Page 9/15 Figure 2 Polar encoding scheme for n=4 Page 10/15 Figure 2 Polar encoding scheme for n=4 Figure 3 General wave pipeline circuit model Figure 2 Polar encoding scheme for n=4 Figure 3 General wave pipeline circuit model General wave pipeline circuit model Page 10/15 Page 11/15 Figure 4 Six stage pipelining Figure 4 Six stage pipelining Six stage pipelining Page 11/15 Figure 5 VHDL Simulation output for Encoder using Modelsim Tool VHDL Simulation output for Encoder using Modelsim Tool Figure 10 Comparative Analysis of Power Dissipation Encoder(8,4)- Synopsis Tool Comparative Analysis of Power Dissipation Encoder(8,4)- Synopsis Tool Comparative Analysis of Power Dissipation Encoder(8,4)- Synopsis Tool Figure 6 Conventional Polar encoding synthesis report Page 12/15 gure 7 Figure 7 Wave pipelined Polar encoding synthesis report Wave pipelined Polar encoding synthesis report Page 13/15 Figure 8 Comparative Analysis of Area Utilization. Figure 9 Cell area utilization of pipe-lined and wave pipelined architecture Figure 8 Comparative Analysis of Area Utilization. Comparative Analysis of Area Utilization. Page 14/15 Figure 9 Cell area utilization of pipe-lined and wave pipelined architecture Cell area utilization of pipe-lined and wave pipelined architecture Figure 10 Comparative Analysis of Power Dissipation Encoder(8,4)- Synopsis Tool Supplementary Files Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. AuthorsBiography.docx AuthorsBiography.docx Page 15/15
https://openalex.org/W4303089650	http://deepblue.lib.umich.edu/bitstream/2027.42/78270/2/1471-2431-9-81.pdf	English	null	Economic and other barriers to adopting recommendations to prevent childhood obesity: results of a focus group study with parents	Deep Blue (University of Michigan)	2,010	cc-by	5,754	Open Research article Economic and other barriers to adopting recommendations to prevent childhood obesity: results of a focus group study with parents Kendrin R Sonneville1,2, Nancy La Pelle3, Elsie M Taveras2,4,5, Matthew W Gillman1,2,4,5 and Lisa A Prosser6 Open Access Address: 1Department of Nutrition, Harvard School of Public Health, Boston, MA, USA, 2Children's Hospital, Boston, Boston, MA, USA, 3Preventive and Behavioral Medicine, University of Massachusetts Medical School, Worcester, MA, USA, 4Obesity Prevention Program, Department of Population Medicine, Harvard Medical School and Harvard Pilgrim Health Care, Boston, MA, USA, 5Division of General Pediatrics, Children's Hospital Boston, Boston, MA, USA and 6Child Health Evaluation and Research Unit, Division of General Pediatrics, University of Michigan Health System, Ann Arbor, MI, USA Email: Kendrin R Sonneville - ksonnevi@hsph.harvard.edu; Nancy La Pelle - Nancy.LaPelle@umassmed.edu; Elsie M Taveras - Elsie_Taveras@harvardpilgrim.org; Matthew W Gillman - Matthew_Gillman@harvardpilgrim.org; Lisa A Prosser - lisapros@med.umich.edu * Corresponding author Received: 5 March 2009 Accepted: 21 December 2009 Published: 21 December 2009 BMC Pediatrics 2009, 9:81 doi:10.1186/1471-2431-9-81 Published: 21 December 2009 BMC Pediatrics 2009, 9:81 doi:10.1186/1471-2431-9-81 This article is available from: http://www.biomedcentral.com/1471-2431/9/81 © 2009 Sonneville et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2009 Sonneville et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cit BioMed Central BioMed Central BioMed Central Background weight is linked to mothers entering the workforce, but only found an effect for upper income families [20]. A possible explanation for this perplexing result is that lower income mothers may be too time-constrained to focus on their child's nutrition and physical activity whether or not they work outside the home. This supports the hypothesis that economic constraints (time and dol- lar) may be especially pertinent to families with children at higher risk for obesity, those of lower socio-economic status. g Childhood obesity is a prominent public health concern that is associated with an increased risk of cardiovascular disease, diabetes, some cancers, depression, discrimina- tion and weight-related bias, and other morbidities [1]. The short- and long-term risks to both physical and psy- chological health of childhood obesity translate into a reduced quality of life [2-5]. As such, intervening early may be necessary to reduce immediate consequences of childhood obesity and engender healthy behaviors before obesogenic lifestyle patterns are deeply established [6,7]. Obesity prevention and management recommendations for children involve changes in parental behavior. As such, understanding parental decision-making related to adopting recommendations is critical and could inform future efforts to reduce childhood obesity. For example, information about parental barriers and facilitators could lead to obesity interventions that are tailored to maximize parental compliance. Parents control many aspects of the nutrition and physical activity environment that contribute to a child's health- related behaviors [8-10]. However, our understanding of the range of barriers parents face when adopting recom- mendations to prevent childhood obesity, and the relative importance of these barriers, is incomplete. Focus group studies with parents have identified barriers to adopting recommendations to increase physical activity which include weather, siblings, finances, time, neighborhood safety, child's preferences for sedentary activities, lack of affordable and accessible recreation facilities, and low car- egiver motivation [11-14]. Findings from two focus group studies suggest that parents believe the external environ- mental and social barriers hold responsibility for some of children's unhealthful eating habits, beliefs which may impact parental receptiveness to interventions aimed at the family level [15,16]. The objective of this study was to explore, through the use of qualitative methods, barriers and facilitators that influ- ence parenting behaviors and decisions that relate to child food choices, activities, and other behaviors that could affect a child's risk of obesity. Background We focused especially on economic barriers, including both time and dollar costs, an understudied yet potentially salient barrier. Focus groups are conducive to identifying constructs of potential interest for future research and in determining whether non-economic barriers generate as much discussion as economic ones. Economic considerations, such as the high cost of nutri- tious foods and affordability of junk foods and fast foods, are commonly assumed determinants of food choice, in part because low-income families exhibit among the high- est prevalence of childhood obesity in the U.S. [17]. In considering economic barriers, however, it is important to consider both out-of-pocket dollar costs and time costs. For example, reducing fast food meals could reduce food costs but would require additional time for food prepara- tion (a decrease in dollar costs but an increase in time costs). If a child enrolled in a sports program, a parent may be required to pay enrollment fees or purchase equip- ment, as well as invest in additional transportation time (an increase in both dollar costs and time costs). Page 2 of 7 (page number not for citation purposes) Abstract Background: Parents are integral to the implementation of obesity prevention and management recommendations for children. Exploration of barriers to and facilitators of parental decisions to adopt obesity prevention recommendations will inform future efforts to reduce childhood obesity. Methods: We conducted 4 focus groups (2 English, 2 Spanish) among a total of 19 parents of overweight (BMI ≥ 85th percentile) children aged 5-17 years. The main discussion focused on 7 common obesity prevention recommendations: reducing television (TV) watching, removing TV from child's bedroom, increasing physically active games, participating in community or school-based athletics, walking to school, walking more in general, and eating less fast food. Parents were asked to discuss what factors would make each recommendation more difficult (barriers) or easier (facilitators) to follow. Participants were also asked about the relative importance of economic (time and dollar costs/savings) barriers and facilitators if these were not brought into the discussion unprompted. Results: Parents identified many barriers but few facilitators to adopting obesity prevention recommendations for their children. Members of all groups identified economic barriers (time and dollar costs) among a variety of pertinent barriers, although the discussion of dollar costs often required prompting. Parents cited other barriers including child preference, difficulty with changing habits, lack of information, lack of transportation, difficulty with monitoring child behavior, need for assistance from family members, parity with other family members, and neighborhood walking safety. Facilitators identified included access to physical activity programs, availability of alternatives to fast food and TV which are acceptable to the child, enlisting outside support, dietary information, involving the child, setting limits, making behavior changes gradually, and parental change in shopping behaviors and own eating behaviors. Conclusions: Parents identify numerous barriers to adopting obesity prevention recommendations, most notably child and family preferences and resistance to change, but also economic barriers. Intervention programs should consider the context of family priorities and how to overcome barriers and make use of relevant facilitators during program development. Page 1 of 7 (page number not for citation purposes) (page number not for citation purposes) BMC Pediatrics 2009, 9:81 http://www.biomedcentral.com/1471-2431/9/81 Barriers The barriers most frequently voiced regarding reducing time watching TV related to monitoring/time costs. After being asked what their children do when they are not watching TV at home, 2 parents in one focus group responded "driving me crazy" and one parent responded "fighting with his little sister". The barriers most fre- quently voiced regarding removing TVs from a child's bed- room related to difficulty with changing habits ("They've had TV since they've had their own room") and parity with other family members ("For me it is kind of hard for me to take the TV out of her bedroom when I have one in my bedroom, you know.") We completed a topic by topic thematic analysis across focus groups within each population segment, defined as English-speaking and Spanish-speaking. Comparison tables with six columns, one for theme code, one for theme description, and one each for the four focus groups, were created and notations were made whenever one or more speakers in each focus group voiced a specific theme. After completing these notations for both English and Spanish speaking focus groups, themes were re- ordered for each focus group major theme in the compar- ison table such that subthemes that were voiced more fre- quently occur in an earlier row of the table. For each theme grouping across the four focus groups, we com- pleted a comparative analysis and comparative cross-pop- ulation segment thematic analysis. The barriers most frequently voiced regarding increasing physical activity related to time costs ("I have a hard time with extracurricular sports activities and homework, because I felt like I couldn't do it all"; "I would have to make compromises in my life, to be able to, say, put him into lacrosse, which is a fabulous activity and I'd love to do it, but I just can't figure out how to do that... timewise. http://www.biomedcentral.com/1471-2431/9/81 http://www.biomedcentral.com/1471-2431/9/81 http://www.biomedcentral.com/1471-2431/9/81 http://www.biomedcentral.com/1471-2431/9/81 A professional focus group facilitator (non-physician), who was bilingual in Spanish and English, conducted all four focus groups. The group facilitator used a semi-struc- tured interview guide for the focus groups that began with an ice-breaker activity and a brief warm-up exercise. The main discussion focused on common obesity prevention recommendations selected by the authors: reducing TV watching time, removing TV from child's bedroom, increasing time spent playing physically active games, par- ticipating in community or school-based athletics, walk- ing to and from school, walking more in general, and eating fewer fast food meals. Parents were asked to assess how difficult or easy each type of advice was for them to follow and to discuss what factors would make each type of advice more difficult (barriers) or easier (facilitators) to implement. Participants were also asked about the relative importance of economic (time and dollar costs/savings) barriers and facilitators if these were not brought into the discussion unprompted. During data reduction, a table was created with one col- umn per each of the four focus groups and one row for each theme code. In each cell, the number of participants within each focus group who explicitly mentioned that particular theme or subtheme in a similar way was recorded along with the participants' initials so that mul- tiple similar responses from the same participant were not overcounted. Themes could then be prioritized by fre- quency of mentions both within and across focus groups. Results When asked about the importance of healthy weight com- pared to other childhood health issues during the warm- up exercise, some parents felt that child safety and aca- demic performance were of higher priority than obesity prevention. Parents went on to identify various barriers and facilitators to adopting obesity prevention recom- mendations throughout the focus group discussion (Table 1). Although "providing a healthier diet" was not one of the recommendations posed by the moderator explicitly, it was raised by participants themselves as advice they often hear. Barriers and facilitators to providing a health- ier diet voiced by parents are also included in Table 1. We audiotaped and transcribed all focus groups to facili- tate analysis and translated Spanish groups into English during transcription. Data were then transcribed into a Microsoft Word table with columns for focus group iden- tifier, theme code, speaker ID, speaker comment and chronological sequence number [21]. We conducted qual- itative analysis using the template organizing style [22- 24]. The qualitative research analyst (NL) read all tran- scripts in detail and constructed a theme codebook that documented themes and subthemes in a logical hierarchy and assigned a numerical theme code to each theme and subtheme to facilitate sorting. Page 3 of 7 (page number not for citation purposes) Methods f We held four focus groups in Boston, MA, during October and November 2007: two in English and two in Spanish. We recruited parents of overweight children (BMI-for-age ≥ 85th percentile) aged 5-17 years attending the Preven- tive Cardiology clinic at Children's Hospital Boston or one of two weight management clinics at the hospital, One Step Ahead and Optimal Weight for Life (n = 19). Three pairs of the English-speaking participants in one focus group were married partners; therefore, data relate to 16 children. Focus group size ranges from 2 to 7 par- ents. Additional demographic data about the parent or the family were not collected at the focus groups. An emerging literature is focusing on the economics of obesity in adults, but there has been less focus on the eco- nomics of childhood obesity. MacInnis & Rausser suggest a role for energy-dense processed foods, which have become less expensive, in contributing to childhood obes- ity or risk of obesity [18]. A related theme of the impact of technological change in food production on adult obesity has been explored by Cutler et al. [19]. Anderson et al. examined whether the likelihood of a child being over- Focus groups lasted two hours. Parents received a $40 gift card incentive for participating in the focus groups. Din- ner was provided at each focus group, as was a parking or taxi voucher. Participants completed written informed consent prior to the start of the focus group. The study protocol was approved by the Institutional Review Boards of Children's Hospital Boston and Harvard Pilgrim Health Care. Page 2 of 7 (page number not for citation purposes) Page 2 of 7 (page number not for citation purposes) BMC Pediatrics 2009, 9:81 BMC Pediatrics 2009, 9:81 Barriers The barrier most fre- quently voiced related to participating in school/commu- nity-based sports was child preference ("Football, hockey and baseball are... very popular. He doesn't like them. He doesn't have an interest in joining a group team."). but all of the equipment involved."), and the need for assistance from family members (parents lack help from extended family to get children to locations where they could engage in physical activity). The barrier most fre- quently voiced related to participating in school/commu- nity-based sports was child preference ("Football, hockey and baseball are... very popular. He doesn't like them. He doesn't have an interest in joining a group team."). ily members ("Nana is going to give them whatever they want. Soda. Because I don't give my kids soda... And, I mean, she sneaks it to them."), child preference ("Going to McDonalds is like a treat for them. She loves to go to McDonalds... It's like the most important thing to go to McDonald."), and time costs ("It is difficult for me to eat fewer fast food meals because I work two shifts, so it's like easier for me to buy them something to eat."). The barriers most frequently voiced regarding providing a healthier diet included parity with other family members ("If I feel like having junk food, she's going to have it too. I can't say that I'm going to have it for myself and not let her have it"; " For me it is a little difficult because my 19- year-old daughter is very skinny. So my problem is they each have their own list. This one wants one thing and the other wants something else."), dollar costs ("When you have a large family to feed, you know, doctor's advice is "eat healthy", but everything that's healthy is very expen- sive."), and difficulty with changing habits (hard to change child's whole diet at once). The barriers most frequently voiced regarding walking to school related to practicality/time cost ("School is about 15 miles away, and the bus stop is only around the corner from our house"; "His school is actually like 2 minutes away, for a start. I'm on my way to work and I just drop him off, so it's not a necessity for him to walk.") and safety ("Going down a busy road is too dangerous for them. Barriers It takes a lot of my time."), lack of information (parents lack information about locations where they could engage in physical activity), lack of transportation ("If there was transportation where I live, I would get on the bus and go to a playground with my girls."), child preference ("She doesn't like to play like basketball, nothing like that."), safety ("If he's home playing video games as opposed to being out, I almost feel like he's safer even though, prob- ably, mentally it's not"), dollar costs ("Hockey is very expensive, football is very expensive, you know, in terms of the equipment. Maybe not so much in terms of the fee, Validation was done after preliminary coding. All simi- larly coded text was reviewed to ensure that text that was not representative of the category definition was not included. Where errors were detected, the inappropriate text was recoded as an instance of the more appropriate theme and regrouped with text representing the corrected theme for data reduction. Page 3 of 7 (page number not for citation purposes) Page 3 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2431/9/81 BMC Pediatrics 2009, 9:81 BMC Pediatrics 2009, 9:81 Table 1: Barriers and facilitators to adopting obesity prevention recommendations cited by parents Obesity Prevention Recommendation Barrier Facilitator Reduce time watching TV Monitoring/Time cost Availability of acceptable alternatives Remove TV from child's bedroom Difficulty with changing habits Parity with other family members Early limit setting Increase physical activity Time cost Lack of information Lack of transportation Child preference Safety Dollar cost Need assistance from family members Availability of acceptable alternatives Enlist outside support Participate in school/community-based sports Practicality/Time cost Child preference Access Walk to school Safety None identified Walk more in general Child preference None identified Eat less fast food Need assistance from family members Child preference/Convenience Time cost Information about dollar costs Parental behavior change Provide a healthier diet Parity with other family members Dollar cost Child preference Difficulty with changing habits Parental behavior change Involve the child Limit setting Make behavior changes gradually e 1: Barriers and facilitators to adopting obesity prevention recommendations cited by parents but all of the equipment involved."), and the need for assistance from family members (parents lack help from extended family to get children to locations where they could engage in physical activity). Barriers Just the security, of someone driving by and being able to grab them, I wouldn't let them do it. And if I have them taking the bus, I know where they are before and after school."). The barrier most frequently voiced regarding walking more in general was child preference ("She doesn't like to walk... She likes her computer."). Facilitators The barriers most frequently voiced regarding eating less fast food related to the need for assistance from other fam- In general, far fewer facilitators than barriers were identi- fied for the obesity prevention recommendations. The Page 4 of 7 (page number not for citation purposes) Page 4 of 7 (page number not for citation purposes) BMC Pediatrics 2009, 9:81 http://www.biomedcentral.com/1471-2431/9/81 facilitator most frequently voiced for reducing time watch- ing TV was the availability of alternative activities. For the recommendation to remove TV from child's bedroom, the most frequently voiced facilitator related to early limit set- ting (tell child they cannot have a TV before they become teenagers). The facilitators most frequently voiced regard- ing increasing physical activity included availability of acceptable alternatives (new active video games that pro- mote exercise inside; make physical activity inside the house more fun) and enlisting outside support (connect- ing children with Big Brothers or Big Sisters who might role model being physically active). The most frequent facilitator about increasing participation in school/com- munity-based sports was access (need more easy-access community centers/local programs that provide physical activity opportunities). For reducing fast food, facilitators most frequently voiced included information about dollar costs (realizing that it costs less to cook a meal than to go out to eat) and parental behavior change (parents have to stop taking kids to fast food restaurants and buying fast food as takeout). The facilitators related to providing a healthier diet included parental behavior change ("I try not to buy like chips and stuff like that, because I know if it's there she's going to eat it."; "I've had bad eating habits for some time. I have to change my lifestyle and my way of eating in order to - and, like I said, I have made some changes, but there are things that I still need to work on."), involve the child (explain to child why diet needs to be changed and present with healthy options), limit setting ("I don't give them options."), and making behav- ior changes gradually ("Well they wanted me to stop eve- rything at once, but I just took it one at a time. The first step was we stopped all the fried foods. Then we started eating more vegetables."). conjunction with other barriers ("They recommended a few [cereals] she should be eating. It's more expensive. My daughter hates it. She says it's horrible."). Facilitators We asked parents in two focus groups how their lives would have to change in order to implement nutrition and physical activity recommendations. Changes that would necessitate time costs were identified. These included cutting back on work/study time to allow par- ents who worked or attended school to be home more to monitor child eating behaviors and spend more time with them ("For me, I also was thinking about making change in my job, so I can be home - to know that I get off at 5 o'clock... So then I can be home to monitor exactly what they eat. I don't know - if I'm at work until 8 o'clock, I don't know if they - they could be up eating M & Ms."). Cultural Issues Related to Weight and Food Parents in all groups voiced cultural or inherited family beliefs/behaviors about food. Parents in the Spanish- speaking groups indicated that country of origin may influence food choices. Cultural issues regarding the per- ceptions of weight were voiced only in the Spanish-speak- ing groups. These included beliefs that being fat or large is "pretty" and that thinness is related to sickness. Parents in the Spanish-speaking groups said that grandparents don't believe that excess weight is unhealthy ("In my house the fatter one was the prettier one."). Parents identified time cost barriers such as time required for transportation, to prepare healthy foods, to allow for participation in physical activity programs, and for behav- ior monitoring, more frequently than dollar cost barriers. Although parents identified dollar costs as barriers in all groups, they were not cited as the most important barriers. This may reflect the relative importance of dollar cost bar- riers or may have resulted because discussions of finances may be considered too sensitive or personal to be brought Discussion Parents in this study identified numerous barriers and some facilitators to adopting obesity prevention recom- mendations. Barriers such as child resistance due to pref- erence for existing behaviors and family member-related barriers were common. Parents acknowledged that expect- ing all family members to adopt recommendations was a considerable obstacle. Parents cited difficulties related to changing their own behaviors (such removing TV from their bedroom) and expressed ambivalence about enforc- ing recommendations that they themselves do not follow. Parents within the focus groups acknowledged that suc- cessful obesity prevention requires making family-based changes. Parents indicated that other family members may not support their efforts to adopt changes related to their child's weight and identified conflicting dietary needs of other children as a barrier, again highlighting the importance of the acceptability of behavior change within the context of the family and household decision-making model. Parents identified few facilitators to implementing recommendations. Parents recognized that changing their own eating habits and shopping habits would help facili- tate their child's adherence to recommendations and acknowledged the role of parenting skills such as limit set- ting. Parents indicated that access to physical activity pro- grams and the availability of alternatives to fast food and TV which are acceptable to the child would facilitate adherence. Economic vs. non-Economic Barriers Economic considerations relating to both dollar and time costs were voiced as barriers to implementing several types of advice. However, parents cited non-economic barriers and time cost barriers more frequently in the focus group discussion than dollar cost barriers and dollar cost barriers were sometimes identified only after prompt- ing. Economic barriers were sometimes cited by parents in Page 5 of 7 (page number not for citation purposes) http://www.biomedcentral.com/1471-2431/9/81 BMC Pediatrics 2009, 9:81 resistance and lack of support from other family mem- bers. up without prompting. Further, dollar costs are not inte- gral to some behavior changes such as removing the TV from the child's bedroom and walking to school so should not be a universally cited barrier. Additional research in this area should include individual survey research in which respondents may feel more comfortable discussing sensitive topics such as financial considera- tions. The frequency with which dollar costs are cited may also suggest that the influence of other barriers, such as child preference or family member-related barriers, dom- inate decisions about implementing recommendations. For example, parents may be less willing to make changes that involve time or dollar costs if their children are highly resistant to the changes. Acknowledgements We are appreciative of the parents who participated in our focus groups for their time and for their candid and thoughtful insights. We would also like to thank Erika Alvarez for moderating the focus groups and Christina Kara for her assistance in coordinating the focus groups. Authors' contributions KS participated in the design and coordination of the study and helped to draft the manuscript. NL participated in the design of the study and conducted all qualitative analyses. ET and MG helped conceive of the study and participated in its design. LP conceived of the study, par- ticipated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript. Competing interests p g The authors declare that they have no competing interests. Conclusions The barriers identified in this study were numerous and varied, suggesting that parents face a unique combination of barriers when adopting recommendations to reduce childhood obesity. Economic considerations, especially time costs, may play a role in parental decisions to imple- ment obesity-related recommendations and should be considered in the context of other priorities for the child and the family. Barriers should be routinely assessed and addressed when obesity-related recommendations are offered to enhance parental compliance. Future research should explore the extent to which identified barriers, including the needs and preferences of other family mem- bers, child preference, and economic considerations, influence parental decision making and should aim to identify approaches to surmounting these barriers. This study was subject to some limitations. Due to recruit- ing challenges, the size of the focus groups varied (from 2 to 7 participants), the sample of Spanish-speaking parents was smaller than the sample of English-speaking parents, and the groups consisted of parents of children of all ages (the original study design called for stratifying focus groups by age). The small sample size limits the analysis of our findings as they may not be representative of the barriers/facilitators faced by all parents. Further, detailed demographic data about the parent and the family such as parental BMI and family size were not collected. Thus, dif- ferences in parental response based on parental or family characteristics were not assessed. Additional research assessing barriers among a large and varied sample of par- ents is warranted to explore the relationship between fam- ily characteristics and barriers faced. In addition, this study explored barriers and facilitators to implementing 7 specific types of obesity prevention recommendations. Although these represent common recommendations made by pediatricians, other possible recommendations related to nutrition and physical activity for overweight children were not included. References Given the limited number of studies that have considered economic factors contributing to physical activity and nutrition choices for children, this study adds to the liter- ature by identifying both time and dollar costs as poten- tial barriers to the adoption of obesity prevention. Further, this study identified that barriers related to all family members including children, parents, and other family members may prevail over these economic barri- ers. Future studies should consider the relative importance of the types of identified barriers and how barriers inter- act. In addition, ongoing research which examines how the entire family participates in decisions about nutrition and physical is needed. Interventions aimed at preventing obesity in childhood should be designed to help parents overcome barriers identified. For example, parents should be given support for changing their own behaviors and parents should be equipped to address issues such as child 1. Katz DL, O'Connell M, Yeh MC, Nawaz H, Njike V, Anderson LM, et al.: Public health strategies for preventing and controlling overweight and obesity in school and worksite settings. MMWR 2005, 54(RR10):1-12. 1. Katz DL, O'Connell M, Yeh MC, Nawaz H, Njike V, Anderson LM, et al.: Public health strategies for preventing and controlling overweight and obesity in school and worksite settings. MMWR 2005, 54(RR10):1-12. 1. Katz DL, O'Connell M, Yeh MC, Nawaz H, Njike V, Anderson LM, et al.: Public health strategies for preventing and controlling overweight and obesity in school and worksite settings. MMWR 2005, 54(RR10):1-12. ( ) 2. Erermis S, Cetin N, Tamar M, Bukusoglu N, Akdeniz F, Goksen D: Is obesity a risk factor for psychopathology among adoles- cents? Pediatrics International 2004, 46(3):296-301. ( ) 2. Erermis S, Cetin N, Tamar M, Bukusoglu N, Akdeniz F, Goksen D: Is obesity a risk factor for psychopathology among adoles- cents? Pediatrics International 2004, 46(3):296-301. ( ) 3. Zeller MH, Modi AC: Predictors of health-related quality of life in obese youth. Obesity (Silver Spring) 2006, 14(1):122-130. ( ) 3. Zeller MH, Modi AC: Predictors of health-related quality of life in obese youth. Obesity (Silver Spring) 2006, 14(1):122-130. y y ( p g) ( ) 4. Pinhas-Hamiel O, Singer S, Pilpel N, Fradkin A, Modan D, Reichman B: Health-related quality of life among children and adoles- cents: associations with obesity. International Journal of Obesity 2006, 30(2):267-272. y y ( p g) ( ) 4. Page 6 of 7 (page number not for citation purposes) References Pinhas-Hamiel O, Singer S, Pilpel N, Fradkin A, Modan D, Reichman B: Health-related quality of life among children and adoles- cents: associations with obesity. International Journal of Obesity 2006, 30(2):267-272. ( ) 5. Schwimmer JB, Burwinkle TM, Varni JW: Health-related quality of life of severely obese children and adolescents. JAMA 2003, 289(14):1813-1819. ( ) 6. Drohan SH: Managing early childhood obesity in the primary care setting: a behavior modification approach. Pediatric Nurs- ing 2002, 28(6):599-610. 7. Koplan JP LC, Kraak VA: Committee on prevention of obesity in children and youth. Executive summary. In Preventing Child- hood Obesity: Health in the Balance Washington, DC: The National Academies Press; 2005:1-20. Page 6 of 7 (page number not for citation purposes) BMC Pediatrics 2009, 9:81 http://www.biomedcentral.com/1471-2431/9/81 http://www.biomedcentral.com/1471-2431/9/81 http://www.biomedcentral.com/1471-2431/9/81 8. Golan M, Weizman A, Apter A, Fainaru M: Parents as the exclu- sive agents of change in the treatment of childhood obesity. The American Journal of Clinical Nutrition 1998, 67(6):1130-1135. J f ( ) 9. Haire-Joshu D, Nanney MS: Prevention of overweight and obes- ity in children: influences on the food environment. The Dia- betes Educator 2002, 28(3):415-423. ( ) 10. Stein RI, Epstein LH, Raynor HA, Kilanowski CK, Paluch RA: The influence of parenting change on pediatrics weight control. Obesity Research 2005, 13(10):1749-1755. 11. Gordon-Larsen P, Griffiths P, Bentley ME, Ward DS, Kelsey K, Shields K, et al.: Barriers to physical activity: qualitative data on car- egiver - daughter perceptions and practices. American Journal of Preventive Medicine 2004, 27(3):218-223. f ( ) 12. Irwin JD, He M, Bouck LM, Tucker P, Pollett GL: Preschoolers' physical activity behaviours: parents' perspectives. Can J Public Health 2005, 96(4):299-303. ( ) 13. Thompson JL, Davis SM, Gittelsohn J: Patterns of physical activity among American Indian children: an assessment of barriers and support. J Community Health 2001, 26(6):423-445. pp J y ( ) 14. Weir LA, Etelson D, Brand DA: Parents' perceptions of neigh- borhood safety and children's physical activity. Preventive Med- icine 2006, 43:212-217. 15. Hart KH, Herriot A, Bishop JA, Truby H: Promoting healthy diet and exercise patterns amongst primary school children: a qualitative investigation of parental perspectives. J Hum Nutr Diet 2003, 16(2):89-96. ( ) 16. Hesketh K, Waters E, Green J, Salmon L, Williams J: Healthy eating, activity and obesity prevention: a qualitative study of parent and child perceptions in Australia. Health Promot Int 2005, 20(1):19-26. ( ) 17. References Boumtje PI, Huang CL, Lee JL, Lin BH: Dietary habits, demo- graphics, and the development of overweight and obesity among children in the United States. Food Policy 2005, 30:115-128. 18. MacInnis B, Rausser G: Does food processing contribute to childhood obesity disparities? Am J Agr Econ 2005, 87(5):1154-1158. ( ) 19. Cutler DM, Glaeser EL, Shapiro JM: Why have Americans become more obese? J Econ Perspectives 2003, 17(3):93-118. 20. Anderson PM, Butcher KF, Levine PB: Economic perspectives on childhood obesity. Economic Perspectives 2003, 3Q:30-48. 21. LaPelle N: Simplifying qualitative data analysis using general purpose software. Field Methods 2004:85-108. 22. Borkan J: Immersion/Crystallization. Thousand Oaks, CA: Sage Publications; 1999. 23. Crabtree BF, Miller WL: Doing Quantitative Research: Using Codes and Code Manuals. Thousand Oaks, CA: Sage Publications; 1999. 24. Miller WL, Crabtree BF: Clinical Research. Thousand Oaks, CA: Sage Publications; 1994. Pre-publication history p y The pre-publication history for this paper can be accessed here: p The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2431/9/81/prepub Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." 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https://openalex.org/W3199328763	https://europepmc.org/articles/pmc8468113?pdf=render	English	null	Efficacy of Combined Therapy with Drug-Eluting Beads-Transcatheter Arterial Chemoembolization Followed by Conventional Transcatheter Arterial Chemoembolization for Unresectable Hepatocellular Carcinoma: A Multi-Center Study	Cancers	2,021	cc-by	8,654	Citation: Morishita, A.; Tani, J.; Nomura, T.; Takuma, K.; Nakahara, M.; Oura, K.; Tadokoro, T.; Fujita, K.; Shi, T.; Yamana, H.; et al. Efﬁcacy of Combined Therapy with Drug-Eluting Beads-Transcatheter Arterial Chemoembolization Followed by Conventional Transcatheter Arterial Chemoembolization for Unresectable Hepatocellular Carcinoma: A Multi-Center Study. Cancers 2021, 13, 4605. https://doi.org/10.3390/ cancers13184605 Academic Editor: Samuel C. Mok Received: 12 July 2021 Accepted: 9 September 2021 Published: 14 September 2021 Citation: Morishita, A.; Tani, J.; Nomura, T.; Takuma, K.; Nakahara, M.; Oura, K.; Tadokoro, T.; Fujita, K.; Shi, T.; Yamana, H.; et al. Efﬁcacy of Combined Therapy with Drug-Eluting Beads-Transcatheter Arterial Chemoembolization Followed by Conventional Transcatheter Arterial Chemoembolization for Unresectable Hepatocellular Carcinoma: A Multi-Center Study. Cancers 2021, 13, 4605. https://doi.org/10.3390/ cancers13184605 Academic Editor: Samuel C. Mok Received: 12 July 2021 Accepted: 9 September 2021 Published: 14 September 2021 Citation: Morishita, A.; Tani, J.; Nomura, T.; Takuma, K.; Nakahara, M.; Oura, K.; Tadokoro, T.; Fujita, K.; Shi, T.; Yamana, H.; et al. Efﬁcacy of Combined Therapy with Drug-Eluting Beads-Transcatheter Arterial Chemoembolization Followed by Conventional Transcatheter Arterial Chemoembolization for Unresectable Hepatocellular Carcinoma: A Multi-Center Study. Cancers 2021, 13, 4605. https://doi.org/10.3390/ cancers13184605 y g jp g jp 2 Department of Radiology, Kagawa University, Kita-gun 761-0793, Japan; sanomura@med.kagawa-u.ac.jp (T.S.); nisiyosi@med.kagawa-u.ac.jp (Y.N.) 3 Department of Medical Technology, Kagawa Prefectural University of Health Sciences, Takamatsu 761-0123, Japan; himoto@chs.pref.kagawa.jp y g jp g jp 2 Department of Radiology, Kagawa University, Kita-gun 761-0793, Japan; sanomura@med.kagawa-u.ac.jp (T.S.); nisiyosi@med.kagawa-u.ac.jp (Y.N.) 3 Department of Medical Technology, Kagawa Prefectural University of Health Sciences, Takamatsu 761-0123, Japan; himoto@chs.pref.kagawa.jp 4 Department of Gastroenterology and Oncology, Institute of Biomedical Sciences, Graduate School of Medicine, Tokushima University, Tokushima 770-8503, Japan; tetsu.tomonari@gmail.com 5 p gy, g Takamatsu 761-0123, Japan; himoto@chs.pref.kagawa.jp 4 Department of Gastroenterology and Oncology, Institute of Biomedical Sciences, Graduate School of Medicine, Tokushima University, Tokushima 770-8503, Japan; tetsu.tomonari@gmail.com 5 Department of Gastroenterology, Mitoyo General Hospital, Kanonji 769-1695, Japan; amoriya@mitoyo-hosp.jp 6 Department of Hepatology, Kagawa Prefectural Central Hospital, Takamatsu 760-8557, Japan; t-senoo@chp-kagawa.jp (T.S.); k.takaguchi@chp-kagawa.jp (K.T.) d h d k l p g jp g p g jp * Correspondence: asahiro@med.kagawa-u.ac.jp; Tel.: +81-87-891-2156; Fax: +81-87-891-2158 p g jp g p g jp * Correspondence: asahiro@med.kagawa-u.ac.jp; Tel.: +81-87-891-2156; Fax: +81-87-891-2158 Simple Summary: Drug-eluting beads-transcatheter chemoembolization (DEB-TACE) has recently been performed. However, local recurrence of HCC at the tumor margins is often observed. cancers cancers cancers cancers Efﬁcacy of Combined Therapy with Drug-Eluting Beads-Transcatheter Arterial Chemoembolization Followed by Conventional Transcatheter Arterial Chemoembolization for Unresectable Hepatocellular Carcinoma: A Multi-Center Study Asahiro Morishita 1,* , Joji Tani 1 , Takako Nomura 1 , Kei Takuma 1, Mai Nakahara 1 , Kyoko Oura 1 , Tomoko Tadokoro 1, Koji Fujita 1 , Tingting Shi 1 , Hiroki Yamana 1, Takanori Matsui 1, Tadayuki Takata 1, Takayuki Sanomura 2, Yoshihiro Nishiyama 2, Takashi Himoto 3, Tetsu Tomonari 4, Akio Moriya 5, Tomonori Senoo 6, Koichi Takaguchi 6 and Tsutomu Masaki 1 1 Department of Gastroenterology and Neurology, Kagawa University, Kita-gun 761 georget@med.kagawa-u.ac.jp (J.T.); takako-n@med.kagawa-u.ac.jp (T.N.); k-takuma@med.kagawa-u.ac.jp (K.T.); m-nakahara@med.kagawa-u.ac.jp (M.N.); kyoko_oura@med.kagawa-u.ac.jp (K.O.); t-nishioka@med.kagawa-u.ac.jp (T.T.); 92m7v9@med.kagawa-u.ac.jp (K.F.); shitingtingc@med.kagawa-u.ac.jp (T.S.); yamanahiroki@med.kagawa-u.ac.jp (H.Y.); s16d729@stu.kagawa-u.ac.jp (T.M.); takata.tadayuki@kagawa-u.ac.jp (T.T.); tmasaki@med.kagawa-u.ac.jp (T.M.) 1 Department of Gastroenterology and Neurology, Kagawa University, Kita-gun 761 georget@med.kagawa-u.ac.jp (J.T.); takako-n@med.kagawa-u.ac.jp (T.N.); k-takuma@med.kagawa-u.ac.jp (K.T.); m-nakahara@med.kagawa-u.ac.jp (M.N.); kyoko_oura@med.kagawa-u.ac.jp (K.O.); t-nishioka@med.kagawa-u.ac.jp (T.T.); 92m7v9@med.kagawa-u.ac.jp (K.F.); shitingtingc@med.kagawa-u.ac.jp (T.S.); yamanahiroki@med.kagawa-u.ac.jp (H.Y.); s16d729@stu.kagawa-u.ac.jp (T.M.); takata.tadayuki@kagawa-u.ac.jp (T.T.); tmasaki@med.kagawa-u.ac.jp (T.M.) The analysis showed that the only factor that increased the CR rate in the cTACE group was the total tumor number (less than four). The OS rate of CR patients was higher than that of non-CR patients in the cTACE group. Adverse events in the cTACE group included severe thrombocytopenia but only in one of twenty-seven patients. Conclusions: The combined therapy with DEB-TACE followed by cTACE may be a new effective therapeutic strategy for the intermediate stage of HCC patients. DEB-TACE and outcomes were analyzed for multiple factors. Results: The complete response rate was signiﬁcantly higher in the cTACE group than in the non-TACE group. The analysis showed that the only factor that increased the CR rate in the cTACE group was the total tumor number (less than four). The OS rate of CR patients was higher than that of non-CR patients in the cTACE group. Adverse events in the cTACE group included severe thrombocytopenia but only in one of twenty-seven patients. Conclusions: The combined therapy with DEB-TACE followed by cTACE may be a new effective therapeutic strategy for the intermediate stage of HCC patients. Keywords: DEB-TACE; cTACE; Barcelona Clinic Liver Cancer staging system; combination therapy; HCC 1. Introduction HCC is the most common cancer [1,2] and the fourth-leading cause of death world- wide [3–6]. Patient prognosis remains poor due to a lack of effective therapy [7]. While surgery is the most effective therapy for HCC [8], most patients are diagnosed at advanced stages precluding surgical therapy. For these patients, the conventional therapies are cTACE [9], radiofrequency ablation [10], molecular-targeting drugs, or these in combina- tion therapies [2,11]. The Barcelona Clinic Liver Cancer Staging System [12] is widely used to evaluate the staging and consequential treatment of HCC. To treat the intermediate stage of HCC, cTACE is often performed [2,13]. Recently, drug-eluting beads-transcatheter chemoem- bolization (DEB-TACE) has been recognized as an alternative therapy for patients with advanced, unresectable liver cancer [14,15]. During DEB-TACE, the patient is injected with chemotherapy-loaded microbeads which embolize the arteries that feed the tumor; the drug-loaded beads slowly release a chemotherapeutic agent into the tumor with a systemic drug concentration peak of less than that in cTACE [16]. DEB-TACE reduces the risk of drug-related adverse events such as post-embolization syndrome [1,17]. Zhiyi et al. demonstrated that DEB-TACE treatment achieved a 19.9% complete response (CR) rate and a 79.6% objective response rate [18]. However, after DEB-TACE, residual areas are often found at the tumor margins, at which increased local recurrence can cause serious damage to the patient. In other words, this residual area, at which drug-eluting beads cannot reach, reduces the CR rate. Therefore, cTACE [19], which is effective for this residual region, is performed as a second line treatment. The aim of our study was to evaluate the efﬁcacy and safety of combination therapy with DEB-TACE followed by cTACE in a short term for the treatment of the intermediate stage of unresectable liver cancer. Con- ventional transcatheter chemoembolization (cTACE) comprises accumulating lipiodol-containing anticancer drugs into the drainage area, which is the ﬁrst invasive site of hepatocellular carcinoma (HCC). We evaluate the therapeutic effect of DEB-TACE followed by cTACE in patients with inter- mediate stage HCC. HCC patients were divided into two groups: one group received DEB-TACE followed by cTACE (cTACE group) and the other group received only DEB-TACE (non-cTACE group). The complete response (CR) rate was signiﬁcantly higher in the cTACE group than in the non-TACE group. The only factor that increased the complete response rate in the cTACE group was the number of tumors. The overall survival (OS) rate of CR patients was higher than that of non-CR patients in the cTACE group. cTACE group adverse events included severe thrombocytopenia but only in one patient. The combined therapy with DEB-TACE followed by cTACE may be useful for HCC patients. Received: 12 July 2021 Accepted: 9 September 2021 Published: 14 September 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Abstract: EB-TACE has recently been performed because of its lower hepatotoxicity compared to cTACE in less advanced HCC. However, local recurrence at the tumor margins is often observed after DEB-TACE. cTACE involves ﬁlling the intratumoral sinusoids with lipiodol-containing anticancer drugs and accumulating in the drainage area, which is the ﬁrst site of HCC recurrence. The aim of this study is to evaluate the therapeutic effect of DEB-TACE followed by cTACE in HCC patients. Between 2014 and 2020, 65 patients with Barcelona clinic liver cancer (BCLC) stage B (intermediate stage) of HCC were enrolled and divided into two groups: one group received DEB-TACE followed by cTACE (cTACE group) and the other group received only DEB-TACE (non-cTACE group). Sixty-ﬁve patients were medically followed. The median observation time was 14 ± 13.1 months after the ﬁrst Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/cancers Cancers 2021, 13, 4605. https://doi.org/10.3390/cancers13184605 Cancers 2021, 13, 4605 2 of 12 DEB-TACE and outcomes were analyzed for multiple factors. Results: The complete response rate was signiﬁcantly higher in the cTACE group than in the non-TACE group. 2.1. Patient Selection and Eligibility This multiple-institution retrospective study was approved by the Institutional Ethics Committee of the Kagawa University, Faculty of Medicine (Kita-gun, Japan), in accordance with the Declaration of Helsinki (approval number 2019-271, approved on March 31, 2020). Sixty-ﬁve patients with HCC who underwent DEB-TACE between 2014 and 2020 at the Kagawa University Hospital (Kita-gun, Japan), Tokushima University Hospital (Tokushima, Japan), Mitoyo General Hospital (Mitoyo-shi, Japan), and Kagawa Prefectural Central Hospital (Takamatsu, Japan) were examined for this retrospective study. The requirement for informed consent from the participants was waived because of the retrospective nature of the study. The study inclusion criteria were: (i) patients diagnosed with the intermediate stage of hepatocellular carcinoma in accordance with the Barcelona Clinic Liver Cancer Staging System [2]; (ii) patients over 18 years old; and (iii) patients who had received DEB-TACE treatment. The exclusion criteria were: (i) patients with advanced stage liver cancer; (ii) patients who were lost to follow-up; (iii) patients with liver or renal failure; and (iv) patients with an allergy to chemoembolization reagents. The patient data collected in Cancers 2021, 13, 4605 3 of 12 our study also included sex, age, etiology of cirrhosis, the Child-Pugh score, clinical tumor stage (cStage), macroscopic classiﬁcation, up-to-7 criteria, alpha fetoprotein (AFP), the third electrophoretic form of lentil lectin-reactive AFP (AFP-L3) [20], and des-gamma-carboxy prothrombin (DCP) [21] (Table 1). Table 1. Baseline characteristics of the patients examined in the study. Table 1. Baseline characteristics of the patients examined in the study. Parameters n = 27, cTACE(+) n = 38, cTACE(−) p-Values Age, median (range) 75 (54–89) 78 (54–90) 0.3286 Sex (male/female) 18/9 31/7 0.1375 Etiology (HBV or HCV/NBNC) 11/16 23/15 0.1155 Child-Pugh score (5, 6/7, or 8) 21/6 23/15 0.1428 cStage (II/III) 8/19 19/19 0.1288 Tumor number (<4/4≤) 20/7 34/4 0.1027 Maximum tumor size (mm), median (range) 59 (30–131) 69 (12–200) 0.7136 MC (SN or SNE/CMN) 23/4 31/7 0.7024 up-to-7 criteria (IN/OUT) 14/13 26/12 0.1760 AFP (ng/mL), median (range) 9192 (4–149,280) 19,672 (2–250,434) 0.9038 AFP-L3 (%), median (range) 19 (0.1–82) 32 (0.5–86) 0.6389 DCP (mAU/mL), median (range) 9594 (30–68,034) 39,178 (8–816,823) 0.3717 Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; cStage, clinical tumor stage; MC, macroscopic classiﬁcation; SN, single nodular type; SNE, single nodular type with extranodular growth; CMN, conﬂuent multinodular type; AFP, α-fetoprotein; and DCP, des-γcarboxy prothrombin. 2.2. DEB-TACE Procedures After the supernatant was extracted from one bottle of drug-eluting beads (DC beads; Eisai Co. Ltd., Tokyo, Japan), one vial (2 mL) of drug-eluting beads with diameters of 100–300 µm was loaded with 50 mg of epirubicin (Nippon Kayaku Co. Ltd., Tokyo, Japan) and diluted 10 times with iopamidol 300 mgI/mL (Iopamiron 300; Bayer Schering Pharma, Osaka, Japan) [22]. Each drug-eluting bead was transferred to a 20-mL syringe in the signiﬁcant diffusion state and injected at 1 mL/min. A microcatheter that was 130 cm in length (Progreat®; Terumo Co. Ltd., Tokyo, Japan) was used. The outer diameters of the catheter tip and shaft were 1.7 and 2.8 Fr, and the inner diameters were 0.016 and 0.026 inches. This procedure was conducted at room temperature. Angiography was performed to detect the tumor-supplying vessels and both the microcatheter and microwire were super-selectively catheterized into the tumor-supplying vessels for embolization. The embolization was discontinued after the ﬂow of the contrast agent stopped. Within 5 min of the chemotherapeutic agent delivery, another angiography was performed to determine if the [blushed/tinted] tumor was still visible and if so, the embolization procedure was repeated. One or two vials (maximum two vials) of DC beads were used in all procedures. Gelpart (1 mg; Nippon Kayaku Co. Ltd., Tokyo, Japan) was administered during arterial embolization if a vascular lake was detected in the tumor. 2.1. Patient Selection and Eligibility Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; cStage, clinical tumor stage; MC, macroscopic classiﬁcation; SN, single nodular type; SNE, single nodular type with extranodular growth; CMN, conﬂuent multinodular type; AFP, α-fetoprotein; and DCP, des-γcarboxy prothrombin. 3.1. Course of Treatment As shown in Figure 1, after seventy-one study participants received their ﬁrst DEB- TACE treatment, six patients were excluded from the study due to having the BCLC C stage of liver cancer or loss to follow-up. The ﬁnal study population consisted of 65 patients. Of these, nine patients received a second DEB-TACE treatment. Three patients from this group of nine received a third cTACE treatment. Twenty-seven patients received a second or third cTACE treatment: fourteen patients using cisplatin, eleven patients using miriplatin, and two patients using epirubicin. Thirty-eight patients underwent non-cTACE treatment, including six patients with a second DEB-TACE treatment. 2.4. Treatment Outcome Assessment Criteria Treatment outcomes were assessed within 1–3 months after the ﬁrst cycle of DEB- TACE and again after the second or third cycle of cTACE according to imaging results and the Response Evaluation Criteria in Cancer of the Liver version 5.0, as follows. (i) A com- plete response: the loss of any intratumoral arterial enhancement in all target nodules; (ii) a partial response: at least a 30% decrease in the sum of the diameters of viable (enhancement in the arterial phase) target nodules relative to the baseline sum of the diameters of target nodules; (iii) a stable disease: absence of partial response or progressive disease; (iv) a progressive disease: an increase of at least 20% in the sum of the diameters of viable (enhancing) target nodules relative to the smallest sum of the diameters of viable (enhanc- ing) target nodules recorded since treatment started; (v) an objective response rate: the percentage of patients who achieved a complete response or partial response; and (vi) a disease control rate: the percentage of patients who achieved a complete response, partial response, or stable disease [24]. 2.5. Liver Function and Safety Assessment Liver function was assessed using liver function-related laboratory parameters in- cluding albumin, total bilirubin, alanine aminotransferase, and aspartate aminotransferase. All adverse events including pain, fever, nausea, and vomiting were recorded using the Common Terminology Criteria for Adverse Events (CTCAE c5.0) [25]. 2.6. Statistical Analyses GraphPad Prism version 8.4.2 (GraphPad Software, San Diego, CA, USA) was used for the statistical analyses. Data are presented as count (%), mean ± standard deviation, or median (25–75th). A comparison between the two treatment groups was performed by the chi-square test. The Student’s t-test was used to compare numerical data for each group. A value of p < 0.05 was considered signiﬁcant. Univariate analyses for continuous variables were undertaken using the Student’s t-test, paired t-test, and one-way ANOVA. For the analysis of categorical variables, the Mann–Whitney U test, Fisher’s exact test, chi- squared test, proportional hazard model test, and Gray’s test with log-rank test results were performed. A multivariate analysis was performed using the Cox proportional hazards model and was applied only to variables that were statistically p < 0.05 in the univariate analysis. A survival analysis was performed using the Kaplan–Meier method. 2.3. cTACE Procedures and Timing of Additional cTACE Angiography was performed to detect the tumor-supplying vessels and percutaneous femoral arterial puncture was performed using the Seldinger technique under topical anesthesia [23]. Subsequently, the microcatheter (1.7Fr; BreakthroughTM, Boston Scientiﬁc, Marlborough, MA, USA) and microwire (0.016 inch; ASAHI Meister, ASAHI INTECC Co., ltd., Seto, Japan) were super-selectively catheterized into the tumor-supplying vessels for the delivery of the chemotherapeutic reagent, namely a solution of cisplatin (Nichi-iko Pharma Co. Ltd., Toyama, Japan), miriplatin (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) or epirubicin and lipiodol (Guerbet Japan Co. Ltd., Tokyo, Japan). Under radiographic guidance, the infusion was discontinued when the ﬂow of lipiodol stopped. Another angiography was performed to ensure that lipiodol had been deposited and to conﬁrm adequate infusion. Gelpart (1 mg; Nippon Kayaku Co. Ltd., Tokyo, Japan) Cancers 2021, 13, 4605 4 of 12 was administered after the transarterial infusion of lipiodol with the chemotherapeutic reagents until adequate embolization. Second or third cTACE was performed in 2 months after DEB-TACE. was administered after the transarterial infusion of lipiodol with the chemotherapeutic reagents until adequate embolization. Second or third cTACE was performed in 2 months after DEB-TACE. g 3.3. Treatment Responses 3.2. Patient Characteristics The baseline characteristics of the patients with HCC are summarized in T There were no significant differences between the cTACE and non-cTACE grou respect to gender, age, etiology (HBV or HCV/NBNC), Child-Pugh score (5 or 6/ clinical stage (II or III/IVA or IVB), tumor number (<4/4≤), maximum tumor size, M or SNE/CMN), up-to-7 criteria (IN/OUT), AFP, AFP-L3, or DCP. The cTACE gro sisted of eighteen male and nine female patients with a median age of seventy-five In the cTACE group, after the ﬁrst cycle of DEB-TACE, no patient and 26 patients (96.3%) achieved a complete response and partial response (Figure 2a–c), respectively, resulting in an overall response rate of 96.3% and a disease control rate of 100% (Table 2). In the non-cTACE group, the numbers of patients who achieved a complete response and partial response were 1 (2.6%) and 32 (84.2%), respectively, resulting in an overall response rate of 86.8% and a disease control rate of 86.8% (Table 2). No statistically signiﬁcant difference was detected between the cTACE and non-cTACE groups after the ﬁrst DEB-TACE. 54–89) years. The non-cTACE group consisted of thirty-one male and seven fem tients with a median age of seventy-eight (range: 54–90) years (Table 1). The num patients with tumors <4 cm and ≥4 cm in size were twenty (74.1%) and seven (25 the cTACE group, respectively, and thirty-four (89.5%) and four (10.5%) in the non group, respectively (Table 1). The median tumor size was 59 mm (range: 30–131 cTACE group and 69 mm (range: 12–200) in the non-cTACE group. In the cTACE twenty-three patients (85.2%) had single nodular-type lesions or those with extran growth and four patients (14.8%) had confluent multinodular-type lesions. In t TACE hi i (81 6%) h d i l d l l i i h We also examined the effect of additional cTACE on the treatment outcome of the ﬁrst DEB-TACE or second DEB-TACE. In the non-cTACE group, four patients (10.5%) achieved a complete response and twenty-four patients (63.2%) achieved a partial response after the ﬁrst DEB-TACE or second DEB-TACE, while fourteen patients (51.9%) and eight patients (29.6%) achieved a complete response (Figure 2d,e,f) and partial response, respectively, in the cTACE group (Table 3). Interestingly, the complete response rate in the cTACE group was signiﬁcantly higher than that in the non-cTACE group (p = 0.0002). 3.2. Patient Characteristics The baseline characteristics of the patients with HCC are summarized in Table 1. There were no signiﬁcant differences between the cTACE and non-cTACE groups with respect to gender, age, etiology (HBV or HCV/NBNC), Child-Pugh score (5 or 6/7 or 8), clinical stage (II or III/IVA or IVB), tumor number (<4/4≤), maximum tumor size, Cancers 2021, 13, 4605 Cancers 2021, 13, x FOR 5 of 12 MC (SN or SNE/CMN), up-to-7 criteria (IN/OUT), AFP, AFP-L3, or DCP. The cTACE group consisted of eighteen male and nine female patients with a median age of seventy- ﬁve (range: 54–89) years. The non-cTACE group consisted of thirty-one male and seven female patients with a median age of seventy-eight (range: 54–90) years (Table 1). The number of patients with tumors <4 cm and ≥4 cm in size were twenty (74.1%) and seven (25.9%) in the cTACE group, respectively, and thirty-four (89.5%) and four (10.5%) in the non-cTACE group, respectively (Table 1). The median tumor size was 59 mm (range: 30–131) in the cTACE group and 69 mm (range: 12–200) in the non-cTACE group. In the cTACE group, twenty-three patients (85.2%) had single nodular-type lesions or those with extranodular growth and four patients (14.8%) had conﬂuent multinodular-type lesions. In the non-cTACE group, thirty-one patients (81.6%) had single nodular-type lesions with or without extranodular growth and seven patients (18.4%) had conﬂuent multinodular-type lesions (Table 1). 3. Results 3.1. Course of Treatment As shown in Figure 1, after seventy-one study participants received their fir TACE treatment, six patients were excluded from the study due to having the B stage of liver cancer or loss to follow-up. The final study population consisted o tients. Of these, nine patients received a second DEB-TACE treatment. Three patien this group of nine received a third cTACE treatment. Twenty-seven patients rec second or third cTACE treatment: fourteen patients using cisplatin, eleven patien miriplatin, and two patients using epirubicin. Thirty-eight patients underwe cTACE treatment, including six patients with a second DEB-TACE treatment. MC (SN or SNE/CMN), up-to-7 criteria (IN/OUT), AFP, AFP-L3, or DCP. The cTACE group consisted of eighteen male and nine female patients with a median age of seventy- ﬁve (range: 54–89) years. The non-cTACE group consisted of thirty-one male and seven female patients with a median age of seventy-eight (range: 54–90) years (Table 1). 3.2. Patient Characteristics The number of patients with tumors <4 cm and ≥4 cm in size were twenty (74.1%) and seven (25.9%) in the cTACE group, respectively, and thirty-four (89.5%) and four (10.5%) in the non-cTACE group, respectively (Table 1). The median tumor size was 59 mm (range: 30–131) in the cTACE group and 69 mm (range: 12–200) in the non-cTACE group. In the cTACE group, twenty-three patients (85.2%) had single nodular-type lesions or those with extranodular growth and four patients (14.8%) had conﬂuent multinodular-type lesions. In the non-cTACE group, thirty-one patients (81.6%) had single nodular-type lesions with or without extranodular growth and seven patients (18.4%) had conﬂuent multinodular-type lesions (Table 1). 3. Results 3.1. Course of Treatment As shown in Figure 1, after seventy-one study participants received their fir TACE treatment, six patients were excluded from the study due to having the B stage of liver cancer or loss to follow-up. The final study population consisted o tients. Of these, nine patients received a second DEB-TACE treatment. Three patien this group of nine received a third cTACE treatment. Twenty-seven patients rec second or third cTACE treatment: fourteen patients using cisplatin, eleven patien miriplatin, and two patients using epirubicin. Thirty-eight patients underwe cTACE treatment, including six patients with a second DEB-TACE treatment. Figure 1 Flow chart of treatment received by the 65 patients enrolled in our study Figure 1. Flow chart of treatment received by the 65 patients enrolled in our study. Figure 1 Flow chart of treatment received by the 65 patients enrolled in our Figure 1. Flow chart of treatment received by the 65 patients enrolled in our study. g 3.3. Treatment Responses Radiographic images showing progress of treatment in 54-year-old female study participant with hepato- cellular carcinoma: (a) dynamic computer tomography image from early treatment phase before transcatheter arterial chemoembolization (TACE) procedures; (b) angiographic image taken during the ﬁrst drug-eluting beads-transcatheter chemoembolization (DEB-TACE) procedure; (c) dynamic computer tomography image from the early treatment phase a month after the ﬁrst DEB-TACE procedure; (d) angiographic image taken during the second conventional TACE (cTACE) procedure; (e) regular computer tomography image taken immediately after the second cTACE procedure; and (f) dynamic computer tomography image taken a month after the second cTACE procedure. Table 2. Treatment response to ﬁrst DEB-TACE. Parameters cTACE(+), n = 27 cTACE(−), n = 38 p-Values Complete response (CR) 0 (0) 1 (2.6) 0.3959 Partial response (PR) 26 (96.3) 32 (84.2) 0.1214 Stable disease (SD) 1 (0) 0 (0) 0.2319 Progressive disease (PD) 0 (0) 5 (13.2) 0.0500 Overall response rate (ORR) 26 (96.3) 33 (86.8) 0.1944 Disease control rate (DCR) 27 (100) 33 (86.8) 0.0500 Data are presented as count n (%). Table 2. Treatment response to ﬁrst DEB-TACE. Table 2. Treatment response to ﬁrst DEB-TACE. Parameters cTACE(+), n = 27 cTACE(−), n = 38 p-Values Complete response (CR) 0 (0) 1 (2.6) 0.3959 Partial response (PR) 26 (96.3) 32 (84.2) 0.1214 Stable disease (SD) 1 (0) 0 (0) 0.2319 Progressive disease (PD) 0 (0) 5 (13.2) 0.0500 Overall response rate (ORR) 26 (96.3) 33 (86.8) 0.1944 Disease control rate (DCR) 27 (100) 33 (86.8) 0.0500 Data are presented as count n (%). Table 3. Treatment response to additional cTACE. Parameters cTACE(+), n = 27 cTACE(−), n = 38 p-Values Complete response (CR) 14 (51.9) 4 (10.5) * 0.0002 Partial response (PR) 8 (29.6) 24 (63.2) * 0.0077 Stable disease (SD) 1 (3.7) 2 (5.3) 0.7678 Progressive disease (PD) 4 (14.8) 8 (21.1) 0.5230 Overall response rate (ORR) 22 (81.5) 28 (73.7) 0.4622 Disease control rate (DCR) 23 (85.2) 30 (78.9) 0.5230 Data are presented as count n (%). cTACE(−) excluded second DEB-TACE (* p < 0.05). Table 3. Treatment response to additional cTACE. Table 3. Treatment response to additional cTACE. g 3.3. Treatment Responses The overall response rate and disease control rate were not signiﬁcantly different between the groups (both p > 0.05; Table 3). 6 of 12 Cancers 2021, 13, 4605 gure 2. Radiographic images showing progress of treatment in 54-year-old female study participant with hepatocellular rcinoma: (a) dynamic computer tomography image from early treatment phase before transcatheter arterial chemoem- lization (TACE) procedures; (b) angiographic image taken during the first drug-eluting beads-transcatheter chemoem- lization (DEB-TACE) procedure; (c) dynamic computer tomography image from the early treatment phase a month er the first DEB-TACE procedure; (d) angiographic image taken during the second conventional TACE (cTACE) proce- re; (e) regular computer tomography image taken immediately after the second cTACE procedure; and (f) dynamic mputer tomography image taken a month after the second cTACE procedure. Figure 2. Radiographic images showing progress of treatment in 54-year-old female study participant with hepato- cellular carcinoma: (a) dynamic computer tomography image from early treatment phase before transcatheter arterial chemoembolization (TACE) procedures; (b) angiographic image taken during the ﬁrst drug-eluting beads-transcatheter chemoembolization (DEB-TACE) procedure; (c) dynamic computer tomography image from the early treatment phase a month after the ﬁrst DEB-TACE procedure; (d) angiographic image taken during the second conventional TACE (cTACE) procedure; (e) regular computer tomography image taken immediately after the second cTACE procedure; and (f) dynamic computer tomography image taken a month after the second cTACE procedure. ure 2. Radiographic images showing progress of treatment in 54-year-old female study participant with hepatocellular cinoma: (a) dynamic computer tomography image from early treatment phase before transcatheter arterial chemoem- ization (TACE) procedures; (b) angiographic image taken during the first drug-eluting beads-transcatheter chemoem- ization (DEB-TACE) procedure; (c) dynamic computer tomography image from the early treatment phase a month er the first DEB-TACE procedure; (d) angiographic image taken during the second conventional TACE (cTACE) proce- re; (e) regular computer tomography image taken immediately after the second cTACE procedure; and (f) dynamic mputer tomography image taken a month after the second cTACE procedure. Figure 2. 3.4. Factors Contributing to Complete Response to DEB-TACE Followed by cTACE Our analysis of various factors related to the complete response to DEB-TACE followed by cTACE is shown in Table 4. Based on our analysis, the following factors did not contribute: age, Child-Pugh score, etiology, tumor size, MC, AFP, AFP-L3, and DCP. No difference between cTACE with cisplatin and that with miriplatin/epirubicin contributed to the complete response. Remarkably, tumor number alone (<4/≥4) contributed to the CR rate, in contrast to non-CR [partial response (PR), stable disease (SD), and progressive disease (PD)] rate (Table 4). These results suggest that DEB-TACE followed by cTACE is highly effective for HCC patients with less than four liver tumors. Table 4. Factors contributing to CR by DEB-TACE following cTACE (CR vs. non-CR). Uni-Variate Analysis Multi-Variate Analysis Parameters OR 95% CI p-Values OR 95% CI p-Values Age (<80/80≤) 0.00772 −0.01404 0.02948 0.4717 0.01196 −0.01671 0.04064 0.3896 Child-Pugh (A/B) 0.02381 −0.47115 0.51876 0.92187 0.10314 −0.59448 0.80076 0.758 Etiology (HBVorHCV/NBNC) 0.04546 −0.373 0.46391 0.82479 0.05984 −0.45035 0.57003 0.8068 Tumor number (<4/4≤) 0.50714 0.08651 0.92777 * 0.02009 0.69165 0.08865 1.29464 * 0.02716 Tumor size (<75/75≤) 0.12143 −0.34555 0.58841 0.59701 0.28535 −0.31613 0.88683 0.32953 MC (SN+SNE/CMN) −0.27174 −0.84018 0.2967 0.33428 0.06001 −0.72434 0.84435 0.8732 AFP (ng/mL) (<9/9≤) 0.1 −0.4282 0.6282 0.69992 −0.81159 −3.33532 1.71213 0.50516 AFP-L3 (%) (<11/11≤) 0.10989 −0.29952 0.51930 0.58531 0.08614 −0.37755 0.54983 0.6989 DCP (mAU/mL) (<3700/3700≤) 0.18681 −0.21784 0.59147 0.35081 0.23685 −0.32434 0.79804 0.3842 cTACE (Cisplatin or Miriplatin/Epirubicin) −0.03846 −0.45007 0.37314 0.84894 0.08178 −0.41276 0.57632 0.7305 Odds-ratios for continuous variables were calculated for one unit. (* p < 0.05). Table 4. Factors contributing to CR by DEB-TACE following cTACE (CR vs. non-CR). 3.5. Comparison of Overall Survival Time between CR and Non-CR (PR + SD + PD) Patients in the cTACE Group 3.5. Comparison of Overall Survival Time between CR and Non-CR (PR + SD + PD) Patients in the cTACE Group The comparison of the overall survival time between CR and non-CR (PR+SD+PD) pa- tients in the cTACE group were analyzed. The median observation time was 14 ± 13.1 months after the ﬁrst DEB-TACE. During the follow-up period, twenty-six patients (96.3%) died and one patient (3.7%) was still alive at the end of the observation. Among the patients with CR, thirteen patients (92.9%) died and one patient (7.1%) was still alive; among the cases with non-CR, thirteen patients (100%) died (Figure 3). 3.4. Factors Contributing to Complete Response to DEB-TACE Followed by cTACE Furthermore, there was a signiﬁcant difference in the overall survival (OS) between CR and non-CR patients in the cTACE group (* p = 0.0403, Figure 3). g 3.3. Treatment Responses Parameters cTACE(+), n = 27 cTACE(−), n = 38 p-Values Complete response (CR) 14 (51.9) 4 (10.5) * 0.0002 Partial response (PR) 8 (29.6) 24 (63.2) * 0.0077 Stable disease (SD) 1 (3.7) 2 (5.3) 0.7678 Progressive disease (PD) 4 (14.8) 8 (21.1) 0.5230 Overall response rate (ORR) 22 (81.5) 28 (73.7) 0.4622 Disease control rate (DCR) 23 (85.2) 30 (78.9) 0.5230 Data are presented as count n (%). cTACE(−) excluded second DEB-TACE (* p < 0.05). Cancers 2021, 13, 4605 7 of 12 3.6. Complications and Adverse Events One week after the additional cTACE, nine patients (33.3%) experienced general fatigue, nine (33.3%) experienced appetite loss, twelve (44.4%) experienced fever, eight (29.6%) experienced pain, two (7.4%) had ascites, three (11.1%) had anemia, ﬁve (18.5%) had thrombocytopenia, four (14.8%) had hyperbilirubinemia, and twenty-seven (100%) had liver dysfunction, while only one (3.7%) patient had severe thrombocytopenia (grade 3) (Table 5). One month after the additional cTACE, two patients (7.4%) had ascites and no other adverse events were observed. 8 of 12 tients Cancers 2021, 13, 4605 . Figure 3. Cumulative rates of overall survival: the Kaplan–Meier method and log-rank test were used to assess the cumulative rates of overall survival. The black line indicates patients within the Figure 3. Cumulative rates of overall survival: the Kaplan–Meier method and log-rank test were used to assess the cumulative rates of overall survival. The black line indicates patients within the cTACE group and the red line indicates patients within the non-cTACE group (* p < 0.05). . Figure 3. Cumulative rates of overall survival: the Kaplan–Meier method and log-rank test were used to assess the cumulative rates of overall survival The black line indicates patients within the Figure 3. Cumulative rates of overall survival: the Kaplan–Meier method and log-rank test were used to assess the cumulative rates of overall survival. The black line indicates patients within the cTACE group and the red line indicates patients within the non-cTACE group (* p < 0.05). cTACE group and the red line indicates patients within the non-cTACE group. (* p < 0.05) 3.6. Complications and Adverse Events One week after the additional cTACE, nine patients (33.3%) experienced general fa- tigue, nine (33.3%) experienced appetite loss, twelve (44.4%) experienced fever, eight (29.6%) experienced pain, two (7.4%) had ascites, three (11.1%) had anemia, five (18.5%) had thrombocytopenia, four (14.8%) had hyperbilirubinemia, and twenty-seven (100%) had liver dysfunction, while only one (3.7%) patient had severe thrombocytopenia (grade 3) (Table 5). One month after the additional cTACE, two patients (7.4%) had ascites and no other adverse events were observed. Table 5. Safety profile following the additional cTACE (n = 27). CTCAE v5.0 All Grades n(%) Grade3 n(%) Table 5. Safety proﬁle following the additional cTACE (n = 27). 3.6. Complications and Adverse Events CTCAE v5.0 All Grades n (%) Grade3 n (%) General fatigue 9 (33.3) 0 Appetite loss 9 (33.3) 0 Fever 12 (44.4) 0 Pain 8 (29.6) 0 Ascites 2 (7.4) 0 Anemia 3 (11.1) 0 Thrombocytopenia 5 (18.5) 1 (3.7) Hyperbilirubinemia 4 (14.8) 0 Liver Dysfunction 27(100) 0 Data are presented as count n (%). Abbreviation: CTCAE v5.0, Common Terminology Criteria for Adverse Events version 5.0. cTACE group and the red line indicates patients within the non-c Table 5. Safety proﬁle following the additional cTACE (n = 27). Gener 4. Discussion This drainage area around the tumor is the ﬁrst metastatic route of hepatocellular carcinoma and micrometastasis is detected at a high rate, and treatment similar to that of the cancerous part is desired [32]. In contrast, in cTACE, lipiodol injected from the hepatic artery ﬁlls the intratumoral sinus according to hemodyamics and accumulates in the drainage area [19]. We addressed that issue by performing an additional cTACE procedure by passing lipiodol through the vasculature to be accumulated in the tumor’s drainage area (Figure 2f), which in turn might lead to an accumulation of lipiodol in the non-tumorous hepatic parenchyma around the liver tumor [19,33–36]. Our results demonstrated that DEB-TACE remarkably increased the complete response rate among the intermediate stage HCC patients (Table 3). Extrapolating from that ﬁnding, combined therapy with DEB-TACE followed by cTACE might also induce necrosis in the intermediate stage of HCC in a complementary manner. g g p y Our data demonstrated that DEB-TACE followed by cTACE may be promising in the treatment of the intermediate stage of HCC, as it achieves a higher complete response rate and induces major tumor necrosis, while reducing the side effects of chemotherapy (Table 5). Additionally, the OS rate of CR patients was higher than that of non-CR patients in the cTACE group (Figure 3). In our study, we chose DEB-TACE as the ﬁrst of two combined therapies because it is associated with less liver toxicity and fewer drug-related adverse events than cTACE, even though its therapeutic effect may be insufﬁcient. In fact, Kalayci et al. showed that the area under the curve and peak concentration levels were the same between systemic chemotherapy and cTACE; thus, the value of cTACE is complicated by the side effects of chemotherapy [37]. With DEB-TACE, a lower amount of chemotherapeutic drugs pass through the patient’s circulatory system, even when these drugs are locally injected in very high doses. Our study conﬁrmed that the area under the curve and the peak concentration values produced by DEB-TACE were signiﬁcantly lower than those produced by cTACE. A therapeutic limitation of DEB-TACE concerns its possible insufﬁcient embolization of the liver tumor. Drug-eluting beads are loaded into the peribiliary plexus, the main feeder of the bile duct wall. This could damage the area by decreasing arterial blood ﬂow or by chemical insult to the vessel walls caused by highly concentrated antitumor drugs [32]. Gener 4. Discussion g ( ) Appetite loss 9 (33.3) 0 Fever 12 (44.4) 0 Pain 8 (29.6) 0 Ascites 2 (7.4) 0 Several clinical trials have demonstrated the efﬁcacy and safety of DEB-TACE. A multicenter phase 2 randomized trial using DEB-TACE with doxorubicin-eluting beads (PRECISION V) showed a marked reduction in liver toxicity and drug-related adverse events as compared to that using cTACE with doxorubicin [16,26,27]. Our study also showed that DEB-TACE was efﬁcient and safe (Table 2). Lammer et al. have also demon- strated that in patients with liver cancer, the tumor response is greater after DEB-TACE than cTACE [26,28]. Golﬁeri and colleagues conducted a randomized trial of DEB-TACE vs. cTACE in 177 patients with HCC. DEB-TACE and cTACE were found to be equally effective and safe, with the only advantage of DEB-TACE concerning the fact that it in- duced less postoperative abdominal pain [29]. The rationale behind DEB-TACE is that drug-loaded embolic microspheres help expose the target tumor to the antineoplastic drug for an extended period of time, while reducing the systemic circulation of the drug, resulting in lower toxicity for the patient [16]. Existing data therefore support our ﬁnding that DEB-TACE is useful for treating the intermediate stage of HCC. Nevertheless, there is no clinical evidence of DEB-TACE’s possible advantage over cTACE. A randomized control study on this topic failed to show that DEB-TACE was superior to cTACE, with Cancers 2021, 13, 4605 9 of 12 the two-year survival rates being 56.8% and 55.4%, respectively [29]. Conversely, the CR rate of DEB-TACE was higher than that of cTACE, but the PR rate also remained high [30]. In addition, OS with DEB-TACE was not prolonged compared to OS with cTACE [31]. In our study, when we performed cTACE 2 months after DEB-TACE, the CR rate was clearly increased. Therefore, we hypothesized that the embolization sites of DEB-TACE and cTACE might be different, and this combination might be effective for the treatment of unresectable hepatocellular carcinoma. In fact, among several cases of liver tumor of the single or multiple nodular type, viable parts of the tumor are often detected after DEB- TACE (Figure 2b). The remaining viable parts are due to the characteristics of DEB-TACE, regarding the fact that no drug-eluting beads reach the peribiliary vascular plexus [32]. 5. Conclusions DEB-TACE has recently been performed for intermediate stage HCC. However, the residual viable lesion located in the drainage area is often observed after DEB-TACE. cTACE involves accumulating lipiodol-containing anticancer drugs in the drainage area, which is the ﬁrst site of HCC recurrence. Therefore, in our present study, DEB-TACE DEB-TACE followed by cTACE was able to reduce the side effects of chemotherapy while increasing the CR rate, which is only affected by the total number of tumors, and prolonge the OS of HCC patients who reached CR. This combination therapy appears to be promising for the treatment of the intermediate stage of HCC. Author Contributions: A.M. (Asahiro Morishita) and T.N. conceived and designed the study. J.T., K.T. (Kei Takuma), M.N. (Mai Nakahara), K.O., T.T. (Tomoko Tadokoro), K.F. (Koji Fujita), T.S. (Tingting Shi), H.Y., T.M. (Takanori Matsui), T.T. (Tadayuki Takata), T.S. (Takayuki Sanomura), Y.N., T.H., T.T. (Tetsu Tomonari), A.M. (Akio Moriya), T.S. (Tomonori Senoo), and K.T. (Koichi Takaguchi). performed the data collection and analyzed the data. A.M. (Asahiro Morishita) and T.M. (Tsutomu Masaki) wrote the paper. All authors read and agreed to the published version of the manuscript. Funding: No funding was received. Institutional Review Board Statement: The present study conformed to the Clinical Research Guide- lines and was approved by the ethical committee of Kagawa University, Faculty of Medicine (2019-271). Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data used in the present study are available from the corresponding author upon reasonable request. Data Availability Statement: The data used in the present study are available from the corresponding author upon reasonable request. Data Availability Statement: The data used in the present study are available from the corresponding author upon reasonable request. Conﬂicts of Interest: All authors declare no conﬂict of interest. Gener 4. Discussion However, no drug-eluting beads accumulate in the tumor’s drainage area, including non-tumorous hepatic parenchyma around the HCC. Therefore, cTACE may be critical for the treatment of the intermediate stage of HCC considering lipiodol in the non-tumorous liver adjacent to the tumor may correspond to the drainage area. j y p g The present study has some limitations. The number of patients enrolled was limited and the follow-up duration was short. However, our ﬁndings concerning DEB-TACE followed by cTACE, which achieved a high CR rate and prolonged OS for CR patients as compared to non-CR patients in the cTACE group, are noteworthy. Therefore, our new combination therapy might be quite valuable for the treatment of the intermediate stage of HCC. Further research for the long-term outcomes with many participants might enable us to obtain the prolonged survival time of patients with intermediate stage HCC. 10 of 12 10 of 12 Cancers 2021, 13, 4605 References 1. Kim, J.Y.; Sinn, D.H.; Gwak, G.Y.; Choi, G.S.; Saleh, A.M.; Joh, J.W.; Cho, S.K.; Shin, S.W.; Carriere, K.C.; Ahn, J.H.; et al. Transarterial chemoembolization versus resection for intermediate-stage (BCLC B) hepatocellular carcinoma. Clin. Mol. Hepatol. 2016, 22, 250–258. [CrossRef] 2. Yang, B.; Li, C.L.; Guo, W.H.; Qin, T.Q.; Jiao, H.; Fei, Z.J.; Zhou, X.; Duan, L.J.; Liao, Z.Y. Intra-arterial ethanol embolization augments response to TACE for treatment of HCC with portal venous tumor thrombus. BMC Cancer 2018, 18, 101. [CrossRef] 3. Global Burden of Disease Cancer, C.; Fitzmaurice, C.; Akinyemiju, T.F.; Al Lami, F.H.; Alam, T.; Alizadeh-Navaei, R.; Allen, C.; Alsharif, U.; Alvis-Guzman, N.; Amini, E.; et al. Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived with Disability, and Disability-Adjusted Life-Years for 29 Cancer Groups, 1990 to 2016: A Systematic Analysis for the Global Burden of Disease Study. JAMA Oncol. 2018, 4, 1553–1568. [CrossRef] y J [ ] 4. Forner, A.; Reig, M.; Bruix, J. Hepatocellular carcinoma. Lancet 2018, 391, 1301–1314. [CrossRef] 4. Forner, A.; Reig, M.; Bruix, J. Hepatocellular carcinoma. Lancet 2018, 391, 1301–1314. [CrossRef] 5. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [CrossRef] , ; g, ; , J p , , [ ] 5. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [CrossRef] 6. Choo, S.P.; Tan, W.L.; Goh, B.K.P.; Tai, W.M.; Zhu, A.X. Comparison of hepatocellular carcinoma in Eastern versus Western populations. Cancer 2016, 122, 3430–3446. [CrossRef] [PubMed] 5. Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2018, 68, 394–424. [CrossRef] 6. Choo, S.P.; Tan, W.L.; Goh, B.K.P.; Tai, W.M.; Zhu, A.X. Comparison of hepatocellular carcinoma in Eastern versus Western populations. Cancer 2016, 122, 3430–3446. [CrossRef] [PubMed] p p 7. Ferenci, P.; Fried, M.; Labrecque, D.; Bruix, J.; Sherman, M.; Omata, M.; Heathcote, J.; Piratsivuth, T.; Kew, M.; Otegbayo, J.A.; et al. Hepatocellular carcinoma (HCC): A global perspective. J. Gastrointestin. Liver Dis. 2010, 19, 311–317. [ ] 12. Llovet, J.M.; Bru, C.; Bruix, J. Prognosis of hepatocellular carcinoma: The BCLC staging classiﬁcation. Semin. Liver Dis. 1999, 19, 329–338. [CrossRef] References [CrossRef] 8 Malkowski, P; Pacholczyk, M ; Lagiewska, B ; Adadynski, L ; Wasiak, D ; Kwiatkowski, A ; Chmura, A ; Czerwinski, J 7. Ferenci, P.; Fried, M.; Labrecque, D.; Bruix, J.; Sherman, M.; Omata, M.; Heathcote, J.; Piratsivuth, T.; Kew, M.; Otegbayo, J.A.; et al. Hepatocellular carcinoma (HCC): A global perspective. J. Gastrointestin. Liver Dis. 2010, 19, 311–317. [CrossRef] 8. Malkowski, P.; Pacholczyk, M.; Lagiewska, B.; Adadynski, L.; Wasiak, D.; Kwiatkowski, A.; Chmura, A.; Czerwinski, J. Hepatocellular carcinoma–epidemiology and treatment. Przegl. Epidemiol. 2006, 60, 731–740. [PubMed] 9. Matsui, O.; Kadoya, M.; Yoshikawa, J.; Gabata, T.; Arai, K.; Demachi, H.; Miyayama, S.; Takashima, T.; Unoura, M.; Kogayashi, K. Small hepatocellular carcinoma: Treatment with subsegmental transcatheter arterial embolization. Radiology 1993, 188, 79–83. [CrossRef] [PubMed] 10. Kainuma, O.; Asano, T.; Aoyama, H.; Shinohara, Y. Recurrent hepatocellular carcinoma suc thermal ablation. J. Hepatobiliary Pancreat. Surg 1999, 6, 190–194. [CrossRef] [PubMed] 11. Katsanos, K.; Kitrou, P.; Spiliopoulos, S.; Maroulis, I.; Petsas, T.; Karnabatidis, D. Comparative effectiveness of different transarterial embolization therapies alone or in combination with local ablative or adjuvant systemic treatments for unresectable hepatocellular carcinoma: A network meta-analysis of randomized controlled trials. PLoS ONE 2017, 12, e0184597. [CrossRef] [PubMed] 11. Katsanos, K.; Kitrou, P.; Spiliopoulos, S.; Maroulis, I.; Petsas, T.; Karnabatidis, D. Comparative effectiveness of different transarterial embolization therapies alone or in combination with local ablative or adjuvant systemic treatments for unresectable hepatocellular carcinoma: A network meta-analysis of randomized controlled trials. PLoS ONE 2017, 12, e0184597. [CrossRef] [PubMed] 12. Llovet, J.M.; Bru, C.; Bruix, J. Prognosis of hepatocellular carcinoma: The BCLC staging classiﬁcation. Semin. Liver Dis. 1999, 19, 329–338. [CrossRef] 11 of 12 11 of 12 Cancers 2021, 13, 4605 13. Raoul, J.L.; Forner, A.; Bolondi, L.; Cheung, T.T.; Kloeckner, R.; de Baere, T. Updated use of TACE for hepatocellular carcinoma treatment: How and when to use it based on clinical evidence. Cancer Treat. Rev. 2019, 72, 28–36. [CrossRef] [PubMed] 14. Kobayashi, S.; Tajiri, K.; Murayama, A.; Entani, T.; Futsukaichi, Y.; Nagata, K.; Takahashi, K.; Yasuda, I. Drug-eluting Bead- Transcatheter Arterial Chemoembolization for Advanced Hepatocellular Carcinoma Refractory to Conventional Lipiodol-based Transcatheter Arterial Chemoembolization. J. Hepatocell. Carcinoma 2020, 7, 181–189. [CrossRef] 15. Chen, P.; Yuan, P.; Chen, B.; Sun, J.; Shen, H.; Qian, Y. Evaluation of drug-eluting beads versus conventional transcatheter arterial chemoembolization in patients with unresectable hepatocellular carcinoma: A systematic review and meta-analysis. Clin. Res. Hepatol. Gastroenterol. 2017, 41, 75–85. References [CrossRef] p 16. Varela, M.; Real, M.I.; Burrel, M.; Forner, A.; Sala, M.; Brunet, M.; Ayuso, C.; Castells, L.; Montana, X.; Llovet, J.M.; et al. Chemoembolization of hepatocellular carcinoma with drug eluting beads: Efﬁcacy and doxorubicin pharmacokinetics. J. Hepatol. 2007, 46, 474–481. [CrossRef] [PubMed] 17. Nakano, M.M.; Yamamoto, A.; Nishida, N.; Hamuro, M.; Hamamoto, S.; Jogo, A.; Sohgawa, E.; Kageyama, K.; Minami, T.; Miki, Y. Risk factors for local recurrence of hepatocellular carcinoma after transcatheter arterial chemoembolization with drug-eluting beads (DEB-TACE). Jpn. J. Radiol. 2019, 37, 543–548. [CrossRef] ( ) Jp J , , [ ] 18. Peng, Z.; Cao, G.; Hou, Q.; Li, L.; Ying, S.; Sun, J.; Zhou, G.; Zhou, J.; Zhang, X.; Ji, W.; et al. The comprehensive analysis of efﬁcacy and safety of CalliSpheres(R) drug-eluting beads transarterial chemoembolization in 367 liver cancer patients: A multiplecenter, cohort study. Oncol. Res. 2019, 28, 249–271. [CrossRef] y [ ] 19. Terayama, N.; Matsui, O.; Gabata, T.; Kobayashi, S.; Sanada, J.; Ueda, K.; Kadoya, M.; Kawamori, Y. Accumulation of iodized oil within the nonneoplastic liver adjacent to hepatocellular carcinoma via the drainage routes of the tumor after transcatheter arterial embolization. Cardiovasc. Interv. Radiol. 2001, 24, 383–387. [CrossRef] 20. Taketa, K.; Endo, Y.; Sekiya, C.; Tanikawa, K.; Koji, T.; Taga, H.; Satomura, S.; Matsuura, S.; Kawai, T.; Hirai, H. A collaborative study for the evaluation of lectin-reactive alpha-fetoproteins in early detection of hepatocellular carcinoma. Cancer Res. 1993, 53, 5419–5423. 21. Murakami, N.; Tamano, M.; Yoneda, M.; Sugaya, H.; Hiraishi, H. Des-gamma-carboxy prothrombi prognostic tumor marker for single nodule hepatocellular carcinoma (HCC). Hepatogastroenterology 200 21. Murakami, N.; Tamano, M.; Yoneda, M.; Sugaya, H.; Hiraishi, H. Des-gamma-carboxy prothrombin (DCP) ratio is a useful prognostic tumor marker for single nodule hepatocellular carcinoma (HCC). Hepatogastroenterology 2008, 55, 197–201. [PubMed] 22. Shiozawa, K.; Watanabe, M.; Ikehara, T.; Ogino, Y.; Umakoshi, T.; Matsukiyo, Y.; Kogame, M.; Matsui, T.; Kikuchi, Y.; Igarashi, Y.; et al. Delayed intratumoral hemorrhage after drug-eluting bead transarterial chemoembolization for hepatocellular carcinoma. Case Rep. Oncol. 2014, 7, 739–745. [CrossRef] [PubMed] prognostic tumor marker for single nodule hepatocellular carcinoma (HCC). Hepatogastroenterology 2008, 55, 197 201. [PubMed] 22. Shiozawa, K.; Watanabe, M.; Ikehara, T.; Ogino, Y.; Umakoshi, T.; Matsukiyo, Y.; Kogame, M.; Matsui, T.; Kikuchi, Y.; Igarashi, Y.; et al. Delayed intratumoral hemorrhage after drug-eluting bead transarterial chemoembolization for hepatocellular carcinoma. Case Rep. Oncol. 2014, 7, 739–745. [CrossRef] [PubMed] p 23. References Wiggermann, P.; Sieron, D.; Brosche, C.; Brauer, T.; Scheer, F.; Platzek, I.; Wawrzynek, W.; Stroszczynski, C. Transarterial Chemoembolization of Child-A hepatocellular carcinoma: Drug-eluting bead TACE (DEB TACE) vs. TACE with cisplatin/lipiodol (cTACE). Med. Sci. Monit. 2011, 17, CR189–CR195. [CrossRef] ( ) [ ] 24. Kudo, M.; Ueshima, K.; Kubo, S.; Sakamoto, M.; Tanaka, M.; Ikai, I.; Furuse, J.; Murakami, T.; Kadoya, M.; Kokudo, N.; et al. Response Evaluation Criteria in Cancer of the Liver (RECICL) (2015 Revised version). Hepatol. Res. 2016, 46, 3–9. [CrossRef] 25. Trotti, A.; Colevas, A.D.; Setser, A.; Basch, E. Patient-reported outcomes and the evolution of adverse event reporting in oncology. J Clin Oncol 2007 25 5121–5127 [CrossRef] ( ) [ ] 24. Kudo, M.; Ueshima, K.; Kubo, S.; Sakamoto, M.; Tanaka, M.; Ikai, I.; Furuse, J.; Murakami, T.; Kadoya, M.; Kokudo, N.; et al. Response Evaluation Criteria in Cancer of the Liver (RECICL) (2015 Revised version). Hepatol. Res. 2016, 46, 3–9. [CrossRef] 25 T tti A C l A D S t A B h E P ti t t d t d th l ti f d t ti i l 25. Trotti, A.; Colevas, A.D.; Setser, A.; Basch, E. Patient-reported outcomes and the evolution of adverse J. Clin. Oncol. 2007, 25, 5121–5127. [CrossRef] 26. Lammer, J.; Malagari, K.; Vogl, T.; Pilleul, F.; Denys, A.; Watkinson, A.; Pitton, M.; Sergent, G.; Pfammatter, T.; Terraz, S.; et al. Prospective randomized study of doxorubicin-eluting-bead embolization in the treatment of hepatocellular carcinoma: Results of the PRECISION V study. Cardiovasc. Interv. Radiol. 2010, 33, 41–52. [CrossRef] 27. Vogl, T.J.; Lammer, J.; Lencioni, R.; Malagari, K.; Watkinson, A.; Pilleul, F.; Denys, A.; Lee, C. Liver, gastrointestinal, and cardiac toxicity in intermediate hepatocellular carcinoma treated with PRECISION TACE with drug-eluting beads: Results from the PRECISION V randomized trial. AJR Am. J. Roentgenol. 2011, 197, W562–W570. [CrossRef] 28. Ikeda, K. Recent advances in medical management of hepatocellular carcinoma. Hepatol. Res. 2019, 49, 14–32. [CrossRef] [PubMed] 29. Golﬁeri, R.; Giampalma, E.; Renzulli, M.; Cioni, R.; Bargellini, I.; Bartolozzi, C.; Breatta, A.D.; Gandini, G.; Nani, R.; Gasparini, D.; et al. Randomised controlled trial of doxorubicin-eluting beads vs conventional chemoembolisation for hepatocellular carcinoma. Br. J. Cancer 2014, 111, 255–264. [CrossRef] 30. Li, H.; Wu, F.; Duan, M.; Zhang, G. Drug-eluting bead transarterial chemoembolization (TACE) vs conventional TACE in treating hepatocellular carcinoma patients with multiple conventional TACE treatments history: A comparison of efﬁcacy and safety. 34. Matsuo, N.; Uchida, H.; Nishimine, K.; Soda, S.; Oshima, M.; Nakano, H.; Nagano, N.; Nishimura, Y.; Yoshioka, T.; Guo, Q.; et al. Segmental transcatheter hepatic artery chemoembolization with iodized oil for hepatocellular carcinoma: Antitumor effect and inﬂuence on normal tissue. J. Vasc. Interv. Radiol. 1993, 4, 543–549. [CrossRef] 35. Nakamura, H.; Hashimoto, T.; Oi, H.; Sawada, S. Iodized oil in the portal vein after arterial embolization. Radiology 1988, 167, 415–417. [CrossRef] 37. Kalayci, C.; Johnson, P.J.; Raby, N.; Metivier, E.M.; Williams, R. Intraarterial adriamycin and lipiodol for inoperable hepatocellular carcinoma: A comparison with intravenous adriamycin. J. Hepatol. 1990, 11, 349–353. [CrossRef] 36. Nakamura, H.; Hashimoto, T.; Oi, H.; Sawada, S. Transcatheter oily chemoembolization of hepatocellular carcinoma. Radiology 1989, 170 Pt 1, 783–786. [CrossRef] References Medicine 2019, 98, e15314. [CrossRef] [PubMed] 31. Kloeckner, R.; Weinmann, A.; Prinz, F.; Pinto dos Santos, D.; Ruckes, C.; Dueber, C.; Pitton, M.B. Conventional transarterial chemoembolization versus drug-eluting bead transarterial chemoembolization for the treatment of hepatocellular carcinoma. BMC Cancer 2015, 15, 465. [CrossRef] [PubMed] 32. Guiu, B.; Deschamps, F.; Aho, S.; Munck, F.; Dromain, C.; Boige, V.; Malka, D.; Leboulleux, S.; Ducreux, M.; Schlumberger, M.; et al. Liver/biliary injuries following chemoembolisation of endocrine tumours and hepatocellular carcinoma: Lipiodol vs. drug-eluting beads. J. Hepatol. 2012, 56, 609–617. [CrossRef] [PubMed] 33. Ueda, K.; Matsui, O.; Kawamori, Y.; Nakanuma, Y.; Kadoya, M.; Yoshikawa, J.; Gabata, T.; Nonomura, A.; Takashima, T. Hypervascular hepatocellular carcinoma: Evaluation of hemodynamics with dynamic CT during hepatic arteriography. Radiology 1998, 206, 161–166. [CrossRef] [PubMed] 12 of 12 Cancers 2021, 13, 4605 12 of 12 J [ ] 35. Nakamura, H.; Hashimoto, T.; Oi, H.; Sawada, S. Iodized oil in the portal vein after arterial embolization. Radiology 1988, 167, 415–417. [CrossRef] 36. Nakamura, H.; Hashimoto, T.; Oi, H.; Sawada, S. Transcatheter oily chemoembolization of hepatocellular carcinoma. Radiology 1989, 170 Pt 1, 783–786. [CrossRef] 37. Kalayci, C.; Johnson, P.J.; Raby, N.; Metivier, E.M.; Williams, R. Intraarterial adriamycin and lipiodol for inoperable hepatocellular carcinoma: A comparison with intravenous adriamycin. J. Hepatol. 1990, 11, 349–353. [CrossRef]
https://openalex.org/W2793401320	https://europepmc.org/articles/pmc5979417?pdf=render	English	null	Role of Inflammation in Diabetic Retinopathy	International journal of molecular sciences	2,018	cc-by	20,733	Received: 15 February 2018; Accepted: 17 March 2018; Published: 22 March 2018 Received: 15 February 2018; Accepted: 17 March 2018; Published: 22 March 2018 Abstract: Diabetic retinopathy is a common complication of diabetes and remains the leading cause of blindness among the working-age population. For decades, diabetic retinopathy was considered only a microvascular complication, but the retinal microvasculature is intimately associated with and governed by neurons and glia, which are affected even prior to clinically detectable vascular lesions. While progress has been made to improve the vascular alterations, there is still no treatment to counteract the early neuro-glial perturbations in diabetic retinopathy. Diabetes is a complex metabolic disorder, characterized by chronic hyperglycemia along with dyslipidemia, hypoinsulinemia and hypertension. Increasing evidence points to inﬂammation as one key player in diabetes-associated retinal perturbations, however, the exact underlying molecular mechanisms are not yet fully understood. Interlinked molecular pathways, such as oxidative stress, formation of advanced glycation end-products and increased expression of vascular endothelial growth factor have received a lot of attention as they all contribute to the inﬂammatory response. In the current review, we focus on the involvement of inﬂammation in the pathophysiology of diabetic retinopathy with special emphasis on the functional relationships between glial cells and neurons. Finally, we summarize recent advances using novel targets to inhibit inﬂammation in diabetic retinopathy. Keywords: diabetic retinopathy; neuroprotection; inflammation; neurodegeneration; pathophysiology; microglia; astrocytes; Müller glial cells; crystallins Anne Rübsam 1, Sonia Parikh 1 and Patrice E. Fort 1,2,* Anne Rübsam 1, Sonia Parikh 1 and Patrice E. Fort 1,2,* 1 Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105, USA; aruebsam@med.umich.edu (A.R.); soniapar@umich.edu (S.P.) 2 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48105, USA * Correspondence: patricef@med.umich.edu; Tel.: +01-734-232-8225 , 1 Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105, USA; aruebsam@med.umich.edu (A.R.); soniapar@umich.edu (S.P.) 2 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48105, USA * Correspondence: patricef@med.umich.edu; Tel.: +01-734-232-8225 International Journal of Molecular Sciences Role of Inﬂammation in Diabetic Retinopathy Anne Rübsam 1, Sonia Parikh 1 and Patrice E. Fort 1,2,* 1. Introduction Diabetic retinopathy (DR) is the primary cause of visual impairment in the working-age population of the Western world [1]. Among microvascular complications related to diabetes mellitus such as nephropathy and neuropathy, DR is the most common. The prevalence rate for DR for all adults with diabetes aged 40 and older is 28.5% in the United States (4.2 million people) while estimated at 34.6% worldwide (93 million people) [2,3]. With the prevalence of diabetes expected to continue to rise, the prevalence of DR in the United States by year 2020 is expected to be 6 million persons with 1.34 million persons having vision-threatening disease [4]. The substantial worldwide public health burden of DR highlights the importance of continuously searching for new approaches beyond current standards of diabetes care. Diabetic retinopathy is categorized based on the presence of ophthalmoscopically visible vascular (and closely associated) lesions. It is staged into a non-proliferative stage, characterized by vascular tortuosity, retinal hemorrhages, microaneurysms and lipid exudates; and a proliferative stage, where fragile new aberrant vessels develop (Figure 1). An important additional categorization in DR is diabetic macular edema (DME), a ﬂuid accumulation into the neural retina, which leads to abnormal retinal thickening and often cystoid edema of the macula. DME could occur across all DR severity levels, of both NPDR and PDR and represents the most common cause of vision loss in Int. J. Mol. Sci. 2018, 19, 942; doi:10.3390/ijms19040942 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 942 2 of 31 patients with DR. While for diabetes the breakdown of the inner blood retinal barrier (BRB) is believed to play a dominant role in vascular leakage [5,6], an important role of the outer BRB has recently gained momentum (for review [7]). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 30 patients with DR. While for diabetes the breakdown of the inner blood retinal barrier (BRB) is belie ed to lay a do i a t ole i a ula leaka e [5 6] a i o ta t ole of the oute BRB ha Clinical evidence indicates that there is a combination of capillary occlusion and an increased capillary permeability. The ability of vascular endothelial growth factor (VEGF) to promote both vascular permeability and angiogenesis made it a likely contributor to the vascular dysfunctions observed in severe DR [8,9]. 1. Introduction In the past decade, a variety of physiologic and molecular changes consistent with a role of inflammation have been found in the retinas or vitreous humor of diabetic animals and patients [12–18]. While the detailed mechanisms remain to be specified, these inflammatory changes seems to play a key role important in the development of DR as their inhibition has been shown to impact the development of retinal alterations in animal models of diabetes [19]. Studies using anti-inflammatory agents such as salicylates or minocycline in patients with DR gave further evidence that regulating the inflammatory response can be beneficial to prevent irreversible vascular and neuronal perturbations over time [20 21] In this review, we analyze evidence that supports the hypothesis, that inﬂammatory-like processes play a critical role in the development of the early and late stages of the retinopathy with special emphasis on the role of inﬂammation in the impaired neuro-glial crosstalk in DR. [20,21]. In this review, we analyze evidence that supports the hypothesis, that inflammatory-like processes play a critical role in the development of the early and late stages of the retinopathy with special emphasis on the role of inflammation in the impaired neuro-glial crosstalk in DR. Figure 1. Clinical features of diabetic retinopathy and causative pro-inflammatory chemokines. (A) A fundus photograph shows the right eye of a 57-year-old man with 20/80 visual acuity and signs of severe non-proliferative diabetic retinopathy with non-significant macular edema (the region of macular edema is indicated by the bracket). Vascular pathologies are depicted in blue, whereas neurodegenerative features are black. Notable features include dot blot hemorrhages, DH; hard exudates, HE; cotton-wool spots, CWS. Each are associated with the upregulation of certain chemokines; (B) Optical coherence tomography with a horizontal scan through the central fovea reveals moderate thickening and edema of the macula with cysts; (C) Ang: Angiopoietin; IL: interleukin; TNF: Tumor Necrosis Factor; NO: nitric oxide; MCP: Monocyte Chemoattractant Protein. Figure 1. Clinical features of diabetic retinopathy and causative pro-inﬂammatory chemokines. (A) A fundus photograph shows the right eye of a 57-year-old man with 20/80 visual acuity and signs of severe non-proliferative diabetic retinopathy with non-signiﬁcant macular edema (the region of macular edema is indicated by the bracket). Vascular pathologies are depicted in blue, whereas neurodegenerative features are black. Notable features include dot blot hemorrhages, DH; hard exudates, HE; cotton-wool spots, CWS. 1. Introduction Although signiﬁcant success could be demonstrated with anti-VEGF therapy, a number of limitations exist ranging from the need for repeated intraocular injections, its efﬁcacy only for late stages of the diseases, and most importantly the fact that only approximately half of the patients respond to the treatment [10]. believed to play a dominant role in vascular leakage [5,6], an important role of the outer BRB has recently gained momentum (for review [7]). Clinical evidence indicates that there is a combination of capillary occlusion and an increased capillary permeability. The ability of vascular endothelial growth factor (VEGF) to promote both vascular permeability and angiogenesis made it a likely contributor to the vascular dysfunctions observed in severe DR [8,9]. Although significant success could be demonstrated with anti-VEGF therapy, a number of limitations exist ranging from the need for repeated intraocular injections, its efficacy only for late stages of the diseases, and most importantly the fact that only approximately h lf f h d h With advancement in diagnostic tools, it has been demonstrated that patients with diabetes exhibit early signs of altered neuroretinal function even before the appearance of microvascular lesions [11]. Currently, there are still no therapies targeting these early stages of the disease to prevent alterations of the neuroretina and thereby preserve visual function. In the past decade, a variety of physiologic and molecular changes consistent with a role of inﬂammation have been found in the retinas or vitreous humor of diabetic animals and patients [12–18]. While the detailed mechanisms remain to be speciﬁed, these inﬂammatory changes seems to play a key role important in the development of DR as their inhibition has been shown to impact the development of retinal alterations in animal models of diabetes [19]. Studies using anti-inﬂammatory agents such as salicylates or minocycline in patients with DR gave further evidence that regulating the inﬂammatory response can be beneﬁcial to prevent irreversible vascular and neuronal perturbations over time [20,21]. half of the patients respond to the treatment [10]. With advancement in diagnostic tools, it has been demonstrated that patients with diabetes exhibit early signs of altered neuroretinal function even before the appearance of microvascular lesions [11]. Currently, there are still no therapies targeting these early stages of the disease to prevent alterations of the neuroretina and thereby preserve visual function. 2. Emergence of the Neurovascular Unit and Implications for Early DR Many DR studies, both clinical and using animal models focused on vascular dysfunction, such as impaired endothelial cells, death of pericytes, thickening of retina capillary basement membrane and altered tight junctions. However, diabetic microvasculopathy does not explain the susceptibility of peripheral nerves, cerebral complications or the early loss of retinal function. Beginning in the late 1990s, the concept of the neurodegenerative aspect of DR started to emerge. Several excellent reviews published in the last decade have summarized the evidence that neurons are vulnerable and die early in diabetic retinopathy, however, the pathophysiological mechanisms underlying this neurodegenerative process remain to be clearly elucidated [22,23]. While retinal ganglion cells (RGCs) are the earliest cells affected and have the highest rate of apoptosis [24,25]. An elevated rate of apoptosis has been also observed in the outer nuclear layer, with a reduction in photoreceptors seen between 4 and 24 weeks after diabetes onset [26,27]. Consistent with this, molecular analyses revealed that proteins important for photoreceptor function, such as rhodopsin, were affected before the appearance of microangiopathy in diabetes [28]. In addition to neuronal cell death, another early feature of diabetic-induced retinal neurodegeneration that has recently gained traction is glial cell activation and dysfunction. Müller glial cells (MGCs), the main glial cells of the retina, play a central role in retinal metabolism, making them highly sensitive to metabolic alterations such as those associated with diabetes. One of the early signs of retinal metabolic stress is the upregulation of glial ﬁbrillary acidic protein (GFAP) by MGCs, an observation classically reported in animal models as well as in tissues from diabetic patients with no to mild NPDR [29,30]. Consequently, the resident immune cells in the retina, called microglia, also become activated and start to produce pro-inﬂammatory mediators, exacerbating neuro-glial and vascular dysfunction [31]. Those retinal abnormalities in neurons and glial cells are believed to drive a variety of functional changes that often precede clinically visible vascular lesions in DR. Among those functional changes are deﬁcits pointing to ganglion cells such as the pattern electroretinogram (ERG), altered microperimetric [32] and perimetric testing [11]; or more generally the inner retina such as increased implicit times and reduction in oscillatory potentials in the multifocal ERG (mfERG) [33]. Other less cellular speciﬁc alterations have also been reported including abnormal dark adaptation, contrast sensitivity [34] and color vision [35]. 1. Introduction Each are associated with the upregulation of certain chemokines; (B) Optical coherence tomography with a horizontal scan through the central fovea reveals moderate thickening and edema of the macula with cysts; (C) Ang: Angiopoietin; IL: interleukin; TNF: Tumor Necrosis Factor; NO: nitric oxide; MCP: Monocyte Chemoattractant Protein. Figure 1. Clinical features of diabetic retinopathy and causative pro-inflammatory chemokines. (A) A fundus photograph shows the right eye of a 57-year-old man with 20/80 visual acuity and signs of severe non-proliferative diabetic retinopathy with non-significant macular edema (the region of macular edema is indicated by the bracket). Vascular pathologies are depicted in blue, whereas neurodegenerative features are black. Notable features include dot blot hemorrhages, DH; hard exudates, HE; cotton-wool spots, CWS. Each are associated with the upregulation of certain chemokines; (B) Optical coherence tomography with a horizontal scan through the central fovea reveals moderate thickening and edema of the macula with cysts; (C) Ang: Angiopoietin; IL: interleukin; TNF: Tumor Necrosis Factor; NO: nitric oxide; MCP: Monocyte Chemoattractant Protein. Figure 1. Clinical features of diabetic retinopathy and causative pro-inﬂammatory chemokines. (A) A fundus photograph shows the right eye of a 57-year-old man with 20/80 visual acuity and signs of severe non-proliferative diabetic retinopathy with non-signiﬁcant macular edema (the region of macular edema is indicated by the bracket). Vascular pathologies are depicted in blue, whereas neurodegenerative features are black. Notable features include dot blot hemorrhages, DH; hard exudates, HE; cotton-wool spots, CWS. Each are associated with the upregulation of certain chemokines; (B) Optical coherence tomography with a horizontal scan through the central fovea reveals moderate thickening and edema of the macula with cysts; (C) Ang: Angiopoietin; IL: interleukin; TNF: Tumor Necrosis Factor; NO: nitric oxide; MCP: Monocyte Chemoattractant Protein. Int. J. Mol. Sci. 2018, 19, 942 3 of 31 2. Emergence of the Neurovascular Unit and Implications for Early DR Optical coherence tomography (OCT) is a widely utilized method for imaging the individual neural layers of the retina in many ophthalmic conditions. Several measures associated with OCT imaging are being explored as potential neuroretinal biomarkers of visual defects in eyes with DME and NPDR. Among those that are currently being assessed are ganglion cell/nerve ﬁber layer thinning, disorganization of the retinal inner layers, and photoreceptor disruption [36–38]. The latest progress in this domain is the technique of OCT angiography, which permits to combine with the anatomical observation the non-invasive study of the different layers of retinal microvasculature and choroid circulation via motion contrast imaging [39]. All these observations strongly suggest that diabetes directly affects the neuroretina rather than being solely secondary to the breakdown of the blood-retinal barrier (BRB). Several interconnected biochemical pathways, activated during diabetes, increase the expression of angiogenic and inﬂammatory mediators and induce aberrant growth factor signaling, all of which have been directly linked to neurodegeneration and vascular dysfunction (Figure 2). Altogether these new insights into retinal physiology have led to the emergence of the concept that the retinal dysfunction associated with diabetes may be viewed as a change in the retinal neurovascular unit [40]. This concept of the neurovascular unit really emphasizes the intimate relationship between retinal neurons, (photoreceptors, horizontal and bipolar cells, amacrine and ganglion cells), their supporting cells (astrocytes and Müller glial cells), and the vascular beds (endothelial cells and pericytes). Proper function of all the elements of this unit is essential for normal retinal function as it allows the neural retina to adapt to varying physiological conditions, therefore, new therapeutic approaches promoting a “healthy” unit should be targeted. 4 of 31 4 of 30 Int. J. Mol. Sci. 2018, 19, 942 Int J Mol Sci 2018 19 x FOR Figure 2. Schematic representation of pathogenic mechanisms leading to sight-threatening endpoints of diabetic retinopathy (DR): proliferative DR (PDR) and diabetic macular edema (DME). Metabolic alterations are first sensed by glial cells resulting in glial dysfunction, which induces inflammation, aberrant signaling of trophic factors and metabolic dysregulation all leading to neuronal apoptosis. Neurodegeneration also participate in blood–retinal barrier (BRB) breakdown, vasoregression and consecutive hypoxia, the main features of early microvascular abnormalities and the end stage neovascularization. According to the vascular lesions DR is staged by ophthalmoscopy in several stages of severity. 2. Emergence of the Neurovascular Unit and Implications for Early DR AGE, advanced glycation end-products; PKC, protein kinase C; RAS, renin–angiotensin system, ROS, reactive oxygen species; NADPH, Nicotinamide adenine dinucleotide phosphate; mETC, mitochondrial electron transport chain; NPDR, non-proliferative DR. 2. Emergence of the Neurovascular Unit and Implications for Early DR AGE, advanced glycation end-products; PKC, protein kinase C; RAS, renin– angiotensin system, ROS, reactive oxygen species; NADPH, Nicotinamide adenine dinucleotide phosphate; mETC, mitochondrial electron transport chain; NPDR, non-proliferative DR. Figure 2. Schematic representation of pathogenic mechanisms leading to sight-threatening endpoints of diabetic retinopathy (DR): proliferative DR (PDR) and diabetic macular edema (DME). Metabolic alterations are ﬁrst sensed by glial cells resulting in glial dysfunction, which induces inﬂammation, aberrant signaling of trophic factors and metabolic dysregulation all leading to neuronal apoptosis. Neurodegeneration also participate in blood–retinal barrier (BRB) breakdown, vasoregression and consecutive hypoxia, the main features of early microvascular abnormalities and the end stage neovascularization. According to the vascular lesions DR is staged by ophthalmoscopy in several stages of severity. AGE, advanced glycation end-products; PKC, protein kinase C; RAS, renin–angiotensin system, ROS, reactive oxygen species; NADPH, Nicotinamide adenine dinucleotide phosphate; mETC, mitochondrial electron transport chain; NPDR, non-proliferative DR. Figure 2. Schematic representation of pathogenic mechanisms leading to sight-threatening endpoints f di b i i h (DR) lif i DR (PDR) d di b i l d (DME) M b li Figure 2. Schematic representation of pathogenic mechanisms leading to sight-threatening endpoints Figure 2. Schematic representation of pathogenic mechanisms leading to sight-threatening endpoints of diabetic retinopathy (DR): proliferative DR (PDR) and diabetic macular edema (DME). Metabolic alterations are first sensed by glial cells resulting in glial dysfunction, which induces inflammation, aberrant signaling of trophic factors and metabolic dysregulation all leading to neuronal apoptosis. Neurodegeneration also participate in blood–retinal barrier (BRB) breakdown, vasoregression and consecutive hypoxia, the main features of early microvascular abnormalities and the end stage neovascularization. According to the vascular lesions DR is staged by ophthalmoscopy in several stages of severity. AGE, advanced glycation end-products; PKC, protein kinase C; RAS, renin– angiotensin system, ROS, reactive oxygen species; NADPH, Nicotinamide adenine dinucleotide phosphate; mETC, mitochondrial electron transport chain; NPDR, non-proliferative DR. Figure 2. Schematic representation of pathogenic mechanisms leading to sight-threatening endpoints of diabetic retinopathy (DR): proliferative DR (PDR) and diabetic macular edema (DME). Metabolic alterations are ﬁrst sensed by glial cells resulting in glial dysfunction, which induces inﬂammation, aberrant signaling of trophic factors and metabolic dysregulation all leading to neuronal apoptosis. Neurodegeneration also participate in blood–retinal barrier (BRB) breakdown, vasoregression and consecutive hypoxia, the main features of early microvascular abnormalities and the end stage neovascularization. According to the vascular lesions DR is staged by ophthalmoscopy in several stages of severity. 3.1. Inflammation 3.1. Inﬂammation Inflammation is a non-specific response to injury or stress that includes a variety of functional and molecular mediators. When invading pathogens are involved, they are recognized by pattern recognition receptors, such as Toll-like receptors (TLRs) and the receptor of advanced glycation end products [36]. The molecules of the pathogens that bind to these receptors are called pathogen- associated molecular patterns (PAMPs) [41]. TLR activation or, in absence of pathogens, tissue stress alone, can lead to de-inhibition of the transcription factor Nuclear factor kappa B (NF-κB), which translocates into the nucleus to stimulate transcription of pro-inflammatory cytokines, acute phase proteins and chemokines, such as IL-6, TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP- 1). All these pro-inflammatory chemokines play a major role in the recruitment and activation of monocytes and leukocytes and the subsequent inflammatory responses. Inflammation normally resolves promptly through a coordinated program that includes resolvins, lipoxins and protectins [42]. When not resolved in a timely fashion the typically beneficial effect of inflammation is lost and dramatic consequences ensues [43]. Inflammation is a non-specific response to injury or stress that includes a variety of functional and molecular mediators. When invading pathogens are involved, they are recognized by pattern recognition receptors, such as Toll-like receptors (TLRs) and the receptor of advanced glycation end products [36]. The molecules of the pathogens that bind to these receptors are called pathogen-associated molecular patterns (PAMPs) [41]. TLR activation or, in absence of pathogens, tissue stress alone, can lead to de-inhibition of the transcription factor Nuclear factor kappa B (NF-κB), which translocates into the nucleus to stimulate transcription of pro-inflammatory cytokines, acute phase proteins and chemokines, such as IL-6, TNF-α, IL-1β, and monocyte chemoattractant protein-1 (MCP-1). All these pro-inflammatory chemokines play a major role in the recruitment and activation of monocytes and leukocytes and the subsequent inflammatory responses. Inflammation normally resolves promptly through a coordinated program that includes resolvins, lipoxins and protectins [42]. When not resolved in a timely fashion the typically beneficial effect of inflammation is lost and dramatic consequences ensues [43]. 3.2. Evidences of Inflammation in Diabetic Retinopathy Pathogenesis 3.2. Evidences of Inﬂammation in Diabetic Retinopathy Pathogenesis 3.2. Evidences of Inflammation in Diabetic Retinopathy Pathogenesis 3.2. Evidences of Inﬂammation in Diabetic Retinopathy Pathogenesis Increasing evidence points to inflammation as a critical contributor to the development of DR [44–46]. Many inflammatory cytokines and chemokines are increased in serum and ocular samples (vitreous and aqueous humor) from diabetic patients with DR (Table 1). Gli l i i li i h b d h i i i h li i l i f DR Increasing evidence points to inflammation as a critical contributor to the development of DR [44–46]. Many inﬂammatory cytokines and chemokines are increased in serum and ocular samples (vitreous and aqueous humor) from diabetic patients with DR (Table 1). 5 of 31 Int. J. Mol. Sci. 2018, 19, 942 Glial activation, or gliosis has been suggested to happen in patients with no clinical signs of DR. Increased levels of GFAP has indeed been reported in the aqueous humor of such patients [47]. DR. Increased levels of GFAP has indeed been reported in the aqueous humor of such patients [47]. Since Müller glial cells are a signiﬁcant source of numerous factors including inﬂammatory modulators, this suggests that retinal glial cell activation might play an early role in the onset of the inﬂammatory process responsible for retinal damage at later stages of the disease. This hypothesis is further supported by the reported elevated levels at this stage of the disease of the adhesion molecules ICAM-1 and the neutrophil chemotactic MCP-2, molecules also produced by MGCs and involved in leukostasis, another phenomenon observed early in DR pathology and associated with inﬂammation [48–50]. Various inﬂammatory cytokines—IL-1β, IL-6, IL-8, TNF-α and MCP-1—have been reported elevated in ocular tissues from non-proliferative DR (NPDR) patients. One study detected even higher levels of IL-8 and TNF-α in diabetic eyes with NPDR than with active PDR [51]. The increase in these cytokines produced by activated microglia, endothelial cells, macroglia, and later even neurons, highlight the increased activity of these inﬂammatory cytokines in the early stages of DR and the progression of the inﬂammatory response throughout all cell types of the retina [52]. The accumulation of these inﬂammatory mediators has been proposed to contribute to early neuronal cell death in the retina in DM. 3.2. Evidences of Inflammation in Diabetic Retinopathy Pathogenesis 3.2. Evidences of Inﬂammation in Diabetic Retinopathy Pathogenesis Some of the cytokines identiﬁed, such as MIP-1, IL-1 and IL-3, are thought to also have a role in angiogenesis, as established in experimental ischemic mouse models suggesting that inﬂammation also contributes and precedes the development of neovascularization in PDR [21,53,54]. Signiﬁcantly higher vitreous levels of neurotrophins (NTs), a group of speciﬁc growth factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BNDF), NT-3, NT-4, ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GNDF) could be detected in DR patients, with even higher levels in NPDR compared to PDR [51]. This might be an attempt by glial cells such as MGCs to rescue compromised neurons during this early stage of DR. In patients with PDR, increased vitreous concentrations of the aforementioned cytokines and neurotrophins along with other growth factors such as VEGF, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), basic ﬁbroblast growth factor (bFGF) and hepatocyte growth factor (HGF) have been reported [55]. Of note, analysis of vitreous samples from PDR patients also revealed increased levels of soluble cytokine receptors, such as sIL-2R [56]. This increase constitutes a known negative regulatory mechanism of cytokine signaling, suggesting that counter-regulatory mechanisms of angiogenesis and inﬂammation exist within the eye. The development of PDR is a multistage event, including angiogenesis, in which basement membrane degradation, endothelial cell migration and proliferation, followed by capillary tube formation, occur. Matrix metalloproteinases (MMPs), which are important regulators of those migratory and tissue remodeling events, have been reported to be upregulated in PDR as well [57]. In patients with DME, levels of angiopoietin-2 (Ang-2), an important modulator of angiogenesis, are signiﬁcantly elevated along with inﬂammatory cytokines and VEGF [12,58]. Currently there are no widely accepted valid biomarkers to monitor DR severity or efﬁciently classify patients for optimal assessment of treatment efﬁcacy, but reports indicate, that VEGF, HGF, IL-6 and MCP-1 intravitreal concentration increase with the progression of DR from the non-proliferative form to active PDR [55,56]. Furthermore, IL-6 levels have been shown to positively correlate with retinal macular thickness [59]. A variety of the inﬂammatory mediators mentioned in the previous paragraphs are activated in DR, but the signaling involved in initiating this response is less clear. One of the caveats of using vitreous or aqueous humor levels to assess the expression of proteins associated with diabetes and DR, is the notion that the changes observed might just represent changes in circulating serum levels. 3.2. Evidences of Inflammation in Diabetic Retinopathy Pathogenesis 3.2. Evidences of Inﬂammation in Diabetic Retinopathy Pathogenesis Of note, it has been demonstrated, that the total vitreous protein content does not differ between NPDR and PDR patients [51], suggesting that increased vitreous protein levels present a real increase in secretion rather than just leakage of serum proteins into the vitreous because of a disrupted BRB. Nevertheless, many features of inﬂammation including leukostasis, neutrophil and macrophage inﬁltration, complement and microglial activation, upregulation of cytokines, increased blood ﬂow, vascular permeability and tissue edema have been described in animal models and human 6 of 31 Int. J. Mol. Sci. 2018, 19, 942 patients with DR [49,60–64]. Furthermore, inhibition or deletion of pro-inﬂammatory molecules has been demonstrated to dampen diabetes-induced vascular and neurodegenerative pathology in animal models of DR [65–67]. Thus, in the next sections we breakdown the speciﬁc contribution of inﬂammation to the main hallmarks of DR: angiogenesis and neurodegeneration. Table 1. Overview over levels of inﬂammation-related cytokines, chemokines and growth factors in the vitreous or aqueous humor of human diabetic patients with different stages of diabetic retinopathy (DR). If only information on serum levels were available, it is mentioned speciﬁcally. ↑upregulation, ↓downregulation, ↔no change Table 1. Overview over levels of inﬂammation-related cytokines, chemokines and growth factors in the vitreous or aqueous humor of human diabetic patients with different stages of diabetic retinopathy (DR). If only information on serum levels were available, it is mentioned speciﬁcally. 3.2. Evidences of Inflammation in Diabetic Retinopathy Pathogenesis 3.2. Evidences of Inﬂammation in Diabetic Retinopathy Pathogenesis ↑upregulation, ↓downregulation, ↔no change Factors Diabetic Retinopathy Stage References Diabetics without DR NPDR PDR DME AQP4 ↑ ↑ not known not known [47] GFAP ↑ ↑ not known not known [47] VEGF ↑serum ↔vitreous ↑ ↑ ↑ [68–70] IFN-γ ↑ ↑ ↑ ↑ [49,52,71,72] IL-1α ↑ ↑ ↑ ↑ [49,69] IL-3 ↑vitreous ↓serum ↓vitreous & serum ↓serum ↑aqueous no change vitreous [49,73–75] s-IL2R ↑ ↑ ↑ not known [56,73] MCP-2 ↑ ↑ not known not known [49] ICAM-1 ↑serum ↑serum ↑ ↑ [68,70,71] IL-1β no ↑ ↑ ↑ [52,76] IL-6 no ↑ ↑ ↑ [52,69,70,77] IL-8 no ↑↑ ↑ ↑ [51,69,77,78] IL-2 no ↑ ↑ not known [52,79] IL-4 no ↑ ↑ not known [52,80] IL-5 no ↑ ↑ not known [52] MCP-1 no ↑ ↑ ↑ [49,77] TNF-α no ↑↑ ↑ ↑ [51,52,70] sTNF-R no ↑ ↑ not known [49] RANTES no ↑ ↑ ↑ [49,81] IP-10 no ↑ ↑ ↑ [49,77,82] GM-CSF no ↑ ↑ not known [49,69] PEDF ↔vitreous ↔serum ↑active PDR ↓inactive PDR ↓vitreous [12,83–86] IGF-1 no conﬂicting results ↑ not known [87–89] PlGF no no ↑ ↑ [90] IL-10 no no ↑ ↑ [52,69,76] Complement factors no no ↑ ↑ [17,63] bFGF not known not known conﬂicting results conﬂicting results [89,91] CD40 no ↑ ↑ not known [92] HIF-1α not known ↑ ↑ not known [93,94] 3.3. 3.2. Evidences of Inflammation in Diabetic Retinopathy Pathogenesis 3.2. Evidences of Inﬂammation in Diabetic Retinopathy Pathogenesis Contribution to Vascular Pathology Factors Diabetic Retinopathy Stage References Diabetics without DR NPDR PDR DME AQP4 ↑ ↑ not known not known [47] GFAP ↑ ↑ not known not known [47] VEGF ↑serum ↔vitreous ↑ ↑ ↑ [68–70] IFN-γ ↑ ↑ ↑ ↑ [49,52,71,72] IL-1α ↑ ↑ ↑ ↑ [49,69] IL-3 ↑vitreous ↓serum ↓vitreous & serum ↓serum ↑aqueous no change vitreous [49,73–75] s-IL2R ↑ ↑ ↑ not known [56,73] MCP-2 ↑ ↑ not known not known [49] ICAM-1 ↑serum ↑serum ↑ ↑ [68,70,71] IL-1β no ↑ ↑ ↑ [52,76] IL-6 no ↑ ↑ ↑ [52,69,70,77] IL-8 no ↑↑ ↑ ↑ [51,69,77,78] IL-2 no ↑ ↑ not known [52,79] IL-4 no ↑ ↑ not known [52,80] IL-5 no ↑ ↑ not known [52] MCP-1 no ↑ ↑ ↑ [49,77] TNF-α no ↑↑ ↑ ↑ [51,52,70] sTNF-R no ↑ ↑ not known [49] RANTES no ↑ ↑ ↑ [49,81] IP-10 no ↑ ↑ ↑ [49,77,82] GM-CSF no ↑ ↑ not known [49,69] PEDF ↔vitreous ↔serum ↑active PDR ↓inactive PDR ↓vitreous [12,83–86] IGF-1 no conﬂicting results ↑ not known [87–89] PlGF no no ↑ ↑ [90] IL-10 no no ↑ ↑ [52,69,76] Complement factors no no ↑ ↑ [17,63] bFGF not known not known conﬂicting results conﬂicting results [89,91] CD40 no ↑ ↑ not known [92] HIF-1α not known ↑ ↑ not known [93,94] 3.3. Contribution to Vascular Pathology 3.3. Contribution to Vascular Pathology 3.3. Contribution to Vascular Pathology Evidence of a chronic low-grade inﬂammation of the retina of patients with advanced DR raised the question of its involvement in the development of edema and pathogenic vascularization [95,96]. Evidence of a chronic low-grade inﬂammation of the retina of patients with advanced DR raised the question of its involvement in the development of edema and pathogenic vascularization [95,96]. 7 of 31 Int. J. Mol. Sci. 2018, 19, 942 Upregulated pro-inﬂammatory cytokines in the diabetic environment may directly induce vessel formation via engagement of target endothelial cells and indirectly, by inducing endothelial cells to produce pro-angiogenic mediators [97–99]. Endothelial cells are extremely susceptible to cytokines, especially IL-1β, TNF-α, and IFN-γ, which then induce production of endothelial cell–derived cytokines IL-8, MCP-1, and Regulated and normal T cell expressed and Secreted (RANTES). In vitro studies demonstrated that endothelial cells respond to cytokines rather than high glucose for induction of inﬂammatory pathways and apoptotic changes, suggesting that diabetes–related endothelial injury is primarily due to glucose-induced cytokine released by neighboring cells rather than a direct effect of hyperglycemia on endothelial cells themselves [100]. Upon cytokine stimulation they also secrete intracellular adhesion molecules such as ICAM-1 and VCAM-1, resulting in attraction of leukocytes, a process called leukostasis [101]. Indeed, animal studies have shown that 4 weeks after diabetes induction by streptozotocin-injection, before any vascular defects can be recorded, leukocytes start to adhere to the retinal capillaries with subsequent migration into the retina [102,103]. As a result, it has been proposed that the vascular wall integrity diminishes leading to increased vascular permeability. This allows extravasation of vascular ﬂuid and migration of additional immune cells (neutrophils, monocytes) into the tissue. Concurrently, loss of capillary pericytes causes endothelial cell degeneration, which has been associated with accumulation of vacuoles and debris in the basement membrane resulting in its thickening, and ultimately vascular lumen occlusion [104–107]. The resulting retinal ischemia and hypoxia are strong stimulus for endothelial and glial cells to promote the expression of VEGF and other pro-inﬂammatory cytokines such as TNF-α, IL-6, and IL-1β, an effect which is in part mediated trough activation of the transcription factor hypoxia-inducible factor 1 (HIF-1) in the cells [108,109]. As inﬂammation increases in the diabetic retina it does so in a self-propagating manner. Indeed, among the ischemia-induced factors are chemokines such as MCP-1, responsible for attracting macrophages into less perfused areas [110]. 3.4. Contribution to Neurodegeneration In addition to the vascular perturbations, the neurosensory retina is profoundly altered in diabetes. An emerging issue in DR research is the focus on the precise relationship between inﬂammatory alterations and the loss of neuronal function. VEGF, which is a mediator of inﬂammation and angiogenesis, positively affects neuronal growth, differentiation and survival [122]. In vitro VEGF stimulates axonal outgrowth, improves the survival of ganglion neurons, and can rescue cerebral neurons from apoptosis induced by serum withdrawal. In vivo VEGF coordinates migration of motor neuron soma and local delivery of VEGF prolongs motor neuron survival [123]. The receptors for VEGF are present in normal retinal neuronal cells, indicating a possible functional role for VEGF in the neural retina [124]. Gene expression studies in the brain and retina also suggest that VEGF is upregulated by hypoxia preconditioning, a brief ischemic episode that protects neurons, against subsequent prolonged ischemia-reperfusion-related damage [123]. Aside from VEGF, insulin is another obvious pro-survival factor involved in diabetes and an interesting target to promote retinal neuron survival. Indeed, several groups have demonstrated that systemic and local administration of insulin can rescue retinal neurons from cell death in the diabetic rat retina [24,125]. Other neuroprotective factors include neuroprotectin D1 (NPD1) [126], brain-derived neurotrophic factor (BDNF) [127], GDNF [128], CNTF [129] and NGF [130], all of which have been suggested to be involved in the neurodegenerative and inﬂammatory process that occurs in DR. Early on, increased cytokine/growth factor expression may serve an adaptive function to maintain neuronal function but, over time, becomes maladaptive, at which point the role of VEGF and other factors is impaired and can lead to alteration of the BRB and induction of inﬂammatory responses. Evidence further indicates that inﬂammatory processes themselves are activated in retinal neurons in response to diabetes. Hyperglycemia has been demonstrated to induce a pro-inﬂammatory phenotype in retinal neurons and their supporting glia in vitro [131,132]. While cytotoxic effects of high glucose treatment itself cannot be completely excluded, it has been demonstrated, that also in vivo hyperglycemia increased circulating cytokine concentrations by an oxidative mechanism in diabetic patients [133]. This suggests a causal role for hyperglycemia in the immune response associated with diabetes. In addition, immunohistochemical studies demonstrated translocation of NF-κB subunits into nuclei of retinal neurons in STZ-induced diabetic rats, suggesting that this pro-inﬂammatory transcription factor is also activated in neurons during diabetes [134]. 3.3. Contribution to Vascular Pathology Hypoxia-activated macrophages, glial cells and microglia cells produce TNF-α and IL-6 and other inﬂammatory cytokines and growth factors, which in turn stimulate further release of MCP-1, IL-6 and VEGF by endothelial cells, potentially resulting in increased vascular permeability as seen in NPDR and DME [21,110]. Angiogenic responses of endothelial cells are regulated by inﬂammatory cytokines and growth factors as well, resulting in the development of new vessel in PDR. The importance of inﬂammation for this phenomenon has been demonstrated by the fact that blocking inﬂammation reduces neovascularization. In a murine model of choroidal neovascularization, inhibition of monocyte recruitment by deleting MCP-1 [111] or deletion of ICAM-1 or CD18 led to signiﬁcant inhibition of neovascularization [112]. Similarly, inhibition of Cyclooxygenase-2 (COX-2), which normally generates prostanoids, reduced the production of VEGF and subsequently vascular leakage in an animal model of DR. It also reduced retinal neovascularization in rodent models of ischemic proliferative retinopathy [113,114]. In addition, High-mobility group box-1 (HMGB1), a DNA-binding protein which facilitates gene transcription and which can be actively secreted by activated monocytes, macrophages and retinal endothelial cells has been linked to inﬂammation and angiogenesis in DR [115]. Extracellular HMGB1 functions as a pro-inﬂammatory cytokine and induces the expression of adhesion molecules and other cytokines and chemokines [116,117]. It also directly induces vessel formation by targeting endothelial cells or indirectly by induction of pro-angiogenic factors in endothelial cells and leukocytes [115]. Furthermore, VEGF itself also serves as pro-inﬂammatory molecule by promoting the expression of other pro-inﬂammatory cytokines such as I-CAM, MIP-1α, MCP-1 and IL-8 [60,118–120]. A role for inﬂammation in the development of pathological retinal neovascularization in DR was also seen in the rat model of oxygen-induced retinopathy (OIR), which utilizes hypoxia to generate neovascularization and capillary non-perfusion, thus mimicking what it seen in human patients with PDR. Gene expression of NF-κB and protein expression of IL-6 and TNF-α were signiﬁcantly increased in the retinas of OIR rats compared to controls [121]. Taken together, studies in the OIR models strongly support an interconnection between vascular alterations and inﬂammation, not only in the early stages of DR, but also in later stages of the disease, including neovascularization and edema. Int. J. Mol. Sci. 2018, 19, 942 8 of 31 3.4. Contribution to Neurodegeneration Recent reports further provided evidence, that photoreceptors contribute to the inﬂammatory response in DR [135]. They are highly oxygen demanding cells and hence have shown to be the main sources oxidative stress in the retina in DR [136]. Moreover, it has been demonstrated, that photoreceptors themselves produce inﬂammatory cytokines such as ICAM-1, COX-2 and iNOS in experimental diabetes. They further release soluble factors in vitro that stimulate TNF-α production in leukocytes and endothelial cells, contributing to the vascular cell death in DR [137]. Consequently, pharmacologic interventions speciﬁcally targeting inﬂammation have been shown to reduce diabetes-induced neurodegeneration in animals studies and patients with DR. Yang et al., demonstrated that treatment with baicalein, a herbal medicine compound, suppressed diabetes-induced inﬂammatory cytokine upregulation, leading to reduced vascular leakage and ganglion cell loss in the retinas of diabetic rats [138]. The neuroprotective actions of the tetracycline derivative minocycline are mediated by the inhibition of microglial activation and of caspase-3 activation in diabetic rats [21]. Furthermore, doxycycline, another tetracycline derivative was able to prevent neuronal function loss, assessed by frequency doubling perimetry, in patients with NPDR [139]. Despite the above success, other studies have demonstrated that diabetes associated neurodegeneration is not exclusively due to inﬂammation. Cluster of differentiation (CD) 40, a TNF receptor, which is expressed on various hematopoietic and non-hematopoietic cells, is expressed by MGCs in the retina [140]. Interaction of CD40 with his ligand CD154 not only regulates cellular and humoral immunity [141], but also activates inﬂammation by activating microglial cells [142]. Of note, inhibition of microglial activation in diabetic CD40 knockout (KO) mice prevented capillary 9 of 31 Int. J. Mol. Sci. 2018, 19, 942 degeneration, but despite a suppressed inﬂammatory response, the mice maintained ERG b-wave defects [143]. Similarly, diabetic mice deﬁcient in nitric oxide generating inducible nitric oxide synthase (iNOS) did not develop the expected degeneration of retinal capillaries, leukostasis as well as ganglion cells loss, however they still presented with retinal functional impairment recorded by ERG [144]. More recently, inhibition of the receptor for advanced glycation end products [36] signaling pathway, which has been demonstrated to induce inﬂammatory changes in DR [145], did not protect from vision loss in diabetic animals [146]. These studies strongly support the notion, that inﬂammatory processes contribute, but not cause the development of DR. 4. Role of Glial Cells in Diabetic Retinopathy The two main retinal glial cells are Müller glial cells (MGCs) and astrocytes. Each cell type differs markedly in distribution, morphology, and function. Together with microglia, the main resident immune cells in the retina, they not only provide structural support, but they are also involved in maintaining the complex homeostasis of the retina by regulating the metabolism, the phagocytosis of debris and the cycling of neurotransmitters and trophic factors (Figure 3). One of the ﬁrst signs of inﬂammation in DR is the activation of glial and microglial cells. The conventional view is that microglia are the ﬁrst responders, initiating an inﬂammatory response that participates to triggering MGCs gliosis [148]. Early histopathological studies reported that both macroglial and microglia cells show early structural changes after 4 weeks of experimental diabetes in rats. These studies originally suggested that diabetes affected the density of microglia and MGCs and that all glial cells sense metabolic changes similarly and at the same time [61]. However, most recently, several studies in animal models and post-mortem human tissues have suggested that the rapid microglial activation and its typical morphological changes can be detected prior to macroglial activation [149]. This notion is also supported by additional histopathologic studies showing that many inﬂammatory molecules, such as TNF-α, can be detected early in the diabetic retina, often in association with microglia [138]. However, using mice with CD40 expression restricted to MGCs, Portillo et al., showed that CD40 in MGCs is sufﬁcient to upregulate retinal inﬂammatory markers by microglia in experimental diabetes [142], supporting that the activation of microglial cells is, at least in part, dependent on other cell types of the retina. Alternatively to the CD40 dependent activation pathway, factors such as free fatty acids that are released by compromised neurons represent another mean for initial microglial activation [149]. Of note, other retinal cell types such as endothelial cells, astrocytes and neurons can respond to the initial activation of microglial cells by themselves participating and amplifying the inﬂammatory response. A support for this theory comes from the observation, that in vitro astrocytes require microglial presence to respond to inﬂammatory stimuli [150]. Early glial cell activation beneﬁts the stressed retina, since activated glia phagocytose apoptotic cells, clear debris and cytotoxins, and secrete neurotrophic factors [151]. However, chronic gliosis in DR is detrimental, as activated glia may not properly regulate retinal blood ﬂow or maintain the BRB. 3.4. Contribution to Neurodegeneration In this context other pathways such as oxidative stress have been shown to be important in the development of DR [147]. Furthermore, the initiation and regulation of inflammation is a considerably complex mechanism, e.g., many cytokines are known to activate NF-κB and other pro-inflammatory mediators; thus, inhibiting one component of this cascade will only be partially effective. Nevertheless, from a therapeutic point, it is likely that identifying and halting key steps of the inflammatory process contributing to tissue destruction will help to control retinopathy. A more in-depth analysis of the role of inflammation in the neuro-glial crosstalk in DR will be discussed thereafter. 4.1. Microglia 4.1. Microglia Microglia cells are the resident immunocompetent cells in the central nervous system. The most reliable characteristics differentiating microglia from invading monocytes are a low CD45 expression and a highly ramified, dendritic morphology [152]. In the developing retina, microglia are involved in the pruning of neuronal and vascular networks through the phagocytic removal of apoptotic cell debris [31,153]. In the adult retina, ramified (‘‘resting”) microglia reside in the inner and outer plexiform layers and they are an important source of neurotrophic factors that support neuronal survival. Resting microglia continuously monitor their environment and when activated, they shift towards an amoeboid morphology [154]. Microglia cells are the resident immunocompetent cells in the central nervous system. The most reliable characteristics differentiating microglia from invading monocytes are a low CD45 expression and a highly ramiﬁed, dendritic morphology [152]. In the developing retina, microglia are involved in the pruning of neuronal and vascular networks through the phagocytic removal of apoptotic cell debris [31,153]. In the adult retina, ramiﬁed (“resting”) microglia reside in the inner and outer plexiform layers and they are an important source of neurotrophic factors that support neuronal survival. Resting microglia continuously monitor their environment and when activated, they shift towards an amoeboid morphology [154]. o a a a oe oi o p o ogy [ ] Although the precise mechanisms of glial activation in DR are not yet fully understood, it is likely that both genotypic variations and genetic susceptibility regulate individual microglial response in the course of DR [148]. Microglia, with their highly motile processes extending into the capillary wall, are assumed to be, together with Müller glia, the first detector of metabolic changes in diabetes [155]. They sense dyslipidemia, increases in ROS and cytokines, and the accumulation of AGEs. Amadori-glycated albumin, an AGE, was shown to specifically stimulate microglia to produce TNF-α in early stages of DR [156]. Once activated, microglia become mobile and migrate to the site of inflammation and will produce a wide range of pro-inflammatory cytokines, glutamate, ROS, nitrous oxide (NO) and proteases. Under chronic activation conditions, this cocktail can be extremely toxic to retinal ganglion cells, greatly exacerbating neuronal cell dysfunction [138,149]. 4. Role of Glial Cells in Diabetic Retinopathy Moreover, gliotic cells secrete inﬂammatory cytokines, cytotoxic molecules and VEGF perpetuating both, vascular dysfunction and neurodegeneration [151]. 10 of 31 10 of 30 Int. J. Mol. Sci. 2018, 19, 942 Int. J. Mol. Sci. 2018, 19, x FO Figure 3. Schematic of a mammalian retina depicting the interactions between glia, neurons and endothelial cells with special regard to the inflammatory chemokines involved. Blood vessels and endothelial cells in pink (9), leukocytes in purple (5), macroglial cells in green (7,8), microglia in brown (2,3) and neurons in blue (6a–c). Scheme showing kalium homeostasis, glutamate metabolism and the secretion of trophic factors, chemokines and interleukins. AGEs, advanced glycation end products. Figure 3. Schematic of a mammalian retina depicting the interactions between glia, neurons and endothelial cells with special regard to the inﬂammatory chemokines involved. Blood vessels and endothelial cells in pink (9), leukocytes in purple (5), macroglial cells in green (7,8), microglia in brown (2,3) and neurons in blue (6a–c). Scheme showing kalium homeostasis, glutamate metabolism and the secretion of trophic factors, chemokines and interleukins. AGEs, advanced glycation end products. Figure 3. Schematic of a mammalian retina depicting the interactions between glia, neurons and endothelial cells with special regard to the inflammatory chemokines involved. Blood vessels and endothelial cells in pink (9), leukocytes in purple (5), macroglial cells in green (7,8), microglia in brown (2,3) and neurons in blue (6a–c). Scheme showing kalium homeostasis, glutamate metabolism and the secretion of trophic factors, chemokines and interleukins. AGEs, advanced glycation end products. Figure 3. Schematic of a mammalian retina depicting the interactions between glia, neurons and endothelial cells with special regard to the inﬂammatory chemokines involved. Blood vessels and endothelial cells in pink (9), leukocytes in purple (5), macroglial cells in green (7,8), microglia in brown (2,3) and neurons in blue (6a–c). Scheme showing kalium homeostasis, glutamate metabolism and the secretion of trophic factors, chemokines and interleukins. AGEs, advanced glycation end products. 4.2. Müller Glial Cells Müller glial cells are the major glial cell type in the mammalian retina, representing 90% of the retinal glia. They form the supporting architectural structure radially stretching across the entire thickness of the neuroretina and participate in forming the outer and inner limiting membranes [159]. Müller glial cells also strongly link with retinal vessels and with the RPE, which establish connections to the subretinal space and choroidal vasculature [160]. Since Müller cells have contact to virtually every cell type in the retina they are uniquely positioned to perform a wide variety of functions necessary to maintaining retinal homeostasis. In the healthy retina, they recycle neurotransmitters, including glutamate thus preventing excitotoxicity; redistribute ions by spatial buffering, participate in the retinoid cycle, and regulate nutrient supplies [160,161]. Any disturbance to the retinal environment will inﬂuence Müller cells, which in turn will affect the entire retina [29]. Müller glia cells are deeply involved in DR, showing morphological and functional alterations from the early phases of the disease. The most striking example being that their size signiﬁcantly increases in rodent models of diabetes, while a progressive increased expression of the stress marker GFAP becomes prominent in tissues from both animal models and diabetic donors [61]. In addition to GFAP upregulation, Müller cells acquire a complex and speciﬁc reactive phenotype, which is also characterized by induction of acute-phase response proteins and other inﬂammatory related genes [22]. In vitro studies have provided compelling evidence that Müller cells are a signiﬁcant source for many growth factors and cytokines, when stimulated with stress conditions, including elevated glucose levels [132]. Considering that most of the growth factors, cytokines, and chemokines released by Müller cells have been identiﬁed in the vitreous of diabetic patients, it has led to the hypothesis that Müller cells contribute to the overall synthesis of these factors in vivo [162]. Non-targeted discovery approaches assessing the gene expression proﬁle of Müller cells after 6 months of STZ induced diabetes, revealed that among 78 altered genes, one third are associated with inﬂammation, including complement factors, VEGF, ICAM-1 and IL-1β [163]. Of note, some of these cytokines in turn have been shown in other studies to stimulate production of other cytokines by Müller glia, suggesting a possible ampliﬁcation effect. [164]. 4.2. Müller Glial Cells Taking into account their central role in maintaining the homeostasis in the retina and their contribution to the inﬂammatory response in DR, understanding Müller cell functions within the retina and restoring such function in DR could become a cornerstone for developing effective therapies to treat diabetic retinopathy. 4.1. Microglia 4.1. Microglia Indeed, prolonged activation of microglia is associated with high levels of glutamate, iNOS and pro- Although the precise mechanisms of glial activation in DR are not yet fully understood, it is likely that both genotypic variations and genetic susceptibility regulate individual microglial response in the course of DR [148]. Microglia, with their highly motile processes extending into the capillary wall, are assumed to be, together with Müller glia, the first detector of metabolic changes in diabetes [155]. They sense dyslipidemia, increases in ROS and cytokines, and the accumulation of AGEs. Amadori-glycated albumin, an AGE, was shown to specifically stimulate microglia to produce TNF-α in early stages of DR [156]. Once activated, microglia become mobile and migrate to the site of inflammation and will produce a wide range of pro-inflammatory cytokines, glutamate, ROS, nitrous oxide (NO) and proteases. Under chronic activation conditions, this cocktail can be extremely toxic to retinal ganglion cells, greatly exacerbating neuronal cell dysfunction [138,149]. Indeed, prolonged activation of microglia is associated with high levels of glutamate, iNOS and pro-inflammatory cytokines Int. J. Mol. Sci. 2018, 19, 942 11 of 31 such as IL-1β, IL-6, IL-12 and TNF-α which can culminate in the activation of caspases, exacerbating retinal neuron cell death retina [157]. Case studies exclusively using histological techniques on retinal cross-sections revealed increas numbers of moderately hypertrophic microglial cells in the plexiform layers of NPDR patients [1 Case studies exclusively using histological techniques on retinal cross-sections revealed increasing numbers of moderately hypertrophic microglial cells in the plexiform layers of NPDR patients [158]. They further showed that in tissues from PDR patients, clusters of microglial cells could be found surrounding ischemic areas and new vessels, while suggesting an even more signiﬁcant rise in their total number [149]. 4.4. Role of Inﬂammation in the Neuro-Glial Crosstalk in DR Glial cells and neurons are intimately associated to make-up the neuro-glial unit. Glial cells are the interface between the neurons and the vasculature and are thus key regulators of neuronal function. Müller glial cells convey circulating glucose into the retina for ATP production and provide intermediary compounds such as lactate to neurons [64]. They play multiple key roles for retinal tissue homeostasis, from storing glycogen for conversion to lactate, to synthesizing retinoic acid from retinol, regulating extracellular ion concentrations to modulate plasma membrane polarization/depolarization, participating with neurons in the glutamate/glutamine cycle to control neurotransmission, and protect neurons from glutamate excitotoxicity [171]. Another important function of macroglial cells is to redistribute the neuronal metabolic by-products such as carbon dioxide and water from glucose metabolism, but also potassium and neurotransmitters associated with neuronal activity, into the blood and vitreous [161]. MGCs maintain proper retinal function by participating in a process known as “potassium spatial buffering”, a process that redistributes and normalizes K+ in the surrounding microenvironment to avoid prolonged accumulation of K+ [172]. This response is coupled to the regulation of water homeostasis by the glial water channels aquaporins (AQP) such as AQP4 [173]. Macroglial cells, besides supporting the synaptic activity, maintain the integrity of the BRB and regulate the vasoconstriction related to neuronal activity, a process named neurovascular coupling by the release of neuroactive and vasoactive substances [174]. Macroglial and microglial cells also regulate the survival of neurons by a molecular network made up of several neurotrophic factors (BNDF, CNTF, GDNF; NGF, NT-3, and bFGF) [148]. Microglial and macroglial cells are further responsible for cellular debris phagocytosis and response to immunological damage [175]. They also play an active role engulﬁng synaptic material in a process termed synaptic pruning and referred to as the innate surveillance function of resident microglia [176,177]. Diabetes also affects the neuro-glial unit by disrupting the communication between neurons and glia. In DR, accumulating evidence over the past decades have revealed that dysfunctional neuro-glial crosstalk, in part associated with inﬂammation, plays a critical role in the early course of the disease [138,178]. One of the ﬁrst altered structural alterations in experimental and human DR is reactive gliosis of macroglial cells, mostly in the initial stages of DM [61]. Reactive gliosis may be interpreted as an effort to limit the extension of tissue damage but sustained inﬂammation in DR lead to the more severe forms of reactive gliosis [179]. 4.3. Astrocytes Like Müller cells, astrocytes are connected to retinal blood vessels and neurons and play a critical role in maintenance of the BRB, including regulation of blood ﬂow [165]. Astrocytes also provide energy substrates to neurons and regulate the production of trophic factors and antioxidants. In contrast to Müller cells, they are largely restricted to the nerve ﬁber layer and ganglion cell layer. The distribution of retinal astrocytes is strikingly correlated with the presence and distribution of retina blood vessels, so vascularized areas of the retina are rich in astrocytes, while avascular zones such as the fovea, contain no astrocytes [166]. In response to injury or disease, they have been shown to upregulate the expression of various genes encoding cytokines, chemokines and elements of the complement Int. J. Mol. Sci. 2018, 19, 942 12 of 31 cascade. Such response has been suggested to participate in compromising the integrity of the BRB and promoting retinal degeneration [167]. In response to the dysmetabolic and hypoxic diabetic environment, astrocytes become activated, also called reactive, and produce a variety of pro-inflammatory cytokines, such as IL-6, IL1β, IL-8, COX-2, transforming growth factor-α (TGF-α), epidermal growth factor (EGF), macrophage inflammatory protein 2α (MIP-2α) and VEGF [168,169]. Apart from inflammatory cytokines reactive astrocytes can secrete chemokines, including MCP-1, C-C motif chemokine ligand (CCL) 5, CCL20, C-X-C motif chemokine ligand (CXCL) 10, CXCL12, CXCL1, CXCL2, and CX3CL1 [170]. These chemokines are involved in the recruitment of microglia, monocytes/macrophages, T-cells, and dendritic cells, amplifying the inflammatory response. Over time astrocyte number seem to decrease in DR patients, as demonstrated by decreased GFAP expression, which then disrupts the inner BRB, contributing to vascular leakage in the retina [61]. 4.4. Role of Inﬂammation in the Neuro-Glial Crosstalk in DR Macroglial cells undergo further hypertrophy, lose their functionality, ultimately forming glial scars that are inhibitory to axonal regeneration and neuronal survival [162,180]. Microglial activation is the main mechanism by which neuroinflammation starts in response to nervous tissue perturbations [15]. Microglial activation in DR acts as a double-edged sword, as its primary function is neuroprotection. On the other hand, activated microglia produce pro-inflammatory mediators such as IL-1β, IL-3, IL-6, TNF-α, VEGF, lymphotoxin, MIP-1α, MMPs, NO, ROS and complement factors, primarily promoting neuronal cell death when sustained. [138,149,157]. The mechanism by 13 of 31 Int. J. Mol. Sci. 2018, 19, 942 which cytokines contribute to neural apoptosis is not clear but may involve the induction of excitotoxicity, oxidative stress or mitochondrial dysfunction [181–183]. A representative example of a neuron-microglia interaction is the FKN/CX3CR1 system, which provides a mechanism to modulate microglial activation by neurons. Fractalkine (CX3CL1 or FKN) exists on neuronal membranes and functions by signaling through its unique receptor CX3CR1 present on microglia. Several reports support the notion that FKN exerts an inhibitory signal on microglia [184]. It has been demonstrated, that increased inﬂammatory microglial responses in absence of the fractalkine receptor contribute to inﬂammatory-mediated damage to neurons in the diabetic retina [65]. Accordingly, microglial receptor proteins appear as important targets for the development of drugs to prevent or manage retinal neuroinﬂammation. Next to microglia, astrocytes react becoming activated during diabetes and undergo a series of changes as mentioned earlier such as secretion various cytokines, chemokines, MMPs and elements of the complement cascade, contributing to neuronal degeneration [138,167,168]. Notably, Müller cells exposed to hyperglycemic conditions also produce increased levels of pro-inﬂammatory molecules such as IL-1β and TNF-α, which then induce expression of pro-death cytokine IL-8 [164]. Similar mechanisms have been proposed for other cytokines secreted by Müller cells such as IL-6, NO and COX-2 [132]. Water accumulation in retinal neurons and glial cells is another pathogenic factor involved in retinal degeneration during DR. Müller cells adapt their morphology to the increased size of activated retinal neurons by extending and thinning the inner stem process. The water ﬂux through AQP4 is involved in the rapid volume regulation of retinal Müller cells in response to osmotic stress. Diabetes is known to enhance the amount of retinal expression of AQP4 and down-regulation of AQP4 exacerbated experimental DR through increased expression of pro-inﬂammatory factors [185]. 5.1. Alpha-Crystallins The two small heat shock proteins α-crystallins, αA and αB, [193,194] have been widely studied for their roles in the lens, but we are just starting to understand their function in the retina. Recent studies revealed that α-crystallins are rapidly upregulated in models of acute retinal injury, including light toxicity and trauma, suggesting that α-crystallins play a role in the retina’s response to cellular stress and damage [195–197]. We recently demonstrated that these chaperones were highly expressed in both RGCs and Müller glial cells of diabetic animals and human patients with DR [198]. Of note, αA-crystallins not only inhibit pro-apoptotic pathways in retinal neurons, but also act as a secreted pro-survival factor [198] and modulator of the expression of inflammatory molecules by glial cells (unpublished data), for the first time establishing a direct link between α-crystallins and neuro-inflammation. Consistent with a critical role in glial cells, α-crystallins have been reported to be upregulated in astrocytes of patients with Alexander disease and microglial cells of patients with Alzheimer’s disease [199–201]. It has been hypothesized that the increased expression of αB-crystallin is part of the normal stress response to regulate aberrant protein aggregation and may prevent apoptosis in the face of the increased phagocytosis and protein aggregate load [202]. Overall, α-crystallin chaperones affect microglial and astrocytic activation in an anti-inflammatory manner resulting in decreased cytokine production and tempering of the innate immune response. Further research, on the protective function of αA-crystallin should govern the development of specifically engineered crystallin proteins, which could be applied to the treatment of diabetic retinopathy and other neurodegenerative conditions. 5.2. Matrix Metalloproteinases Matrix metalloproteinases constitute a large family of secreted and membrane associated zinc-dependent proteinases that degrade at least one component of the extracellular matrix (ECM). They are known to execute functions in motility, cell growth, injury response, and remodeling of the ECM by both, degrading it and controlling proteolysis of specific targets including cytokines, growth factors and adhesion molecules [203,204]. MMPs are highly sensitive to oxidative stress in the diabetic environment, with MMP-2 and MMP-9 being induced by increased ROS [57,205,206]. It has been suggested that these MMPs, especially MMP-9, can increase the inflammatory response by degrading some of the major components of the basement membrane, enabling invasion of the injured tissue by circulating immune cells. MMP-9 knockout-mice indeed showed significant reductions in the expression of inflammatory markers such as MIP-1α, MIP-1β, and MCPs after spinal cord injury [207]. In addition, activated MMPs also contribute directly to angiogenesis and the increase in vascular permeability in DR by proteolytic degradation of occludin, disruption of the tight junction complex [57,208], and increased tissue levels of VEGF [209], making them interesting potential therapeutic targets. 4.4. Role of Inﬂammation in the Neuro-Glial Crosstalk in DR Therefore, regulation of retinal function by AQP4 may attenuate diabetic retinopathy, offering a promising therapeutic strategy for diabetic retinopathy [185]. Neurodegeneration leads to the alteration of expression of growth factors (e.g., VEGF) by glial cells [186]. The release of VEGF by Müller cells under conditions stimulating gliosis is especially interesting, as it acts in two directions. VEGF signaling in Müller cells may represent a stress-responsive neuroprotective mechanism, particularly under hypoxic conditions. VEGF receptor 2 (VEGFR2)-mediated signaling in MGCs has been also shown to stimulate the production of BDNF and GDNF [178]. As a result, the loss of VEGFR2-mediated signaling in MGCs caused a signiﬁcant elevation of apoptotic MGCs and neurons during experimental DR [178]. On the other hand sustained VEGF release also acts as a pro-inﬂammatory molecule by inducing the expression of other cytokines, thus exacerbating retinal neurodegeneration [187]. Conversely a growing body of evidence indicates that hyperglycemia-induced downregulation of a number of important neurotrophic factors, such as NGF, Pigment epithelium-derived factor (PEDF), CNTF, interphotoreceptor retinoid-binding protein (IRBP) and somatostatin, the loss of which may have a direct impact on neuronal survival [188]. Factors, such as PEDF, functions as an anti-inflammatory and anti-oxidative agent by inhibiting the activation of the NF-kB and Extracellular signal-regulated kinase 1 (ERK1) pathways. When secreted by Müller cells, this factor acts directly on PEDF receptors at the surface of RGCs [189,190]. PEDF levels were found to be decreased in cultured MGCs subjected to hyperglycemia and mild hypoxia [191,192]. However, under severe hypoxic conditions, PEDF was upregulated, potentially reflecting an attempt to rescue retinal neurons [192]. Why this observation does not translate in vivo, where decreased vitreous levels of PEDF are demonstrated also at later stages of DR such as PDR is not clear yet [83]. CNTF has been shown to directly prevent photoreceptor apoptosis and indirectly rescue neurons by stimulation of Müller cells to produce photoreceptor survival factors. Altogether, studies clearly show how modulation of the inﬂammatory response by glial cells might act as an important target to prevent early neuronal damage in DR. Int. J. Mol. Sci. 2018, 19, 942 14 of 31 6.2.1. Nonsteroidal Anti-Inﬂammatory Drugs (NSAID) Clinically the beneﬁcial effect of NSAIDs, that inhibit COX enzyme-mediated ICAM-1 expression and leukostasis [222,223], has been suggested in a study reporting reduced incidence of DR in patients taking salicylates for the treatment of rheumatoid arthritis [20]. This ﬁnding encouraged the launch of clinical trials, which showed the development of retinal microaneurysms is only signiﬁcantly attenuated in patients with early stage of DR when treated with high dose of aspirin (900 mg/day) [224,225]. Alternatively, while systemic administration of speciﬁc COX-2 inhibitors is made impossible by increased incidence of heart attacks and strokes [226], preclinical studies with topical administration were shown to reduce signs of DR [227,228]. 5.3. Toll-Like Receptors Toll-like receptors are pathogen-associated molecular pattern receptors that have been found on the surface of all cell types in the retina. Their activation results in the production of cytokines/chemokines in an NF-κB dependent manner [210]. Among the various TLRs, TLR-2 and TLR-4 have been most consistently associated with type 1 and type 2 diabetes. For example, genetic polymorphism in the TLR-4 gene is strongly associated with insulin resistance levels in patients with type 2 diabetes [211]. Additionally, TLRs also promoted the secretion of angiogenic growth factors in arterial endothelial cells [212] and led to choroidal neovascularization in vivo [213]. Human microvascular retinal endothelial cells demonstrated increased TLR-2 and TLR-4 expression when exposed to high glucose, an observation also associated with increased expression of pro-inﬂammatory molecules. Supporting a regulatory role of TLRs in this process, this effect was blunted by using a TLR4/2 inhibitory peptide or antioxidant treatment [214]. Int. J. Mol. Sci. 2018, 19, 942 15 of 31 15 of 31 5.4. Complement System While diabetes is not considered a complement-mediated disease, some data suggest a link between DR progression and complement dysregulation. Evidence for such a link comes from the observation of increased expression of several complement factors in the vitreous of patients with DR. Among over 30 small proteins and protein fragments identiﬁed as part of the complement pathway, some of the key regulators have been reported increased in PDR patients: C3, C4b, C9, and factor B (CFB) [215,216]. C5 cleavage, along with C3, is considered one of the two critical steps for the full activation of the complement pathways. A fully activated complement system results in the formation of the membrane attack complex (MAC or C5b-9) that can kill pathogens but also under certain conditions host cells, thus could play a role in neurodegeneration and vascular permeability. Additionally, cleavage products C3a and C5a have a chemotactic effect on neutrophils, mast cells, and lymphocytes, potentially exacerbating the inﬂammatory response. Finally, Müller glial cells constitutively express C5aR, the expression of which is further increased by high glucose exposure, and associated with release of IL-6 and VEGF by these cells, a mechanisms that could be involved in promoting the overt-inﬂammatory response observed in diabetes [217]. 6.1. Current Therapies Several therapeutic approaches are in use clinically to inhibit the development or progression of DR. Metabolic control (tight glycemic control, regulation of blood pressure and hyperlipidemia) is associated with a lower risk of retinopathy [218,219]. Specific therapeutic options for DR include laser coagulation, surgery and intravitreal injection of anti-VEGF agents. Alternatively, intravitreal injections of steroids have been shown to reduce vascular permeability, reduce the breakdown of the BRB, inhibit leukostasis, inhibit VEGF gene transcription and translation [220]. Because of an increased rate of elevated IOP and cataract formation with steroids, the use of intravitreal steroids in clinical practice is currently reserved as the second-line treatment in patients with previous cataract surgery [221]. Unfortunately, all these therapeutic interventions have signiﬁcant side effects and are only useful for patients with more advanced stages of DR, when vision is already compromised, and neurodegeneration already occurred. Considering the role of inﬂammation in the pathogenesis of DR, inhibiting the inﬂammatory pathway has been an appealing treatment option for DR in future practices. 6.2. Inﬂammation-Targeting Therapies under Development and Validation 6.2.4. The Renin-Angiotensin System (RAS) Specific blockade of the RAS with the Angiotensin II receptor type 1 (AT1R) blocker (Losartan, Candesartan) and angiotensin-converting enzyme inhibitor (Enalapril) has been shown to reduce the risk of onset and the progression of retinopathy in patients with type 1 and 2 diabetes [44,237,238]. In another clinical trial involving 285 type 1 diabetic patients with normotensive and normoalbuminuria, blockade of RAS with Losartan or Enalapril significantly reduced the progression of retinopathy by 70% and 65% respectively [238]. However, the Diabetic Retinopathy Candesartan Trials which enrolled 3326 patients with type 1 diabetes and 1905 patients with type 2 diabetes, did not observe such a dramatic effect. It has been demonstrated, that Candesartan treatment reduced the incidence of DR but did not prevent progression of DR [237]. Further studies are needed to resolve the discrepancy between the above trials and to fully evaluate the beneficial effect of RAS blockade before its clinical usefulness is fully demonstrated. 6.2.3. Inﬂammatory Growth Factors Angiopoietins represent a family of inflammatory growth factors that bind to the receptor tyrosine kinase Tie2 and are important modulators of angiogenesis. A recent study also reported that Ang-2 promotes adhesion of monocytes and the subsequent tissue inflammation by modulating the TNF-α-induced expression of ICAM-1 [235]. Thus, the Ang-2 pathway is currently being targeted in a recent ongoing clinical trial with a Tie-2 activator (AKB-9778, Aerpio Therapeutics, Cincinnati, OH, USA) in patients with NPDR, where improvements in DR severity will be evaluated. A previous study already demonstrated a significant benefit of AKB-9778 combined with anti-VEGF therapy over anti-VEGF monotherapy in the reduction of DME with a trend toward improved visual acuity [236]. 6.2.2. Blocking Inﬂammatory Molecules The effect of anti-TNFα therapy has been studied in a few clinical cases. Patients with refractory DME treated with intravitreal Etanercept (TNFα inhibitor) showed no statistically signiﬁcant improvement [229]. Inﬂiximab, a TNFα neutralizing antibody on the other hand improved visual acuity and reduced macular thickness in 4 patients who failed to improve in response to laser Int. J. Mol. Sci. 2018, 19, 942 16 of 31 photocoagulation treatment [230]. Larger trials are needed to determine the efﬁcacy of these drugs in DME patients. Alternatively, a pilot study on the efﬁcacy and safety of a selective IL-1β antibody, Canakinumab (Novartis, Basel, Switzerland), for the treatment of patients with proliferative DR showed stabilization but no regression of retinal neovascularizations, along with promising effects on diabetic macular edema reduction [231]. Similarly, a clinical trial in DME patients comparing an oral inhibitor targeting the receptors for MCP-1, CCR2/CCR5 (Pﬁzer, New York, NY, USA) to intravitreal anti-VEGF therapy showed non-inferiority of the study drug regarding gain in visual acuity and reduction in central retinal thickness [232]. Leukostasis, one of the early steps of inﬂammation, is dependent on speciﬁc integrins on the endothelium to trigger leukocyte adhesion and inﬁltration [233]. The DEL MAR Phase 2b trial using an integrin antagonist, Luminate, ALG-1001 (Allegro Ophthalmics, San Juan Capistrano, CA, USA) as sequential therapy to a single anti-VEGF injections in DME patients compared to anti-VEGF monotherapy reported sustained and equal visual acuity gains for both treatments [234]. 6.2.6. Photobiomodulation Photobiomodulation (PBM), also known as low level laser therapy or far-red to near-infrared (FR/NIR) light therapy, consists of series of brief illumination with speciﬁc wavelengths of light (600–1000 nm) from a laser or a light emitting diode [243] and results in the activation of signalling pathways within the cell. It has been demonstrated, that it effectively inhibits diabetes induced increase in superoxide production, leukostasis and ICAM-1 expression in STZ induced rats, which resulted in a signiﬁcant reduction in ganglion cell death and improvement of the photopic b wave ERG amplitude [244]. A subsequent case series by the same group including 4 patients with type 2 diabetes with non-center-involving DME underwent treatment with PBM twice a day for 2–9 months, demonstrated statistically signiﬁcant decrease in macular thickness by an average of 20%, while the non-treated eye of the same patients featured a slight increase in thickness [245]. Further investigations, using larger patient populations, will be necessary to conﬁrm the efﬁcacy, establish the optimal treatment dose and duration, as well as identify and address potential safety concerns. 6.2.5. Tetracyclines In addition to their antimicrobial activity, minocycline and doxycycline are known to possess several immunomodulatory and neuroprotective properties [239,240], such as inhibiting the production of NO, cyclooxygenases, prostaglandins, IL-1β and TNF-α and caspases [21,241]. A single-center phase I/II clinical trial in 5 patients with DME showed that minocycline as primary treatment was associated with improved visual function, reduced central macular edema, and vascular leakage [242]. Another clinical trial demonstrated an improvement of perimetric parameters in patients with severe non-proliferative or non-high-risk proliferative DR treated with doxycycline compared to patients who received placebo. Of note, visual acuity and anatomical parameters (such as DR severity levels) were equal among both groups, which may relate to a differential effect of tetracyclines on different stages of DR dysfunction [139]. Int. J. Mol. Sci. 2018, 19, 942 17 of 31 17 of 31 7. Conclusions This review summarizes recent clinical and laboratory ﬁndings suggesting a pivotal role of inﬂammation in the pathophysiology of early DR. Many studies carried out in diabetic patients and diabetic animal models have shown that the diabetic milieu provoke increased local expression of inﬂammatory molecules, such as cytokines, chemokines, and growth factors involved in the development of DR. Glial cells are critically located between vasculature and neurons of the retina, having a key role in closely regulating the retinal microenvironment, which is often responsible for early harm of neuroretina in the beginning stages of DR. Recent ﬁndings implicate that these cells also initiate the inﬂammatory cascade. New technologies able to detect early alterations of the neuroglial unit support the idea that changes in the metabolism of glia cells and subsequent damages of retinal neurons precede microvascular impairment. Additional work has now been performed that suggests that in this manner anti-inﬂammatory drugs are promising future therapies to address both, vascular alterations and neurodegeneration. However, studies are still needed to better understand the exact molecular mechanisms underlying ocular inﬂammation in patients with DR. Acknowledgments: This project and its authors were supported by a fellowship from the Deutsche Forschungsgemeinschaft RU 2020/2-1 (Anne Rübsam), Fight for Sight Grant-in-Aid, and NIH EY020895 (Patrice E. Fort). Patrice E. Fort is the guarantor of this work. We thank David Murrel for his assistance with the illustration. Author Contributions: Anne Rübsam and Sonia Parikh wrote the manuscript. Patrice E. Fort obtained funding, wrote and edited the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Klein, B.E. Overview of epidemiologic studies of diabetic retinopathy. Ophthalmic Epidemiol. 2007, 14, 179–183. [CrossRef] [PubMed] 1. Klein, B.E. Overview of epidemiologic studies of diabetic retinopathy. Ophthalmic Epidemiol. 2007, 14, 179–183. [CrossRef] [PubMed] 2. Kempen, J.H.; O’Colmain, B.J.; Leske, M.C.; Haffner, S.M.; Klein, R.; Moss, S.E.; Taylor, H.R.; Hamman, R.F.; Eye Diseases Prevalence Research Group. The prevalence of diabetic retinopathy among adults in the United States. Arch. Ophthalmol. 2004, 122, 552–563. [PubMed] 2. Kempen, J.H.; O’Colmain, B.J.; Leske, M.C.; Haffner, S.M.; Klein, R.; Moss, S.E.; Taylor, H.R.; Hamman, R.F.; Eye Diseases Prevalence Research Group. The prevalence of diabetic retinopathy among adults in the United States. Arch. Ophthalmol. 2004, 122, 552–563. [PubMed] p 3. Zhang, X.; Saaddine, J.B.; Chou, C.F.; Cotch, M.F.; Cheng, Y.J.; Geiss, L.S.; Gregg, E.W.; Albright, A.L.; Klein, B.E.; Klein, R. Prevalence of diabetic retinopathy in the United States, 2005–2008. JAMA 2010, 304, 649–656. [CrossRef] [PubMed] 4. AAO PPP Retina/Vitreous Panel, Hoskins Center for Quality Eye Care. Diabetic Retinopathy Ppp—Updated. 2016. Available online: http://www.aao.org/preferred-practice-pattern/diabetic-retinopathy-ppp--2014 (accessed on 30 October 2017). 5. Frey, T.; Antonetti, D.A. Alterations to the blood-retinal barrier in diabetes: Cytokines and reactive oxygen species. Antioxid. Redox Signal 2011, 15, 1271–1284. [CrossRef] [PubMed] 6. Zhang, X.; Zeng, H.; Bao, S.; Wang, N.; Gillies, M.C. Diabetic macular edema: New concepts in patho-physiology and treatment. Cell Biosci. 2014, 4, 27. [CrossRef] [PubMed] 7. Daruich, A.; Matet, A.; Moulin, A.; Kowalczuk, L.; Nicolas, M.; Sellam, A.; Rothschild, P.R.; Omri, S.; Gelize, E.; Jonet, L.; et al. Mechanisms of macular edema: Beyond the surface. Prog. Retin. Eye Res. 2017. [CrossRef] [PubMed] 8. Miller, J.W.; Adamis, A.P.; Shima, D.T.; D’Amore, P.A.; Moulton, R.S.; O’Reilly, M.S.; Folkman, J.; Dvorak, H.F.; Brown, L.F.; Berse, B.; et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am. J. Pathol. 1994, 145, 574–584. [CrossRef] [PubMed] 9. Ferrara, N.; Davis-Smyth, T. The biology of vascular endothelial growth factor. Endocr. Rev. 1997, 18, 4–25. [CrossRef] [PubMed] 10. Singer, M.A.; Kermany, D.S.; Waters, J.; Jansen, M.E.; Tyler, L. Diabetic macular edema: It is more than just VEGF. F1000Research 2016, 5. [CrossRef] [PubMed] 11. Gardner, T.W.; Abcouwer, S.F.; Barber, A.J.; Jackson, G.R. An integrated approach to diabetic retinopathy research. Arch. Ophthalmol. 2011, 129, 230–235. [CrossRef] [PubMed] 12. Funatsu, H.; Noma, H.; Mimura, T.; Eguchi, S.; Hori, S. Association of vitreous inﬂammatory factors with diabetic macular edema. Abbreviations AGE Advanced Glycation End-product AH Aqueous Humor Ang Angiopoietin AT1R Angiotensin II Receptor Type 1 AQP Aquaporin BDNF Brain-derived Neurotrophic factor bFGF Basic Fibroblast Growth Factor BRB Blood-retinal Barrier CCL C-C motif ligand CCR C-C receptor CD Cluster of differentiation CNS Central Nervous System CNTF Ciliary Neurotrophic Factor COX Cyclooxygenase 18 of 31 Int. J. Mol. Sci. 2018, 19, 942 CRP C-reactive Protein CXCL C-X-C motif ligand DR Diabetic Retinopathy DCCT Diabetes Control and Complications Trial DME Diabetic Macular Edema EGF Epidermal Growth factor ERG Electroretinogram ERK Extracellular Signal-Regulated Kinase FDA US Food and Drug Administration GDNF Glial-derived Neurotrophic factor GFAP Glial Fibrillary Acidic Protein GM-CSF Granulocyte Macrophage Colony-Stimulating Factor HGF Hepatocyte Growth Factor HIF Hypoxia-inducible Factor HMGB High Mobility Group Box ICAM Intracellular Adhesion Molecule IGF Insulin-like Growth Factor IFN Interferon IL Interleukin iNOS Inducible Nitrous Oxide Synthetase IP Interferon γ-induced Protein IRBP Interphotoreceptor Retinoid Binding Protein IRMA Intraretinal Microvascular Abnormality IVTA Intravitreal Triamcinolone Acetonide LIF Leukemia inhibitory factor LPS Lipopolysaccharide MCP Monocyte Chemoattractant Protein MIP Macrophage Inﬂammatory Protein MMP Matrix Metalloproteinase NFG Nerve Growth Factor NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NO Nitric Oxide NPDR Non-proliferative Diabetic Retinopathy NSAID Nonsteroidal Anti-Inﬂammatory Drug PAMP Pathogen-associated Molecular Pattern PDGF Platelet-derived Growth Factor PDR Proliferative Diabetic Retinopathy PEDF Pigment Epithelium-derived Factor PGE Prostaglandin PGF Placental growth factor PKC Protein Kinase C PPAR Peroxisome Proliferator-Activated Receptor PPV Pars Plana Vitrectomy RAS Renin-Angiotensin System RAGE Receptor for Advanced Glycation End-product RANTES Regulated and normal T cell expressed and secreted RGC Retinal Ganglion Cell ROS Reactive Oxygen Species RPE Retinal Pigment Epithelium SDF Stromal-derived Factor sTNF-R Soluble Tumor Necrosis Factor-Receptor STZ Streptozotocin TGF Transforming Growth Factor TLR Toll-like Receptor TNF Tumor Necrosis Factor UKPDS United Kingdom Prospective Diabetes Study VEGF Vascular Endothelial Growth Factor 19 of 31 19 of 31 Int. J. Mol. Sci. 2018, 19, 942 References Ophthalmology 2009, 116, 73–79. [CrossRef] [PubMed] 13. Elner, S.G.; Elner, V.M.; Jaffe, G.J.; Stuart, A.; Kunkel, S.L.; Strieter, R.M. Cytokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy. Curr. Eye Res. 1995, 14, 1045–1053. [CrossRef] [PubMed] 14. Mitamura, Y.; Takeuchi, S.; Matsuda, A.; Tagawa, Y.; Mizue, Y.; Nishihira, J. Monocyte chemotactic protein-1 in the vitreous of patients with proliferative diabetic retinopathy. Ophthalmologica 2001, 215, 415–418. [CrossRef] [PubMed] 15. Hernandez, Y.; Arora, S.; Erb-Downward, J.R.; McDonald, R.A.; Toews, G.B.; Huffnagle, G.B. Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis. J. Immunol. 2005, 174, 1027–1036. [CrossRef] [PubMed] 16. Abu El-Asrar, A.M.; Struyf, S.; Kangave, D.; Geboes, K.; Van Damme, J. Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy. Eur. Cytokine Netw. 2006, 17, 155–165. [CrossRef] [PubMed] 17. Muramatsu, D.; Wakabayashi, Y.; Usui, Y.; Okunuki, Y.; Kezuka, T.; Goto, H. Correlation of complement fragment C5a with inﬂammatory cytokines in the vitreous of patients with proliferative diabetic retinopathy. Graefes Arch. Clin. Exp. Ophthalmol. 2013, 251, 15–17. [CrossRef] [PubMed] 18. Wakabayashi, Y.; Usui, Y.; Okunuki, Y.; Kezuka, T.; Takeuchi, M.; Goto, H.; Iwasaki, T. Correlation of vascular endothelial growth factor with chemokines in the vitreous in diabetic retinopathy. Retina 2010, 30, 339–344. [CrossRef] [PubMed] 19. Joussen, A.M.; Murata, T.; Tsujikawa, A.; Kirchhof, B.; Bursell, S.E.; Adamis, A.P. Leukocyte-mediated endothelial cell injury and death in the diabetic retina. Am. J. Pathol. 2001, 158, 147–152. [CrossRef] 19. Joussen, A.M.; Murata, T.; Tsujikawa, A.; Kirchhof, B.; Bursell, S.E.; Adamis, A.P. Leukocyte-mediated endothelial cell injury and death in the diabetic retina. Am. J. Pathol. 2001, 158, 147–152. [CrossRef] 20. Powell, E.D.; Field, R.A. Diabetic retinopathy and rheumatoid arthritis. Lancet 1964, 2, 17–18. [CrossRef] 0. Powell, E.D.; Field, R.A. Diabetic retinopathy and rheumatoid arthritis. Lancet 1964, 2, 17–18. [CrossRe Int. J. Mol. Sci. 2018, 19, 942 20 of 31 20 of 31 21. Krady, J.K.; Basu, A.; Allen, C.M.; Xu, Y.; LaNoue, K.F.; Gardner, T.W.; Levison, S.W. Minocycline reduces proinﬂammatory cytokine expression, microglial activation, and Caspase-3 activation in a rodent model of diabetic retinopathy. Diabetes 2005, 54, 1559–1565. [CrossRef] [PubMed] 22. Lorenzi, M.; Gerhardinger, C. Early cellular and molecular changes induced by diabetes in the Diabetologia 2001, 44, 791–804. [CrossRef] [PubMed] 23. Lieth, E.; Gardner, T.W.; Barber, A.J.; Antonetti, D.A.; Penn State Retina Research, G. Retinal neurodegeneration: Early pathology in diabetes. Clin. Exp. Ophthalmol. 2000, 28, 3–8. [CrossRef] [PubMed] 24. References Barber, A.J.; Lieth, E.; Khin, S.A.; Antonetti, D.A.; Buchanan, A.G.; Gardner, T.W. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin. J. Clin. Investig. 1998, 102, 783–791. [CrossRef] [PubMed] 26. Park, S.H.; Park, J.W.; Park, S.J.; Kim, K.Y.; Chung, J.W.; Chun, M.H.; Oh, S.J. Apoptotic death of photoreceptors in the streptozotocin-induced diabetic rat retina. Diabetologia 2003, 46, 1260–1268. [CrossRef] [PubMed] 27. Enzsoly, A.; Szabo, A.; Kantor, O.; David, C.; Szalay, P.; Szabo, K.; Szel, A.; Nemeth, J.; Lukats, A. Pathologic alterations of the outer retina in streptozotocin-induced diabetes. Investig. Ophthalmol. Vis. Sci. 2014, 55, 3686–3699. [CrossRef] [PubMed] 28. Ostroy, S.E.; Frede, S.M.; Wagner, E.F.; Gaitatzes, C.G.; Janle, E.M. Decreased rhodopsin regeneration in diabetic mouse eyes. Investig. Ophthalmol. Vis. Sci. 1994, 35, 3905–3909. 29. Mizutani, M.; Gerhardinger, C.; Lorenzi, M. Muller cell changes in human diabetic retinopathy. Diabetes 1998, 47, 445–449. [CrossRef] [PubMed] 30. Abu-El-Asrar, A.M.; Dralands, L.; Missotten, L.; Al-Jadaan, I.A.; Geboes, K. Expression of apoptosis markers in the retinas of human subjects with diabetes. Investig. Ophthalmol. Vis. Sci. 2004, 45, 2760–2766. [CrossRef] [PubMed] 31. Langmann, T. Microglia activation in retinal degeneration. J. Leukoc. Biol. 2007, 81, 1345–1351. [CrossRef] [PubMed] 32. Verma, A.; Rani, P.K.; Raman, R.; Pal, S.S.; Laxmi, G.; Gupta, M.; Sahu, C.; Vaitheeswaran, K.; Sharma, T. Is neuronal dysfunction an early sign of diabetic retinopathy? Microperimetry and spectral domain optical coherence tomography (Sd-Oct) study in individuals with diabetes, but no diabetic retinopathy. Eye (London) 2009, 23, 1824–1830. [CrossRef] [PubMed] 33. Van der Torren, K.; van Lith, G. Oscillatory potentials in early diabetic retinopathy. Doc. Ophthalmol. 1989, 71, 375–379. [CrossRef] [PubMed] 34. Sokol, S.; Moskowitz, A.; Skarf, B.; Evans, R.; Molitch, M.; Senior, B. Contrast sensitivity in diabetics with and without background retinopathy. Arch. Ophthalmol. 1985, 103, 51–54. [CrossRef] [PubMed] 35. Roy, M.S.; Gunkel, R.D.; Podgor, M.J. Color vision defects in early diabetic retinopathy. Arch. Ophthalmol. 1986, 104, 225–228. [CrossRef] [PubMed] 36. Sun, J.K.; Lin, M.M.; Lammer, J.; Prager, S.; Sarangi, R.; Silva, P.S.; Aiello, L.P. Disorganization of the retinal inner layers as a predictor of visual acuity in eyes with center-involved diabetic macular edema. JAMA Ophthalmol. 2014, 132, 1309–1316. [CrossRef] [PubMed] 37. Bonnin, S.; Tadayoni, R.; Erginay, A.; Massin, P.; Dupas, B. Correlation between ganglion cell layer thinning and poor visual function after resolution of diabetic macular edema. Investig. Ophthalmol. Vis. Sci. 2015, 56, 978–982. [CrossRef] [PubMed] 38. References Shin, H.J.; Lee, S.H.; Chung, H.; Kim, H.C. Association between photoreceptor integrity and visual outcome in diabetic macular edema. Graefes Arch. Clin. Exp. Ophthalmol. 2012, 250, 61–70. [CrossRef] [PubMed] 39. Agemy, S.A.; Scripsema, N.K.; Shah, C.M.; Chui, T.; Garcia, P.M.; Lee, J.G.; Gentile, R.C.; Hsiao, Y.S.; Zhou, Q.; Ko, T.; et al. Retinal vascular perfusion density mapping using optical coherence tomography angiography in normals and diabetic retinopathy patients. Retina 2015, 35, 2353–2363. [CrossRef] [PubMed] 40. Antonetti, D.A.; Klein, R.; Gardner, T.W. Diabetic retinopathy. N. Engl. J. Med. 2012, 366, 1227–1239. [CrossRef] [PubMed] 41. Jialal, I.; Kaur, H. The role of toll-like receptors in diabetes-induced inﬂammation: Implications for vascular complications. Curr. Diabetes Rep. 2012, 12, 172–179. [CrossRef] [PubMed] 21 of 31 Int. J. Mol. Sci. 2018, 19, 942 21 of 31 42. Serhan, C.N.; Brain, S.D.; Buckley, C.D.; Gilroy, D.W.; Haslett, C.; O’Neill, L.A.; Perretti, M.; Rossi, A.G.; Wallace, J.L. Resolution of inﬂammation: State of the art, deﬁnitions and terms. FASEB J. 2007, 21, 325–332. [CrossRef] [PubMed] Hotamisligil, G.S. Inﬂammation and metabolic disorders. Nature 2006, 444, 860–867. [CrossRef] [PubMed] 43. Hotamisligil, G.S. Inﬂammation and metabolic disorders. Nature 2006, 444, 860–867. [CrossRef] [PubMed] 44. Adamis, A.P. Is diabetic retinopathy an inﬂammatory disease? Br. J. Ophthalmol. 2002, 86, 363–365. [CrossRef] [PubMed] 44. Adamis, A.P. Is diabetic retinopathy an inﬂammatory disease? Br. J. Ophthalmol. 2002, 86, 363–365. [CrossRef] [PubMed] 45. Kern, T.S. Contributions of inﬂammatory processes to the development of the early stages of diabetic retinopathy. Exp. Diabetes Res. 2007, 2007, 95103. [CrossRef] [PubMed] 46. Noda, K.; Nakao, S.; Ishida, S.; Ishibashi, T. Leukocyte adhesion molecules in diabetic retinopathy. J. Ophthalmol. 2012, 2012, 279037. [CrossRef] [PubMed] 47. Vujosevic, S.; Micera, A.; Bini, S.; Berton, M.; Esposito, G.; Midena, E. Aqueous humor biomarkers of muller cell activation in diabetic eyes. Investig. Ophthalmol. Vis. Sci. 2015, 56, 3913–3918. [CrossRef] [PubMed] 48. Khalfaoui, T.; Lizard, G.; Ouertani-Meddeb, A. Adhesion molecules (ICAM-1 and VCAM-1) and diabetic retinopathy in type 2 diabetes. J. Mol. Histol. 2008, 39, 243–249. [CrossRef] [PubMed] 49. Vujosevic, S.; Micera, A.; Bini, S.; Berton, M.; Esposito, G.; Midena, E. Proteome analysis of retinal glia cells-related inﬂammatory cytokines in the aqueous humour of diabetic patients. Acta Ophthalmol. 2016, 94, 56–64. [CrossRef] [PubMed] 50. Sun, W.; Gerhardinger, C.; Dagher, Z.; Hoehn, T.; Lorenzi, M. Aspirin at low-intermediate concentrations protects retinal vessels in experimental diabetic retinopathy through non-platelet-mediated effects. Diabetes 2005, 54, 3418–3426. [CrossRef] [PubMed] 51. References Boss, J.D.; Singh, P.K.; Pandya, H.K.; Tosi, J.; Kim, C.; Tewari, A.; Juzych, M.S.; Abrams, G.W.; Kumar, A. Assessment of neurotrophins and inﬂammatory mediators in vitreous of patients with diabetic retinopathy. Investig. Ophthalmol. Vis. Sci. 2017, 58, 5594–5603. [CrossRef] [PubMed] 52. Wu, H.; Hwang, D.K.; Song, X.; Tao, Y. Association between aqueous cytokines and diabetic retinopathy stage. J. Ophthalmol. 2017, 2017, 9402198. [CrossRef] [PubMed] 53. Demircan, N.; Safran, B.G.; Soylu, M.; Ozcan, A.A.; Sizmaz, S. Determination of vitreous interleukin-1 (IL-1) and tumour necrosis factor (TNF) levels in proliferative diabetic retinopathy. Eye (London) 2006, 20, 1366–1369. [CrossRef] [PubMed] 54. Yoshida, S.; Yoshida, A.; Ishibashi, T.; Elner, S.G.; Elner, V.M. Role of MCP-1 and MIP-1alpha in retinal neovascularization during postischemic inﬂammation in a mouse model of retinal neovascularization. J. Leukoc. Biol. 2003, 73, 137–144. [CrossRef] [PubMed] 55. Rusnak, S.; Vrzalova, J.; Sobotova, M.; Hecova, L.; Ricarova, R.; Topolcan, O. The measurement of intraocular biomarkers in various stages of proliferative diabetic retinopathy using multiplex xmap technology. J. Ophthalmol. 2015, 2015, 424783. [CrossRef] [PubMed] 56. Doganay, S.; Evereklioglu, C.; Er, H.; Turkoz, Y.; Sevinc, A.; Mehmet, N.; Savli, H. Comparison of serum no, TNF-alpha, IL-1beta, SIL-2r, IL-6 and il-8 levels with grades of retinopathy in patients with diabetes mellitus. Eye (London) 2002, 16, 163–170. [CrossRef] [PubMed] 57. Giebel, S.J.; Menicucci, G.; McGuire, P.G.; Das, A. Matrix metalloproteinases in early diabetic retinopathy and their role in alteration of the blood-retinal barrier. Lab. Investig. 2005, 85, 597–607. [CrossRef] [PubMed] 57. Giebel, S.J.; Menicucci, G.; McGuire, P.G.; Das, A. Matrix metalloproteinases in early diabetic retinopathy and their role in alteration of the blood-retinal barrier. Lab. Investig. 2005, 85, 597–607. [CrossRef] [PubMed] 58. Patel, J.I.; Hykin, P.G.; Gregor, Z.J.; Boulton, M.; Cree, I.A. Angiopoietin concentrations in diabetic retinopathy. 57. Giebel, S.J.; Menicucci, G.; McGuire, P.G.; Das, A. Matrix metalloproteinases in early diabetic retinopathy and their role in alteration of the blood-retinal barrier. Lab. Investig. 2005, 85, 597–607. [CrossRef] [PubMed] 58. Patel, J.I.; Hykin, P.G.; Gregor, Z.J.; Boulton, M.; Cree, I.A. Angiopoietin concentrations in diabetic retinopathy. Br. J. Ophthalmol. 2005, 89, 480–483. [CrossRef] [PubMed] 58. Patel, J.I.; Hykin, P.G.; Gregor, Z.J.; Boulton, M.; Cree, I.A. Angiopoietin concentrations in diabetic retinopathy. Br. J. Ophthalmol. 2005, 89, 480–483. [CrossRef] [PubMed] 59. Chalam, K.V.; Grover, S.; Sambhav, K.; Balaiya, S.; Murthy, R.K. Aqueous interleukin-6 levels are superior to vascular endothelial growth factor in predicting therapeutic response to bevacizumab in age-related macular degeneration. References J. Ophthalmol. 2014, 2014, 502174. [CrossRef] [PubMed] 60. Joussen, A.M.; Poulaki, V.; Qin, W.; Kirchhof, B.; Mitsiades, N.; Wiegand, S.J.; Rudge, J.; Yancopoulos, G.D.; Adamis, A.P. Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo. Am. J. Pathol. 2002, 160, 501–509. [CrossRef] 61. Rungger-Brandle, E.; Dosso, A.A.; Leuenberger, P.M. Glial reactivity, an early feature of diabetic retinopathy. Investig. Ophthalmol. Vis. Sci. 2000, 41, 1971–1980. 62. Cusick, M.; Chew, E.Y.; Chan, C.C.; Kruth, H.S.; Murphy, R.P.; Ferris, F.L., 3rd. Histopathology and regression of retinal hard exudates in diabetic retinopathy after reduction of elevated serum lipid levels. Ophthalmology 2003, 110, 2126–2133. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 942 22 of 31 63. Zhang, J.; Gerhardinger, C.; Lorenzi, M. Early complement activation and decreased levels of glycosylphosphatidylinositol-anchored complement inhibitors in human and experimental diabetic retinopathy. Diabetes 2002, 51, 3499–3504. [CrossRef] [PubMed] 64. Antonetti, D.A.; Barber, A.J.; Bronson, S.K.; Freeman, W.M.; Gardner, T.W.; Jefferson, L.S.; Kester, M.; Kimball, S.R.; Krady, J.K.; LaNoue, K.F.; et al. Diabetic retinopathy: Seeing beyond glucose-induced microvascular disease. Diabetes 2006, 55, 2401–2411. [CrossRef] [PubMed] 65. Cardona, S.M.; Mendiola, A.S.; Yang, Y.C.; Adkins, S.L.; Torres, V.; Cardona, A.E. Disruption of fractalkine signaling leads to microglial activation and neuronal damage in the diabetic retina. ASN Neuro 2015, 7. [CrossRef] [PubMed] 66. Vallejo, S.; Palacios, E.; Romacho, T.; Villalobos, L.; Peiro, C.; Sanchez-Ferrer, C.F. The interleukin-1 receptor antagonist anakinra improves endothelial dysfunction in streptozotocin-induced diabetic rats. Cardiovasc. Diabetol. 2014, 13, 158. [CrossRef] [PubMed] 67. Li, Q.; Verma, A.; Han, P.Y.; Nakagawa, T.; Johnson, R.J.; Grant, M.B.; Campbell-Thompson, M.; Jarajapu, Y.P.; Lei, B.; Hauswirth, W.W. Diabetic enos-knockout mice develop accelerated retinopathy. Investig. Ophthalmol. Vis. Sci. 2010, 51, 5240–5246. [CrossRef] [PubMed] 68. Funatsu, H.; Yamashita, H.; Sakata, K.; Noma, H.; Mimura, T.; Suzuki, M.; Eguchi, S.; Hori, S. Vitreous levels of vascular endothelial growth factor and intercellular adhesion molecule 1 are related to diabetic macular edema. Ophthalmology 2005, 112, 806–816. [CrossRef] [PubMed] 69. Bromberg-White, J.L.; Glazer, L.; Downer, R.; Furge, K.; Boguslawski, E.; Duesbery, N.S. Identiﬁcation of VEGF-independent cytokines in proliferative diabetic retinopathy vitreous. Investig. Ophthalmol. Vis. Sci. 2013, 54, 6472–6480. [CrossRef] [PubMed] 70. Adamiec-Mroczek, J.; Oficjalska-Mlynczak, J.; Misiuk-Hojlo, M. Proliferative diabetic retinopathy-the influence of diabetes control on the activation of the intraocular molecule system. Diabetes Res. Clin. Pract. 2009, 84, 46–50. [CrossRef] [PubMed] 71. References Jain, A.; Saxena, S.; Khanna, V.K.; Shukla, R.K.; Meyer, C.H. Status of serum VEGF and ICAM-1 and its association with external limiting membrane and inner segment-outer segment junction disruption in type 2 diabetes mellitus. Mol. Vis. 2013, 19, 1760–1768. [PubMed] 72. Burgos, R.; Simo, R.; Audi, L.; Mateo, C.; Mesa, J.; Garcia-Ramirez, M.; Carrascosa, A. Vitreous levels of vascular endothelial growth factor are not inﬂuenced by its serum concentrations in diabetic retinopathy. Diabetologia 1997, 40, 1107–1109. [CrossRef] [PubMed] 73. Hang, H.; Yuan, S.; Yang, Q.; Yuan, D.; Liu, Q. Multiplex bead array assay of plasma cytokines in type 2 diabetes mellitus with diabetic retinopathy. Mol. Vis. 2014, 20, 1137–1145. [PubMed] 74. Jonas, J.B.; Jonas, R.A.; Neumaier, M.; Findeisen, P. Cytokine concentration in aqueous humor of eyes with diabetic macular edema. Retina 2012, 32, 2150–2157. [CrossRef] [PubMed] 75. Ghodasra, D.H.; Fante, R.; Gardner, T.W.; Langue, M.; Niziol, L.M.; Besirli, C.; Cohen, S.R.; Dedania, V.S.; Demirci, H.; Jain, N.; et al. Safety and feasibility of quantitative multiplexed cytokine analysis from ofﬁce-based vitreous aspiration. Investig. Ophthalmol. Vis. Sci. 2016, 57, 3017–3023. [CrossRef] [PubMed] 76. Mao, C.; Yan, H. Roles of elevated intravitreal IL-1beta and IL-10 levels in proliferative diabetic retinopathy. Indian J. Ophthalmol. 2014, 62, 699–701. [PubMed] 77. Kim, M.; Kim, Y.; Lee, S.J. Comparison of aqueous concentrations of angiogenic and inﬂammatory cytokines based on optical coherence tomography patterns of diabetic macular edema. Indian J. Ophthalmol. 2015, 63, 312–317. [CrossRef] [PubMed] 78. Lee, W.J.; Kang, M.H.; Seong, M.; Cho, H.Y. Comparison of aqueous concentrations of angiogenic and inﬂammatory cytokines in diabetic macular oedema and macular oedema due to branch retinal vein occlusion. Br. J. Ophthalmol. 2012, 96, 1426–1430. [CrossRef] [PubMed] 79. Tang, S.; Le-Ruppert, K.C. Activated t lymphocytes in epiretinal membranes from eyes of patients with proliferative diabetic retinopathy. Graefes Arch. Clin. Exp. Ophthalmol. 1995, 233, 21–25. [CrossRef] [PubMed] 79. Tang, S.; Le-Ruppert, K.C. Activated t lymphocytes in epiretinal membranes from eyes of patients with proliferative diabetic retinopathy. Graefes Arch. Clin. Exp. Ophthalmol. 1995, 233, 21–25. [CrossRef] [PubMed] 80. Takeuchi, M.; Sato, T.; Tanaka, A.; Muraoka, T.; Taguchi, M.; Sakurai, Y.; Karasawa, Y.; Ito, M. Elevated l l f t ki i t d ith TH2 d TH17 ll i it ﬂid f lif ti di b ti ti th 80. Takeuchi, M.; Sato, T.; Tanaka, A.; Muraoka, T.; Taguchi, M.; Sakurai, Y.; Karasawa, Y.; Ito, M. References Elevated levels of cytokines associated with TH2 and TH17 cells in vitreous ﬂuid of proliferative diabetic retinopathy patients. PLoS ONE 2015, 10, e0137358. [CrossRef] [PubMed] 81. Meleth, A.D.; Agron, E.; Chan, C.C.; Reed, G.F.; Arora, K.; Byrnes, G.; Csaky, K.G.; Ferris, F.L., 3rd; Chew, E.Y. Serum inﬂammatory markers in diabetic retinopathy. Investig. Ophthalmol. Vis. Sci. 2005, 46, 4295–4301. [CrossRef] [PubMed] y [CrossRef] [PubMed] 23 of 31 Int. J. Mol. Sci. 2018, 19, 942 82. Noma, H.; Mimura, T.; Yasuda, K.; Motohashi, R.; Kotake, O.; Shimura, M. Aqueous humor levels of soluble vascular endothelial growth factor receptor and inﬂammatory factors in diabetic macular edema. Ophthalmologica 2017, 238, 81–88. [CrossRef] [PubMed] 83. Ogata, N.; Nishikawa, M.; Nishimura, T.; Mitsuma, Y.; Matsumura, M. Unbalanced vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic retinopathy. Am. J. Ophthalmol. 2002, 134, 348–353. [CrossRef] 84. Duh, E.J.; Yang, H.S.; Haller, J.A.; De Juan, E.; Humayun, M.S.; Gehlbach, P.; Melia, M.; Pieramici, D.; Harlan, J.B.; Campochiaro, P.A.; et al. Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor: Implications for ocular angiogenesis. Am. J. Ophthalmol. 2004, 137, 668–674. [PubMed] 85. Wang, S.; Gottlieb, J.L.; Sorenson, C.M.; Sheibani, N. Modulation of thrombospondin 1 and pigment epithelium-derived factor levels in vitreous ﬂuid of patients with diabetes. Arch. Ophthalmol. 2009, 127, 507–513. [CrossRef] [PubMed] 86. Funatsu, H.; Yamashita, H.; Nakamura, S.; Mimura, T.; Eguchi, S.; Noma, H.; Hori, S. Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor are related to diabetic macular edema. Ophthalmology 2006, 113, 294–301. [CrossRef] [PubMed] 87. Meyer-Schwickerath, R.; Pfeiffer, A.; Blum, W.F.; Freyberger, H.; Klein, M.; Losche, C.; Rollmann, R.; Schatz, H. Vitreous levels of the insulin-like growth factors I and II, and the insulin-like growth factor binding proteins 2 and 3, increase in neovascular eye disease. Studies in nondiabetic and diabetic subjects. J. Clin. Investig. 1993, 92, 2620–2625. [CrossRef] [PubMed] 88. Grant, M.; Russell, B.; Fitzgerald, C.; Merimee, T.J. Insulin-like growth factors in vitreous. Studies in control and diabetic subjects with neovascularization. Diabetes 1986, 35, 416–420. [CrossRef] [PubMed] 89. Sivalingam, A.; Kenney, J.; Brown, G.C.; Benson, W.E.; Donoso, L. Basic ﬁbroblast growth factor levels in the vitreous of patients with proliferative diabetic retinopathy. Arch. Ophthalmol. 1990, 108, 869–872. [CrossRef] [PubMed] 90. Al Kahtani, E.; Xu, Z.; Al Rashaed, S.; Wu, L.; Mahale, A.; Tian, J.; Abboud, E.B.; Ghazi, N.G.; Kozak, I.; Gupta, V.; et al. References Joussen, A.M.; Poulaki, V.; Le, M.L.; Koizumi, K.; Esser, C.; Janicki, H.; Schraermeyer, U.; Kociok, N.; Fauser, S.; Kirchhof, B.; et al. A central role for inﬂammation in the pathogenesis of diabetic retinopathy. FASEB J. 2004, 18, 1450–1452. [CrossRef] [PubMed] 103. Miyamoto, K.; Khosrof, S.; Bursell, S.E.; Rohan, R.; Murata, T.; Clermont, A.C.; Aiello, L.P.; Ogura, Y.; Adamis, A.P. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc. Natl. Acad. Sci. USA 1999, 96, 10836–10841. [CrossRef] [PubMed] 104. Stitt, A.W.; Curtis, T.M.; Chen, M.; Medina, R.J.; McKay, G.J.; Jenkins, A.; Gardiner, T.A.; Lyons, T.J.; Hammes, H.P.; Simo, R.; et al. The progress in understanding and treatment of diabetic retinopathy. Prog. Retin. Eye Res. 2016, 51, 156–186. [CrossRef] [PubMed] 105. Durham, J.T.; Herman, I.M. Microvascular modiﬁcations in diabetic retinopathy. Curr. Diabetes Rep. 2011, 11, 253–264. [CrossRef] [PubMed] 106. Valle, A.; Giamporcaro, G.M.; Scavini, M.; Stabilini, A.; Grogan, P.; Bianconi, E.; Sebastiani, G.; Masini, M.; Maugeri, N.; Porretti, L.; et al. Reduction of circulating neutrophils precedes and accompanies type 1 diabetes. Diabetes 2013, 62, 2072–2077. [CrossRef] [PubMed] 107. Engerman, R.L. Pathogenesis of diabetic retinopathy. Diabetes 1989, 38, 1203–1206. [CrossRef] [PubM 107. Engerman, R.L. Pathogenesis of diabetic retinopathy. Diabetes 1989, 38, 1203–1206. [CrossRef] [PubMed] 108. Dell’omo, R.; Semeraro, F.; Bamonte, G.; Cifariello, F.; Romano, M.R.; Costagliola, C. Vitreous mediators in retinal hypoxic diseases. Mediat. Inﬂamm. 2013, 2013, 935301. [CrossRef] [PubMed] 108. Dell’omo, R.; Semeraro, F.; Bamonte, G.; Cifariello, F.; Romano, M.R.; Costagliola, C. Vitreous mediators in retinal hypoxic diseases. Mediat. Inﬂamm. 2013, 2013, 935301. [CrossRef] [PubMed] 109. Costagliola, C.; Romano, V.; De Tollis, M.; Aceto, F.; dell’Omo, R.; Romano, M.R.; Pedicino, C.; Semeraro, F. TNF-alpha levels in tears: A novel biomarker to assess the degree of diabetic retinopathy. Mediat. Inﬂamm. 2013, 2013, 629529. [CrossRef] [PubMed] 110. Yoshida, S.; Yoshida, A.; Ishibashi, T. Induction of IL-8, MCP-1, and BFGF by TNF-alpha in retinal glial cells: Implications for retinal neovascularization during post-ischemic inﬂammation. Graefes Arch. Clin. Exp. Ophthalmol. 2004, 242, 409–413. [CrossRef] [PubMed] 111. Tsutsumi, C.; Sonoda, K.H.; Egashira, K.; Qiao, H.; Hisatomi, T.; Nakao, S.; Ishibashi, M.; Charo, I.F.; Sakamoto, T.; Murata, T.; et al. The critical role of ocular-inﬁltrating macrophages in the development of choroidal neovascularization. J. Leukoc. Biol. 2003, 74, 25–32. [CrossRef] [PubMed] 112. Sakurai, E.; Taguchi, H.; Anand, A.; Ambati, B.K.; Gragoudas, E.S.; Miller, J.W.; Adamis, A.P.; Ambati, J. References Vitreous levels of placental growth factor correlate with activity of proliferative diabetic retinopathy and are not inﬂuenced by bevacizumab treatment. Eye (London) 2017, 31, 529–536. [CrossRef] [PubMed] 91. Cassidy, L.; Barry, P.; Shaw, C.; Duffy, J.; Kennedy, S. Platelet derived growth factor and ﬁbroblast growth factor basic levels in the vitreous of patients with vitreoretinal disorders. Br. J. Ophthalmol. 1998, 82, 181–185. [CrossRef] [PubMed] 92. Yngen, M.; Ostenson, C.G.; Hu, H.; Li, N.; Hjemdahl, P.; Wallen, N.H. Enhanced p-selectin expression and increased soluble cd40 ligand in patients with type 1 diabetes mellitus and microangiopathy: Evidence for platelet hyperactivity and chronic inﬂammation. Diabetologia 2004, 47, 537–540. [CrossRef] [PubMed] 93. Loukovaara, S.; Koivunen, P.; Ingles, M.; Escobar, J.; Vento, M.; Andersson, S. Elevated protein carbonyl and HIF-1alpha levels in eyes with proliferative diabetic retinopathy. Acta Ophthalmol. 2014, 92, 323–327. [CrossRef] [PubMed] 94. Wang, X.; Wang, G.; Wang, Y. Intravitreous vascular endothelial growth factor and hypoxia-inducible factor 1A in patients with proliferative diabetic retinopathy. Am. J. Ophthalmol. 2009, 148, 883–889. [CrossRef] [PubMed] 95. Tang, J.; Kern, T.S. Inﬂammation in diabetic retinopathy. Prog. Retin. Eye Res. 2011, 30, 343–358. [CrossRef] [PubMed] 96. Rangasamy, S.; McGuire, P.G.; Das, A. Diabetic retinopathy and inﬂammation: Novel therapeutic targets. Middle East Afr. J. Ophthalmol. 2012, 19, 52–59. [PubMed] 97. Aplin, A.C.; Gelati, M.; Fogel, E.; Carnevale, E.; Nicosia, R.F. Angiopoietin-1 and vascular endothelial growth factor induce expression of inﬂammatory cytokines before angiogenesis. Physiol. Genom. 2006, 27, 20–28. [CrossRef] [PubMed] 98. Voronov, E.; Shouval, D.S.; Krelin, Y.; Cagnano, E.; Benharroch, D.; Iwakura, Y.; Dinarello, C.A.; Apte, R.N. IL-1 is required for tumor invasiveness and angiogenesis. Proc. Natl. Acad. Sci. USA 2003, 100, 2645–2650. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 942 24 of 31 24 of 31 99. Leali, D.; Dell’Era, P.; Stabile, H.; Sennino, B.; Chambers, A.F.; Naldini, A.; Sozzani, S.; Nico, B.; Ribatti, D.; Presta, M. Osteopontin (ETA-1) and ﬁbroblast growth factor-2 cross-talk in angiogenesis. J. Immunol. 2003, 171, 1085–1093. [CrossRef] [PubMed] 00. Busik, J.V.; Mohr, S.; Grant, M.B. Hyperglycemia-induced reactive oxygen species toxicity to endothelial is dependent on paracrine mediators. Diabetes 2008, 57, 1952–1965. [CrossRef] [PubMed] 101. Chen, W.; Esselman, W.J.; Jump, D.B.; Busik, J.V. Anti-inflammatory effect of docosahexaenoic acid on cytokine-induced adhesion molecule expression in human retinal vascular endothelial cells. Investig. Ophthalmol. Vis. Sci. 2005, 46, 4342–4347. [CrossRef] [PubMed] 102. References Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization. Investig. Ophthalmol. Vis. Sci. 2003, 44, 2743–2749. [CrossRef] 113. Sennlaub, F.; Valamanesh, F.; Vazquez-Tello, A.; El-Asrar, A.M.; Checchin, D.; Brault, S.; Gobeil, F.; Beauchamp, M.H.; Mwaikambo, B.; Courtois, Y.; et al. Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy. Circulation 2003, 108, 198–204. [CrossRef] [PubMed] 114. Ayalasomayajula, S.P.; Kompella, U.B. Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozotocin-induced diabetic rat model. Eur. J. Pharmacol. 2003, 458, 283–289. [CrossRef] 115. Abu El-Asrar, A.M.; Mohammad, G.; Nawaz, M.I.; Siddiquei, M.M. High-mobility group box-1 modulates the expression of inﬂammatory and angiogenic signaling pathways in diabetic retina. Curr. Eye Res. 2015, 40, 1141–1152. [CrossRef] [PubMed] 116. Fiuza, C.; Bustin, M.; Talwar, S.; Tropea, M.; Gerstenberger, E.; Shelhamer, J.H.; Suffredini, A.F. Inﬂammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood 2003, 101, 2652–2660. [CrossRef] [PubMed] 117. Treutiger, C.J.; Mullins, G.E.; Johansson, A.S.; Rouhiainen, A.; Rauvala, H.M.; Erlandsson-Harris, H.; Andersson, U.; Yang, H.; Tracey, K.J.; Andersson, J.; et al. High mobility group 1 b-box mediates activation of human endothelium. J. Intern. Med. 2003, 254, 375–385. [CrossRef] [PubMed] 25 of 31 Int. J. Mol. Sci. 2018, 19, 942 118. Croll, S.D.; Ransohoff, R.M.; Cai, N.; Zhang, Q.; Martin, F.J.; Wei, T.; Kasselman, L.J.; Kintner, J.; Murphy, A.J.; Yancopoulos, G.D.; et al. VEGF-mediated inﬂammation precedes angiogenesis in adult brain. Exp. Neurol. 2004, 187, 388–402. [CrossRef] [PubMed] 119. Marumo, T.; Schini-Kerth, V.B.; Busse, R. Vascular endothelial growth factor activates nuclear factor-kappab and induces monocyte chemoattractant protein-1 in bovine retinal endothelial cells. Diabetes 1999, 48, 1131–1137. [CrossRef] [PubMed] 120. Lee, T.H.; Avraham, H.; Lee, S.H.; Avraham, S. Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells. J. Biol. Chem. 2002, 277, 10445–10451. [CrossRef] [PubMed] 121. Cai, M.; Zhang, X.; Li, Y.; Xu, H. Toll-like receptor 3 activation drives the inﬂammatory response in oxygen-induced retinopathy in rats. Br. J. Ophthalmol. 2015, 99, 125–132. [CrossRef] [PubMed] 122. Lange, C.; Storkebaum, E.; de Almodovar, C.R.; Dewerchin, M.; Carmeliet, P. Vascular endothelial growth factor: A neurovascular target in neurological diseases. Nat. Rev. Neurol. 2016, 12, 439–454. [CrossRef] [PubMed] 123. Imai, H.; Singh, R.S.; Fort, P.E.; Gardner, T.W. Neuroprotection for diabetic retinopathy. Dev. Ophthalmol. 2009, 44, 56–68. [PubMed] 124. References Gilbert, R.E.; Vranes, D.; Berka, J.L.; Kelly, D.J.; Cox, A.; Wu, L.L.; Stacker, S.A.; Cooper, M.E. Vascular endothelial growth factor and its receptors in control and diabetic rat eyes. Lab. Investig. 1998, 78, 1017–1027. [PubMed] 125. Barber, A.J.; Nakamura, M.; Wolpert, E.B.; Reiter, C.E.; Seigel, G.M.; Antonetti, D.A.; Gardner, T.W. Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3. J. Biol. Chem. 2001, 276, 32814–32821. [CrossRef] [PubMed] 126. Gordon, W.C.; Bazan, N.G. Mediator lipidomics in ophthalmology: Targets for modulation in inﬂammation, neuroprotection and nerve regeneration. Curr. Eye Res. 2013, 38, 995–1005. [CrossRef] [PubMed] 127. Seki, M.; Tanaka, T.; Nawa, H.; Usui, T.; Fukuchi, T.; Ikeda, K.; Abe, H.; Takei, N. Involvement of brain-derived neurotrophic factor in early retinal neuropathy of streptozotocin-induced diabetes in rats: Therapeutic potential of brain-derived neurotrophic factor for dopaminergic amacrine cells. Diabetes 2004, 53, 2412–2419. [CrossRef] [PubMed] 128. Wang, L.; Deng, Q.Q.; Wu, X.H.; Yu, J.; Yang, X.L.; Zhong, Y.M. Upregulation of glutamate-aspartate transporter by glial cell line-derived neurotrophic factor ameliorates cell apoptosis in neural retina in streptozotocin-induced diabetic rats. CNS Neurosci. Ther. 2013, 19, 945–953. [CrossRef] [PubMed] 129. Aizu, Y.; Katayama, H.; Takahama, S.; Hu, J.; Nakagawa, H.; Oyanagi, K. Topical instillation of ciliary neurotrophic factor inhibits retinal degeneration in streptozotocin-induced diabetic rats. Neuroreport 2003, 14, 2067–2071. [CrossRef] [PubMed] 130. Ali, T.K.; Al-Gayyar, M.M.; Matragoon, S.; Pillai, B.A.; Abdelsaid, M.A.; Nussbaum, J.J.; El-Remessy, A.B. Diabetes-induced peroxynitrite impairs the balance of pro-nerve growth factor and nerve growth factor, and causes neurovascular injury. Diabetologia 2011, 54, 657–668. [CrossRef] [PubMed] 131. Costa, G.N.; Vindeirinho, J.; Cavadas, C.; Ambrosio, A.F.; Santos, P.F. Contribution of tnf receptor 1 to retinal neural cell death induced by elevated glucose. Mol. Cell. Neurosci. 2012, 50, 113–123. [CrossRef] [PubMed] 132. Zong, H.; Ward, M.; Madden, A.; Yong, P.H.; Limb, G.A.; Curtis, T.M.; Stitt, A.W. Hyperglycaemia-induced pro-inﬂammatory responses by retinal muller glia are regulated by the receptor for advanced glycation end-products (RAGE). Diabetologia 2010, 53, 2656–2666. [CrossRef] [PubMed] 133. Esposito, K.; Nappo, F.; Marfella, R.; Giugliano, G.; Giugliano, F.; Ciotola, M.; Quagliaro, L.; Ceriello, A.; Giugliano, D. Inﬂammatory cytokine concentrations are acutely increased by hyperglycemia in humans: Role of oxidative stress. Circulation 2002, 106, 2067–2072. [CrossRef] [PubMed] 134. Zheng, L.; Howell, S.J.; Hatala, D.A.; Huang, K.; Kern, T.S. Salicylate-based anti-inﬂammatory drugs inhibit the early lesion of diabetic retinopathy. Diabetes 2007, 56, 337–345. [CrossRef] [PubMed] 135. 136. Du, Y.; Veenstra, A.; Palczewski, K.; Kern, T.S. Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inﬂammation in the retina. Proc. Natl. Acad. Sci. USA 2013, 110, 16586–16591. [CrossRef] [PubMed] References Kern, T.S.; Berkowitz, B.A. Photoreceptors in diabetic retinopathy. J. Diabetes Investig. 2015, 6, 371–380. [CrossRef] [PubMed] 136. Du, Y.; Veenstra, A.; Palczewski, K.; Kern, T.S. Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inﬂammation in the retina. Proc. Natl. Acad. Sci. USA 2013, 110, 16586–16591. [CrossRef] [PubMed] 26 of 31 Int. J. Mol. Sci. 2018, 19, 942 26 of 31 137. Tonade, D.; Liu, H.; Kern, T.S. Photoreceptor cells produce inﬂammatory mediators that contribute to endothelial cell death in diabetes. Investig. Ophthalmol. Vis. Sci. 2016, 57, 4264–4271. [CrossRef] [PubMed] 138. Yang, L.P.; Sun, H.L.; Wu, L.M.; Guo, X.J.; Dou, H.L.; Tso, M.O.; Zhao, L.; Li, S.M. Baicalein reduces inﬂammatory process in a rodent model of diabetic retinopathy. Investig. Ophthalmol. Vis. Sci. 2009, 50, 2319–2327. [CrossRef] [PubMed] 139. Scott, I.U.; Jackson, G.R.; Quillen, D.A.; Larsen, M.; Klein, R.; Liao, J.; Holfort, S.; Munch, I.C.; Gardner, T.W. Effect of doxycycline vs placebo on retinal function and diabetic retinopathy progression in patients with severe nonproliferative or non-high-risk proliferative diabetic retinopathy: A randomized clinical trial. JAMA Ophthalmol. 2014, 132, 535–543. [CrossRef] [PubMed] 140. Peters, A.L.; Stunz, L.L.; Bishop, G.A. Cd40 and autoimmunity: The dark side of a great activator. Semin. Immunol. 2009, 21, 293–300. [CrossRef] [PubMed] 141. van Kooten, C.; Banchereau, J. CD40-CD40 ligand. J. Leukoc. Biol. 2000, 67, 2–17. [CrossRef] [PubMed] 142. Portillo, J.C.; Lopez Corcino, Y.; Miao, Y.; Tang, J.; Sheibani, N.; Kern, T.S.; Dubyak, G.R.; Subauste, C.S. CD40 in retinal muller cells induces P2X7-dependent cytokine expression in macrophages/microglia in diabetic mice and development of early experimental diabetic retinopathy. Diabetes 2017, 66, 483–493. [CrossRef] [PubMed] 143. Samuels, I.S.; Portillo, J.C.; Miao, Y.; Kern, T.S.; Subauste, C.S. Loss of CD40 attenuates experimental diabetes-induced retinal inflammation but does not protect mice from electroretinogram defects. Vis. Neurosci. 2017, 34. [CrossRef] [PubMed] 144. Zheng, L.; Du, Y.; Miller, C.; Gubitosi-Klug, R.A.; Kern, T.S.; Ball, S.; Berkowitz, B.A. Critical role of inducible nitric oxide synthase in degeneration of retinal capillaries in mice with streptozotocin-induced diabetes. Diabetologia 2007, 50, 1987–1996. [CrossRef] [PubMed] 145. Li, G.; Tang, J.; Du, Y.; Lee, C.A.; Kern, T.S. Beneﬁcial effects of a novel rage inhibitor on early diabetic retinopathy and tactile allodynia. Mol. Vis. 2011, 17, 3156–3165. [PubMed] 146. Lee, C.A.; Li, G.; Patel, M.D.; Petrash, J.M.; Benetz, B.A.; Veenstra, A.; Amengual, J.; von Lintig, J.; Burant, C.J.; Tang, J.; et al. References Diabetes-induced impairment in visual function in mice: Contributions of p38 mapk, rage, leukocytes, and aldose reductase. Investig. Ophthalmol. Vis. Sci. 2014, 55, 2904–2910. [CrossRef] [PubMed] 147. Calderon, G.D.; Juarez, O.H.; Hernandez, G.E.; Punzo, S.M.; De la Cruz, Z.D. Oxidative stress and retinopathy: Development and treatment. Eye (London) 2017, 31, 1122–1130. [CrossRef] [PubMed] 148. Sorrentino, F.S.; Allkabes, M.; Salsini, G.; Bonifazzi, C.; Perri, P. The importance of glial cells in the homeostasis of the retinal microenvironment and their pivotal role in the course of diabetic retinopathy. Life Sci. 2016, 162, 54–59. [CrossRef] [PubMed] 149. Zeng, H.Y.; Green, W.R.; Tso, M.O. Microglial activation in human diabetic retinopathy. Arch. Ophthalmol. 2008, 126, 227–232. [CrossRef] [PubMed] 150. Holm, T.H.; Draeby, D.; Owens, T. Microglia are required for astroglial toll-like receptor 4 response and for optimal TLR2 and TLR3 response. Glia 2012, 60, 630–638. [CrossRef] [PubMed] 151. Cuenca, N.; Fernandez-Sanchez, L.; Campello, L.; Maneu, V.; De la Villa, P.; Lax, P.; Pinilla, I. Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases. Prog. Retin. Eye Res. 2014, 43, 17–75. [CrossRef] [PubMed] g y 152. Greter, M.; Lelios, I.; Croxford, A.L. Microglia versus myeloid cell nomenclature during brain inﬂammation. Front. Immunol. 2015, 6, 249. [CrossRef] [PubMed] 153. Fernandes, A.; Miller-Fleming, L.; Pais, T.F. Microglia and inﬂammation: Conspiracy, controversy or control? Cell. Mol. Life Sci. 2014, 71, 3969–3985. [CrossRef] [PubMed] 154. Davis, E.J.; Foster, T.D.; Thomas, W.E. Cellular forms and functions of brain microglia. Brain Res. Bull. 1994, 34, 73–78. [CrossRef] 155. Graeber, M.B.; Li, W.; Rodriguez, M.L. Role of microglia in cns inﬂammation. FEBS Lett. 2011, 585, 3798–3805. [CrossRef] [PubMed] 156. Ibrahim, A.S.; El-Remessy, A.B.; Matragoon, S.; Zhang, W.; Patel, Y.; Khan, S.; Al-Gayyar, M.M.; El-Shishtawy, M.M.; Liou, G.I. Retinal microglial activation and inﬂammation induced by amadori-glycated albumin in a rat model of diabetes. Diabetes 2011, 60, 1122–1133. [CrossRef] [PubMed] 157. Grigsby, J.G.; Cardona, S.M.; Pouw, C.E.; Muniz, A.; Mendiola, A.S.; Tsin, A.T.; Allen, D.M.; Cardona, A.E. The role of microglia in diabetic retinopathy. J. Ophthalmol. 2014, 2014, 705783. [CrossRef] [PubMed] 27 of 31 Int. J. Mol. Sci. 2018, 19, 942 158. Karlstetter, M.; Scholz, R.; Rutar, M.; Wong, W.T.; Provis, J.M.; Langmann, T. Retinal microglia: Just bystander or target for therapy? Prog. Retin. Eye Res. 2015, 45, 30–57. [CrossRef] [PubMed] 159. Coorey, N.J.; Shen, W.; Chung, S.H.; Zhu, L.; Gillies, M.C. The role of glia in retinal vascular disease. Clin. Exp. Optom. References 2012, 95, 266–281. [CrossRef] [PubMed] 160. Bringmann, A.; Pannicke, T.; Grosche, J.; Francke, M.; Wiedemann, P.; Skatchkov, S.N.; Osborne, N.N.; Reichenbach, A. Muller cells in the healthy and diseased retina. Prog. Retin. Eye Res. 2006, 25, 397–424. [CrossRef] [PubMed] 161. Newman, E.; Reichenbach, A. The muller cell: A functional element of the retina. Trends Neurosci. 1996, 19, 307–312. [CrossRef] 162. Eastlake, K.; Banerjee, P.J.; Angbohang, A.; Charteris, D.G.; Khaw, P.T.; Limb, G.A. Muller glia as an important source of cytokines and inﬂammatory factors present in the gliotic retina during proliferative vitreoretinopathy. Glia 2016, 64, 495–506. [CrossRef] [PubMed] 163. Gerhardinger, C.; Costa, M.B.; Coulombe, M.C.; Toth, I.; Hoehn, T.; Grosu, P. Expression of acute-phase response proteins in retinal muller cells in diabetes. Investig. Ophthalmol. Vis. Sci. 2005, 46, 349–357. [CrossRef] [PubMed] 164. Liu, X.; Ye, F.; Xiong, H.; Hu, D.; Limb, G.A.; Xie, T.; Peng, L.; Yang, W.; Sun, Y.; Zhou, M.; et al. Il-1beta upregulates IL-8 production in human muller cells through activation of the p38 mapk and erk1/2 signaling pathways. Inﬂammation 2014, 37, 1486–1495. [CrossRef] [PubMed] p y 165. Ridet, J.L.; Malhotra, S.K.; Privat, A.; Gage, F.H. Reactive astrocytes: Cellular and molecular cues to biological function. Trends Neurosci. 1997, 20, 570–577. [CrossRef] 166. Rungger-Brandle, E.; Messerli, J.M.; Niemeyer, G.; Eppenberger, H.M. Confocal microscopy and computer-assisted image reconstruction of astrocytes in the mammalian retina. Eur. J. Neurosci. 1993, 5, 1093–1106. [CrossRef] [PubMed] 167. Kim, J.H.; Kim, J.H.; Park, J.A.; Lee, S.W.; Kim, W.J.; Yu, Y.S.; Kim, K.W. Blood-neural barrier: Intercellular communication at glio-vascular interface. J. Biochem. Mol. Biol. 2006, 39, 339–345. [CrossRef] [PubMed] 168. Pekny, M.; Wilhelmsson, U.; Pekna, M. The dual role of astrocyte activation and reactive gliosis. Neurosci. Lett. 2014, 565, 30–38. [CrossRef] [PubMed] 169. Shin, E.S.; Huang, Q.; Gurel, Z.; Sorenson, C.M.; Sheibani, N. High glucose alters retinal astrocytes phenotype through increased production of inﬂammatory cytokines and oxidative stress. PLoS ONE 2014, 9, e103148. [CrossRef] [PubMed] 170. Rothhammer, V.; Quintana, F.J. Control of autoimmune cns inﬂammation by astrocytes. Semin. Immunopathol. 2015, 37, 625–638. [CrossRef] [PubMed] 171. Lieth, E.; LaNoue, K.F.; Berkich, D.A.; Xu, B.; Ratz, M.; Taylor, C.; Hutson, S.M. Nitrogen shuttling between neurons and glial cells during glutamate synthesis. J. Neurochem. 2001, 76, 1712–1723. [CrossRef] [PubMed] 171. Lieth, E.; LaNoue, K.F.; Berkich, D.A.; Xu, B.; Ratz, M.; Taylor, C.; Hutson, S.M. Nitrogen shuttling between neurons and glial cells during glutamate synthesis. J. Neurochem. 2001, 76, 1712–1723. References [CrossRef] [PubMed] 172. Orkand, R.K.; Nicholls, J.G.; Kufﬂer, S.W. Effect of nerve impulses on the membrane potential of glial cells in 172. Orkand, R.K.; Nicholls, J.G.; Kufﬂer, S.W. Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia. J. Neurophysiol. 1966, 29, 788–806. [CrossRef] [PubMed] 173. Uckermann, O.; Wolf, A.; Kutzera, F.; Kalisch, F.; Beck-Sickinger, A.G.; Wiedemann, P.; Reichenbach, A.; Bringmann, A. Glutamate release by neurons evokes a purinergic inhibitory mechanism of osmotic glial cell swelling in the rat retina: Activation by neuropeptide y. J. Neurosci. Res. 2006, 83, 538–550. [CrossRef] [PubMed] Metea, M.R.; Newman, E.A. Glial cells dilate and constrict blood vessels: A mechanism of neurovascular coupling. J. Neurosci. 2006, 26, 2862–2870. [CrossRef] [PubMed] 175. Chang, M.L.; Wu, C.H.; Jiang-Shieh, Y.F.; Shieh, J.Y.; Wen, C.Y. Reactive changes of retinal astrocytes and muller glial cells in kainate-induced neuroexcitotoxicity. J. Anat. 2007, 210, 54–65. [CrossRef] [PubMed] 176. Kettenmann, H.; Kirchhoff, F.; Verkhratsky, A. Microglia: New roles for the synaptic stripper. Neuron 2013, 77, 10–18. [CrossRef] [PubMed] 177. Bilimoria, P.M.; Stevens, B. Microglia function during brain development: New insights from animal models. Brain Res. 2015, 1617, 7–17. [CrossRef] [PubMed] 178. Fu, S.; Dong, S.; Zhu, M.; Sherry, D.M.; Wang, C.; You, Z.; Haigh, J.J.; Le, Y.Z. Muller glia are a major cellular source of survival signals for retinal neurons in diabetes. Diabetes 2015, 64, 3554–3563. [CrossRef] [PubMed] 179. Bringmann, A.; Iandiev, I.; Pannicke, T.; Wurm, A.; Hollborn, M.; Wiedemann, P.; Osborne, N.N.; Reichenbach, A. Cellular signaling and factors involved in muller cell gliosis: Neuroprotective and detrimental effects. Prog. Retin. Eye Res. 2009, 28, 423–451. [CrossRef] [PubMed] 28 of 31 28 of 31 Int. J. Mol. Sci. 2018, 19, 942 180. Roy, S.; Amin, S.; Roy, S. Retinal ﬁbrosis in diabetic retinopathy. Exp. Eye Res. 2016, 142, 71–75. [CrossRef] [PubMed] 181. Fogal, B.; Hewett, S.J. Interleukin-1beta: A bridge between inﬂammation and excitotoxicity? J. Neurochem. 2008, 106, 1–23. [CrossRef] [PubMed] 182. Tezel, G. Tnf-alpha signaling in glaucomatous neurodegeneration. Prog. Brain Res. 2008, 173, 409–421. [PubMed] 183. Abcouwer, S.F.; Shanmugam, S.; Gomez, P.F.; Shushanov, S.; Barber, A.J.; Lanoue, K.F.; Quinn, P.G.; Kester, M.; Gardner, T.W. Effect of IL-1beta on survival and energy metabolism of R28 and RGC-5 retinal neurons. Investig. Ophthalmol. Vis. Sci. 2008, 49, 5581–5592. [CrossRef] [PubMed] 184. Sheridan, G.K.; Murphy, K.J. Neuron-glia crosstalk in health and disease: Fractalkine and CX3CR1 take centre stage. Open Biol. References 2013, 3, 130181. [CrossRef] [PubMed] 185. Cui, B.; Sun, J.H.; Xiang, F.F.; Liu, L.; Li, W.J. Aquaporin 4 knockdown exacerbates streptozotocin-induced diabetic retinopathy through aggravating inﬂammatory response. Exp. Eye Res. 2012, 98, 37–43. [CrossRef] [PubMed] Gariano, R.F.; Gardner, T.W. Retinal angiogenesis in development and disease. Nature 2005, 438, 960–966 [CrossRef] [PubMed] 187. Tolentino, M.J.; McLeod, D.S.; Taomoto, M.; Otsuji, T.; Adamis, A.P.; Lutty, G.A. Pathologic features of vascular endothelial growth factor-induced retinopathy in the nonhuman primate. Am. J. Ophthalmol. 2002, 133, 373–385. [CrossRef] 188. Simo, R.; Hernandez, C. Novel approaches for treating diabetic retinopathy based on recent pathogenic evidence. Prog. Retin. Eye Res. 2015, 48, 160–180. [CrossRef] [PubMed] 189. Unterlauft, J.D.; Claudepierre, T.; Schmidt, M.; Muller, K.; Yafai, Y.; Wiedemann, P.; Reichenbach, A.; Eichler, W. Enhanced survival of retinal ganglion cells is mediated by muller glial cell-derived PEDF. Exp. Eye Res. 2014, 127, 206–214. [CrossRef] [PubMed] 190. Unterlauft, J.D.; Eichler, W.; Kuhne, K.; Yang, X.M.; Yafai, Y.; Wiedemann, P.; Reichenbach, A.; Claudepierre, T. Pigment epithelium-derived factor released by muller glial cells exerts neuroprotective effects on retinal ganglion cells. Neurochem. Res. 2012, 37, 1524–1533. [CrossRef] [PubMed] 91. Mu, H.; Zhang, X.M.; Liu, J.J.; Dong, L.; Feng, Z.L. Effect of high glucose concentration on VEGF and PE expression in cultured retinal muller cells. Mol. Biol. Rep. 2009, 36, 2147–2151. [CrossRef] [PubMed] 192. Lange, J.; Yafai, Y.; Reichenbach, A.; Wiedemann, P.; Eichler, W. Regulation of pigment epithelium-derived factor production and release by retinal glial (muller) cells under hypoxia. Investig. Ophthalmol. Vis. Sci. 2008, 49, 5161–5167. [CrossRef] [PubMed] 193. Sakaguchi, H.; Miyagi, M.; Darrow, R.M.; Crabb, J.S.; Hollyﬁeld, J.G.; Organisciak, D.T.; Crabb, J.W. Intense light exposure changes the crystallin content in retina. Exp. Eye Res. 2003, 76, 131–133. [CrossRef] 194. Sax, C.M.; Piatigorsky, J. Expression of the alpha-crystallin/small heat-shock protein/molecular chaperone genes in the lens and other tissues. Adv. Enzymol. Relat. Areas Mol. Biol. 1994, 69, 155–201. [PubMed] 195. Rao, N.A.; Saraswathy, S.; Wu, G.S.; Katselis, G.S.; Wawrousek, E.F.; Bhat, S. Elevated retina-speciﬁc expression of the small heat shock protein, alphaa-crystallin, is associated with photoreceptor protection in experimental uveitis. Investig. Ophthalmol. Vis. Sci. 2008, 49, 1161–1171. [CrossRef] [PubMed] 196. Steele, M.R.; Inman, D.M.; Calkins, D.J.; Horner, P.J.; Vetter, M.L. Microarray analysis of retinal gene expression in the DBA/2J model of glaucoma. Investig. Ophthalmol. Vis. Sci. 2006, 47, 977–985. [CrossRef] [PubMed] 197. Vazquez-Chona, F.; Song, B.K.; Geisert, E.E., Jr. References Temporal changes in gene expression after injury in the rat retina. Investig. Ophthalmol. Vis. Sci. 2004, 45, 2737–2746. [CrossRef] [PubMed] 198. Ruebsam, A.; Dulle, J.E.; Myers, A.M.; Sakrikar, D.; Green, K.M.; Khan, N.W.; Schey, K.; Fort, P.E. A speciﬁc phosphorylation regulates the protective role of alphaa-crystallin in diabetes. JCI Insight 2018, 3. [CrossRef] [PubMed] 199. Lowe, J.; Landon, M.; Pike, I.; Spendlove, I.; McDermott, H.; Mayer, R.J. Dementia with beta-amyloid deposition: Involvement of alpha b-crystallin supports two main diseases. Lancet 1990, 336, 515–516. [CrossRef] 200. Renkawek, K.; Voorter, C.E.; Bosman, G.J.; van Workum, F.P.; de Jong, W.W. Expression of alpha b-crystallin in Alzheimer’s disease. Acta Neuropathol. 1994, 87, 155–160. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 942 29 of 31 29 of 31 201. Iwaki, T.; Wisniewski, T.; Iwaki, A.; Corbin, E.; Tomokane, N.; Tateishi, J.; Goldman, J.E. Accumulation of alpha b-crystallin in central nervous system glia and neurons in pathologic conditions. Am. J. Pathol. 1992, 140, 345–356. [PubMed] 02. Kegel, K.B.; Iwaki, A.; Iwaki, T.; Goldman, J.E. Alphab-crystallin protects glial cells from hypertonic st Am. J. Physiol. 1996, 270, C903–C909. [CrossRef] [PubMed] 203. Parks, W.C.; Wilson, C.L.; Lopez-Boado, Y.S. Matrix metalloproteinases as modulators of inﬂammation and innate immunity. Nat. Rev. Immunol. 2004, 4, 617–629. [CrossRef] [PubMed] 204. Stamenkovic, I. Extracellular matrix remodelling: The role of matrix metalloproteinases. J. Pathol. 2003, 200, 448–464. [CrossRef] [PubMed] 205. Jin, M.; Kashiwagi, K.; Iizuka, Y.; Tanaka, Y.; Imai, M.; Tsukahara, S. Matrix metalloproteinases in human diabetic and nondiabetic vitreous. Retina 2001, 21, 28–33. [CrossRef] [PubMed] 206. Yang, R.; Liu, H.; Williams, I.; Chaqour, B. Matrix metalloproteinase-2 expression and apoptogenic activity in retinal pericytes: Implications in diabetic retinopathy. Ann. N. Y. Acad. Sci. 2007, 1103, 196–201. [CrossRef] [PubMed] 207. Zhang, H.; Trivedi, A.; Lee, J.U.; Lohela, M.; Lee, S.M.; Fandel, T.M.; Werb, Z.; Noble-Haeusslein, L.J. Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood-borne monocytes into the injured spinal cord. J. Neurosci. 2011, 31, 15894–15903. [CrossRef] [PubMed] 208. Navaratna, D.; McGuire, P.G.; Menicucci, G.; Das, A. Proteolytic degradation of VE-cadherin alters the blood-retinal barrier in diabetes. Diabetes 2007, 56, 2380–2387. [CrossRef] [PubMed] 209. Lee, C.Z.; Xue, Z.; Zhu, Y.; Yang, G.Y.; Young, W.L. Matrix metalloproteinase-9 inhibition attenuates vascular endothelial growth factor-induced intracerebral hemorrhage. Stroke 2007, 38, 2563–2568. [CrossRef] [PubMed] 210. Kawasaki, T.; Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 2014, 5, 461. [CrossRef] [PubMed] 211. References Kim, J.J.; Sears, D.D. TLR4 and insulin resistance. Gastroenterol. Res. Pract. 2010, 2010, 212563. [CrossRef] [PubMed] 212. Van Beijnum, J.R.; Buurman, W.A.; Grifﬁoen, A.W. Convergence and ampliﬁcation of toll-like receptor (TLR) and receptor for advanced glycation end products (rage) signaling pathways via high mobility group b1 (HMGB1). Angiogenesis 2008, 11, 91–99. [CrossRef] [PubMed] 213. Feng, L.; Ju, M.; Lee, K.Y.V.; Mackey, A.; Evangelista, M.; Iwata, D.; Adamson, P.; Lashkari, K.; Foxton, R.; Shima, D.; et al. A proinﬂammatory function of toll-like receptor 2 in the retinal pigment epithelium as a novel target for reducing choroidal neovascularization in age-related macular degeneration. Am. J. Pathol. 2017, 187, 2208–2221. [CrossRef] [PubMed] 214. Rajamani, U.; Jialal, I. Hyperglycemia induces toll-like receptor-2 and -4 expression and activity in human microvascular retinal endothelial cells: Implications for diabetic retinopathy. J. Diabetes Res. 2014, 2014, 790902. [CrossRef] [PubMed] 215. Gao, B.B.; Chen, X.; Timothy, N.; Aiello, L.P.; Feener, E.P. Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy. J. Proteome Res. 2008, 7, 2516–2525. [CrossRef] [PubMed] 216. Garcia-Ramirez, M.; Canals, F.; Hernandez, C.; Colome, N.; Ferrer, C.; Carrasco, E.; Garcia-Arumi, J.; Simo, R. Proteomic analysis of human vitreous ﬂuid by ﬂuorescence-based difference gel electrophoresis (DIGE): A new strategy for identifying potential candidates in the pathogenesis of proliferative diabetic retinopathy. Diabetologia 2007, 50, 1294–1303. [CrossRef] [PubMed] 217. Cheng, L.; Bu, H.; Portillo, J.A.; Li, Y.; Subauste, C.S.; Huang, S.S.; Kern, T.S.; Lin, F. Modulation of retinal muller cells by complement receptor c5ar. Investig. Ophthalmol. Vis. Sci. 2013, 54, 8191–8198. [CrossRef] [PubMed] 218. King, P.; Peacock, I.; Donnelly, R. The uk prospective diabetes study (UKPDS): Clinical and therapeutic implications for type 2 diabetes. Br. J. Clin. Pharmacol. 1999, 48, 643–648. [CrossRef] [PubMed] 219. The effect of intensive diabetes treatment on the progression of diabetic retinopathy in insulin-dependent diabetes mellitus. The diabetes control and complications trial. Arch. Ophthalmol. 1995, 113, 36–51. 220. Cunningham, M.A.; Edelman, J.L.; Kaushal, S. Intravitreal steroids for macular edema: The past, the present, and the future. Surv. Ophthalmol. 2008, 53, 139–149. [CrossRef] [PubMed] 30 of 31 30 of 31 Int. J. Mol. Sci. 2018, 19, 942 221. Jonas, J.B. Intravitreal triamcinolone acetonide for diabetic retinopathy. Dev. Ophthalmol. 2007, 39, 96–110. [PubMed] 222. Joussen, A.M.; Poulaki, V.; Mitsiades, N.; Kirchhof, B.; Koizumi, K.; Dohmen, S.; Adamis, A.P. Nonsteroidal anti-inﬂammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression. FASEB J. References 2002, 16, 438–440. [CrossRef] [PubMed] 223. Du, Y.; Sarthy, V.P.; Kern, T.S. Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2004, 287, R735–R741. [CrossRef] [PubMed] 224. Early Treatment Diabetic Retinopathy Study Research Group. Effects of aspirin treatment on diabetic retinopathy. Etdrs report number 8. Ophthalmology 1991, 98, 757–765. 225. The Damad Study Group. Effect of aspirin alone and aspirin plus dipyridamole in early diabetic retinopathy. A multicenter randomized controlled clinical trial. Diabetes 1989, 38, 491–498. 226. Kim, S.J.; Flach, A.J.; Jampol, L.M. Nonsteroidal anti-inﬂammatory drugs in ophthalmology. Surv. Ophthalmol. 2010, 55, 108–133. [CrossRef] [PubMed] 227. Kern, T.S.; Miller, C.M.; Du, Y.; Zheng, L.; Mohr, S.; Ball, S.L.; Kim, M.; Jamison, J.A.; Bingaman, D.P. Topical administration of nepafenac inhibits diabetes-induced retinal microvascular disease and underlying abnormalities of retinal metabolism and physiology. Diabetes 2007, 56, 373–379. [CrossRef] [PubMed] 228. Amrite, A.C.; Ayalasomayajula, S.P.; Cheruvu, N.P.; Kompella, U.B. Single periocular injection of celecoxib-plga microparticles inhibits diabetes-induced elevations in retinal PGE2, VEGF, and vascular leakage. Investig. Ophthalmol. Vis. Sci. 2006, 47, 1149–1160. [CrossRef] [PubMed] 229. Tsilimbaris, M.K.; Panagiotoglou, T.D.; Charisis, S.K.; Anastasakis, A.; Krikonis, T.S.; Christodoulakis, E. The use of intravitreal etanercept in diabetic macular oedema. Semin. Ophthalmol. 2007, 22, 75–79. [CrossRef] [PubMed] 230. Sfikakis, P.P.; Markomichelakis, N.; Theodossiadis, G.P.; Grigoropoulos, V.; Katsilambros, N.; Theodossiadis, P.G. Regression of sight-threatening macular edema in type 2 diabetes following treatment with the anti-tumor necrosis factor monoclonal antibody infliximab. Diabetes Care 2005, 28, 445–447. [CrossRef] [PubMed] 230. Sfikakis, P.P.; Markomichelakis, N.; Theodossiadis, G.P.; Grigoropoulos, V.; Katsilambros, N.; Theodossiadis, P.G. Regression of sight-threatening macular edema in type 2 diabetes following treatment with the anti-tumor 231. Stahel, M.; Becker, M.; Graf, N.; Michels, S. Systemic interleukin 1beta inhibition in proliferative diabetic retinopathy: A prospective open-label study using canakinumab. Retina 2016, 36, 385–391. [CrossRef] [PubMed] 232. A Phase 2, Multi-Center Study to Compare the Efﬁcacy and Safety of a Chemokine CCR2/5 Receptor Antagonist with Ranibizumab in Adults Withdiabetic Macular Edema. Available online: https://clinicaltrials. gov/ct2/show/NCT01994291 (accessed on 30 October 2017). 233. Ulbrich, H.; Eriksson, E.E.; Lindbom, L. Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inﬂammatory disease. Trends Pharmacol. Sci. 2003, 24, 640–647. [CrossRef] [PubMed] 234. Allegro Ophthalmics Announces Positive Topline Results from Del Mar Phase 2b Trial Evaluating Luminate®in Patients with Diabetic Macular Edema. References Available online: http://www.allegroeye.com/press (accessed on 30 October 2017). 235. Fiedler, U.; Reiss, Y.; Scharpfenecker, M.; Grunow, V.; Koidl, S.; Thurston, G.; Gale, N.W.; Witzenrath, M.; Rosseau, S.; Suttorp, N.; et al. Angiopoietin-2 sensitizes endothelial cells to tnf-alpha and has a crucial role in the induction of inﬂammation. Nat. Med. 2006, 12, 235–239. [CrossRef] [PubMed] 236. The Time-2b Study: A Study of Akb-9778, a Novel Tie 2 Activator, in Patients with Non-Proliferative Diabetic Retinopathy (NPDR) (Time-2b). Available online: https://clinicaltrials.gov/ct2/show/NCT03197870 (accessed on 30 October 2017). 237. Sjolie, A.K.; Klein, R.; Porta, M.; Orchard, T.; Fuller, J.; Parving, H.H.; Bilous, R.; Chaturvedi, N.; DIRECT Programme Study Group. Effect of candesartan on progression and regression of retinopathy in type 2 diabetes (direct-protect 2): A randomised placebo-controlled trial. Lancet 2008, 372, 1385–1393. [CrossRef] 238. Mauer, M.; Zinman, B.; Gardiner, R.; Suissa, S.; Sinaiko, A.; Strand, T.; Drummond, K.; Donnelly, S.; Goodyer, P.; Gubler, M.C.; et al. Renal and retinal effects of enalapril and losartan in type 1 diabetes. N. Engl. J. Med. 2009, 361, 40–51. [CrossRef] [PubMed] 239. Garrido-Mesa, N.; Zarzuelo, A.; Galvez, J. Minocycline: Far beyond an antibiotic. Br. J. Pharmacol. 2013, 169, 337–352. [CrossRef] [PubMed] Int. J. Mol. Sci. 2018, 19, 942 31 of 31 31 of 31 240. Domercq, M.; Matute, C. Neuroprotection by tetracyclines. Trends Pharmacol. Sci. 2004, 25, 609–612. [CrossRef] [PubMed] 241. Bernardino, A.L.; Kaushal, D.; Philipp, M.T. The antibiotics doxycycline and minocycline inhibit the inﬂammatory responses to the lyme disease spirochete borrelia burgdorferi. J. Infect. Dis. 2009, 199, 1379–1388. [CrossRef] [PubMed] 242. Cukras, C.A.; Petrou, P.; Chew, E.Y.; Meyerle, C.B.; Wong, W.T. Oral minocycline for the treatment of diabetic macular edema (DME): Results of a phase I/II clinical study. Investig. Ophthalmol. Vis. Sci. 2012, 53, 3865–3874. [CrossRef] [PubMed] 243. Lachin, J.M.; Genuth, S.; Nathan, D.M.; Zinman, B.; Rutledge, B.N.; Group, D.E.R. Effect of glycemic exposure on the risk of microvascular complications in the diabetes control and complications trial—Revisited. Diabetes 2008, 57, 995–1001. [CrossRef] [PubMed] 244. Tang, J.; Du, Y.; Lee, C.A.; Talahalli, R.; Eells, J.T.; Kern, T.S. Low-intensity far-red light inhibits early lesions that contribute to diabetic retinopathy: In vivo and in vitro. Investig. Ophthalmol. Vis. Sci. 2013, 54, 3681–3690. [CrossRef] [PubMed] 245. Tang, J.; Herda, A.A.; Kern, T.S. Photobiomodulation in the treatment of patients with non-center-involving diabetic macular oedema. Br. J. Ophthalmol. 2014, 98, 1013–1015. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4233660278	https://europepmc.org/articles/pmc6876880?pdf=render	English	null	Expression of Concern: The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast	PloS one	2,019	cc-by	1,525	EXPRESSION OF CONCERN a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS The authors apologize for the Fig 5 issues and noted that they were due to figure preparation errors. They commented that the image of JY948(cyr1-, sxa2-, pmp1-) cells in Fig 5B was placed on a black background in preparing the figure, but that the cell image accurately reflects data from the original slide. The incorrect data were included in Fig 5B, JY1716(sxa2-, pmp1-) panel, and in Fig 5D, JY710(sxa2-, pyp2-) and JY1717(sxa2-, pyp2-, cpc2+) panels. In the updated Fig 5, these panels are replaced with the correct images from the original experiments. The underlying raw image for the original Fig 5B, 1716(sxa2-, pmp1-) panel is included in S2 File. Citation: The PLOS ONE Editors (2019) Expression of Concern: The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast. PLoS ONE 14(11): e0225013. https://doi.org/10.1371/journal. pone.0225013 The PLOS ONE Editors After this article [1] was published, concerns were raised about the following calcofluor white staining results reported in Figs 1, 2 and 5: • In the 8h, 16h, and 32h panels for JY546(cyr1-, sxa2-) in Fig 2A, and the 8h panel for JY1628 (cyr1-, sxa2-, cpc2-) in Fig 2B, there appear to be discontinuities in the background image when levels are adjusted. • In the 8h, 16h, and 32h panels for JY546(cyr1-, sxa2-) in Fig 2A, and the 8h panel for JY1628 (cyr1-, sxa2-, cpc2-) in Fig 2B, there appear to be discontinuities in the background image when levels are adjusted. • In the JY1578(cyr1-, sxa2-, oe-cpc2+) panels shown in Fig 2C, when levels are adjusted it appears that contrast levels differ between the cells and the background in the 0 hour, 8 hour and 16 hour panels. There is also a rectangle in the 0h panel and an area where background appears discontinuous with surrounding area in the 8h panel. • In Fig 5B, the image for JY948(cyr1-, sxa2-, pmp1-) appears to have different contrast levels between the cells and surrounding background. • The image for strain JY1716(sxa2-, pmp1-) in Fig 5B shows a strong similarity to the 16h panel in Fig 2C for strain JY1711(sxa2-,oe-cpc2+). • The image for strain JY710(sxa2-, pyp2-) in Fig 5D shows a strong similarity to the panel in Fig 1C for strain JY1711(sxa2-,oe-cpc2+). a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 The authors are unable to clarify the concerns raised about background irregularities in Fig 2A–2C but acknowledge the apparent discontinuities. The authors noted that in preparing Fig 2C the cell images were mounted on black backgrounds and a cell which was out of focus in the 0h panel was covered with an overlying rectangle. According to the authors, except for this instance, no cells were obscured or cropped out of the images. In the updated figure, the panels in question in Fig 2C have been replaced with alternative images from the original experi- ments; Fig 2A and 2B are unchanged. The underlying raw image for Fig 2A, JY546(cyr1-, sxa2-), 16h panel is provided in S1 File. PLOS ONE \| https://doi.org/10.1371/journal.pone.0225013 November 25, 2019 Fig 2. Cpc2p has profound morphological effects upon pheromone-stimulated cells. The strains (A) JY448 (h−, sxa2−) and JY546 (h−, cyr1−, sxa2>lacZ); (B) JY1712 (h−, sxa2−, cpc2−) and JY1628 (h−, cyr1−, sxa2>lacZ, cpc2−); (C) JY1711 (h−, sxa2−+oe-cpc2+), JY1578 (h−, cyr1−, sxa2>lacZ+oe-cpc2+), were grown to mid-exponential phase over 32 h in minimal media. Cells were then imaged using bright field microscopy on pads containing 10 μM of pheromone (see methods). Cells were also stained with calcofluor white (lower panels A-C) to visualize septation. Scale bars 10 μm. Prolonged exposure to pheromone for cells overexpressing Cpc2p (oe-cpc2+) results in multiple projection tips and a failure to undergo cytokinesis. Cells lacking Cpc2p fail to generate the classical shmoo formation as observed for control cells. Published: November 25, 2019 Copyright: © 2019 The PLOS ONE Editors. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Original data are no longer available for the other figure panels discussed above and other results reported in the article. Due to the nature and extent of image issues and lack of underlying image data for the figures in question, the PLOS ONE Editors issue this Expression of Concern. Some of the above concerns were raised in 2014. The PLOS ONE Editors apologize for our delay in resolving this matter and thank the authors for replying promptly to journal queries. PLOS ONE \| https://doi.org/10.1371/journal.pone.0225013 November 25, 2019 1 / 5 2 / 5 PLOS ONE \| https://doi.org/10.1371/journal.pone.0225013 November 25, 2019 Fig 2. Cpc2p has profound morphological effects upon pheromone-stimulated cells. The strains (A) JY448 (h−, sxa2−) and JY546 (h−, cyr1−, sxa2>lacZ); (B) JY1712 (h−, sxa2−, cpc2−) and JY1628 (h−, cyr1−, sxa2>lacZ, cpc2−); (C) JY1711 (h−, sxa2−+oe-cpc2+), JY1578 (h−, cyr1−, sxa2>lacZ+oe-cpc2+), were grown to mid-exponential phase over 32 h in minimal media. Cells were then imaged using bright field microscopy on pads containing 10 μM of pheromone (see methods). Cells were also stained with calcofluor white (lower panels A-C) to visualize septation. Scale bars 10 μm. Prolonged exposure to pheromone for cells overexpressing Cpc2p (oe-cpc2+) results in multiple projection tips and a failure to undergo cytokinesis. Cells lacking Cpc2p fail to generate the classical shmoo formation as observed for control cells. Fig 2. Cpc2p has profound morphological effects upon pheromone-stimulated cells. The strains (A) JY448 (h−, sxa2−) and JY546 (h−, cyr1−, sxa2>lacZ); (B) JY1712 (h−, sxa2−, cpc2−) and JY1628 (h−, cyr1−, sxa2>lacZ, cpc2−); (C) JY1711 (h−, sxa2−+oe-cpc2+), JY1578 (h−, cyr1−, sxa2>lacZ+oe-cpc2+), were grown to mid-exponential phase over 32 h in minimal media. Cells were then imaged using bright field microscopy on pads containing 10 μM of pheromone (see methods). Cells were also stained with calcofluor white (lower panels A-C) to visualize septation. Scale bars 10 μm. Prolonged exposure to pheromone for cells overexpressing Cpc2p (oe-cpc2+) results in multiple projection tips and a failure to undergo cytokinesis. Cells lacking Cpc2p fail to generate the classical shmoo formation as observed for control cells. Supporting information S1 File. Underlying image for Fig 2A, JY546(cyr1-, sxa2-), 16h panel. (PDF) S2 File. Underlying image for Fig 5B, 1716(sxa2-, pmp1-) panel. (PDF) PLOS ONE \| https://doi.org/10.1371/journal.pone.0225013 November 25, 2019 1. Mos M, Esparza-Franco MA, Godfrey EL, Richardson K, Davey J, Ladds G (2013) The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast. PLoS ONE 8(7): e65927. https://doi.org/10.1371/journal.pone.0065927 PMID: 23843946 Published: November 25, 2019 https://doi.org/10.1371/journal.pone.0225013.g001 3 / 5 ps://doi.org/10.1371/journal.pone.0225013 November 25, 2019 4 / PLOS ONE \| https://doi.org/10.1371/journal.pone.0225013 November 25, 2019 4 / 5 Fig 5. Overexpression of Cpc2 in pheromone stimulated cells mimics prolonged pheromone stimulation. (A) Cell morphology and size, at division (micrometers ± S.D.) for the strains JY1716 (h−, sxa2−, pmp1−) and JY948 (h−, cyr1−, sxa2>lacZ, pmp1−) grown in minimal medium at 29˚C and stained with calcofluor white. (B) Strains from A strains were exposure to 10 μM of pheromone for 32 h and stained with calcofluor white. (C) The percentage of cells containing a 1C content (arrested in G1) for the strains JY448, JY1716, JY546 and JY948 as determined using flow cytometry. Cells lacking Pmp1p show a failure to exit from a G1 arrest analogous to strains where the cpc2 ORF has been deleted. (D) Cell morphology and size, at division (micrometers ± S.D.) for strains JY710 (h−, sxa2−pyp2−) and JY1717 (h−, sxa2−, pyp2−+oe-cpc2+) grown in minimal medium at 29˚C and stained with calcofluor white (top panel). Cell morphology and size at division (micrometers ± S.D.) for the strains JY709 (h−, cyr1−, sxa2>lacZ, pyp2−) and JY1661 (h−, cyr1−, sxa2>lacZ, pyp2−+oe-cpc2+) grown in minimal medium at 29˚C and stained with calcofluor white (bottom panel). (E) Numbers of non-septated, septated and multiple septa containing cells for the strains JY448, JY1714, JY1715, JY546, JY987 and JY1661 were determined from 400 individual cells. Values shown correspond to the percentages of the total population. Cells were stained with calcofluor white, to enable visualization of septum material. (F) Pheromone-dependent transcription for the strains JY546, JY709, JY1661 and JY948 was determined using the sxa2>lacZ reporter. Cells were stimulated with pheromone for 16 h in minimal media and assayed for β-galactosidase production using ONPG. Activity is expressed as OD420 units per 106 cells. Values are means of triplicate determinations ± S.E.M. (G) The strains JY709 and JY1661 were grown in minimal medium containing 10 μM of pheromone for the times indicated. Cells were harvested and fixed prior to staining with propidium iodide prior to analysis using flow cytometry (see methods). The proportion of cells exhibiting 1C or 2C DNA content was determined using FACSDiva v4.1 software for the assigned gates indicated by the blue and red shapes. (H) The percentage of cells containing a 1C content (arrested in G1) as determined for the strains JY448, JY546, JY710, JY1717, JY709 and JY1661. https://doi.org/10.1371/journal.pone.0225013.g002 Reference 1. Mos M, Esparza-Franco MA, Godfrey EL, Richardson K, Davey J, Ladds G (2013) The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast. PLoS ONE 8(7): e65927. https://doi.org/10.1371/journal.pone.0065927 PMID: 23843946 5 / 5
https://openalex.org/W4382539386	https://nhess.copernicus.org/preprints/nhess-2023-53/nhess-2023-53.pdf	English	null	Reply on RC1	null	2,023	cc-by	24,001	Inform@Risk. The Development of a Prototype for an Integrated Landslide Early Warning System in an Informal Settlement: the Case of Bello Oriente in Medellín, Colombia 5 Christian Werthmann1, Marta Sapena2, Marlene Kühnl3, John Singer4, Carolina Garcia1, Bettina Menschik5, Heike Schäfer1, Sebastian Schröck6, Lisa Seiler1, Kurosch Thuro5, Hannes Taubenböck2 1Leibniz Universität Hannover, Institut für Landschaftsarchitektur, Herrenhäuserstrasse 2a, 30419 Hannover, Germany 2Deutsches Zentrum für Luft- und Raumfahrt (DLR), Deutsches Fernerkundungsdatenzentrum (DFD), Münchner Str. 20, 82234 Wessling & Universität Würzburg, Institut für Geographie und Geologie, Am Hubland, 97074 Würzburg, Germany 10 3Company for Remote Sensing and Environmental Research (SLU), Kohlsteiner Str. 5, 81243 München, Germany 4AlpGeorisk, Lorenz-Hübner-Str. 15, 86609 Donauwörth, Germany 5Technische Universität München, Lehrstuhl für Ingenieurgeologie, Arcisstraße 21, 80333 München, Germany 6Technische Hochschule Deggendorf (THD), Technologie Campus Freyung (TCF), Institut für Angewandte Informatik, Grafenauerstr. 22, D-94078 Freyung, Germany 15 Correspondence to: Christian Werthmann (werthmann@ila.uni-hannover.de) ORCID ID: 0000-0002-8275-1558 Correspondence to: Christian Werthmann (werthmann@ila.uni-hannover.de) ORCID ID: 0000-0002-8275-1558 20 Abstract. The global number of vulnerable citizens in areas of landslide risk is expected to increase due to the twin forces of climate change and growing urbanization. Self-constructed or informal settlements are, due to shortage of urban land, frequently built in hazardous terrain such as landslide-prone slopes. They are characterized by high dynamics of growth, simple construction methods, strong social dynamics, and are exposed to unsteady political approaches. Landslide Early 20 20 Abstract. The global number of vulnerable citizens in areas of landslide risk is expected to increase due to the twin forces of climate change and growing urbanization. Self-constructed or informal settlements are, due to shortage of urban land, frequently built in hazardous terrain such as landslide-prone slopes. They are characterized by high dynamics of growth, simple construction methods, strong social dynamics, and are exposed to unsteady political approaches. Landslide Early 20 Abstract. The global number of vulnerable citizens in areas of landslide risk is expected to increase due to the twin forces of climate change and growing urbanization. Self-constructed or informal settlements are, due to shortage of urban land, frequently built in hazardous terrain such as landslide-prone slopes. They are characterized by high dynamics of growth, simple construction methods, strong social dynamics, and are exposed to unsteady political approaches. Landslide Early Warning Systems (LEWS) can contribute to decrease their vulnerability, but precise, affordable and culturally integrated 25 LEWS need to be further developed. In this paper, we present a four-year living lab research project that aimed to design, implement, and evaluate a LEWS in the neighborhood of Bello Oriente, located in the urban-rural border of Medellín, Colombia. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Inform@Risk. The Development of a Prototype for an Integrated Landslide Early Warning System in an Informal Settlement: the Case of Bello Oriente in Medellín, Colombia 5 Its research team is composed of landscape architects, geo-engineers, remote sensing and geo-informatic experts. The research team collaborated with a multitude of stakeholders: civil society, private enterprises, non-governmental agencies Warning Systems (LEWS) can contribute to decrease their vulnerability, but precise, affordable and culturally integrated 25 LEWS need to be further developed. In this paper, we present a four-year living lab research project that aimed to design, implement, and evaluate a LEWS in the neighborhood of Bello Oriente, located in the urban-rural border of Medellín, Colombia. Its research team is composed of landscape architects, geo-engineers, remote sensing and geo-informatic experts. The research team collaborated with a multitude of stakeholders: civil society, private enterprises, non-governmental agencies and various branches of government. A prototypical LEWS has been designed, implemented and handed over to the 30 government. It has entered a test and calibration phase. First findings indicate that the integrative development of technical aspects of a LEWS in informal settlements can be challenging, but manageable; whereas, the social and political support can vary and be beyond the control of the designer. It can be concluded that a resilient LEWS for informal settlements has to achieve sufficient social and technical redundancy to maintain basic functionality even in a reduced support scenario. and various branches of government. A prototypical LEWS has been designed, implemented and handed over to the 30 government. It has entered a test and calibration phase. First findings indicate that the integrative development of technical aspects of a LEWS in informal settlements can be challenging, but manageable; whereas, the social and political support can vary and be beyond the control of the designer. It can be concluded that a resilient LEWS for informal settlements has to achieve sufficient social and technical redundancy to maintain basic functionality even in a reduced support scenario. and various branches of government. A prototypical LEWS has been designed, implemented and handed over to the 30 government. It has entered a test and calibration phase. First findings indicate that the integrative development of technical aspects of a LEWS in informal settlements can be challenging, but manageable; whereas, the social and political support can vary and be beyond the control of the designer. It can be concluded that a resilient LEWS for informal settlements has to achieve sufficient social and technical redundancy to maintain basic functionality even in a reduced support scenario. Inform@Risk. The Development of a Prototype for an Integrated Landslide Early Warning System in an Informal Settlement: the Case of Bello Oriente in Medellín, Colombia 5 Keywords: landslides, early warning systems, risk, natural hazard, informal settlements, living lab 35 1 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 1 Introduction The global number of vulnerable people in areas of landslide risk is expected to increase due to the twin forces of climate change (IPCC, 2023) and rising urbanization rates (Taubenböck et al., 2012). For example, in the Andes region, about 1.4 million households are exposed to natural hazards (Peduzzi et al., 2009; UN-Habitat, 2015; UN-Habitat, 2016). Citizens living 40 in informal settlements (neighborhoods that are constructed outside of governmental planning regulations) are disproportionally affected. Alone in the Colombian city of Medellín, 81% of the 290,000 residents exposed to landslide hazard dwell in substandard housing areas (Kühnl et al., 2021). And this is not only true for the Medellín example: Müller et al. (2020) showed that informal settlers tend to settle or have to settle in highly exposed landslide-prone locations. This exposure follows The global number of vulnerable people in areas of landslide risk is expected to increase due to the twin forces of climate change (IPCC, 2023) and rising urbanization rates (Taubenböck et al., 2012). For example, in the Andes region, about 1.4 million households are exposed to natural hazards (Peduzzi et al., 2009; UN-Habitat, 2015; UN-Habitat, 2016). Citizens living 40 in informal settlements (neighborhoods that are constructed outside of governmental planning regulations) are disproportionally affected. Alone in the Colombian city of Medellín, 81% of the 290,000 residents exposed to landslide hazard dwell in substandard housing areas (Kühnl et al., 2021). And this is not only true for the Medellín example: Müller et al. (2020) showed that informal settlers tend to settle or have to settle in highly exposed landslide-prone locations. This exposure follows 40 the global trend of socioeconomically vulnerable populations being disproportionally affected by the effects of climate change. 45 Therefore, the sixth IPCC AR6 Synthesis Report (2023) stresses the urgency to prioritize issues of equity, climate justice, social justice, and inclusion when it comes to mitigation and climate resilient development. The fundamental goal of risk reduction, as for example articulated in the Sendai Framework for Disaster Risk Reduction 2015- 2030 (UNISDR, 2015), is for instance to be achieved by means of EWSs, in our case against landslide hazards. This is a 50 scientific and technical challenge on the one hand and a social challenge on the other. 1 Introduction In this paper, the goals, the technical- social challenges and experiences with the implementation of a LEWS are presented - a possible way between abstract goals and local implementation in the field of risk reduction is to be shown. The city of Medellín was chosen as a case study, because of a large number of residents (over 230,000) living in landslide-prone informal settlements (Kühnl et al., 2021), a pro-active 2030 (UNISDR, 2015), is for instance to be achieved by means of EWSs, in our case against landslide hazards. This is a 50 scientific and technical challenge on the one hand and a social challenge on the other. In this paper, the goals, the technical- social challenges and experiences with the implementation of a LEWS are presented - a possible way between abstract goals and local implementation in the field of risk reduction is to be shown. The city of Medellín was chosen as a case study, because of a large number of residents (over 230,000) living in landslide-prone informal settlements (Kühnl et al., 2021), a pro-active 2030 (UNISDR, 2015), is for instance to be achieved by means of EWSs, in our case against landslide hazards. This is a 50 scientific and technical challenge on the one hand and a social challenge on the other. In this paper, the goals, the technical- social challenges and experiences with the implementation of a LEWS are presented - a possible way between abstract goals and local implementation in the field of risk reduction is to be shown. The city of Medellín was chosen as a case study, because of a large number of residents (over 230,000) living in landslide-prone informal settlements (Kühnl et al., 2021), a pro-active government with a tracking history of innovative urban improvement (Drummond et al., 2012), and the existence of a unique 55 early warning agency in Latin America (SIATA: Sistema de Alerta Temprana del Valle de Aburrá). In Medellín, a resettlement of citizens out of landslide hazard zones is infeasible given the large numbers of people at-risk and lack of urban land in low hazard areas in the city. government with a tracking history of innovative urban improvement (Drummond et al., 2012), and the existence of a unique 55 early warning agency in Latin America (SIATA: Sistema de Alerta Temprana del Valle de Aburrá). 1 Introduction In Medellín, a resettlement of citizens out of landslide hazard zones is infeasible given the large numbers of people at-risk and lack of urban land in low hazard areas in the city. In cases where resettlement or landslide mitigation efforts are infeasible or only partially effective, Landslide Early Warning 60 Systems (LEWS) can decrease human vulnerability, especially in informal settlements characterized by informal governance, insecure tenure, substandard housing and infrastructure conditions. This is one of the reasons why Early Warning Systems (EWS) are considered key tools for reducing disaster risks and supporting climate adaptation (UNFCCC, 2022). Consequently, several integrated LEWS have been implemented throughout the world in recent years (Fathani et al., 2016, Fathani et al., In cases where resettlement or landslide mitigation efforts are infeasible or only partially effective, Landslide Early Warning 60 Systems (LEWS) can decrease human vulnerability, especially in informal settlements characterized by informal governance, insecure tenure, substandard housing and infrastructure conditions. This is one of the reasons why Early Warning Systems (EWS) are considered key tools for reducing disaster risks and supporting climate adaptation (UNFCCC, 2022). Consequently, several integrated LEWS have been implemented throughout the world in recent years (Fathani et al., 2016, Fathani et al., In cases where resettlement or landslide mitigation efforts are infeasible or only partially effective, Landslide Early Warning 60 Systems (LEWS) can decrease human vulnerability, especially in informal settlements characterized by informal governance, insecure tenure, substandard housing and infrastructure conditions. This is one of the reasons why Early Warning Systems (EWS) are considered key tools for reducing disaster risks and supporting climate adaptation (UNFCCC, 2022). Consequently, several integrated LEWS have been implemented throughout the world in recent years (Fathani et al., 2016, Fathani et al., 2023; Peters et al., 2022). Based on these experiences the international standard ISO 22327 (2018), which provides a 65 framework for the implementation of community-based LEWS, has been developed. This general framework however needs to be tailored to the specific situation of informal settlements. 2 2 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. The research project Inform@Risk sought to develop an integrated LEWS in informal settlements with a socio-technical approach following a living lab approach. 1 Introduction As a learning and test case, an informal neighborhood exposed to landslides at the 70 fringes of the Colombian city of Medellín was selected. The goal was to develop an integrated LEWS that is 1) precise 2) affordable 3) socially integrated 4) multi-sectoral 5) spatially integrated 6) multi-scalar and 7) replicable. In detail, these goals mean: 1) precise 75 Existing LEWSs in the Andes region rely on calculated probabilities based on the amount of rainfall in relation to previous events (Sirangelo and Versace, 1996; Segoni et al., 2009). In several cities, including Medellín, geosensors that allow a precise monitoring of earth movements are placed in active mass movements (Michoud et al., 2013), especially in areas with critical infrastructure. In contrast, we seek to develop a self-learning automatic monitoring system also in non-active mass movement zones The system should consist of sensors monitoring soil movement and water pressure and use neuronal networks to 80 zones. The system should consist of sensors monitoring soil movement and water pressure, and use neuronal networks to 80 increase the specificity of warnings and warning times. 2) affordable Precise landslide monitoring systems are available, but they can be cost-intensive. Therefore, the goal is to develop a LEWS that increases affordability in materials, installation and maintenance using a cost-effective Internet of Things solution for the 85 sensor system (e.g. Prakasam et al., 2021; Esposito et al., 2022). 2) affordable Precise landslide monitoring systems are available, but they can be cost-intensive. Therefore, the goal is to develop a LEWS that increases affordability in materials, installation and maintenance using a cost-effective Internet of Things solution for the 85 sensor system (e.g. Prakasam et al., 2021; Esposito et al., 2022). ) Precise landslide monitoring systems are available, but they can be cost-intensive. Therefore, the goal is to develop a LEWS that increases affordability in materials, installation and maintenance using a cost-effective Internet of Things solution for the 85 sensor system (e.g. Prakasam et al., 2021; Esposito et al., 2022). 3) socially integrated An integrated LEWS needs to be accepted, jointly developed with and supported by the residents of vulnerable areas (Baudoin et al., 2016; Marchezini et al., 2018). Within the setting of the living lab, we seek to actively engage at-risk residents of the 90 informal settlement in the design of the LEWS and its training activities. 1 Introduction 7) replicable Direct transference of a successful approach from one informal settlement to another can be counterproductive as individual conditions in informal settlements do vary widely (Perlman, 2010). The gained knowledge in the development of the LEWS 15 should yield process knowledge (not what-to, but how-to) that can be adapted to other locations with similar challenges. ) p Direct transference of a successful approach from one informal settlement to another can be counterproductive as individual conditions in informal settlements do vary widely (Perlman, 2010). The gained knowledge in the development of the LEWS 115 should yield process knowledge (not what-to, but how-to) that can be adapted to other locations with similar challenges. as follows: In section 2 the methodology is introduced, followed in section 3 by findings that are considered preliminary as 120 the socio-technical LEWS is running in a test phase for a year now, but has to prove its durability over longer time periods. In section 4 conclusions are drawn and section 5 ends this paper with a final statement. 1 Introduction An integrated LEWS needs to be accepted, jointly developed with and supported by the residents of vulnerable areas (Baudoin et al., 2016; Marchezini et al., 2018). Within the setting of the living lab, we seek to actively engage at-risk residents of the 90 informal settlement in the design of the LEWS and its training activities. 4) multi-sectoral An integrated LEWS relies on the successful collaboration of a variety of actors: residents, authorities, academic researchers, An integrated LEWS relies on the successful collaboration of a variety of actors: residents, authorities, academic researchers, social organizations, entrepreneurs and non-governmental agencies (UNEP, 2012; García and Fearnley, 2012; Thapa and 95 Adhikari, 2019). Thus, the design seeks the eye-to-eye collaboration with the identified actors in order to develop a fully integrated LEWS. It specifically tries to connect the civil society organizations and the residents of the informal neighborhood with the responsible risk management authorities. social organizations, entrepreneurs and non-governmental agencies (UNEP, 2012; García and Fearnley, 2012; Thapa and 95 Adhikari, 2019). Thus, the design seeks the eye-to-eye collaboration with the identified actors in order to develop a fully integrated LEWS. It specifically tries to connect the civil society organizations and the residents of the informal neighborhood with the responsible risk management authorities. 5) socio-spatial integrated 100 3 3 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. The socio-spatial integration of a LEWS is especially relevant for informal neighborhoods characterized by strong social dynamics and alternative governance models (Brillembourg et al., 2005; Padilla et al., 2009). The constructed technical components of the LEWS should not be incomprehensible by solely installing foreign objects in the neighborhood, but should through a co-creative development process be familiar and comprehensible to the residents, serve as a natural reminder of landslide risk, increase the quality of public spaces in their daily use, the acceptance of the technical system and decrease 105 vandalism. The socio-spatial integration of a LEWS is especially relevant for informal neighborhoods characterized by strong social dynamics and alternative governance models (Brillembourg et al., 2005; Padilla et al., 2009). 1 Introduction The constructed technical components of the LEWS should not be incomprehensible by solely installing foreign objects in the neighborhood, but should through a co-creative development process be familiar and comprehensible to the residents, serve as a natural reminder of landslide risk, increase the quality of public spaces in their daily use, the acceptance of the technical system and decrease 105 vandalism. 105 6) multi-scalar The research aims to gain and connect the knowledge of risk management on the scale of the neighborhood, the district lev and the whole city. The consideration of these three scales should combine the specific situation of the particular neighborhoo 110 with the more general situation of the district and the whole city. 7) replicable Direct transference of a successful approach from one informal settlement to another can be counterproductive as individu conditions in informal settlements do vary widely (Perlman, 2010). The gained knowledge in the development of the LEW 115 should yield process knowledge (not what-to, but how-to) that can be adapted to other locations with similar challenges. This paper aims to show one of many possible paths from global risk reduction goals to local implementation. It shows th developed socio-technical implementation path and reflects on the experiences made. The remainder of this paper is organize as follows: In section 2 the methodology is introduced, followed in section 3 by findings that are considered preliminary 120 the socio-technical LEWS is running in a test phase for a year now, but has to prove its durability over longer time periods. section 4 conclusions are drawn and section 5 ends this paper with a final statement. The research aims to gain and connect the knowledge of risk management on the scale of the neighborhood, the district level and the whole city. The consideration of these three scales should combine the specific situation of the particular neighborhood 110 with the more general situation of the district and the whole city. The research aims to gain and connect the knowledge of risk management on the scale of the neighborhood, the district level and the whole city. The consideration of these three scales should combine the specific situation of the particular neighborhood 110 with the more general situation of the district and the whole city. 2 Methodology and Process The living lab approach in this study integrates the following basic stakeholders: 1) scientific institutions, 2) residents, 3) 140 governmental agencies, 4) local civil-society and non-governmental organizations (CBO’s and NGO’s) and 5) private companies (Fig. 1). 5 Figure 1. Capacities of actors in the living lab approach. The living lab was structured into three phases: 1) investigation, 2) transformation and 3) implementation (Fig. 2). In order 45 gain an understanding of the goals of the actors, the instruments of action, the technical, social and political preconditions well as the interfaces between the multi- and transdisciplinary actors, intensive communication is required. Throughou monthly meetings of all stakeholders in Medellín, quarterly progress meetings of the scientific teams, as well numerous workshops with residents and local experts were held. Extensive field work was pursued in form of physic Figure 1. Capacities of actors in the living lab approach. 2 Methodology and Process The research project follows a living lab methodology, which has been applied in many d The research project follows a living lab methodology, which has been applied in many different research contexts, but has internationally gained increasing relevance for research in the sustainable transformation of cities (Hossain et al., 2019; von 125 Wirth et al., 2019). As the profound understanding of complex socio-technical processes plays a key role, the living lab methodology has been chosen for the development of an integrated LEWS in an informal settlement. There is no uniform definition of living labs, because all have different goals and ways of working. However, a common characteristic is the horizontal partnership between scientists, various institutions and citizens, working together in a bounded 130 space for intentional collaborative experience for a study case (Voytenco et al., 2015). Living Labs promise to produce a broader diversity of solutions as unexpected results are integral to the method (Schneidewind, 2014). Thereby, the living lab characteristic is the horizontal partnership between scientists, various institutions and citizens, working together in a bounded 130 space for intentional collaborative experience for a study case (Voytenco et al., 2015). Living Labs promise to produce a broader diversity of solutions as unexpected results are integral to the method (Schneidewind, 2014). Thereby, the living lab 4 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. methodology is understood as a learning process, where all stakeholders are actively involved from the initial stage to jointly identify challenges and different mind-sets. Transformation can be co-created and prototypes can be developed, which are then tested and improved in a real live environment. While the methodology can produce tailored innovation for social systems and 135 governmental structures (Yasuoka et al., 2018), the transferability of its gained knowledge is context specific and varies. The more the living lab is dependent from its specific physical, social and political context, the lesser are its results transferable. In some cases, only patterns that might serve as useful guides for similar cases can be deduced (Schneidewind, 2014). The living lab approach in this study integrates the following basic stakeholders: 1) scientific institutions, 2) residents, 3) 140 governmental agencies, 4) local civil-society and non-governmental organizations (CBO’s and NGO’s) and 5) private companies (Fig. 1). Figure 1. Capacities of actors in the living lab approach. 5 The living lab was structured into three phases: 1) investigation, 2) transformation and 3) implementation (Fig. 2). In order to 145 gain an understanding of the goals of the actors, the instruments of action, the technical, social and political preconditions as well as the interfaces between the multi- and transdisciplinary actors, intensive communication is required. Throughout, monthly meetings of all stakeholders in Medellín, quarterly progress meetings of the scientific teams, as well as numerous workshops with residents and local experts were held. Extensive field work was pursued in form of physical, 5 The living lab was structured into three phases: 1) investigation, 2) transformation and 3) implementation (Fig. 2). In order to 145 gain an understanding of the goals of the actors, the instruments of action, the technical, social and political preconditions as well as the interfaces between the multi- and transdisciplinary actors, intensive communication is required. Throughout, monthly meetings of all stakeholders in Medellín, quarterly progress meetings of the scientific teams, as well as numerous workshops with residents and local experts were held. Extensive field work was pursued in form of physical, 5 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. geological and geo-electric surveys, drillings, drone flights, community and expert workshops, meetings, interviews, 150 questionnaires and two representative surveys. External as internal events shaped the individual outcome of the study. Figure 2 gives a detailed overview of the research process in chronological order. Figure 2. Time line of the living lab approach in relation to external and internal events that impacted the research. 5 Figure 2. Time line of the living lab approach in relation to external and internal events that impacted the research. 155 Figure 2. Time line of the living lab approach in relation to external and internal events that impacted the research. 155 In the first investigative phase, hazard and vulnerability assessments to landslides were conducted on the city, district and neighborhood level of Medellín. For the selection of a pilot-neighborhood, 16 informal settlements with high landslide risk were examined based on 20 qualitative and quantitative technical, social and administrative parameters, such as risk, type of past mass movements or willingness of the community to participate. Finally, the informal neighborhood of Bello Oriente on the northeastern fringe of Medellín (Fig 3) was selected as case study based on several factors being the four most critical: 160 Figure 2. Figure 1. Capacities of actors in the living lab approach. Time line of the living lab approach in relation to external and internal events that impacted the research. 155 In the first investigative phase, hazard and vulnerability assessments to landslides were conducted on the city, district and neighborhood level of Medellín. For the selection of a pilot-neighborhood, 16 informal settlements with high landslide risk were examined based on 20 qualitative and quantitative technical, social and administrative parameters, such as risk, type of past mass movements or willingness of the community to participate. Finally, the informal neighborhood of Bello Oriente on the northeastern fringe of Medellín (Fig. 3) was selected as case study based on several factors, being the four most critical: 160 Figure 2. Time line of the living lab approach in relation to external and internal events that impacted the research. 155 In the first investigative phase, hazard and vulnerability assessments to landslides were conducted on the city, district and neighborhood level of Medellín. For the selection of a pilot-neighborhood, 16 informal settlements with high landslide risk were examined based on 20 qualitative and quantitative technical, social and administrative parameters, such as risk, type of past mass movements or willingness of the community to participate. Finally, the informal neighborhood of Bello Oriente on the northeastern fringe of Medellín (Fig. 3) was selected as case study based on several factors, being the four most critic 160 1. landslides have recently occurred in the neighborhood with a consequent risk awareness of the population 2. the community voluntarily embraces the pilot project 1. landslides have recently occurred in the neighborhood with a consequent risk awareness of 1. landslides have recently occurred in the neighborhood with a consequent risk awareness of the population 2. the community voluntarily embraces the pilot project 6 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3. the community is well organized and has experience in working with non-profit organizations 4. field work can be performed in an acceptably safe environment 3. the community is well organized and has experience in working with non-profit organizations 165 Figure 3 illustrates the pilot-neighborhood. It shows the steep terrain in the upper reaches of Bello Oriente as well as the location of buildings and their usages as recorded based on drone data. 170 Figure 3. 3D model of Bello Oriente based on a 2021 drone flight. Figure 1. Capacities of actors in the living lab approach. The second transformative phase was characterized by the design of the LEWS and its subsequent installation in Bello Oriente. A two-pronged approach of landslide monitoring by a technical sensor system and residents was developed leading to multiple warning and dissemination options that will be explained in more detail later (Fig. 4). In general, both warning chains report to the risk management authority of the city of Medellín. There, the information is processed to different warning types and 175 170 Figure 3. 3D model of Bello Oriente based on a 2021 drone flight. The second transformative phase was characterized by the design of the LEWS and its subsequent The second transformative phase was characterized by the design of the LEWS and its subsequent installation in Bello Oriente. A two-pronged approach of landslide monitoring by a technical sensor system and residents was developed leading to multiple warning and dissemination options that will be explained in more detail later (Fig. 4). In general, both warning chains report to the risk management authority of the city of Medellín. There, the information is processed to different warning types and 75 communicated via various channels. p y g q A two-pronged approach of landslide monitoring by a technical sensor system and residents was developed leading to multiple warning and dissemination options that will be explained in more detail later (Fig. 4). In general, both warning chains report to the risk management authority of the city of Medellín. There, the information is processed to different warning types and 175 communicated via various channels. 7 Figure 4. Landslide monitoring and warning design. The implementation, the third phase, was characterized by community training and technical improvement. It concluded with 0 the handover of a preliminary and prototypical LEWS to the disaster risk management authorities of Medellín at the end of 2022. After the handover, a reduced research team continues in 2023 with the calibration of the sensor components and the further development of a mobile application as part of a one-year test and training phase to include the prototype into the disaster risk management practices of the municipality and further evaluate its usefulness. Figure 4. Landslide monitoring and warning design. Figure 4. Landslide monitoring and warning design. The implementation, the third phase, was characterized by community training and technical improvement. It concluded with 180 the handover of a preliminary and prototypical LEWS to the disaster risk management authorities of Medellín at the end of 2022. After the handover, a reduced research team continues in 2023 with the calibration of the sensor components and the further development of a mobile application as part of a one-year test and training phase to include the prototype into the disaster risk management practices of the municipality and further evaluate its usefulness. The implementation, the third phase, was characterized by community training and technical improvement. It concluded with 180 the handover of a preliminary and prototypical LEWS to the disaster risk management authorities of Medellín at the end of 2022. After the handover, a reduced research team continues in 2023 with the calibration of the sensor components and the further development of a mobile application as part of a one-year test and training phase to include the prototype into the disaster risk management practices of the municipality and further evaluate its usefulness. 8 8 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.1.1 Landslide Risk at City Scale The city of Medellín is located in the Aburrá valley with steep slopes on both sides. By combining heterogeneous geodata sources - official geodata of the city and remote sensing - it becomes possible to map and analyze both, the topography and the exposed landslide zones as well as the settlement structures, their development, and the amount of people living in specific 190 parts of the city. This topographical situation in combination with heavy rain fall events and the increasing population numbers makes many of the 2.32 million inhabitants of Medellín exposed to landslide hazards due to their residential location. On the one hand, the available landslide hazard map (Plan de ordenamiento territorial, POT 2014), which includes the zoning of landslide hazards (Alcaldía de Medellín, 2014), was used to map landslide susceptibility areas across entire Medellín. These the exposed landslide zones as well as the settlement structures, their development, and the amount of people living in specific 190 parts of the city. This topographical situation in combination with heavy rain fall events and the increasing population numbers makes many of the 2.32 million inhabitants of Medellín exposed to landslide hazards due to their residential location. On the one hand, the available landslide hazard map (Plan de ordenamiento territorial, POT 2014), which includes the zoning of landslide hazards (Alcaldía de Medellín, 2014), was used to map landslide susceptibility areas across entire Medellín. These are areas of slopes mostly steeper than 17° predominantly formed by debris flow deposits and Medellín Dunite. On the other 195 hand, census data from 2018 was used for a very-high-resolution disaggregation of the population on a three-dimensional building model (cf. for methodological details, see Sapena et al., 2022). Beyond, the building morphologies and patterns were used to assess the character of the neighborhood into precarious or non-precarious settlements (Kühnl et al., 2021) – a physical approach that is capable to proxy the social group of deprived populations to a certain degree (Wurm and Taubenböck, 2018). are areas of slopes mostly steeper than 17° predominantly formed by debris flow deposits and Medellín Dunite. On the other 195 hand, census data from 2018 was used for a very-high-resolution disaggregation of the population on a three-dimensional building model (cf. for methodological details, see Sapena et al., 2022). 3.1.1 Landslide Risk at City Scale Beyond, the building morphologies and patterns were used to assess the character of the neighborhood into precarious or non-precarious settlements (Kühnl et al., 2021) – a physical approach that is capable to proxy the social group of deprived populations to a certain degree (Wurm and Taubenböck, 2018). are areas of slopes mostly steeper than 17° predominantly formed by debris flow deposits and Medellín Dunite. On the other 195 hand, census data from 2018 was used for a very-high-resolution disaggregation of the population on a three-dimensional building model (cf. for methodological details, see Sapena et al., 2022). Beyond, the building morphologies and patterns were used to assess the character of the neighborhood into precarious or non-precarious settlements (Kühnl et al., 2021) – a physical approach that is capable to proxy the social group of deprived populations to a certain degree (Wurm and Taubenböck, 2018). Various characteristics such as small building sizes, low building heights, complex, dense and organic patterns were used for 200 classification of precarious settlements (see Kühnl et al., 2021; Taubenböck et al., 2018). Lastly, medium and high-resolution satellite data was used to monitor the development of Medellín and with it the increase of landslide exposure figures. Overall, more than 87,000 inhabitants (about 4%) were found to live in high risk landslide hazard zones in Medellín. Interestingly, around 70,000 of them are precarious dwellers (Fig. 5). This reveals inequality for this social group due to Various characteristics such as small building sizes, low building heights, complex, dense and organic patterns were used for 200 classification of precarious settlements (see Kühnl et al., 2021; Taubenböck et al., 2018). Lastly, medium and high-resolution satellite data was used to monitor the development of Medellín and with it the increase of landslide exposure figures. Overall, more than 87,000 inhabitants (about 4%) were found to live in high risk landslide hazard zones in Medellín. Interestingly, around 70,000 of them are precarious dwellers (Fig. 5). This reveals inequality for this social group due to residential locations, as for the entire city only 38% of the population are classified precarious, while in landslide prone areas 205 it is more than 80%. These findings are particularly relevant since we measured an annual growth of 150 built-up hectares per year since 2016, mainly taking place in precarious neighborhoods, which resulted in an increase of 1% of exposed areas in a period of only five years. 3.1.1 Landslide Risk at City Scale residential locations, as for the entire city only 38% of the population are classified precarious, while in landslide prone areas 205 it is more than 80%. These findings are particularly relevant since we measured an annual growth of 150 built-up hectares per year since 2016, mainly taking place in precarious neighborhoods, which resulted in an increase of 1% of exposed areas in a period of only five years. 9 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 5. Exposure of built-up areas in Medellin with the location of precarious settlements (in yellow), as well as areas at high ris 10 of landslides (in red). In the background, a hill shade map displays the topography. Using remote sensing methods, census, and official data sets, it thus becomes possible to locate landslide-exposed areas an Figure 5. Exposure of built-up areas in Medellin with the location of precarious settlements (in yellow), as well as areas at high risk 210 of landslides (in red). In the background, a hill shade map displays the topography. Using remote sensing methods, census, and official data sets, it thus becomes possible to locate landslide-exposed areas and quantify the exposed population and social groups. This large-scale approach allows for an approximation, but locally very specific conditions arise, for which higher resolutions of data are needed. Figure 5. Exposure of built-up areas in Medellin with the location of precarious settlements (in yellow), as well as areas at high risk 210 of landslides (in red). In the background, a hill shade map displays the topography. Figure 5. Exposure of built-up areas in Medellin with the location of precarious settlements (in yellow), as well as areas at high risk 210 of landslides (in red). In the background, a hill shade map displays the topography. Using remote sensing methods, census, and official data sets, it thus becomes possible to locate landslide-exposed areas and quantify the exposed population and social groups. This large-scale approach allows for an approximation, but locally very specific conditions arise, for which higher resolutions of data are needed. 10 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.1.2 Landslide Hazard at Pilot-Neighborhood The official data used in the city-wide analysis are highly relevant; however, their aggregated The official data used in the city-wide analysis are highly relevant; however, their aggregated information often does not satisfy the local specifics, thus a higher level of spatial and thematic detail and accuracy is required. This can be achieved by field 220 investigations. A detailed geological map of Bello Oriente and its vicinity has firstly been generated in 2020 and was re- evaluated in 2022. Additionally, several electrical resistivity tomography (ERT) profiles as well as four exploratory drillings were conducted to achieve a better understanding of the geological conditions. As Fig. 6 shows, a major part of the pilot- neighborhood is composed of colluvium material (green and turquoise colors), which is prone to landslide activity. The bedrock the local specifics, thus a higher level of spatial and thematic detail and accuracy is required. This can be achieved by field 220 investigations. A detailed geological map of Bello Oriente and its vicinity has firstly been generated in 2020 and was re- evaluated in 2022. Additionally, several electrical resistivity tomography (ERT) profiles as well as four exploratory drillings were conducted to achieve a better understanding of the geological conditions. As Fig. 6 shows, a major part of the pilot- neighborhood is composed of colluvium material (green and turquoise colors), which is prone to landslide activity. The bedrock consists of the Medellín Dunite Formation. Parts of the rock mass which are in-situ weathered and not dislocated due to 225 landslide processes can be named as Saprolite. According to earlier studies (Tobón-Hincapié et al., 2011) and as was confirmed by the geological investigations carried out in this project, the Medellín Dunite shows signs of so called pseudokarst processes. The dissolution processes taking place in the rock mass go back to weathering, generating a strongly indented relief of dunite top, caves and joints filled with clay material of up to 1.0 m width, which reach a depth of up to about 60 m (Rendón-Giraldo, consists of the Medellín Dunite Formation. Parts of the rock mass which are in-situ weathered and not dislocated due to 225 landslide processes can be named as Saprolite. According to earlier studies (Tobón-Hincapié et al., 2011) and as was confirmed by the geological investigations carried out in this project, the Medellín Dunite shows signs of so called pseudokarst processes. 3.1.2 Landslide Hazard at Pilot-Neighborhood The dissolution processes taking place in the rock mass go back to weathering, generating a strongly indented relief of dunite top, caves and joints filled with clay material of up to 1.0 m width, which reach a depth of up to about 60 m (Rendón-Giraldo, 2020; Breuninger et al 2021) 230 11 11 Figure 6. Geological Map of the pilot-neighborhood. The major part above the settlement of Bello Oriente is composed of colluvium material (green and turquoise colors), which is prone to landslides (Breuninger et al., 2021). https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 6. Geological Map of the pilot-neighborhood. The major part above the settlement of Bello Oriente is composed of colluvium material (green and turquoise colors), which is prone to landslides (Breuninger et al., 2021). The combination of weathering processes and tectonics has a major influence on the landslide haz 7 shows the tectonic setting of Bello Oriente and its near vicinity. The map was compiled by combining the results of hillshade 235 analyses and structural information collected during detailed field investigations and scanline analyses. The orange-colored joints (E-W strike) belong to a fault system which, based on the ERT profiles, seems to create a graben-structure in the region between the drillings B2 and A1. Due to this graben-structure, a slight increase in the colluvium thickness (up to 10 m) at the center of this structure can be recognized. The green-colored joint set (NNE-SSW strike) is oriented roughly perpendicular to 7 shows the tectonic setting of Bello Oriente and its near vicinity. The map was compiled by combining the results of hillshade 235 analyses and structural information collected during detailed field investigations and scanline analyses. The orange-colored joints (E-W strike) belong to a fault system which, based on the ERT profiles, seems to create a graben-structure in the region between the drillings B2 and A1. Due to this graben-structure, a slight increase in the colluvium thickness (up to 10 m) at the center of this structure can be recognized. The green-colored joint set (NNE-SSW strike) is oriented roughly perpendicular to the E-W striking fault set and runs nearly parallel to the slope. 3.1.2 Landslide Hazard at Pilot-Neighborhood This joint set could promote landslides as it might act as a zone 240 of weakness in the rock mass, favoring water infiltration and possibly back scarps. the E-W striking fault set and runs nearly parallel to the slope. This joint set could promote landslides as it might act as a zone 240 of weakness in the rock mass, favoring water infiltration and possibly back scarps. 12 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 7. Tectonic map at the neighborhood scale of Bello Oriente. The E-W as well as the NNE-SSW striking fault systems ar suggested to influence the rock mass mechanics and thus the landslide hazard in the current project area. Figure 7. Tectonic map at the neighborhood scale of Bello Oriente. The E-W as well as the NNE-SSW striking fault systems are suggested to influence the rock mass mechanics and thus the landslide hazard in the current project area. Figure 7. Tectonic map at the neighborhood scale of Bello Oriente. The E-W as well as the NNE-SSW striking fault systems are suggested to influence the rock mass mechanics and thus the landslide hazard in the current project area. Figure 7. Tectonic map at the neighborhood scale of Bello Oriente. The E-W as well as the NNE-SSW striking fault systems are suggested to influence the rock mass mechanics and thus the landslide hazard in the current project area. The most relevant landslide process has been identified as rotational slides of different sizes in the soil cover above the dunite. 245 Thereby in general, the depth of the soil cover controls the size of the probable landslide events. As rainfall is the most important trigger for landslides in the region, the hydrogeological conditions are key to understand where future landslides are likely to occur. Due to the high heterogeneity of the subsurface it however is impossible to gain detailed local knowledge of the subsurface water flow. The most relevant landslide process has been identified as rotational slides of different sizes in the soil cover above the dunite. 245 Thereby in general, the depth of the soil cover controls the size of the probable landslide events. As rainfall is the most important trigger for landslides in the region, the hydrogeological conditions are key to understand where future landslides are likely to occur. 250 The hazard map for Bello Oriente was established by combining the information of the geological investigation with mapped landslide processes on-site and the recognized events of the SIMMA database, which contains landslide events in the Aburrá Valley, classified according to their type of process, their location, and the event date. The analysis showed, that the frequency - magnitude distribution of landslides in the region of Bello Oriente can be characterized by three scenarios: 255 3.1.2 Landslide Hazard at Pilot-Neighborhood Due to the high heterogeneity of the subsurface it however is impossible to gain detailed local knowledge of the subsurface water flow. The most relevant landslide process has been identified as rotational slides of different sizes in the soil cover above the dunite. 245 Thereby in general, the depth of the soil cover controls the size of the probable landslide events. As rainfall is the most important trigger for landslides in the region, the hydrogeological conditions are key to understand where future landslides are likely to occur. Due to the high heterogeneity of the subsurface it however is impossible to gain detailed local knowledge of the subsurface water flow. 250 The hazard map for Bello Oriente was established by combining the information of the geological investigation with mapped landslide processes on-site and the recognized events of the SIMMA database, which contains landslide events in the Aburrá Valley, classified according to their type of process, their location, and the event date. The analysis showed, that the frequency - magnitude distribution of landslides in the region of Bello Oriente can be characterized by three scenarios: 255 250 13 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. • small landslides (annuality of 30 years), an area of up to 4000 m2 and a depth of 5-10 m, • medium landslides (annuality of 100 years) with areas of 4000-15000 m2 and depths of 20-30 m and • large landslides (annuality of 300 years or more) with areas of more than 100000 m2 and depths of up to 30 m. The areas, where these scenarios might occur within the project area were determined based on the existing landslide 260 phenomena, information of the soil cover thickness and a critical slope angle for landslides. The latter was determined by 2D limit equilibrium analyses using the code SLIDE-2D (Rocscience) and shear tests on soil samples. Both indicate that the critical slope angle for landslides varies between 22° and 24° depending on the water saturation. Using the “Fahrböschung” approach with an approximated worst-case runout angle of 20°, the potential runout of the landslides was estimated. The areas, where these scenarios might occur within the project area were determined based on the existing landslide 260 phenomena, information of the soil cover thickness and a critical slope angle for landslides. • small landslides (annuality of 30 years), an area of up to 4000 m2 and a depth of 5-10 m, • medium landslides (annuality of 100 years) with areas of 4000-15000 m2 and depths of 20-30 m and • large landslides (annuality of 300 years or more) with areas of more than 100000 m2 and depths of up to 30 m. 3.1.2 Landslide Hazard at Pilot-Neighborhood The grey shaded overlays represent landslides, which were mapped in the field and are suggested to belong to the 30-year event-category. It was estimated that 72% of the population of the pilot-neighborhood are exposed to some landslide hazard. This finding 280 roughly correlates with the hazard perception of the residents of Bello Oriente. In a representative survey in 2020 (259 participants) over 60% of the interviewees rated the likelihood of a landslide nearby their home as high to very high and 65% of participants were very concerned about it. It was estimated that 72% of the population of the pilot-neighborhood are exposed to some landslide hazard. This finding 280 roughly correlates with the hazard perception of the residents of Bello Oriente. In a representative survey in 2020 (259 participants) over 60% of the interviewees rated the likelihood of a landslide nearby their home as high to very high and 65% of participants were very concerned about it. 3.1.2 Landslide Hazard at Pilot-Neighborhood The latter was determined by 2D limit equilibrium analyses using the code SLIDE-2D (Rocscience) and shear tests on soil samples. Both indicate that the critical slope angle for landslides varies between 22° and 24° depending on the water saturation. Using the “Fahrböschung” approach with an approximated worst-case runout angle of 20°, the potential runout of the landslides was estimated. Figure 8 shows the hazard map for the most frequent 30-year event. The map contains not only the information about the hazard and the run-out area, but also the location and dimensions of the mapped landslides in Bello Oriente. Figure 8 shows the hazard map for the most frequent 30-year event. The map contains not only the information about the hazard and the run-out area, but also the location and dimensions of the mapped landslides in Bello Oriente. The local-level analysis revealed a high vulnerability in Bello Oriente, particularly in the LEWS pilot- neighborhood (38ha), where 49% of the roughly 4,600 residents (in 2021) live in an area with a “high” landslide hazard, 23 % 270 in the “residual hazard” area and 28 % in an area, where up to now no landslide risk could be detected (Table 1) Landslide Hazard Number of buildings Population (%) category “high” 680 49 category “residual” 258 23 no hazard/risk 347 28 Table 1: Statistics summary of the vulnerability analysis for the LEWS pilot-neighborhood including the exposed buildings and the population in %. 275 Table 1: Statistics summary of the vulnerability analysis for the LEWS pilot-neighborhood including the exposed buildings and the population in %. 275 275 14 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 8. Hazard map (30-year event) of Bello Oriente and its vicinity at the neighborhood scale. The orange area marks the hazard area, where scarps could occur, the red shaded area represents the potential runout areas. The grey shaded overlays represent landslides, which were mapped in the field and are suggested to belong to the 30-year event-category. Figure 8. Hazard map (30-year event) of Bello Oriente and its vicinity at the neighborhood scale. The orange area marks the hazard area, where scarps could occur, the red shaded area represents the potential runout areas. 3.2 Landslide Monitoring While the results of the detailed landslide hazard and risk analysis characterizes the causes for likelihood of initiation and the 285 effect of a landslide at any point in the area of the test-site, they do not allow to make a prediction where and when the next landslide will occur. To achieve this, the slope needs to be continuously monitored. Two complementary monitoring concepts were pursued in the living lab approach: automatic sensor-based monitoring and manual community monitoring. While the results of the detailed landslide hazard and risk analysis characterizes the causes for likelihood of initiation and the 285 effect of a landslide at any point in the area of the test-site, they do not allow to make a prediction where and when the next landslide will occur. To achieve this, the slope needs to be continuously monitored. Two complementary monitoring concepts were pursued in the living lab approach: automatic sensor-based monitoring and manual community monitoring. 15 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.2.1 Automatic Sensor-Based Monitoring While the CSM and wire-exten- 305 someter systems provide continuous, spatially highly resolved deformation observations along measurement profiles, the wireless sensor nodes add punctual observations of deformation and triggering mechanisms based on the integrated MEMS sensors and other external geotechnical and hydrological sensors (e.g. piezometers). implemented in Bello Oriente (check Fig. A1, Gamperl et al., 2021; Gamperl et al., 2023). While the CSM and wire-exten- 305 someter systems provide continuous, spatially highly resolved deformation observations along measurement profiles, the wireless sensor nodes add punctual observations of deformation and triggering mechanisms based on the integrated MEMS sensors and other external geotechnical and hydrological sensors (e.g. piezometers). The Measurement Nodes (Fig. 9a and b) are based on a newly developed sensor PCB and the Arduino MKR1310 310 microcontroller, which provides a LoRa® (Long Range) radio transmission link. The new LoRa® wireless technology allows the transmission of small data packets across large distances of several kilometers while only requiring very little power. This makes the development of sensor networks possible, which can be distributed in a wide area with little additional infrastructure required. The nodes can be operated with six standard AA batteries for a very long period (up to about 2 years) and by using The Measurement Nodes (Fig. 9a and b) are based on a newly developed sensor PCB and the Arduino MKR1310 310 microcontroller, which provides a LoRa® (Long Range) radio transmission link. The new LoRa® wireless technology allows the transmission of small data packets across large distances of several kilometers while only requiring very little power. This makes the development of sensor networks possible, which can be distributed in a wide area with little additional infrastructure required. The nodes can be operated with six standard AA batteries for a very long period (up to about 2 years) and by using The Measurement Nodes (Fig. 9a and b) are based on a newly developed sensor PCB and the Arduino MKR1310 310 microcontroller, which provides a LoRa® (Long Range) radio transmission link. The new LoRa® wireless technology allows the transmission of small data packets across large distances of several kilometers while only requiring very little power. This makes the development of sensor networks possible, which can be distributed in a wide area with little additional infrastructure required. 3.2.1 Automatic Sensor-Based Monitoring The general concept of the sensor-based monitoring system is to predict the future behavior of the observed landslide prone 290 area. This is achieved based on detailed hydrogeological and geotechnical models, which have been calibrated by observational data from hydrogeological field tests, geotechnical laboratory tests and a dense low-cost geosensor network. By including the triggering process in the models (e.g. intense precipitation leading to high ground water levels), it is feasible to issue first general notifications several days to hours in advance of a critical phase concerning the stability of the slope. When the onset of slow, but increasing movement is detected, spatially precise early warnings can be issued. In case of further or sudden strong 295 acceleration of the slope, (evacuation-)alarms can be disseminated at least hours to minutes prior to a catastrophic event, allowing people to leave the endangered area in time. of slow, but increasing movement is detected, spatially precise early warnings can be issued. In case of further or sudden strong 295 acceleration of the slope, (evacuation-)alarms can be disseminated at least hours to minutes prior to a catastrophic event, allowing people to leave the endangered area in time. In order to reliably detect the initiation of landslides – especially in the “small” scenario within a 30-years repeat period described above (rotational slides, usually 10–100 m wide) – without previous knowledge of the exact location, area-wide 300 spatially and temporally highly resolved and accurate deformation observations are required. To achieve this high measure- ment density at an affordable cost, a new geosensor network consisting of a combination of horizontally installed Continuous Shear Monitor (CSM) (Singer et al., 2009) and wire-extensometer measurement systems combined with low cost wireless sensor nodes (Inform@Risk Measurement Nodes) based on Internet of Things technology has been developed and implemented in Bello Oriente (check Fig. A1, Gamperl et al., 2021; Gamperl et al., 2023). While the CSM and wire-exten- 305 someter systems provide continuous, spatially highly resolved deformation observations along measurement profiles, the wireless sensor nodes add punctual observations of deformation and triggering mechanisms based on the integrated MEMS sensors and other external geotechnical and hydrological sensors (e.g. piezometers). implemented in Bello Oriente (check Fig. A1, Gamperl et al., 2021; Gamperl et al., 2023). 3.2.1 Automatic Sensor-Based Monitoring The nodes can be operated with six standard AA batteries for a very long period (up to about 2 years) and by using small solar panels continuous operation is possible. By using affordable hardware and materials and utilizing simple 315 manufacturing and installation techniques (e.g. 3D printing), the cost of the complete system is comparably low. small solar panels continuous operation is possible. By using affordable hardware and materials and utilizing simple 315 manufacturing and installation techniques (e.g. 3D printing), the cost of the complete system is comparably low. While the Measurement Node already has a suite of MEMS sensors on-board (high-quality tilt sensor, thermometer and baro- meter), additional analog and digital sensors can be connected via the onboard 24 bit A/D converter and several digital interfaces. 320 16 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Above fallows the first inclination sensor (orange), which - when installed - is followed by another ball joint (2nd reference point 35 located exactly 1 m above first joint). If required, further inclination sensors can be added. (b) installation of subsurface probe into a brick seating cube. Figure 10. (a) Sensor Probes ready for installation. The piezometer (blue) is located at the bottom of the probe. Above follows a ball joint (black) which exactly fits into the used installation pipes and functions as reference points for the inclination measurements. Above fallows the first inclination sensor (orange), which - when installed - is followed by another ball joint (2nd reference point 5 located exactly 1 m above first joint). If required, further inclination sensors can be added. (b) installation of subsurface probe into a brick seating cube. 335 The Measurement Node and the Subsurface Probe have been developed as open source. Details on the sensor system can be found on the Inform@Risk Wiki page (www.informatrisk.com). The Measurement Node and the Subsurface Probe have been developed as open source. Details on the sensor system can be found on the Inform@Risk Wiki page (www.informatrisk.com). In total 115 measurement nodes, of which 45 have a subsurface probe attached to them, as well as about 1 km of CSM/EXT 340 measurement lines have been installed in the project area (check Fig. A2). The sensor system is operational since fall 2022 and has been in operation without major complications. q y g y g the pilot-neighborhood. These via internet connected gateways receive the LoRa® transmissions of the measurement nodes 345 and the data of the CSM/EXT system on site and transmit the data to the data cloud. In the living lab approach the AlpGeorisk ONLINE (www.alpgeorisk.com) data management platform is used to manage the large amounts of data (several GB per month) which are generated by the geosensor network. This platform is responsible for data quality management, calibration, storage, analysis and visualization via web-portal as well as system management and control and notifications. In order to achieve an optimal effectiveness of the system in terms of risk reduction, the density of the observations was varied 350 throughout the project area based on the results of the risk assessment. In less risk prone areas, the sensor density is significantly reduced. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 9. (a) Sensor Node with mounted solar panel for autarkic operation (b) Inside view on four sensor nodes. The Arduino microprocessor MKR WAN 1310 (blue PCB) is connected to the Inform@Risk sensor PCB (large green PCB), which contains a suite of different sensors including a thermometer, barometer, 4-channel 24 bit ADC and an accurate tiltmeter, which is mounted on an orange rotatable base to make leveling of the sensor independent of the box orientation possible. Figure 9. (a) Sensor Node with mounted solar panel for autarkic operation (b) Inside view on four sensor nodes. The Arduino microprocessor MKR WAN 1310 (blue PCB) is connected to the Inform@Risk sensor PCB (large green PCB), which contains a suite of different sensors including a thermometer, barometer, 4-channel 24 bit ADC and an accurate tiltmeter, which is mounted on an orange rotatable base to make leveling of the sensor independent of the box orientation possible. 325 One example of an external sensor is the Subsurface Probe (Fig.10a and b), which consists of a combination of one or more inclination sensors and a water pressure sensor. The subsurface probe is installed into a small diameter borehole (1 1/4 inch) and can function either as a chain inclinometer system (multiple inclination sensors) or as a simple motion detection system (one inclination sensor). The water pressure sensor gives information on the ground water conditions at installation depth. 30 17 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 10. (a) Sensor Probes ready for installation. The piezometer (blue) is located at the bottom of the probe. Above follows a ball joint (black) which exactly fits into the used installation pipes and functions as reference points for the inclination measurements. Above fallows the first inclination sensor (orange), which - when installed - is followed by another ball joint (2nd reference point 35 located exactly 1 m above first joint). If required, further inclination sensors can be added. (b) installation of subsurface probe into a brick seating cube. Figure 10. (a) Sensor Probes ready for installation. The piezometer (blue) is located at the bottom of the probe. Above follows a ball joint (black) which exactly fits into the used installation pipes and functions as reference points for the inclination measurements. 3.2.3 Socio-spatial Integration of Monitoring System 370 3.2.3 Socio-spatial Integration of Monitoring System 370 In order to be accepted, any spatial intervention in informal settlements has to be developed together with residents (Imparato et al., 2003; Werthmann, 2021). This is especially true for Bello Oriente, as many of the 4,600 residents display a fundamental distrust against the government due to feared resettlement measures. A main objective of the socio-spatial integration of the technical elements of the LEWS was to serve as a natural reminder of landslide risk, while increasing the quality of public 3.2.3 Socio-spatial Integration of Monitoring System 370 In order to be accepted, any spatial intervention in informal settlements has to be developed together with residents (Imparato et al., 2003; Werthmann, 2021). This is especially true for Bello Oriente, as many of the 4,600 residents display a fundamental distrust against the government due to feared resettlement measures. A main objective of the socio-spatial integration of the technical elements of the LEWS was to serve as a natural reminder of landslide risk, while increasing the quality of public spaces in their daily use, the acceptance of the technical system and decrease vandalism (goal 5). Accordingly, particular 375 attention was paid to involve the residents right from the beginning and develop trust through transparent communication, while gradually building up risk awareness and monitoring skills by joint activities on site. From 2019 to 2022 over 40 workshops and community meetings with over 1,000 participants and 19 organizations (NGO’, CBO’s) were held (an example for one typical workshop setting can be seen in Fig. 11a). Different types of designs for the sensor locations were developed, spaces in their daily use, the acceptance of the technical system and decrease vandalism (goal 5). Accordingly, particular 375 attention was paid to involve the residents right from the beginning and develop trust through transparent communication, while gradually building up risk awareness and monitoring skills by joint activities on site. From 2019 to 2022 over 40 workshops and community meetings with over 1,000 participants and 19 organizations (NGO’, CBO’s) were held (an example for one typical workshop setting can be seen in Fig. 11a). Different types of designs for the sensor locations were developed, ranging from simple sensor covers and markers in remote areas to painted cube seats and benches with information boards in 380 the public spaces, which can be used as meeting places and recreational spaces. 3.2.2 Manual and People-Based Monitoring 355 3.2.2 Manual and People-Based Monitoring 355 The inhabitants of Bello Oriente have a good empiric knowledge of the different landslide signs. During a landslide in 2017 for example, residents detected changes in the slope such as cracks, water ponds, new bumps in the road, tilted trees, among others. After monitoring the evolution of those signs over the course of a week, people of the community alerted the neighbors who were able to evacuate just in time. The inhabitants of Bello Oriente have a good empiric knowledge of the different landslide signs. During a landslide in 2017 for example, residents detected changes in the slope such as cracks, water ponds, new bumps in the road, tilted trees, among others. After monitoring the evolution of those signs over the course of a week, people of the community alerted the neighbors who were able to evacuate just in time. Therefore, it is a fact that the inhabitants of the high hazard areas, especially those who have lived there the longest, 360 perform visual monitoring of their surroundings. The community also looks for water pipe leaking, which is one of the main anthropic landslide triggers, together with informal house, road and path construction. The importance of visual monitoring was discussed and preliminary experiences were exchanged in numerous workshops with the community. Moreover, some manual instruments with recycled materials were constructed (Fig. 11b shows a simple Therefore, it is a fact that the inhabitants of the high hazard areas, especially those who have lived there the longest, 360 perform visual monitoring of their surroundings. The community also looks for water pipe leaking, which is one of the main anthropic landslide triggers, together with informal house, road and path construction. The importance of visual monitoring was discussed and preliminary experiences were exchanged in numerous workshops with the community. Moreover, some manual instruments with recycled materials were constructed (Fig. 11b shows a simple Therefore, it is a fact that the inhabitants of the high hazard areas, especially those who have lived there the longest, 360 perform visual monitoring of their surroundings. The community also looks for water pipe leaking, which is one of the main anthropic landslide triggers, together with informal house, road and path construction. The importance of visual monitoring was discussed and preliminary experiences were exchanged in numerous workshops with the community. Moreover, some manual instruments with recycled materials were constructed (Fig. 3.2.2 Manual and People-Based Monitoring 355 11b shows a simple inclinometer) in order to show that it is possible to manually monitor and measure deformations (UNGRD, 2013), equivalent 365 to the functionality of automatic sensors. In deviation of the international standard ISO 22327 (2018), community leaders of the pilot-neighborhood opted against the creation of a specific landslide monitoring group, and preferred a decentralized integration of monitoring activities in existing community groups. inclinometer) in order to show that it is possible to manually monitor and measure deformations (UNGRD, 2013), equivalent 365 to the functionality of automatic sensors. In deviation of the international standard ISO 22327 (2018), community leaders of the pilot-neighborhood opted against the creation of a specific landslide monitoring group, and preferred a decentralized integration of monitoring activities in existing community groups. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. While this approach ensures an optimal cost-benefit ratio of the entire system, it must be emphasized that especially 18 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. small low intensity and very unlikely events might not be detected. To fill these gaps, the sensor-based monitoring was complemented with people-based monitoring. small low intensity and very unlikely events might not be detected. To fill these gaps, the sensor-based monitoring was complemented with people-based monitoring. 3.2.3 Socio-spatial Integration of Monitoring System 370 Tree plantings and micro gardens were co- created to support the self-proclaimed goal of the pilot-neighborhood to transform into an ‘eco-barrio’. 19 Figure 11. (a) Typical community workshop setting and (b) manual monitoring installation of a simple inclination meter by residents. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 11. (a) Typical community workshop setting and (b) manual monitoring installation of a simple inclination meter by residents. Figure 11. (a) Typical community workshop setting and (b) manual monitoring installation of a simple inclination meter by residents. 385 Prototypes of cubes and benches were tested on site with different materials which are weather resistant and have low susceptibility to vandalism and theft. Finally, because of its low cost, local availability, and its familiarity to local craftsmen, brick was chosen (Fig.12a, b). In addition, an overall signage and information system was developed to explain landslide risk and the LEWS (Fig. 12c). Continuous information campaigns were organized using a graphic language with comic characters that provide easy to understand information and allow for emotional attachment (Fig. 12d). 390 Sensor locations were discussed and continuously adjusted in joint field walks with the residents. Although the sites are located in public spaces, the permission of landowners and neighboring residents was considered of importance to gain acceptance and avoid future conflicts. Thereby, the local expertise on the territory was essential to cope with complicated landownership and criminal activities of gangs controlling parts of the informal settlement. Before the construction works were carried out by professional firms, tree plantings were organized with the community to 395 mark the sensor locations. Moreover, a concept of sensor ‘godparents’ was established, where nearby residents agreed to care for the sites. Some bench locations were refused by the residents, being concerned that members of criminal groups would use them as meeting places. Finally, 15 cube seats, six brick benches, nine information boards, and two murals painted by local artists (Fig. 12e) were installed in the upper part of the pilot-neighborhood by mid 2022 (Fig. A3 shows all locations in plan). Before the construction works were carried out by professional firms, tree plantings were organized with the community to 395 mark the sensor locations. Moreover, a concept of sensor ‘godparents’ was established, where nearby residents agreed to care for the sites. knowledge about its function. 410 28) living nearby the enclosures have been conducted shortly after installation. It was found that the degree of socio-spatial 405 integration of the LEWS varies. In the survey, 50% of the residents were not even aware of the enclosures. This can be partly explained by the fact that the installations are located in the upper half of the pilot-neighborhood and that the survey was done shortly after completion of installation. 28% liked the new elements while only 17% of the residents knew about their special function. The interviewed residents living near the enclosures generally liked them, but many also displayed only rudimentary knowledge about its function. 410 3.2.3 Socio-spatial Integration of Monitoring System 370 Some bench locations were refused by the residents, being concerned that members of criminal groups would use them as meeting places. Finally, 15 cube seats, six brick benches, nine information boards, and two murals painted by local artists (Fig. 12e) were installed in the upper part of the pilot-neighborhood by mid 2022 (Fig. A3 shows all locations in plan). 20 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 12. (a) Collaborative painting of a brick seating cube with a subsurface probe inside (b) a micro garden next to a brick bench sheltering a Subsurface Probe (c) Brick bench housing the central monitoring station and an information board (d) Inclination sensor on a private home with the Inform@Risk mascot “Lora” (e) murals painted by local artists with the help of children. 400 Figure 12. (a) Collaborative painting of a brick seating cube with a subsurface probe inside (b) a micro garden next to a brick bench sheltering a Subsurface Probe (c) Brick bench housing the central monitoring station and an information board (d) Inclination sensor on a private home with the Inform@Risk mascot “Lora” (e) murals painted by local artists with the help of children. Figure 12. (a) Collaborative painting of a brick seating cube with a subsurface probe inside (b) a micro garden next to a brick bench sheltering a Subsurface Probe (c) Brick bench housing the central monitoring station and an information board (d) Inclination sensor on a private home with the Inform@Risk mascot “Lora” (e) murals painted by local artists with the help of children. 28) living nearby the enclosures have been conducted shortly after installation. It was found that the degree of socio-spatial 405 integration of the LEWS varies. In the survey, 50% of the residents were not even aware of the enclosures. This can be partly explained by the fact that the installations are located in the upper half of the pilot-neighborhood and that the survey was done shortly after completion of installation. 28% liked the new elements while only 17% of the residents knew about their special function. The interviewed residents living near the enclosures generally liked them, but many also displayed only rudimentary 21 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.2.3 Socio-spatial Integration of Monitoring System 370 A different indicator of socio-spatial integration is the degree of vandalism. Here, also a mixed picture emerges. After six months of use, most of the communal tree plantings were cut. In contrast, the micro-gardens are actively tended to and the cubes and benches reveal only minor traces of damage (mostly caused by children). 3.3.1 Warning Levels 415 In the first quarter of 2023, the LEWS is still in a “learning phase”, where the sensor data, on site observations, and numerical modeling results are used to define the warning thresholds for the different individual sensors and sensor combinations. In the first quarter of 2023, the LEWS is still in a “learning phase”, where the sensor data, on site observations, and numerical modeling results are used to define the warning thresholds for the different individual sensors and sensor combinations. In order to assess the short- to medium-term hazard level, mainly the triggering factors rainfall and groundwater height are considered. On the one hand, as soon as enough data is gathered time series analyses will be performed to identify causal and considered. On the one hand, as soon as enough data is gathered time series analyses will be performed to identify causal and temporal relationships between short-, medium- and long-term rainfall and groundwater levels. On the other hand, the hazard 420 level is determined using groundwater level thresholds at e.g. 50, 75 and 90 percent of the critical water table derived from the geological/geomechanical models created during the hazard analysis. The threshold values thereby are determined individually for different geological homogeneous zones throughout the pilot-neighborhood and are applied to the according sensors in this area. temporal relationships between short-, medium- and long-term rainfall and groundwater levels. On the other hand, the hazard 420 level is determined using groundwater level thresholds at e.g. 50, 75 and 90 percent of the critical water table derived from the geological/geomechanical models created during the hazard analysis. The threshold values thereby are determined individually for different geological homogeneous zones throughout the pilot-neighborhood and are applied to the according sensors in this area. 425 Four basic warning levels have been developed in collaboration with the Medellín risk management agency DAGRD and the civil society organizations, combining both qualitative and quantitative thresholds (see table 2). As an augmentation to the three proposed warning levels of the international standard ISO 22327 (2018), a first level (green) has been added, in order to promote constant monitoring and to avoid increasing risk activities such as incorrect construction practices and leaking water 425 Four basic warning levels have been developed in collaboration with the Medellín risk management agency DAGRD and the civil society organizations, combining both qualitative and quantitative thresholds (see table 2). 3.3.1 Warning Levels 415 As an augmentation to the three proposed warning levels of the international standard ISO 22327 (2018), a first level (green) has been added, in order to promote constant monitoring and to avoid increasing risk activities such as incorrect construction practices and leaking water pipes. The second level (yellow) promotes more detailed monitoring, when any changes in weather conditions and/or soil 430 movement are detected. The third level (orange) promotes to prepare for evacuation. The fourth level (red) means immediate evacuation. The third and fourth level are critical warning levels that are only reached when significant deformation is detected. pipes. The second level (yellow) promotes more detailed monitoring, when any changes in weather conditions and/or soil 430 movement are detected. The third level (orange) promotes to prepare for evacuation. The fourth level (red) means immediate evacuation. The third and fourth level are critical warning levels that are only reached when significant deformation is detected. Based on how many and which neighboring sensor nodes show deformation, the affected area and landslide mass are estimated 435 and reported to the risk management authorities. If a further or sudden strong acceleration is detected, it is planned that the system can issue an immediate (evacuation)-alarm using acoustic signals. Based on how many and which neighboring sensor nodes show deformation, the affected area and landslide mass are estimated 435 and reported to the risk management authorities. If a further or sudden strong acceleration is detected, it is planned that the system can issue an immediate (evacuation)-alarm using acoustic signals. Based on how many and which neighboring sensor nodes show deformation, the affected area and landslide mass are estimated 435 and reported to the risk management authorities. If a further or sudden strong acceleration is detected, it is planned that the system can issue an immediate (evacuation)-alarm using acoustic signals. Depending on the exposed population, deformation rate and the affected area, different actors (experts, trained community members, first responders, whole population) are informed. Usually, warnings will be checked by an expert before they are 440 sent to the inhabitants. Only when at least two neighboring sensor nodes show strong acceleration in similar direction and at the same time the warnings are issued without review. Depending on the exposed population, deformation rate and the affected area, different actors (experts, trained community members, first responders, whole population) are informed. 3.3.2 Warning Dissemination channels Several channels are used to assure that the warnings reach the people at risk. First, direct communication via a WhatsApp group with members of the community and representatives of governmental risk management organizations has been installed. Photos, written and audio messages are shared in the group to describe the situation and alert in case of a possible emergency. Second, in the framework of the project two instruments were installed to send alert messages and alarm sounds when needed. 450 They consist in one sound system with eight speakers and one siren, both with solar panels to avoid disruption in case of failure in the cable energy grid Fig.13a). The third alert channel is the Inform@Risk App. Second, in the framework of the project two instruments were installed to send alert messages and alarm sounds when needed. 450 They consist in one sound system with eight speakers and one siren, both with solar panels to avoid disruption in case of failure in the cable energy grid Fig.13a). The third alert channel is the Inform@Risk App. 3.3.1 Warning Levels 415 Usually, warnings will be checked by an expert before they are 440 sent to the inhabitants. Only when at least two neighboring sensor nodes show strong acceleration in similar direction and at the same time the warnings are issued without review. 22 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.3.3 Warning App 455 Smart phone use is limited and most inhabitants do not have access to reliable internet; therefore, there is greater need than usual for the app’s functionality to be available offline. Smart phones in use have limited memory space 465 and processing power, imposing challenges for interactive content like large dynamic maps. In addition, inhabitants are worried about tracking through personal data. The app is currently in a testing phase. 460 3.3.4 Alert Network The landslide that occurred in 2017 is strongly present in the memory of the inhabitants of The landslide that occurred in 2017 is strongly present in the memory of the inhabitants of Bello Oriente. Having caused no The landslide that occurred in 2017 is strongly present in the memory of the inhabitants of Bello Oriente. Having caused no casualties due to the timely alert disseminated by the community itself and an evacuation just seconds before the landslide, 470 this event serves as a baseline for future landslides. However, future events might not show preliminary signs and might happen at night, when it is more difficult to visually identify the movement, therefore increasing the risk of having casualties. Considering that the population is highly dynamic, with constant new incomers in the pilot-neighborhood, the residents of g p p g y y , p g , Bello Oriente decided not to create specific alert networks but to promote for every neighbor to be responsible to disseminate 475 the alert among the closest neighbors. In order to achieve redundancy, other alert methods were suggested as using WhatsApp groups, the sound and siren system, the mobile App, and also to involve the Local Community Council and local civil society organizations in the alert dissemination. The effectiveness of this alert network has to be evaluated through regular emergency drills in the future. Bello Oriente decided not to create specific alert networks but to promote for every neighbor to be responsible to disseminate 475 the alert among the closest neighbors. In order to achieve redundancy, other alert methods were suggested as using WhatsApp groups, the sound and siren system, the mobile App, and also to involve the Local Community Council and local civil society organizations in the alert dissemination. The effectiveness of this alert network has to be evaluated through regular emergency drills in the future. Bello Oriente decided not to create specific alert networks but to promote for every neighbor to be responsible to disseminate 475 the alert among the closest neighbors. In order to achieve redundancy, other alert methods were suggested as using WhatsApp groups, the sound and siren system, the mobile App, and also to involve the Local Community Council and local civil society organizations in the alert dissemination. The effectiveness of this alert network has to be evaluated through regular emergency drills in the future. 3.3.3 Warning App 455 The Inform@Risk Smartphone App serves as a hub of both general and immediate information concerning the current state of landslide risk. It displays a range of relevant information for inhabitants of the study area like the currently active warning level as defined by the LEWS, status and data of individual sensors in the project area and pre-defined meeting points in case 23 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. of alert. In case of emergency, users of the app receive push notifications and detailed information about spatial constraints of the ongoing event. The app also encourages users to participate in the warning dissemination and decision-making process by 460 enabling them to upload reports containing photos and descriptions of local developments (e.g. new sinkholes or cracks in infrastructure) to notify authorities directly (Fig. A4). of alert. In case of emergency, users of the app receive push notifications and detailed information about spatial constraints of the ongoing event. The app also encourages users to participate in the warning dissemination and decision-making process by 460 enabling them to upload reports containing photos and descriptions of local developments (e.g. new sinkholes or cracks in infrastructure) to notify authorities directly (Fig. A4). Additional challenges for the development of the smart phone app were added due to the location of the pilot-neighborhood in an informal settlement. Smart phone use is limited and most inhabitants do not have access to reliable internet; therefore, there is greater need than usual for the app’s functionality to be available offline. Smart phones in use have limited memory space 465 and processing power, imposing challenges for interactive content like large dynamic maps. In addition, inhabitants are worried about tracking through personal data. The app is currently in a testing phase. of alert. In case of emergency, users of the app receive push notifications and detailed information about spatial constraints of the ongoing event. The app also encourages users to participate in the warning dissemination and decision-making process by 460 enabling them to upload reports containing photos and descriptions of local developments (e.g. new sinkholes or cracks in infrastructure) to notify authorities directly (Fig. A4). Additional challenges for the development of the smart phone app were added due to the location of the pilot-neighborhood in an informal settlement. 3.4.1 Evacuation Planning In Bello Oriente, the first respondents are the community members themselves, which is fairly common for informal settlements in fringe areas (Heffer and ELLA Brazil, 2013). Bello Oriente, which is located in the upper hills of Medellín, can settlements in fringe areas (Heffer and ELLA Brazil, 2013). Bello Oriente, which is located in the upper hills of Medellín, can only be reached through steep and heavily trafficked narrow streets. It is almost impossible for the emergency response teams 490 of the nearest firefighter station to swiftly reach the area (in one emergency drill, it took the firefighters 40 minutes to arrive). Therefore, the training of residents for a rapid evacuation scenario is crucial. Accordingly, several community workshops have been held to identify best evacuation routes and meeting points. The evacuation strategy was designed for different scenarios of day/night time and weather conditions. Basic evacuation actions were established for each warning level. Afterwards, the participants of the workshops were asked to share the gained knowledge with the other inhabitants of the pilot- 495 neighborhood. Afterwards, the participants of the workshops were asked to share the gained knowledge with the other inhabitants of the pilot- 495 neighborhood. 3.4 Response Capacity 480 According to the risk management Colombian Law 1523 of 2012, risk management is the responsibility of every inhabitant of Colombia. Before, all the responsibility was with the government. Following this concept, the Inform@risk project has been working on strengthening the risk management capacities of the community and the local organizations, regarding risk knowledge, basic risk reduction and mitigation measures, as well as capacity reaction. Despite the expected increase in monitoring and dissemination capacities of the LEWS prototype there remains the possibility of a fast onset landslide with 485 According to the risk management Colombian Law 1523 of 2012, risk management is the responsibility of every inhabitant of Colombia. Before, all the responsibility was with the government. Following this concept, the Inform@risk project has been working on strengthening the risk management capacities of the community and the local organizations, regarding risk knowledge, basic risk reduction and mitigation measures, as well as capacity reaction. Despite the expected increase in monitoring and dissemination capacities of the LEWS prototype, there remains the possibility of a fast-onset landslide with 485 monitoring and dissemination capacities of the LEWS prototype, there remains the possibility of a fast-onset landslide with 485 just minutes of warning time. 24 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3.4.2 Evacuation Training The first two community landslide evacuation drills ever held in Medellín have been performed in 2022 with about 100 participants each in Bello Oriente (Fig. 13b). They were developed with the support of the local risk management authority, firefighters and volunteers from different organizations, together with volunteers of the community. In order to execute the 00 drill, the area was divided into several sub-sectors, where the evacuation was led by the volunteers, who were promoting the evacuation door to door Afterwards participants evaluated the drills and identified various improvements For example The first two community landslide evacuation drills ever held in Medellín have been performed in 2022 with about 100 participants each in Bello Oriente (Fig. 13b). They were developed with the support of the local risk management authority, y p participants each in Bello Oriente (Fig. 13b). They were developed with the support of the local risk management authority, firefighters and volunteers from different organizations, together with volunteers of the community. In order to execute the 500 drill, the area was divided into several sub-sectors, where the evacuation was led by the volunteers, who were promoting the evacuation door to door. Afterwards, participants evaluated the drills and identified various improvements. For example, residents with permanent and temporary disabilities need to be identified in order to assign neighbors, who could assist them in case of an emergency; or additional sound systems and megaphones are needed to reach all residents. firefighters and volunteers from different organizations, together with volunteers of the community. In order to execute the 500 drill, the area was divided into several sub-sectors, where the evacuation was led by the volunteers, who were promoting the evacuation door to door. Afterwards, participants evaluated the drills and identified various improvements. For example, residents with permanent and temporary disabilities need to be identified in order to assign neighbors, who could assist them in case of an emergency; or additional sound systems and megaphones are needed to reach all residents. g y y g p 505 Overall, it became clear that the participation of civil society organizations needs to be strengthened and the numbers of participating residents increased. The risk management authorities need to continue regular emergency trainings and biannual drills in order to establish a familiar routine for the residents at risk. As these drills are a novelty for the risk management authorities, proper protocols need to be further developed. 3.4.2 Evacuation Training Overall, it became clear that the participation of civil society organizations needs to be strengthened and the numbers of participating residents increased. The risk management authorities need to continue regular emergency trainings and biannual drills in order to establish a familiar routine for the residents at risk. As these drills are a novelty for the risk management authorities, proper protocols need to be further developed. 510 One of the unresolved challenges that remains is the best timing of evacuation, when a landslide is imminent. Landslides in the pilot-neighborhood usually take days, or weeks, to completely move after first signs have been detected. This makes the evacuation timing problematic as populations in densely populated informal areas rarely have private options (neighbors or friends) to evacuate to for longer periods, and public agencies are not allowed to build temporary shelters before a landslide has occurred. Therefore, better evacuation procedures and shelter options still need to be developed. 515 One of the unresolved challenges that remains is the best timing of evacuation, when a landslide is imminent. Landslides in the pilot-neighborhood usually take days, or weeks, to completely move after first signs have been detected. This makes the evacuation timing problematic as populations in densely populated informal areas rarely have private options (neighbors or friends) to evacuate to for longer periods, and public agencies are not allowed to build temporary shelters before a landslide has occurred. Therefore, better evacuation procedures and shelter options still need to be developed. 515 25 Figure 13. (a) siren system that can be manually operated and (b) residents congregate at one of the meeting points after an emergency drill. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure 13. (a) siren system that can be manually operated and (b) residents congregate at one of the meeting points after an emergency drill. Figure 13. (a) siren system that can be manually operated and (b) residents congregate at one of the meeting points after an emergency drill. 4 Living Lab Experiences and Conclusions 520 The following conclusions have been drawn from the four-year research and development phase with the addition of some cautious prognostic statements: firm understanding of its usefulness and potential replicability. The following conclusions have been drawn from the four-year research and development phase with the addition of some cautious prognostic statements: firm understanding of its usefulness and potential replicability. The following conclusions have been drawn from the four-year research and development phase with the addition of some cautious prognostic statements: 1) precise 540 The automatic sensor-based monitoring system delivers temporally and spatially very dense data on deformation and trigger process activity. Based on the collected data, we believe that it will be possible to generate landslide activity warnings with to date unparalleled precision in terms of location and level of hazard. As even small movements in the range of millimeters can be detected by the sensor system, landslides can be identified and 1) precise 540 The automatic sensor-based monitoring system delivers temporally and spatially very dense data on deformation and trigger process activity. Based on the collected data, we believe that it will be possible to generate landslide activity warnings with to date unparalleled precision in terms of location and level of hazard. As even small movements in the range of millimeters can be detected by the sensor system, landslides can be identified and monitored at a very early stage of development. This allows to detect critical changes in the landslide activity early 545 on giving more time to react appropriately. Based on the experiences made from past landslides in the region it is believed that an early warning (without certainty of critical failure) can be issued several days in advance, an alert (with near certainty of critical failure) hours in advance to the event. Currently, the warning generation procedure is in development and still has to prove its capability in the coming months and years. It also has to be noted that monitored at a very early stage of development. This allows to detect critical changes in the landslide activity early 545 on giving more time to react appropriately. Based on the experiences made from past landslides in the region it is believed that an early warning (without certainty of critical failure) can be issued several days in advance, an alert (with near certainty of critical failure) hours in advance to the event. 4 Living Lab Experiences and Conclusions 520 The development of an LEWS is a socio-technical challenge. The living lab approach revealed one possible pathway to integrate the multiple objectives of stakeholders, as well as the technical, scientific, and social and planning expertise, in such a way that the risk of a precarious settlement to landslides could be addressed. It is precisely the integration of such diverse issues as scientific concept, data and results, technical implementation, social acceptance, and political will that has to align. As the gained knowledge of living labs is generally context 525 specific and not universally transferable to other cases (Schneidewind et al., 2014), we will critically reflect on the findings in the specific context of the study and try to deduct useful patterns for the development of LEWS in informal urbanization: social acceptance, and political will that has to align. As the gained knowledge of living labs is generally context 525 specific and not universally transferable to other cases (Schneidewind et al., 2014), we will critically reflect on the findings in the specific context of the study and try to deduct useful patterns for the development of LEWS in informal urbanization: In the presented living lab approach seven goals for an integrated LEWS against landslides in informal 530 settlements have been formulated at the outset. The LEWS has to be 1) precise, 2) affordable, 3) socially integrated, 4) multi-sectoral, 5) spatially integrated, 6) multi-scalar and 7) replicable. In the course of the research an eighth goal was found to be of high relevance and was thus added: 8) redundant. A prototype of a LEWS has been developed and handed over to the responsible risk management agency of Medellín (DAGRD), at the end of 2022. This prototype is not yet a fully functioning warning system; it has entered a test and calibration phase. It needs to 535 be further refined and integrated into the disaster prevention procedures of the city of Medellín in order to get a 26 firm understanding of its usefulness and potential replicability. The following conclusions have been drawn from the four-year research and development phase with the addition of some cautious prognostic statements: https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. firm understanding of its usefulness and potential replicability. 4 Living Lab Experiences and Conclusions 520 Currently, the warning generation procedure is in development and still has to prove its capability in the coming months and years. It also has to be noted that the automatic sensor-based monitoring system can despite increased accuracies only sense deformation, where a 550 sensor is actually located. It is designed for medium to large landslides with potential catastrophic impact. Smaller landslides (smaller than about 30m) cannot be detected and community-based monitoring remains essential (also in case of technical failure). the automatic sensor-based monitoring system can despite increased accuracies only sense deformation, where a 550 sensor is actually located. It is designed for medium to large landslides with potential catastrophic impact. Smaller landslides (smaller than about 30m) cannot be detected and community-based monitoring remains essential (also in case of technical failure). 2) affordable 555 The low-cost technologies used to build up the sensor-based monitoring system and the risk-based sensor placement strategy lead to an overall very efficient system with comparably low installation cost (about € 5 to €41 per protected inhabitant, Sapena et al., 2023). This provides a basis for installing such a system in other areas as well. Nevertheless, the political will and thus also the financing must first be given. 2) affordable 555 The low-cost technologies used to build up the sensor-based monitoring system and the risk-based sensor placement strategy lead to an overall very efficient system with comparably low installation cost (about € 5 to €41 per protected inhabitant, Sapena et al., 2023). This provides a basis for installing such a system in other areas as well. Nevertheless, the political will and thus also the financing must first be given. 2) affordable 555 The low-cost technologies used to build up the sensor-based monitoring system and the risk-based sensor placement strategy lead to an overall very efficient system with comparably low installation cost (about € 5 to €41 per protected inhabitant, Sapena et al., 2023). This provides a basis for installing such a system in other areas as well. Nevertheless, the political will and thus also the financing must first be given. Installation costs can be de- or increased by choosing the density of the sensors depending on the desired monitoring 560 precision. 4 Living Lab Experiences and Conclusions 520 The pilot installation in Bello Oriente will expose the medium to long-term running costs (maintenance, operation and community training) and direct benefits in terms of avoided costs (protection of life and assets in case of an event) of the implemented system. Considering also the indirect benefits, which include the increase in hazard knowledge, awareness, response capability and reduced anxiety levels, and community bonding of the i h bit t th l t t t b fit ti i t d t b iti 565 Installation costs can be de- or increased by choosing the density of the sensors depending on the desired monitoring 560 precision. The pilot installation in Bello Oriente will expose the medium to long-term running costs (maintenance, operation and community training) and direct benefits in terms of avoided costs (protection of life and assets in case of an event) of the implemented system. Considering also the indirect benefits, which include the increase in hazard knowledge, awareness, response capability and reduced anxiety levels, and community bonding of the inhabitants, the long-term cost to benefit ratio is expected to be very positive. 565 27 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 3) socially integrated The living lab approach reached about 12% of the population in about 40 workshops and community meetings in the course of four years. A low vandalism rate (after six months) indicates a satisfactory level of social acceptance. the course of four years. A low vandalism rate (after six months) indicates a satisfactory level of social acceptance. However, a representative survey conducted in 2022 showed that only about half of the residents have even heard 570 of the project. Given the intense community outreach and prominent installations in public space, this number seems low at first glance. However, in low-income neighborhoods, other more acute life risks (sudden income loss, bad health, gang activity, etc.) do command more attention than preparing for an eventual landslide that might never happen. Additionally, a high fluctuation of residents and the Corona pandemic during the research phase further aggravated community outreach. Regular community training and information campaigns by experienced 575 social workers are further needed to sustain the LEWS and maintain its acceptance in the community. The long- term success of the LEWS will depend on the sufficient funding of governmental agencies to maintain community training. 4 Living Lab Experiences and Conclusions 520 A further critical issue for the future acceptance of the LEWS in Bello Oriente is, how well in the perception of residents, the risk management agencies will handle evacuation and eviction of households threatened by an 580 imminent landslide. Current governmental options for the evacuated families (which usually only includes low economic support for 3-6 months of rent with no housing replacement options) are perceived as not satisfactory up to the point that residents have reacted negatively in the past to DAGRD personnel entering the pilot-neighborhood (the research project has improved that bias). Therefore, the concern that better landslide monitoring and reaction residents, the risk management agencies will handle evacuation and eviction of households threatened by an 580 imminent landslide. Current governmental options for the evacuated families (which usually only includes low economic support for 3-6 months of rent with no housing replacement options) are perceived as not satisfactory up to the point that residents have reacted negatively in the past to DAGRD personnel entering the pilot-neighborhood (the research project has improved that bias). Therefore, the concern that better landslide monitoring and reaction capacities will result in more evictions has been voiced quite frequently by local committees. If better shelter and 585 housing solutions are not found, residents might perceive the LEWS as an enabler of eviction and reject it in the future. capacities will result in more evictions has been voiced quite frequently by local committees. If better shelter and 585 housing solutions are not found, residents might perceive the LEWS as an enabler of eviction and reject it in the future. 4) multi-sectoral ) The living lab approach developed the LEWS with five stakeholder groups: residents, academics, government, civil 590 society organizations and private companies. A substantial amount of time and effort (about 200 coordination meetings) was spent to engage the various stakeholders in a collaborative process. While collaboration and outreach efforts with the community at risk (40 workshops, 2 emergency drills) were expected to be resource intensive, the collaboration with governmental stakeholders bound more challenges than expected due to frequent leadership The living lab approach developed the LEWS with five stakeholder groups: residents, academics, government, civil 590 society organizations and private companies. A substantial amount of time and effort (about 200 coordination meetings) was spent to engage the various stakeholders in a collaborative process. While collaboration and outreach efforts with the community at risk (40 workshops, 2 emergency drills) were expected to be resource intensive, the collaboration with governmental stakeholders bound more challenges than expected due to frequent leadership changes, bureaucratic and legal hindrances and not formally allocated budgets to the project. While the voluntary 595 collaboration and perseverance of Medellín’s municipal agencies, especially of the municipal risk management agency DAGRD, was exceptional, the continuation of the prototype beyond the research phase was for the longest 28 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. time not assured. As a bad coincidence, Medellín’s premier agency responsible for early warnings (SIATA) was heavily impaired by political infighting shortly after the research commenced. While this incapacitation was even exceptional for Medellín’s dynamic political landscape, these unforeseen events are the most important lessons of 600 the living lab methodology. It forced the research team to think about a LEWS that still can offer benefits even under a reduced government support scenario, which can be often the case for municipalities with a high rate of informal settlements (Werthmann, 2021; Perlman, 2010). In the end, the experimental operation of the LEWS prototype beyond the research phase has been confirmed by 600 In the end, the experimental operation of the LEWS prototype beyond the research phase has been confirmed by the city of Medellín. The strong will of the Colombian partners to support the project throughout all phases was 605 decisive and grounded its design deeper into the political and social realities. However, a funding situation where Colombian and German partners are equally financed from the beginning would be strongly preferable. 5) spatially integrated 5) spatially integrated The living lab approach integrated the sensor system of the LEWS as miniature public spaces in form of 610 individually painted brick benches and brick cubes, tree plantings, ground markers and information signs. In a representative survey only 17% of the residents were aware that these elements housed the sensors of the LEWS. About 19% actively used and liked them as recreational spaces. These relatively low numbers can be partially explained by the fact that the installations can only be enjoyed in the upper half of the settlement. Overall, the The living lab approach integrated the sensor system of the LEWS as miniature public spaces in form of 610 individually painted brick benches and brick cubes, tree plantings, ground markers and information signs. In a representative survey only 17% of the residents were aware that these elements housed the sensors of the LEWS. About 19% actively used and liked them as recreational spaces. These relatively low numbers can be partially explained by the fact that the installations can only be enjoyed in the upper half of the settlement. Overall, the public installations are well used and only in a few cases, the installation of the benches is perceived by some 615 residents as problematic as they can be used by criminal gangs to execute territorial control. Vandalism of the miniature public spaces is low. These assessments are preliminary and further evaluation is needed to assess the long-term impact of spatial integration. public installations are well used and only in a few cases, the installation of the benches is perceived by some 615 residents as problematic as they can be used by criminal gangs to execute territorial control. Vandalism of the miniature public spaces is low. These assessments are preliminary and further evaluation is needed to assess the long-term impact of spatial integration. 6) multi-scalar 620 Remote sensing data with spatial resolutions of around 10cm allow to localize the exposed buildings with high precision and indirectly the exposed population. Thus, these data provide crucial quantitative and spatio-temporal information to support the planning of the LEWS. 4) multi-sectoral the city of Medellín. The strong will of the Colombian partners to support the project throughout all phases was 605 decisive and grounded its design deeper into the political and social realities. However, a funding situation where Colombian and German partners are equally financed from the beginning would be strongly preferable. 5) spatially integrated 7) replicable 630 The living lab developed a prototype of a LEWS which relies on 1) the precise analysis of landslide risk by academic researchers or specialized firms, 2) the risk monitoring through observation by residents and through ground sensors monitored by experts 24/7, 3) the warning dissemination and 4) response by residents and governmental authorities. About 32 sites have been identified in Medellín, where variations of the LEWS could be 7) replicable 630 The living lab developed a prototype of a LEWS which relies on 1) the precise analysis of landslide risk by academic researchers or specialized firms, 2) the risk monitoring through observation by residents and through ground sensors monitored by experts 24/7, 3) the warning dissemination and 4) response by residents and governmental authorities. About 32 sites have been identified in Medellín, where variations of the LEWS could be useful (cf. Sapena et al., 2023). In the moment, a test and calibration phase is conducted in order to investigate the 635 practicability and subsequently the replicability potential of the prototype. As a first insight, it can be stated that this type of LEWS is confined to urban areas where there is sufficient technical expertise to operate the complex sensor-based instrumentation. Secondly, the whole system requires increased political will to expand the capacities and funding of local risk management authorities for the construction, operation and maintenance of the LEWS. Thirdly, risk management authorities will have to find better procedures for housing evacuees. If not, informal 640 residents will reject the LEWS out of fear of eviction. ) Redundancy has not been listed as a specific goal at the outset of the research as it is a commonly acknowledged strategy for achieving greater resilience in systems especially EWS (Michoud et al., 2013). During the course of 645 the Inform@Risk project, it was necessary to increase aspects of redundancy to maintain basic functions of the LEWS in case of wavering government support or infrastructure failure. Therefore, the low-tech, self-help capacities regarding landslide monitoring, warning dissemination and response capacity of the at-risk community came to the foreground as the first line of defense in case everything else fails; even if budgets are low, community capacity needs to be still fostered as the most relevant ingredient and expense of any LEWS. 650 650 5) spatially integrated However, these high-resolution data from drones, flight campaigns or now even satellites are very costly in time and resources and thus are not yet an option for large area 6) multi-scalar 620 Remote sensing data with spatial resolutions of around 10cm allow to localize the exposed buildings with high precision and indirectly the exposed population. Thus, these data provide crucial quantitative and spatio-temporal information to support the planning of the LEWS. However, these high-resolution data from drones, flight campaigns or now even satellites are very costly in time and resources and thus are not yet an option for large area 6) multi-scalar 620 Remote sensing data with spatial resolutions of around 10cm allow to localize the exposed buildings with high precision and indirectly the exposed population. Thus, these data provide crucial quantitative and spatio-temporal information to support the planning of the LEWS. However, these high-resolution data from drones, flight campaigns or now even satellites are very costly in time and resources and thus are not yet an option for large area or even global applications. However, beyond this capability on local levels (i.e. to work on individual building 625 level) lower resolution data free of cost are available globally. They allow - although thematically less detailed and 29 less accurate - to locate exposed areas in terms of topography or built environments or indirectly also population. This enables a city-wide approach to assess where further LEWS can be deployed most effectively, if desired. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. less accurate - to locate exposed areas in terms of topography or built environments or indirectly also population. This enables a city-wide approach to assess where further LEWS can be deployed most effectively, if desired. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. less accurate - to locate exposed areas in terms of topography or built environments or indirectly also population. This enables a city-wide approach to assess where further LEWS can be deployed most effectively, if desired. less accurate - to locate exposed areas in terms of topography or built environments or indirectly also population. This enables a city-wide approach to assess where further LEWS can be deployed most effectively, if desired. 5 Final Statement The research has shown that precise, affordable and socially integrated LEWS in informal settlements are not out of reach. Based on the preliminary experiences with the LEWS in Bello Oriente, an adaptation of the system to other locations with different conditions is conceivable as the modular technical components of the LEWS can be down or upgraded as needed. As a first-time project, the technical aspects of the LEWS in Bello Oriente were more 655 655 30 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. time and cost intensive than an off-the-shelf application. While the cost for technical equipment will decrease with more wide-spread application, the effort for social outreach, collaboration and training of residents will not. As the LEWS is both, a technical and a social enterprise, the two elements need constant care by trained personnel. Based on these preliminary findings, four fundamental prerequisites for a municipality to start an LEWS in an 660 informal settlement can be formulated: Based on these preliminary findings, four fundamental prerequisites for a municipality to start an LEWS in an 660 informal settlement can be formulated: 1) LEWS make only sense in areas where the resettlement of vulnerable residents is infeasible or will last for decades. 2) government authorities and its representatives need to already have or regain the trust of the residents of 665 landslide-prone informal settlements. Trust can be achieved through consistency of policies, steady social engagement over longer time periods or during implementation of an LEWS. 3) the government has to keep a constant open and honest interaction with the civil organizations of the territory since they are key to understand and solve challenges and conflicts with the community 3) the government has to keep a constant open and honest interaction with the civil organizations of the territory since they are key to understand and solve challenges and conflicts with the community 3) government authorities have to have a long-term commitment independent of election cycles to support an 670 LEWS in informal settlements. Financial allowances and personnel to socially and technically sustain the LEWS have to be allocated long-term in the governmental budget. The sustenance and expansion of low-tech, self-help capacities of the at-risk community has to be an inherent part of this budget. LEWS in informal settlements. 5 Final Statement The understanding that the physical presence of an LEWS is more than just an assemblage of black boxes only begins (Mazereeuw and Yarina, 2017). In our case its usefulness needs to be further evaluated in the coming years. If successful, the deliberate design of risk technology in public spaces could become a further design parameter for LEWS in urban areas. can contribute to the risk awareness of affected residents while improving livelihood. The understanding that the physical presence of an LEWS is more than just an assemblage of black boxes only begins (Mazereeuw and Yarina, 2017). In our case its usefulness needs to be further evaluated in the coming years. If successful, the deliberate design of risk technology in public spaces could become a further design parameter for LEWS in urban areas. 690 A second aspect of innovation of the developed LEWS is the technical monitoring of not-yet-active ground movements in an informal settlement. In Medellín, ground sensors are usually placed only in an active landslide or near critical infrastructure. The preemptive placement of sensors promises to give more precise monitoring capacities, but is also more maintenance intensive and needs a refined process of data interpretation in order not to A second aspect of innovation of the developed LEWS is the technical monitoring of not-yet-active ground movements in an informal settlement. In Medellín, ground sensors are usually placed only in an active landslide or near critical infrastructure. The preemptive placement of sensors promises to give more precise monitoring capacities, but is also more maintenance intensive and needs a refined process of data interpretation in order not to cause false alarms. Through the connection of the sensors with the app, residents will be able to see the status of 695 sensors in their neighborhood in the future. Therefore, the correct interpretation of the sensor readings and its continuous surveillance is the most important task of the ensuing calibration phase of the system. 695 cause false alarms. Through the connection of the sensors with the app, residents will be able to see the status of 695 sensors in their neighborhood in the future. Therefore, the correct interpretation of the sensor readings and its continuous surveillance is the most important task of the ensuing calibration phase of the system. 5 Final Statement Financial allowances and personnel to socially and technically sustain the LEWS have to be allocated long-term in the governmental budget. The sustenance and expansion of low-tech, self-help capacities of the at-risk community has to be an inherent part of this budget. 4) a commonly accepted process of community resettlement in case of imminent landslide threat has to be in place. 675 The presented living lab approach is not the first LEWS to be installed in an informal settlement; other attempts have been made, for example in Rio de Janeiro (Heffer and ELLA, 2013), Honduras (Peters et al.; 2022), or in Nepal (Thapa and Adhikari, 2019). The interim results of the living lab confirm the common notion that an LEWS is a predominantly social system with embedded technical elements, even more so in an informal settlement with its strong social and political dynamics (Kelman and Glantz, 2014). 680 4) a commonly accepted process of community resettlement in case of imminent landslide threat has to be in place. 675 The presented living lab approach is not the first LEWS to be installed in an informal settlement; other attempts 4) a commonly accepted process of community resettlement in case of imminent landslide threat has to be in place. 675 The presented living lab approach is not the first LEWS to be installed in an informal settlement; other attempts have been made, for example in Rio de Janeiro (Heffer and ELLA, 2013), Honduras (Peters et al.; 2022), or in Nepal (Thapa and Adhikari, 2019). The interim results of the living lab confirm the common notion that an LEWS is a predominantly social system with embedded technical elements, even more so in an informal settlement with its strong social and political dynamics (Kelman and Glantz, 2014). 680 The presented living lab approach expands this socio-technical understanding by a spatial component. The interaction between the technology of the monitoring system (the technical) and the residents and social organizations of the informal neighborhood (the social) has deliberately transformed the physical space of the neighborhood (the spatial). The new miniature public spaces test the notion that the spatial dimension of an LEWS 685 31 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. can contribute to the risk awareness of affected residents while improving livelihood. 5 Final Statement As the living lab approach developed a prototype of an integrative and participatory LEWS that introduces novel technology, additional time for calibration, development and adjustment is needed in order to reach a certain level 700 of functionality. Its usefulness is not yet proven. The continued operation and further development of the system by the risk management agencies of Medellín is therefore crucial in order to obtain more knowledge in the future 32 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Appendices Figure A1. Schematic layout of the Inform@Risk sensor based monitoring system. The system achieves a 05 observations by combining linear deformation measurements (Continuous Shear Monitor and Extensometer detection based on tilt measurements (wireless (LoRa(R)) measurement nodes). Additionally the landslide tr and groundwater table are monitored. The acquired data is processed in a cloud system utilizing methods of s the reliability of the system (e.g. only if several sensors indicate a landslide (red dots), an automated public wa et al., 2020, copyright Ernst & Sohn GmbH. Reproduced with permission) 0 Figure A1. Schematic layout of the Inform@Risk sensor based monitoring system. The system achieves a high spatial density of 705 observations by combining linear deformation measurements (Continuous Shear Monitor and Extensometers) with punctual motion detection based on tilt measurements (wireless (LoRa(R)) measurement nodes). Additionally the landslide triggering factors rainfall and groundwater table are monitored. The acquired data is processed in a cloud system utilizing methods of sensor fusion to improve the reliability of the system (e.g. only if several sensors indicate a landslide (red dots), an automated public warning is issued). (Thuro et al., 2020, copyright Ernst & Sohn GmbH. Reproduced with permission) 710 33 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. gure A2. Map showing the spatial distribution of the elements of the sensor system in the project area. Red lines indicate landslide enomena. Background: orthomosaic of drone images acquired in Nov. 2019. Figure A2. Map showing the spatial distribution of the elements of the sensor system in the project area. Red lines indicate landslide phenomena. Background: orthomosaic of drone images acquired in Nov. 2019. 34 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A3. Map with location of LEWS elements in public space. 715 Figure A3. Map with location of LEWS elements in public space. 35 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Figure A4. Interfaces of the warning app (from left to right): main menu of the App, map of sensor distribution, and information provided by sensor. Figure A4. Interfaces of the warning app (from left to right): main menu of the App, map of sensor distribution, and information provided by sensor. Competing Interests Dr. John Singer (AlpGeorisk) has concluded a contract with the municipal risk management agency of Medellín, DAGRD to continue the development and testing of the sensor system in future. The used data management and analysis platform (AlpGeorisk ONLINE) is developed and commercially distributed by AlpGeorisk. 735 The other authors have no relevant financial or non-financial interests to disclose. Author Contributions 720 All authors contributed to the study conception and design. The first outline and final version of the manuscript was developed by Christian Werthmann. In the section “3 Preliminary Findings”, the following authors were mainly responsible for the research and content of: the section “3.1.1 Landslide Risk at City Scale” by Marta Sapena and Marlene Kühnl, “3.1.2 Landslide Hazard at Pilot-Neighborhood” by Bettina Menschik and Kurosch Thuro. “3.2.1 Automatic Sensor-Based All authors contributed to the study conception and design. The first outline and final version of the manuscript was developed by Christian Werthmann. In the section “3 Preliminary Findings”, the following authors were mainly responsible for the research and content of: the section “3.1.1 Landslide Risk at City Scale” by Marta Sapena and Marlene Kühnl, “3.1.2 Landslide Hazard at Pilot-Neighborhood” by Bettina Menschik and Kurosch Thuro. “3.2.1 Automatic Sensor-Based Monitoring” by John Singer, 3.2.2 Manual and People-Based Monitoring by Carolina Garcia, and “3.2.3 Socio-spatial 725 Integration of Monitoring System” by Christian Werthmann and Heike Schäfer. “3.3.1 Warning Levels”, “3.3.2 Warning Dissemination channels”, “3.3.4 Alert Network” and 3.4 Response Capacity by Carolina Garcia, the “3.3.3 Warning App” by Sebastian Schröck. The abstract and the section “4 Living Lab Experiences and Conclusions” was a joint effort. All other sections were written by Christian Werthmann. All authors commented on previous versions of the manuscript and all authors 36 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Acknowledgements Living Lab Participants: German partners: Leibniz Universität Hannover (Coordinator), Technische Hochschule Deggendorf, Technische Universität München, Deutsches Zentrum für Luft- und Raumfahrt e.V., AlpGeorisk, Sachverständigenbüro für Luftbildauswertung und 750 Umweltfragen; Colombian partners: EAFIT University - urbam, Departamento , Administrativo de Gestión de Riesgos – DAGRD, Alcaldía de Medellín, Área Metropolitana del Valle de Aburrá, Departamento Administrativo de Planeación, Secretaría de Medio Ambiente, Secretaría de Infraestructura Física, Sistema de Alerta Temprana del Valle de Aburrá, Sociedad Colombiana de München, Deutsches Zentrum für Luft- und Raumfahrt e.V., AlpGeorisk, Sachverständigenbüro für Luftbildauswertung und 750 Umweltfragen; Colombian partners: EAFIT University - urbam, Departamento , Administrativo de Gestión de Riesgos – DAGRD, Alcaldía de Medellín, Área Metropolitana del Valle de Aburrá, Departamento Administrativo de Planeación, Secretaría de Medio Ambiente, Secretaría de Infraestructura Física, Sistema de Alerta Temprana del Valle de Aburrá, Sociedad Colombiana de Geología Colectivo Tejearañas Corporación Convivamos Fundación Palomá Red Barrial Bello Oriente Residents of Bello 755 Geología, Colectivo Tejearañas, Corporación Convivamos, Fundación Palomá, Red Barrial Bello Oriente, Residents of Bello 755 Oriente. Geología, Colectivo Tejearañas, Corporación Convivamos, Fundación Palomá, Red Barrial Bello Oriente, Residents of Bello 755 Oriente. Acknowledgements This research was funded by the German Federal Ministry of Education and Research as part of the FONA Client II initiative, 740 grant number 03G0883A-F, Inform@Risk – Strengthening the Resilience of Informal Settlements against Slope Movements. Many thanks go to Ms. Fretzdorff and Ms. Putbrese from Project Management Jülich for their great support and guidance. The Departamento Administrativo de Gestión del Riesgo de Desastres of Medellín (DAGRD) provided funding for several on-site installations. All the listed Colombian partners volunteered to invest time and effort. Special thanks go to Alejandro This research was funded by the German Federal Ministry of Education and Research as part of the FONA Client II initiative, 740 grant number 03G0883A-F, Inform@Risk – Strengthening the Resilience of Informal Settlements against Slope Movements. Many thanks go to Ms. Fretzdorff and Ms. Putbrese from Project Management Jülich for their great support and guidance. The Departamento Administrativo de Gestión del Riesgo de Desastres of Medellín (DAGRD) provided funding for several on-site installations. All the listed Colombian partners volunteered to invest time and effort. Special thanks go to Alejandro This research was funded by the German Federal Ministry of Education and Research as part of the FONA Client II initiative, 740 grant number 03G0883A-F, Inform@Risk – Strengthening the Resilience of Informal Settlements against Slope Movements. Many thanks go to Ms. Fretzdorff and Ms. Putbrese from Project Management Jülich for their great support and guidance. The Departamento Administrativo de Gestión del Riesgo de Desastres of Medellín (DAGRD) provided funding for several on-site installations. All the listed Colombian partners volunteered to invest time and effort. Special thanks go to Alejandro Echeverrri, director from urbam at EAFIT university and Laura Duarte director from DAGRD for their continuous support of 745 the project. Many thanks go to the residents of Bello Oriente for their support and collaboration. Echeverrri, director from urbam at EAFIT university and Laura Duarte director from DAGRD for their continuous support of 745 the project. Many thanks go to the residents of Bello Oriente for their support and collaboration. Alcaldía de Medellín: Plan de ordenamiento territorial. Acuerdo 48 de 2014. Medellín, 2014. References 37 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. 760 Basher, R. Global early warning systems for natural hazards: systematic and people-centred, Philos. Trans. R. Soc. Math. Phy Eng. Sci., vol. 364, no. 1845, pp. 2167-2182, Aug. 2006. Baudoin, MA., Henly-Shepard, S., Fernando, N. et al. From Top-Down to “Community-Centric” Approaches to Early Warni Systems: Exploring Pathways to Improve Disaster Risk Reduction Through Community Participation. Int J Disaster Risk S 765 7, 163–174, https://doi.org/10.1007/s13753-016-0085-6, 2016. Breuninger, T., Menschik, B., Demharter, A., Gamperl, M., and Thuro, K.: Investigation of critical geotechnical, petrologic and mineralogical parameters for landslides in deeply weathered dunite rock (Medellín, Colombia), International journal environmental research and public health, 18, 11141, 2021. 770 Brillembourg, A., Feireiss, K., and Klumpner, H.: Informal city : Caracas case ; urban think tank, Prestel, Munich, 360 p 2005. 760 Basher, R. Global early warning systems for natural hazards: systematic and people-centred, Philos. Trans. R. Soc. Math. Phys. Eng. Sci., vol. 364, no. 1845, pp. 2167-2182, Aug. 2006. Baudoin, MA., Henly-Shepard, S., Fernando, N. et al. From Top-Down to “Community-Centric” Approaches to Early Warning Systems: Exploring Pathways to Improve Disaster Risk Reduction Through Community Participation. Int J Disaster Risk Sci 765 7, 163–174, https://doi.org/10.1007/s13753-016-0085-6, 2016. Colombian Law 1523/2012, Congress of Colombia, url: 775 https://www.funcionpublica.gov.co/eva/gestornormativo/norma.php?i=47141 (in Spanish, consulted on 28/03/2023). Drummond, H., Dizgun, J., and Keeling, D. J.: Medellin: A city reborn?, FOCUS on Geography, 55, 146–154, 2012. Drummond, H., Dizgun, J., and Keeling, D. J.: Medellin: A city reborn?, FOCUS on Geography, 55, 146–154, 2012. Fathani, T. F., Karnawati, D., and Wilopo, W.: An integrated methodology to develop a standard for landslide early warning 780 systems, Natural Hazards and Earth System Sciences, 16, 2123–2135, 2016. Fathani, T. F., Karnawati, D., and Wilopo, W.: An integrated methodology to develop a standard for landslide early warning 780 systems, Natural Hazards and Earth System Sciences, 16, 2123–2135, 2016. Fathani, T.F., Karnawati, D., Wilopo, W. and Setiawan, H.: Strengthening the Resilience by Implementing a Standard for Landslide Early Warning System, In: Sassa, K., Konagai, K., Tiwari, B., Arbanas, Ž., Sassa, S. (eds): Progress in Landslide Fathani, T. F., Karnawati, D., and Wilopo, W.: An integrated methodology to develop a standard for landslide early warning 780 systems, Natural Hazards and Earth System Sciences, 16, 2123–2135, 2016. Research and Technology, Volume 1 Issue 1, 2022. https://doi.org/10.1007/978-3-031-16898-7_20, 2023. Esposito, M.; Palma, L.; Belli, A.; Sabbatini, L.; Pierleoni, P. Recent Advances in Internet of Things Solutions for Early 790 Warning Systems: A Review. Sensors, 22, 2124. https://doi.org/10.3390/s22062124, 2022. References 785 Hossain, M., Leminen, S., and Westerlund, M.: A systematic review of living lab literature, Journal of cleaner production, 213, 976–988, 2019. Esposito, M.; Palma, L.; Belli, A.; Sabbatini, L.; Pierleoni, P. Recent Advances in Internet of Things Solutions for Early 790 Warning Systems: A Review. Sensors, 22, 2124. https://doi.org/10.3390/s22062124, 2022. 38 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Gamperl, M.; Singer, J.; Thuro, K. Internet of Things Geosensor Network for Cost-Effective Landslide Early Warning Systems. Sensors 2021, 21, 2609. https://doi.org/10.3390/s21082609, 2021. 795 Gamperl, M., Singer, J., Garcia-Londoño, C., Seiler, L., Castañeda, J., Cerón-Hernandez, D., and Thuro, K.: An integrated, replicable Landslide Early Warning System for informal settlements – case study in Medellín, Colombia, Nat. Hazards Earth Syst. Sci. https://doi.org/10.5194/nhess-2023-20, https://doi.org/10.5194/nhess-2023-20, in review, 2023. Gamperl, M.; Singer, J.; Thuro, K. Internet of Things Geosensor Network for Cost-Effective Landslide Early Warning Systems. Sensors 2021, 21, 2609. https://doi.org/10.3390/s21082609, 2021. 795 Gamperl, M., Singer, J., Garcia-Londoño, C., Seiler, L., Castañeda, J., Cerón-Hernandez, D., and Thuro, K.: An integrated, replicable Landslide Early Warning System for informal settlements – case study in Medellín, Colombia, Nat. Hazards Earth Syst. Sci. https://doi.org/10.5194/nhess-2023-20, https://doi.org/10.5194/nhess-2023-20, in review, 2023. Gamperl, M.; Singer, J.; Thuro, K. Internet of Things Geosensor Network for Cost-Effective Landslide Early Warning Systems. Sensors 2021, 21, 2609. https://doi.org/10.3390/s21082609, 2021. Garcia, C. & Fearnley, C. Evaluating critical links in early warning systems for natural hazards, Environmental Hazards, 11:2, 800 123-137, DOI: 10.1080/17477891.2011.609877, 2012. Garcia, C. & Fearnley, C. Evaluating critical links in early warning systems for natural hazards, Environmental Hazards, 11:2, 800 123-137, DOI: 10.1080/17477891.2011.609877, 2012. Imparato, I. and Ruster, J.: Slum upgrading and participation: Lessons from Latin America, World Bank Publications, 2003. IPCC: AR6 Synthesis Report: Climate Change 2023, Headlline Statements C.5, 2023. 805 ISO 22327: Security and resilience — Emergency management — Guidelines for implementation of a community based landslide early warning system, 2018. IPCC: AR6 Synthesis Report: Climate Change 2023, Headlline Statements C.5, 2023. 805 ISO 22327: Security and resilience — Emergency management — Guidelines for implementation of a community based landslide early warning system, 2018. Heffer, C. & ELLA Brazil: Rio de Janeiro city’s early warning system for heavy rain. Policy Brief ELLA. 7 p. 810 http://ella.practicalaction.org/wp-content/uploads/files/131101_ENV_CitAdaMit_BRIEF4.pdf, 2013. Hossain, M., Leminen, S., and Westerlund, M.: A systematic review of living lab literature, 213, 976–988, https://doi.org/https://doi.org/10.1016/j.jclepro.2018.12.257, 2019. Kelman, I. and Glantz, M. References H.: Early Warning Systems Defined, in: Reducing Disaster: Early Warning Systems For Climate 815 Change, edited by: Singh, A. and Zommers, Z., Springer Netherlands, Dordrecht, 89–108, https://doi.org/10.1007/978-94-017- 8598-3_5, 2014. Kühnl, M., Sapena, M., and Taubenböck, H.: Categorizing Urban Structural Types using an Object-Based Local Climate Zone Classification Scheme in Medellín, Colombia, in: Proceedings of the REAL CORP 2021, 26th International Conference on 820 Urban Development, Regional Planning and Information Society, Vienna, Austria, 7-10 September 2021, 173-182, 2021. Kühnl, M., Sapena, M., Wurm, M., Geiß, C., and Taubenböck, H.: Multitemporal landslide exposure and vulnerability assessment in Medellín, Colombia, Nat Hazards , https://doi.org/10.1007/s11069-022-05679-z, 2022. 825 825 39 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Marchezini V, Horita FEA, Matsuo PM, Trajber R, Trejo-Rangel MA and Olivato D. A Review of Studies on Participatory Early Warning Systems (P-EWS): Pathways to Support Citizen Science Initiatives. Front. Earth Sci. 6:184. doi: 10.3389/feart.2018.00184, 2018. Marchezini V, Horita FEA, Matsuo PM, Trajber R, Trejo-Rangel MA and Olivato D. A Review of Studies on Participatory Early Warning Systems (P-EWS): Pathways to Support Citizen Science Initiatives. Front. Earth Sci. 6:184. doi: 10.3389/feart.2018.00184, 2018. Mazereeuw, M. and Yarina, E.: Emergency Preparedness Hub: Designing Decentralized Systems for Disaster Resilience, 71, 830 65–72, https://doi.org/10.1080/10464883.2017.1260928, 2017. Michoud, C., Bazin, S., Blikra, L. H., Derron, M.-H., and Jaboyedoff, M.: Experiences from site-specific landslide early warning systems, 13, 2659–2673, https://doi.org/10.5194/nhess-13-2659-2013, 2013. 835 Müller, I., Taubenböck, H., Kuffer, M., and Wurm, M.: Misperceptions of predominant slum locations? Spatial analysis of slum locations in terms of topography based on earth observation data, Remote sensing, 12, 2474, 2020. Mazereeuw, M. and Yarina, E.: Emergency Preparedness Hub: Designing Decentralized Systems for Disaster Resilience, 71, 830 65–72, https://doi.org/10.1080/10464883.2017.1260928, 2017. Mazereeuw, M. and Yarina, E.: Emergency Preparedness Hub: Designing Decentralized Systems for Disaster Resilience, 71, 830 65–72, https://doi.org/10.1080/10464883.2017.1260928, 2017. Michoud, C., Bazin, S., Blikra, L. H., Derron, M.-H., and Jaboyedoff, M.: Experiences from site-specific landslide early warning systems, 13, 2659–2673, https://doi.org/10.5194/nhess-13-2659-2013, 2013. 835 Michoud, C., Bazin, S., Blikra, L. H., Derron, M.-H., and Jaboyedoff, M.: Experiences from site-specific landslide early warning systems, 13, 2659–2673, https://doi.org/10.5194/nhess-13-2659-2013, 2013. Müller, I., Taubenböck, H., Kuffer, M., and Wurm, M.: Misperceptions of predominant slum locations? Spatial analysis of slum locations in terms of topography based on earth observation data, Remote sensing, 12, 2474, 2020. References Padilla Galicia, S., Redondo Gómez, M., Ribbeck, E., Espinosa Dorantes, E., Bedregal Villanuev Padilla Galicia, S., Redondo Gómez, M., Ribbeck, E., Espinosa Dorantes, E., Bedregal Villanueva, J. F., Villareal Molina, H. de J., Misselwitz, P., Roettchen, T., Wemhöener, A., and Weisel, A.: Urbanismo informal, Universidad Autónoma 840 Metropolitana, Unidad Azcapotzalco, División de …, 2009. de J., Misselwitz, P., Roettchen, T., Wemhöener, A., and Weisel, A.: Urbanismo informal, Universidad Autónoma 840 Metropolitana, Unidad Azcapotzalco, División de …, 2009. de J., Misselwitz, P., Roettchen, T., Wemhöener, A., and Weisel, A.: Urbanismo informal, Universidad Autónoma 840 Metropolitana, Unidad Azcapotzalco, División de …, 2009. Peduzzi, P., Deichmann, U., Dao, Q.-H., Herold, C., Chatenoux, B., and De Bono, A.: 2009 Global Assessment Report on Disaster Risk Reduction: patterns, trends and drivers, 2009. Peduzzi, P., Deichmann, U., Dao, Q.-H., Herold, C., Chatenoux, B., and De Bono, A.: 2009 Global Assessment Report on Disaster Risk Reduction: patterns, trends and drivers, 2009. Peduzzi, P., Deichmann, U., Dao, Q.-H., Herold, C., Chatenoux, B., and De Bono, A.: 2009 Global Assessment Report on Disaster Risk Reduction: patterns, trends and drivers, 2009. 845 Perlman, J.: Favela: Four decades of living on the edge in Rio de Janeiro, Oxford University Press, 2010. Peters, L.E., Clark-Ginsberg, A., McCaul, B., Cáceres, G., Nuñez, A.L., Balagna, J., López, A., Patel, S.S., Patel, R.B. and Van Den Hoek, J.: Informality, violence, and disaster risks: Coproducing inclusive early warning and response systems in urban informal settlements in Honduras. Frontiers in Climate 4:937244. https://doi.org/10.3389/fclim.2022.937244, 2022. 850 Prakasam C., Aravinth R., Varinder S. Kanwar, Nagarajan B.: Design and Development of Real-time landslide early warning system through low cost soil and rainfall sensors, Materials Today: Proceedings, Volume 45, Part 6, Pages 5649-5654, ISSN 2214-7853, https://doi.org/10.1016/j.matpr.2021.02.456, 2021. 855 Rendón-Giraldo, D.A.: Departamento de Geociencias y Medio Ambiente, Universidad Nacional de Colombia, Medellín, Antioquia, Colombia, Personal Communication, 18th of November, 2020. 845 Perlman, J.: Favela: Four decades of living on the edge in Rio de Janeiro, Oxford University Press, 2010. Peters, L.E., Clark-Ginsberg, A., McCaul, B., Cáceres, G., Nuñez, A.L., Balagna, J., López, A., Patel, S.S., Patel, R.B. and Van Den Hoek, J.: Informality, violence, and disaster risks: Coproducing inclusive early warning and response systems in urban informal settlements in Honduras. Frontiers in Climate 4:937244. https://doi.org/10.3389/fclim.2022.937244, 2022. 850 Prakasam C., Aravinth R., Varinder S. Schneidewind, U.: Urbane Reallabore: ein Blick in die aktuelle Forschungswerkstatt, 2014 Schneidewind, U.: Urbane Reallabore: ein Blick in die aktuelle Forschungswerkstatt, 2014. Segoni, S., Leoni, L., Benedetti, A., Catani, F., Righini, G., Falorni, G., Gabellani, S., Rudari, R., Silvestro, F., and Rebora, 870 N.: Towards a definition of a real-time forecasting network for rainfall induced shallow landslides, Natural Hazards and Earth System Sciences, 9, 2119–2133, 2009. SIMMA Sistema de Información de Movimientos en Masa. References Kanwar, Nagarajan B.: Design and Development of Real-time landslide early warning system through low cost soil and rainfall sensors, Materials Today: Proceedings, Volume 45, Part 6, Pages 5649-5654, ISSN 2214-7853, https://doi.org/10.1016/j.matpr.2021.02.456, 2021. 855 Rendón-Giraldo, D.A.: Departamento de Geociencias y Medio Ambiente, Universidad Nacional de Colombia, Medellín, Antioquia, Colombia, Personal Communication, 18th of November, 2020. Perlman, J.: Favela: Four decades of living on the edge in Rio de Janeiro, Oxford University Press, 2010. Peters, L.E., Clark-Ginsberg, A., McCaul, B., Cáceres, G., Nuñez, A.L., Balagna, J., López, A., Patel, S.S., Patel, R.B. and Van Den Hoek, J.: Informality, violence, and disaster risks: Coproducing inclusive early warning and response systems in urban informal settlements in Honduras. Frontiers in Climate 4:937244. https://doi.org/10.3389/fclim.2022.937244, 2022. 850 40 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Sapena, M., Kühnl, M., Wurm, M., Patino, J.E., Duque, J.C., Taubenböck, H.: Empiric recommendations for population 860 disaggregation under different data scenarios. PLoS ONE 17(9): e0274504. https://doi.org/10.1371/journal.pone.0274504, 2022. Sapena, M., Gamperl, M., Kühnl, M., Garcia-Londoño, C., Singer, J., and Taubenböck, H.: Co instrumentalization of Landslide Early Warning Systems, Nat. Hazards Earth Syst. Sci. Discuss. [preprint], 865 https://doi.org/10.5194/nhess-2023-41, in review, 2023. SIMMA Sistema de Información de Movimientos en Masa. https://simma.sgc.gov.co. Accessed: 30.03.2023. 875 Singer J., Schuhbäck S., Wasmeier P., Thuro K., Heunecke O., Wunderlich Th., Glabsch J., Festl J.: Monitoring the Aggenalm landslide using economic deformation measurement techniques. Austrian J Earth Sci 102(2):20–34, 2009. https://simma.sgc.gov.co. Accessed: 30.03.2023. 875 Sirangelo, B. and Versace, P.: A real time forecasting model for landslides triggered by rainfall, Meccanica, 31, 73–85, 1996. 880 Thapa P.S., Adhikari B.R.: Development of community-based landslide early warning system in the earthquake-affected areas of Nepal Himalaya. J. Mt. Sci.16, 2701–2713, https://doi.org/10.1007/s11629-019-5586-5, 2019. Thuro K., Singer J., Menschik B., Breuninger T., Gamperl M.: Development of an early warning system for landslides in the 885 tropical Andes (Medellín; Colombia). Geomechanics and Tunnelling, 13 (1), 103-115, 2020. Taubenböck, H., Kraff, N. J., and Wurm, M.: The morphology of the Arrival City-A global categorization based on literature surveys and remotely sensed data, Applied Geography, 92, 150–167, 2018. 890 Thuro K., Singer J., Menschik B., Breuninger T., Gamperl M.: Development of an early warning system for landslides in the 885 tropical Andes (Medellín; Colombia). Geomechanics and Tunnelling, 13 (1), 103-115, 2020. Taubenböck, H., Kraff, N. J., and Wurm, M.: The morphology of the Arrival City-A global categorization based on literature surveys and remotely sensed data, Applied Geography, 92, 150–167, 2018. 890 Taubenböck H., Esch T., Felbier A., Wiesner M., Roth A. & Dech S.: Monitoring of mega cities from space. Remote Sensing of Environment, vol. 117, pp. 162-176, 2012. Taubenböck H., Esch T., Felbier A., Wiesner M., Roth A. & Dech S.: Monitoring of mega cities from space. Remote Sensing of Environment, vol. 117, pp. 162-176, 2012. 41 https://doi.org/10.5194/nhess-2023-53 Preprint. Discussion started: 17 April 2023 c⃝Author(s) 2023. CC BY 4.0 License. Tobón-Hincapié M.P., Joya-Camacho A.M., Marcías-Torres C.E. Gomez-Gallo M.H., Zapata-Wills C., Velásquez A..: Medidas de Mitigacíon de la Ladera Oriental del Valle de Aburrá entra las Quebradas La Poblada y La Sanín Alcaldía de 895 Medellín, Secretaria del Medio Ambiente Medellín, Colombia, p. 218., 2011. Tobón-Hincapié M.P., Joya-Camacho A.M., Marcías-Torres C.E. Gomez-Gallo M.H., Zapata-Wills C., Velásquez A..: Medidas de Mitigacíon de la Ladera Oriental del Valle de Aburrá entra las Quebradas La Poblada y La Sanín Alcaldía de 895 Medellín, Secretaria del Medio Ambiente Medellín, Colombia, p. 218., 2011. UN-Habitat.: World Cities Report 2016: Urbanization and Development–Emerging Futures, Publisher: UN-Habitat, 2016. 900 UN-Habitat.: World Cities Report 2016: Urbanization and Development–Emerging Futures, Publisher: UN-Habitat, 2016. SIMMA Sistema de Información de Movimientos en Masa. 900 UNEP - United Nations Environment Programme: Early Warning Systems: A State of the Art Analysis and Future Directions. Division of Early Warning and Assessment (DEWA), Nairobi, 2012. UNEP - United Nations Environment Programme: Early Warning Systems: A State of the Art Analysis and Future Directions. Division of Early Warning and Assessment (DEWA), Nairobi, 2012. UNFCCC - United Nations Climate Change: UN: Early Warning Systems Must Protect Everyone Within Five Years. 905 https://unfccc.int/news/un-early-warning-systems-must-protect-everyone-within-five-years, 2022. UNGRD:Cartilla monitoreo comunitario de movimientos en masa. Unidad Nacional de Gestión de Riesgos, Colombia. https://docplayer.es/169347016-Cartilla-monitoreo-comunitario-de-movimientos-en-masa-2013.html, 2013. p p y , 910 UNISDR: Sendai framework for disaster risk reduction 2015–2030. Geneva: UNISDR, 2015. von Wirth, T., Fuenfschilling, L., Frantzeskaki, N., and Coenen, L.: Impacts of urban living labs on sustainability transitions: mechanisms and strategies for systemic change through experimentation, European Planning Studies, 27, 229–257, https://doi.org/10.1080/09654313.2018.1504895, 2019. 915 Voytenko, Y., McCormick, K., Evans, J., and Schliwa, G.: Urban living labs for sustainability and low carbon cities in Europe: Towards a research agenda, Journal of cleaner production, 123, 45–54, 2016. UNISDR: Sendai framework for disaster risk reduction 2015–2030. Geneva: UNISDR, 2015. Werthmann, C.: Informal Urbanization in Latin America: Collaborative Transformations of Public Spaces, Routledge Taylor 920 & Francis Group, 2021. Wurm, M. and Taubenböck, H.; Detecting social groups from space – Remote sensing-based mapping of morphological slums and assessment with income data, Remote Sens. Lett., 9(1), 41-50. https://doi.org/10.1080/2150704X.2017.1384586, 2018. Yasuoka, M., Akasaka, F., Kimura, A., and Ihara, M.: Living labs as a methodology for service design-An analysis based on cases and discussions from a systems approach viewpoint, DS 92: Proceedings of the DESIGN 2018 15th International Design 925 Conference, 127–136, 2018. 42
https://openalex.org/W3173048371	https://iiste.org/Journals/index.php/JHMN/article/download/32657/33548	English	null	The Delivery of Service in Forming the Image of the Hospital and Patients Satisfaction to Gain Patients Loyality in Kajuruhan Kepanjen Hospital Malang Regency	Global Journal of Business & Social Review/Global Journal of Business & Social Science Review	2,015	cc-by	4,710	Journal of Health, Medicine and Nursing ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 Journal of Health, Medicine and Nursing ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 1. INTRODUCTION 1.1. Background of The Problem The industry of hospital service is basically socioeconomic, i.e. in addition to emphasizing the application of social values; it must also consider economic principles in carrying out its activities. This is clearly shown in in a private hospital in which the financial resources or the funding come from donors or investors who expect that their investment can be returned, and possibly it is expected to gain profits. The success go a hospital is determined by the service provided mainly tools quality, means, and resources. When the quality of treatment and professional human resources is better, the hospital utilization tends to increase (Health Profile of Malang Regency, 2005-2007) Preliminary results of a survey on patients who seek treatment in Kanjuruhan Kepanjen Hospital Malang Regency, which is determined at the beginning of treatment, was received by the clerk at the front lines until completed and allowed to go home or otherwise to be hospitalized, show that only 50% of respondents stated that the hospital cares about the society. This means that patients/ the society perceive that the image of the hospital is not good. This shows that the service delivery system which includes physical support and contact personnel influence the company’s image. The quality of doctors, nursing facilities and technology, diagnostics facilities, the overall quality of care, interpersonal attention, awareness of staff to the personal needs of the patient and the hospital experience, location and cost, and also ease of location, influence the image of the hospital, the patient's satisfaction and loyalty. 1.2. Limitations of The Problem 1. Does the delivery of service which consists of physical support and contact personnel influence t of Kajuruhan Kepanjen Hospital Malang Regency? 2. Does the delivery of service which consists of physical support and contact personnel influence th satisfaction of Kajuruhan Kepanjen Hospital Malang Regency? 3. Does the image of the hospital influence the patients satisfaction of Kajuruhan Kepanjen Hospital Malang Regency? 4. Does the image of the hospital influence the patients loyalty of Kajuruhan Kepanjen Hospital Malang Regency? g y 5. Does the patients satisfaction influence the patients loyalty of Kajuruhan Kepanjen Hospital Malang Regency? The Delivery of Service in Forming the Image of the Hospital and Patients Satisfaction to Gain Patients Loyality in Kajuruhan Kepanjen Hospital Malang Regency Mamik Ferry Kriswandana Umi Rahayu Department of Environmental Health, Health Polytechnic of Surabaya, Indonesia Abstract Kajuruhan Kepanjen Hospital Malang Regency is the only hospital possessed by Malang Regency. The competition among hospitals is getting tighter along with the appearance of private hospitals. The success of hospitals is caused mainly by the delivery of service, especially the service, tolls, and personals because hospitals as service industries have given good services for their patients so that the patients will be loyal. Data collecting method is by a field observation, i.e. distributing the questionnaires and interviewing 210 respondents (patients of Kajuruhan Kepanjen Hospital Malang Regency). Then the data was processed and analyzed by using Structural Equation Modeling (SEM) method, with AMOS software. The result of data analysis shows that the delivery of service has positive and significant influence to the image of the hospital but it does not have significant influence to patients satisfaction. The image of Kajuruhan Kepanjen Hospital Malang Regency has positive and significant influence to patients satisfaction and loyalty. Patients satisfaction has positive and significant influence to patients loyalty. Keywords: Delivery of Service, Image, Satisfaction, Loyalty. 1.3. Study Objectives The objectives of the study are to identify and review the influence of the delivery of service to the image of the hospital and patients satisfaction, the influence of the hospital image to patients satisfaction, and also the influence of the hospital image and patients loyalty of Kajuruhan Kepanjen Hospital Malang Regency. 13 Journal of Health, Medicine and Nursing Journal of Health, Medicine and Nursing ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 2.3. Customer Satisfaction Customer satisfaction depends on the estimate product performance in giving the grade, relatively to the customer expectation. The customer is satisfied when the performance meets the expectation. The customer is dissatisfied when the product performance is much lower than what is expected (Kotler and Armstrong, 2009). The satisfaction felt by the customer consists of satisfaction of the product and satisfaction to the information used to choose the product. Each form is called satisfaction indicator and information satisfaction that influence the customer loyalty ( Spreng et al., 1996). 2. 1. Service Delivery System According to Lovelock and Wright (2007), a service business is viewed as a service operation system and a service delivery system. A service operation system is a component which is included in the whole service business system, where the inputs are processed and the service product elements are created by human resources component and physical component. The important components of service marketing namely people, process, and physical evidence are important elements in delivering the service offered and play a significant role in doing “tangibilize in the intangible”. The service quality in a hospital service outside the related competences that forms the patients experience in health care is by giving service excellence, i.e. an attitude or a way of an employee or a health worker in serving the patients satisfactorily in line with the goals and benefits of service results i.e. Speed, accuracy, hospitality, and comfort. 2.2. Image Keller (1993) defined a company image as a perception of a company as a company picture restrained in the customer’s memory. The image and awareness are directed to two components of image knowledge. The whole formed perception (the conclusion of the image/ brand) in the customer’s mind are caused by brand physic, name, symbol, display, and reputation for bank service. The factors of company image variable according to Keller (1993) are three factors namely corporate image, user image, and product image. 2. Theoretical Review 2. 1. Service Delivery System . 3. RESEARCH METHODS The population of this study is all patients of Kanjuruhan Kepanjen Malang Regency. The number of samples is based on Hair et al. (2010) who stated that the minimum data for multi-variant analysis technique is at least 100 samples. Thus, the samples used in this study are 210 patients. The determination of samples in this study is purposive sampling i.e. The researcher uses his/her own consideration (using adequate knowledge of population) to choose the samples. The analysis data technique used is Structural Equation Modeling (SEM). 2.4. Loyalty Customer loyalty is a concept that is studied more in customer behavior study. Customer loyalty has two elements namely behavior element and attitude. In the approach of behavior element, the definition of loyalty becomes shallow. Loyalty is is based on buying pattern only, and this approach defines loyalty in accordance with buying level of certain product or brand in a period. Moven and Minor (2007) defined brand loyalty as how far a customer shows a positive attitude toward a brand, has a commitment to a brand, and wants to keep buying in the future. Brand loyalty is influenced directly by the satisfaction or dissatisfaction to a product which is accumulated in a period as the perception of a product quality. 2.5. Study Model The proposed study model is shown in Picture 1. Picture 1. Study Model Patients satisfaction Hospital image Patients loyalty Service delivery system 2.5. Study Model The proposed study model is shown in Picture 1. Patients satisfaction Service delivery system Patients loyalty Hospital image Picture 1. Study Model 2.6. Study Hypothesis H1 : the delivery service system has significant influence to the hospital image of Kanjuruhan Kepanjen Hospital Malang Regency. H2 : the delivery service system has significant influence to patients satisfaction of Kanjuruhan Kepanjen Hospital Malang Regency. H3 : the hospital image has significant influence to patients satisfaction of Kanjuruhan Kepanjen Hospital 14 Journal of Health, Medicine and Nursing Journal of Health, Medicine and Nursing www.iiste.org iste.org ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 Malang Regency. H4 : the hospital image has significant influence to patients loyalty of Kanjuruhan Kepanjen Hospit Malang Regency. g g y the hospital image has significant influence to patients loyalty of Kanjuruhan Kepanjen Hospital Malang Regency. H4 : the hospital image has significant influence to patients loyalty of Kanjuruhan Kepanjen Hospital Malang Regency. H5 : the patients satisfaction has significant influence to patients loyalty of Kanjuruhan Kepanjen Hospital Malang Regency H5 : the patients satisfaction has significant influence to patients loyalty of Kanjuruhan Kepanjen Hospital Malang Regency 4. 3. Hypothesis Test Result 4. 3. Hypothesis Test Result yp The hypothesis test is done by comparing probability grade (p) of each latent variable with 0,05 (α = significant) in which it is stated as significant if pv latent variable is smaller or the same with 0,05 (p ≤ 0,05). Table 3 shows: 1. Service delivery system variable (X) has positive and significant influence to hospital image (Y1), with PV grade = 0,000 < 0,05 (α), and has a path coefficient 0,968. yp The hypothesis test is done by comparing probability grade (p) of each latent variable with 0,05 (α = significant) in which it is stated as significant if pv latent variable is smaller or the same with 0,05 (p ≤ 0,05). Table 3 shows: The hypothesis test is done by comparing probability grade (p) of each latent variable with 0,05 (α = significant) in which it is stated as significant if pv latent variable is smaller or the same with 0,05 (p ≤ 0,05). Table 3 shows: 1. Service delivery system variable (X) has positive and significant influence to hospital image (Y1), with PV grade = 0,000 < 0,05 (α), and has a path coefficient 0,968. 1. Service delivery system variable (X) has positive and significant influence to hospital image (Y1), with PV grade = 0,000 < 0,05 (α), and has a path coefficient 0,968. g , , ( ), p , 2. Service delivery system variable (X) has positive but not significant influence to patients satisfaction (Y2), with PV grade = 0,378 > 0,05 (α), and has a path coefficient -0,732. g ( ) p 2. Service delivery system variable (X) has positive but not significant influence to patients satisfaction (Y2), with PV grade = 0,378 > 0,05 (α), and has a path coefficient -0,732. 3. Hospital Image variable (Y1)has positive and significant influence to patients satisfaction (Y2), with PV grade = 0,05 ≤ 0,05 (α), and has a regression coefficient 1,679. 3. Hospital Image variable (Y1)has positive and significant influence to patients satisfaction (Y2), with PV grade = 0,05 ≤ 0,05 (α), and has a regression coefficient 1,679. i l i i bl ( ) h i i d i ifi i fl i l l ( ) i h d 4. Hospital image variable (Y1) has positive and significant influence to patients loyalty (Y3), with = 0,047 < 0,05 (α), and has a regression coefficient 1,379. 4. RESULTS AND DISCUSSION 4. 1. Model Analysis Result This study applies Structural Equation Modeling (SEM) analysis. The analysis results are shown in Picture 1 and Table 1. 4. 1. Model Analysis Result This study applies Structural Equation Modeling (SEM) analysis. The analysis results are shown in Picture 1 and Table 1. y This study applies Structural Equation Modeling (SEM) analysis. The analysis results are shown in Picture 1 and Table 1 Picture 1. SEM Beginning Step Y2 Y2.1 e15 1 1 Y2.2 e1 61 Y2.3 e171 Y1 Y1.3 e14 1 1 Y1.2 e13 1 Y1.1 e12 1 ey11 ey2 1 Y3 Y3.1 e18 1 1 Y3.2 e19 1 Y3.4 e21 1 Y3.5 e22 1 ey3 1 X1 X1.11 e11 1 1 X1.10 e10 1 X1.9 e9 1 X1.8 e8 1 X1.7 e7 1 X1.4 e4 1 X1.2 e2 1 X1.1 e1 1 ex1 1 Picture 1. SEM Beginning Step X1 Table 1. Goodness of Fit Beginning Model Test Criteria Cut-of Value Model Result Information Chi-Square Expected low 299,207 Not accepted Probability ≥0.05 0,000 Not accepted RMSEA ≤0.08 0,079 Accepted GFI ≥0.90 0,865 Not accepted AGFI ≥0.90 0,823 Not accepted CFI ≥0.95 0,901 Not accepted Source: analysis result SEM (2012) The analysis result of SEM early step shows that the beginning model is proposed based on the unacceptable theory, because there is only one good requirement (not supported by empirical data in the field) so the result is not fit. Then, the beginning model is modified with a guidance of modification indices which must be supported by relevant theory. The result is shown in Picture 2 Table 2. 15 Journal of Health, Medicine and Nursing g ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 Picture 2. SEM Modification Step Y2 Y2.1 e15 1 1 Y2.2 e1 61 Y2.3 e171 Y1 Y1.3 e14 1 1 Y1.2 e13 1 Y1.1 e12 1 ey1 1 ey2 1 Y3 Y3.1 e18 1 1 Y3.2 e19 1 Y3.4 e21 1 Y3.5 e22 1 ey3 1 X1 X1.11 e11 1 1 X1.10 e10 1 X1.9 e9 1 X1.8 e8 1 X1.7 e7 1 X1.4 e4 1 X1.2 e2 1 X1.1 e1 1 ex1 1 Picture 2. SEM Modification Step Picture 2. 4. RESULTS AND DISCUSSION SEM Modification Step Y2 Y2.1 e15 1 1 Y2.2 e1 61 Y2.3 e171 Y1 Y1.3 e14 1 1 Y1.2 e13 1 Y1.1 e12 1 ey1 1 ey2 1 Y3 Y3.1 e18 1 1 Y3.2 e19 1 Y3.4 e21 1 Y3.5 e22 1 ey3 1 X1 X1.11 e11 1 1 X1.10 e10 1 X1.9 e9 1 X1.8 e8 1 X1.7 e7 1 X1.4 e4 1 X1.2 e2 1 X1.1 e1 1 ex1 1 Picture 2. SEM Modification Step Table 2. Goodness of Fit Modification Model Test Criteria Cut-of Value Model Result Information Chi-Square Expected low 179,651 Accepted Probability ≥0.05 0,000 Not accepted RMSEA ≤0.08 0,048 Accepted GFI ≥0.90 0,917 Accepted AGFI ≥0.90 0,882 Quite Accepted CFI ≥0.95 0,966 Accepted Source: analysis result SEM (2012) Table 2. Goodness of Fit Modification Model Test Table 2 shows that six criteria used to assess whether a model is fit or not, only one criteria that is quite adequate. This can be said that the model can be accepted, meaning that there is suitability between the model and the data. 4.2. Causal Relationship between Latent Variables T bl 3 C l R l ti 4.2. Causal Relationship between Latent Variables Table 3. Causal Relationship between Latents Variables Causal Relationship P value Loading Factor Hospital Image <--- Service Delivery 0,000 0.968 Patients Satisfaction <--- Hospital Image 0,050 1.679 Patients Satisfaction <--- Service Delivery 0,378 -0.732 Patients Loyalty <--- Hospital Image 0.047 1.379 Patients Loyalty <--- Patients Satisfaction 0,004 2.148 Source: Analysis Result SEM (2012) Source: Analysis Result SEM (2012) 5. Discussion 1. The Influence of Service Delivery to Hospital Image (H1) The first hypothesis is proven that the service delivery system with room design, waiting room comfort, ease of being informed, service tools, other supporting means (public telephone, ATM, Bank) indicators, and the availability of parking area, timeliness of service, patient acceptance procedure, and the communication between the health workers and the patients has significant influence to hospital image of Kanjuruhan Kepanjen Hospital Malang Regency. Tis conclusion is based on the grade of PV = 0,000 < 0,05 (significant level α = 0,05), with path coefficient 0, 968. p This is in accordance with Nguyen and Leblanc (2002) that the service delivery system which includes physical support and contact personnel has influence to company image. In addition, this is also in line with Cooper (1994), where doctor quality, care and technology facilities, diagnostics facilities, whole care qualities, interpersonal attention, staff awareness to patients personal needs and hospital experience, location, costs, ease of patient location influence the hospital image. The service delivery system is a communication system formed by physical support dimension and contact personnel dimension. Both physical support and contact personnel become very important in supporting the formation of hospital image. The service quality of hospital service outside the related competences that forms the patient experience in health care is by giving service excellence, i.e. an attitude or way of an employee in giving service to patients satisfactorily, according to the goals and benefits of the service results namely speed, accuracy, hospitality, and comfort. The four basic elements are a unit of integrated service, meaning that a service becomes imperfect if there is one of elements that is ignored. p g luence of Service Delivery to Patients Satisfaction (H2) p g . The Influence of Service Delivery to Patients Satisfaction (H2) The second hypothesis is not proven that the service delivery system does not have significant influence to patients satisfaction of Kanjuruhan Kepanjen Hospital Malang Regency. This conclusion is based on the grade of PV = 0,378 > 0,05 (significant level α = 0,05), with path coefficient -0,732. This result is not in line with Shamdasani and Balakrishnan (2000), who concluded that physical support and contact personnel influence the customer satisfaction. 4. 3. Hypothesis Test Result ( ) g 5. Patients satisfaction variable (Y2) has positive and significant influence to patients loyalty (Y3), with PV grade 0,004 < 0,05 (α), and has a regression coefficient 2,148. 5. Patients satisfaction variable (Y2) has positive and significant influence to patients loyalty (Y3), with PV grade 0,004 < 0,05 (α), and has a regression coefficient 2,148. 16 Journal of Health, Medicine and Nursing www.iiste.org 4. 4. Indirect Influence Analysis Table 4. Indirect Influence of Latent Variables Latent Variable Patients Satisfaction (Y2) Patients Loyalty (Y3) 1. X  Y2 (via Y1) 2. X  Y3 (via Y1) 3. X  Y3 (via Y1 & Y2) 0,968 x 1,679 = 1,625 (S) - - - 0,968 x 1,379 = 1,335 (S) 0,968 x 1,679 x 2,148 = 3,491 (S) Th l i f i di i fl f l i bl f ll 4. 4. Indirect Influence Analysis 0,968 x 1,379 = 1,335 (S) 0,968 x 1,679 x 2,148 = 3,491 (S) The explanation of indirect influence of latent variables are as follows: p 1. The indirect effect of service delivery system (X) to patients satisfaction (Y2) via hospital image (Y1) has indirect positive and significant influence 1, 625 (0,968 x 1,697). This shows that the service delivery system variable (X) indirectly (via hospital image) has significant influence to patients satisfaction of Kanjuruhan Kepanjen Hospital Malang Regency (Y2) whereas the direct influence is not significant. 2. The indirect effect of service delivery system (X) to patients loyalty (Y3) via hospital image (Y1) has indirectly positive and significant influence that is 1, 335 (0, 968 x 1, 379) y ( ) y ( p g ) g p Kanjuruhan Kepanjen Hospital Malang Regency (Y2) whereas the direct influence is not significant. 2. The indirect effect of service delivery system (X) to patients loyalty (Y3) via hospital image (Y1) has indirectly positive and significant influence that is 1, 335 (0, 968 x 1, 379) Kanjuruhan Kepanjen Hospital Malang Regency (Y2) whereas the direct influence is not significant. 2. 4. 3. Hypothesis Test Result The indirect effect of service delivery system (X) to patients loyalty (Y3) via hospital image (Y1) has indirectly positive and significant influence that is 1, 335 (0, 968 x 1, 379) y p g ( ) The indirect effect of service delivery system (X) to patients loyalty (Y3) via hospital image (Y1) and patients satisfaction (Y2) has indirectly positive and significant influence that is 3, 491 (0, 968 x 1, 679 x 2, 148). 5. Discussion This study result indicates the support to the theories which stated that there is a significant influence of image to patients loyalty. Patients will be loyal if the patients view a hospital as a good hospital. The influence of company image to the patients which is shown in Bloemer et al. (2002) who concluded that an image does not have direct influence to customers loyalty, but it is only a moderator variable between quality and customers. This study shows that a company image has direct influence to customers loyalty and also acts as a moderator variable between service delivery system and customers loyalty, meaning that image variable can be either endogenous variable or exogenous variable. This study is also in line with Nguyen and Leblanc (2002) who stated that a company image has positive influence to customers loyalty in three sectors (telecommunication, retail, and education). ) fluence of Patients Satisfaction to Patients Loyalty (H5) ) 5. The Influence of Patients Satisfaction to Patients Loyalty (H5) y y ( ) The fifth hypothesis is proven that patients satisfaction has significant influence to patients loyalty of Kanjuruhan Kepenjen Hospital Malang Regency. This conclusion is based on the grade of PV = 0,004 ≤ 0,50 (significant level α = 0,05), with path coefficient 2,148. Athis study is in line with Bloemer et al. (2002) and Spreng et al. (1996). Bloemer et al. (2002) concluded that image influences customers satisfaction, satisfaction influences trust, and trust influences customers commitment (loyalty). Spreng et al. (1996) stated that overall customers satisfaction consists of the satisfaction to the product itself and the satisfaction to the information used to choose the product. Each form is named satisfaction indicator and information satisfaction which influence customers loyalty. Customer satisfaction is a key for business profit and customers loyalty is a key for continuing satisfaction. The satisfied customers tend to be loyal. This is also applied in health agency. The patients who are satisfied with the hospital service will tend to be loyal to the hospital. Based on the result of this study, patients loyalty is much influenced by patients satisfaction. Besides the direct influence of patients satisfaction, patients loyalty is also influenced directly by image and indirectly by service delivery system through image as a moderator variable. 5. Discussion In this study, physical support owned by the hospital less influence the forming of hospital service delivery system, meaning that the patients think that reliability is more important than facilities and infrastructures owned by the hospital. The same opinion is also put forward by Lupiyoadi (2006) that in hospital service, the reliability and responsiveness of the health workers are much more dominant that the hospital interiors. 3. The Influence of Hospital Image to Patients Satisfaction (H3) 3. The Influence of Hospital Image to Patients Satisfaction (H3) 3. The Influence of Hospital Image to Patients Satisfaction (H3) The third hypothesis is proven that the hospital image- with the indicators of patients impressions to the hospital, the patients trust to the hospital, and the health workers ability - has significant influence to the satisfaction of patients of Kanjuruhan Kepanjen Hospital Malang Regency. This conclusion is based on the grade of PV = 0,050 ≤ 0,50 (significant level α = 0,05), with path coefficient 1,679. The study result is in line with the result of the research done by Selnes (1993) and Bloemer et al. (2002). Selnes (1993) concluded that a company image influences the customer satisfaction and loyalty. Bloemer et al. (2002) stated that an image influences customer satisfaction. Besides, this study also supports Mc Daniel et al. (2007) who stated that the purchase of a brand results in satisfaction for a customer, that is compared to the expectation of a brand strength potential. Also the expectation becomes the foundation which underlies the first decision made by a customer. 17 Journal of Health, Medicine and Nursing www.iiste.org iste.org ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 4. The Influence of Hospital Image to Patients Loyalty (H4) 4. The Influence of Hospital Image to Patients Loyalty (H4) The fourth hypothesis is proven that hospital image has significant influence to patients loyalty of Kanjuruhan Kepanjen Hospital Malang Regency. This conclusion is based on the grade of PV = 0,047 ≤ 0,50 (significant level α = 0,05), with path coefficient 1,379. The study result is in line with the result of the research done by Selnes (1993) and Bloemer et al. (2002). Selnes (1993) showed that a company image influences the customers satisfaction and loyalty, meaning that the company (Kanjuruhan Kepanjen Hospital Malang Regency) influences the patients loyalty. 6. CONCLUSION The image of Kajuruhan Kepanjen Hospital Malang Regency has positive and significant influence to patients satisfaction and loyalty. Patients satisfaction has positive and significant influence to patients loyalty. 7. SUGGESTION Physical support owned by the hospital can change patients orientation about reliability hospital than facilities and infrastructures owned by the hospital. 7. SUGGESTION Physical support owned by the hospital can change patients orientation about reliability hospital than facilities and infrastructures owned by the hospital. 5. Discussion Patients loyalty is a point which is expected by service company because customers loyalty is a benchmark of the success of service delivery system and company image. ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 red, 1993, An Examination of The Effect of Product Performance on Brand Reputaion, Satisfaction and lt E J l f M k ti V l 27 (M ) 19 36 f g ( ) pp Richard A., Scott B. MacKenzie, and Richard W. Olshavsky, 1996, A Reexamination of the Determinants of Customer Satisfaction, Journal of Marketing, Vol. 9 (July), pp. 15-32. y y p f g ( y) pp ani, Prem N., and Audrey A. Balakrishnan, 2000, Determinants of Relatioship Quality and Loyalty in rsonalized Services, Journal of Marketing, Vol. 6 (June) pp.390-407. , , p , yalty, European Journal of Marketing, Vol. 27 (May) pp.19-36. Journal of Health, Medicine and Nursing An Investigating in Financial Service. The International Journal Marketing. Vol. 16. pp 52-74. g g g pp , 1993, An Examination of The Effect of Product Performance on Brand Reputaion, Satisfaction and ty, European Journal of Marketing, Vol. 27 (May) pp.19-36. An Investigating in Financial Service. The International Journal Marketing. Vol. 16. pp 52-74. Selnes, Fred, 1993, An Examination of The Effect of Product Performance on Brand Reputaion, Satisfaction and Loyalty, European Journal of Marketing, Vol. 27 (May) pp.19-36. Shamdasani, Prem N., and Audrey A. Balakrishnan, 2000, Determinants of Relatioship Quality and Loyalty in Personalized Services, Journal of Marketing, Vol. 6 (June) pp.390-407. Spreng, Richard A., Scott B. MacKenzie, and Richard W. Olshavsky, 1996, A Reexamination of the Determinants of Customer Satisfaction, Journal of Marketing, Vol. 9 (July), pp. 15-32. ISSN 2422-8419 An International Peer-reviewed Journal Loyalty, European Journal of Marketing, Vol. 27 (May) pp.19 36. Shamdasani, Prem N., and Audrey A. Balakrishnan, 2000, Determinants of Relatioship Quality and Loyalty in Personalized Services, Journal of Marketing, Vol. 6 (June) pp.390-407. Spreng, Richard A., Scott B. MacKenzie, and Richard W. Olshavsky, 1996, A Reexamination of the Determinants of Customer Satisfaction Journal of Marketing Vol 9 (July) pp 15 32 Journal of Health, Medicine and Nursing Selnes, Fred, 1993, An Examination of The Effect of Product Performance on Brand Reputaion, Satisfa Journal of Health, Medicine and Nursing ISSN 2422-8419 An International Peer-reviewed Journal Vol.29, 2016 REFERENCES Bloemer, Ruyter, and Peter, 2002, Investigating Drivers of Bank Loyalty: the Complex Relationship between Image, Service Quality and Satisfaction, Journal of Marketing, Vol. 9, No.3, pp. 23-42. Bloemer, Ruyter, and Peter, 2002, Investigating Drivers of Bank Loyalty: the Complex Relationship between Image, Service Quality and Satisfaction, Journal of Marketing, Vol. 9, No.3, pp. 23-42. Cooper, Joseph, 1994, Measuring Service Quality: A Reexamination and Extension. Journal of Marketing, Vol. Cooper, Joseph, 1994, Measuring Service Quality: A Reexamination and Extension. Journal of Ma 56 (July), pp. 55-68. Hair, J.F., Anderson R.E., Tatham R.L., and Black W.C., 2010, Multivariate Data Analysis, Seventh Edition, Pearson Prentice-Hall International Inc., New Jersey. Keller, K. L., 1993, A Conceptualizing, Measuring, and Managing Customer-Based Brand Equity, Journal of Marketing Research, Vol. 29, pp.1-22. r, Philip, and Gary Armstrong, 2009, Prinsip-Prinsip Pemasaran, Edisi Terjemahan, Erlangga, Jakarta. p y g p p j gg Lovelock, Christopher H., and Lauren K. Wright, 2007, Manajemen Pemasaran Jasa, Cetakan Kedua, Indeks, Jakarta. Lupiyoadi, Rambat, 2006, Manajemen Pemasaran Jasa, Edisi Kedua, Penerbit Salemba Empat, Jakarta. aran Jasa, Edisi Kedua, Penerbit Salemba Empat, Jak McDaniel C., Hair J.F., dan Lamb C.W. Jr, 2007, Pemasaran, Edisi Terbatas (Terjemahan). Prentice H york. Mowen, John C., dan Michael Minor, 2007, Customer Loyalty, Edisi Ketiga, Erlangga, Jakarta. Nguyen, N., and Leblanc G., 2002, The Mediating Role of Corporate Image on Customers’ Retention Decisions: 18 www.iiste.org 19
W105995516.txt	https://figshare.com/articles/conference_contribution/Beauty_leaf_Calophyllum_inophyllum_L_tree_a_tree_with_great_economic_potential/13423097/1/files/25827437.pdf	en		Beauty Leaf (Calophyllum inophyllum L.), tree: a tree with great economic potential	Proceedings of the International Forestry and Enviroment symposium	2,012	cc-by	640	Session II - Forest Resources Utilization 207 Beauty Leaf (Calophyllum inophyllum L.), tree: a tree with great economic potential Subhash Hathurusingha and N. Ashwath Centre for Plant and Water Sciences, Central Queensland University, Rockhampton QLD 4702, Australia Calophyllum inophyllum L. (Clusiaceae) commonly known as Alexandrian laurel or beauty leaf or Domba (in Sri Lanka) is essentially a littoral tree of the tropics, occurring above the high-tide mark along sea coasts of northern Australia and extending throughout Southeast Asia and southern India (Agroforesrey Database 2007).The tree is native to both Australia and Sri Lanka. For many years it has been used by indigenous communities and alternative medicine practitioners. It also has a high demand for its seed oil from cosmetics and pharmaceutical industries. It is a high potential bio-diesel plant. Seed yields 65% oil from its dry weight 3744 kg/ha from 400 trees (Azam et al. 2005). It has the highest per tree oil yield from 75 plant species tested so far. It can be used in conventional diesel engines (without any alterations) in its pure form or as a blend with mineral oil (Agarwal 2006). It is also a durable multi-purpose timber (density 560-900kg/m3) (Timber Species Notes, DPI Queensland 2007). With recently discovered plant properties (anti-HIV and anti-cancer active compounds), Calophyllum inophyllum can be placed amongst the most important multi-purpose trees. This paper reviews various economically important uses and services of Calophyllum inophyllum L. and areas for future research. This also outlines an ongoing research project "Provenance variations in ecophysioIogy, growth, performance, seed oil and Calocoumarin-A (anti-cancer agent) content ofCalophyllum inophyllum L." that is being carried out in Queensland, Australia and in Sri Lanka. 208 Sustainable Management of Forest Resources in India Through Criteria and Indicator AlJproach G M Devagiri, T Nethravathi, Sathish and H S Vasudev University of Agricultural Sciences, Bangalore, India College of Forestry, Ponnampet, Kodagu, Karnataka, India The concept of Sustainable Forest Management (SFM) is not new, but has become popular in recent years. SFM encompasses environmental dimension relating to maintenance of the natural resources in perpetuity, economic dimensions for production of goods and services and social dimensions describing involvement of people in decision-making process and equitable benefit sharing. For operationalising SFM it is imperative to have assessment system such that deviations towards or away from sustainability can be ascertained and corrective actions can be taken. The system should evaluate the two main conditions i.e., maintenance of ecosystem integrity and maintenance and enhancement of well being of people. Criteria and indicators (C & I) are the tools for assessing the magnitude and the direction of change in a given forestry situations and provide a forest management information system, which is important for forest managers and other actors for forestry related decision making. C & I can be used to encourage more holistic thinking when planning forest management activities, and to bring about greater vigor, openness, transparency and accountability in forest management planning, monitoring and reporting. The important eight criteria adopted in some forest divisions of India includes increase in the extent of forest and tree cover, maintenance, conservation and enhancement of biodiversity, maintenance and enhancement of ecosystem function and vitality, conservation and maintenance of soil and water resources, maintenance and enhancement of forest resource productivity, optimization offorest resource utilization, maintenance and enhancement of social, cultural and spiritual benefits and adequacy of policy, legal and institutional framework. Under each of these criteria such as for example under ecosystem function and vitality, the status of natural regeneration is used as an indicator. Mishra et al. (2004) observed that the people are mostly dependent on trees outside forest, hence reducing the pressure on the natural forest. The sustainability index shows comprehensive impact of various indicators on , Proceedings of the International Forestry and Environment Symposium 2007 of the Department Forestry and Environmental Science, University of Sri Jayewardenepura, Sri Lanka. of
https://openalex.org/W2906060991	https://cdr.lib.unc.edu/downloads/js956n16m	English	null	Longitudinal patterns of unmet need for contraception among women living with HIV on antiretroviral therapy in South Africa	PloS one	2,018	cc-by	8,014	RESEARCH ARTICLE Editor: Rena C. Patel, University of Washington, UNITED STATES Received: September 12, 2018 Accepted: November 29, 2018 Published: December 20, 2018 Copyright: © 2018 Rucinski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: September 12, 2018 Accepted: November 29, 2018 Published: December 20, 2018 Abstract Citation: Rucinski KB, Powers KA, Schwartz SR, Pence BW, Chi BH, Black V, et al. (2018) Longitudinal patterns of unmet need for contraception among women living with HIV on antiretroviral therapy in South Africa. PLoS ONE 13 (12): e0209114. https://doi.org/10.1371/journal. pone.0209114 Objectives Fertility intentions and contraceptive use are often not assessed in the context of clinical HIV care, representing a possible programming gap if women’s family planning needs change over time. We aimed to identify longitudinal patterns of unmet need for contraception over a 12-month period among women living with HIV taking antiretroviral therapy (ART). Editor: Rena C. Patel, University of Washington, UNITED STATES Study design 850 non-pregnant, HIV-positive women aged 18–35 on or initiating ART in Johannesburg, South Africa, were enrolled into a prospective cohort study in 2009–2010. Fertility intentions and contraceptive use were assessed during routine HIV care visits via an interviewer- administered questionnaire, and women were referred for on-site contraceptive counseling. We used group-based trajectory modeling to identify patterns of unmet need for contracep- tion over 12 months, first in the entire population and then in a subset of recent ART initiators. Copyright: © 2018 Rucinski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are available from within the manuscript as well as a supplemental information file. Longitudinal patterns of unmet need for contraception among women living with HIV on antiretroviral therapy in South Africa Katherine B. RucinskiID1,2¤, Kimberly A. Powers1, Sheree R. Schwartz2,3, Brian W. Pence1, Benjamin H. Chi4, Vivian Black2,5, Helen Rees2, Audrey E. Pettifor1 1 Department of Epidemiology, University of North Carolina Gillings School of Global Public Health, Chapel Hill, NC, United States of America, 2 Wits Reproductive Health and HIV Institute, University of the Witwatersrand, Johannesburg, South Africa, 3 Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States of America, 4 Department of Obstetrics and Gynecology, University of North Carolina School of Medicine, Chapel Hill, NC, United States of America, 5 Clinical Microbiology and Infectious Diseases, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ¤ Current address: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States of America rucinski@jhu.edu ¤ Current address: Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States of America * rucinski@jhu.edu Conclusions SRS. KBR was supported by an award through the Society of Family Planning Research Fund (SFPRF11-T8) and through NIH/NIAID T32 AI102623. BHC was supported through NIH/NIAID (K24 AI120796). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Half of women were estimated to have a high probability of unmet need that persisted over time, and more than a quarter were estimated to experience patterns of changing unmet need over 12 months. Family planning needs should be assessed more regularly in HIV- positive women to prevent unintended pregnancies and support safer conception among women trying to conceive. Competing interests: The authors have declared that no competing interests exist. Longitudinal patterns of unmet need for contraception Introduction Prevention of unintended pregnancy through reliable contraceptive methods improves wom- en’s health and reduces both maternal and infant mortality among women living with HIV [1,2]. Prevention of unintended pregnancy is also an effective strategy to reduce mother-to- child HIV transmission [3], and is the second prong of WHO’s four-pronged framework for prevention of mother-to-child transmission (PMTCT) [4]. For these reasons and to ensure women’s reproductive rights, South African national contraception and family planning guidelines promote the availability of accessible and comprehensive contraceptive services to meet the family planning needs of women living with HIV [5]. In practice, however, women living with HIV in South Africa still experience tremendous difficulties meeting their family planning needs [6–9]. Family planning needs of women living with HIV are likely to change over time, yet these changes may go undetected due to facility- and provider-level factors that preclude providers from routinely assessing fertility preferences and contraceptive use in the context of HIV clini- cal care. Comprehensive programs to reduce unmet need for contraception in HIV-positive women, broadly defined as a discrepancy between expressed fertility preferences and con- traceptive use [10], have traditionally focused on improving availability of services, increasing education and information dissemination around contraceptive use, and strengthening con- traceptive counseling at the provider level [7,11–16]. However, even in settings where these enhanced family planning services are offered within HIV clinics, women may be reluctant to seek contraceptive counseling, and health-facility factors such as high patient volume and lim- ited number of providers can affect patient-provider interaction time and quality of care [17– 19]. To the extent that fertility intentions and contraceptive use are dynamic, this presents a missed opportunity for intervention. Delineating patterns of unmet contraceptive need among women living with HIV could help providers better understand the extent to which the risk for unintended pregnancy is likely to change over time. Furthermore, characterization of temporal trends or predictors of unmet need for contraception could inform more targeted screening practices within inte- grated reproductive health and antiretroviral therapy (ART) services, including both contra- ception provision and safer conception interventions. We assessed longitudinal patterns of unmet need for contraception over a 12-month period within a cohort of women living with HIV taking ART in South Africa. Results In the full population we identified four patterns of unmet need for contraception over one year. Half of the enrolled women were predicted to have a consistently high probability of unmet need, 22.9% a consistently low probability, 16.7% a decreasing probability, and 10.4% an increasing probability over time. Contraceptive method discontinuation and rap- idly changing fertility intentions were the primary drivers of changing (increasing or decreas- ing) unmet need over follow-up. Results were similar in recent ART initiators. Funding: This research was conducted in clinics supported by PEPFAR (President’s Emergency Plan For AIDS Relief) and the United States Agency for International Development. The original parent study received funding from the United States Centers for Disease Control and Prevention (CDC) public health dissertation grant R36 PS001584 to PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 1 / 14 Study setting, population and design Data were prospectively collected from four public-sector HIV outpatient clinics in inner city Johannesburg between 2009 and 2011 as part of a larger study to estimate the 12-month 2 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Longitudinal patterns of unmet need for contraception incidence of pregnancy among women living with HIV on ART. Full study procedures and eli- gibility criteria have been previously described [8,17,20]. In brief, all HIV-positive, non-preg- nant, sexually active women between the ages of 18–35 on or initiating ART were eligible for participation if they had not given birth in the last three months, were not breastfeeding, had not had a previous tubal ligation, hysterectomy, bi-lateral oophorectomy, and had not received a diagnosis of permanent infertility. Pregnancy was determined using urine-based tests admin- istered by study staff (One Step hCG Urine Pregnancy Test, Atlas Link Technology, Beijing) and confirmed through repeat testing. ART regimen information, CD4 cell count and viral load data were confirmed through medical record review and pharmacy and laboratory rec- ords. After providing written informed consent, women meeting eligibility criteria completed an interviewer-administered questionnaire to assess demographic and behavioral characteris- tics including sexual risk behaviors, fertility history and intentions, and contraceptive use. Women were referred for antenatal care, on-site contraceptive counseling, or other services as needed. Outcome definition Our outcome of interest was unmet need for contraception (yes/no), which we assessed at the time of each routine HIV care visit. Our assessment was guided by the algorithm for popula- tion-level unmet need for contraception as proposed by Bradley et al. [21], which we used as a framework for determining each woman’s individual-level unmet need during study visits (see S1 Questionnaire). Specifically, women were considered to have unmet need for contraception at a given study visit if they reported 1) being married, living with a partner, or being sexually active in the three months preceding study enrollment; 2) not currently trying to become preg- nant (at time of interview); and 3) not currently using oral contraceptives, injectable, a hor- monal implant or an intrauterine device. Women who met the first two criteria and reported condom use as their primary family planning method were considered to have unmet need, as rates of unintended pregnancy are comparable among women who do not use family planning methods and among those who report condom use alone [8,22]. We assessed unmet need for contraception at monthly intervals after enrollment, corre- sponding to the routine care visits, for a total of 12 time points. In cases where a woman had two routine care visits within a given month, we employed a two-stage process for measure- ment selection. If another visit occurred in the subsequent interval, we retained only the first measurement for the month in question. If, however, no visit occurred in the next interval, we retained both measurements for the month in question provided they occurred at least 21 days apart, and assigned the second value forward to the next interval. This process accounted for the heterogeneity in clinic visit frequency among women, minimized missing data, and reflected any conflicting, individual measurement within a given interval. Women were cen- sored at pregnancy, loss to follow-up, death, or the completion of 12 months of follow-up. The original study protocol was approved by the University of the Witwatersrand Human Research Ethics Committee, Johannesburg, South Africa. Written informed consent was obtained from all participants. The University of North Carolina at Chapel Hill Institutional Review Board also approved the secondary analysis presented here. Statistical analysis We used group-based trajectory modeling to identify distinct longitudinal patterns of unmet need for contraception over the 12-month period [23,24]. Group-based trajectory modeling is an application of finite mixture modeling designed to identify clusters of individuals who fol- low a similar trajectory for a given outcome over time [24]. We first fit a series of predictor- 3 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Longitudinal patterns of unmet need for contraception free trajectory models to identify the optimal number of trajectories describing the population patterns of unmet need for contraception, fitting models with 2, 3, 4, or 5 trajectory groups to allow for heterogeneity while ensuring interpretability. Given the dichotomous nature of the outcome, we specified a logit link and binomial distribution, and we decided a priori to con- sider both quadratic and cubic specifications of each model to enable flexible trajectory shapes. We chose the optimal model based on optimum Bayesian information criterion (BIC) using Jeffreys’s scale of evidence for Bayes factors [25,26]. As unmet need for contraception is a function of both fertility intentions and contraceptive use, changes in unmet need may reflect changes in fertility intentions, contraceptive use, or both. Therefore, to delineate changes in the separate phenomena contributing to the compos- ite outcome of unmet need for contraception, we conducted a descriptive analysis in which we graphically characterized both fertility intentions and contraceptive use over the study period. At each interval we assigned women to one of four mutually exclusive categories:1) those who had unmet need according to the definition above; 2) those who were married, living with a partner, or sexually active and using a reliable method of contraception to prevent pregnancy; 3) those who were married, living with a partner, or sexually active and trying to conceive, and 4) those who were not married, living with a partner, or sexually active. We conducted this descriptive analysis overall and by trajectory group (assigned on the basis of the maximum posterior probability of group membership [23,24]). To identify predictors of trajectory group membership, we first constructed a categorical mea- sure for group membership based on the final predictor-free model, assigning each woman to a group based on her maximum posterior membership probability in each model [23,24]. After assigning women to groups, we conducted descriptive, within-group analyses of clinical and behavioral features assessed at enrollment. Statistical analysis These features, which we identified through a literature review of factors associated with unmet need for contraception, were related to demographics, measures of fertility history and contraceptive use, partner-associated characteristics, measures of patient/provider interaction and HIV-related clinical outcomes. We then fit a multinomial logistic regression model with the categorical measure for group membership as the outcome and the aforementioned predictors of interest as explanatory variables. In a second stage of the predictor analysis, we constructed a new group-based trajectory model for unmet need for contraception, retaining all predictors that were found to be signifi- cant (α = 0.1) for at least one trajectory group comparison in the first stage [23]. We included this second stage to ensure that our coefficients and standard errors properly accounted for covariance between parameter estimates and the probabilities of membership in each trajec- tory group [23]. We conducted all analyses in the full cohort and repeated them in a subgroup of women ini- tiating ART within 3 months of enrollment, given the likelihood that fertility intentions may change rapidly immediately after ART initiation [27–29], as well as the distinctness of ART ini- tiation as a clearly identifiable developmental origin and intervention point. In sensitivity anal- yses for both the full cohort and recent initiators, we re-estimated trajectories using quarterly interval assessments (every three months). All models were fit using Proc Traj (https://www. andrew.cmu.edu/user/bjones/index.htm), a free downloadable SAS add-on package (SAS, ver- sion 9.4, Cary, NC). Longitudinal patterns of unmet need for contraception Table 1. Characteristics at enrollment of 850 women living with HIV taking ART in Johannesburg by ART initiation status, 2009–2011. Overall N = 850 ART Experienced N = 693 Recent ART Initiators† N = 157 Median IQR Median IQR Median IQR Age (years) 30.4 27–33 30.5 28–33 30.0 26–32 Monthly income (ZAR) 2000.0 1000–3480 2000.0 1000–3500 1700.0 1000–3000 No. living children 1.0 1–2 1.0 1–2 1.0 0–2 Months since last pregnancy 48.0 20–96 48.0 18–84 72.0 26–96 Months since HIV diagnosis 24.0 12–48 28.0 15–48 11.0 3–24 CD4 count at enrollment (cells/μl) 312.0 178–462 356.0 242–510 149.0 87–179 Months on ART 13.0 5–24 16.0 9–29 1.0 0–2 n % n % n % Education completed None-Grade 10 55 6.5 44 6.4 11 7.0 Grade 11-Grade 12 671 78.9 542 78.2 129 82.2 Post-grad degree or certificate 124 14.6 107 15.4 17 10.8 Employed 510 60.0 427 61.6 83 52.9 Married/cohabitating with a partner 378 44.5 300 43.3 78 49.7 In a relationship 789 92.8 655 94.5 134 85.4 Sexually active, last three months 754 89.4 627 91.0 127 82.5 Partner HIV status‡ Negative 200 25.3 165 25.2 35 25.7 Positive 312 39.4 260 39.7 52 38.2 Unknown 279 35.3 230 35.1 49 36.0 Ever pregnant 760 89.4 630 90.9 130 82.8 Pregnant at HIV diagnosis 271 31.9 248 35.8 23 14.7 Trying to conceive, currently 105 12.4 83 12.0 22 14.0 Trying to conceive, next year 396 46.6 299 43.2 97 61.8 Taking hormonal contraception 224 26.4 203 29.3 21 13.4 Using condoms for family planning 410 48.2 325 46.9 85 54.1 Unmet need for contraception 500 58.8 415 59.9 85 54.1 Abb i i IQR I il R ART A i i l h nrollment of 850 women living with HIV taking ART in Johannesburg by ART initiation status, 2009–2011. Table 1. Characteristics at enrollment of 850 women living with HIV taking ART in Johannesburg by ART initiation sta 9) of follow-up before they were censored at time of pregnancy. Among the 701 women who did not become pregnant, median follow-up time was 11.4 months (IQR 9, 12), with 28 (4.0%) not returning to care after their initial study visit. Median time between follow-up visits was 1.7 months (IQR 1, 2) for women who became pregnant and 1.8 months (IQR 1, 2) for those who did not. Study population characteristics A total of 850 women were enrolled and followed for up to 12 months. Over the 12-month period, 149 women (17.5%) became pregnant and contributed a median of 6.2 months (IQR 4, 4 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 No deaths were reported over the study period. Women were on average 30 years of age (IQR 27, 33), and the majority were in a relation- ship (789/850, 92.8%) (Table 1). Most women (760/850, 89.4%) had previously been pregnant, with a median time since last pregnancy of 48 months (IQR 20, 96). The median time since HIV diagnosis was 24 months (IQR 12, 48), and median time since ART initiation was 13 months (IQR 5, 24). Of the 850 women in the full cohort, 157 (18.5%) had initiated ART within three months of study enrollment (“recent initiators”) and 693 (81.5%) had initiated ART more than three 5 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Longitudinal patterns of unmet need for contraception months prior to study enrollment (“ART experienced”). Demographics, behavioral character- istics and clinical characteristics differed by ART experience. Compared to ART-experienced women, a larger proportion of recent initiators reported at enrollment that they planned to become pregnant in the next 12 months (97/157 (61.8%) vs. 299/693 (43.2%)) and a smaller proportion were using hormonal contraception (21/157 (13.4%) vs. 203/693 (29.3%)) to pre- vent pregnancy. Unmet need was assessed at study enrollment in both the full cohort and in recent ART ini- tiators. Of the 850 women included in the full cohort, 500 (58.8%) had unmet need. The pro- portion of women with unmet need was similar among recent initiators of ART compared to women who were ART experienced (54.1% vs. 59.9%). Longitudinal assessment of unmet need for contraception A four-group quadratic model was selected as the optimal predictor-free model to identify pat- terns of unmet need over 12 months (Fig 1). Half (50.0%) of the cohort was predicted to have a consistently high probability of unmet need (predicted probability of unmet need between 79.0% and 98.0%) over the study period. Nearly a quarter (22.9%) were predicted to have a consistently low probability of unmet need (1.2%-8.0% probability of unmet need over time). Additionally, 16.7% had a steadily decreasing probability of unmet need (from 86.9% at month 0 to 30.8% at month 9) and 10.4% exhibited a sharp increase in this probability (from 6.0% at month 0 to 81.4% at month 12). Based on these trends for unmet need, we qualitatively described these groups as “consistently high,” “consistently low,” “decreasing,” and “increas- ing,” respectively. In the sub-analysis of recent ART initiators (n = 157), a four-group, predictor-free cubic model was selected to identify patterns of unmet need (Fig 1). Similar to the model selected for the full cohort, nearly half (40.8%) of recent initiators were predicted to have a consistently high probability of unmet need (predicted probability of unmet need between 91.1% and 96.9% throughout). Women who were predicted to have a consistently low probability of unmet need (between 0.0% and 14.6% across the 12 months) accounted for 24.4% of recent initiators. Nearly one-fifth (17.5%) of recent initiators were predicted to have a slowly increas- ing probability of unmet need (from 25.1% at month 3 to 94.5% at month 12), and a nearly identical proportion (17.4%) were predicted to have a decreasing probability of unmet need (from 88.3% at month 1 to 16.9% at month 9). In sensitivity analyses where we re-estimated trajectories using quarterly intervals assess- ments, results were generally similar (S1 Fig). PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Fertility intentions and contraceptive use over time https://doi.org/10.1371/journal.pone.0209114.g001 https://doi.org/10.1371/journal.pone.0209114.g001 https://doi.org/10.1371/journal.pone.0209114.g001 https://doi.org/10.1371/journal.pone.0209114.g001 Fertility intentions and contraceptive use over time Temporal patterns in the two distinct contributors to unmet need–fertility intentions and con- traceptive use–varied dramatically across trajectory groups (Fig 2). In those who followed an increasing trajectory of unmet need (based on assignment to the increasing group using a maximum posterior membership probability), the proportion trying to conceive decreased over time (from 35.6% in month 0 to 10.3% in month 12), as did the proportion using reliable contraception to prevent pregnancy (from 61.6% in month 0 to 13.8% in month 12). In those who followed a decreasing trajectory of unmet need, the proportion trying to conceive increased over time (from 3.5% at month 0 to 45.5% at month 12) and the proportion using reliable contraception to prevent pregnancy also increased (between 4.3% and 16.9% over the 12 months). For women who followed trajectories of unmet need that were relatively constant (i.e. consistently low or consistently high), we observed little change in fertility intentions and contraceptive use over the 12 months. Results were similar in recent initiators. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 6 / 14 Longitudinal patterns of unmet need for contraception Fig 1. Predicted trajectories of unmet need for contraception among women living with HIV taking ART in Johannesburg, 2009–2011. Panel A represents predicted trajectories for the four-group quadratic model estimated for the full cohort (N = 850). Panel B represents predicted trajectories for the four-group cubic model estimated for recent initiators of ART (N = 157). Dots represent the observed proportion of women with unmet need for contraception among those assigned to a given trajectory group on the basis of their maximum posterior group membership probability. Curves represent the proportion of women with unmet need for contraception as estimated by the model for a given trajectory group. Fig 1. Predicted trajectories of unmet need for contraception among women living with HIV taking ART in Johannesburg, 2009–2011. Panel A represents predicted trajectories for the four-group quadratic model estimated for the full cohort (N = 850). Panel B represents predicted trajectories for the four-group cubic model estimated for recent initiators of ART (N = 157). Dots represent the observed proportion of women with unmet need for contraception among those assigned to a given trajectory group on the basis of their maximum posterior group membership probability. Curves represent the proportion of women with unmet need for contraception as estimated by the model for a given trajectory group. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Predictors of trajectory group membership Our multinomial logistic regression analysis allowed comparison of predictors for each of the four trajectory groups relative to each other group. For simplicity, we focus here on the two main comparisons of greatest apparent clinical relevance. Specifically, based on our observa- tion of similar “starting points” for the consistently high and decreasing trajectories (and for the consistently low and increasing trajectories), we describe predictors associated with mem- bership in the decreasing group (compared to the consistently high group) and those associ- ated with membership in the increasing group (compared to the consistently low group), as found using the two-stage process outlined above. Compared to the consistently high unmet need group, those in the decreasing group were likely to have fewer children (OR 0.4, 95% CI 0.3, 0.5 per one-unit increase in the categorical variable describing number of living children) and were more likely to have a partner who desired a/another child (OR 4.2 95% CI 1.6, 10.9) PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 7 / 14 Longitudinal patterns of unmet need for contraception Fig 2. Fertility intentions and contraceptive use over time, stratified by assigned trajectory group based on each participant’s highest posterior group-membership probability for the full cohort (N = 850). At each monthly assessment women were either 1) not married, co-habitating, or sexually active, 2) trying to conceive, 3) using a reliable method of contraception to prevent pregnancy (contraceptive users) and 4) not using a reliable method of contraception to prevent pregnancy (unmet need for contraception). https://doi org/10 1371/journal pone 0209114 g002 Fig 2. Fertility intentions and contraceptive use over time, stratified by assigned trajectory group based on each participant’s highest posterior group-membership probability for the full cohort (N = 850). At each monthly assessment women were either 1) not married, co-habitating, or sexually active, 2) trying to conceive, 3) using a reliable method of contraception to prevent pregnancy (contraceptive users) and 4) not using a reliable method of contraception to prevent pregnancy (unmet need for contraception). https://doi.org/10.1371/journal.pone.0209114.g002 https://doi.org/10.1371/journal.pone.0209114.g002 (Table 2). Compared to the consistently low group, women in the increasing group were more likely to have reported a problem with their contraceptive method (i.e., heavy bleeding, irregu- lar bleeding and spotting, amenorrhea, rash, or thrush as well as common side effects such as weight gain or headaches) (OR 3.0, 95% CI 1.4, 6.6) (Table 3). Predictors of trajectory group membership In analyses confined to recent initiators, we were unable to identify meaningful predictors of group membership in stage one due to the limited number of women assigned to each tra- jectory group, and models did not converge in stage two. Additional descriptive profiles of tra- jectory group membership for both the full cohort and recent initiators are reported in S1 and S2 Tables. Longitudinal patterns of unmet need for contraception Table 3. Odds ratios (95% confidence intervals) associated with membership in the increasing unmet need trajectory group (compared to membership in the con- sistently low unmet need group) (N = 850). OR (95% CI) Characteristic Consistently Low Increasing Decreasing Consistently High Number of living children† 1 1.2 (0.8, 1.9) 0.7 (0.5, 1.0) 2.9 (1.5, 2.6) Employed 1 1.1 (0.6, 2.2) 2.1 (1.2, 3.8) 1.2 (0.8, 1.8) Main partner desires a/another child 1 1.3 (0.5, 3.4) 2.0 (0.8, 4.9) 0.6 (0.3, 1.0) Problems with contraceptive method‡ 1 3.0 (1.4, 6.6) 1.2 (0.5, 2.8) 1.3 (0.7, 2.4) Abbreviations; OR: Odds Ratio, CI: Confidence Interval † Per increase in category (corresponding to an increase in total number of children), with 1 = 0 children, 2 = 1 child, 32 children ‡ Includes heavy bleeding, irregular bleeding and spotting, amenorrhea, and rash or thrush as well as common side effects such as weight gain or headaches htt //d i /10 1371/j l 0209114 t003 vals) associated with membership in the increasing unmet need trajectory group (compared to membership in the con- Abbreviations; OR: Odds Ratio, CI: Confidence Interval † Per increase in category (corresponding to an increase in total number of children), with 1 = 0 children, 2 = 1 child, 32 children ‡ Includes heavy bleeding, irregular bleeding and spotting, amenorrhea, and rash or thrush as well as common side effects such as weight gain or headaches absence of compensatory contraceptive use. In this study, we demonstrated that women living with HIV on ART in South Africa have a high probability of unmet need for contraception, and that rapidly changing fertility intentions and decisions about contraceptive use contribute to changing unmet need in a non-trivial subset of women. Half of women in our full cohort were predicted to have a consistently high probability of unmet need that persisted over time. Approximately one in ten women in our full cohort and almost 20% of those recently initiating ART were predicted to have unmet contraceptive need that increased over time, indicating they were at higher risk of unintended pregnancy as time under observation increased. Notably, among women with increasing unmet contraceptive need, few were observed to have unmet need at study enrollment (S1 Table). Discussion Changes in fertility intentions following ART initiation, coupled with improvements in fecun- dity [22,30] and renewed sexual desire and greater coital frequency [27] are thought to contrib- ute to increasing incidence of pregnancies in women living with HIV after starting ART in the Table 2. Odds ratios (95% confidence intervals) associated with membership in the decreasing unmet need trajectory group (compared to membership in the con- sistently high unmet need group) (N = 850). OR (95% CI) Characteristic Consistently High Decreasing Increasing Consistently Low Number of living children† 1 0.4 (0.3, 0.5) 0.6 (0.4, 0.9) 0.6 (0.4, 0.7) Employed 1 1.7 (0.9, 3.0) 1.9 (0.8, 4.4) 1.5 (0.9, 2.7) Main partner desires a/another child 1 4.2 (1.6, 10.9) 0.5 (0.1, 2.9) 0.8 (0.5, 1.2) Problems with contraceptive method‡ 1 0.7 (0.3, 1.4) 1.9 (1.0, 3.7) 0.7 (0.4, 1.3) Abbreviations; OR: Odds Ratio, CI: Confidence Interval † Per increase in category (corresponding to an increase in total number of children), with 1 = 0 children, 2 = 1 child, 32 children ‡ Includes heavy bleeding, irregular bleeding and spotting, amenorrhea, and rash or thrush as well as common side effects such as weight gain or headaches https://doi.org/10.1371/journal.pone.0209114.t002 associated with membership in the decreasing unmet need trajectory group (compared to membership in the con- atios (95% confidence intervals) associated with membership in the decreasing unmet need trajectory group (compared t nmet need group) (N = 850). PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 8 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 We observed that family planning needs of women living with HIV changed frequently over time in this South African setting, yet in clinical practice fertility intentions and con- traceptive use were infrequently assessed. While current national service delivery guidelines promote the integration of sexual and reproductive health services with HIV prevention, care and treatment in South Africa [5], implementation of these guidelines in clinical practice has been limited [35,36]. Women in our study, for example, received on-site referrals for con- traceptive counseling that technically constitute integrated services. However, providers were frequently located in different rooms or wards, contributing to long queues and lengthy wait times. Preliminary results from the Tsepamo Study in Botswana indicating an increased risk for neural tube defects among women taking dolutegravir at conception have spurred further guidance around increasing contraceptive uptake for women initiating ART [37]. However, the extent to which this additional guidance will ultimately streamline delivery of family plan- ning services is yet to be determined. Routine HIV clinical care visits represent a potential opportunity to regularly assess family planning needs, particularly among women on ART who may be unaware that their fertility will increase once started on ART [22,30,38]. Furthermore, regularly assessing the family plan- ning needs of women who have trouble achieving viral suppression is critical for both preven- tion of horizontal transmission to HIV-uninfected partners and PMTCT. Previous studies have suggested that multidimensional measures of fertility intentions (e.g., measures that incorporate questions around childbearing preferences, feelings about a pregnancy/child, and perceived consequence of a pregnancy/child) are needed to reliably determine women’s family planning needs [39,40]. However, operationalizing more complex tools within the context of high patient volume and limited provider resources may pose further challenges. More suc- cinct measures of fertility intentions, such as those used to inform this study’s assessment of unmet need, are demonstrably reliable in predicting pregnancy outcomes [8,17,41] and may be more readily integrated into clinical practice to evaluate the immediate family planning needs of women living with HIV. In one high-volume facility in Malawi, integration of a short family planning assessment using an electronic medical record-based tool prompted health- care workers to more routinely offer contraceptives during HIV care visits, increasing con- traceptive prevalence over a four-year period [42]. Our study is not without limitations. Our findings are compatible with previous research showing a single assessment of contraceptive need is insuf- ficient in identifying those at high risk for unintended pregnancy, even within a 12-month period [8]. Consistent with other estimates of contraceptive use in South Africa, few women in our study were using a reliable method of contraception to prevent pregnancy at enrollment. Low uptake of hormonal contraception may reflect fear of side effects or health risks (e.g., heavy menstrual bleeding, irregular bleeding, bleeding between periods, lower abdominal pain), which are strongly associated with contraceptive discontinuation or switching in HIV-positive women [31,32,33]. Limited uptake or method discontinuation may also reflect structural barri- ers such as long clinic wait times [17,18] or other unknown factors, as well as additional fears around side effects resulting from ART/contraceptive drug interactions [34]. However, only one woman in our study reported discontinuing her method for this reason. Compared to women in our study who followed a trajectory of consistently low unmet contraceptive need, those with increasing unmet need were significantly more likely to report problems or side effects with their method of contraception. Furthermore, a significant proportion of those who followed an increasing trajectory of unmet need discontinued their method of contraception over the 12-month period without a corresponding change in fertility intentions. Changes in unmet need for contraception also reflect changes in fertility intentions. We found that among women with a decreasing unmet need for contraception over follow-up, the primary driver of reduced unmet need was increasing fertility intentions, not an increase in uptake of contraception. Previous studies have found that fertility intentions increase rapidly in the period following ART initiation [27–29], though evidence is limited. Increasing fertility intentions among women on ART may also reflect partner fertility desires, and we found that women with a decreasing unmet contraceptive need were more likely to report a partner who desired a/another child. To the extent that fertility intentions reflect decisions around PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 9 / 14 Longitudinal patterns of unmet need for contraception childbearing in both partners, comprehensive screening of family planning needs that includes asking women about their partner’s fertility intentions may offer additional opportunities to identify women who require safer conception counseling. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Acknowledgments We thank the participants, clinic staff and study team for their support of the study. We thank the participants, clinic staff and study team for their support of the study. Conclusions We have demonstrated that in this South African setting, a high proportion of women living with HIV experience a persistently high probability of unmet need for contraception. We have also shown that the family planning needs of women living with HIV may change rapidly, even over a 12-month period. As more and more women initiate ART through South Africa’s universal testing and treatment initiative, routine screening of fertility intentions combined with rapid service referral for reliable contraception and safer conception methods will be crit- ical in ensuring that women living with HIV are able to meet their reproductive needs. Supporting information S1 Questionnaire. Fertility intentions and contraceptive use assessment items. (PDF) S1 Fig. Predicted trajectories of unmet need for contraception using quarterly measure- ments among women living with HIV taking ART in Johannesburg, 2009–2011. Figure that illustrates trajectories of unmet need for contraception estimated using quarterly assessments for A) the full cohort (N = 850) and B) recent ART initiators (N = 157). (TIFF) S1 Table. Characteristics of Women Assigned to “Consistently Low,” “Increasing,” “Decreasing,” and “Consistently High” Unmet Need Trajectory Groups using a Maxi- mum-Probability Assignment Rule (N = 850). (DOCX) S2 Table. Characteristics of Recent ART Initiators Assigned to “Consistently Low,” “Increasing,” “Decreasing,” and “Consistently High” Unmet Need Trajectory Groups using a Maximum-Probability Assignment Rule (N = 157). (DOCX) S1 Dataset. (ZIP) First, nearly half of women in this analysis reported condoms as their primary method of family planning at enrollment but were considered to have unmet need for contraception. As some of these women may have used condoms consis- tently and correctly, we may have overestimated the total number of women in our cohort with unmet need. However, women in this study who reported using condoms had nearly identical pregnancy rates as women who reported using no contraceptive method [8], suggest- ing that the majority of women using condoms were not doing so effectively enough to prevent pregnancy. Second, all behavioral outcomes were measured by self-report and are subject to potential misclassification; however, questionnaires were administered in private by trained study staff who developed a strong rapport with the participants to minimize social desirability bias. Third, data included in this study predate South African national service delivery guide- lines for integrated family planning and HIV care and earlier thresholds for treatment initia- tion. At the time of study enrollment, non-pregnant women were only eligible for lifelong ART initiation with a CD4 count 200 cells/μl or WHO clinical stage 4 diagnosis [43], and tra- jectories of unmet need (and predictors thereof) may differ in the current era. We were also unable to confirm the original indication for treatment among women who initiated ART PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 10 / 14 Longitudinal patterns of unmet need for contraception prior to enrolling in this study, and trajectories of unmet need may also differ among women who initiated ART through PMTCT compared to women who initiated ART for their own health. However, we saw very little difference in CD4 count by trajectory group overall, and trajectories were largely similar between the whole cohort and the subgroup of recent ART ini- tiators who were known to have started treatment outside of pregnancy. Furthermore, opera- tional standards around the assessment of fertility intentions and the provision of effective methods of contraception (e.g., long-acting reversible contraception) within the ART clinic setting remain relatively unchanged in current clinical practice. References 1. Cleland J, Conde-Agudelo A, Peterson H, Ross J, Tsui A. Contraception and health. Lancet. 2012; 380 (9837): 149–156. https://doi.org/10.1016/S0140-6736(12)60609-6 PMID: 22784533 2. Singh S, Darroch JE. Adding it up: Costs and benefits of contraceptive services. New York, United States of America: 2012; Guttmacher Institute and United States Population Fund. Available at: http:// www.who.int/woman_child_accountability/ierg/reports/Guttmacher_AIU_2012_estimates.pdf?ua=1 3. Reynolds HW, Janowitz B, Wilcher R, Cates W. Contraception to prevent HIV-positive births: current contribution and potential cost savings in PEPFAR countries. Sex Transm Infect. 2008; 84 Suppl 2:ii49– 53. 4. Joint United Nations Programme on HIV/AIDS (UNAIDS). 2015 Progress Report on the Global Plan towards the elimination of new HIV infections among children and keeping their mothers alive. Geneva, Switzerland: 2015. Available at: http://www.unaids.org/sites/default/files/media_asset/JC2774_ 2015ProgressReport_GlobalPlan_en.pdf 5. Republic of South Africa Department of Health. South African National Contraception and Fertility Plan- ning Policy and Service Delivery Guidelines: A Companion to the National Contraception Clinical Guide- lines. Pretoria, South Africa: 2014. Available at: http://www.partners-popdev.org/wp-content/uploads/ 2015/08/National-contraception-family-planning-policy.pdf 6. Yotebieng M, Norris A, Chalachala JL, Matumona Y, Ramadhani HO, Behets F. Fertility desires, unmet need for family planning, and unwanted pregnancies among HIV-infected women in care in Kinshasa, DR Congo. Pan African Medical Journal. 2015; 20(235). 7. Sarnquist CC, Rahangdale L, Maldonado Y. Reproductive health and family planning needs among HIV-infected women in Sub-Saharan Africa. Curr HIV Res. 2013; 11(2): 160–168. PMID: 23432491 8. Schwartz SR, Rees H, Mehta S, Venter WD, Taha TE, Black V. High incidence of unplanned pregnancy after antiretroviral therapy initiation: findings from a prospective cohort study in South Africa. PLoS One. 2012; 7(4): e36039. https://doi.org/10.1371/journal.pone.0036039 PMID: 22558319 9. Chersich MF, Wabiri N, Risher K, Shisana O, Celentano D, Rehle T, et al. Contraception coverage and methods used among women in South Africa: A national household survey. Samj S Afr Med J. 2017; 107(4): 307–314. https://doi.org/10.7196/SAMJ.2017.v107i4.12141 PMID: 28395681 10. Machiyama K, Casterline JB, Mumah JN, Huda FA, Obare F, Odwe G, et al. Reasons for unmet need for family planning, with attention to the measurement of fertility preferences: protocol for a multi-site cohort study. Reprod Health. 2017; 14(1): 23. https://doi.org/10.1186/s12978-016-0268-z PMID: 28183308 11. Kosgei RJ, Lubano KM, Shen C, Wools-Kaloustian KK, Musick BS, Siika AM, et al. Impact of integrated family planning and HIV care services on contraceptive use and pregnancy outcomes: a retrospective cohort study. J Acquir Immune Defic Syndr. 2011; 58(5): e121–126. https://doi.org/10.1097/QAI. 0b013e318237ca80 PMID: 21963940 12. Author Contributions Conceptualization: Katherine B. Rucinski, Kimberly A. Powers, Sheree R. Schwartz, Brian W. Pence, Benjamin H. Chi, Audrey E. Pettifor. Conceptualization: Katherine B. Rucinski, Kimberly A. Powers, Sheree R. Schwartz, Brian W. Pence, Benjamin H. Chi, Audrey E. Pettifor. Data curation: Katherine B. Rucinski, Sheree R. Schwartz, Vivian Black. Data curation: Katherine B. Rucinski, Sheree R. Schwartz, Vivian Black. 11 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Longitudinal patterns of unmet need for contraception Formal analysis: Katherine B. Rucinski. Formal analysis: Katherine B. Rucinski. Investigation: Sheree R. Schwartz, Vivian Black, Helen Rees. Methodology: Katherine B. Rucinski, Kimberly A. Powers, Sheree R. Schwartz, Audrey E. Pettifor. Supervision: Kimberly A. Powers, Sheree R. Schwartz, Brian W. Pence, Benjamin H. Chi, Audrey E. Pettifor. Validation: Sheree R. Schwartz. Visualization: Katherine B. Rucinski, Kimberly A. Powers. Writing – original draft: Katherine B. Rucinski, Kimberly A. Powers, Sheree R. Schwartz, Brian W. Pence, Benjamin H. Chi, Vivian Black, Helen Rees, Audrey E. Pettifor. Writing – review & editing: Katherine B. Rucinski, Kimberly A. Powers, Sheree R. Schwartz, Brian W. Pence, Benjamin H. Chi, Vivian Black, Helen Rees, Audrey E. Pettifor. 12. Grossman D, Onono M, Newmann SJ, Blat C, Bukusi EA, Shade SB, et al. Integration of family planning services into HIV care and treatment in Kenya: a cluster-randomized trial. AIDS. 2013; 27: S77–S85. https://doi.org/10.1097/QAD.0000000000000035 PMID: 24088687 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 References Grossman D, Onono M, Newmann SJ, Blat C, Bukusi EA, Shade SB, et al. Integration of family planning services into HIV care and treatment in Kenya: a cluster-randomized trial. AIDS. 2013; 27: S77–S85. https://doi.org/10.1097/QAD.0000000000000035 PMID: 24088687 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 12 / 14 Longitudinal patterns of unmet need for contraception 13. Bachanas P, Kidder D, Medley A, Pals SL, Carpenter D, Howard A, et al. Delivering Prevention Inter- ventions to People Living with HIV in Clinical Care Settings: Results of a Cluster Randomized Trial in Kenya, Namibia, and Tanzania. AIDS Behav. 2016; 20(9): 2110–2118. https://doi.org/10.1007/s10461- 016-1349-2 PMID: 26995678 14. Lopez LM, Hilgenberg D, Chen M, Denison J, Stuart G. Behavioral interventions for improving con- traceptive use among women living with HIV. Cochrane Database Syst Rev. 2013(1): Cd010243. https://doi.org/10.1002/14651858.CD010243.pub2 PMID: 23440846 15. Cohen CR, Grossman D, Onono M, Blat C, Newmann SJ, Burger RL, et al. Integration of family plan- ning services into HIV care clinics: Results one year after a cluster randomized controlled trial in Kenya. PLoS One. 2017; 12(3): e0172992. https://doi.org/10.1371/journal.pone.0172992 PMID: 28328966 16. Shade SB, Kevany S, Onono M, Ochieng G, Steinfeld RL, et al. Cost, cost-efficiency and cost effective- nessof integrated family planning and HIV services. AIDS. 2016; 27 Suppl 1: S87–S92. 17. Schwartz SR, Mehta SH, Taha TE, Rees HV, Venter F, Black V. High pregnancy intentions and missed opportunities for patient-provider communication about fertility in a South African cohort of HIV-positive women on antiretroviral therapy. AIDS Behav. 2012; 16(1): 69–78. https://doi.org/10.1007/s10461-011- 9981-3 PMID: 21656145 18. Marlow HM, Maman S, Moodley D, Curtis S. Postpartum family planning service provision in Durban, South Africa: client and provider perspectives. Health Care Women Int. 2014; 35(2):175–199. https:// doi.org/10.1080/07399332.2013.815753 PMID: 23998760 19. Tang JH, Phiri S, Ng’ambi W, Krashin JW, Mlundira L, Chaweza T, et al. Factors Associated with Hor- monal and Intrauterine Contraceptive Use among HIV-Infected Men and Women in Lilongwe, Malawi: A Cross-Sectional Study. Infectious diseases in obstetrics and gynecology. 2016. 20. Steiner RJ, Black V, Rees H, Schwartz SR. Low receipt and uptake of safer conception messages in routine HIV care: Findings from a prospective cohort of women living with HIV in South Africa. J Acquir Immune Defic Syndr. 2016. 21. Bradley S, Croft TN, Fishel JD, Westoff CF. Revising unmet need for family planning. Maryland, United States of America: United States Agency for International Development; 2012. Available at: http://pdf. usaid.gov/pdf_docs/Pnady130.pdf 22. References Myer L, Carter RJ, Katyal M, Toro P, El-Sadr WM, Abrams EJ. Impact of antiretroviral therapy on inci- dence of pregnancy among HIV-infected women in Sub-Saharan Africa: a cohort study. PLoS Med. 2010; 7(2):e1000229. https://doi.org/10.1371/journal.pmed.1000229 PMID: 20161723 23. Nagin D. Group-based modeling of development [electronic resource]. Cambridge, Mass.: Harvard University Press; 2005. 24. Nagin DS, Odgers CL. Group-based trajectory modeling in clinical research. Annu Rev Clin Psychol. 2010; 6:109–138. https://doi.org/10.1146/annurev.clinpsy.121208.131413 PMID: 20192788 25. Gideon S. Estimating the dimension of a model. The annals of statistics. 1978; 6(2):461–464. 26. Kass RE, Wasserman L. A Reference Bayesian Test for Nested Hypotheses and Its Relationship to the Schwarz Criterion. J Am Stat Assoc. 1995; 90(431):928–934. 27. Kaida A, Gray G, Bastos FI, Andia I, Maier M, McIntyre J, et al. The relationship between HAART use and sexual activity among HIV-positive women of reproductive age in Brazil, South Africa, and Uganda. AIDS Care. 2008; 20(1): 21–25. https://doi.org/10.1080/09540120701426540 PMID: 18278611 28. Maier M, Andia I, Emenyonu N, Guzman D, Kaida A, Pepper L, et al. Antiretroviral therapy is associated with increased fertility desire, but not pregnancy or live birth, among HIV+ women in an early HIV treat- ment program in rural Uganda. AIDS and Behavior. 2009; 13(1): 28–37. 29. Cooper D, Moodley J, Zweigenthal V, Bekker LG, Shah I, Myer L. Fertility intentions and reproductive health care needs of people living with HIV in Cape Town, South Africa: implications for integrating reproductive health and HIV care services. AIDS Behav. 2009; 13 Suppl 1: 38–46. 30. Burgos-Soto J, Balestre E, Minga A, Ajayi S, Sawadogo A, Zannou MD, et al. Incidence of pregnancy after antiretroviral therapy initiation and associated factors in 8 West African countries. J Acquir Immune Defic Syndr. 2014; 67(2): e45–54. https://doi.org/10.1097/QAI.0000000000000279 PMID: 25216079 31. Haddad L, Wall KM, Vwalika B, Khu NH, Brill I, Kilembe W, et al. Contraceptive discontinuation and switching among couples receiving integrated HIV and family planning services in Lusaka, Zambia. AIDS. 2013; 27 Suppl 1: S93–103. 32. Todd CS, Anderman TC, Long S, Myer L, Bekker LG, Petro GA, et al. A systematic review of contracep- tive continuation among women living with HIV. Contraception. 2018 [epub ahead of print]. 33. Haider TL, Sharma M. Barriers to family planning and contraception uptake in sub-Saharan Africa: a systematic review. Int Q Community Health Educ. 2012; 33(4): 403–413. https://doi.org/10.2190/IQ.33. PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 References 4.g PMID: 24044930 13 / 14 PLOS ONE \| https://doi.org/10.1371/journal.pone.0209114 December 20, 2018 Longitudinal patterns of unmet need for contraception 34. Robinson JA, Jamshidi R, Burke AE. Contraception for the HIV-positive woman: a review of interactions between hormonal contraception and antiretroviral therapy. Infect Dis Obstet Gynecol. 2012;2012:890160. 35. Cooper D, Mantell JE, Moodley J, Mall S. The HIV epidemic and sexual and reproductive health policy integration: views of South African policymakers. BMC Public Health. 2015; 15: 217. https://doi.org/10. 1186/s12889-015-1577-9 PMID: 25879464 36. Haberlen SA, Narasimhan M, Beres LK, Kennedy CE. Integration of Family Planning Services into HIV Care and Treatment Services: A Systematic Review. Stud Fam Plann. 2017; 48(2): 153–177. https:// doi.org/10.1111/sifp.12018 PMID: 28337766 37. Zash R, Makhema J, Shapiro RL. Neural-Tube Defects with Dolutegravir Treatment from the Time of Conception. New England Journal of Medicine. 2018; 379(10): 979–981. https://doi.org/10.1056/ NEJMc1807653 PMID: 30037297 38. Gray RH, Wawer MJ, Serwadda D, Sewankambo N, Li C, Wabwire-Mangen F, et al. Population-based study of fertility in women with HIV-1 infection in Uganda. Lancet. 1998; 351(9096): 98–103. https://doi. org/10.1016/S0140-6736(97)09381-1 PMID: 9439494 39. Rocca CH, Ralph LJ, Gould H, Barar R, Foster DG. Measuring Pregnancy Preferences Prospectively: a Multidimensional Psychometric Instrument. Population Association of America Annual Meeting. Chi- cago, IL, April 27–29 2017. Available at: https://paa.confex.com/paa/2017/meetingapp.cgi/Paper/ 16597 40. Mumford SL, Sapra KJ, King RB, Louis JF, Buck Louis GM. Pregnancy intentions-a complex construct and call for new measures. Fertility and sterility. 2016: 106(6), 1453–1462. https://doi.org/10.1016/j. fertnstert.2016.07.1067 PMID: 27490044 41. Wilson KS, Wanje G, Masese L, Simoni JM, Shafi J, Adala L, et al. A prospective cohort study of fertility desire, unprotected sex, and detectable viral load in HIV-positive female sex workers in Mombasa, Kenya. JAIDS. 2018. Jul 1; 78(3):276–82. https://doi.org/10.1097/QAI.0000000000001680 PMID: 29543635 42. Tweya H, Feldacker C, Haddad LB, Munthali C, Bwanali M, Speight C, et al. Integrating family planning services into HIV care: Use of a point-of-care electronic medical record system in Lilongwe, Malawi. Glob Health Action. 2017; 10(1): 1383724 https://doi.org/10.1080/16549716.2017.1383724 PMID: 29039263 43. Republic of South Africa Department of Health. Implementation of the Universal Test and Treat Strategy for HIV Positive Patients and Differentiated Care for Stable Patients. Pretoria, South Africa: 2016. Avail- able at: http://www.sahivsoc.org/Files/22%208%2016%20Circular%20UTT%20%20%20Decongestion %20CCMT%20Directorate.pdf 14 / 14
https://openalex.org/W2788281384	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1005967&type=printable	English	null	Optimizing homeostatic cell renewal in hierarchical tissues	PLOS computational biology/PLoS computational biology	2,018	cc-by	15,344	RESEARCH ARTICLE Cesar Alvarado1, Nicole A. Fider2, Helen J. Wearing1,3, Natalia L. Komarova2,4* Cesar Alvarado1, Nicole A. Fider2, Helen J. Wearing1,3, Natalia L. Komarova2,4* 1 Department of Mathematics and Statistics, University of New Mexico, Albuquerque, New Mexico, United States of America, 2 Department of Mathematics, University of California Irvine, Irvine, California, United States of America, 3 Department of Biology, University of New Mexico, Albuquerque, New Mexico, United States of America, 4 Department of Ecology and Evolutionary Biology, University of California Irvine, Irvine, California, United States of America * komarova@uci.edu * komarova@uci.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Alvarado C, Fider NA, Wearing HJ, Komarova NL (2018) Optimizing homeostatic cell renewal in hierarchical tissues. PLoS Comput Biol 14(2): e1005967. https://doi.org/10.1371/journal. pcbi.1005967 Citation: Alvarado C, Fider NA, Wearing HJ, Komarova NL (2018) Optimizing homeostatic cell renewal in hierarchical tissues. PLoS Comput Biol 14(2): e1005967. https://doi.org/10.1371/journal. pcbi.1005967 Editor: Arne Traulsen, Max-Planck-Institute for Evolutionary Biology, GERMANY Editor: Arne Traulsen, Max-Planck-Institute for Evolutionary Biology, GERMANY Copyright: © 2018 Alvarado et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract In order to maintain homeostasis, mature cells removed from the top compartment of hierar- chical tissues have to be replenished by means of differentiation and self-renewal events happening in the more primitive compartments. As each cell division is associated with a risk of mutation, cell division patterns have to be optimized, in order to minimize or delay the risk of malignancy generation. Here we study this optimization problem, focusing on the role of division tree length, that is, the number of layers of cells activated in response to the loss of terminally differentiated cells, which is related to the balance between differentiation and self-renewal events in the compartments. Using both analytical methods and stochastic simulations in a metapopulation-style model, we find that shorter division trees are advanta- geous if the objective is to minimize the total number of one-hit mutants in the cell popula- tion. Longer division trees on the other hand minimize the accumulation of two-hit mutants, which is a more likely evolutionary goal given the key role played by tumor suppressor genes in cancer initiation. While division tree length is the most important property determin- ing mutant accumulation, we also find that increasing the size of primitive compartments helps to delay two-hit mutant generation. Optimizing homeostatic cell renewal in hierarchical tissues a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Introduction Many tissues in complex organisms are hierarchically organized, such as the human colon, the small intestine, the skin, and the haematopoietic system. Hierarchically organized tissues are renewed constantly by means of a balance of cell deaths and cell divisions. Understanding the mechanisms that regulate cell self-renewal and differentiation patterns is key to explaining the robust nature of homeostasis [1]. Because of the imminent risk of mutation acquisition, tissue organization also plays an important role in preventing cancer [2–9]. At the root of hierarchically organized tissues are a small number of stem cells (SCs), capa- ble of both self renewal and differentiation into more specialized cells [10]. Downstream from SCs there are intermediate cells of increasing degrees of maturity, which can undergo a certain number of divisions. Finally, there are fully mature (terminally) differentiated cells, which per- form their function and are discarded and replenished through divisions of less differentiated cells. For instance, evidence suggests that on or near the bottom of the colon crypts and the small intestine crypts, there are between 4 −6 SCs [11–13], and the remaining compartments are composed of transit amplifying (TA) cells and finally mature differentiated cells that are discarded, so that the entire crypt is renewed every 2 −7 days [14]. A similar situation occurs in the haematopoietic system, where a stem cell pool of about 400 cells is required to replenish a daily bone marrow output of about 3.5 × 1011 cells [15, 16]. Stem cells are thought to be capable of symmetric or asymmetric divisions. Asymmetric divisions of a stem cell result in one daughter cell that is a stem cell and the other daughter cell that is a more differentiated cell. Symmetric divisions result in two identical daughter cells. The two types of symmetric divisions are illustrated in Fig 1(a) in a more general context: dif- ferentiation divisions result in two offspring that are both more differentiated than the divid- ing cell, and self-renewal divisions result in two offspring whose differentiation status is identical to that of the parent. Because experiments to track cell divisions and mutations are in general difficult, or not feasible in some hierarchically organized tissues, mathematical models have been used to understand cellular dynamics or homeostasis and mutations. In particular, the origin and development of colorectal cancer have been extensively studied. Optimizing homeostatic cell renewal in hierarchical tissues everyday tissue maintenance (homeostasis). For example, tumor suppressor gene inactiva- tion (two consecutive mutations) is an early rate-limiting step in many cancers. Using mathematical and computational methods, we find that the length of division trees is anti- correlated with the likelihood of double mutations, and lengthening the trees may provide an evolutionary advantage to the organism by delaying the onset of cancer. Author summary Cells in multicellular organisms are organized hierarchically. A stem cell gives rise to a chain of dividing and progressively differentiating offspring. At the end of this chain (called a lineage) are terminally differentiated cells that perform their function and undergo programmed cell death, to be replaced by new divisions of less differentiated cells. Here we are interested in the design of such lineages. At one extreme, one can imag- ine that a loss of terminally differentiated cells only results in divisions of cells in close hierarchical proximity to them, giving rise to very short division trees. On the other hand, it is possible that a long chain of increasingly primitive cells gets activated in response to the loss of differentiated cells. We expect that an important type of selection pressure act- ing upon tissue design is the minimization of mutations that happen in the course of Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: NK gratefully acknowledges the support of the National Institute of Health grant 1 U01 CA187956-01, https://www.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 1 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Introduction It has been demonstrated the- oretically that mutations leading to colorectal cancer can originate in either the stem cell com- partment or TA cells [3, 5, 7, 17]. Computational models, such as virtual crypts, have helped to understand the process of self renewal in hierarchically organized tissues, for instance the organization of the colon [18–21]. Several studies have investigated tissue architecture with the goal of understanding its utility in protection against mutation accumulation. Traulsen, Wer- ner and colleagues used mathematical models to study mutations in the haematopoietic sys- tem, and found theoretical evidence that tissue architecture and the process of self renewal were a protection mechanism against cancer [6, 9, 22, 23]. Rodriguez-Brenes et al. [8] pro- posed that an optimal tissue architecture that minimized the replication capacity of cells was one where the less differentiated cells had a larger rate of self-renewal. Another study [2] showed that having symmetric stem cell divisions (self-renewals and differentiations) rather PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 2 / 24 Optimizing homeostatic cell renewal in hierarchical tissues Fig 1. Schematics showing key concepts of the paper. (a) Two types of symmetric divisions: self-renewals and differentiations. Each circle represents a cell, and i denotes the ith compartment, while i + 1 denotes the (i + 1)th compartment. Panels (b) and (c) demonstrate the division chains that replenish 8 differentiated cells eliminated from the top compartment. Dead cells are denoted by X’s and arrows show divisions. Cells are arranged in horizontal layers corresponding to compartments. Only the dividing cells are shown (for example, there may be more than 4 cells in the second to top compartment in panel (b)). In (b), the dead cells are replaced by a longer division tree, and in (c) by four shorter division trees. than asymmetric stem cell divisions minimized the risk of two-hit mutant generation. Further- more, Dingli et al. [24] considered the question of mutation generation by stem cells and found that mutations that increased the probability of asymmetric replication could lead to rapid expansion of mutant stem cells in the absence of a selective fitness advantage. Pepper et al. [25] examined a tissue undergoing serial differentiation patterns originating with self- renewing somatic stem cells, continuing with several TA cell differentiations, and showed that such patterns lowered the rate of somatic evolution. Finally, Sprouffske et al. https://doi.org/10.1371/journal.pcbi.1005967.g001 The top-down approach To illustrate the questions we are interested in solving, consider a hierarchical tissue where symmetric divisions are prevalent, such as the human colon [28–31]. When mature differenti- ated cells in the most downstream compartment (the “top” layer of tissue) are discarded, cells from the adjacent upstream compartment divide to replace the eliminated cells. This gives rise to a chain of differentiations, because the cells that differentiated and migrated from the upstream compartment also need to be replaced, in order to keep the tissue at homeostasis. Fig 1(b) and 1(c) schematically illustrates two opposite trends. Both panels depict 8 mature cells that are eliminated. These cells are replenished by 4 differentiation events, whereby cells from the neighboring compartment differentiate and migrate downstream to replace the dead cells. In panel (b), the 4 cells are, in turn, replenished by two differentiation events from the adjacent upstream layer (representing a compartment in the hierarchy), leaving two cells to be replaced. This is done by a single differentiation event from the bottom layer, and the remaining cell is then replaced by a self-renewal event from the same layer. These processes comprise a differen- tiation cascade, or a differentiation chain, which in this case includes differentiation events in 3 consecutive compartments. A different scenario is shown in panel (c) where the 4 cells that dif- ferentiate and migrate from the second-to-top layer are all replaced by self-renewal divisions of cells in the same layer. In this case we have 4 shorter differentiation chains which only include differentiation events in one compartment. As will be shown, a range of intermediate scenarios is possible. Biologically, differentiation chains are shaped by a system of feedback loops determined by signals altering self-renewal, differentiation, apoptosis, migration, and adhesion in both the stem cell compartment and the transit amplifying cells [1, 8, 32]. The self-renewal and differ- entiation activity is a well controlled system to guarantee tissue homeostasis in hierarchically organized tissues, thus, changes in self-renewal and differentiation patterns might be associ- ated with an increased risk of cancer development, as found, for example, by Merrit et al. [33] in the context of colorectal cancer. Fig 1(b) and 1(c) suggests that in terms of tissue architec- ture, and in the presence of hierarchical lineages, there are multiple arrangements that are all, in principle, capable of maintaining balanced tissue turnover. Introduction [26] emphasized the importance of spatial considerations in the modeling of stem cell hierarchies and division patterns. Despite significant progress reported in the literature, there are still unanswered questions regarding tissue renewal and cancer development in hierarchically organized tissues. In partic- ular, the optimal mechanisms of self renewal and self-renewal to maintain homeostasis is a crucial process which is not completely understood. In a recent paper, [27] present an elegant model that allows one to calculate the optimal lineage structure that minimizes the divisional load of cells. The premise of this paper is that to limit the accumulation of somatic mutations, renewing tissues must minimize the number of times each cell divides during differentiation. On the other hand, as was discovered by Werner et al. in their analysis of mutant dynamics [23], the occurrence of a mutant and the compartment of origin and its subsequent clonal dynamics are all of importance. In the present study we consider an optimization problem, where the objective is to optimize observables that are important for cancer prevention/delay. 3 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues Namely, our aim is to minimize the number of one-hit mutants accumulated in the tissue, and to maximize the expected time until two-hit mutants are generated. We proceed by first formulating a top-down, hierarchical stochastic model of tissue self- renewal, and then deriving analytical expressions for the expected number of mutants in each compartment. This informs a deterministic approximation resulting in a set of differential equations describing mutant dynamics in different compartments. It turns out that this meth- odology can be further adapted to describe not only the approximately deterministic regime of large populations and large mutation rates, but a more relevant regime of small populations and small mutation rates. We investigate the dynamics of our model in different scenarios, focusing on different self-renewal/differentiation probabilities and different compartment size arrangements. In addition, we perform stochastic simulations to study the accumulation of mutations in a stochastic regime. We describe both one-hit and two-hit mutant generation, and find the parameters that can be tuned to delay cancer initiation in hierarchically organized tissues. The stochastic process Assume that there are n + 1 compartments, C0, . . ., Cn, with total constant cell numbers N0, . . ., Nn. Inside individual compartments, we assume complete mixing, and the compart- ments are arranged linearly from the least mature, C0, to the most mature, Cn. At each time step, 2n cells from the most differentiated compartment Cn are removed. These cells must be replaced by 2n divisions in different compartments. This is done by going through the compartments consecutively in the upstream direction (Cn−1, Cn−2, . . ., C0) and probabilistically assigning the division event types that take place. The parameter that determines the division type is 0 vi 1, which is the probability for a cell in compartment Ci to be replaced by means of a self-renewing division in the same com- partment. We assume that vn = 0, that is, terminally differentiated cells do not self-renew. Fur- ther, because there is no compartment below the stem cell layer, removed stem cells can only be replaced by self-renewal and v0 = 1 necessarily. In the stochastic simulations, we let the size of each compartment Ci be constant (Ni, for each i = 0, 1, . . ., n) and we track the numbers of mutants mi in each of the compartments. The number of healthy cells is therefore Ni −mi, where each such cell can be thought of as a biolog- ical “wild card” in the sense that, if the cell is chosen for cell division, it will mutate with proba- bility u. The initial number of mutants is zero, and the simulation proceeds as a sequence of discrete updates. Each update starts with 2n cells removed from compartment Cn. A mutant is dis- carded with probability mn Nn, otherwise, the cell discarded is a wild type cell. The next step is to replace the 2n cells removed in compartment Cn. Because cells in compartment Cn cannot pro- liferate, 2n−1 cells differentiate out from compartment Cn−1. In each division, a mutant is cho- sen with probability mn1 Nn1, which increases the number of mutants in compartment Cn by two while the number of mutants in compartment Cn−1 decreases by one. If a wild type cell is cho- sen for differentiation in compartment Cn−1, a de-novo mutant is produced with probability u, such that the number of mutants in Cn increases by one. Optimizing homeostatic cell renewal in hierarchical tissues turnover and/or compartment sizes in order to minimize the number of mutants generated and accumulated. We note that in our top-down approach for the optimization problem, we assume that the number and the turnover rate of the terminally differentiated cells is a given constraint, dictated by the needs of the organ. It is the upstream arrangement of the less differentiated compartments that is under the evolutionary pressure to delay cancer. A precise formulation of this concept is presented next. The top-down approach Each arrangement preserves the number of cells in each compartment in the face of divisions triggered by cell deaths in the top compartment. Because each cell division is associated with a probability of harmful muta- tions, the question then becomes if there is a preferred strategy for a tissue to arrange its PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 4 / 24 Trees and their probabilities Here we will build all possible division trees that can result from removing 2n cells from com- partment Cn. Then, given the values vi, we will calculate the probability for each possible divi- sion tree. Since cells in compartment Cn do not proliferate, following an 2n removals from Cn, there will be an−1 = 2n−1 differentiation divisions in compartment Cn−1. Next, an−1 cells in this com- partment must be replaced, either by differentiation divisions from compartment Cn−2 or by self-renewal divisions from compartment Cn−1. Suppose there are an−2 differentiations and an−1 −2an−2 self-renewals. Next, an−2 cells from compartment Cn−2 must be replaced. This pro- cess gives rise to a division tree, which is uniquely characterized by the numbers of differentia- tion divisions in each compartment: fa0; . . . ; an1g; ð1Þ ð1Þ fa0; . . . ; an1g; where an−1 = 2n−1 and 0 ai 2i; i ¼ 0; . . . ; n 1: The number of self-renewals in compartment Ci is given by ai −2ai−1. The length of the tree is the number of compartments that have nonzero numbers of differentiation events, and it var- ies from 1 to n. In other words, the length of the tree is the number of cell levels that activated as a response to cell death in the top level. The number of self-renewals in compartment Ci is given by ai −2ai−1. The length of the tree is the number of compartments that have nonzero numbers of differentiation events, and it var- ies from 1 to n. In other words, the length of the tree is the number of cell levels that activated as a response to cell death in the top level. Self-renewal and differentiation events are assigned probabilistically, by means of the fol- lowing process. Suppose ai cells must be replaced in compartment Ci, that is, there are i open- ings to fill. Each cell is replaced by a self-renewal division in this compartment with probability vi. If the number of remaining openings is odd, then an additional self-renewal division hap- pens, such that the number of remaining openings is even, and they are then filled by differen- tiation divisions from compartment Ci−1. This allows us to calculate the probability of a given tree defined by string (1). Optimizing homeostatic cell renewal in hierarchical tissues is a differentiation from compartment Cn−2, mutants and wild type cells are updated in the same way as described above for differentiations from compartment Cn−1. After this, there are a number of cells “missing” from compartment Cn−2 that have to be replaced in a similar way. Every time a cell is chosen for division from compartment Ci, it is a mutant with probability mi Ni. The process of cell replacement proceeds until compartment C0 is reached. Cells “missing” from this compartment are replaced by self-renewal divisions in the same compartment with probability v0 = 1. The stochastic process The number of wild-type cells in Cn−1 is decreased by one. There are altogether 2n−1 differentiations from Cn−1 performed in this way. Next, 2n−1 cells have to be replaced in compartment Cn−1. Each of these cells can be replaced by a self-renewal event in Cn−1 with probability vn−1. Cells that are not replaced by a self- renewal event are replaced by a differentiation event from compartment Cn−2 with probability 1 −vn−1. If the number of cells to be replaced by differentiation events is odd, we add an extra self-renewal event in compartment Cn−1 so that the number of remaining openings is even. If the division event is a self-renewal, a mutant is chosen with probability mn1 Nn1 which increases the mutant population in compartment Cn−1 by one. If the proliferating cell is a wild type cell, it produces a de-novo mutant with probability u such that the number of mutants in compart- ment Cn−1 increases by one. If the wild type cell did not mutate or a mutant was not chosen for reproduction, the number of wild type cells in compartment Cn−1 increases by one. If the event 5 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 where an−1 = 2n−1 and Trees and their probabilities First we calculate the probability of having ai differentiations in com- partment Ci given that there are ai+1 differentiations in compartment Ci+1: pðaiÞ ¼ C2ai aiþ1 ð1 viþ1Þ 2aiv aiþ12ai iþ1 þ C2aiþ1 aiþ1 ð1 viþ1Þ 2aiþ1v aiþ12ai1 iþ1 ; 0 i n 1; where formally an = 2n and we use the following notation for the binomial coefficients: Ck n ¼ n! k!ðn kÞ! : We then have the probability of string (1): We then have the probability of string (1): Pða0; . . . ; an1Þ ¼ Y n1 i¼0 pðaiÞ: Pða0; . . . ; an1Þ ¼ Y n1 i¼0 pðaiÞ: In particular, since vn = 0, we obtain that an−1 = 2n−1 with certainty. In particular, since vn = 0, we obtain that an−1 = 2n−1 with certainty. 6 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues Fig 2. Properties of division trees as a function of v, the probability of self-renewal in compartments 1, . . ., n −1. (a) Mean tree length, defined as the mean number of compartments involved in the division trees. (b) Mean division position. For an individual tree, sequence (1), this is defined as 2n Pn i¼0 2iðai ai1Þ; plotted is the expectation of this quantity across all the trees. In this example, n = 4. https://doi.org/10.1371/journal.pcbi.1005967.g002 Fig 2. Properties of division trees as a function of v, the probability of self-renewal in compartments 1, . . ., n −1. (a) Mean tree length, defined as the mean number of compartments involved in the division trees. (b) Mean division position. For an individual tree, sequence (1), this is defined as 2n Pn i¼0 2iðai ai1Þ; plotted is the expectation of this quantity across all the trees. In this example, n = 4. https://doi.org/10.1371/journal.pcbi.1005967.g002 In Fig A in S1 Text and Fig B in S1 Text (Section 1 in S1 Text) examples are presented where probabilities of all possible division trees are calculated for a particular value of n. In general, decreasing the probability of self-renewal, v, increases the mean length of differentia- tion trees, meaning that divisions occur in the more primitive cell levels, see Fig 2. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues probability, we note that ak −i wild type cells differentiating in compartment Ck will produce 2(ak −i) offspring, each of which is a mutant with probability u. Therefore, l mutants are generated with probability Pdiff i;l ¼ Cl 2ðakiÞulð1 uÞ 2ðakiÞl: The total change in compartment Ck+1 is then 2i + l, the total change in compartment Ck is −i, and this happens with probability Pdiff i Pdiff i;l . These changes must be summed up for 0 i ak and 0 l 2(ak −i). The total change in compartment Ck+1 is then 2i + l, the total change in compartment Ck is −i, and this happens with probability Pdiff i Pdiff i;l . These changes must be summed up for 0 i ak and 0 l 2(ak −i). 0 i ak and 0 l 2(ak −i). (b) When ak+1 −2ak cells proliferate in compartment Ck+1, 0 k n −1, with probability P pro i ¼ Ci akþ12akmi kþ1ð1 mkþ1Þ akþ12aki; i of them are mutants. This means that i new mutants are added to compartment Ck+1. The remaining ak+1 −2ak −i proliferating cells are wild type, and it is possible that l of the offspring are new mutants. This happens with probability i of them are mutants. This means that i new mutants are added to compartment Ck+1. The remaining ak+1 −2ak −i proliferating cells are wild type, and it is possible that l of the offspring are new mutants. This happens with probability P pro i;l ¼ Cl 2ðakþ12akiÞulð1 uÞ 2ðakþ12akiÞl: The total change in compartment Ck+1 is then given by i + l and happens with probability P pro i P pro i;l . Again, these changes must be summed up for 0 i ak and 0 l 2(ak −i). (c) A special case is compartment C0. Here, a0 cells will proliferate, and the number of mutants is calculated similar to (b). (c) A special case is compartment C0. Here, a0 cells will proliferate, and the number of mutants is calculated similar to (b). (d) Another special case is compartment Cn. Deterministic dynamics of the expected number of mutants We can formulate a metapopulation-style, ordinary differential equation (ODE) model of the renewal dynamics described above. Let us suppose that in each compartment, the number of mutants is given by mi, 0 i n. We would like to calculate the expected change in the num- ber of mutants after 2n cells are removed in compartment Cn. To do this, let us determine the expected change in the number of mutants associated with a particular tree, string (1). This can be done by considering the change resulting from ak differentiations from compartment Ck to compartment Ck+1 and ak+1 −2ak self-renewals in compartment Ck+1. These events will affect the numbers of mutants in compartments Ck and Ck+1. Note that in the equations in this section we used combinatorial expressions obtained from sampling with replacement. In the stochastic simulations below, sampling without replacement was used, but it was established that sampling with replacement led to similar results. (a) When ak cells differentiate out of compartment Ck, 0 k n −1, with probability Pdiff i ¼ Ci akmi kð1 mkÞ aki; (a) When ak cells differentiate out of compartment Ck, 0 k n −1, with probability Pdiff i ¼ Ci akmi kð1 mkÞ aki; (a) When ak cells differentiate out of compartment Ck, 0 k n −1, with probability Pdiff i ¼ Ci akmi kð1 mkÞ aki; i of them may be mutants, where mk ¼ mk Nk mk ¼ mk Nk is the probability to pick a mutant among Nk cells. In this case, i mutants are removed from compartment Ck and 2i mutants are added to compartment Ck+1. Further, among the remaining ak −i differentiations, there may be l mutations which will increase the number of mutants in compartment Ck+1 by l. To calculate the corresponding PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 7 / 24 Cells do not proliferate in this compartment, but 2n cells are removed, and we need to calculate the expected number of mutants removed, similar to Pdiff i in (a). (d) Another special case is compartment Cn. Cells do not proliferate in this compartment, but 2n cells are removed, and we need to calculate the expected number of mutants removed, similar to Pdiff i in (a). For each tree, the expected change in the number of mutants can be expressed as a vector whose components are obtained as the expected change in each compartment, as calculated in (a-d) above. These vectors must be added together, weighted by the probability of each tree. The resulting vector gives the time derivatives of the quantities m0, . . ., mn. he resulting vector gives the time derivatives of the quantities m0, . . ., mn. When ak cells differentiate out of compartment Ck, 0 ak n −1, the expected gain of mutants in compartment Ck+1 is given by adiff kþ1 ¼ X ak i¼0 X 2ðakiÞ l¼0 ð2i þ lÞPdiff i Pdiff i;l : When ak+1 −2ak cells proliferate in compartment Ck+1, 0 k n −1, the expected gain of mutants in compartment Ck+1 is When ak+1 −2ak cells proliferate in compartment Ck+1, 0 k n −1, the expected gain of mutants in compartment Ck+1 is a pro kþ1 ¼ X ak i¼0 X 2ðakiÞ l¼0 ði þ lÞPpro i P pro i;l : The compartment Ck+1 loses mutants when ak+1 cells differentiate and migrate into compart- ment Ck+2, and the expected loss is bdiff kþ1 ¼ X akþ1 i¼0 X 2ðakþ1iÞ l¼0 ð2i þ lÞPdiff i Pdiff i;l : bdiff kþ1 ¼ X akþ1 i¼0 X 2ðakþ1iÞ l¼0 ð2i þ lÞPdiff i Pdiff i;l : 8 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues Thus the total change in the number of mutants in compartment Ck+1 is Tkþ1 ¼ adiff kþ1 þ a pro kþ1 bdiff kþ1: Tkþ1 ¼ adiff kþ1 þ a pro kþ1 bdiff kþ1: The value Tk+1 represents the expected number of mutants in compartment Ck+1 produced by a single tree or chain of differentiation; because more than one tree could affect the expected number of mutants in each compartment, we need to sum the contributions from all trees to compartment Ck+1 weighted by their probability. This weighted sum defines the rate of change of the number of mutants in each compartment: _mk ¼ X All trees ProbtreeTtree k ; 0 k n: ð2Þ ð2Þ For the simplest cases, we list the ODEs derived by computing the expected number of mutants produced by all possible chains of differentiation in each compartment. We will use the assumption that vi = v for 1 i n −1, which has been assumed in other studies [7, 9, 23]. For the case n = 1 we have For the case n = 1 we have _m1 ¼ u þ m0ð1 uÞ m1; _m0 ¼ 2uð1 m0Þ: For n = 2 we have For n = 2 we have _m2 ¼ 4ðu þ m1ð1 uÞ m2Þ; _m1 ¼ 2ð1 vÞ 2ðu þ m0ð1 uÞ m1Þ þ 4uð2 vÞvð1 m1Þ; _m0 ¼ 2uð1 vÞ 2ð1 m0Þ: For n = 3 we have _m3 ¼ 8ðu þ m2ð1 uÞ m3Þ; ð3Þ _m3 ¼ 8ðu þ m2ð1 uÞ m3Þ; ð3Þ _m3 ¼ 8ðu þ m2ð1 uÞ m3Þ; ð3Þ _m2 ¼ 4½uð1 m1 þ vð2 3v þ 4v2 2v3Þð1 þ m1 2m2ÞÞ þð1 vÞ 2ð1 þ 2v2Þðm1 m2Þ; ð4Þ ð4Þ _m1 ¼ 2ð1 vÞ 6ðu þ m0ð1 uÞ m1Þ þ 4uð2 vÞð1 vÞ 4vð1 m1Þ þ4uvð2 3v þ v3Þð1 m1Þ; ð5Þ _m0 ¼ 2uð1 vÞ 6ð1 m0Þ: ð6Þ ð6Þ To create a system for a general value of n, and also for general, non constant values of vi, we have created a program in Mathematica, see S1 File. In Section 1.2 in S1 Text, we present fur- ther details of these calculations. To create a system for a general value of n, and also for general, non constant values of vi, we have created a program in Mathematica, see S1 File. In Section 1.2 in S1 Text, we present fur- ther details of these calculations. The n + 1 linear ordinary differential equations derived here are applicable in the high mutation rate / large population limit (uN 1). The unique fixed point of the system, for all n, which is always stable, is mi ¼ Ni; 0 i n: In Fig 3, we set n = 3 and show the number of mutants in each of 4 compartments as a function of time, obtained from numerical solution of deterministic system (3–6) (solid lines) and sto- chastic simulations (dashed lines). The dynamics for two different values of v are presented, v = 0.9 and v = 0.1; the numerical and simulated results in both scenarios are very close and for PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 9 / 24 Optimizing homeostatic cell renewal in hierarchical tissues Fig 3. Comparison between the average number of mutants from 100 stochastic simulations without replacemen and the number of mutants predicted by the ODE approximation. (a) Comparing the number of mutants for a hig value of the proliferation probability, v = 0.9. (b) Comparing the number of mutants when the proliferation probabilit is small, v = 0.1. Solid lines correspond to the number of mutants in compartment Ci, i = 1, . . ., 3, predicted by the ODE approximation and dashed lines correspond to the mean number of mutants in compartment Ci from the stochastic simulations. We assume n = 3, a mutation rate of u = 10−3, and the compartment sizes are N0 = 1063, N1 = 104, N2 = 105, N3 = 106. https://doi.org/10.1371/journal.pcbi.1005967.g003 Fig 3. Comparison between the average number of mutants from 100 stochastic simulations without replacement and the number of mutants predicted by the ODE approximation. (a) Comparing the number of mutants for a high value of the proliferation probability, v = 0.9. (b) Comparing the number of mutants when the proliferation probability is small, v = 0.1. Solid lines correspond to the number of mutants in compartment Ci, i = 1, . . Results The central question we address is what values of the self-renewal probability, v, produce fewer mutants; related to this, we investigate how the length of differentiation chains affects the accu- mulation of cancer cells. If low values of v produce fewer mutants, this would imply that long chains of differentiation are favorable, as they are likely to decrease the number of cancer cells; on the other hand, if high values of v produce fewer mutants, short chains of differentiation would decrease the number of cancer cells. In addition, we investigate how tissue architecture combined with the self-renewal probabilities affects mutant production. Optimizing homeostatic cell renewal in hierarchical tissues ODE’s as a deterministic tool to study the stochastic system at hand. An alternative approach was adopted by [23] who used ODEs as the basic model and studied the stochastic effects by formulating a related Gillespie process. ODE’s as a deterministic tool to study the stochastic system at hand. An alternative approach was adopted by [23] who used ODEs as the basic model and studied the stochastic effects by formulating a related Gillespie process. De-novo mutant generation In the ODE model derived here, mutations are generated constantly at a rate u. This is a cor- rect assumption with high mutation rates and high population numbers. In smaller systems with low mutation rates, the processes of mutant (de novo) generation and their clonal spread can be treated as separate. Mutant generation becomes a rare event, and a single mutant that is generated gives rise to a clone that does not interfere with the creation of other mutant clones. This regime can also be studied with the help of the probabilities calculated above. The removal of 2n mature cells from compartment Cn gives rise to a chain of 2n cell divi- sions that replenishes the lost cells. The probability of each such chain is calculated above. Further, every chain gives rise to an expected change in the mutant numbers in each of the compartments. This information can be used to study the de-novo generation of mutants and their subsequent clonal spread. To calculate the probability that a mutant will be generated in each of n + 1 compartments, we evaluate the right hand side of system Eq (2) with mi = 0 for all 0 i n (that is, no prior mutations exist in the system). We obtain a vector proportional to the mutation rate u. Normalizing this vector to create a probability distribution, we obtain the probability to generate a mutant in each of the compartments. This vector only depends on the quantities (v1, . . ., vn−1), and not on the population sizes of individual compartments. We can study the vector of probabilities of mutant generation by setting vi = v for all 1 i n −1 and examining the dependence on v. Smaller values of v give rise to longer trees, and consequently higher probabilities of generating mutants in the stem cell compartment. Larger values of v correspond to shorter trees, such that mutants are not generated in the stem cell compartment. These trends are illustrated in Fig 4(a). ., 3, predicted by the ODE approximation and dashed lines correspond to the mean number of mutants in compartment Ci from the stochastic simulations. We assume n = 3, a mutation rate of u = 10−3, and the compartment sizes are N0 = 1063, N1 = 104, N2 = 105, N3 = 106. https://doi org/10 1371/journal pcbi 1005967 g003 https://doi.org/10.1371/journal.pcbi.1005967.g003 the most part are impossible to distinguish. The number of mutants reaches saturation, Ni, in each compartment. The larger the value of v, the longer it takes for the lower compartments to reach their equilibrium values. This is because for large self-renewal probabilities, long trees are unlikely and divisions rarely happen in the less differentiated compartments. Further details of the deterministic regime are presented in S1 Text. Note that in this paper, we use the most part are impossible to distinguish. The number of mutants reaches saturation, Ni, in each compartment. The larger the value of v, the longer it takes for the lower compartments to reach their equilibrium values. This is because for large self-renewal probabilities, long trees are unlikely and divisions rarely happen in the less differentiated compartments. Further details of the deterministic regime are presented in S1 Text. Note that in this paper, we use 10 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues Fig 4. The role of the self-renewal rate on mutant generation and mutant dynamics (the analytical approach). (a) The probability of generating a mutant in each of the compartments for 6 different values of v: v = 0, 0.05, 0.1, 0.4, 0.7, 1. (b) The expected number of mutants produced from a single mutant cell in the absence of further de-novo mutations, plotted as a function of time for three different values of v. (c,d) The expected dynamics of mutants generated in different compartments at t = 0, in the absence of new mutations, for (c) v = 0.1 and (d) v = 0.5. Other parameters are: n = 4, and the compartment sizes are, from C0 to Cn: 40, 80, 120, 160, 200. htt //d i /10 1371/j l bi 1005967 004 Fig 4. The role of the self-renewal rate on mutant generation and mutant dynamics (the analytical approach). (a) The probability of generating a mutant in each of the compartments for 6 different values of v: v = 0, 0.05, 0.1, 0.4, 0.7, 1. (b) The expected number of mutants produced from a single mutant cell in the absence of further de-novo mutations, plotted as a function of time for three different values of v. (c,d) The expected dynamics of mutants generated in different compartments at t = 0, in the absence of new mutations, for (c) v = 0.1 and (d) v = 0.5. Other parameters are: n = 4, and the compartment sizes are, from C0 to Cn: 40, 80, 120, 160, 200. Fig 4. The role of the self-renewal rate on mutant generation and mutant dynamics (the analytical approach). (a) The probability of generating a mutant in each of the compartments for 6 different values of v: v = 0, 0.05, 0.1, 0.4, 0.7, 1. (b) The expected number of mutants produced from a single mutant cell in the absence of further de-novo mutations, plotted as a function of time for three different values of v. (c,d) The expected dynamics of mutants generated in different compartments at t = 0, in the absence of new mutations, for (c) v = 0.1 and (d) v = 0.5. Other parameters are: n = 4, and the compartment sizes are, from C0 to Cn: 40, 80, 120, 160, 200. Clonal dynamics of mutants Once generated, a mutant undergoes clonal expansion and is also subject to being flushed out of the system (depending on its location). Let us consider the clonal propagation of mutants, in the absence of new mutations, which is achieved by setting u = 0 in the main system of ODEs, Eq (2). We obtain the following system: _m0 ¼ 0; _mk ¼ K ðvÞ k mk1 Nk1 mk Nk ; k 1; ð7Þ ð7Þ where the constants K ðvÞ k , which we can call the “flush-out rates”, depend on the vector (v0, . . ., vn). One can show that these rates scale with powers of (1 −v). For example, for PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 11 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues • The remainder of the cells, Q(1 −vk), will be replaced by differentiations from compartment Ck−1. There will be Q(1 −vk)/2 such differentiations, resulting in Q(1 −vk)μk−1 mutants added on average to compartment Ck. • The remainder of the cells, Q(1 −vk), will be replaced by differentiations from compartment Ck−1. There will be Q(1 −vk)/2 such differentiations, resulting in Q(1 −vk)μk−1 mutants added on average to compartment Ck. The net change in the mean number of mutants is then given by Q(1 −vk)(−μk + μk−1), which agrees with the above equations. The steady state of system Eq (7) is given by mk ¼ m0 Nk N0 : ð9Þ ð9Þ The interpretation of this result is quite straightforward: the probability of fixation of the mutant in compartment C0 is m0/N0, and the expected number of mutants in all the down- stream compartments is given by the corresponding compartment size multiplied by this fixa- tion probability. Depending on the origin of the mutation, the behavior of this system (and therefore, mutant clonal dynamics) will be different. Only in the case where the mutant is introduced at C0, is the steady state Eq (9) nontrivial, that is, mutations will persist in the system if the stem cell compartment acquires a mutation. If a mutant is introduced in any other compartment, there is transient dynamics, and then the mutation will be flushed out, since m0 = 0 in Eq (9). y q The diagonal entries of the lower-triangular matrix in the linear system Eq (7) define the time-scale of the transient dynamics. The first diagonal entry is zero and corresponds to the neutrality of the number of mutants in the stem cell compartment. This is a consequence of modeling the behavior of mutants deterministically: in a stochastic system, the probability of increasing and decreasing the number of mutants in C0 are equal to each other, leading to a symmetric Markov chain. The mean behavior however is correctly captured by the present sys- tem. The rest of the diagonal entries of the matrix in system Eq (7) have quantities Ni in the denominators (that is, larger compartments are characterized by slower mutant dynamics), and the numerators are products of powers of 1 −vk. For small values of the self-renewal prob- abilities, the mutants are flushed out quickly because of a high rate of differentiations leading to upward motion of cells through the compartments. For larger values of v, mutants are flushed out slowly so they tend to accumulate in compartments, giving rise to very long-lived mutant populated states. In the extreme case of v = 1, only compartment Cn−1 divides, and mutants accumulate in that compartment (the right hand side of Eq (7) is zero in this case, except for the equation for mn). These trends are illustrated in Fig 4(c) and 4(d), where the dynamics of mutant clones are shown depending on the compartment of mutant origin. These results are obtained from the ODEs in Eq (7). https://doi.org/10.1371/journal.pcbi.1005967.g004 compartment C1 and for constant v, the flush-out rate is given by K ðvÞ 1 ¼ 2ð1 vÞ 2n2; ð8Þ ð8Þ see Section 1 in S1 Text. Generally, for constant v, we have the following expansion of the flush-out rates in the vicinity of v = 1: see Section 1 in S1 Text. Generally, for constant v, we have the following expansion of the flush-out rates in the vicinity of v = 1: K ðvÞ i ¼ Aið1 vÞ gi þ Oð1 vÞ giþ1; 1 i n; where γi = 2n−i+1 −2 and Ai does not depend on v. where γi = 2n−i+1 −2 and Ai does not depend on v. The structure of Eq (7) can be understood intuitively. Suppose that Q cells are removed from compartment Ck. This results in three effects: The structure of Eq (7) can be understood intuitively. Suppose that Q cells are removed from compartment Ck. This results in three effects: • The expected number of mutants removed from Ck by differentiating out is Qμk. • On average, Qvk cells will be replaced by self-renewals in compartment Ck, resulting in Qvkμk mutants added on average to Ck. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 12 / 24 Optimizing homeostatic cell renewal in hierarchical tissues mutants in steady state (that is, a good chance that the mutants fixate). For larger values of v, mutants tend to accumulate (as shown by the transient increase in the number of mutants), but in the long run, the expected number of mutants is smaller (a smaller chance that there is fixation in the system). While the above examples assume a constant value of v, a more general result holds (see Section 1.2 in S1 Text): the flush-out rates K ðvÞ i are non-increasing functions of all self-renewal probabilities vi (see Fig B in S1 Text), and the probability of mutant creation in compartment C0 is a decreasing function of the values vi. In S1 Text we also explore how compartment sizes influence mutant generation and expan- sion (Section 7 in S1 Text), the effect of asymmetric divisions (Section 5 in S1 Text) and the developmental stage (Section 4 in S1 Text). Stochastic simulations and a summary of trends As v increases and the mean tree length decreases, there is a trade-off between decreasing the probability of mutant creation in the SC compartment and also decreasing the flush-out rate. In the long run, however, we observe that the latter tendency becomes less important, and larger values of v result in a smaller overall number of mutants. This can be seen again in Fig 4(b). The same tendency is observed in stochastic simulations of Fig 5(c), where blue lines (v = 0.9) are below green lines (v = 0.1) for large times. The typical time-scale before the equilibrium is reached scales as 2−γ with γ = 2n −2. 2. Self-renewal probability and mutations in stem cell compartment. Larger values of v are again advantageous from the viewpoint of minimizing the number of mutants in the stem cell compartment. With a shorter division tree length, divisions in the stem cell compart- ment happen less often leading to a lower number of mutations. This can be seen for exam- ple in Fig 3 by comparing the black lines (the number of mutants in compartment C0) and in Fig 4(a); see also Fig 5, where for the time scale of the simulations, no mutants have appeared in C0 in (a) for v = 0.9, and there is a nonzero expected number of mutants in C0 in (b) for v = 0.1 (depicted by black lines). 2. Self-renewal probability and mutations in stem cell compartment. Larger values of v are again advantageous from the viewpoint of minimizing the number of mutants in the stem cell compartment. With a shorter division tree length, divisions in the stem cell compart- ment happen less often leading to a lower number of mutations. This can be seen for exam- ple in Fig 3 by comparing the black lines (the number of mutants in compartment C0) and in Fig 4(a); see also Fig 5, where for the time scale of the simulations, no mutants have appeared in C0 in (a) for v = 0.9, and there is a nonzero expected number of mutants in C0 in (b) for v = 0.1 (depicted by black lines). 3. Compartment sizes and accumulation of mutants. The role of self-renewal in mutant generation and dynamics As the value of v increases, there is a trade-off between two different trends associated with the probability of self-renewal: the shortening of the division trees on the one hand, and a decrease in the mutant flush-out rate. More precisely, small values of v imply longer trees and a higher probability of generating mutants in the stem cell compartment. When the mutants arise how- ever, they will be flushed out quickly. On the contrary, large values of v correspond to shorter trees, such that mutants are not likely to be generated in the stem cell compartment. Once gen- erated, however, the mutants accumulate and persist for longer. These trends are illustrated in Fig 4(b). There, we plot the expected dynamics of mutants, where the initial condition reflects the probabilities of producing a mutant in the different compartments, Fig 4(a). We assume that the initial condition is a probability vector from Fig 4(a), no further mutants are generated, and clonal dynamics are according to Eq (7). We observe that for small values of v, mutant accumulation is initially the least, but for larger times, there is a significant expected number of PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 13 / 24 Stochastic simulations and a summary of trends In this study, the number of mutants produced by a stem cell lineage system is influenced by different factors. The two factors that we focused our investigation on are the probability of self-renewal in the compartments, v, and the size of different compartments. Recall that low probability of self-renewal translates into activation of many upstream cell compartments, which is equivalent to having longer division trees, while high probability of self-renewal translates into activation of few upstream cell compartments, which is equivalent to having shorter division trees. Here we summarize our findings and further illustrate them with sto- chastic simulations, Fig 5. In this figure, we present the mean dynamics of mutations in differ- ent compartments, corresponding to two different values of v and two different architectures, increasing and constant. As shown below, the behavior of the stochastic system is consistent with our predictions based on the ODEs, which were described above. 1. Self-renewal probability and accumulation of mutants. In the short term, lower probabil- ities of cell self-renewal (lower values of v) tend to be advantageous, as they result in a lower overall number of mutants; for larger values of v mutants tend to accumulate quickly, which temporarily increases the number of mutants beyond what is observed in low-v sys- tems, see Figs 5(c) and 4(b). As v increases and the mean tree length decreases, there is a trade-off between decreasing the probability of mutant creation in the SC compartment and also decreasing the flush-out rate. In the long run, however, we observe that the latter tendency becomes less important, and larger values of v result in a smaller overall number of mutants. This can be seen again in Fig 4(b). The same tendency is observed in stochastic simulations of Fig 5(c), where blue lines (v = 0.9) are below green lines (v = 0.1) for large times. The typical time-scale before the equilibrium is reached scales as 2−γ with γ = 2n −2. 1. Self-renewal probability and accumulation of mutants. In the short term, lower probabil- ities of cell self-renewal (lower values of v) tend to be advantageous, as they result in a lower overall number of mutants; for larger values of v mutants tend to accumulate quickly, which temporarily increases the number of mutants beyond what is observed in low-v sys- tems, see Figs 5(c) and 4(b). PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Stochastic simulations and a summary of trends When the value of v is fixed, constant architectures (where all compartment sizes are equal, marked “constant” in the figures) will ultimately produce fewer mutants than increasing architectures; see Fig 5(c) here in and Fig J(b) in S1 Text. However, as shown in Fig J(a) in S1 Text, increasing architectures (when compartment size increases in the downstream direction) will appear to perform better 3. Compartment sizes and accumulation of mutants. When the value of v is fixed, constant architectures (where all compartment sizes are equal, marked “constant” in the figures) will ultimately produce fewer mutants than increasing architectures; see Fig 5(c) here in and Fig J(b) in S1 Text. However, as shown in Fig J(a) in S1 Text, increasing architectures (when compartment size increases in the downstream direction) will appear to perform better 3. Compartment sizes and accumulation of mutants. When the value of v is fixed, constant architectures (where all compartment sizes are equal, marked “constant” in the figures) will ultimately produce fewer mutants than increasing architectures; see Fig 5(c) here in and Fig J(b) in S1 Text. However, as shown in Fig J(a) in S1 Text, increasing architectures (when compartment size increases in the downstream direction) will appear to perform better PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 14 / 24 Optimizing homeostatic cell renewal in hierarchical tissues than constant architectures for a short initial period with large v values. Thus, while con- Fig 5. Mean number of mutants from 1000 stochastic simulations. We compare two arrangements of the compartment sizes for n = 3: constant from C0 to C3 (N0 = 65, N1 = 65, N2 = 65, N3 = 65) and increasing from C0 to C3 (N0 = 20, N1 = 40, N2 = 80, N3 = 120). (a) The mean number of mutants produced in compartments C1, C2 and C3 for v = 0.9. Note that no mutants were produced in compartment C0 over the time scale shown. (b) The mean number of mutants produced in compartments C0, C1, C2 and C3 for v = 0.1. (c) The mean of the total number of mutants comparing both architectures for v = 0.9 (blue line) and v = 0.1 (green line). For all panels, solid lines correspond to constant architecture and dashed lines to increasing architecture. In these simulations u = 0.001. https://doi.org/10.1371/journal.pcbi.1005967.g005 Fig 5. Mean number of mutants from 1000 stochastic simulations. Generation of two-hit mutants Stochastic simulations developed here were also used to investigate the dynamics of two-hit mutant generation. In this setting, one-hit mutants were allowed to undergo a secondary mutation process, and the simulations were stopped as soon as the first two-hit mutant was generated. This situation corresponds to the scenario where two-hit mutants are advantageous and do not obey the same homeostatic control as the wild type cells or one-hit mutants. Fig 6 shows the histograms of times when the first two-hit mutant was generated under different parameters. It presents a comparison between different tissue architectures (panels (a) and (b)) and different self-renewal probabilities (panels (c) and (d)) s. In particular, we notice that the largest difference in two-hit mutant generation times is experienced when we change v; see Fig 6(c) and 6(d). For both architecture types, low values of v lead to longer two-hit mutant generation times. Therefore, we can say that from the viewpoint of two-hit mutant generation, low values of v are advantageous. This can again be explained by thinking about flush-out rates. Larger values of v lead to long-lived (although transient) mutant accumulation in tissue compartments, which in turn results in a higher chance of two-hit mutant generation. As a consequence, having lower self-renewal rates and longer differentiation trees results in slower two-hit mutant generation. It has been observed that small differences in fitness can be acted upon by selection [34, 35]. The differences in tissue architectures result in smaller differences in two-hit mutant genera- tion time, but the trends we observe coincide with those seen in Fig J in S1 Text: for small val- ues of v, increasing architecture leads to a faster production of two-hit mutants (because it results in the fastest accumulation of one-hit mutants), and for larger values of v, increasing architecture corresponds to the largest delay of two-hit mutant generation. This effect becomes even stronger if asymmetric divisions are included in the model, see Fig I in S1 Text. Fig 7 further explores the differences between increasing and constant architecture by assuming that the self-renewal probability is defined by the compartment sizes. We observe that the constant architecture tends to delay the production of two-hit mutants compared to an increasing architecture. This result follows from the fact that lower values of v slow down two-hit mutant generation, as demonstrated above. Optimizing homeostatic cell renewal in hierarchical tissues small compartments is favored. In this case, increasing architecture may give rise to fewer mutants, see Fig J(b) in S1 Text. 4. Compartment sizes and mutations in stem cell compartment. Increasing architecture always minimizes the number of mutants in the stem cell compartment. This follows from the ODE description, see for example, the equation for the number of mutants in compart- ment C0, Eq (6). The same trend is observed in the stochastic dynamics, see the black lines in Fig 5(b), where the dashed line corresponding to the increasing architecture shows fewer mutants in C0 compared to the solid line for constant architecture (please note that in this figure, in the long run, the number of mutants in each compartment in the constant archi- tecture is very similar, such that all the solid lines are superimposed). 4. Compartment sizes and mutations in stem cell compartment. Increasing architecture always minimizes the number of mutants in the stem cell compartment. This follows from the ODE description, see for example, the equation for the number of mutants in compart- ment C0, Eq (6). The same trend is observed in the stochastic dynamics, see the black lines in Fig 5(b), where the dashed line corresponding to the increasing architecture shows fewer mutants in C0 compared to the solid line for constant architecture (please note that in this figure, in the long run, the number of mutants in each compartment in the constant archi- tecture is very similar, such that all the solid lines are superimposed). Finally we note that a model that includes asymmetric divisions is presented in Section 5 in S1 Text. Generation of two-hit mutants Since the constant architecture results in lower values of v, it results in the longest two-hit mutant generation times. Stochastic simulations and a summary of trends We compare two arrangements of the compartment sizes for n = 3: constant from C0 to C3 (N0 = 65, N1 = 65, N2 = 65, N3 = 65) and increasing from C0 to C3 (N0 = 20, N1 = 40, N2 = 80, N3 = 120). (a) The mean number of mutants produced in compartments C1, C2 and C3 for v = 0.9. Note that no mutants were produced in compartment C0 over the time scale shown. (b) The mean number of mutants produced in compartments C0, C1, C2 and C3 for v = 0.1. (c) The mean of the total number of mutants comparing both architectures for v = 0.9 (blue line) and v = 0.1 (green line). For all panels, solid lines correspond to constant architecture and dashed lines to increasing architecture. In these simulations u = 0.001. https://doi.org/10.1371/journal.pcbi.1005967.g005 Fig 5. Mean number of mutants from 1000 stochastic simulations. We compare two arrangements of the compartment sizes for n = 3: constant from C0 to C3 (N0 = 65, N1 = 65, N2 = 65, N3 = 65) and increasing from C0 to C3 (N0 = 20, N1 = 40, N2 = 80, N3 = 120). (a) The mean number of mutants produced in compartments C1, C2 and C3 for v = 0.9. Note that no mutants were produced in compartment C0 over the time scale shown. (b) The mean number of mutants produced in compartments C0, C1, C2 and C3 for v = 0.1. (c) The mean of the total number of mutants comparing both architectures for v = 0.9 (blue line) and v = 0.1 (green line). For all panels, solid lines correspond to constant architecture and dashed lines to increasing architecture. In these simulations u = 0.001. https://doi.org/10.1371/journal.pcbi.1005967.g005 https://doi.org/10.1371/journal.pcbi.1005967.g005 https://doi.org/10.1371/journal.pcbi.1005967.g005 than constant architectures for a short initial period with large v values. Thus, while con- stant architectures are more beneficial in the long-term, increasing architectures are benefi- cial in the short-term when v is large. Another scenario where increasing compartment sizes are advantageous arises if we assume that the self-renewal probabilities in compart- ments are correlated with compartment sizes, such that differentiation from large to PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 15 / 24 Discussion (a) The time to observe a second mutant for both architectures and a high value of the self-renewal probability, v = 0.9. The mean time for constant and increasing architectures is 3.33 and 3.39 respectively; the p-value obtained by two-tailed t-test is p = 0.078, indicating that the means are different only at the 10% level (size effect is 0.04). (b) The time to observe a second mutant for both architectures and a small value of the self-renewal probability, v = 0.1. The mean time for constant and increasing architectures is 3.67 and 3.60 respectively; the p-value is p 0.001, indicating that the means are different; the effect size is 0.16. (c) and (d) The time to observe a second mutant for a constant and increasing architecture, respectively, for small and high v. In these simulations u = 0.001, and the effect size is 0.68 and 0.46 respectively. https://doi org/10 1371/journal pcbi 1005967 g006 Fig 6. Distribution of the generation times to second mutation from 5000 stochastic simulations. We consider two Fig 6. Distribution of the generation times to second mutation from 5000 stochastic simulations. We consider two arrangements of the compartment sizes for n = 3: constant from C0 to C3 (N0 = 65, N1 = 65, N2 = 65, N3 = 65) and increasing from C0 to C3 (N0 = 20, N1 = 40, N2 = 80, N3 = 120). (a) The time to observe a second mutant for both architectures and a high value of the self-renewal probability, v = 0.9. The mean time for constant and increasing architectures is 3.33 and 3.39 respectively; the p-value obtained by two-tailed t-test is p = 0.078, indicating that the means are different only at the 10% level (size effect is 0.04). (b) The time to observe a second mutant for both architectures and a small value of the self-renewal probability, v = 0.1. The mean time for constant and increasing architectures is 3.67 and 3.60 respectively; the p-value is p 0.001, indicating that the means are different; the effect size is 0.16. (c) and (d) The time to observe a second mutant for a constant and increasing architecture, respectively, for small and high v. In these simulations u = 0.001, and the effect size is 0.68 and 0.46 respectively. https://doi.org/10.1371/journal.pcbi.1005967.g006 https://doi.org/10.1371/journal.pcbi.1005967.g006 viewpoint of minimizing mutations? Discussion In this paper we considered the dynamics of mutant accumulation in hierarchical tissues under homeostatic turnover. What tissue architecture can be considered optimal from the 16 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues Fig 6. Distribution of the generation times to second mutation from 5000 stochastic simulations. We consider two arrangements of the compartment sizes for n = 3: constant from C0 to C3 (N0 = 65, N1 = 65, N2 = 65, N3 = 65) and increasing from C0 to C3 (N0 = 20, N1 = 40, N2 = 80, N3 = 120). (a) The time to observe a second mutant for both architectures and a high value of the self-renewal probability, v = 0.9. The mean time for constant and increasing architectures is 3.33 and 3.39 respectively; the p-value obtained by two-tailed t-test is p = 0.078, indicating that the means are different only at the 10% level (size effect is 0.04). (b) The time to observe a second mutant for both architectures and a small value of the self-renewal probability, v = 0.1. The mean time for constant and increasing architectures is 3.67 and 3.60 respectively; the p-value is p 0.001, indicating that the means are different; the effect size is 0.16. (c) and (d) The time to observe a second mutant for a constant and increasing architecture, respectively, for small and high v. In these simulations u = 0.001, and the effect size is 0.68 and 0.46 respectively. https://doi.org/10.1371/journal.pcbi.1005967.g006 Fig 6. Distribution of the generation times to second mutation from 5000 stochastic simulations. We consider two arrangements of the compartment sizes for n = 3: constant from C0 to C3 (N0 = 65, N1 = 65, N2 = 65, N3 = 65) and increasing from C0 to C3 (N0 = 20, N1 = 40, N2 = 80, N3 = 120). (a) The time to observe a second mutant for both Fig 6. Distribution of the generation times to second mutation from 5000 stochastic simulations. We consider two arrangements of the compartment sizes for n = 3: constant from C0 to C3 (N0 = 65, N1 = 65, N2 = 65, N3 = 65) and increasing from C0 to C3 (N0 = 20, N1 = 40, N2 = 80, N3 = 120). Discussion We have focused our attention on two aspects of tissue architecture: (1) probability of self-renewals/differentiations of cells in compartments; and (2) compartment size. The first aspect is the probability of cells in each compartment to proliferate. A self-renewal division is one way of replenishing cells that have been removed (by differentiation) from the compartment; the other way is to replace “missing” cells by a differentiation division from an PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 17 / 24 Optimizing homeostatic cell renewal in hierarchical tissues Fig 7. Distribution of the generation times to second mutation, obtained from stochastic simulations, in the case where v depends on the compartment sizes. We perform 5000 stochastic simulations and compare two arrangements of the compartment sizes: (a) constant compartment size (red bars) and increasing (blue bars); (b) decreasing (red bars) and increasing (blue bars). Compartment sizes are as in Fig 6. The self-renewal probabilities are v1 = 1, v3 = 0, vi ¼ Ni NiþNi1, i = 1, 2. We assume n = 3 and u = 0.001. In part (a), the mean time to a two-hit mutant for constant and increasing architecture is 3.65 and 3.59 respectively; the p value obtained by a two-tailed t-test is p 0.001, the effect size is 0.12. In part (b), the mean time to a two-hit mutant for decreasing architecture is 3.73; p 0.001; the effect size is 0.30. https://doi.org/10.1371/journal.pcbi.1005967.g007 Fig 7. Distribution of the generation times to second mutation, obtained from stochastic simulations, in the case whe Fig 7. Distribution of the generation times to second mutation, obtained from stochastic simulations, in the case where v depends on the compartment sizes. We perform 5000 stochastic simulations and compare two arrangements of the compartment sizes: (a) constant compartment size (red bars) and increasing (blue bars); (b) decreasing (red bars) and increasing (blue bars). Compartment sizes are as in Fig 6. The self-renewal probabilities are v1 = 1, v3 = 0, vi ¼ Ni NiþNi1, i = 1, 2. We assume n = 3 and u = 0.001. In part (a), the mean time to a two-hit mutant for constant and increasing architecture is 3.65 and 3.59 respectively; the p value obtained by a two-tailed t-test is p 0.001, the effect size is 0.12. In part (b), the mean time to a two-hit mutant for decreasing architecture is 3.73; p 0.001; the effect size is 0.30. Optimizing homeostatic cell renewal in hierarchical tissues concentrated near the stem cell compartment. This finding coincides with in vivo measure- ments performed in [36, 37], where divisions were more often observed near the bottom of the hierarchical compartments. In Section 7 in S1 Text, we compare the cell division data pre- sented in [37] to the expected number of divisions generated by our model. The model output is only consistent with the data under the assumption of relatively low values of the self- renewal probability v, supporting our prediction that low values of v should be selected for. Other, indirect pieces of evidence pointing toward the protective potential of cell division regulation come from studies that investigate the connection between loss of differentiation and cancer. Our result that small values of the self-renewal probability v lead to double-hit mutant suppression is consistent with the findings of [38], who reported that overexpression of protein kinase CβII induced colonic hyperproliferation, and thus, increased the risk of colon carcinogenesis. This behavior has also been observed in other types of cancers, where a disrup- tion in differentiation patterns might increase the risk of cancer [33, 39–42]. Tenen [43] explained that the loss of differentiation is an important component of many cancers, specifi- cally, haematopoietic transcription factors are crucial for differentiation to particular lineages, and their disruption is critical in acute myeloid leukemia (AML) development. Researchers have also found that mutations related to brain cancer and leukemia cause the production of an enzyme that can reconfigure on–off switches across the genome and stop cells from differ- entiating [44–46]. To test model predictions about the accumulation of mutations more directly, we propose measuring the natural flush-out rate of cells in a hierarchical tissue, such as the colonic crypt. One approach to this is to use an experimental animal model and label a single cell (in the stem or other compartment). The cell’s progeny should then be tracked in the hierarchy through time using prospective lineage tracing. While often this is done at a single time point, observations at multiple time points would allow for a comparison with the clonal expansion of mutant (“labeled”) cells predicted by the model. Discussion https://doi.org/10.1371/journal.pcbi.1005967.g007 https://doi.org/10.1371/journal.pcbi.1005967.g007 upstream compartment. Therefore, by changing the probability of self-renewal v (as opposed to differentiation) we change the probability that an upstream compartment will be engaged, thus changing the length of a typical hierarchical division tree. We have found that increasing v results in two clear tends: (i) the trees get shorter and it becomes unlikely that mutations are acquired in the more primitive compartments, and (ii) mutations that are generated are flushed out more slowly from the non-stem cell compartments, and tend to accumulate (at least, transiently). This interesting trade-off results in different “optimal” solutions, depending on the objective of optimization. If the goal is to minimize the total number of one-hit mutants residing long-term in all compartments, relatively large values of v are desirable. If on the other hand we want to protect the stem cell compartment, then low values of v are superior. Finally, from the point of view of two-hit mutant generation, again lower values of v are advantageous. Although the optimization problem solved in this paper is formulated differently from the work presented by De´renyi et al. in [27], there is an interesting point of convergence between our results and the results of [27]. The optimal architecture for delaying 2nd mutations corre- sponds to low values of v. Therefore, it is characterized by the longest possible active trees. These trees look like binary trees, which are (nearly) the perfect solution found by [27]. y , ( y) p y [ ] Maximally delaying the generation of two-hit mutants is important in the context of tumor suppressor gene inactivation, which is a common event in carcinogenesis. In tumor suppressor genes, such as the APC gene that is inactivated in a large fraction of colorectal cancers, or Rb gene responsible for retinoblastoma, both copies must be inactivated for the resulting cell to acquire a phenotypic change that eventually may lead to cancer. Tumor suppressor gene inac- tivation is often considered an early event in cancer progression, and therefore a delay in this event is of crucial importance for evolution. Our analysis indicates that in order to delay tumor suppression gene inactivation in a hierarchical tissue, mitotic activities must be PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 18 / 24 Optimizing homeostatic cell renewal in hierarchical tissues models or metapopulation models on the other, see also [54, 55] for discussion of these issues. Mutant spread is greatly restricted in one-dimensional geometry, whereas there are different ways to spread in higher dimensions or in metapopulations. The second aspect of architecture investigated here is the arrangement of compartment sizes. It is physiologically determined that the top compartment, containing the most mature cells performing their function in the organ, must be the largest. Under this restriction, there are a great variety of ways in which sizes of consecutive compartments can be arranged. In this paper we show that the size of the upstream compartments matter when it comes to delaying mutant production in a hierarchical tissue. We found that the smaller the differences in the rel- ative compartment size, the longer it takes on average to generate a double-hit mutant. This suggests the existence of evolutionary pressure to equalize compartment sizes to facilitate the flushing out of mutant cells. Next, we would like to interpret our model and its implications in the broader context of tissue architecture and the development of multicellular organisms. We can view tissue as the “functional unit” consisting of the physiologically necessary, fully mature cells, and a “support unit” consisting of less differentiated cells that do not perform the same function but are there to support and replenish the cells from the functional unit. The question then becomes, how to best organize the support unit that maintains a certain number of “functional” cells, see also [5, 56]. As one extreme scenario, one can envisage a great number of very short division trees where more or less each mature cell is replenished by divisions of its own stem cell. At the other extreme, it could happen that all the mature cells are replenished by means of an enor- mously long division tree, and are all part of the same large lineage stemming from a common stem cell. Our model is capable of distinguishing between these two extreme possibilities (and the whole range of intermediate possibilities). If 2n cells are removed, the longest division tree has length n and the shortest has length 1. If v = 0, then the longest tree is always utilized. If v = 1, then only the shortest trees are activated, and only cells in one upstream compartment (compartment Cn−1) divide. While experimentally intensive, there are a variety of genetic techniques for prospective cell lineage tracing in model organisms that could be used to generate such data (see [29] for an example in the mouse intestinal crypt and [47] for a recent review). We should also discuss this result in the context of the previous theoretical work by [7] and [48]. In both of these papers, variants of a linear model of the colon were investigated, where individual cells were arranged in a linear array and where dividing cells pushed out their neighbors upstream [49]. Interestingly, while both papers found that divisions near the stem cell compartment (the “bottom-high” division distribution) resulted in lowering the risk of secondary mutations experienced by existing one-hit mutants, the overall two-hit mutant gen- eration was found by [48] to be accelerated in this case (and the “top-high” division distribu- tion, where most divisions happen near the top, was shown to lead to a delay in two-hit mutant production). This pattern is different from the result reported in the present model, and the difference stems from the modeling assumptions: the linear model was adopted by [7, 48], whereas a model with separate compartments that experience a certain degree of mixing was considered here (similar in spirit to metapopulation models that are often used in popula- tion biology, see e.g. [50–53]). In the present model, the linear arrangement is not 1-cell thick, but instead, there is a possibility that mutants can accumulate, or “get stuck”, in a compart- ment that has a size greater than one cell. This is in contrast to the linear model, where such a possibility does not exist, and each mutant will inevitably be pushed toward death at the top of the crypt in a regular fashion. The tendency of mutants to accumulate in the presence of a large self-renewal probability discovered in the present setting, points toward a possibility that is ignored in the linear models. In a sense, this is similar to the differences between one-dimen- sional spatial population models on the one hand, and two-/three-dimensional population PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 19 / 24 Acknowledgments CA and NLK would like to thank the 2015 q-bio Summer school (Albuquerque, NM), which provided an opportunity for collaborations, and where this project was started. CA and HJW thank the Wearing Lab for their helpful comments and suggestions while working on this manuscript. Supporting information S1 Text. A document containing additional calculations, numerical simulations, and fig- ures, that further illustrate points made in the main text. S1 File. A Mathematica file that allows to generate, for a given value of N, a list of all divi- sion trees, to calculate their probabilities, and evaluate the expected change in the number of mutants resulting from cell divisions that proceed according to each tree. (NB) Optimizing homeostatic cell renewal in hierarchical tissues to each compartment, which would definitely have an impact on the type of chains of differen- tiation observed and therefore, on mutant development. It has been shown that a finite, and relatively small, replication capacity (between 50 and 70 divisions [57]) is a mechanism of pro- tection against cancer in hierarchically organized tissues [32]. Finally, we note that the present model takes the metapopulation approach to spatial rela- tionships among cells. It can be refined by adopting a fully spatial, two- or three-dimensional description, for example, a cellular automaton or a hybrid model with a degree of cell migra- tion included. It will be interesting to see how the properties of this model change under such a refinement. The main finding of the present model is that lowering the probability of self- renewal in each compartment and compensating by differentiations in longer division trees leads to a delay in two-hit mutant generation. We predict that this result should continue to hold in the fully spatial case. If this was the evolutionarily preferable scenario, this would mean that compartment Cn−1 is essentially the SC compartment, and that only one division step sep- arates SCs from fully differentiated cells. If an intermediate value of v was selected, then the size of stem cell lineage would be defined by the length of a typical division tree corresponding to this value of v, and the upstream compartments that hardly ever divide would eventually be eliminated. Our results point towards the evolutionary utility of lengthening of stem cell line- ages, because decreasing v leads to an increase in the two-hit mutant generation time. This process of lengthening of stem cell lineages however has to be limited by other factors, such as geometric constraints and the necessity of spatial distribution of lineages (for example colon crypts). Incidentally, our result whereby lengthening of the SC lineages is preferred, is again differ- ent from the previous one obtained in [56], for the same reason that was highlighted above: the present model allows for mixing in the compartments, whereas the older model of [56] is rigid in the same way as the linear model of [7] (although it is not a linear model but rather a sequence of deterministic divisions on a binary tree, where randomness only comes about when creating de-novo mutations, see [17]). The hierarchical model considered here, although less rigid in terms of division patterns compared with the linear model of [7] and the binary model of [56], is obviously still a simpli- fication of reality. There are many extensions that can be introduced into this model. Somatic mutations are not necessarily neutral, they might be disadvantageous, or advantageous. In this context, a fitness advantage or disadvantage might be assigned to mutants to study how it impacts the mutant population. Another important feature that could be added to our model is a replication capacity. This could be done by assigning a maximum number of divisions PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 20 / 24 References 1. Komarova NL. Principles of regulation of self-renewing cell lineages. PLoS ONE. 2013; 8(9):1–12. https://doi.org/10.1371/journal.pone.0072847 2. Shahriyari L, Komarova N. Symmetric vs. asymmetric stem cell divisions: an adaptation against can- cer? PLoS One. 2013; 8(10):1–16. https://doi.org/10.1371/journal.pone.0076195 3. Nowak MA, Michor F, Iwasa Y. The linear process of somatic evolution. Proceedings of the National Academy of Sciences. 2003; 100(25):14966–14969. https://doi.org/10.1073/pnas.2535419100 4. Michor F, Iwasa Y, Nowak MA. Dynamics of cancer progression. Nature Reviews Cancer. 2004; 4:197– 205. https://doi.org/10.1038/nrc1295 5. Komarova NL. Cancer, aging and the optimal tissue design. Seminars in Cancer Biology. 2005; 15 (6):494–505. https://doi.org/10.1016/j.semcancer.2005.07.003 6. Dingli D, Traulsen A, Pacheco JM. Compartmental architecture and dynamics of hematopoiesis. PLoS One. 2007;(4):2–5. 7. Zhao R, Michor F. Patterns of proliferative activity in the colonic crypt determine crypt stability and rates of somatic evolution. PLoS Computational Biology. 2013; 9(6):1–15. https://doi.org/10.1371/journal. pcbi.1003082 8. Rodriguez-Brenes IA, Wodarz D, Komarova NL. Minimizing the risk of cancer: tissue architecture and cellular replication limits. Journal of the Royal Society Interface. 2013; 10(86):1–12. https://doi.org/10. 1098/rsif.2013.0410 9. Werner B, Dingli D, Traulsen A. A deterministic model for the occurrence and dynamics of multiple mutations in hierarchically organized tissues. Journal of the Royal Society. 2013; 10:1–9. 10. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001; 414:105–111. https://doi.org/10.1038/35102167 11. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature. 2007; 449:1003–1007. https://doi.org/ 10.1038/nature06196 12. Sangiorgi E, Capecchi M. Bmi1 is expressed in vivo in intestinal stem cells. Nature Genetics. 2010; 40 (7):915–920. https://doi.org/10.1038/ng.165 13. Wright NA. Epithelial stem cell repertoire in the gut: clues to the origin of cell lineages, proliferative units and cancer. Journal of Experimental Pathology. 2000; 181:117–143. 14. Brittan M, Wright NA. Stem cell in gastrointestinal structure and neoplastic development. Gut. 2004; 53:899–910. https://doi.org/10.1136/gut.2003.025478 15. Buescher ES, Alling DW, Gallin JI. Use of an X-linked human neutrophil marker to estimate timing of lyonization and size of the dividing stem cell pool. The Journal of Clinical Investigation. 1985; 76 (4):1581–1584. https://doi.org/10.1172/JCI112140 16. Dingli D, Pacheco JM. Allometric scaling of the active hematopoietic stem cell pool across mammals. PLoS One. 2006; 1(1):e2. https://doi.org/10.1371/journal.pone.0000002 17. Komarova NL, Wang L. Initiation of colorectal cancer: Where do the two hits hit? Cell Cycle. 2004; 3 (12):1558–1565. 18. Optimizing homeostatic cell renewal in hierarchical tissues Author Contributions Conceptualization: Natalia L. Komarova. p Data curation: Natalia L. Komarova. Formal analysis: Cesar Alvarado, Natalia L. Komarova. Funding acquisition: Helen J. Wearing, Natalia L. Komarova. Investigation: Cesar Alvarado, Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Methodology: Cesar Alvarado, Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Project administration: Natalia L. Komarova. Software: Cesar Alvarado, Helen J. Wearing, Natalia L. Komarova. Supervision: Helen J. Wearing, Natalia L. Komarova. Validation: Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Visualization: Helen J. Wearing, Natalia L. Komarova. Writing – original draft: Cesar Alvarado, Helen J. Wearing, Natalia L. Komarova. Writing – review & editing: Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Data curation: Natalia L. Komarova. Formal analysis: Cesar Alvarado, Natalia L. Komarova. Funding acquisition: Helen J. Wearing, Natalia L. Komarova. Investigation: Cesar Alvarado, Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Methodology: Cesar Alvarado, Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Project administration: Natalia L. Komarova. Formal analysis: Cesar Alvarado, Natalia L. Komarova. Investigation: Cesar Alvarado, Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Methodology: Cesar Alvarado, Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Project administration: Natalia L. Komarova. Software: Cesar Alvarado, Helen J. Wearing, Natalia L. Komarova. Software: Cesar Alvarado, Helen J. Wearing, Natalia L. Komarova. Validation: Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. Visualization: Helen J. Wearing, Natalia L. Komarova. Writing – original draft: Cesar Alvarado, Helen J. Wearing, Natalia L. Komarova. Writing – review & editing: Nicole A. Fider, Helen J. Wearing, Natalia L. Komarova. 21 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues 24. Dingli D, Traulsen A, Michor F. (A)symmetric stem cell replication and cancer. PLoS Computational Biology. 2007; 3(3):0482–0487. https://doi.org/10.1371/journal.pcbi.0030053 25. Pepper JW, Sprouffske K, Maley CC. Animal cell differentiation patterns suppress somatic evolution. PLoS Computational Biology. 2007; 3(12):2532–2545. https://doi.org/10.1371/journal.pcbi.0030250 26. Sprouffske K, Aktipis CA, Radich JP, Carroll M, Nedelcu AM, Maley CC. An evolutionary explanation for the presence of cancer nonstem cells in neoplasms. Evolutionary Applications. 2012; 6:92–101. https:// doi.org/10.1111/eva.12030 27. Dere´nyi I, Szo¨llősi GJ. Hierarchical tissue organization as a general mechanism to limit the accumula- tion of somatic mutations. Nature Communications. 2017; 8:14545. https://doi.org/10.1038/ ncomms14545 28. Lopez-Garcia C, Klein AM, Simons BD, Winton DJ. Intestinal stem cell replacement follows a pattern of neutral drift. Science. 2010; 330(6005):822–825. https://doi.org/10.1126/science.1196236 29. Snippert HJ, Van Der Flier LG, Sato T, Van Es JH, Van Den Born M, Kroon-Veenboer C, et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell. 2010; 143(1):134–144. https://doi.org/10.1016/j.cell.2010.09.016 30. Simons BD, Clevers H. Strategies for homeostatic stem cell self-renewal in adult tissues. Cell. 2011; 145(6):851–862. https://doi.org/10.1016/j.cell.2011.05.033 31. Klein AM, Simons BD. Universal patterns of stem cell fate in cycling adult tissues. Development. 2011; 138(15):3103–3111. https://doi.org/10.1242/dev.060103 32. Rodriguez-Brenes IA, Komarova NL, Wodarz D. Cancer-associated mutations in healthy individuals: assessing the risk of carcinogenesis. Cancer Research. 2014; 74(6):1661–1669. https://doi.org/10. 1158/0008-5472.CAN-13-1452 33. Merritt AJ, Potten CS, Watson AJM, Loh DY, Nakayama Ki, Nakayama K, et al. Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the inci- dence of colonic neoplasia. Journal of Cell Science. 1995; 108:2261–2271. 34. Lewontin RC, et al. The genetic basis of evolutionary change. vol. 560. Columbia University Press New York; 1974. 35. Ohta T. The nearly neutral theory of molecular evolution. Annual Review of Ecology and Systematics. 1992; 23(1):263–286. https://doi.org/10.1146/annurev.es.23.110192.001403 36. Potten CS, Kellett M, Rew D, Roberts SA. Proliferation in human gastrointestinal epithelium using bro- modeoxyuridine in vivo: data for different sites, proximity to a tumour, and polyposis coli. Gut. 1992; 33 (4):524–529. https://doi.org/10.1136/gut.33.4.524 37. Buske P, Galle J, Barker N, Aust G, Clevers H, Loeffler M. A comprehensive model of the spatio-tempo- ral stem cell and tissue organisation in the intestinal crypt. PLoS Comput Biol. 2011; 7(1):e1001045. https://doi.org/10.1371/journal.pcbi.1001045 38. Murray NR, Davidson LA, Chapkin RS, Gustafson WC, Schattenberg DG, Fields AP. References Dunn SJ, Appleton PL, Nelson SA, Na¨thke IS, Gavaghan DJ, Osborne JM. A two-dimensional model of the colonic crypt accounting for the role of the basement membrane and pericryptal fibroblast sheath. PLoS Computational Biology. 2012; 8(5):1–20. https://doi.org/10.1371/journal.pcbi.1002515 19. Buske P, Galle J, Barker N, Aust G, Clevers H, Loeffler M. A comprehensive model of the spatio-tempo- ral stem cell and tissue organisation in the intestinal crypt. PLoS Computational Biology. 2011; 7(1):1– 13. https://doi.org/10.1371/journal.pcbi.1001045 20. van der Wath RC, Gardiner BS, Burgess AW, Smith DW. Cell Organisation in the colonic crypt: a theo- retical comparison of the pedigree and niche concepts. PLoS ONE. 2013; 8(9):1–15. https://doi.org/10. 1371/journal.pone.0073204 21. Bravo R, Axelrod DE. A calibrated agent-based computer model of stochastic cell dynamics in normal human colon crypts useful for in silico experiments. Theoretical Biology and Medical Modelling. 2013; 10(66):1–24. 22. Traulsen A, Lenaerts T, Pacheco JM, Dingli D. On the dynamics of neutral mutations in a mathematical model for a homogeneous stem cell population. Journal of the Royal Society Interface. 2012; 10:1–8. https://doi.org/10.1098/rsif.2012.0810 23. Werner B, Dingli D, Lenaerts T, Pacheco JM, Traulsen A. Dynamics of mutant cells in hierarchical orga- nized tissues. PLoS Computational Biology. 2011; 7(12):1–9. https://doi.org/10.1371/journal.pcbi. 1002290 22 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Overexpression of protein kinase C bII induces colonic hyperproliferation and increased sensitivity to colon carcinogene- sis. Journal of Cell Biology. 1999; 145(4):699–711. https://doi.org/10.1083/jcb.145.4.699 39. Pabst T, Mueller BU, Harakawa Nari, Schoch Claudia Haferlach T, Behre G, Hiddeman W, et al. AML1- ETO downregulates the granulocytic differentiation factor C/EBP [alpha] in t (8; 21) myeloid leukemia. Nature Medicine. 2001; 7(4):444–451. https://doi.org/10.1038/86515 40. Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Behre G, et al. Dominant-negative muta- tions of CEBPA, encoding CCAAT / enhancer binding protein-α (C/ EBPα), in acute myeloid leukemia. Nature Genetics. 2001; 27:263–270. https://doi.org/10.1038/85820 41. Pabst T, Mueller BU. Complexity of CEBPA dysregulation in human acute myeloid leukemia. Clinical Cancer Research. 2009; 15(17):5303–5307. https://doi.org/10.1158/1078-0432.CCR-08-2941 42. Van Der Flier L, Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annual Review of Physiology. 2009; 71:241–260. https://doi.org/10.1146/annurev.physiol.010908.163145 43. Tenen DG. Disruption of differentiation in human cancer: AML shows the way. Nature Reviews Cancer. 2003; 3:89–101. https://doi.org/10.1038/nrc989 44. Koivunen P, Lee S, Duncan CG, Lopez G, Lu G, Ramkissoon S, et al. Transformation by the (R)-enan- tiomer of 2-hydroxyglutarate linked to EGLN activation. Nature. 2012; 483:484–488. https://doi.org/10. 1038/nature10898 45. Turcan S, Rohle D, Goenka A, Walsh LA, Fang F, Yilmaz E, et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature. 2012; 483:479–483. https://doi.org/10.1038/nature10866 46. Lu C, Ward PS, Kapoor GS, Rohle D, Turcan S, Abdel-wahab O, et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature. 2012; 483:474–478. https://doi.org/ 10.1038/nature10860 23 / 24 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1005967 February 15, 2018 Optimizing homeostatic cell renewal in hierarchical tissues 47. Woodworth MB, Girskis KM, Walsh CA. Building a lineage from single cells: genetic techniques for cell lineage tracking. Nature Reviews Genetics. 2017; 18(4):230–44. https://doi.org/10.1038/nrg.2016.159 48. Shahriyari L, Komarova NL, Jilkine A. The role of cell location and spatial gradients in the evolutionary dynamics of colon and intestinal crypts. Biology Direct. 2016; 11(42):1–17. 49. Michor F, Iwasa Y, Rajagopalan H, Lengauer C, Nowak MA. Linear model of colon cancer initiation. Cell Cycle. 2004; 3(3):356–360. https://doi.org/10.4161/cc.3.3.690 50. Levins R. Some demographic and genetic consequences of environmental heterogeneity for biological control. Bulletin of the Entomological Society of America. 1969; 15(3):237–240. https://doi.org/10.1093/ besa/15.3.237 51. Hanski I. Single-species metapopulation dynamics: concepts, models and observations. Biological Journal of the Linnean Society. 1991; 42(1-2):17–38. https://doi.org/10.1111/j.1095-8312.1991. tb00549.x 52. Hanski I. Metapopulation ecology. Oxford University Press; 1999. 52. Hanski I. Metapopulation ecology. Oxford University Press; 1999. 53. Gilpin M. Metapopulation dynamics: empirical and theoretical investigations. Academic Press; 2012. 54. Wodarz D, Sun Z, Lau JW, Komarova NL. Nearest-neighbor interactions, habitat fragmentation, and the persistence of host-pathogen systems. The American Naturalist. 2013; 182(3):E94–E111. https:// doi.org/10.1086/671185 55. Komarova NL, Shahriyari L, Wodarz D. Complex role of space in the crossing of fitness valleys by asex- ual populations. Journal of The Royal Society Interface. 2014; 11(95):20140014. https://doi.org/10. 1098/rsif.2014.0014 56. Komarova NL, Cheng P. Epithelial tissue architecture protects against cancer. Mathematical Biosci- ences. 2006; 200(1):90–117. https://doi.org/10.1016/j.mbs.2005.12.001 57. Huffman KE, Levene SD, Tesmer VM, Shay JW, Wright WE. Telomere shortening is proportional to the size of the G-rich telomeric 3’-overhang. The Journal of Biological Chemistry. 2000; 275(26):19719– 19722. https://doi.org/10.1074/jbc.M002843200 24 / 24
https://openalex.org/W4393193243	https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-024-18274-7	English	null	Changes in social mixing and attitudes and practices to precautionary measures in a maturing COVID-19 pandemic in six communities in Sudan: a qualitative study	BMC public health	2,024	cc-by	13,067	BMC Public Health BMC Public Health Ahmed et al. BMC Public Health (2024) 24:895 https://doi.org/10.1186/s12889-024-18274-7 Open Access Abstract Introduction With low COVID-19 vaccination coverage, non-pharmaceutical interventions were critical to mitigating the COVID-19 pandemic in Sudan. We explored changes in social contact patterns, risk perception, attitudes, and practices toward protective measures during an evolving COVID-19 outbreak in six illustrative communities in Sudan. Methods This qualitative study took place in six communities in five Sudanese states using focus group discussions with community members and non-participant structured observations in public spaces between March 2021 and April 2021. A total of 117 participants joined 24 group discussions. We used a two-stage thematic analysis. Results The perceived importance of compliance with individual preventative measures among those who believe in COVID-19 was higher than observed compliance with behaviors in most study sites. Adherence was consistently low and mainly driven by enforced movement restrictions. As restrictions were lifted, social contacts outside the household resumed pre-COVID-19 levels, and risk perception and individual and institutional adherence to protective measures diminished. We identified an environment that is socially and economically unsupportive of preventive practices, compounded by widespread rumours, misinformation, and mistrust in the government-led response. However, we identified new social habits that can contribute to reducing COVID-19 transmission. Conclusion The unfavourable social and economic environment, coupled with the low visibility of the pandemic and pandemic response, has likely modulated the influence of higher risk perception on adopting precautionary behaviours by individuals. Governments and non-governmental actors should increase the visibility of the pandemic †Omama Abdalla, Omnia Kamal Hassan, Ahmed Tom, Ahmed Eldirdiri, and Drieg Ismael, contributed equally to this work while Maysoon Dahab and Nada Abdelmagid contributed equally to this work. Correspondence: Salma A.E. Ahmed eljailanis@gmail.com Correspondence: Salma A.E. Ahmed eljailanis@gmail.com Full list of author information is available at the end of the article Changes in social mixing and attitudes and practices to precautionary measures in a maturing COVID-19 pandemic in six communities in Sudan: a qualitative study Salma A.E. Ahmed1,6* , Rahaf AbuKoura2,6 , Abd Elhameed Ahmed3,6, Omama Abdalla3,6†, Omnia Kamal Hassan3,6†, Ahmed Tom3,6†, Ahmed Eldirdiri3,6†, Drieg Ismaeil3,6†, Israa Zainalabdeen3,6, Nazik Nurelhuda4,6 , Aljaile Ahmed3,6, Afrah Abdan5, Maysoon Dahab2,6† and Nada Abdelmagid2,6† Backgroundh in successive surveys conducted since August 2020 [18]. Key barriers to the uptake of key protective behaviours in Sudan are poorly understood. However, financial and logistical barriers, high levels of stigma, denialism, and misinformation are all suspected to play a role [19, 20]. The effectiveness of future COVID-19 mitigation efforts relies on understanding and addressing these barriers to increase the acceptance and feasibility of key preventa tive measures [21]. g The Sudanese national response to COVID-19 has largely fallen short in addressing the challenges posed by suc cessive waves, as official figures for both incidence and mortality are widely recognised as significantly underes timated [1]. Literature reports suggest that only 0.2% of coronavirus infections were identified in Sudan, echoing the significant underestimation of both the prevalence and incidence of COVID-19 across Sub-Saharan Africa [2]. Soon after the first COVID-19 case in Sudan was confirmed on 13 March 2020, the government quickly declared a series of restrictions including school and uni versity closures, bans of mass gatherings, international border closures and partial curfews, culminating with a complete lockdown between April and July 2020 [3, 4]. According to Sudan’s Ministry of Health COVID-19 situation reports, the second and third waves peaked in December 2020 and April 2021 [5], respectively. How ever, the national response beyond the first wave primar ily relied on promoting individual protective measures, and no wide-scale movement restrictions were enforced. This stems from the fact that the initial imposition of movement restrictions has been widely criticised, pri marily due to their adverse effects on the economy and the disruption of essential health services, despite their role in decreasing the incidence of COVID-19 [6–10]. The lockdown has also greatly hampered access to pub lic health services, routine vaccination programs and the livelihoods of those at the margins of society [11]. To our knowledge, there are no published studies that explored social contact patterns in Sudan in general and in the targeted communities in particular. Understanding how different population groups mix and interact socially during the pandemic is a crucial indicator of how viral transmission will occur and critical in informing social distancing interventions tailored to contact patterns unique to Sudanese contexts. It will also allow a more rig orous assessment of the effectiveness of different social distancing interventions. Methods Against this background, non-pharmaceutical inter ventions and preventative measures were the cornerstone of efforts to mitigate COVID-19 in Sudan and simi lar resource-limited settings [12–14]. This is especially important considering limited COVID-19 vaccination access and uptake [15, 16]. Among non-pharmaceutical interventions, social distancing and mask-wearing have been specially promoted as an alternative to wide-scale movement restrictions [12]. However, their acceptabil ity and uptake in resource-limited settings are subject to desensitisation to infection risk [17]. For example, self-reported adherence to individual COVID-19 pre ventive measures overall among 1,371 individuals in Sudan September 2021 was 42%, with only 49% report ing avoiding physical greetings and handshakes [11]. Self-reported compliance with avoiding places of wor ship, public gatherings and staying at home was 41%, 58% and 38% respectively, with a consistent downward trend © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. (2024) 24:895 Page 2 of 15 d et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health and pandemic response, enforce and incentivise infection control measures in public areas, promote emerging preventive social habits, and actively track and address rumours and misinformation related to COVID-19 and COVID- 19 vaccines. Keywords COVID-19, Social distancing, Social mixing, Non-pharmaceutical interventions, Resource-poor, Participatory analysis, Sudan Backgroundh This study explored changes in social contact patterns relevant to COVID-19 transmis sion and the extent, barriers, and enablers of adherence to recommended protective behaviours and measures during the COVID-19 outbreak in six illustrative com munities in Sudan. We aimed to inform policymakers and non-governmental COVID-19 responders of appro priate adaptations and contextualisation of social dis tancing interventions to encourage higher uptake in the targeted communities. Study designh This is an exploratory qualitative study using a combina tion of focus group discussions (FGDs) and non-partici pant structured observations in public spaces. The design draws on the protection motivation theory [22], which assumes that an individual’s decision to adopt and com ply with preventative health behaviours is based on their motivation to protect themselves from health threats after weighing the risks and potential benefits [23]. The FGDs provide insight into how people appraise the threat of COVID-19 to their health and their beliefs about the necessity and feasibility of the recommended behaviours (coping appraisal). On the other hand, non-participant observations were used to objectively identify adher ence to protective behaviors, practices, and measures in public areas, and allow us to compare reported and Page 3 of 15 (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health observed adherence to protective measures in the study population. COVID-19 research, advocacy and awareness-raising activities across Sudan. Y-peers were community mem bers living and working in the study sites. We trained male and female Y-peers remotely to col lect qualitative data through live and pre-recorded short audio/video training sessions via WhatsApp and You Tube. The Y-peers received training on study procedures (data collection tools and informed consent process), qualitative research, and preventative strategies devel oped and delivered by co-authors NA, SAEA, and RA. Each training session was followed by a live questions & answers online session with trainers, and a WhatsApp group was used to facilitate communication and provide ongoing support. We asked Y-peers to pilot the FGD guide and observation checklists in their communities as part of the interactive training. A weekly online meeting took place to troubleshoot and share updates on data col lection and field experiences. Study setting We conducted the study in six illustrative communities in five Sudanese states, representing different settings. In Khartoum state, we chose Ombadda locality, a high- density poor urban population with pockets of long- term internally displaced populations, and Tuti Island, a close-knit agricultural community. We selected two other urban, and peri-urban cities in North Kordofan and Blue Nile states, El-Obeid and Damazin, respec tively. In addition, we included one internally displaced persons’ (IDP) community in South Darfur, Dereij IDP camp, and one rural community in the south of Gezira state (Abu Haraz). Ombadda is a high-density poor urban community in Khartoum State, where the main sources of income are low-wage jobs and micro-businesses. The education level is generally high, particularly for girls, as boys tend to leave secondary school to support their fam ilies. Tuti Island is an island in the heart of the capital city Khartoum. Its inhabitants are close-knit and historically descend from a few families and tribes, and homes tend to house extended families. The community is a low- to -middle economic one, where most household incomes coming from freelance trade and businesses. Inhabit ants typically have high educational levels. El-Obeid is an urban middle-class community and the capital of North Kordofan state. Most residents work as public servants or traders. Our study site in Damazin, the capital city of Blue Nile state, is a poor peri-urban community, where most residents work as manual laborers, small-scale farmers or traders. Abu Haraz village is a rural village on the east bank of the Blue Nile river and is located approx imately 17 km from Wad Madani, Gezira State’s capital. Its inhabitants are mainly farmers, and Abu Haraz is host to a large Sufi community and Sufist shrines. Dereij IDP camp is situated 4 km from Nyala city, the capital of South Darfur state. Its residents are long-standing IDPs, mostly poor and dependent on humanitarian aid. Data management and analysis Audio-recorded interviews were transcribed verbatim in Arabic, and then transcripts were verified by the Y-peers for accuracy. Field notes and filled checklists from observation were typed in Arabic. All transcripts and checklist notes were analysed in Arabic. We con ducted a preliminary analysis using a site-specific sum mary report prepared by each site team, summarising key themes, describing the context of data collection and identifying immediate insights. After that, the co- authors invited the observers and FGD facilitators to participate in a group discussion. We used the reports to prompt follow-up questions and use the interper sonal dynamic of group discussions to generate new insights. These FGDs with data collectors helped for mulate initial and preliminary ideas and obtain the study team’s reflections on the extent to which sup portive measures facilitated social distancing in public areas and to which people adhered to such measures. They also generated preliminary ideas for detailed analysis. The discussions were recorded and employed by the co-authors to develop preliminary findings report for quick dissemination to Y-PEER Sudan, the Ministry of Health, other COVID-19 responders and targeted communities. Y-peers organised sessions with the targeted communities to get their feedback on research findings as part of the awareness-raising campaigns. Study participants Y-peers purposively identified eligible participants for the FGDs through their existing community contacts. Partic ipants were approached by the Y-peers either face-to-face or through the phone. At each study site, four FGDs were conducted: adolescent/young women, adolescent/young men, adult women, and adult men, to achieve a balanced sample of age and gender among participants. Inclusion criteria for FGD participants included being males or females aged 18–24 years (younger women and men) or 25 − 59 years (older women and men) and living in the study community. We excluded the following: those who did not provide informed consent, those aged less than18 years or more than 59 years of age, those who lived in the same household as another participant selected for the study, persons of any age with bronchial asthma, dia betes, hypertension or have immunosuppression, and those who had COVID-19-like symptoms in the past two weeks. Those older than 59 were excluded as they are at higher risk of severe COVID-19, and we did not want to expose them to the risk of COVID-19 transmission dur ing FGDs. Each FGD session comprised 4 to 8 partici pants. Youth peers purposively identified initial eligible participants through their existing community contacts and used snowball sampling to capitalise on naturally occurring social networks in the community to construct the focus groups. This ensured we minimise exposure to COVID-19 through bringing people together into a focus group who were already in regular social contact with each other. It also ensured that groups were fairly homogenous in socioeconomic status, as neighbours, friends and family members. All those who were invited to participate joined the FGD sessions and no refusals or drop-outs occurred. Sudan’s Youth Peer Education Network (Y-PEER Sudan) was actively engaged in promoting COVID-19 prevention, through its member volunteers, or “Y-peers”, in the sites at the time of the study. The selection of com munities was mainly operational based on the accessibil ity of the community (existence of Y-peers in a specific community) and the existence of activities of awareness- raising campaigns as the research was meant to inform these interventions. The Sudan COVID-19 Research Group, in partner ship with trained volunteers from Y-PEER Sudan, a nationwide network of youth volunteers and researchers, designed and implemented the study. Since the start of the pandemic in Sudan, Y-PEER Sudan has implemented Page 4 of 15 Ahmed et al. Study participants BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Observations and FGD sessions were conducted in paral lel between March 2021 and April 2021. The places for structured observations of public spaces were purposively selected to reflect various functions, layouts, outdoor versus indoor settings and use by differ ent population subgroups. In each study site, observation locations were a public transportation journey, a place of worship, the nearest health facility or pharmacy, the local open market, and a bakery. We selected the place of wor ship, health facility or pharmacy, and bakery known to be most used by the study population or closest to the geo graphical centre of the study site, after discussion with existing community contacts. We also observed the clos est local open market, which in Sudan is most commonly one market serving a large neighborhood, or one market serving several small neighborhoods. For public trans portation observation, the Y-peers took a 15- to 30-min ute journey on any local transportation vehicle departing from the study community between 10 am and 4 pm. Before finalisation, the FGD guide and observation checklists were piloted in one focus group and two pub lic spaces by two different data collectors to check for suitability of content, translation and sequence/flow of questions and duration of the data collection. After a preliminary analysis of data for each site, we reviewed the data and revisited the need to increase the num ber of group discussions or observations to reach data saturation. Results A total of 122 participants attended 24 focus group dis cussion groups in the six study sites (El-Obeid, Tuti Island, Dereij IDP camp, Ombadda, Damazin, Gezira). The number of participants and age breakdown of FGDs is presented in Table 1. Our study aimed to explore the social interaction patterns during COVID-19, self- reported individual and communal protective behaviors adopted, and to understand the factors that affect com pliance with recommended protective behaviours and measures during the COVID-19 outbreak in Sudan. We present the findings according to six broad categories (Fig. 1). Concerns over the risk of COVID-19 seemed to vary by gender and age, with adult women seeming more con cerned while adult men seemed least concerned. “W Allah [I swear] I worry a lot, especially for our offspring who work outside the house, we are staying at home. My son once had flu; I thought this would be corona [COVID-19]. I kept going to his room to check on him at night” [older woman, Tuti Island]. Data collectionh However, during the second wave, I t i d t ll” [ O b dd ] Table 1 FGD participants across the six study sites Site Number of participants Age range of participants per group (years) Younger women Younger men Older women Older men Younger women Younger men Older women Older men El-Obeid 4 4 5 4 22–24 19–24 28–54 25–30 Tuti Island 5 4 5 4 21–24 20–22 28–45 26–49 Dereij IDP camp 6 6 6 6 19–23 19–22 23–38 28–50 Ombadda 8 4 5 6 18–26 20–24 25–53 25–30 Damazin 5 6 6 4 19–29 18–24 28–39 25–59 Gezira 5 6 4 4 19–24 18–24 29–38 25–27 Fig. 1 Main categories of study findings Age range of participants per group (years) Fig. 1 Main categories of study findings Fig. 1 Main categories of study findings “In the beginning, I was worried, like 90% worried, as the media was a big driver of that fear; currently I am not worried because the situation is normal” [younger man, Dereij IDP camp]. were extracted by NA, translated into English by SAEA and back-translated to Arabic by RA. This article adheres to the Consolidated Criteria for Reporting Qualitative Research (COREQ) reporting guidelines [26] (Supple mentary file 3). “I was very worried about the illness, because I have seen people who were sick, and the number of cases was increasing. However, during the second wave, I was not worried at all” [younger woman, Ombadda]. Data collectionh Explanatory and corroborative quotes Page 5 of 15 Page 5 of 15 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health were extracted by NA, translated into English by SAEA and back-translated to Arabic by RA. This article adheres to the Consolidated Criteria for Reporting Qualitative Research (COREQ) reporting guidelines [26] (Supple mentary file 3). “In the beginning, I was worried, like 90% worried, as the media was a big driver of that fear; currently I am not worried because the situation is normal” [younger man, Dereij IDP camp]. “I was very worried about the illness, because I have Table 1 FGD participants across the six study sites Site Number of participants Age range of participants per group (years) Younger women Younger men Older women Older men Younger women Younger men Older women Older men El-Obeid 4 4 5 4 22–24 19–24 28–54 25–30 Tuti Island 5 4 5 4 21–24 20–22 28–45 26–49 Dereij IDP camp 6 6 6 6 19–23 19–22 23–38 28–50 Ombadda 8 4 5 6 18–26 20–24 25–53 25–30 Damazin 5 6 6 4 19–29 18–24 28–39 25–59 Gezira 5 6 4 4 19–24 18–24 29–38 25–27 Fig. 1 Main categories of study findings were extracted by NA, translated into English by SAEA and back-translated to Arabic by RA. This article adheres to the Consolidated Criteria for Reporting Qualitative Research (COREQ) reporting guidelines [26] (Supple mentary file 3). Results A total of 122 participants attended 24 focus group dis h d ( l b d “In the beginning, I was worried, like 90% worried, as the media was a big driver of that fear; currently I am not worried because the situation is normal” [younger man, Dereij IDP camp]. “I was very worried about the illness, because I have seen people who were sick, and the number of cases was increasing. Data collectionh The data collectors conducted four FGD sessions with 6 to 8 participants per session at each study site. Each site coordinator telephoned the eligible participants identi fied by the volunteers. After receiving informed consent and permission for the group discussion to be recorded, the focus group discussions took place at a pre-agreed date, time, and location chosen by and convenient to participants. The FGDs happened within few days of recruitment of the participants (2 to 4 days). Data collec tors used a pretested FGD guide to facilitate the discus sion (Supplementary file 1). The FGD guide covered the following topics: [1] risk perception, [2] social contact patterns, [3] individual protective behaviors outside the home, [4] communal distancing measures, and [5] fac tors affecting compliance with preventative measures. Finally, participants were asked to recommend mea sures for encouraging and supporting people to success fully adopt and maintain protective behaviors outside the home for COVID-19 prevention. The FGD sessions were conducted in the Sudanese dialect of the Arabic language, the native language of the participants, inter viewers and authors. The FGD sessions lasted between 40 and 80 min. The non-participant structured obser vations were conducted using set time and duration at pre-defined public locations using a checklist specific to each observation site (supplementary file 2). The obser vation checklist comprised of four sections: implemen tation of COVID-19 related health and safety measures, (ii) adherence to COVID-19 prevention behaviours, (iii) description of people present in the observed location, and (iv) a description of the environment at the observa tion site. The observation periods lasted between 20 and 30 min. We considered this sample size a manageable workload for the Y-PEER volunteers during an outbreak. We conducted an in-depth analysis of the transcripts and observation checklists using a thematic analysis approach based on the framework method [24]. We used deductive and inductive approaches to derive possible themes from study questions and discussion guides while allowing new themes to emerge from the data [25]. Data for each site was independently analyzed for possible themes by the co-authors SAEA, NA, RA, IZ, NN, and A Ahmed. NA then collated the analysis in a single spread sheet and a narrative document. Co-authors reached a consensus on themes and subthemes, and categorisation was agreed upon. The themes were interpreted further and compared to the objectives of the study to gener ate conclusions. Risk perception of COVID-19 Almost all participants reported worrying about others rather than themselves. Fear was driven by cultural ten dencies to protect the family and the vulnerable, with participants mentioning concerns for the elderly, chil dren, and those with chronic illnesses. Varying levels of risk perception were reported within sites, except for Gezira, where risk perception seemed consistently low throughout the pandemic, regardless of gender and age (Fig. 2). Participants reported that risk perception decreased as the pandemic evolved and their perceived familiarity with the disease grew. However, in two sites (El-Obeid and Tuti Island), adult women expressed that worry increased once schools re-opened and government mea sures were lifted in July and August 2020 (Fig. 2). “One needs to be careful because you have an old person at home. My father, may Allah grant him health and well-being, is sick. He has diabetes and hypertension. We interact with him in a way that protects him from infection, that means you Ahmed et al. BMC Public Health (2024) 24:895 (2024) 24:895 Page 6 of 15 Ahmed et al. BMC Public Health shouldn’t be causing any harm to someone else” [older man, Tuti Island]. perception. Despite the prolonged nature of the pan demic, COVID-19 denialism was not uncommon. Fig. 2 Grouping of study sites by similarity of findings Fig. 2 Grouping of study sites by similarity of findings perception. Despite the prolonged nature of the pan demic, COVID-19 denialism was not uncommon. shouldn’t be causing any harm to someone else” [older man, Tuti Island]. “Until now, I’m not convinced of the existence of COVID-19 in Sudan, and my lifestyle hasn’t changed, like socialising and handshaking”[younger woman, Damazin]. Participants reported several factors that influenced the levels of risk perception at different stages of the pandemic. Increased COVID-19 cases and deaths reported by the Ministry of Health or observed in their local communities increased risk perception. In addi tion, the high-profile and prominent response at the beginning of the pandemic increased risk perception, for example, intensive media messaging and scaling up of isolation centres. Similarly, the fall in cases and deaths and the response’s waning have reduced risk perception. “By God, I haven’t changed a thing. Believe me, I didn’t believe a word of it. I was doubtful whether there was even a coronavirus, to begin with. in mosques– you don’t know their whereabouts” [younger man, Tuti Island]. ing support didn’t come [from abroad].” [younger woman, Damazin]. ing support didn’t come [from abroad].” [younger woman, Damazin]. Participants reported changes in some different types of social contacts during periods of severe movement restrictions. Contacts in public facilities were reduced due to enforced closures and infection prevention mea sures in banks, pharmacies, and hospitals. Bans of large gatherings during the first wave also reduced contacts in social events, especially weddings and funerals. Although these events did not wholly stop despite the ban, they were adapted to be shorter and smaller. In fact, some respondents in Damazin reported that some people took advantage of the ban on gatherings to hold more celebra tory events, thereby avoiding the higher costs of tradi tionally more extensive and longer events. Respondents also reported that contacts related to economic and edu cational activities were reduced during periods of severe restrictions when schools, markets and public services were either closed or operated at reduced hours. A few respondents also reported that informal social gatherings in the neighborhood, such as regular pre-pandemic cof fee meetups and social house visits between families and neighbours, became smaller, shorter and less frequent. In addition, there was a reduction in the frequency and number of attendees of informal neighborhood social gatherings, which before the pandemic, would have been daily or weekly gatherings between neighbours, friends or extended family members. Patterns of social interactions at different stages of the pandemic For this study, relevant social contacts were either physi cal contact or a two-way conversation with three or more words within one meter of another person but no physi cal contact. Prior to COVID-19, prevailing cultural tra ditions included weddings and funerals that lasted for days or weeks, with people expected to attend daily and spend hours or days at these events. Generally, relevant social contacts outside the household decreased during the first wave when severe movement restrictions were enforced and gradually resumed pre-COVID-19 lev els once the restrictions were lifted in July 2020. How ever, even after the first wave, women in Tuti Island and El-Obeid reported some sustained reductions in social contacts, such as staying less at social events such as weddings and funerals (Fig. 2). Also, and in line with con sistently low-risk perception, hardly any changes in social contacts occurred at any pandemic stage in rural Gezira. During movement restrictions, this rural community collectively installed measures to maintain social inter action and bypass movement restrictions, e.g., opening a new ‘Corona’ market when usual markets were closed by public orders, continued mass prayers outside closed mosques and continued gatherings in homes rather than closed public social clubs (Fig. 2). Sudanese culture is strongly collectivistic, with a very strong sense of community, mutual reliance and a sense of duty towards relatives and neighbours. An individu al’s social capital is, to a large extent, determined by the extent to which they participate in their community. In our study, participants reported that the primary influ ence for reducing social interactions outside households was government enforcement of movement restrictions rather than a personal drive to reduce contacts. How ever, a few participants found the enforced measures a welcomed opportunity to reduce social contacts without losing social capital. They felt conflicted between a sense of necessity to protect themselves from COVID-19 and a firm rejection of restrictions by people in their social circle. “No change happened. Even in Eid, people went out and visited each other and everything.” Risk perception of COVID-19 Every time I discussed it with people, I said, “Brothers, this whole thing they show on television is about sick peo ple in America, and other Arab countries, etc. Hey, these things are not happening in Sudan, do you see anyone in Sudan?” [older man, Tuti Island]. “I was afraid of severe illness Because I saw that the numbers were increasing, especially in Sudan. But during this second wave, I wasn’t afraid at all.”[Younger woman, Ombada]. Participants reported that rumours about COVID-19 continue to drive disbelief in COVID-19 and low-risk perception. Participants reported that rumours about COVID-19 continue to drive disbelief in COVID-19 and low-risk perception. “Various channels, including the media, the Ministry of Health, governmental initiatives, and the intensity of awareness-raising campaigns have dwindled. This is compounded by societal negligence. Consequently, there still exists people who deny the existence of COVID-19” [Older woman, Obaid]. “ [they say] no, no, it’s just a normal flu,’ and they pass it on to the people with them.. This is nonsense; these are just government matters.” [younger woman, Gezira]. “Come on, the society itself feels there’s a lack of credibility from the government. Because in every COVID case, there was supposed to be [external] financial support [to the government], but where did the support go? I can’t believe any official claim Furthermore, as the disease became familiar over time, the severity of the disease, seemingly less than seen in other countries, has contributed to lower risk Page 7 of 15 Page 7 of 15 Ahmed et al. BMC Public Health (2024) 24:895 (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health in mosques– you don’t know their whereabouts” [younger man, Tuti Island]. [younger woman, Gezira] Pharmacies - Three sites included an observation session in a local pharmacy. - Physical distancing between customers was rarely observed. - Wearing of face coverings and avoidance of physical contact were rare among costumers - Mask-wearing was fairly common among pharmacists. Places of worship - All six sites included observation in a local mosque. - Few worshipers wore face masks, and few brought their prayer mats. - In all mosques, worshipers did not observe physical distancing during prayers and stood in lines shoulder to shoulder. - The majority of worshipers did not abstain from physical greetings. Public Transportation - All six sites included observation during a public bus trip. - Very few passengers wore face masks, whereas none of the drivers wore face masks. - No physical greetings were observed. - Physical distancing was not observed in all sites, with some passengers sharing seats. - In two of the sites, the bus station was overcrowded Public markets - All six sites included an observation session of a local public market. - Physical distancing between customers was rarely observed. - Wearing face coverings was rare (most wearers were women who covered their face using their traditional dress ‘toub’, and a few wore medical face masks). - Avoidance of physical contact was rare among customers and vendors. - Markets were crowded. Bakeries - All six sites included an observation session of a local bakery. - There were long queues of customers for hours. - Physical distancing was not observed in all sites, and it was rare to find customers wearing a mask or using hand sanitizer. - No avoidance of physical greetings. Perceived importance and adoption of COVID-19 protective behaviors Individual protective behaviors explored in this study include avoiding physical greetings, wearing face cover ings, handwashing, sanitising, physical distancing, avoid ing contact with people with COVID-19-like symptoms and vaccination. Our findings indicate the perceived importance of compliance among those who believe in COVID-19 is higher than reported and observed com pliance with behaviors in most study sites, but especially in Tuti Island and Ombadda. Based on observation of public spaces in study areas, there were varying lev els of compliance with individual protective behaviors within sites (except Gezira, which showed hardly any compliance), but a generally low level of compliance across sites (Fig. 2). A detailed description of the adop tion of COVID-19 individual protective measures is illus trated in Table 2. Participants discussed that abstaining from physical greetings was, by far, the most challeng ing behavior to comply with, even among those worried about COVID-19. Politeness is strongly embedded in Sudanese culture, and therefore participants reported that abstaining from physical greetings presented the most significant social challenge, as well as being a habit that is difficult to break. As a result, physical greetings continued even if it meant incurring additional financial costs of using hand sanitizer after handshakes. - All six sites included an observation session of a local public market. “I cannot embarrass girls who want to greet or say to them physically do not shake hands with me because of corona. I just sanitize my hands after handshak ing” [younger woman, Ombadda]. “As our brother mentioned, women did not aban don handshake and physical greetings– everywhere! In the market or street, 24/7, mostly with hugs and kisses and the like” [older man, Ombadda]. “I cannot embarrass girls who want to greet or say to them physically do not shake hands with me because of corona. I just sanitize my hands after handshak ing” [younger woman, Ombadda]. - Physical distancing between customers was rarely observed. - Wearing face coverings was rare (most wearers were women who covered their face using their traditional dress ‘toub’, and a few wore medical face masks). “As our brother mentioned, women did not aban don handshake and physical greetings– everywhere! In the market or street, 24/7, mostly with hugs and kisses and the like” [older man, Ombadda]. - Avoidance of physical contact was rare among customers and vendors. - Markets were crowded. [younger woman, Gezira] Regardless of the stage of the pandemic, people tended to maintain the same pre-pandemic close social contacts with people they are familiar and comfortable with, such as family, friends and neighbors. Even during the lock down, most close contacts in people’s immediate social circles remained unchanged. “The people whom I interacted with the most during COVID-19 time were those in my neighborhood. We were in lockdown, but we usually sat down in our street or played football close to sunset time. We sat outside until 11 pm or midnight and so forth” [older man, Ombadda]. “I personally see it as a considerable change. We are not the kind of people who live according to a spe cific pattern of social interaction. For me, socialis ing is something that comes naturally or sponta neously. We did not have any previous experience with social reduction or isolation, and this was the first time for us to experience such measures. On a personal level, I exerted a lot of effort to get used to this idea. Although deep down I know the necessity “I mean, even in Ramadan, we used to pray in a group although it was not allowed to do so, we tended to pray with the people we could trust. You can see the person in front of you washing and sanitising his hands which means he may not get COVID-19, unlike those whom you meet Page 8 of 15 Ahmed et al. BMC Public Health (2024) 24:895 (2024) 24:895 Ahmed et al. BMC Public Health of these precautions and the severity of the illness, I find myself conflicted between these personal beliefs of the need for isolation and the societal and family customs.” [younger man, Damazin]. of these precautions and the severity of the illness, I find myself conflicted between these personal beliefs of the need for isolation and the societal and family customs.” [younger man, Damazin]. Table 2 Observed compliance with individual protective measures Adherence to COVID-19 prevention behaviors Health Facilities - Five sites included an observation session of a health centre in the neighbourhood. - Social distancing and individual protective mea sures such as mask-wearing and refraining from physical greetings were rarely implemented. - Persons wearing masks tend to remove them once they leave the health facility. - Most health professionals did not wear face masks or protective equipment or observe physi cal distancing. Restrictions on public gatherings and institutional implementation of prevention measures in communal spacesh “If we say that this was left to people themselves to implement health precautions and they failed, the authorities need to have a role. For instance, the government needs to have a prompt response in restricting gatherings or obliging people to comply with protective measures in markets, mosques, or bakeries. The government needs to have an obvious role in this” [older man, El-Obeid]. The perceived importance of communal distancing measures among respondents who believe in COVID- 19 was higher than reported and observed preventive measures in public areas. Implementing measures in public areas and bans on public gatherings were higher during the first wave and gradually stopped once severe movement restrictions were lifted. Mandates for restric tions on gatherings and implementation of prevention measures occurred mainly during the lockdown period. Outside this period, and without enforceable mandates, there were only rare examples in three communities (Tuti Generally, respondents had a low tolerance for the clo sure of mosques and bans on social events such as wed dings and funerals. However, for a minority, such bans Table 3 Observed implementation of COVID-19 communal preventive measures Implementation of COVID-19 related health and safety measures Health Facilities - There were no stations for measuring the temperature of those entering the facilities or hand sanitizers in all facilities observed. - There were no markings on the floor or seating areas to indicate physical distancing. - Waiting areas in all but one health facility (Damazin) were poorly ventilated. - There were no educational flyers and posters about COVOD-19, social distancing, or the use of face masks in all facilities but one - None of the health facilities had control over the number of visitors, and the waiting areas were crowded in all facilities observed Pharmacies - At two sites (Dereij and Gezira), there were educational posters about COVID-19 and written instructions mandating mask- wearing and physical distancing for customers. - Safe distancing was observed between pharmacists and customers. However, two sites did not have a physical barrier between customers and service providers. - Only one of the sites had a station for hand sanitizer. Places of worship “Mosques” - All mosques observed except one were closed, and they were opened 10 to 15 min before prayers and closed immediately after - All mosques were kept clean and well ventilated either by keeping the windows open during prayers or using fans. Perceived importance and adoption of COVID-19 protective behaviors - All six sites included an observation session of a local bakery. At the time of data collection, COVID-19 vaccination rollout in Sudan had begun for high-risk groups in 2 of the 5 states in which there were study sites (Khartoum and Gezira). At the time, there were no publicly-declared plans to offer the vaccine to the wider population. Par ticipants expressed various attitudes towards COVID-19 vaccination, including welcoming attidues, scepticism about benefit versus harm, and refusal of vaccination. Some respondents reported rumours circulating in their communities of the side-effects of the vaccine. - There were long queues of customers for hours. - Physical distancing was not observed in all sites, and it was rare to find customers wearing a mask or using hand sanitizer. - No avoidance of physical greetings. Page 9 of 15 Page 9 of 15 Page 9 of 15 (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 (2024) 24:895 Ahmed et al. BMC Public Health “Personally, if the [COVID-19] vaccine is [offered to me], I will get vaccinated” [older woman, El-Obeid]. Island, Ombadda and Damazin) of private pharmacies or banks implementing social distancing measures. Based on observation of communal spaces in study sites, there were hardly any educational materials about COVID- 19, hand washing or sanitisation stations, or markings on seats or the floor to facilitate social distancing in observed sites. Please see Table 3 for a detailed descrip tion of the implementation of communal prevention measures for COVID-19 in observed locations. “There were many countries that stopped the vac cine, including some European Union countries, because it causes blood clots. But if it does not cause problems, [COVID-19] vaccination can be useful” [older man, Damazin]. “During this period, we got used to Corona. We are taking our precautions. At the moment, we are refus ing to go get vaccinated” [older woman, Tuti Island]. In general, most participants saw these measures in public spaces as supportive of or incentivising compli ance with individual behaviors. However, most also felt that the onus was on the government and institutions providing services to mandate these measures and not depend on individuals’ decisions to comply. Restrictions on public gatherings and institutional implementation of prevention measures in communal spacesh - In all mosques except one (Tuti Island), there were no educational flyers and posters about COVID-19, no written instructions about physical distancing during prayers, bringing own holy book, using face masks or hand washing. - Imams included a speech on COVID-19 precautions in two of the observed mosques. - No stations for hand sanitizers, and handwashing stations did not have soap. Public Transportation - At all sites, buses were well-ventilated, and windows were open. - None of the buses had markings on seats to indicate physical distancing, and there were no educational flyers or posters about COVID-19, no written instructions about physical distancing or wearing masks in public transportation Public markets - All of the sites observed had no or few billboards about COVID-19 prevention. - There were no posters for written instructions mandating mask-wearing and physical distancing. - None of the markets observed except one (Tuti Island) had a dirty environment with safe distancing rarely observed. - There were no markings to indicate physical distancing, and handwashing and sanitizer stations were rare. - There were no physical barriers between customers and vendors. Bakeries - There were no educational posters about COVID-19 or written instruction mandating mask-wearing and social distancing. - There were no markings to indicate physical distancing, and handwashing and sanitizer stations were absent. Table 3 Observed implementation of COVID-19 communal preventive measures Implementation of COVID 19 related health and safety measures ere were no stations for measuring the temperature of those entering the facilities or hand sanitizers in all facilities observed. ere were no markings on the floor or seating areas to indicate physical distancing. - Waiting areas in all but one health facility (Damazin) were poorly ventilated. g y y - There were no educational flyers and posters about COVOD-19, social distancing, or the use of face masks in all facilities but - None of the health facilities had control over the number of visitors, and the waiting areas were crowded in all facilities obse - At two sites (Dereij and Gezira), there were educational posters about COVID-19 and written instructions mandating mask- wearing and physical distancing for customers. - Safe distancing was observed between pharmacists and customers. However, two sites did not have a physical barrier betw customers and service providers. afe distancing was observed between pharmacists and customers. However, two sites did not have a physical barrier between stomers and service providers. Barriers and facilitators of compliance with COVID-19 protective measuresh There were barriers and facilitating factors to adherence to COVID-19 protective measures outside the household (See Table 4). “My friend was harassed while using public trans portation because she was wearing a mask; people literally walked behind her and verbally harassed her” [younger woman, Gezira]. Restrictions on public gatherings and institutional implementation of prevention measures in communal spacesh l f h h d f h d - All mosques observed except one were closed, and they were opened 10 to 15 min before prayers and closed immediately - All mosques were kept clean and well ventilated either by keeping the windows open during prayers or using fans. - In all mosques except one (Tuti Island), there were no educational flyers and posters about COVID-19, no written instruction about physical distancing during prayers, bringing own holy book, using face masks or hand washing. - Imams included a speech on COVID-19 precautions in two of the observed mosques. p one of the buses had markings on seats to indicate physical distancing, and there were no educational flyers or posters about VID-19, no written instructions about physical distancing or wearing masks in public transportation - All of the sites observed had no or few billboards about COVID-19 prevention. - There were no posters for written instructions mandating mask-wearing and physical distancing. - None of the markets observed except one (Tuti Island) had a dirty environment with safe distancing rarely ob - There were no markings to indicate physical distancing, and handwashing and sanitizer stations were rare. - There were no physical barriers between customers and vendors. Bakeries - There were no educational posters about COVID-19 or written instruction mandating mask-wearing and social distancing. - There were no markings to indicate physical distancing, and handwashing and sanitizer stations were absent. - There were no educational posters about COVID-19 or written instruction mandating mask-wearing and social distancing. - There were no markings to indicate physical distancing, and handwashing and sanitizer stations were absent. Page 10 of 15 (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health were considered a more tolerable alternative to lock downs should COVID-19 cases and deaths increase. were considered a more tolerable alternative to lock downs should COVID-19 cases and deaths increase. include the absence of supportive measures in pub lic areas, e.g. larger/ventilated public transportation, handwashing/sanitizers stations, and masks-wearing mandates. Socially, a discouraging attitude towards com pliance with protective measures and the widespread stigma of COVID-19 prevailed, which created social pressure on compliers to abandon preventative measures. Socioeconomic and religious context Due to a lack of compatibility with Sudanese customs and habits, such as community participation and collec tive interdependence, politeness, humility, generosity and hospitality, compliance was challenging with pro tective measures such as social distancing and acts of social withdrawal (stopping physical greetings, and not attending social events). However, the deeply-rooted Sudanese culture of protecting the vulnerable and duty of care towards the elderly motivated household mem bers to comply with protective measures when outside the household. Participants also discussed that barriers “People would say you are arrogant, but this is not arrogance. I said to them that I am following the ministry of health guidelines. Also, one time I walked by and I said hi from a distance, they told me to come and greet them (physically), and so I told them that this illness has become common, so I won’t shake hands with them; they said to me “you are arrogant, and we no longer want to talk with you”” [older man, Ombadda]. Table 4 Barriers and facilitators of adherence to COVID-19 protective measures in six Sudanese communities Facilitators of adherence to COVID-19 protective measures Barriers to adherence to COVID-19 protective measures i. Socioeconomic context Youth seemingly more resilient/rebellious in the face of social pressures Negative impact on business revenues and household income Supportive community measures and normalisation of new/ adapted behaviors Poverty and the high cost of essential supplies Shortages in necessities such as fuel and bread (long queues) The stigma associated with COVID-19 Bullying and harassment of compliers Denialism, rumors and misinformation about COVID-19 and the vaccine ii. Cultural context Cultural tendency to protect the vulnerable and the elderly Preventive measures are seen as not compatible with Sudanese customs and habits Cultural tendency to bypass or manipulate gov ernment restrictions iv. Religious beliefs Recommendations to comply from well-regarded religious experts/institutions Conflicts with scripture or religious practices (e.g. physical distancing between worshippers during prayer in mosques is seen as unacceptable) v. Visibility of the pandemic and pandemic response Enforcement of restrictions/protective measures in pub lic areas and transportation means and penalties for non-compliers Regular bulletins and information about COVD-19 in media The waning intensity of awareness-raising activities Consistent and sustained risk communication activities (ob served local languages and dialects and context-specific) Mistrust in government information Visibility of pandemic locally (confirmed cases and deaths) vi. Visibility of the pandemic and pandemic response Visibility of the pandemic and pandemic response Increased visibility of the pandemic response locally and nationally was associated with increased risk percep tion levels and higher adherence to individual protective behaviors. Indicators of pandemic response mentioned by respondents included enforced restrictions or protec tive measures, presence of risk communication activities, compliance of local health workers with protective mea sures (mainly mask-wearing), and closure and opening of local isolation wards in their communities. Enforce ment of restrictions and protective measures in com munal areas and transportation means was mentioned as a facilitator for compliance. Some participants recom mended the enforcement of penalties for non-compliers in public areas since, for many, a penalty was a primary driver of adherence, especially for physical distancing and mask-wearing. fi The majority of the Sudanese population identify as Muslim, and people express their religious beliefs on a daily basis through regular prayers, including group prayers at mosques. According to participants in most study sites, social distancing during prayers in mosques and the closure of mosques during the pandemic were unacceptable compromises. However, due to trust in well-regarded religious scholars and institutions, social distancing during prayers can be implemented if sup ported by those entities. “I think that enforcement of restrictions support compliance with positive behaviors; also, penalties, even if small, for those who don’t comply can make people get used to these measures” [older man, Dam azin]. “Social distancing during prayer can be done, but it is honestly challenging; during prayers, it is difficult” [older man, El-Obeid]. “This (physical distancing during group prayers) needs consultations with religious experts. If a decision came from above, people would imple ment social distancing during prayer, no question” [younger man, Damazin]. “Social distancing during prayer can be done, but it is honestly challenging; during prayers, it is difficult” [older man, El-Obeid]. “This (physical distancing during group prayers) needs consultations with religious experts. If a decision came from above, people would imple ment social distancing during prayer, no question” [younger man, Damazin]. The participants confirmed that the reduced risk per ception and diminished compliance with protective measures during the third wave were due to the lack of regular news coverage about COVID-19 and the waning of awareness-raising activities about the disease– both creating an impression that COVID-19 was no longer a threat. Visibility of the pandemic and pandemic response Furthermore, increased visibility of the pandemic locally was also associated with higher levels of perceived risk, increases in risk perception levels, and acceptance of bans on public gatherings (and vice versa). Participants’ indicators of an ongoing pandemic included personal experiences and losses due to COVID-19 and the over all number and trend of cases and deaths in their local communities. Another obstacle to compliance with protective mea sures was persistent COVID-19 denialism and rumors. According to participants, many rumors and misinfor mation were circulating about COVID-19. For instance, some participants mentioned that Africans are less prone to severe COVID-19, and others said the third wave was milder than the previous two waves. Another participant mentioned that the hot climate is protective as it weakens the virus. “Most housewives are scared of using hand sani tizer. Because it contains alcohol and if she used the gas cooker…there are number of fire casualties that happened because of this” [younger woman, Damazin]. “I mean, you see a person sitting on a chair in a (telecommunications’ company) office, and when Socioeconomic and religious context Feasibility Provision of economic and in-kind support Discomfort associated with mask-wearing Fatigue due to the long duration of the pandemic vii. Perceived importance of behaviours and measures Belief in the effectiveness of prevention measures Inconvenience caused by compliance with pro tective measures in communal areas, e.g., long waiting times COVID-19 preventive behaviors and measures are also protec tive against other diseases viii. Perceived health system accessibility/capacity Concerns because of difficulty in accessing health services Recognizing the need to protect the weak health system from being overwhelmed with COVID-19 cases The waning intensity of awareness-raising activities Mistrust in government information Discomfort associated with mask-wearing Fatigue due to the long duration of the pandemic Inconvenience caused by compliance with pro tective measures in communal areas, e.g., long waiting times Page 11 of 15 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 (2024) 24:895 Ahmed et al. BMC Public Health Ahmed et al. BMC Public Health he gets up, you find written on the chair that is not allowed to sit on it” [younger man, Ombadda]. he gets up, you find written on the chair that is not allowed to sit on it” [younger man, Ombadda]. Due to the current economic situation and hyperinfla tion, the high cost of supplies and competing priorities for expenditures hinder compliance with mask-wear ing, hand sanitizers and frequent hand washing. More over, the economic costs of reducing working hours or opening hours for businesses were reported as an intol erable compromise. In addition, the limited availability of necessities, such as long queues in petrol stations and bakeries, meant people spent long hours outside the household and in crowded areas, making compli ance with social distancing measures in public areas difficult. Recommendations for communities Given the high perceived importance of protective behav iors and measures, study participants recommended that communities gear local efforts towards supporting masks wearing and using hand sanitizers/disinfectants for those most at risk of severe or fatal COVID-19. In addi tion, they emphasised the need to capitalise on emerging social habits such as shorter and smaller social events and no-contact greeting trends among the youth. “Compliance with protective measures is essential, I swear, for all diseases not only corona, but one also needs to protect oneself and prevention is better than cure” [younger woman, Ombadda]. “It will also be helpful for the health system, I mean, corona make us pay more [health expenditure]” [younger woman, El-Obeid]. Expectations of the government’s response p g p Many recommended that the government or the Min istry of Health provide regular and transparent updates about the COVID-19 situation nationally and locally to maintain a high level of public risk perception needed to increase compliance. Many participants mentioned specific expectations of the government’s role, such as enforcing physical distancing measures in public trans portation and communal areas and ending shortages in necessities to eliminate lengthy and crowded queues at bakeries and other service facilities. Given the deterio rating economic situation in most people’s lives, partici pants also recommended governmental financial support for hard-hit families to support compliance. Finally, par ticipants recommended accelerating COVID-19 vaccina tion campaigns to expedite the return to a pre-COVID state and reduce pressure on health services. “I need to know the side effects of these chemicals in the long term as I use it every 10 minutes. If we want to continue using sanitisers, they need to be envi ronmentally friendly or have no chemicals because alcohol is strong on hands” [younger woman, Tuti Island]. Perceived importance of behaviours and measures Many participants reported that they complied with pre ventive measures to protect themselves, others and loved ones who are elderly or have chronic illnesses, reflecting their belief in the effectiveness of prevention measures. Furthermore, some participants mentioned that COVID- 19 preventive measures are also protective against other diseases and that it is, therefore, efficient to comply. Other participants commented that compliance is vital to protect an already-weak health system from collapsing under the pressure of an overwhelming load of COVID- 19 cases and having to increase health expenditures to cope. Operational feasibilityh The economic costs of masks, sanitisers, and reduced working hours when self-isolating were considered intol erable compromises for many people. Many mentioned the need for financial support to comply with these pro tective measures. Due to the long pandemic duration, many expressed their concerns about compliance fatigue, discomfort associated with mask-wearing and the harms of long-term use of hand sanitizers. Other participants commented on a cultural tendency to bypass or manipulate restrictions. “I mean, you see a person sitting on a chair in a (telecommunications’ company) office, and when Page 12 of 15 Page 12 of 15 (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health “As my friend said, I do not wear a mask unless nec essary. If I am not obliged to wear it, I would never wear it” [older man, Gezira]. Expectations of the government’s response Discussion COVID-19 transmission is highly sensitive to the levels of social interaction and adherence to preventive mea sures. In the presence of low COVID-19 vaccine cover age in resource-poor settings [27], non-pharmaceutical interventions continue to be an essential pillar of the pandemic response. Our study explored changes in social contact patterns during an evolving COVID-19 pandemic in six communities in Sudan and identified barriers and enablers to adherence to selected individual and commu nal preventive measures. Understanding the evolution of the population’s interaction with the pandemic and pan demic response is essential to inform interventions by pandemic responders to encourage uptake of non-phar maceutical interventions in the study communities.i Inconvenience caused by social distancing measures, such as long queues or waiting times, was reported as discouraging compliance to many people. Some partici pants mentioned they would refrain from using facilities that strictly enforce these measures if they could. Risk communication of Washington’s Institute for Health Metrics and Evalu ation, was at its highest in December 2020, at 34% and dropped to 21% in April 2021 [28]. However, in contrast to our findings, a worldwide assessment of changes in adher ence to COVID-19 protective behaviors reported low lev els of initial adherence in similar low-income countries, followed by a later uptick in adherence [29]. Petherick et al. posit that adjustments in risk assessment by individuals may explain the behavioral changes they observed in the study. The assessment used changes in national COVID-19 case and death rates and citizens’ internet searches related to COVID-19 as proxies for variations in individuals’ risk perceptions. The assessment revealed a smaller rebound in adherence when controlling for these variations. In our study, we were able to contrast direct risk perception reports with adherence levels within each site and at differ ent pandemic stages. Therefore, the absence of a later uptick in adherence to precautionary measures may be explained by individual risk assessments remaining unchanged after a decrease from initial higher levels of worry. Pandemic responders in Sudan need to increase the visibility of the local epidemic through strengthened surveillance and regu lar updates on the number of cases and deaths. 34]. Those who believe rumors and conspiracy theories are less likely to adopt health-protective behaviors such as wearing masks or social distancing [33, 35]. Pandemic responders in Sudan should actively track rumors and misinformation related to COVID-19 and COVID-19 vaccines in their communities and use tailored and con text-specific interventions to address them. i Research has also shown that rumours and misinforma tion in other African countries tend to proliferate where there is low trust in government and mainstream media outlets [34]. There was widespread mistrust in government information about COVID-19 among our study popula tion. An international survey examining the association of government trust regarding COVID-19 control with recommended health behaviours in 23 countries revealed that trust in governments is positively associated with the adoption of recommended prevention measures [36]. Par ticipants in our study also implied that the government-led response was sub-optimal and largely invisible after the first epidemic wave, which further reduced their trust in the government. Risk communication The same survey indicated that trust in governments during the COVID-19 pandemic, and hence compliance of the public with recommended health mea sures, was associated with perceived impartiality, transpar ency in conveying official statistics to the public, clear and organised information about COVID-19 and a perceived systematised response [34, 36]. Nonetheless, our study population had expectations of government action, includ ing incentives, such as free distribution of masks and sani tizers and the enforcement of penalties for non-compliant individuals, public services and private institutions. The government should enforce incentive and penalty systems for individuals and institutions to both support those who intend to adopt preventive behaviors and increase the pan demic response’s visibility. p An individual’s appraisal of their capacity to adhere and sustain to adherence precautionary health behaviors influences their intent to adopt them [22]. We identified a lack of a supportive environment for protective behav iors, particularly once severe movement restrictions were lifted. Specifically, we identified social pressures that disincentivise adherence and extend to compliance- related bullying and harassment. Previous research has described COVID-19-related bullying directed towards COVID-19 patients [30] or healthcare workers [31]. However, evidence is scarce on the impact of bullying and harassment related to compliance with COVID-19 pre ventive measures in low-income settings. We also found that the limited affordability and availability of necessities meant that masks and sanitisers were perceived as inac cessible by many in the study population. This balancing act between the costs of, for example, masks and other necessities is experienced by populations in other African countries [32]. Despite these challenges, we identified a few instances of adaptations in social habits that are con ducive to COVID-19 prevention. Pandemic responders in Sudan should acknowledge the social and economic pressures of preventive measures and use a solution- based approach in their risk communications activities. Responders should also actively promote and normalise new protective behaviors emerging in their communities. S i l d i i t d l Risk communication To improve compliance with protective measures, par ticipants recommended that the Ministry of Health have ongoing awareness-raising campaigns using multiple and parallel channels, such as championing local role mod els, religious sermons, giant billboards in public areas, and two-way dialogue methods like home visits. They also recommended having interventions that target the elderly tailored to their needs and lifestyle with practi cal tips for implementation. Participants emphasised the need to engage and train youth for awareness-raising activities in local communities and use local languages and dialects and context-specific messages. We identified consistent trends in risk perception, social contacts and adherence to protective measures in our study population. As the pandemic progressed, restrictions were relaxed, the visibility of the pandemic response waned, social interactions increased, and risk perception and indi vidual and institutional adherence to protective measures diminished. However, reported adherence to protective measures was consistently low throughout the pandemic, and adherence seemed to be primarily driven by the brief period of enforced movement restrictions. For example, reported mask use in Sudan, according to the University Ahmed et al. BMC Public Health (2024) 24:895 Page 13 of 15 Ahmed et al. BMC Public Health of Washington’s Institute for Health Metrics and Evalu ation, was at its highest in December 2020, at 34% and dropped to 21% in April 2021 [28]. However, in contrast to our findings, a worldwide assessment of changes in adher ence to COVID-19 protective behaviors reported low lev els of initial adherence in similar low-income countries, followed by a later uptick in adherence [29]. Petherick et al. posit that adjustments in risk assessment by individuals may explain the behavioral changes they observed in the study. The assessment used changes in national COVID-19 case and death rates and citizens’ internet searches related to COVID-19 as proxies for variations in individuals’ risk perceptions. The assessment revealed a smaller rebound in adherence when controlling for these variations. In our study, we were able to contrast direct risk perception reports with adherence levels within each site and at differ ent pandemic stages. Therefore, the absence of a later uptick in adherence to precautionary measures may be explained by individual risk assessments remaining unchanged after a decrease from initial higher levels of worry. Pandemic responders in Sudan need to increase the visibility of the local epidemic through strengthened surveillance and regu lar updates on the number of cases and deaths. Data availability The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request. Author contributions SAEA and NA conceptualised the study and developed data collection tools in discussion with other co-authors. IZ and AEA coordinated and supervised data collection while OA, OKH, AT, AE and DI implemented data collection at study sites. SAEA, NA, NN, MD, IZ, and A Ahmed analysed and interpreted the data. SAEA, RA, MD and NA drafted the manuscript. All authors read, reviewed and approved the final manuscript. Funding The study was funded by the United Kingdom’s Foreign Commonwealth and Development Office (FCDO) via The British Council as part of the ‘Community- led COVID-19 Mitigation in Sudan: A Research and Advocacy Intervention’ project. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Study strengths and limitations Our study has strengths and limitations. Firstly, the tim ing of the data collection, not being during an active wave, would have influenced the current level of risk perception and implementation of prevention measures among tar geted communities. Secondly, the competing priorities in the Sudanese context and possible pandemic fatigue might have affected the interest of the community members in the study. Data collectors were part of the communities, which helped reach the targeted participants. Having com munity members collecting the data was excellent in reach ing communities and familiarity with the context. However, at the same time, that might have influenced the way they perceived information shared by the community members as being normal or taken for granted. Frequent discus sions with team members from other sites and the research group were helpful. Finally, we acknowledge that the eli gible age range for the older adult groups (25–59 years) is Social and economic pressures in our study popula tion were aggravated by persistent disbelief, rumors and misinformation about COVID-19. Research has shown that the public can easily distrust scientific evi dence and believe rumors and conspiracy theories [33, Ahmed et al. BMC Public Health (2024) 24:895 Ahmed et al. BMC Public Health (2024) 24:895 Page 14 of 15 Ahmed et al. BMC Public Health (2024) 24:895 Acknowledgements wide and cultural age-related dynamics may have affected the FGD dynamics between participants in these groups. The study has strengths that include a detailed descrip tion of the study context and methods, which can facilitate transferability to other similar contexts. Secondly, the trian gulation of data collection methods observation and FGDs and triangulation of sources of information that included women and men and the different age groups provides a comprehensive understanding of the phenomenon. We are grateful to the volunteers from Y-PEER Sudan for their substantial role in the data collection and preliminary analysis of the study. They secured access to targeted communities and provided a rich understanding of the context at study sites. We thank Ms Sian White for her thoughtful feedback on the data collection tools and study protocol. We thank Dr Reem Gaafar for formulating policy recommendations from the study findings. We are grateful to the community members who participated in this study. Conclusioni We identified generally low adherence to precautionary behaviours throughout the pandemic, driven primarily by low self-efficacy among the study population. How ever, we found variations in risk perception levels within and between study sites and at different pandemic stages. We argue that the unfavourable social and economic environment, coupled with the low visibility of the pan demic and pandemic response, have likely modulated the influence of risk perception on adopting precautionary behaviours by individuals, even in instances where risk perception was moderate or high. However, we also iden tified new social habits that can contribute to reducing COVID-19 transmission. References 1. Watson O, Abdelmagid N, Ahmed A, Ahmed Abd Elhameed A, Whittaker C, Brazeau N et al. Report 39: Characterising COVID-19 epidemic dynamics and mortality under-ascertainment in Khartoum, Sudan. School of Public Health, Imperial College London COVID-19: Imperial College London COVID-19; 2020 1-Dec-2020. Author details 1 1Independent public health researcher, Khartoum, Sudan 2London School of Hygiene and Tropical Medicine, Department of Infectious Disease Epidemiology, London, UK 3Y-PEER Sudan, Khartoum, Sudan 4University of Khartoum, Khartoum, Sudan 5The Federal Ministry of Health, Khartoum, Sudan 6Sudan COVID-19 Research Group, Khartoum, Sudan Abbreviations COREQ Consolidated Criteria for Reporting Qualitative Research COVID-19 Coronavirus disease IDP Internally displaced persons FGDs Focus group discussions Y-PEER Sudan Sudan’s Youth Peer Education Network Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12889-024-18274-7. Supplementary Material 1 Supplementary Material 2 Supplementary Material 3 Abbreviations COREQ Consolidated Criteria for Reporting Qualitative Research COVID-19 Coronavirus disease IDP Internally displaced persons FGDs Focus group discussions Y-PEER Sudan Sudan’s Youth Peer Education Network Received: 28 May 2022 / Accepted: 4 March 2024 Received: 28 May 2022 / Accepted: 4 March 2024 Supplementary Information h l l The online version contains supplementary material available at https://doi. org/10.1186/s12889-024-18274-7. Consent for publication Consent for publication Not applicable. Consent for publication Not applicable. Ethics approval and consent to participate This study was implemented in compliance with the Declaration of Helsinki. Before the data collection, ethical clearance was obtained from Sudan’s National Health Research Ethics Committee based at the Federal MoH (number 9–1–21) and the London School of Hygiene and Tropical Medicine’s Research Ethics Committee (Ref: 22794–2). Data collectors approached potential participants and explained study objectives, possible risks and benefits, and their rights before obtaining their written informed consent, including explicit permission to record the interview and publish anonymised quotes from the interview. Participation was strictly voluntary; no financial remuneration was paid to participants, although refreshments and COVID-19 prevention supplies were provided during the focus group discussions. Written informed consent was obtained from all the participants for study participation. We recommend that COVID-19 responders increase the visibility of the local and national COVID-19 pan demic through consistent, regular and high-profile public updates on the number of cases and deaths. In addition, we recommend that governments and non-governmental actors increase the visibility of the pandemic response through continuous and appropriate health education interventions and highly-publicised coverage of treatment and vaccination programs. The government should also incentivise infection control measures in public areas and health facilities. Rumors and misinformation related to COVID-19 and COVID-19 vaccines must be actively tracked and addressed through tailored interventions. Finally, we recommend interventions to actively normalise emerging healthy behaviours, such as shorter social gath erings and no-contact physical greetings. We recommend that COVID-19 responders increase the visibility of the local and national COVID-19 pan demic through consistent, regular and high-profile public updates on the number of cases and deaths. In addition, we recommend that governments and non-governmental actors increase the visibility of the pandemic response through continuous and appropriate health education interventions and highly-publicised coverage of treatment and vaccination programs. The government should also incentivise infection control measures in public areas and References Effec tiveness of isolation, testing, contact tracing, and physical distancing on reducing transmission of SARS-CoV-2 in different settings: a mathematical modelling study. Lancet Infect Dis. 2020;20(10):1151–60. 32. Aloui-Zarrouk Z, El Youssfi L, Badu K, Francis Fagbamigbe A, Matoke-Muhia D, Ngugi C et al. The wearing of face masks in African countries under the COVID-19 crisis: luxury or necessity? [version 1; peer review: 1 approved, 1 approved with reservations]. Open Res Afr. 2020;3(36). y 15. Aborode AT, Fajemisin EA, Ekwebelem OC, Tsagkaris C, Taiwo EA, Uwishema O, et al. Vaccine hesitancy in Africa: causes and strategies to the rescue. Thera peutic Adv Vaccines Immunotherapy. 2021;9:25151355211047514. 33. Rieger MO, Wang M. Trust in government actions during the COVID-19 crisis. Soc Indic Res. 2021:1–23. 33. Rieger MO, Wang M. Trust in government actions during the COVID-19 crisis. Soc Indic Res. 2021:1–23. 16. Musa MB, Osman Elmahi OK, Modber MAKA, Abuelgasim Mohammed Yagoub FE, Elkabashi Dafallah AA, Musa SS, et al. The fight against the COVID-19 pandemic: vaccination challenges in Sudan. Public Health Pract. 2021;2:100205. 34. Ogola G. Africa and the Covid-19 Information Framing Crisis. Media Com munication. 2020;8:440. 35. Karić T, Međedović J. COVID-19 conspiracy beliefs and containment-related behaviour: the role of political trust. Pers Indiv Differ. 2021;175:110697. 17. Ilesanmi OS, Bello AE, Afolabi AA. COVID-19 pandemic response fatigue in Africa: causes, consequences, and counter-measures. Pan Afr Med J. 2020;37(Suppl 1):37.f 36. Han Q, Zheng B, Cristea M, Agostini M, Belanger JJ, Gützkow B et al. Trust in government and its associations with health behaviour and prosocial behav iour during the COVID-19 pandemic. PsyArXiv Preprints. 2020. 18. Partnership for Evidence-Based. Response to COVID-19. Effective Implemen tation of Public Health and Social Measures in Sudan: Situational Analysis. 2020. References Page 15 of 15 Ahmed et al. BMC Public Health (2024) 24: Page 15 of 15 Ahmed et al. BMC Public Health Ahmed et al. BMC Public Health (2024) 24:895 (2024) 24:895 20. RT Arabic. Sudanese doctors threaten a comprehensive strike 2020 [updated 21.05.2020. Available from: https://arabic.rt.com/features/1116495- ءابطأ-نادوسلا-نوددهي-بارضإب-لماش- نع-لمعلا/.f 2. Barber RM, Sorensen RJ, Pigott DM, Bisignano C, Carter A, Amlag JO, et al. Estimating global, regional, and national daily and cumulative infec tions with SARS-CoV-2 through Nov 14, 2021: a statistical analysis. Lancet. 2022;399(10344):2351–80. 2. Barber RM, Sorensen RJ, Pigott DM, Bisignano C, Carter A, Amlag JO, et 3. Reuters Staff. UPDATE 1-Sudan closes airports and borders over coronavirus fears. Reuters. 2020. 21. Talic S, Shah S, Wild H, Gasevic D, Maharaj A, Ademi Z, et al. Effectiveness of public health measures in reducing the incidence of covid-19, SARS-CoV-2 transmission, and covid-19 mortality: systematic review and meta-analysis. BMJ. 2021;375:e068302. 4. Dabanga. Coronavirus measures: Sudan’s Council of Ministers issues direc tions. Dabanga. 2020. 22. Rogers RW, Prentice-Dunn S. Protection motivation theory. 1997 5. Health RoSFMo. Corona in Sudan https://fmoh.gov.sd2021 [Available from: https://fmoh.gov.sd. 5. Health RoSFMo. Corona in Sudan https://fmoh.gov.sd2021 [Available from: https://fmoh.gov.sd. 23. Prentice-Dunn S, Rogers RW. Protection Motivation Theory and preventive health: beyond the Health Belief Model. Health Educ Res. 1986;1(3):153–61. 6. Watson O, Abdelmagid N, Ahmed A, Ahmed Abd Elhameed A, Whittaker C, Brazeau N et al. Report 39: characterising COVID-19 epidemic dynamics and mortality under-ascertainment in Khartoum, Sudan 2020 [Available from: https://www.imperial.ac.uk/mrc-global-infectious-disease-analysis/covid-19/ report-39-sudan/. 6. Watson O, Abdelmagid N, Ahmed A, Ahmed Abd Elhameed A, Whittaker C, Brazeau N et al. Report 39: characterising COVID-19 epidemic dynamics and mortality under-ascertainment in Khartoum, Sudan 2020 [Available from: https://www.imperial.ac.uk/mrc-global-infectious-disease-analysis/covid-19/ report-39-sudan/. 24. Gale NK, Heath G, Cameron E, Rashid S, Redwood S. Using the framework method for the analysis of qualitative data in multi-disciplinary health research. BMC Med Res Methodol. 2013;13(1):1–8. 25. Fereday J, Muir-Cochrane E. Demonstrating rigor using thematic analysis: a hybrid approach of inductive and deductive coding and theme develop ment. Int J Qualitative Methods. 2006;5(1):80–92. p 7. Mukhtar MM, Khogali M. The accelerating COVID-19 epidemic in Sudan. Nat Immunol. 2021;22(7):797–8. p 7. Mukhtar MM, Khogali M. The accelerating COVID-19 epidemic in Sudan. Nat Immunol. 2021;22(7):797–8. 26. Booth A, Hannes K, Harden A, Noyes J, Harris J, Moher D, et al. Consolidated Criteria for Reporting Qualitative Studies COREQ. Guidelines for reporting health research: a user’s manual. Oxford: Wiley; 2014. References 8. Fadul N, Hussein ME, Fadul AA. Re-opening Sudan: the Balance between maintaining Daily Living and avoiding the next peak of COVID-19. Curr Trop Med Rep. 2021:1–7. 8. Fadul N, Hussein ME, Fadul AA. Re-opening Sudan: the Balance between maintaining Daily Living and avoiding the next peak of COVID-19. Curr Trop Med Rep. 2021:1–7. 27. Chen Z, Zheng W, Wu Q, Chen X, Peng C, Tian Y, et al. Global diversity of policy, coverage, and demand of COVID-19 vaccines: a descriptive study. BMC Med. 2022;20(1):130. 9. Dabanga. Coronavirus measures: Sudan’s Council of Minis ters issues directions 2020 [updated 15.03.2021. Available from: https://www.dabangasudan.org/en/all-news/article/ coronavirus-measures-sudan-s-council-of-ministers-issue-directions. 28. Institute for Health Metrics and Evaluation. COVID-19 Projections: University of Washington,; 2022 [Available from: https://covid19.healthdata.org/ sudan?view=mask-use&tab=trend. 10. GaradaWorld S. Authorities ban mass gatherings due to COVID-19 March 20 /update 2 2020 [Available from: https://www.garda.com/crisis24/news-alerts/325211/ sudan-authorities-ban-mass-gatherings-due-to-covid-19-march-20-update-2 29. Petherick A, Goldszmidt R, Andrade EB, Furst R, Hale T, Pott A, et al. A worldwide assessment of changes in adherence to COVID-19 protec tive behaviours and hypothesized pandemic fatigue. Nat Hum Behav. 2021;5(9):1145–60. 11. Partnership for Evidence-Based. Response to COVID-19. Finding the Balance: Public Health and Social Measures in Sudan. 2020. 12. Dahab M, van Zandvoort K, Flasche S, Warsame A, Ratnayake R, Favas C, et al. COVID-19 control in low-income settings and displaced populations: what can realistically be done? Confl Health. 2020;14(1):54. 30. Haddad C, Sacre H, Bou Malhab S, Malaeb D, Saadeh D, Abou Tayeh C, et al. A cross-sectional study of COVID-19-related bullying in a sample of Lebanese adults: scale validation, correlates, and mediating effect of fear and anxiety. BMC Psychol. 2021;9(1):137. l 13. Brauner JM, Mindermann S, Sharma M, Johnston D, Salvatier J, Gavenčiak T et al. The effectiveness of eight nonpharmaceutical interventions against COVID-19 in 41 countries. medRxiv. 2020:2020.05.28.20116129. 31. Dye TD, Alcantara L, Siddiqi S, Barbosu M, Sharma S, Panko T, et al. Risk of COVID-19-related bullying, harassment and stigma among health care workers: an analytical cross-sectional global study. BMJ Open. 2020;10(12):e046620.i 14. Kucharski AJ, Klepac P, Conlan AJK, Kissler SM, Tang ML, Fry H, et al. Effec tiveness of isolation, testing, contact tracing, and physical distancing on reducing transmission of SARS-CoV-2 in different settings: a mathematical modelling study. Lancet Infect Dis. 2020;20(10):1151–60. 14. Kucharski AJ, Klepac P, Conlan AJK, Kissler SM, Tang ML, Fry H, et al. Publisher’s Note 19. Abdelmagid N, Ahmed SAE, Nurelhuda N, Zainalabdeen I, Ahmed A, Fadlallah MA, et al. Acceptability and feasibility of strategies to shield the vulnerable during the COVID-19 outbreak: a qualitative study in six Sudanese communi ties. BMC Public Health. 2021;21(1):1153. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4367860604	https://www.research-collection.ethz.ch/bitstream/20.500.11850/629347/2/086110_1_5.0156861.pdf	English	null	Coherent Control of Mid-Infrared Frequency Comb by Optical Injection of Near-Infrared Light	arXiv (Cornell University)	2,023	cc-by	7,983	ETH Library AFFILIATIONS 1 Laboratoire Temps-Fréquence, Institut de Physique, Université de Neuchâtel, Avenue de Bellevaux 51, 2000 Neuchâtel, Switzerland 2Institute for Quantum Electronics, ETH Zurich, Auguste-Piccard-Hof 1, 8093 Zurich, Switzerland a)Author to whom correspondence should be addressed: kenichi.komagata@unine.ch ABSTRACT We demonstrate the use of a low power near-infrared laser illuminating the front facet of a quantum cascade laser (QCL) as an optical actuator for the coherent control of a mid-infrared frequency comb. We show that with appropriate current control of the QCL comb and intensity modulation of the near-infrared laser, a tight phase lock of a comb line to a distributed feedback laser is possible with 2 MHz of locking bandwidth and 200 mrad of residual phase noise. A characterization of the whole scheme is provided, showing the limits of the electrical actuation, which we bypassed using the optical actuation. Both comb degrees of freedom can be locked by performing electrical injection locking of the repetition rate in parallel. However, we show that the QCL acts as a fast near-infrared light detector such that injection locking can also be achieved through modulation of the near-infrared light. These results on the coherent control of a QCL frequency comb are particularly interesting for coherent averaging in dual-comb spectroscopy and for mid-infrared frequency comb applications requiring high spectral purity. 05 September 2023 08:16:36 © 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0156861 Coherent control of mid-infrared frequency comb by optical injection of near-infrared light Coherent control of mid-infrared frequency comb by optical injection of near-infrared light Cite as: APL Photon. 8, 086110 (2023); doi: 10.1063/5.01568 Submitted: 3 May 2023 • Accepted: 25 July 2023 • Published Online: 17 August 2023 Kenichi N. Komagata,1,a) Alexandre Parriaux,1 Mathieu Bertrand,2 Johannes H Mattias Beck,2 Valentin J. Wittwer,1 Jérôme Faist,2 and Thomas Südmeyer1 Journal Article Funding acknowledgement: 176584 - High-resolution QCL frequency comb spectrometer for the detection of trace gases and their isotopes (SNF) This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. APL Photonics ARTICLE pubs.aip.org/aip/app I. INTRODUCTION and expensive instruments.11,12 DCS has been well demonstrated with mode-locked lasers in the near-infrared (NIR), but the MIR is more interesting as molecules generally have stronger absorption features, which is advantageous for many applications such as trace gas detection13 or isotope ratio measurements.14 DCS with QCLs is then highly interesting as it provides a compact, low footprint, and high-resolution spectrometer.4–6 and expensive instruments.11,12 DCS has been well demonstrated with mode-locked lasers in the near-infrared (NIR), but the MIR is more interesting as molecules generally have stronger absorption features, which is advantageous for many applications such as trace gas detection13 or isotope ratio measurements.14 DCS with QCLs is then highly interesting as it provides a compact, low footprint, and high-resolution spectrometer.4–6 Quantum cascade lasers (QCLs) emitting frequency combs were first demonstrated in 20121 and have since become an established technology for fast and broadband mid-infrared (MIR) spectroscopic applications, such as time-resolved studies of microsecond-scale molecular dynamics,2 high-pressure and temper- ature thermometry in shock tubes,3 or high-resolution measure- ments of molecular spectra.4–6 Some of their key assets are their low footprint and large optical power compared to other types of MIR combs, especially in the 8–10 μm spectral range that is usu- ally reached via nonlinear frequency conversion.7–9 Indeed, QCLs are electrically driven devices that directly emit frequency combs in the MIR with a power that can reach 1 W.10 In DCS, comb stabilization is not strictly necessary thanks to the availability of computational phase correction of free-running lasers.15 Nevertheless, stabilization of the combs allows accurate frequency referencing and arguably more flexibility, e.g., smaller repetition rates. Moreover, to properly establish QCLs as a source of choice for MIR comb applications such as optical frequency synthesis,16–18 demonstrating high spectral purity and coherent con- trol can be considered as important as increasing their bandwidth and detecting their offset frequency.19 In the context of comb spectroscopy, the use of two mutu- ally coherent combs with slightly different repetition rates, namely dual-comb spectroscopy (DCS), has shown great potential for fast and high resolution measurements without the need for complex For QCLs, actuation on the drive-current is the most straight- forward way to phase-lock a comb line,20–22 while the other degree of APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 8, 086110-1 ARTICLE pubs.aip.org/aip/app FIG. 1. I. INTRODUCTION p Indeed, light illumination at a different wavelength can influ- ence various parameters with different strengths, for example, to allow pure frequency modulation of a DFB-QCL.33 Moreover, multi- ple locking schemes could be possible for combs, such as the locking of the repetition rate via current actuation34,35 combined with the locking of the comb line frequency with NIR light. Finally, the NIR is supported by mature technologies, allowing a wide range of possi- bilities. Among others, the high modulation bandwidths reaching a few tens of GHz could enable the injection locking of the QCL comb repetition frequency. In light of the above, there is a compelling interest in investigating the potential of NIR light illumination for the coherent control of MIR QCL combs.l 05 September 2023 08:16:36 waveguides on a printed circuit board to efficiently inject and extract the repetition rate independently of the drive current. A custom-made dichroic mirror produced by ion beam sput- tering is placed in the optical comb path to direct NIR light from a continuous wave (CW) laser at 1.55 μm (Optilab, DFB-1550- EAM-12-K) to the front facet of the QCL via the collimation lens (Thorlabs, C037TME-F). The NIR laser has an output power of around 1 mW and can be modulated up to 12 GHz using an inte- grated electro-absorption modulator (EAM). The beam is aligned into the QCL so as to maximize the frequency response (see Sec. III). The amount of light effectively reaching the front facet is 55% of the emitted power of the NIR laser, and these losses are mainly due to the transmission of the QCL collimation lens at 1.55 μm. The polar- ization of the NIR light was fixed; however, it did not seem to change the experimental results. In this work, we characterize the influence of NIR light illu- minating the front facet of a MIR QCL comb emitting in the 8 μm range. We measure its transfer function at the comb frequencies and compare it to the more conventional electrical actuation. We demonstrate that the limits of electrical actuation for phase-locks can be bypassed using intensity modulated NIR light by tightly locking a QCL comb to a DFB-QCL. Finally, we show that the repetition rate of the QCL can also be injection-locked by intensity modulation of the NIR light. I. INTRODUCTION Schematic showing the experimental setup used to phase lock a line from a QCL comb to a DFB-QCL using an intensity modulated NIR CW laser and for characterization. The electrical blue wires represent the path for characterizing the repetition frequency and a comb line, whereas the orange wires represent the path for phase locking. CW: Continuous wave, EAM: Electro-absorption modula- tor, DM: Dichroic mirror, FD: Frequency discriminator, PD: Photodetector, FTIR: Fourier transform infrared spectrometer. APL Photonics pubs.aip.org/aip/app freedom, namely the repetition rate, is locked by electrically injecting a radio-frequency (RF) signal close to the round-trip frequency.23 These two handles allow the coherent control of QCL combs and can be used together.18 However, as for distributed feedback (DFB) QCLs,24,25 we expect the intrinsic frequency noise of the QCL comb and the stabilization by drive current to involve the same physical process, i.e., current to temperature to refractive index change. In that case, the time scale of the stabilization will always be close to the cutoff of the noise process, thus limiting the performance of the lock. A solution adopted for other types of lasers is to employ another actuator bound by time scales much faster than the noise source such as opto-optical modulation.26 Light illumination at another wavelength also enables con- trol of QCLs and can act as an actuator. The technique has been used on DFB-QCLs emitting a single wavelength for applications such as fast switching,27 gain enhancement,28 stabilization,29 and frequency modulation.30 More recently, a QCL comb emitting in the THz range was locked via the intensity modulation of a white light-emitting diode, although with a relatively low bandwidth of 800 Hz.31 We also note that the illumination of resonant light enabled the mutual lock between two MIR QCL combs via injec- tion locking.32 However, this only allows mutual locking at the same optical frequency, whereas modulation by off-resonant light offers more possibilities.l FIG. 1. Schematic showing the experimental setup used to phase lock a line from a QCL comb to a DFB-QCL using an intensity modulated NIR CW laser and for characterization. The electrical blue wires represent the path for characterizing the repetition frequency and a comb line, whereas the orange wires represent the path for phase locking. CW: Continuous wave, EAM: Electro-absorption modula- tor, DM: Dichroic mirror, FD: Frequency discriminator, PD: Photodetector, FTIR: Fourier transform infrared spectrometer. I. INTRODUCTION After passing through the dichroic mirror, the comb is mixed on a 50/50 beam splitter with CW MIR light generated by a DFB- QCL (Alpes Laser). The latter is driven at a current of 191 mA and a temperature of 0 ○C to emit at a frequency fcw = 1309.79 cm−1, which is within the spectral range of the frequency comb. A typical optical spectrum of the comb and the DFB recorded with a Fourier transform infrared spectrometer (Bristol 771A- MIR) is presented in Fig. 2. The beating fb between the comb and the DFB-QCL is recorded on a fast photodetector (VIGO, PV-4TE-10.6). II. EXPERIMENTAL SETUP The experimental setup considered here is presented in Fig. 1 and pivots around a QCL comb. The laser is controlled in current and temperature with a custom-made driver that sets the operation point of the laser to 1200 mA (1.45 times the lasing threshold) and 0 ○C. A frequency comb centered around 1305 cm−1 is emitted, with ∼80 lines, a total power of 126 mW, and a repetition frequency of 11.057 GHz. In QCL combs, the comb modes beating together in the Fabry–Perot cavity lead to a measurable voltage oscillating at the repetition frequency.36 Therefore, two wire bonds connect the top of the QCL waveguide near the front and back facets of the laser to RF In Sec. III, we will start by studying the frequency response of the QCL comb when NIR light illuminates its front facet. 8, 086110-2 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 APL Photonics ARTICLE pubs.aip.org/aip/app FIG. 2. Typical optical spectrum generated by the QCL comb and the continuous wave generated by the DFB-QCL. For small variations (1 mA) of the QCL drive currents, the frequencies shift linearly with a superimposed sinusoidal modu- lation on fr due to back-reflections.37 The fitted functions to the experimental data (dashed lines in Fig. 3) using a linear model sup- plemented by a sine wave give an average slope of −210 MHz/mA and −85 kHz/mA for fn and fr, respectively, although the slope for fr is dependent on the drive current (position within the modula- tion) due to the back reflections. The response to low power NIR light suggests a quadratic trend. Moreover, the shift ratio between fn and nf r depends on the alignment of the NIR light on the active region of the QCL comb. The main alignment method used in this article (alignment 1, described in Sec. III B) is to maximize the dynamic response of fn at modulation frequencies of 100 kHz. Another method would be to maximize the static shift of fn (align- ment 2). In the latter case, fr shows a sinusoidal modulation that is nearly nonexistent for alignment 1 and varies far less than for align- ment 1. For alignment 1, the slopes interpolated at zero power are −315 MHz/mW and −570 kHz/mW for fn and fr, respectively. FIG. 2. III. RESPONSE CHARACTERIZATION We now take an interest in the frequency-dependent response of the QCL comb. A lock-in amplifier (LIA, Zurich Instruments, UHFLI) modulates the power of the NIR light via the EAM or the comb drive current through the laser driver (see the blue path in Fig. 1). We study the response of three comb characteristic frequen- cies, namely, the offset frequencies f0, fr, and fn. Naturally, the three frequencies are coupled to each other through the comb equation We start with the static response before moving on to the frequency dependent response. II. EXPERIMENTAL SETUP Typical optical spectrum generated by the QCL comb and the continuous wave generated by the DFB-QCL. A. Static response 05 September 2023 08:16:36 First, we slowly vary the NIR power reaching the QCL from 0 to 0.6 mW and measure on an RF spectrum and phase noise ana- lyzer (Rohde & Schwarz, FSWP26) the frequency shift of a comb line fn via its beating with the DFB-QCL and of the repetition rate fr, measured directly via the independent channel for RF extraction on the QCL comb. For comparison, we also measure the frequency shifts when the drive current of the QCL comb is varied over 1 mA. These results are presented in Figs. 3(a) and 3(b), where the shift in fr is scaled by the mode number n = 3550 for better comparability with fn. fn = f0 + nfr, (1) (1) where n is an integer. Due to the modulation of the drive current or the NIR power set by the LIA at frequency ω, the comb, i.e., its frequencies, respond as fi = f (0) i + Δfi sin (ωt + θi), (2) (2) where i = {0; r; n} indexes the three comb frequencies under study, f (0) i is the average value of fi, Δfi is the peak amplitude of fi due to the modulation, and θi is the phase of the response. To measure the amplitudes and phases, we convert the frequency modulation of fi to a voltage modulation using a frequency discriminator (FD) and demodulate this voltage on the LIA. FIG. 3. Static response of the QCL comb frequencies to a change in (a) drive current or (b) illuminated NIR power. The shift in fr is scaled by n. In (b), the response is reported for two alignment conditions (see main text). Dashed lines are fitted functions to the experimental data. Error bars are plotted when they are larger than the data marker. For this purpose, the repetition rate of the comb is extracted electrically as before, amplified, and down-mixed to 60 MHz. As Δfr is only on the order of 20 kHz, we take the tenth harmonic and down-mix it to 21 MHz before sending it to FD-1 (MITEQ, FMDM- 21.4/4-2, see Fig. 1), whose output is connected to the LIA. As for the comb line n, the amplitude and phase response of the comb line fn sre encoded in the beating signal with the DFB-QCL as fb = f (0) n + Δfn sin (ωt + θn) −fcw. A. Static response The (quasi-) fix point38,39 increases with modulation frequency from mode number 1850 at 1 Hz to mode number 2200 at 100 kHz. Regarding f0, this frequency cannot be directly detected in practice since f –to–2f interferometry19 is currently unavailable to QCL combs, but fluctuations thereof can be detected,35 which allows the measurement of its transfer function. However, for experimen- tal simplicity, we can compute it from the transfer functions of fn and fr. Indeed, according to Eqs. (1) and (2) and the elastic tape model,38,39 we have Δf0 sin (ωt + θ0) = Δfn sin (ωt + θn) −nΔfr sin (ωt + θr). (4) Therefore, Eq. (4) yields that Δf0 and θ0 are, respectively, the mod- ulus and phase of the complex value Δfn exp(iθn) −nΔfr exp(iθr). Here, n = 3551 is set by the wavenumber of the DFB-QCL at 1309.79 cm−1 and by the repetition rate. Although the transfer function of a MIR QCL frequency comb has already been reported in Ref. 35, our measurements highlight a 90○modulation bandwidth, one order of magnitude above what was previously shown. This is in agreement with the response of DFB-QCLs25,40,41 and other QCL combs, measured directly42 or demonstrated in a phase-lock loop.20,22 This discrepancy with the measured modulation bandwidth could be attributed to the fre- quency response of the bias-tee used in Ref. 35. Furthermore, we observed in Fig. 3 that modulations of fr due to back reflections locally change the slope, such that the ratio between Δf0 and Δfn and the fix points are expected to be modulated as well. Figure 4 shows the resulting phase θi and amplitude responses Δfi of the frequencies (fn, nfr, and f0) to modulation of the electri- cal drive current, ΔI = 200 μA, and the NIR power, ΔP = 50 μW, where Δ represents the peak amplitude of the modulation. Note that Δfr is scaled by n for better comparison with Δfn. In addition, the contributions of various components of the characterization scheme (i.e., the FDs and the laser driver) were measured and deducted in order to obtain the laser response as faithfully as possible. More- over, the responses were measured with two different settings for the ranges [1 Hz, 100 Hz] and [100 Hz, 10 MHz], leading to negligible mismatches at 100 Hz.i i We now turn to the optical actuation shown in Figs. 4(b) and 4(d). A. Static response (3) (3) The signal fb detected on the photodetector is then filtered, ampli- fied, divided by 15 and 3 (RF bay, FPS-15-8, FPS-3-8), up-mixed to 60 MHz (not shown), and fed to a FD (MITEQ, FMDM-60/16-4BC), whose output is connected to the LIA, as shown in Fig. 1. The divi- sion step allows a larger frequency excursion to be measured than the linear range of the FD. The frequency-to-voltage conversion ratios, The signal fb detected on the photodetector is then filtered, ampli- fied, divided by 15 and 3 (RF bay, FPS-15-8, FPS-3-8), up-mixed to 60 MHz (not shown), and fed to a FD (MITEQ, FMDM-60/16-4BC), whose output is connected to the LIA, as shown in Fig. 1. The divi- sion step allows a larger frequency excursion to be measured than the linear range of the FD. The frequency-to-voltage conversion ratios, FIG. 3. Static response of the QCL comb frequencies to a change in (a) drive current or (b) illuminated NIR power. The shift in fr is scaled by n. In (b), the response is reported for two alignment conditions (see main text). Dashed lines are fitted functions to the experimental data. Error bars are plotted when they are larger than the data marker. APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 8, 086110-3 APL Photonics ARTICLE pubs.aip.org/aip/app including all division and multiplication steps, for Δfr and Δfn are, respectively, 8.2 ± 0.4 V/MHz and 5.93 ± 0.06 mV/MHz. Moreover, the response of fn closely mimics that of DFB-QCLs.25,40 fr follows a similar behavior as fn with a 3-dB and 90○modula- tion bandwidth of 80 and 840 kHz, respectively. The dashed line in Fig. 4(c) is the phase response of fn with the laser driver and has a 90○modulation bandwidth of 280 kHz. The difference between Δfn and nΔfr gives the response f0, which has a flatter response in amplitude with a 3-dB modulation bandwidth of 160 kHz. The uncertainty in the measurements of Δfn and nΔfr, in particular the slope of the FDs, which depend on the input RF power, induces a large absolute uncertainty on Δf0. As for the phase, its response is also flatter, reaching −70 near 1 MHz, after which the measurement is no longer accurate due to the lack of sensitivity. A. Static response For this measurement, the NIR laser was aligned on the QCL to maximize the dynamic response of fn at a 100 kHz modulation. We observe a nearly flat phase response for fr up to 1 MHz, with a small resonance near 33 Hz and the start of a roll-off near 1 MHz. At the resonance near 33 Hz, the amplitude response decreases by a factor 2 and then remains nearly flat apart from the onset of the roll- off near 1 MHz. The 33-Hz resonance also marks a change of regime for fn due to the crossing of nΔfr with Δf0, the latter having a simi- lar response as fr, although a smaller relative change in amplitude response and opposite phase response. Therefore, for modulation frequencies lower (higher) than 33 Hz, Δf0 is smaller (larger) than nΔfr, such that the response of fn flips sign from 180○to 0○from 1 Hz to 1 kHz. The respective (quasi-) fixed points are at mode num- bers 3250 and 3950. An associated dip in the amplitude response of fn is also visible. Above 1 kHz, the response is flat up to about 50 kHz, where the response increases in phase and magnitude before showing a signature of a roll-off near 1 MHz. IV. MUTUAL STABILIZATION We now seek to implement a mutual lock between one comb line fn of the QCL comb and a DFB-QCL as a proof-of-principle demonstration. The electrical part of the setup is adapted accord- ing to the orange path in Fig. 1. The phase fluctuations between the two lasers are obtained by mixing the signal fb after division by 15 with a synthesized reference frequency locked to the maser. This error signal is injected into two proportional integral differ- ential (PID) controllers (Vescent, D2-125). The output of one PID controller is fed back to the driver of the QCL comb to modulate the electrical current. The output of the second PID controller is fed back, after high-pass filtering with a 2nd-order custom-designed fil- ter with a cutoff at 1 kHz, to the EAM to modulate the intensity of the NIR CW laser. We also monitor the beat note with the RF spectrum and phase noise analyzer. In this way, slow (<100 kHz) corrections of fn, which cannot be handled by the NIR light due to the sign reversal, are performed by the drive current, while the NIR light extends the available bandwidth and cancels added noise from the drive current, i.e., the servo bump. p The addition of the optical actuation reduces the phase noise PSD to a value in the order of −80 dBc/Hz, below the β-line,43 for all Fourier frequencies. As a result, the integrated phase noise between 1 Hz and 10 MHz is decreased from 2.16 rad to 200 mrad [see inset in Fig. 5(a)]. The electrical servo bump is eliminated, while a new servo bump appears at 2 MHz. This fast bandwidth allows us to employ a lower division ratio of 4: the residual phase noise from 1 Hz to 3 kHz is set by the reference oscillator. At higher frequencies, we believe that the mismatch between the reference voltages of the two different servo controllers added noise to the system. Moreover, the bumps near 3 kHz coincide with the cutoff frequency of the high- pass filter. Furthermore, optimization of the electronic components could improve the lock and decrease the integrated phase noise fur- ther, including the use of a single dual-output servo controller. We also believe the stabilization bandwidth could be increased by short- ening all the cables, fibers, and free-space paths. In terms of spectrum [Fig. 05 September 2023 08:16:36 05 September 2023 08:16:36 Focusing first on the electrical actuation in Figs. 4(a) and 4(c), we observe that the amplitude response Δfn decreases steadily with the modulation frequency, setting the 3-dB modulation bandwidth to 30 kHz before decreasing sharply after about 200 kHz. The phase response remains flat up to 10 kHz with a 90○modulation bandwidth at 420 kHz, which is coherent with previous tight-locking results.20 FIG. 4. Response of the comb frequencies (fn, nf r, f0) to modulation of the drive current [(a,c)] and of the intensity of the NIR light [(b,d)]. Panels (a) and (b) present the frequency excursions Δfi, while panels (c) and (d) show the phase response θi. The dashed line in (c) is the phase response of fn with the laser driver. The mechanism causing frequency modulation due to the drive current change is identical to DFB-QCLs, a change of the refrac- tive index.25 In the case of a NIR injection, the strong inter-band absorption in the InGaAs quantum wells (the absorption coefficient α = 6000 cm−1 at 1550 nm) that are part of the active region of the QCL is responsible for the modulation of the refractive index through the generation of carriers in the vicinity of the NIR injec- tion point. In addition, we believe that the response below 30 Hz is dominated by thermal processes (heating of the QCL), as the sign of the responses is equal to drive current modulation and as the opti- mization of the maximum static shift of fn causes modulation of fr as with drive current changes. FIG. 4. Response of the comb frequencies (fn, nf r, f0) to modulation of the drive current [(a,c)] and of the intensity of the NIR light [(b,d)]. Panels (a) and (b) present the frequency excursions Δfi, while panels (c) and (d) show the phase response θi. The dashed line in (c) is the phase response of fn with the laser driver. From the perspective of locking the QCL comb, the actuation via the NIR power is advantageous as it offers a higher bandwidth than the drive current actuation for all comb frequencies, even when APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 8, 086110-4 8, 086110-4 APL Photonics ARTICLE pubs.aip.org/aip/app bumps in the phase noise power spectral density (PSD). 05 September 2023 08:16:36 05 September 2023 08:16:36 p Figures 5(a) and 5(b), respectively, show the results of the stabilization of fb in terms of phase noise and the RF power spec- trum. When only electrical actuation is considered, we observe two FIG. 5. Tight-locking of a QCL comb to a DFB-QCL. (a) Phase noise power spectral density (PSD) of the mutual beat note in free-running mode, electrical actuation, and both electrical and optical actuation. Inset: Integrated phase noise between f cut and 10 MHz. (b) RF power spectrum at 100 Hz RBW of the beat note between one line of the QCL comb and the DFB-QCL, depending on the stabilization scheme used. Inset: Zoom on the beat note at 1 Hz RBW. In parallel, the repetition frequency of the comb is stabi- lized by RF injection locking using a resonant RF signal with 15 dBm of power delivered by a signal generator (Rohde & Schwarz, SMF100A) referenced to a maser. This RF signal is sent to the QCL via the dedicated channel for RF injection. Therefore, both degrees of freedom are tightly locked simultaneously, with low residual phase noise. The electrical injection of the repetition rate is the usual approach to its stabilization. However, the previous results suggest that the repetition rate could also be injection-locked by modulating the NIR light. 05 September 2023 08:16:36 The first is due to the limit of the integrator at about 100 kHz, while the sec- ond near 300 kHz is the servo bump, which nearly coincides with the 90○modulation bandwidth of the combined driver and laser [see dashed blue line in Fig. 4(c)]. The white phase noise from 1 Hz to about 100 Hz is due to the reference oscillator, which, due to the division by 15, has its contribution increased by 23 dB. The result- ing RF spectrum shows a Gaussian shape topped with a coherent peak. the driver response has been accounted for. It is especially well suited for the stabilization of f0 if it can be measured. The stabilization of fn will require a countermeasure against the sign reversal as detailed in Sec. IV. As for fr, the increased actuation bandwidth is not as interesting given that it can be tightly locked by actuation on the drive current.35 IV. MUTUAL STABILIZATION 5(b)], the power in the coherent peak improves by 20 dB. The height of the pedestal is decreased by 15 dB, such that the difference between the coherent peak and the top of the pedestal is 50 dB at a resolution bandwidth (RBW) of 100 Hz, or about 25 dB more than in Ref. 20 at a 500 Hz RBW. The inset shows a zoom over the peak at a 1 Hz RBW with a signal-to-noise ratio (SNR) of 70 dB. V. REPETITION RATE INJECTION VIA NIR ILLUMINATION We thus modulated the EAM of the NIR laser near the repeti- tion frequency of 11.06 GHz while monitoring the generated voltage modulation of the QCL via the dedicated RF extraction port on an RF spectrum analyzer. At low NIR power and close to resonance (1 MHz offset), the modulation frequency was picked up by the QCL, which thus acted as a NIR detector [see Fig. 6(a)]. By slightly increas- ing the average NIR power with an amplifier to 1.5 mW at the QCL and the RF power on the EAM to 17 dBm (estimated modulation depth close to 100%), we were able to injection-lock the repetition frequency to the synthesizer and obtain the resolution-limited signal shown in Fig. 6(b) at 50 Hz RBW. We obtained a lock range of a few kHz when scanning the modulation frequency across the free run- ning repetition frequency [see Fig. 6(c)]. The phase noise PSD was FIG. 5. Tight-locking of a QCL comb to a DFB-QCL. (a) Phase noise power spectral density (PSD) of the mutual beat note in free-running mode, electrical actuation, and both electrical and optical actuation. Inset: Integrated phase noise between f cut and 10 MHz. (b) RF power spectrum at 100 Hz RBW of the beat note between one line of the QCL comb and the DFB-QCL, depending on the stabilization scheme used. Inset: Zoom on the beat note at 1 Hz RBW. APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 8, 086110-5 APL Photonics ARTICLE pubs.aip.org/aip/app FIG. 6. Injection locking of the repetition rate by the NIR laser. (a) and (b) Electri- cally measured RF spectrum of the QCL with (a) the free running intermode beat and the detection of a weak off-resonant NIR modulation near −1 MHz and (b) the injection-locked intermode beat using 1.5 mW of NIR light with 100% modu- lation depth. (c) Stacked RF spectra (50 Hz RBW) with the increasing modulation frequency crossing the natural repetition frequency and causing injection-locking over a range of a few kHz. The dashed horizontal line indexes the acquisition shown in (b). (d) Phase noise power spectral density (PN PSD) of the intermode beat signal in the free-running and injection locked regimes. V. REPETITION RATE INJECTION VIA NIR ILLUMINATION locking of enhancement cavities for further improvement of sensi- tivity.45 Moreover, high spectral purity in the MIR is relevant for a variety of applications such as quantum control of molecules,46 tests of fundamental physics47,48 and, generally the study of molecules via high-resolution spectroscopy.5,49,50 Therefore, we expect our results to facilitate the application of QCL frequency combs in the MIR. As a further outlook, we anticipate that other wavelengths33 could be more suitable for orthogonal control without sign reversal of the comb properties such as the offset and repetition frequen- cies, while perhaps they could also be used to dynamically tune laser parameters such as dispersion, gain, or nonlinearity. Faster mod- ulations from 10 MHz to a few GHz could be investigated as a way to achieve (pure) frequency modulation of the comb. To keep the compactness of the device and its low footprint, light could be delivered by fiber or directly generated and modulated on the same chip if low power is sufficient. Then, full optical control of the QCL comb merely driven by a battery could be envisaged. Optical control could also be investigated for interband cascade lasers.51 Moreover, the recent work on free-space communications using QCL combs42 inspires the adaptation of injection locking of the QCL by NIR light for direct conversion of NIR telecommunication streams to MIR signals. We believe that increasing the injected optical power could increase the locking range of the repetition rate to a sufficient level for this application. Further work including theoretical and numerical investigations of the laser dynamics52,53 is necessary to understand the full potential of controlling QCLs via optical means. FIG. 6. Injection locking of the repetition rate by the NIR laser. (a) and (b) Electri- cally measured RF spectrum of the QCL with (a) the free running intermode beat and the detection of a weak off-resonant NIR modulation near −1 MHz and (b) the injection-locked intermode beat using 1.5 mW of NIR light with 100% modu- lation depth. (c) Stacked RF spectra (50 Hz RBW) with the increasing modulation frequency crossing the natural repetition frequency and causing injection-locking over a range of a few kHz. The dashed horizontal line indexes the acquisition shown in (b). (d) Phase noise power spectral density (PN PSD) of the intermode beat signal in the free-running and injection locked regimes. 05 September 2023 08:16:36 Conflict of Interest In this article, we used a low power NIR CW light illuminating the front facet of a MIR QCL frequency comb as an optical actuator for the phase stabilization of a comb line and as a means to achieve coherent injection locking of the repetition rate. First, by character- izing the response of the QCL, we showed that intensity modulation of the NIR light offers a higher modulation bandwidth compared to conventional drive current modulation. Then, we implemented a stabilization scheme exploiting the NIR light to extend the locking- bandwidth from 300 kHz to over 2 MHz, which resulted in an increase of the SNR by 35 dB and an integrated phase noise as low as 200 mrad. Finally, we showed that the QCL can act as a detector of NIR light modulated at a frequency of 11 GHz and that it can be injection-locked in such a way. The authors have no conflicts to disclose. ACKNOWLEDGMENTS We acknowledge Stéphane Schilt for his support in the early stage of the investigation. We acknowledge Alpes Laser for pro- viding the DFB-QCL used in this work. We acknowledge fund- ing from the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Grant No. 40B2-1_176584). reduced up to a Fourier frequency of about 3 kHz compared to the free-running case [Fig. 6(d)]. We expect that larger locking ranges could be achieved when using more NIR power, which will be one of the studies we present in another dedicated article. REFERENCES 21J. Westberg, L. A. Sterczewski, and G. Wysocki, “Mid-infrared multiheterodyne spectroscopy with phase-locked quantum cascade lasers,” Appl. Phys. Lett. 110, 141108 (2017). 1A. Hugi, G. Villares, S. Blaser, H. C. Liu, and J. Faist, “Mid-infrared frequency comb based on a quantum cascade laser,” Nature 492, 229–233 (2012). 22K. Komagata, A. Shehzad, G. Terrasanta, P. Brochard, R. Matthey, M. Gianella, P. Jouy, F. Kapsalidis, M. Shahmohammadi, M. Beck, V. J. Wittwer, J. Faist, L. Emmenegger, T. Südmeyer, A. Hugi, and S. Schilt, “Coherently-averaged dual comb spectrometer at 7.7 μm with master and follower quantum cascade lasers,” Opt. Express 29, 19126 (2021). 2J. L. Klocke, M. Mangold, P. Allmendinger, A. Hugi, M. Geiser, P. Jouy, J. Faist, and T. Kottke, “Single-shot sub-microsecond mid-infrared spectroscopy on pro- tein reactions with quantum cascade laser frequency combs,” Anal. Chem. 90, 10494–10500 (2018). gg , y , g , , y g comb spectrometer at 7.7 μm with master and follower quantum cascade lasers,” Opt. Express 29, 19126 (2021). 3N. H. Pinkowski, P. Biswas, J. Shao, C. L. Strand, and R. K. Hanson, “Thermometry and speciation for high-temperature and -pressure methane pyrol- ysis using shock tubes and dual-comb spectroscopy,” Meas. Sci. Technol. 32, 125502 (2021). 23J. Hillbrand, A. M. Andrews, H. Detz, G. Strasser, and B. Schwarz, “Coherent injection locking of quantum cascade laser frequency combs,” Nat. Photonics 13, 101–104 (2019). 24L. Tombez, S. Schilt, J. Di Francesco, P. Thomann, and D. Hofstetter, “Temperature dependence of the frequency noise in a mid-IR DFB quantum cas- cade laser from cryogenic to room temperature,” Opt. Express 20, 6851–6859 (2012). 4M. Lepère, O. Browet, J. Clément, B. Vispoel, P. Allmendinger, J. Hayden, F. Eigenmann, A. Hugi, and M. Mangold, “A mid-infrared dual-comb spectrom- eter in step-sweep mode for high-resolution molecular spectroscopy,” J. Quant. Spectrosc. Radiat. Transfer 287, 108239 (2022). 25L. Tombez, F. Cappelli, S. Schilt, G. Di Domenico, S. Bartalini, and D. Hofstet- ter, “Wavelength tuning and thermal dynamics of continuous-wave mid-infrared distributed feedback quantum cascade lasers,” Appl. Phys. Lett. 103, 031111 (2013). 5J. A. Agner, S. Albert, P. Allmendinger, U. Hollenstein, A. Hugi, P. Jouy, K. Keppler, M. Mangold, F. Merkt, and M. Quack, “High-resolution spectroscopic measurements of cold samples in supersonic beams using a QCL dual-comb spectrometer,” Mol. Phys. 120, e2094297 (2022). 05 September 2023 08:16:36 26M. Hoffmann, S. Schilt, and T. REFERENCES Südmeyer, “CEO stabilization of a femtosec- ond laser using a SESAM as fast opto-optical modulator,” Opt. Express 21, 30054–30064 (2013). 6K. N. Komagata, V. J. Wittwer, T. Südmeyer, L. Emmenegger, and M. Gianella, “Absolute frequency referencing for swept dual-comb spectroscopy with midinfrared quantum cascade lasers,” Phys. Rev. Res. 5, 013047 (2023). 27C. Zervos, M. D. Frogley, C. C. Phillips, D. O. Kundys, L. R. Wilson, M. Hopkin- son, and M. S. Skolnick, “All-optical switching in quantum cascade lasers,” Appl. Phys. Lett. 90, 053505 (2007). 7K. Iwakuni, G. Porat, T. Q. Bui, B. J. Bjork, S. B. Schoun, O. H. Heckl, M. E. Fer- mann, and J. Ye, “Phase-stabilized 100 mW frequency comb near 10 μm,” Appl. Phys. B 124, 128 (2018). 28G. Chen, C. G. Bethea, and R. Martini, “Quantum cascade laser gain enhancement by front facet illumination,” Opt. Express 17, 24282 (2009). 29 8K. Krzempek, D. Tomaszewska, A. Głuszek, T. Martynkien, P. Mergo, J. Sotor, A. Foltynowicz, and G. Sobo´n, “Stabilized all-fiber source for generation of tunable broadband fCEO-free mid-IR frequency comb in the 7–9 μm range,” Opt. Express 27, 37435 (2019). 29L. Tombez, S. Schilt, D. Hofstetter, and T. Südmeyer, “Active linewidth- narrowing of a mid-infrared quantum cascade laser without optical reference,” Opt. Lett. 38, 5079–5082 (2013). 9N. Hoghooghi, S. Xing, P. Chang, D. Lesko, A. Lind, G. Rieker, and S. Did- dams, “Broadband 1-GHz mid-infrared frequency comb,” Light Sci. Appl. 11, 264 (2022). 30C. Peng, G. Chen, J. Tang, L. Wang, Z. Wen, H. Zhou, and R. Martini, “High-speed mid-infrared frequency modulation spectroscopy based on quantum cascade laser,” IEEE Photonics Technol. Lett. 28, 1727–1730 (2016). 31 10P. Jouy, J. M. Wolf, Y. Bidaux, P. Allmendinger, M. Mangold, M. Beck, and J. Faist, “Dual comb operation of λ ∼8.2 μm quantum cascade laser frequency comb with 1 W optical power,” Appl. Phys. Lett. 111, 141102 (2017). 31L. Consolino, A. Campa, M. De Regis, F. Cappelli, G. Scalari, J. Faist, M. Beck, M. Rösch, S. Bartalini, and P. De Natale, “Controlling and phase-locking a THz quantum cascade laser frequency comb by small optical frequency tuning,” Laser Photonics Rev. 15, 2000417 (2021). 11I. Coddington, N. Newbury, and W. Swann, “Dual-comb spectroscopy,” Optica 3, 414 (2016). 32J. Hillbrand, M. Bertrand, V. Wittwer, N. Opaˇcak, F. Kapsalidis, M. Gianella, L. Emmenegger, B. Schwarz, T. Südmeyer, M. Beck, and J. Faist, “Synchronization of frequency combs by optical injection,” Opt. Express 30, 36087–36095 (2022). Author Contributions Kenichi N. Komagata: Conceptualization (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Validation (equal); Visualization (lead); Writing – original draft (lead). Alexandre Par- riaux: Conceptualization (supporting); Formal analysis (support- ing); Investigation (equal); Methodology (equal); Validation (equal); Visualization (equal); Writing – original draft (supporting); Writ- ing – review & editing (lead). Mathieu Bertrand: Formal analysis (equal); Validation (equal); Writing – review & editing (equal). Johannes Hillbrand: Resources (equal); Writing – review & editing (supporting). Mattias Beck: Resources (equal); Writing – review & editing (supporting). Valentin J. Wittwer: Funding acquisition (equal); Resources (equal); Supervision (equal); Validation (support- ing); Writing – review & editing (equal). Jérôme Faist: Funding acquisition (equal); Resources (equal); Supervision (supporting); We believe that the tighter mutual stabilization enabled by the high bandwidth of the NIR light could lead to higher sensitivities through coherent averaging in DCS,22,44 which is currently one of the main applications of QCL combs. In this regard, a comprehen- sive comparison with the performance of computational coherent averaging15 is necessary. The high bandwidth could also allow the APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 8, 086110-6 APL Photonics ARTICLE pubs.aip.org/aip/app Writing – review & editing (supporting). Thomas Südmeyer: Fund- ing acquisition (equal); Supervision (equal); Writing – review & editing (equal). 18K. N. Komagata, M. Gianella, P. Jouy, F. Kapsalidis, M. Shahmohammadi, M. Beck, R. Matthey, V. J. Wittwer, A. Hugi, J. Faist, L. Emmenegger, T. Süd- meyer, and S. Schilt, “Absolute frequency referencing in the long wave infrared using a quantum cascade laser frequency comb,” Opt. Express 30, 12891 (2022). 19 Writing – review & editing (supporting). Thomas Südmeyer: Fund- ing acquisition (equal); Supervision (equal); Writing – review & editing (equal). 19H. R. Telle, G. Steinmeyer, A. E. Dunlop, J. Stenger, D. H. Sutter, and U. Keller, “Carrier-envelope offset phase control: A novel concept for absolute optical fre- quency measurement and ultrashort pulse generation,” Appl. Phys. B 69, 327–332 (1999). DATA AVAILABILITY The data that support the findings of this study are openly available in EUDAT B2SHARE at http://doi.org/ 10.23728/b2share.4c30e6bcadd3443d8d49e00200cbd7b5.54 20F. Cappelli, G. Campo, I. Galli, G. Giusfredi, S. Bartalini, D. Mazzotti, P. Cancio, S. Borri, B. Hinkov, J. Faist, and P. De Natale, “Frequency stability characterization of a quantum cascade laser frequency comb,” Laser Photonics Rev. 10, 623–630 (2016). REFERENCES 33 12N. Picqué and T. W. Hänsch, “Frequency comb spectroscopy,” Nat. Photonics 13, 146–157 (2019). 13F. R. Giorgetta, J. Peischl, D. I. Herman, G. Ycas, I. Coddington, N. R. New- bury, and K. C. Cossel, “Open-path dual-comb spectroscopy for multispecies trace gas detection in the 4.5–5 μm spectral region,” Laser Photonics Rev. 15, 2000583 (2021). 33C. Peng, H. Zhou, L. Zhu, T. Chen, Q. Liu, D. Wang, J. Li, Q. Peng, G. Chen, and Z. Li, “Purified frequency modulation of a quantum cascade laser with an all-optical approach,” Opt. Lett. 42, 4506–4509 (2017). 34F. Cappelli, L. Consolino, G. Campo, I. Galli, D. Mazzotti, A. Campa, M. Sicil- iani de Cumis, P. Cancio Pastor, R. Eramo, M. Rösch, M. Beck, G. Scalari, J. Faist, P. De Natale, and S. Bartalini, “Retrieval of phase relation and emission profile of quantum cascade laser frequency combs,” Nat. Photonics 13, 562–568 (2019). 14A. Parriaux, K. Hammani, C. Thomazo, O. Musset, and G. Millot, “Isotope ratio dual-comb spectrometer,” Phys. Rev. Res. 4, 023098 (2022). 15 15L. A. Sterczewski, J. Westberg, and G. Wysocki, “Computational coherent aver- aging for free-running dual-comb spectroscopy,” Opt. Express 27, 23875–23893 (2019). 35A. Shehzad, P. Brochard, R. Matthey, F. Kapsalidis, M. Shahmohammadi, M. Beck, A. Hugi, P. Jouy, J. Faist, T. Südmeyer, and S. Schilt, “Frequency noise correlation between the offset frequency and the mode spacing in a mid-infrared quantum cascade laser frequency comb,” Opt. Express 28, 8200–8210 (2020). 16J. D. Jost, J. L. Hall, and J. Ye, “Continuously tunable, precise, single frequency optical signal generator,” Opt. Express 10, 515–520 (2002). 36M. Piccardo, D. Kazakov, N. A. Rubin, P. Chevalier, Y. Wang, F. Xie, K. Lascola, A. Belyanin, and F. Capasso, “Time-dependent population inversion gratings in laser frequency combs,” Optica 5, 475–478 (2018). 17B. Argence, B. Chanteau, O. Lopez, D. Nicolodi, M. Abgrall, C. Chardonnet, C. Daussy, B. Darquié, Y. Le Coq, and A. Amy-Klein, “Quantum cascade laser frequency stabilization at the sub-Hz level,” Nat. Photonics 9, 456–460 (2015). APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 8, 086110-7 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 APL Photonics ARTICLE pubs.aip.org/aip/app 47P. Jansen, H. L. Bethlem, and W. Ubachs, “Perspective: Tipping the scales: Search for drifting constants from molecular spectra,” J. Chem. Phys. 140, 010901 (2014). 37C. C. Teng, J. Westberg, and G. REFERENCES Wysocki, “Gapless tuning of quantum cas- cade laser frequency combs with external cavity optical feedback,” Opt. Lett. 48, 363–366 (2023). 48A. Cournol, M. Manceau, M. Pierens, L. Lecordier, D. B. A. Tran, R. Santa- gata, B. Argence, A. Goncharov, O. Lopez, M. Abgrall, Y. Le Coq, R. Le Targat, H. Alvarez Martinez, W. K. Lee, D. Xu, P. E. Pottie, R. J. Hendricks, T. E. Wall, 38H. Telle, B. Lipphardt, and J. Stenger, “Kerr-lens, mode-locked lasers as transfer oscillators for optical frequency measurements,” Appl. Phys. B: Lasers Opt. 74, 1–6 (2002). J. M. Bieniewska, B. E. Sauer, M. R. Tarbutt, A. Amy-Klein, S. K. Tokunaga, and B. Darquié, “A new experiment to test parity symmetry in cold chiral molecules using vibrational spectroscopy,” Quantum Electron. 49, 288–292 (2019). 39V. Dolgovskiy, N. Bucalovic, P. Thomann, C. Schori, G. Di Domenico, and S. Schilt, “Cross-influence between the two servo loops of a fully stabilized Er:Fiber optical frequency comb,” J. Opt. Soc. Am. B 29, 2944–2957 (2012). 49P. B. Changala, M. L. Weichman, K. F. Lee, M. E. Fermann, and J. Ye, “Rovibrational quantum state resolution of the C60 fullerene,” Science 363, 49–54 (2019). 40A. Hangauer, G. Spinner, M. Nikodem, and G. Wysocki, “High frequency mod- ulation capabilities and quasi single-sideband emission from a quantum cascade laser,” Opt. Express 22, 23439–23455 (2014). 50M. Germann, A. Hjältén, V. Boudon, C. Richard, K. Krzempek, A. Hudzikowski, A. Głuszek, G. Sobo´n, and A. Foltynowicz, “A methane line list with sub-MHz accuracy in the 1250 to 1380 cm−1 range from optical frequency comb Fourier transform spectroscopy,” J. Quant. Spectrosc. Radiat. Transfer 288, 108252 (2022). 41S. Borri, S. Bartalini, P. C. Pastor, I. Galli, G. Giusfredi, D. Mazzotti, M. Yaman- ishi, and P. De Natale, “Frequency-noise dynamics of mid-infrared quantum cascade lasers,” IEEE J. Quantum Electron. 47, 984–988 (2011). 42N. Corrias, T. Gabbrielli, P. De Natale, L. Consolino, and F. Cappelli, “Analog FM free-space optical communication based on a mid-infrared quantum cascade laser frequency comb,” Opt. Express 30, 10217 (2022). 51M. Bagheri, C. Frez, L. A. Sterczewski, I. Gruidin, M. Fradet, I. Vurgaftman, C. L. Canedy, W. W. Bewley, C. D. Merritt, C. S. Kim, M. Kim, and J. R. Meyer, “Passively mode-locked interband cascade optical frequency combs,” Sci. Rep. 8, 51M. Bagheri, C. Frez, L. A. Sterczewski, I. Gruidin, M. Fradet, I. Vurgaftman, C. L. Canedy, W. W. Bewley, C. D. Merritt, C. S. Kim, M. REFERENCES Kim, and J. R. Meyer, “Passively mode-locked interband cascade optical frequency combs,” Sci. Rep. 8, 3322 (2018). 43G. Di Domenico, S. Schilt, and P. Thomann, “Simple approach to the relation between laser frequency noise and laser line shape,” Appl. Opt. 49, 4801–4807 (2010). “Passively mode-locked interband cascade optical frequency combs,” Sci. Rep. 8, 3322 (2018). 52G. Villares and J. Faist, “Quantum cascade laser combs: Effects of modulation and dispersion,” Opt. Express 23, 1651 (2015). 53 44Z. Chen, M. Yan, T. W. Hänsch, and N. Picqué, “A phase-stable dual-comb interferometer,” Nat. Commun. 9, 3035 (2018). 53N. Opaˇcak and B. Schwarz, “Theory of frequency-modulated combs in lasers with spatial hole burning, dispersion, and Kerr nonlinearity,” Phys. Rev. Lett. 123, 243902 (2019). 45A. Foltynowicz, P. Masłowski, A. J. Fleisher, B. J. Bjork, and J. Ye, “Cavity- enhanced optical frequency comb spectroscopy in the mid-infrared application to trace detection of hydrogen peroxide,” Appl. Phys. B 110, 163–175 (2013).i 54K. N. Komagata, A. Parriaux, M. Bertrand, J. Hillbrand, M. Beck, V. J. Wit- twer, J. Faist, and T. Südmeyer (2023). “Coherent control of mid-infrared fre- quency comb by optical injection of near-infrared light,” EUDAT B2SHARE. http://doi.org/10.23728/b2share.4c30e6bcadd3443d8d49e00200cbd7b5. 46M. Sinhal, Z. Meir, K. Najafian, G. Hegi, and S. Willitsch, “Quantum- nondemolition state detection and spectroscopy of single trapped molecules,” Science 367, 1213–1218 (2020). 05 September 2023 08:16:36 APL Photon. 8, 086110 (2023); doi: 10.1063/5.0156861 © Author(s) 2023 8, 086110-8 8, 086110-8
https://openalex.org/W2996067255	https://europepmc.org/articles/pmc6957120?pdf=render	English	null	Clinical Results After Single-fraction Radiosurgery for 1,002 Vestibular Schwannomas	Curēus	2,019	cc-by	5,369	Schwannomas Paul Y. Windisch , Joerg-Christian Tonn , Christoph Fürweger , Berndt Wowra , Markus Kufeld , Christian Schichor , Alexander Muacevic 1 2 3 4 1 5 6 1. Radiation Oncology, European CyberKnife Center, Munich, DEU 2. Neurosurgery, Ludwig Maximilian University of Munich, Munich, DEU 3. Medical Physics, European CyberKnife Center, Munich, DEU 4. Oncology, European CyberKnife Center, Munich, DEU 5. Neurosurgery, University Hospital of Munich, Munich, DEU 6. Neurosurgery, Radiosurgery, European CyberKnife Center, Munich, DEU  Corresponding author: Alexander Muacevic, alexander.muacevic@cureus.com DOI: 10.7759/cureus.6390 Results Median follow-up was 3·6 years (1-12·5 years). The three, five, and 10-year Kaplan-Meier estimated local tumor control was 96·6%, 92·3%, and 90·8%, respectively. The median hearing loss of the affected ear as compared to its healthy counterpart was 17 dB at treatment start and increased to 23 and 29 dB at one and five years. Six patients (0·6%) developed symptomatic hydrocephalus and underwent the placement of a ventriculoperitoneal shunt. In 30 patients (3·0%), trigeminal sensory dysfunction developed, five patients (0·5%) had a mild transient weakness, and nine patients (0·9%) had a permanent facial weakness (House-Brackmann Grade > II) after SRS. Methods Clinical data and imaging follow-up were stored in a database of 1,378 patients, with 1,384 VS treated consecutively between 2005 and 2018 and analyzed retrospectively. A total of 996 patients with 1,002 tumors with at least one year of follow-up were included for analysis. Background Herein, we report clinical results for patients treated with stereotactic radiosurgery (SRS) for vestibular schwannomas (VS) over a period of 10 years. Open Access Original Article Open Access Original Article Open Access Original Article Categories: Radiation Oncology, Neurosurgery, Neurology Categories: Radiation Oncology, Neurosurgery, Neurology Keywords: vestibular schwannoma, radiosurgery, acoustic neuroma, radiotherapy, neurofibromatosis © Copyright 2019 © Copyright 2019 Windisch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 3.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Conclusion Received 12/10/2019 Review began 12/10/2019 Review ended 12/14/2019 Published 12/16/2019 Single fraction SRS proves to be highly effective and shows low treatment-related toxicity for VS. SRS should be considered a primary treatment option for small and middle-sized VS. Materials And Methods The treatment records of 1,378 patients with 1,384 VS treated with CyberKnife-based SRS (Accuray Inc., Sunnyvale, CA) at the European CyberKnife Center in Munich between 2005 and 2018 were collected in a database for SRS [8]. CyberKnife is a frameless, image-guided robotic SRS system [9]. The therapeutic radiation is generated by a 6-MV compact linear accelerator mounted on a six-axis robotic manipulator. In a typical VS treatment, 100-200 non-isocentric, non-coplanar beams are directed at the tumor. Intra-fraction patient motion is compensated by the automatic adaptation of beam directions based on stereoscopic X-ray images of the patient’s skull acquired periodically during treatment. Patients who received SRS as a treatment for recurrence after previous radiotherapy were excluded. Two cases where the tumor was considered a surgery-induced metastasis and four cases where patients were treated in more than a single fraction were excluded as well. A total of 996 patients with 1,002 tumors had at least one year of follow-up after SRS and were included for analysis. Follow-ups consisted of a clinical examination and magnetic resonance imaging (MRI). Audiograms were recorded by otorhinolaryngologists elsewhere and added to our database during follow-up. Follow-ups were performed after six months, every year for two years, and every two years thereafter. Tumor response was assessed by MRI. Shrinkage and no change in size were scored as a locally controlled disease. Increased size in two consecutive follow-ups was interpreted as a local recurrence. Facial nerve palsy was assessed using the House-Brackmann (HB) score. Hearing function and ototoxicity were assessed using bilateral serial pure tone audiometry as described previously [10]. Only patients with testable hearing prior to SRS (defined as Gardner-Robertson Class 1-4) were included in the analysis. First, to determine the overall hearing loss, bilateral serial pure tone audiometry was performed including the frequencies 0·5, 1, 2, 4, and 8 kHz. Then, the net hearing loss was calculated at each frequency as the difference between the hearing thresholds of the healthy ear and the affected ear. The mean of the net hearing loss values at the frequencies of 0·5, 1, 2, 4, and 8 kHz defined the overall hearing loss in decibels (dB). Hearing loss attributable to radiosurgery was calculated as the difference between the overall hearing loss at the time of radiosurgery and during follow-up. Worsening of hearing loss attributable to radiosurgery between SRS and follow-up by more than 20 dB was defined as ototoxicity. Introduction Stereotactic radiosurgery (SRS) becomes increasingly popular for mostly small vestibular schwannomas (VS) due to its treatment efficiency and ease of use as compared to surgical How to cite this article Windisch P Y, Tonn J, Fürweger C, et al. (December 16, 2019) Clinical Results After Single-fraction Radiosurgery for 1,002 Vestibular Schwannomas. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 How to cite this article Windisch P Y, Tonn J, Fürweger C, et al. (December 16, 2019) Clinical Results After Single-fraction Radiosurgery for 1,002 Vestibular Schwannomas. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 tumor resection. However, long-term data with reasonably large patient cohorts are missing and few quality-of-life evaluations after SRS for VS have been presented to date [1-2]. Moreover, most published series comprise smaller heterogeneous patient groups treated with inconsistent radiosurgical techniques and doses [3-5]. While there has been a tendency to treat increasingly smaller and sometimes asymptomatic tumors, concerns have been raised regarding long-term hearing toxicity and cases of suspected malignant transformation [6-7]. The purpose of this study was to analyze the functional outcome and the local tumor control after SRS for VS of a large patient group treated with the same technique in a dedicated treatment center over a period of 10 years. 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 Materials And Methods Statistical analysis was performed with the Statistical Package for Social Sciences (SPSS) v. 23.0 (IBM SPSS Statistics, Armonk, NY) and Prism v. 8.0 (GraphPad, San Diego, CA). The significance of time to event data was assessed using the Cox proportional hazards model and the log-rank test. Variables tested for predictive significance concerning local recurrence were age, sex, side of the tumor, NF2 status, prior surgery, tumor volume, and radiosurgical prescription dose. In the case of a toxicity analysis, tumor recurrence was also included as a variable in the models and multivariate analysis was performed accordingly. Local control was plotted as a Kaplan- Meier survival curve for each variable of interest. Continuous variables were split into two 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 2 of 13 groups at their respective median. All data was gathered in accordance with the World Medical Association Declaration of Helsinki. 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 Patient characteristics Patient characteristics are depicted in Table 1. The median age at SRS was 55.1 years (range: 15.1 - 85.2 years) and the median follow-up was 3.6 years (1 - 12.5 years). All tumors were treated in a single fraction, with a median prescription dose of 13 Gy (11.5 - 15 Gy). The median prescription isodose line was 65% (55% - 80%). Tumors that had undergone surgical resection prior to SRS received a median dose of 13.5 Gy. While 827 tumors (82.5%) had not been treated previously, 175 tumors (17.5%) had undergone surgical resection. Of those 175 tumors, 39 received SRS due to subtotal resection while the remaining 136 schwannomas had recurred. Median tumor volume was 0.61 ccm (0.03 - 13.5 ccm). Thirty-one tumors (3.1%) were NF2- associated. 3 of 13 Number of patients 996 Number of tumors 1002 Localization left 530 52.9% right 472 47.1% Sex male 459 46.1% female 537 53.7% Median age [yr] 55.1 (15.1 - 85.2) Pretreatment none 827 82.2% Surgery (Residual Tumor) 39 3.9% Surgery (Local Recurrence) 136 13.5% Follow-up Median [yr] 3.6 (1.0 - 12.5) >= 1 yr 1002 >= 3 yr 609 >= 5 yr 321 >= 10 yr 48 NF2-associated tumors 31 3.1% Median tumor volume [cc] 0.61 (0.03 - 13.5) Median dose [Gy] 13 (11.5 - 15) Median Isodose [%] 65 (55 - 80) TABLE 1: Patient characteristics Numbers in parentheses denote ranges if not specified otherwise TABLE 1: Patient characteristics Numbers in parentheses denote ranges if not specified otherwise Tumor control Three, five, and 10-year follow-up data were available for 609, 321, and 48 tumors, respectively, showing Kaplan-Meier estimates for local control of 96.6% (95% CI: 94.9% - 97.7%), 92.3% (95% CI: 89.8% - 94.3%), and 90.8% (95% CI: 87.2% - 93.9%) (Figure 1). Three, five, and 10-year follow-up data were available for 609, 321, and 48 tumors, respectively, showing Kaplan-Meier estimates for local control of 96.6% (95% CI: 94.9% - 97.7%), 92.3% (95% CI: 89.8% - 94.3%), and 90.8% (95% CI: 87.2% - 93.9%) (Figure 1). 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 4 of 13 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 FIGURE 1: Kaplan-Meier estimates of local control over time The three, five, and 10-year local tumor control was 96.6%, 92.3%, and 90.8%, respectively. Thin lines in the 'Local control over time' plot indicate 95% CI intervals. The number of tumors at risk for each subgroup is depicted below each graph. Larger tumors are associated with significantly reduced local control. Tumor volume was a significant predictor of local control, with larger volumes being associated with worse control in both the Cox proportional hazards model and the log-rank test (Table 2). When splitting the tumors into two groups at the median (0.61 ccm), Kaplan-Meier estimated local control at three, five, and 10 years was 97.4%, 94.4%, and 94.4% for smaller and 95.7%, 90.3%, and 87.7% for larger tumors (Figure 1). Age, sex, side, NF2 status, and dose were not predictive of local control (Figure 2). Surgery prior to SRS was only significant in the univariate analysis. FIGURE 1: Kaplan-Meier estimates of local control over time The three, five, and 10-year local tumor control was 96.6%, 92.3%, and 90.8%, respectively. Thin lines in the 'Local control over time' plot indicate 95% CI intervals. The number of tumors at risk for each subgroup is depicted below each graph. Larger tumors are associated with significantly reduced local control. The three, five, and 10-year local tumor control was 96.6%, 92.3%, and 90.8%, respectively. Thin lines in the 'Local control over time' plot indicate 95% CI intervals. The number of tumors at risk for each subgroup is depicted below each graph. Larger tumors are associated with significantly reduced local control. Tumor volume was a significant predictor of local control, with larger volumes being associated with worse control in both the Cox proportional hazards model and the log-rank test (Table 2). When splitting the tumors into two groups at the median (0.61 ccm), Kaplan-Meier estimated local control at three, five, and 10 years was 97.4%, 94.4%, and 94.4% for smaller and 95.7%, 90.3%, and 87.7% for larger tumors (Figure 1). Age, sex, side, NF2 status, and dose were not predictive of local control (Figure 2). Surgery prior to SRS was only significant in the univariate analysis. Tumor volume was a significant predictor of local control, with larger volumes being associated with worse control in both the Cox proportional hazards model and the log-rank test (Table 2). 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 When splitting the tumors into two groups at the median (0.61 ccm), Kaplan-Meier estimated local control at three, five, and 10 years was 97.4%, 94.4%, and 94.4% for smaller and 95.7%, 90.3%, and 87.7% for larger tumors (Figure 1). Age, sex, side, NF2 status, and dose were not predictive of local control (Figure 2). Surgery prior to SRS was only significant in the univariate analysis. 5 of 13 Hazard ratio 95% CI p Log-rank Age (years) 0.99 0.97 - 1.01 0.169 0.414 Sex (f/m) 1.36 0.76 - 2.42 0.299 0.217 Side (r/l) 1.23 0.70 - 2.17 0.464 0.309 NF2 0.25 0.03 - 1.97 0.19 0.486 Surgery 1.72 0.93 - 3.16 0.086 0.035 Tumor vol (cc) 1.16 1.02 - 1.33 0.033 0.026 Dmin (Gy) 0.82 0.65 - 1.02 0.072 0.262 TABLE 2: Cox proportional hazards model predicting local control reveals tumor volume as the only significant predictive variable Numbers in parentheses denote ranges if not specified otherwise TABLE 2: Cox proportional hazards model predicting local control reveals tumor volume as the only significant predictive variable Numbers in parentheses denote ranges if not specified otherwise 6 of 13 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 1.94 cc (range: 0.59 - 5.34 cc). FIGURE 2: Kaplan-Meier estimates of local control over time Number of tumors at risk for each subgroup is depicted below each graph Creating three subgroups of tumor volumes (< 0.5 ccm, 0.5 - 2 ccm, > 2 ccm) revealed that the smallest tumors showed significantly improved local control as compared to both, the middle- sized (p = 0.0153) and larger schwannomas (p = 0.038), which, in turn, did not differ FIGURE 2: Kaplan-Meier estimates of local control over time Creating three subgroups of tumor volumes (< 0.5 ccm, 0.5 - 2 ccm, > 2 ccm) revealed that the smallest tumors showed significantly improved local control as compared to both, the middle- sized (p = 0.0153) and larger schwannomas (p = 0.038), which, in turn, did not differ significantly from each other. Of the 49 patients who experienced tumor recurrence, 13 received an additional CyberKnife treatment at a median of 3.2 years (2 - 8.5 years) after initial SRS while 16 underwent surgery. The remaining 20 cases were either very recent so that the additional therapy had not been documented at the time of this study or the patients were lost to follow-up. The median follow- up for SRS retreatment was 4.6 years (range: 0.5 - 8.1 years). Local control was achieved in all cases while no grave toxicity was observed. The median volume of the re-irradiated tumors was Of the 49 patients who experienced tumor recurrence, 13 received an additional CyberKnife treatment at a median of 3.2 years (2 - 8.5 years) after initial SRS while 16 underwent surgery. The remaining 20 cases were either very recent so that the additional therapy had not been documented at the time of this study or the patients were lost to follow-up. The median follow- up for SRS retreatment was 4.6 years (range: 0.5 - 8.1 years). Local control was achieved in all cases while no grave toxicity was observed. The median volume of the re-irradiated tumors was 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 7 of 13 Toxicity The HB score prior to treatment was available for 997 tumors. Of the 943 cases with good facial function (HB grades I-II) before SRS, 14 (1.5%) experienced worsening to HB grade III-V, which was transient in five cases. In six of the nine cases where worsening of facial nerve function was permanent, the tumor had recurred and three of these recurrences had already been treated with surgical resection. No patient experienced total facial nerve palsy (HB VI) following SRS. Of the 54 patients with HB grade III-VI prior to SRS, four patients (7.4%) experienced an improvement of facial nerve function to HB I-II, which was permanent in all cases. Valid audiograms prior to treatment and at one year (6 - 18 months) post-treatment were available for 210 patients. Fifty-five patients had valid pre-treatment and five-year (48 - 72 months) post-treatment audiograms. Median hearing loss prior to SRS was 17 dB and increased to 23 dB at one year and 29 dB at five years post-treatment. At one year, 63 ears (30%) experienced an improvement in hearing as compared to the healthy ear while five patients (2.4%) had no change in hearing deficit and 142 patients (67.6%) experienced worsening. However, only 23 of these patients (10.9%) experienced ototoxicity as defined by an increase of hearing loss >= 20 dB. Results of audiograms at one-year post-SRS are depicted in Table 3. Number of patients 210 Median HL pre-treatment [dB] 17 (-45 - 72) Median HL at 1 year [dB] 23 (-40 - 90) Patients with ototoxicity (HL >= 20 dB) 23 10.9% Patients with improved hearing 63 30.0% Patients with worsened hearing 142 67.7% Patients with unchanged hearing 5 2.4% TABLE 3: Hearing toxicity at year one post-SRS Numbers in parentheses denote ranges if not specified otherwise SRS: stereotactic radiosurgery At five years, 12 patients (22.8%) experienced an improvement in hearing as compared to the healthy ear while the remaining 43 patients (78.2%) experienced worsening. However, only 13 (23.6%) of these patients experienced ototoxicity as defined by an increase of hearing loss >= 20 dB post-SRS. The results of audiograms at five years post-SRS are depicted in Table 4. TABLE 4: Hearing toxicity at year five post-SRS Numbers in parentheses denote ranges if not specified otherwise.\ Two patients had seizures during their follow-up period, without any hints suggesting an association with the tumor or the treatment. Treatment-associated hydrocephalus requiring shunt implantation could be observed in five patients (0.5%). One patient developed hydrocephalus due to local recurrence and received shunt implantation combined with microsurgical resection. The median tumor volume of patients with treatment-associated hydrocephalus requiring shunt implantation was 3.38 ccm (0.27 - 7.88 ccm) with five of the six tumors being larger than 2.4 ccm. Thirty-one patients (3.1%) who reported no trigeminal sensory dysfunction at treatment start presented symptoms during follow-up examinations. However, these were permanent in only five patients (0.5%) as defined by having trigeminal sensory dysfunction at each patient’s most recent respective follow-up. No case of malignant tumor transformation was observed. Toxicity Number of patients 210 Median HL pre-treatment [dB] 17 (-45 - 72) Median HL at 1 year [dB] 23 (-40 - 90) Patients with ototoxicity (HL >= 20 dB) 23 10.9% Patients with improved hearing 63 30.0% Patients with worsened hearing 142 67.7% Patients with unchanged hearing 5 2.4% At five years, 12 patients (22.8%) experienced an improvement in hearing as compared to the healthy ear while the remaining 43 patients (78.2%) experienced worsening. However, only 13 (23.6%) of these patients experienced ototoxicity as defined by an increase of hearing loss >= 20 dB post-SRS. The results of audiograms at five years post-SRS are depicted in Table 4. 8 of 13 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 Number of patients 55 Median HL pre-treatment [dB] 17 (-64 - 53) Median HL at 5 years [dB] 29 (-7 - 91) Patients with ototoxicity (HL >= 20 dB) 13 23.6% Patients with improved hearing 12 21.5% Patients with worsened hearing 43 78.2% Patients with unchanged hearing 0 0.0% TABLE 4: Hearing toxicity at year five post-SRS Numbers in parentheses denote ranges if not specified otherwise.\ HL: hearing loss Number of patients 55 Median HL pre-treatment [dB] 17 (-64 - 53) Median HL at 5 years [dB] 29 (-7 - 91) Patients with ototoxicity (HL >= 20 dB) 13 23.6% Patients with improved hearing 12 21.5% Patients with worsened hearing 43 78.2% Patients with unchanged hearing 0 0.0% TABLE 4: Hearing toxicity at year five post-SRS Local tumor control As most of the studies on the long-term safety and efficacy of SRS for VS are based on different treatment technologies, such as the Gamma Knife (Elekta, Stockholm, Sweden), this study, to the best of our knowledge, analyzes the largest patient collectives treated exclusively with CyberKnife, as well as the largest collective treated with SRS in general. The findings concerning local control > 90% even 10 years after treatment are in line with other studies on the long-term efficacy of SRS for VS using Gamma Knife radiosurgery [11]. Given that, in some cases, local recurrence was diagnosed within less than two years following treatment, the actual tumor control might be even higher, as pseudoprogression is a frequent cause of volume change after SRS for VS, especially in the first 24 months post-treatment [12]. The association of reduced local control with increased tumor volumes has been the subject of 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 9 of 13 ongoing discussion [13]. Analyzing local control for tumors smaller and larger than the median volume (0.61 ccm) resulted in local control of 97.4%, 94.4%, and 94.4% at three, five and 10 years for the smaller and 95.7%, 90.3%, and 87.7% for the larger group, which corresponds to a recent study by Ruess et al. where, for a group of 335 patients with a median tumor volume of 1.1 ccm, local control was 89% and 87% at five and 10 years, respectively [14]. These differences, especially in long-term control, should be considered when deciding to place smaller tumors under surveillance. The finding that prior surgery was a significant factor predicting local control in the univariate, but not in the multivariate, analysis might be due to the fact that tumors, which had undergone surgery prior to SRS, had significantly larger volumes (median volume 1.31 ccm, p < 0.0001). The missing dose-effect on tumor control that has been reported by previous publications, including one from this institution, and could be explained by the narrow dose range as 963 of 1,002 tumors were irradiated with 12.5 - 13.5 Gy [10,15]. Contrary to most research on SRS for VS, NF2 was not associated with reduced local control. Given the limited number of NF2-associated tumors (n = 31) in this study (but also in many other publications), drawing definite conclusions from this finding is difficult [16-18]. However, the median tumor volume for NF2-associated tumors was 0.82 ccm, which is considerably smaller than in many existing publications [18]. Mathieu et al. report local control rates of 85% and 81% at five and 10 years following Gamma Knife radiosurgery of NF2-associated VS for 74 tumors with a mean volume of 5.7 ccm [17]. One could, therefore, hypothesize that the effect of reduced local control associated with NF2 loses predictive significance once the tumors are irradiated while at a sufficiently small volume. As many studies on NF2-associated schwannomas already report tumor volume as a predictor of local control, subgroup analyses of these collectives could answer the question, whether treatment of small NF2-associated tumors may result in equally good local control rates as treatment of sporadic VS [16-17]. Retreatment In several cases where the initial SRS treatment could not stop tumor growth, patients were suitable to receive SRS retreatment. While there is still very little data on retreatment with SRS for VS (which makes it difficult to assess the risk associated with the accumulation of radiation dose), retreatment seems to be a safe and effective option. Others have reported good tumor control upon Gamma Knife retreatment as well, but noted a slightly higher rate of facial nerve toxicity, at least compared to the patient group that received SRS as the initial treatment in this study [19]. 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 Hydrocephalus A study by Lee et al. reported a hydrocephalus incidence of 4.1% for 702 patients treated with Gamma Knife radiosurgery and found age, tumor origin, and tumor volume as significant predictors [25]. The higher incidence could be due to a higher mean tumor volume of 3.6 ccm as compared to 1.25 ccm in this study. The median age of the patients who developed hydrocephalus in this study was 55.9 years (48.4 - 74.6 years), only marginally higher than the median age of the whole collective. Hydrocephalus is also a rare complication when treating VS surgically. tumor has been left untreated [21]. Therefore, in the case of small tumors and good hearing, we suggest monitoring the hearing function closely with six-month intervals and suggest treatment only when a hearing decline can be documented and/or the tumor is growing. Hearing A meta-analysis of hearing outcomes following SRS for VS and a study by Santa Maria et al. comprising 344 patients with audiograms and more than three years of follow-up reported hearing preservation of 51% and 50%, respectively, at three years post-treatment [7,20]. Even though ototoxicity, as defined in this study, occurred in only 23.2% of cases, there is a risk of underestimating the extent of ototoxicity, as patients who have no remaining hearing on the treated (or both) ears might stop doing audiograms as part of their follow-up and as a reduction in hearing capacity of the healthy ear reduces the difference between healthy and affected ear. This should be considered when irradiating very small tumors that have not shown significant growth in an attempt to save the patient’s hearing. However, if growth is present, the hearing function has been reported to decline fairly quickly, with patients often losing serviceable hearing within the first five years in cases where the 10 of 13 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 Conclusions SRS is a safe and effective treatment option for treating VS with tolerable toxicities. Though additional and particularly prospective studies are desirable, SRS should be considered a primary treatment option for small and middle-sized vestibular schwannomas. Facial nerve toxicity While facial nerve toxicity is rare as compared to hearing toxicity, it can severely impact the patient’s quality of life. A meta-analysis covering 1,908 patients by Yang et al. described an association between lower marginal doses of 13 Gy or less and reduced facial nerve toxicity for Gamma Knife radiosurgery [22]. However, reducing the dose may result in tumor recurrence, which was seen in the majority of cases with facial nerve toxicity in this study. Additionally, increased tumor volume was associated with higher rates of facial nerve toxicity. Overall, the authors report a facial nerve preservation rate of 96.2%. In a study of facial nerve function after translabyrinthine vestibular schwannoma surgery on 392 patients, 81% had HB grade I-II one year after surgery while 12 patients experienced total facial nerve palsy (HB grade VI) [23]. Falcioni et al. reported anatomical interruption of the facial nerve in 48 out of 1151 cases. Thirty-five percent of the remaining cases where the facial nerve could be preserved had postoperative HB grade III or worse. Smaller tumors had a better facial nerve outcome with postoperative HB grade III or worse occurring in 14% of 444 patients with tumor diameters of less than 1 cm [24]. 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 References 1. Berkowitz O, Han Y-Y, Talbott EO, et al.: Gamma Knife radiosurgery for vestibular schwannomas and quality of life evaluation. Stereotact Funct Neurosurg. 2017, 95:166-173. 10.1159/000472156 1. Berkowitz O, Han Y-Y, Talbott EO, et al.: Gamma Knife radiosurgery for vestibular schwannomas and quality of life evaluation. Stereotact Funct Neurosurg. 2017, 95:166-173. 10.1159/000472156 2. Myrseth E, Pedersen P-H, Møller P, Lund-Johansen M: Treatment of vestibular schwannomas Why, when and how?. Acta Neurochir. 2007, 149:647-660. 10.1007/s00701-007-1179-0 3. Watanabe S, Yamamoto M, Kawabe T, Koiso T, Yamamoto T, Matsumura A, Kasuya H: Stereotactic radiosurgery for vestibular schwannomas: average 10-year follow-up results focusing on long-term hearing preservation. J Neurosurg. 2016, 125:64-72. 10.3171/2016.7.GKS161494 4. Rutten I, Baumert BG, Seidel L, et al.: Long-term follow-up reveals low toxicity of radiosurgery for vestibular schwannoma. Radiother Oncol. 2007, 82:83-89. 4. Rutten I, Baumert BG, Seidel L, et al.: Long-term follow-up reveals low toxicity of radiosurgery for vestibular schwannoma. Radiother Oncol. 2007, 82:83-89. 10.1016/j.radonc.2006.11.019 4. Rutten I, Baumert BG, Seidel L, et al.: Long-term follow-up reveals low toxicity of radiosurgery for vestibular schwannoma. Radiother Oncol. 2007, 82:83-89. 10.1016/j.radonc.2006.11.019 5. Mahboubi H, Sahyouni R, Moshtaghi O, et al.: CyberKnife for treatment of vestibular schwannoma: a meta-analysis. Otolaryngol Head Neck Surg. 2017, 157:7-15. 10.1177/0194599817695805 5. Mahboubi H, Sahyouni R, Moshtaghi O, et al.: CyberKnife for treatment of vestibular schwannoma: a meta-analysis. Otolaryngol Head Neck Surg. 2017, 157:7-15. 10.1177/0194599817695805 6. Maducdoc MM, Ghavami Y, Linskey ME, Djalilian HR: Evaluation of reported malignant transformation of vestibular schwannoma: de novo and after stereotactic radiosurgery or surgery. Otol Neurotol. 2015, 36:1301-1308. 10.1097/MAO.0000000000000801 7. Santa Maria PL, Shi Y, Gurgel RK, et al.: Long-term hearing outcomes following stereotactic radiosurgery in vestibular schwannoma patients—a retrospective cohort study. Neurosurgery. 2018, 85:550-559. 10.1093/neuros/nyy407 8. Kufeld M, Fürweger C, Drexler CG, Wowra B, Muacevic A: Implementation of a medical database system for a radiosurgery center. Cureus. 2009, 1:e4. 10.7759/cureus.4 9. Adler JR Jr, Chang SD, Murphy MJ, Doty J, Geis P, Hancock SL: The Cyberknife: a frameless robotic system for radiosurgery. Stereotact Funct Neurosurg. 1997, 69:124-128. 10.1159/000099863 10. Wowra B, Muacevic A, Fürweger C, Schichor C, Tonn J-C: Therapeutic profile of single- fraction radiosurgery of vestibular schwannoma: unrelated malignancy predicts tumor control. Neuro Oncol. 2012, 14:902-909. 10.1093/neuonc/nos085 11. Disclosures Human subjects: Consent was obtained by all participants in this study. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: Joerg- Christian Tonn declare(s) personal fees from Brainlab, Medac. JT received speaker honoraria from Brainlab and Medac outside of the submitted work. Christoph Fuerweger declare(s) personal fees from Accuray. CF received speaker honoraria from Accuray outside of the submitted work. Other relationships: All authors have declared that there are no other 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 11 of 13 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 relationships or activities that could appear to have influenced the submitted work. References Hasegawa T, Kida Y, Kato T, Iizuka H, Kuramitsu S, Yamamoto T: Long-term safety and efficacy of stereotactic radiosurgery for vestibular schwannomas: evaluation of 440 patients more than 10 years after treatment with Gamma Knife surgery. J Neurosurg. 2013, 118:557- 565. 10.3171/2012.10.JNS12523 12. Hayhurst C, Zadeh G: Tumor pseudoprogression following radiosurgery for vesti schwannoma. Neuro Oncol. 2012, 14:87-92. 10.1093/neuonc/nor171 13. Huang C-W, Tu H-T, Chuang C-Y, Chang C-S, Chou H-H, Lee M-T, Huang C-F: Gamma Knife radiosurgery for large vestibular schwannomas greater than 3 cm in diameter. J Neurosurg. 2018, 128:1380-1387. 10.3171/2016.12.JNS161530 14. Rueß D, Pöhlmann L, Hellerbach A, et al.: Acoustic neuroma treated with stereotactic radiosurgery: follow-up of 335 patients. World Neurosurg. 2018, 116:194-202. 10.1016/j.wneu.2018.04.149 15. Anniko M, Arndt J, Norén G: The human acoustic neurinoma in organ culture. II. Tissue changes after gamma irradiation. Acta Otolaryngol. 1981, 91:223-235. 10 3109/00016488109138503 16. Kruyt IJ, Verheul JB, Hanssens PEJ, Kunst HPM: Gamma Knife radiosurgery for treatment of growing vestibular schwannomas in patients with neurofibromatosis type 2: a matched cohort study with sporadic vestibular schwannomas. J Neurosurg. 2018, 128:49-59. 10 3171/2016 9 JNS161463 17. Mathieu D, Kondziolka D, Flickinger JC, Niranjan A, Williamson R, Martin JJ, Lunsford D: Stereotactic radiosurgery for vestibular schwannomas in patients with neurofibromatosis type 2: an analysis of tumor control, complications, and hearing preservation rates. Neurosurgery. 2007, 60:460-470. 10.1227/01.NEU.0000255340.26027.53 18. Sharma MS, Singh R, Kale SS, Agrawal D, Sharma BS, Mahapatra AK: Tumor control and hearing preservation after Gamma Knife radiosurgery for vestibular schwannomas in 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 12 of 13 2019 Windisch et al. Cureus 11(12): e6390. DOI 10.7759/cureus.6390 neurofibromatosis type 2. J Neurooncol. 2010, 98:265-270. 10.1007/s11060-010-0181-1 19. Fu VX, Verheul JB, Beute GN, Kunst HPM, Mulder JJS, Hanssens PEJ: Retreatment of vestibular schwannoma with Gamma Knife radiosurgery: clinical outcome, tumor control, and review of literature. J Neurosurg. 2018, 129:137-145. 10.3171/2017.3.JNS162033 20. Yang I, Sughrue ME, Han SJ, Aranda D, Pitts LH, Cheung SW, Parsa AT: A comprehensive analysis of hearing preservation after radiosurgery for vestibular schwannoma. J Neurosurg. 2010, 112:851-859. 10.3171/2009.8.JNS0985 21. Kondziolka D, Mousavi SH, Kano H, Flickinger JC, Lunsford LD: The newly diagnosed vestibular schwannoma: radiosurgery, resection, or observation?. Neurosurg Focus. 2012, 33:8. 10.3171/2012.6.FOCUS12192 22. Yang I, Sughrue ME, Han SJ, et al.: Facial nerve preservation after vestibular schwannoma Gamma Knife radiosurgery. J Neurooncol. 2009, 93:41-48. 10.1007/s11060-009-9842-3 23. Brackmann DE, Cullen RD, Fisher LM: Facial nerve function after translabyrinthine vestibular schwannoma surgery. Otolaryngol Head Neck Surg. 2007, 136:773-777. 10.1016/j.otohns.2006.10.009 24. Falcioni M, Fois P, Taibah A, Sanna M: Facial nerve function after vestibular schwannoma surgery. J Neurosurg. 2011, 115:820-826. 10.3171/2011.5.JNS101597 25. Lee S, Seo S-W, Hwang J, Seol HJ, Nam D-H, Lee J-II, Kong D-S: Analysis of risk factors to predict communicating hydrocephalus following gamma knife radiosurgery for intracranial schwannoma. Cancer Med. 2016, 5:3615-3621. 10.1002/cam4.955 13 of 13
https://openalex.org/W2012298606	https://zenodo.org/records/1978389/files/article.pdf	English	null	No. VIII.-AVES, WITH SOME NOTES ON THE DISTRIBUTION OF THE LAND-BIRDS OF THE SEYCHELLES.	Transactions of the Linnean Society of London. Zoology/Transactions of the Linnean Society of London. Zoology.	1,907	public-domain	3,949	* Vide ‘The Fauns and Geography of the Maldive and Laccadive Archipelagoes,’ ~01. i. p. 368. INTRODU BIRDS were only collected on the coral-islands visited by the expedition, no attempt having been made to obtain those of Mauritius and the Seychelles. The common land- birds at present found in the latter group are of little interest, being all recent introductions, Of the indigenous forms probably more than half are now extinct, owing to the exertions of paid collectors. There was, it is true, a law for the protection of birds, but it was copied from that of Maiiritius ; and the birds protected were those found in Mauritius and not in the Seychelles. y Most are marine or waders, or of recent introduction by man. A pigeon and a few harriers and hawks, probably those found in the Malclive Group, are said to visit the Chagos Archipelago about December, i. e. during the prevalence of the north-east monsoon in the northern hemisphere. Guinea-fowl have been released in several islands and breed in Salonion, Chagos. None of the birds found in the list below are peculiar to the islands. From the point of view of distribution none of the birds happen to be oE any interest, except the little green bittern, Bzctorides atricapilla. This genus has a wide circum- tropical distribution-B. virescens in North and Central America, B. striata in South America, B. plzcmbea i n Galapagos, B. stagnatilis in Australia, Papuasia, and Pacific islands, B.javanica in India and Malaya (also said to live on Mascarenes and Diego Garcia), and B. atricapilla in Africa and Madagascar. The Providence and Darros birds are distinctly smaller than B. javanica and in the younger specimens the throat is streaked as in B. africapilla; they are decidedly more African than Indian, but the Chagos specimens are more intermediate. Bzlbulcus ibis is African against the Indian B. coromalzdzcs. The annexed list serves to illustrate the character of the avifauca of the Seychelles as compared with that of the neighbouring Mascarene Islands, Madagascar being included for comparison. It was prepared by Dr. Gadow for the International Congress of O m i t h o l o v 4 . . PERCY SLADEN TRUST EXPEDITION. 104! I Falculia palliata .................... Pregilnpus varius .................... Necropsar rodericanus ................ Foudia conaobriria. ................... algondse .................... aldabrana .................... madagascar .................. bruante.. .................... erythrocephala ................ flavicans .................... seychellarnm .................. Cinnyris comorensis .................. aldabrensis .................. humblot'i ................... angaladiana ................ souinianga .................. dussumieri .................. Zosterops anjuanensis ................ kirki ...................... aldabrensis ................ madagascariensis ............ INTRODU borbonica .................. hresitata .................. E. newtoni ................ mauritiana ................ chloronotus ................ semiflava .................. modesta .................. borbonica.. ................ Pratincola sibylla .................... Copsgchus pioa ...................... eeychellarum .............. Hypsipetes rostratua .................. parviroetrie ................ ourovang ................ borbonicus ................ olivacsus ................ crassirostris .............. comorensis .............. mutata .................. borbonica ............... corvina ................. Oxynotus rufiventris ............... newtoni ................. Phedina madagascariensis ........... borbonica ................. Necropsittacue rodericanus ........... Lophopeittacus mauritianus ........... Coracopsis comorensis ............... Terpsiphone vulpina ................. , sibilans ................. vaza ................... n i p ................... barklya ................. Masaarinus duboisi ................. Palreornis equcs ................... exsul .................... wardi .................... Ilesidenl. Land-Birds only. '_ X .. .. .. X .. .. .. .. X X .. .. .. .. .. .. X X .. .. .. X X .. .. . .. .. 9 . .. * . . d .. .. .. .. .. X .. .. X X .. .. .. . I .. .. .. .. .. X X .. .. * . .. I . .. .. .. .. .. X .. .. .. .. .. X .. .. .. X .. .. .. .. .. ,. * . .. .. .. .. .. X .. .. -. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. X .. .. X .. .. .. .. X X .. X .. .. * . * . .. .. .. X X .. .. X .. .. .. X .. .. .. .. X .. .. .. X X .. .. .. .. .. .. x l x X X .. .. .. .. .. X - .. X .. X x X X X - X x GADOW -4ND QABDIR'EH-AVES. 105 .. .. X .. I .......... I - * ErythrcEna madagaacariensiis sauzieri ........... .. pulcberrima .............. ' * * * * * ! * . nitidissima .............. .. Turtur comorensis ................. . I x aldebranus ................. ' .. piaturatus .................... .. meyeri .................... . . I ' * . ..I * . Scops rutilus ........................ .. capnodes ..................... ./ x Gymnoscops insularis ................ .. Tinnunculus newtoni ................ .. punctatus ................ ' .. gracilis * I ' * . . I .................. rostratus ................. I X I .. '. I .. ! .. .. ' X .. , ... .. x .. .. .. 1 . .. * * ~ x l x :: I ,. .. .. .. INTRODU X .. 1 x .. .. X x ! i I ~ ! I X I .. 1 x , This list in the first place illustrates the effects of insularity on birds, the genera Fozcdia, Zosterops, Hyps@etes, Terpsiphone, and Twtur having peculiar species in all or most of the Mascarenes. It serves also to show the close relationship between the avifaunas of all these islands. None of the islands in question are situated at any great distance from the others, and it would necessarily follow that the dispersal of land-birds to island groups is a much less frequent incident than usually supposed, Otherwise it must be assumed that land-birds are extraordinarily plastic, capable indeed of adaptation in a, very few generations to their environment. Is it possible that all the genera specially mentioned above, represented each by one or two species, could at one time have been spread over all the islands, and have subsequently become isolated in the different localities by the disappearance ot' intervening lands ? INTRODU If the genera and species be examined so as to trace the affinity of the avifauna, we get the following facts :-Fulculiu acd Fregilupus are local genera of indefinite relationships ; Foudicc is essentially Madagascan and African ; Cinnyris has many species in India, but those in question are all of A f i h n affinity; Zouterops and Pmtincolu are found both in India and Africa, but the species are allied to those of the latter country ; Copsychz~s and Hypsipetes are essentia!ly Indian genera, having no species in Africa ; Terpsiphone is an Indian-Austral-African genus and its species show no special affinities ; Oxynotus is a local genus, allied in Africa and India ; Phedirta is a local subgenus of swallows and is useless for our purpose ; Corncopsis is a Malagasy genus, its allies being two species of African Psittacus (a grnus which certainly came I from the east) and one Dasyptih of Xew Guinea; Xuscj iainus is a local genus allied to the last, its only other close relationship being Auq tralasian ; Necropsittuczcs and Lophopsittacus were possibly Cacatuine, and in this :case their affinities would be Australasian ; Pula?ornis is African a and Indian, but its spel-2~ exsul probably and epzces and wardi certainly are related to Iudirtn; Erythrcena is a genus of the Malagasy district with absolute Malay and Australasian relationship (Ptilinopzcs) against I're~on which is African, Indian, and E C O N DS S OO OG XII 15 ~ E C O N D SERIES.-ZOOLOGY, VOL. XII. 15 106 PERCT SLADEN TRUST EXPEDITION. Australian; Turtur and 5!'V!nnunculus are useless for our purpose; #cops ~ U t i l l l 8 is a subspecies of magicue and is certainly Indo-Malayan or Moluccan, but the genus is also African and of no use; and, lastly, Gymnoscops ilzezclaris is at best a race of Xt&x Rammea, which is cosmopolitan. Including the herons, we find the following to be western or African : Fozcdia in its genus, and Bzcbulczcs, Butorides, Cinnyris, Zosterops, and Prcltincola in their species. Eastern or Iado-Malayan would be in species Palmorrbis and Scops, and in genera Copy C h 8 , Hyp8ipetC8, Erythrcena, and the parrots ~ecropsittacus, Lophopsittacus, Coiwcopsis, and Mascarinus. The ultimate result of this investigation is rather startling. INTRODU African and Indo-Malayan influences seem to balance, but it so happens that (I) the western immigrants are all birds of poor flight, either bitterns, 11 hich do not make long voyages, or small birds like the weavers, sunbirds, and girdle-eyes, all of which have come from the nearest available laud, i. e. Africa and Madagascar, by the natural island bridges, and (2) the eastern immigrants are all birds of good flight with no tiny forms amongst them. Copsychzcs and Hypsipetes fly well, as do parrots, pigeons, and owls, though the latter perhaps may be ruled out as belonging to an indifferent genus (although its species is eastern). Finally, it is interesting to observe that parrots and pigeons must be regarded as ancient groups, while Cinnyris, Zosterops, and Foudia are as certainly of very recent date. One parrot, Lophopsittacus, extinct now, is supposed to have been flightless ; in this respect it has parallels in the extinct dodo and solitaire and in tLe flightless rail, Erytlwomccchzcs. 3. Foudia rmadagascar. Weaver. This little weaver is found in all the inhabited islands or groups we visited, except It must be regarded every- Cargados Cltrajos, feeding on scraps of coconuts, &c. where as an intentional introduction of man. 2. Cr i t hag r a c h r y 8 op y g a. ,4 small finch intentionally carried to Desroches, Amirantes, and now found wild all o\ er the island. 1. Passer domesticzce. Sparrow. 1. Passer domesticzce. Sparrow. Found around habitations in all the islands visited. t The names employed throughout aro those of the British Museum Cataloguen. See also The Fauna of British * The names of birds with an asterisk prefixed aa abow were species observed br the expeditiou, but specimens India: Aves,' by R. T. Blanford. were not brought home, * The names of birds with an asterisk prefixed aa abow were species observed br the expeditiou, but specimens , y were not brought home, t The names employed throughout aro those of the British Museum Cataloguen. See also ' The Fauna * India: Aves,' by R. T. Blanford. 9. Pramcolinzcs pon.diceriamus. Grey Partridge. 9. Pramcolinzcs pon.diceriamus. Grey Partridge. 9. Pramcolinzcs pon.diceriamus. Grey Partridge. This bird, which is really a native of India, has been introdncsd into Mauritius and practically all the islands between Seychelles and Madagasmr for sporting purposes. 10. Tiling a 8th bar qu a t a. Very common between Madagascar and Seychelles in September and October, feeding Curlew-Stint. on the mud-flats ; a single specimen obtained on Salomon, Chagos, i u May. 10. Tiling a 8th bar qu a t a. Very common between Madagascar and Seychelles in September and October, feeding Curlew-Stint. on the mud-flats ; a single specimen obtained on Salomon, Chagos, i u May. Avocet-Sandpiper. 11. Ter e k i Q ciner ea. A single specimen out of a small flock of four from the southern islands of Providence. Avocet-Sandpiper. 11. Ter e k i Q ciner ea. A single specimen out of a small flock of four from the southern islands of Providence. 12. Gallinago ccelestis. Full Snipe. A small wisp, probably helonging to this species, was seen by a freshwater pool in one of the islands of S. Joseph Atoll, Amiruntes. 13. Totunus stagnatilis. Little Greenshank. A single specimen from the southern islands of Providence at the beqinning of October ; not found elsewhere, except in the Seychelles. 13. Totunus stagnatilis. Little Greenshank. A single specimen from the southern islands of Providence at the beqinning of October ; not found elsewhere, except in the Seychelles. 141. T o t a n u s fwczcs. Spotted Redshank. Several flocks were seen during August and September at Coetivy and Curgados Carajos, but not found elsewhere. 141. T o t a n u s fwczcs. Spotted Redshank. Several flocks were seen during August and September at Coetivy and Curgados Carajos, but not found elsewhere. 16. ATu?nenius asaquata. Curlew. 16. Nu?nenius phceopus. Whimlurel. The Curlew and Whinlbrel are regular inhabitants of the marshes and shores of all islands between India and Madagascar. They were seen and co:istitntly shot by different members of our par@ from May to December, but they are not known to breed in any of the islands. 4. "Hiru9hdo rzcstica, 8wallow. b. Hirando zcrbica. Martin. Isolated birds belonging to two species of Hiivndo, probably the above, were seen flying about the different atolls of the Chaqos from May to July. Isolated birds belonging to two species of Hiivndo, probably the above, were seen flying about the different atolls of the Chaqos from May to July. t The names employed throughout aro those of the British Museum Cataloguen. See also ' The Fauna of British India: Aves,' by R. T. Blanford. * The names of birds with an asterisk prefixed aa abow were species observed br the expeditiou, but specimens were not brought home, 107 GADOW AXD GABDINER-AYES. 6. * 0 i w c u l u religiosa. Mynah-Starlinp. Escaped from captivity, and now found wild all over Egmont Atoll, Chagos. 6. * 0 i w c u l u religiosa. Mynah-Starlinp. Escaped from captivity, and now found wild all over Egmont Atoll, Chagos. 7. T w t u r pictwpatzcs. Turtle-Dove. Very common in Faiqiihar and Seychelles, but not in the intervening islands. Probably intentionally introduced everywhere from Madagascar. 7. T w t u r pictwpatzcs. Turtle-Dove. 7. T w t u r pictwpatzcs. Turtle-Dove. 7. T w t u r pictwpatzcs. Turtle-Dove. Very common in Faiqiihar and Seychelles, but not in the intervening islands. Probably intentionally introduced everywhere from Madagascar. 7. T w t u r pictwpatzcs. Turtle Dove. Very common in Faiqiihar and Seychelles, but not in the intervening islands. Probably intentionally introduced everywhere from Madagascar. 8. Geopelicc striata. Barred Ground-Dove. 8. Geopelicc striata. Barred Ground-Dove. 8. Geopelicc striata. Barred Ground-Dove. h r g e numbers of these bii-ds live in the trees around the settlement at Parquhar. It is an Indo-Malayan species, and was no doubt intentionally introduced by natives from India by way of Mauritius. 8. Geopelicc striata. Barred Ground Dove. h r g e numbers of these bii-ds live in the trees around the settlement at Parquhar. It is an Indo-Malayan species, and was no doubt intentionally introduced by natives from India by way of Mauritius. 23. Xterlza aneatheta. Panayan Tern. 23. Xterlza aneatheta. Panayan Tern. 23. Xterlza aneatheta. Panayan Tern. These two terns were constantly seen during September and October in the coral- islands to the north of ‘5ladagascar; they often came on board the Sealark,’ resting on the masts. The Wide-awake breeds in enormous numbers on the islands of Cargaclos Carajos, laying their single large eggs upon the ground within a foot or eighteen inches of one another. We took large numbers of eggs in August, but saw no young birds. The same species also breeds on certain rocky islets in the Seychelles. So far as our observations go, it never occurs on the same islands as S, fuliginoacc, ant1 indeed rather shuns the locality of its “ Fairs.” The same is also true to a large extent of S. melanauchen in respect to Gygis cunclida, and of Anous stoliclus to A . Zetwocupillus, but these species mny select different habitats (shore and trees) on the same island. 8. anmthetu breeds on some of the islands of Providence and of the Amirantes. Neither species was seen in the Chagos. 30. ~ ’ g i a Zit i s cn ri t ia n 4 = u lexu n d r i n u. Kentish Plover. A single specimcn from Providence ; observed also in Farquhar. 24. St ern a II erks t e ini. Small flocks of these birds were seen on every island we visited and often met with at sea. We only brought specimens from Peros Banhos, Chagoa, and Darros, Amirantes. .It was never definitely found breeding, though two or three pairs appeared to be occupying certain trees in Ile Lubine, Egmont, Chagos. 25. S t e m u enulzder8i. Black-shafted Ternlet. 25. S t e m u enulzder8i. Black-shafted Ternlet. Large numbers of these birds were seen on the southern islets of Providence, but not elsewhere. 18. Lirnosa lapponicn. Bar-tailed Godwit. A single specimen from Farquliar ; seen also on Providence 18. Lirnosa lapponicn. Bar-tailed Godwit. A single specimen from Farquliar ; seen also on Providence. 19. Squaturoln helvetica. Grey Plover. 19. Squaturoln helvetica. Grey Plover. q y Only two specimens were obtained, an immature bird from Peros Banhos, Chagos, and an adult from Farquhar. It was observed also at Salomon, Chegos, and at Eagle Island, Amirantes. Only two specimens were obtained, an immature bird from Peros Banhos, Chagos, and an adult from Farquhar. It was observed also at Salomon, Chegos, and at Eagle Island, Amirantes. 21. Drornus urdeola. Crab-Plover. 21. Drornus urdeola. Crab-Plover. Small flocks of four to ten of these birds followed the receding tide over every reef we We saw one flock near Ile d’Ambre, Mauritius, and one off Cbte d’Or, Praslin, visited. Seychelles. They are said to breed in the Chagos about the month of December. Small flocks of four to ten of these birds followed the receding tide over every reef we We saw one flock near Ile d’Ambre, Mauritius, and one off Cbte d’Or, Praslin, visited. Seychelles. They are said to breed in the Chagos about the month of December. 22. Xterna fmliginoso. Sooty Tern or ‘‘ Wide-awako.” 22. Xterna fmliginoso. Sooty Tern or ‘‘ Wide-awako.” 17. St r ep B i 1 as i n t e rp 1- e 8. Turnstone. 17. St r ep B i 1 as i n t e rp 1- e 8. Turnstone. Flocks of six or eight of these birds were commou on the shores of all the islands we visited, being particularly abundant at Diego Garcia, Chagos, where they fly from barachois to barachois. 108 PERCY SLADEN TRUST EXPEDITION. 18. Lirnosa lapponicn. Bar-tailed Godwit. A single specimen from Farquliar ; seen also on Providence. 26. Sterna melanauchen. Black-naped Tern. 26. Sterna melanauchen. Black-naped Tern. 26. Sterna melanauchen. Black-naped Tern. Found over the whole Indian Ocean. It was found breeding on the trees of Yey&, Yeros, in June, its single egg balanced on the bare tbrunks of Z’ournefo~tiu or t.he leaf- bases of coconuts. On Providence and Farquhar it was, however, a sliorsbreeder. . 109 GADOW ASD GARDIXER-ATE% 27. Gygis cntidicla. Wliite Noddy. 28. A no us 1 euc ocap i I lus = t enuiros t ?is. White-headed Noddy. 28. A no us 1 euc ocap i I lus = t enuiros t ?is. White-headed Noddy. 29. An.ous stolidus. Noddy. 29. An.ous stolidus. Noddy. These two species of noddies were found all over the western half of the Indian Ocean which we visited, breeding everywhere from May to November. They breed indifferently on the ground or in trees, commonly selecting the bases of the coconut-leaves. The two species never seem to occupy the same breeding-habitnt in any island or locality. Thus on Petite Coquillage, Peros Banhos, in June, though it is evidently not the season of the large '' Fair," a few A. stolidus were found breeding on t h ground and some A. leucocapillus in the trees. The latter bird, with Xterna fuliginosa, is the chief guano- former of Cargados Carajos ; as the ground is occupied, it lays its single eggs on the tops of the low bushes, which are all matted together by CassytAa filiformis ('' liane sans f euilles " ) . A, stolidus was not found by us in the Maldives in 1899-1900. A, stolidus was not found by us in the Maldives in 1899-1900. 30. Bubulcus ibis. Egret. 27. Gygis cntidicla. Wliite Noddy. This bird ranges over the wliole western part of the Indian Ocean. We found it breeding on the diores of Petite Coqnillage, Peros Banhos, in June, and on those of Cargados Carajjos in August. In Diego Garcia, Chagos, and in Coetivy it is a tree- breeder. 33. P u ff in u s t e nu i r o s t r is. 33. P u ff in u s t e nu i r o s t r is. This bird was found breeding in large numbers on several islands of S. Joseph Atoll, Amirante Group, in October 1905. It is very similar in its breeding-habits to P.persicus (Fauna and Geog. of the Mald. and Lacc. Archip. vol. i. p. 369), breeding under the coconut-trees in Cardiosoma-holes, but forming its own burrows in the mop0 open sandy islands of the reefs. 32. "Brdea cinerea. Common Grey Heron. 32. "Brdea cinerea. This bird was only found on the southern islands of Providence reef, where considerable iiuinbers were breeding at the end of September in the low Xccevola and Tournefortia bushes. Common Grey Heron. 32. Brdea cinerea. This bird was only found on the southern islands of Providence reef, where considerable iiuinbers were breeding at the end of September in the low Xccevola and Tournefortia bushes. Common Grey Heron. 31. Butorides atricapilla. Little Green Heron. 31. Butorides atricapilla. Little Green Heron. 31. Butorides atricapilla. This heron was seen in every group of islands which we visited. It feeds along tho shore, darting off into the densest bush when disturbed. It is particularly common on the islands where terns breed, feeding on their eggs. Young birds were seen in June on Petite Coquillage, Peros Banhos. The four specimens brought home from the Chagos all belong to the above species. Little Green Heron. 31. Butorides atricapilla. This heron was seen in every group of islands which we visited. It feeds along tho shore, darting off into the densest bush when disturbed. It is particularly common on the islands where terns breed, feeding on their eggs. Young birds were seen in June on Petite Coquillage, Peros Banhos. The four specimens brought home from the Chagos all belong to the above species. Little Green Heron. 30. Bubulcus ibis. Egret. A single specimen from Cargados Carajos, seen also in Mauritius. It is an African species, replacing the Indian B. coroinundua in this region. A single specimen from Cargados Carajos, seen also in Mauritius. It is an African species, replacing the Indian B. coroinundua in this region. 34. Xula piscator. Red-legged Booby. 16 PERCY SLADEN TRUST EXPEDITION. 110 Reef. They were apparently collecting for breedingpurposes at the beginning oE October 1905, their nests being placed in the low mapou (Pieonia) and tanghain shrub covering the island, They were also seen at Farquhar, Alphonse, and the Amirantes. The guano of Bird Island, Seychelles, was principally formed by a great colony of this species. 35. Pelecanue criepzce. These large birds were found breeding in a colony in the coconut and other large trees Young birds were seen in of tlie eastern island OP 5. Joseph Atoll, Amirante Group. October 1905. 36. Phaethon lepturue= f lavirostrie. Tropic-bird. A single pair of these birds were seen on Diego Garcia, Chagos, where they were stated to have bred for some years in a large mapou (Pieonia) tree. Tropic-birds were next seen in the Seychelles, flying around the mountain-summits of MahB. 37. Fregata ariel. Smaller Frigate-bird. 37. Fregata ariel. Smaller Frigate-bird. Nelson Island, to the north of the Great Chagos Bank, is a regular breedingplace for these birds, the eggs and young being often taken in December and January by boats from Salomon Atoll. From thence they are daily visitors to every islaiid of the Chagos Archipelago, always apparently returning at night.
W4390792920.txt	https://www.nature.com/articles/s41598-024-51195-4.pdf	en		Numerical investigation of heat and mass transfer in three-dimensional MHD nanoliquid flow with inclined magnetization	Scientific reports	2,024	cc-by	9,696	www.nature.com/scientificreports OPEN Numerical investigation of heat and mass transfer in three‑dimensional MHD nanoliquid flow with inclined magnetization Ahmed M. Galal 1,2, Fahad M. Alharbi 3, Mubashar Arshad 4,5,6, Mohammad Mahtab Alam 7, Thabet Abdeljawad 8,9,10 & Qasem M. Al‑Mdallal 11* Heat and mass transfer rate by using nanofluids is a fundamental aspect of numerous industrial processes. Its importance extends to energy efficiency, product quality, safety, and environmental responsibility, making it a key consideration for industries seeking to improve their operations, reduce costs, and meet regulatory requirements. So, the principal objective of this research is to analyze the heat and mass transfer rate for three-dimensional magneto hydrodynamic nanoliquid movement with thermal radiation and chemical reaction over the dual stretchable surface in the existence of an inclined magnetization, and viscous dissipation. The flow is rotating with constant angular speed ω∗ about the axis of rotation because such flows occur in the chemical processing industry and the governing equations of motion, energy, and concentration are changed to ODEs by transformation. The complex and highly nonlinear nature of these equations makes them impractical to solve analytically so tackled numerically at MATLAB. The obtained numerical results are validated with literature and presented through graphs and tables. Increasing the Eckert number from 5 ≤ Ec ≤ 10, a higher Nusselt and Sherwood number was noted for the hybrid nanofluid. By changing the angle of inclination α, the Nux performance is noted at 8% for nanofluid and 33% for hybrid nanofluid. At the same time, Shx performance of 0.5% and 2.0% are observed respectively. Additionally, as the angle of inclination increases the skin friction decreases and the chemical reaction rate increases the mass transmission rate. List of symbols ω∗ Angular velocity Cp Specific heat φ1 , φ2 Volume fraction for nanoparticles G1 , G2 , G3 , G4 Constants for nanofluid T, C Temperature and concentration 1 Department of Mechanical Engineering, College of Engineering in Wadi Alddawasir, Prince Sattam Bin Abdulaziz University, Al‑Kharj, Saudi Arabia. 2Production Engineering and Mechanical Design Department, Faculty of Engineering, Mansoura University, P. O 35516, Mansoura, Egypt. 3Department of Mathematics, Al‑Qunfudah University College, Umm Al-Qura University, Mecca, Saudi Arabia. 4Department of Mathematics, University of Gujrat, Gujrat 50700, Pakistan. 5Institute for Numerical and Applied Mathematics, University of Göttingen, 37083 Göttingen, Germany. 6Department of Mathematics, Abbottabad University of Science & Technology, Abbottabad, 22500, Pakistan. 7Department of Basic Medical Sciences, College of Applied Medical Science, King Khalid University, 61421 Abha, Saudi Arabia. 8Department of Mathematics and Sciences, Prince Sultan University, P.O. Box 66833, 11586 Riyadh, Saudi Arabia. 9Department of Medical Research, China Medical University, Taichung 40402, Taiwan. 10Department of Mathematics and Applied Mathematics, Sefako Makgatho Health Sciences University, Garankuwa 0204, Medusa, South Africa. 11Department of Mathematical Sciences, UAE University, P.O. Box 15551, Al Ain, United Arab Emirates. *email: imbashrii@gmail.com; tabdeljawad@ psu.edu.sa; q.almdallal@uaeu.ac.ae Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ Z Porosity parameter a, b Stretching rate along x and y axis Pr Prandtl number π Thermal radiation parameter α Inclination angle B0 Magnetic field Nt , Nb Thermophoresis and Brownian motion parameter qw Heat flux R Reynolds number Kc Chemical reaction g ∗ Gravitational acceleration αhnf Thermal diffusivity Rotational velocity ρ Density γ Stretching ratio parameter k Thermal conductivity Nux , Shx Nusselt and Sherwood number coefficient η Similarity variable u, v, w Velocity components in x,y,z direction Cf x , Cf y Skin frictions p′, q′ Dimensionless velocity H1 , H2 , H3 , H4 Constants for hybrid nanofluid Sc Schmidt number DB Brownian diffusion βt Thermal expansion vf Kinematic viscosity µf Dynamic viscosity r, s Temperature and concentration profile 3D Three dimensional σ Electrical conductivity τw Shear stress ǫ Mixed convection parameter In a myriad of industrial applications, ranging from nuclear reactors and automobiles to electronics, the role of fluids is pivotal in enhancing heat transfer rates (HTR). Traditional fluid options like water, oils, and ethylene glycol, however, exhibit limited thermal conductivity. This limitation has spurred extensive research endeavors aimed at elevating HTR for improved efficiency and performance. First, Choi and E astman1 coined the term "nanofluid (NF)" by mixing non-metallic or metallic nanoparticles (NPs) in host fluid which dramatically augments HTR. The characteristics of such fluids depend upon different factors like the shape and size of suspended nanoparticles in host fluid, thermal conduction, etc. Later, many i nvestigators2–4 followed his idea and worked on the enhancement of HTR. Similarly, the mixing of two or more nano-sized particles in the host fluid is known as a hybrid nanofluid (HNF). Continuous and different strategies have been adopted to enhance HTR. Jena et al.5 explored the recent development in heat transfer (HT) characteristics of NF by considering the non-uniform heat source and inclined magnetization. Parida et al.6 computationally discussed the dust particles in water and kerosene-based nanoliquid for HTR. Pattnaik et al.7,8 used different NPs like copper, aluminum, gold, and singlewall carbon nanotubes to explain HTR by using water as host fluid over permeable surfaces. In industrial sectors like aerodynamics, plastic sheet extrusion, continuous metallic plate extrusion, artificial fiber synthesis, and plastic film magnification, the motion of an incompressible fluid over an expanding surface is a frequently observed occurrence. The HTR at the deformable surface plays a substantial role in determining the overall quality of the product in each of these applications. S akiadis9 gave the thought of boundary layer (BL) enhancement during the movement of the surface and attracted researchers’ attention. The Sakiadis’ problem was expanded by Erickson et al.10 who also looked at the causes of puffing or sucking at the moving sheet on HMT in the BL flow. Baag et al.11 for exploration of MHD boundary layer flow over porous exponentially SS with a uniform heat source. Nayak et al.12 considered the radially stretched sheet for discussion by incorporating the variable magnetic field. Mishra et al.13 investigated the HT influence on the MHD movement of micropolar liquid passing from a permeable medium considering a similar heat source. Upreti et al.14 used the Casson NF over SS utilizing the Cattaneo-Christov model in stagnation point flow to examine the shape factor. Seini and Makinde15 explored the magnetohydrodynamic (MHD) BL flow above the exponentially stretched sheet by considering the chemical reaction (CR). Arshad et al.16 considered radiative heat and mass transfer (HMT) for HNF incorporating the inclined magnetic field. Upreti et al.17 explored the influence of shape factor on Casson goldblood nanofluid flow through SS incorporating magnetic effect. Also, considered the impact of Ohmic heating, convective heating, and suction/injection on heat transfer rate. Singh et al.18 investigated the influence of Melting and CR on the immobility point flow of a micropolar fluid over a Porous SS Medium. Pandey et al.19,20 presented the multiple slip mechanism and volumetric heat generation over porous SS and cone respectively. Sreedevi and Reddy21 considered 3D NF flow above SS with radiation and CR for the exploration of thermo-diffusion and Brownian movement. They concluded that enhancing the Deborah number increases the temperature profile. In recent years, significant research attention has been directed toward Heat and Mass Transfer flows due to their essential nature, prevalent in various engineering and industrial sectors. An example is the extrudate Scientific Reports \| Vol:.(1234567890) (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 2 www.nature.com/scientificreports/ from the die in a melt spinning process. Researchers have investigated multiple flow scenarios, including those involving a stretching surface (SS), chemical reaction (CR), mixed convection, and thermal radiation, to address HMT-related issues. Rao et al.22 delved into HMT aspects in the context of a thermally oscillating fluid. Arshad et al.23,24 explored HMT by considering thermal radiation and chemical reactions for different types of Newtonian fluids (NFs) and Hybrid Nanofluids (HNFs). Mathur et al.25 examined the Darcy–Forchheimer skin coefficients and explored the velocity slip properties of a micropolar NF. Upreti et al.26 explored thermodynamics and HT using the Riga plate for the magnetized Casson HNF. They also discussed the entropy generation. Jayavel et al.27 provided a discussion on heat transfer analysis and irreversibility in MHD Darcy-Forchheimer movement of Casson HNF flow over wedge and cone. They used nanofluid and hybrid nanofluid for their discussion. Hassan et al.28 used the molybdenum di-sulfide NPs to explore the HMT incorporating the non-linear and linear radiation. Hussain et al.29 computationally investigated the thermal radiation to find the HTR over a stretchy surface. Different related r esearch30–38 in literature can be found. Arshad and H assan39 studied the heat and mass transmission rate in a rotating permeable system using hybrid nanofluids. Nowadays, chemical reactions, thermal radiation, and the presence of heat source/sink are fundamental components in the study of HMT phenomena. In various engineering applications, the interplay of chemical reactions, thermal radiation, and heat source/sink mechanisms plays a pivotal role in shaping the thermal behavior of systems. Understanding the effects of chemical reactions and thermal radiation, along with heat source/ sink interactions, is essential for optimizing processes in fields such as materials science and chemical engineering. Heidary et al.40 numerically investigated the magnetic field effect with forced convection in a duct for NF flow. Sheikholeslami and R okni41 gave a review on the simulation for the HT phenomenon of nanoliquid in the existence of a magnetic field. Makinde and Mishra42 examined the MHD mixed convection with non-uniform viscosity Blasius flow inserted in a permeable medium incorporating the chemical reaction. Using the CR and heat source, a semi-analytical solution for MHD Jeffery fluid flow is provided by Nisar et al.43. Mishra et al.44 explained the influence of nonlinear radiation and cross-diffusion effects on the flow of micropolar nanoliquid over a stretching sheet with an exponential heat source. Mehrizi et al.45 reported a new analysis of natural convection BL flow with variable wall temperature on a horizontal plate. Reddy et al.46 explored the HMT flow of NF at inclined plates with thermal radiation and magnetic fields under enhanced boundary conditions. Nayak et al.47 investigated the flow and HTR non-Newtonian fluid with hybrid nanoparticles by employing a magnetic field. The literature review conducted indicates a notable gap in research, as there has been no investigation into comparing different types of nanofluids over dual stretchable surfaces while factoring in the existence of an inclined magnetic field and accounting for viscous dissipation. Such applications involving nanofluids occur in advanced cooling systems, enhanced heat exchangers, biomedical devices, material processing, renewable energy, etc. Jena et al.5 considered only the temperature profile in their study but ignored the concentration profile. Jayavel et al.27 made their analysis over the wedge and cone but ignored the inclined magnetization. Pattanaik et al.7 examined the uniform heat source and ignored the chemical reaction. Gupta et al.32 used the kerosene oil for their investigations over exponentially SS. Arshad and Hassan39 deliberated hybrid nanofluids using different NPs. Singh et al.18 considered the non-uniform heat source but ignored the thermal radiation. Similarly, Pattanaik et al.38, Mohanty et al.31, and P arida6 presented their research in different aspects but none of these considered the inclined magnetic field. Based on the literature survey conducted above, the novelty of this study aims to provide a comparative analysis of different water-based NFs. These NFs, namely Cu/H2 O ; NF, andCu − Al2 O3 /H2 O HNF, are investigated concerning their HMT rates. This report is prepared for a dual stretching surface placed within a porous medium. Viscous dissipation, thermal radiation, chemical reaction, and most importantly inclined magnetic field are considered because they involve semiconductor manufacturing, solar energy, food processing, etc. The governing equalities are changed into the ODEs by employing a transformation and tackled a MATLAB by BVP-4c algorithm by setting the tolerance 10−6 for solutions. The results are obtained for increasing values of different parameters involved in this research. This comparative analysis serves to provide insights into the central research queries outlined below: 1. How does the rotation parameter affect the velocity, temperature, and concentration profile? 2. What is the influence of increasing behavior of mixed convection, stretching ratio, magnetic force, and inclination angle on velocity and temperature profile? 3. Does the increased thermal radiation and viscous dissipation reduce skin friction and enhance the HTR? 4. How does the chemical reaction affect the concentration profile and Sherwood number? 5. What do we get numerical outcomes for skin frictions along thex − axis , y − axis, Nusselt, and Sherwood numbers versus different parameters? Problem formulation Consider a steady three-dimensional boundary layer, the MHD flow of a fluid over a dual stretching sheet within a porous medium, accompanied by a chemical reaction, thermal radiation, and the existence of a nonuniform heat source. The conceptual representation of the problem, inclusive of the flow design and coordinate system, is presented in Fig. 1. The horizontal direction is represented by the x-axis, the upward direction by the z-axis, and the y-axis is perpendicular to both axes. The fluid is undergoing a steady rotation at a constant speed denoted as ω∗ around the z-axis . The surface is stretching along the x-axis with velocity Uw = ax and along the y-axis with velocity Vw = by as shown. Inclined magnetic field B0 with angle α from the x-axis to the axis of rotation is working. By these assumptions, the flow conservation, momentum along the x-axis, and y-axis, temperature and concentration in the existence of joule heating, thermal radiation, and viscous dissipation equation are: Equation of continuity48: Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 3 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Flow configuration of the problem. ∂ u ∂ v ∂w + + = 0, ∂x ∂y ∂z (1) Momentum equations along the x and y axis48: u µhnf ∂ u ∂ ∂ u u v= v + +w − 2ω∗ ∂x ∂y ∂z ρhnf µhnf ∂ v ∂ ∂ v v u u= + v +w − 2ω∗ ∂x ∂y ∂z ρhnf u ∂ 2 u u ∂ 2 ∂ 2 + 2 + 2 2 ∂x ∂y ∂z ∂ 2 ∂ 2 v v v ∂ 2 + 2 + 2 ∂x 2 ∂y ∂z + + g ∗ (ρBt )hnf ρhnf (T − T∞ ) − µhnf σhnf 2 2 u u− , B0 sin (α) ρhnf ρhnf ko (2) g ∗ (ρBt )hnf ρhnf σhnf 2 2 µhnf v , B0 sin (α) v− (T − T∞ ) − ρhnf ρhnf ko Energy equation without qr relation48,49: � � � � � � � � ∂T ∂T ∂ 2T ∂ 2T 1 ∂ 2T ∂qr ∂T � � � +� v +w = αhnf + 2 + 2 −� u ∂x ∂y ∂z ∂z ∂x 2 ∂y ∂z ρC p hnf �� 2 � � � 2 µhnf σhnf ∂� u 2 ∂� v � � � B0 2 sin2 (α) � u +� v2 + + +� ∂z ∂z ρC p ρC p hnf hnf � � � � � �2 � �2  �� 2  ∂T � ∂C � ∂C � ∂C � � �  � � � ∂ T ∂T ∂T T T ∂ ∂ D T , + τ  DB + + . + . + . +  ∂x ∂x ∂y ∂y ∂z ∂z T∞  ∂x ∂y ∂z (3) (4) Concentration equation49: ∂C ∂C ∂C + +w = DT v u ∂x ∂y ∂z ∂ 2C ∂ 2C ∂ 2C + 2 + 2 2 ∂x ∂y ∂z DT + D∞ ∂ 2T ∂ 2T ∂ 2T + 2 + 2 2 ∂x ∂y ∂z − kc (C − C∞ ). (5) The respective boundary c onditions48 for the current problem are: =T w , C =C w , at z = 0 = 0, T v = Vw = by, w u = Uw = ax, →T ∞ , C →C ∞ , as z → ∞ u → 0, v → 0, T (6) Here g ∗—gravitational acceleration, B0—magnetic field, T —temperature, C—concentration, T∞—ambient temperature, qr—radiative heat flux, µ—dynamic viscosity, ρ—density, DB—mass diffusion, DT —temperature diffusion, k—thermal conductivity, Cp—specific heat, σ —electrical conductivity, Bt—thermal volumetric coefficient, α—the angle of inclination, u, v, w are velocity components in x, y, z respectively, the subscript hnf represents the hybrid nanofluid. The specified boundary conditions for the fluid flow and thermal transport problem outline the physical behavior near and far from the solid surface. At the origin (z = 0), the prescribed velocities Uw and Vw denote a stretching wall, while w = 0 enforces a no stretching. Temperature (T) and concentration (C) conditions Tw and Cw at the wall capture heat and mass transfer interactions. As z → ∞, the velocity components approach zero (u → 0, v → 0), signifying a quiescent state, while temperature and concentration (T∞ , C∞ ) represent Scientific Reports \| Vol:.(1234567890) (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 4 www.nature.com/scientificreports/ Properties Nanofluid relations50 Density ρnf = (1 − (φ 1 ))ρf + φ1 ρs1 , G1 = Dynamicviscosity µnf = Heatcapacity Thermalconductivity Thermal expansion Electrical conductivity ρCp knf kf µf [1−(φ1 )]5/2 = K1 (ρCp ) = [1 − (φ1 )] ρcp f + φ1 (ρcp )s1 , G2 = (ρC )nf p f nf ks1 +2kf −2φ1 (kf −ks1 ) ks1 +2kf +φ1 ×(kf −ks1 ) , G3 = ρnf ρf = knf kf (ρBt )nf = (1 − (φ 1 ))ρBt f + φ1 ρBt s1 , G4 = σnf σf =1+ 3 σs1 −σf σs1 +2σf − σs1 −σf φ1 , G5 = (ρBt )nf (ρBt )f σnf σf Table 1. Thermophysical relations of nanofluid. Properties Hybrid nanofluid relations39 Density ρhnf = (1 − (φ 1 + φ2 ))ρf + φ1 ρs1 + φ2 ρs2 , H1 = Dynamicviscosity µhnf = Heatcapacity Thermalconductivity ρCp khnf knf hnf = Here µf [1−(φ1 +φ2 )]5/2 knf kf ρhnf ρf = K2 (ρCp ) = [1 − (φ1 + φ2 )] ρcp f + φ1 (ρcp )s1 + φ2 (ρcp )s2 , H2 = (ρC )hnf p f ks2 +2×knf −2×φ2 × knf −ks2 , ks2 +2×knf +φ2 × knf −ks2 = ks1 +2×kf −2×φ1 ×(kf −ks1 ) ks1 +2×kf +φ1 ×(kf −ks1 ) ,H3 = khnf kf Thermal expansion (ρBt )hnf = (1 − (φ 1 + φ2 ))(ρBt )f + φ1 (ρBt )s1 + φ2 (ρBt )s2 , H4 = Electrical conductivity σhnf σf =1+ σ φ −σ φ 3 s1 1σ s2 2 −(φ 1 +φ2 ) f , H5 σ +σ σ φ −σ φ 2+ s1σ s2 − s1 1σ s2 2 +(φ 1 +φ2 ) f = (ρBt )hnf (ρBt )f σhnf σf f Table 2. Thermophysical relations of hybrid nanofluid. Physical properties Electrical conductivity 0.05 Water Density Specific heat Thermal conductivity Thermal expansion 997 4179 0.614 21 × 10−5 Copper(s1 ) 5.96 × 107 8933 385 400 1.67 × 10−5 Aluminumoxide(s2 ) 6.27 × 10−5 3970 765 40 0.85 × 10−5 Table 3. Thermophysical properties of base fluid51 and nanoparticles. Wang52 Present outcomes Nazar et al.53 p′′ (0) q′ (0) p′′ (0) q′ (0) p′′ (0) q′ (0) 0.0 −1.0 0.0 −1.013 0.0 −1.0 0.0 0.5 −1.13 −0.51 −1.141 −0.518 −1.13 −0.51 1.0 −1.32 −0.83 −1.332 −0.831 −1.32 −0.83 2.0 −1.65 −1.28 −1.660 −1.292 −1.65 −1.28 Table 4. Comparison of present outcomes with literature. the free-stream values far from the surface. These conditions collectively provide interactions and asymptotic behavior of fluid flow, heat transfer, and mass transport in the given problem. There are numerous models in literature for characterizing the effective properties of NF and HNF. So, the thermophysical relations of NF and HNF are given in Tables 1 and 2 correspondingly. Table 3 shows the thermophysical values for used NPs and base fluid. Table 4 shows the comparison of literature and present outcomes. Here φ-volume fraction of NPs, subscripts f , s, nf , hnf are representing fluid, solid nanoparticles, nanofluid and hybrid nanofluid respectively. Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 5 Vol.:(0123456789) www.nature.com/scientificreports/ The term qr in the right side of Eq. (4) presents the heat radiation effect. Utilizing the Rosseland approximation, the radiative heat flux is computed in the following: qr = − 4σ ∗ ∂T 4 . 3k1 ∂z (7) Here σ ∗ represents the Stefan–Boltzmann coefficient and k1 represents the mean absorption constant. The radiation is optically thick considered. Considering that sufficiently small temperature difference in the flow, the term T 4 by employing the Taylor series as follows: (8) T 4 = T 4 ∞ + 4T 3 ∞ (T − T∞ ) + 6T 2 ∞ (T − T∞ )2 + . . . Therefore, by abandoning higher-order terms above the first degree in (T − T∞ ), we get. T 4 = 4T 3 ∞ T − 3T 4 ∞ . (9) 16σ ∗ T∞ 3 ∂ 2 T ∂qr , =− ∂z 3k1 ∂z 2 (10) By using Eqs. (7) and (8) So, Eq. (4) takes the following form: � � � � � � � � ∂T ∂T ∂ 2T ∂ 2T 16σ ∗ T∞ 3 ∂ 2 T ∂ 2T ∂T � � � � +� v +w = αhnf + + + u 2 2 2 ∂x ∂y ∂z ∂x ∂y ∂z ∂z 2 3k1 ρC p hnf �� 2 � � � 2 µhnf σhnf ∂� u 2 ∂� v � � B0 2 sin2 (α) � u +� v2 +� + +� ∂z ∂z ρC p ρC p hnf hnf � � � � � �2 � �2  �� 2  ∂T � ∂C � ∂C � ∂C � � �  � � � ∂ T ∂T ∂T T T ∂ ∂ D T , + τ  DB + + . + . + . +  ∂x ∂x ∂y ∂y ∂z ∂z T∞  ∂x ∂y ∂z (11) Similarity transformation: In the present problem, the following similarity transformations are taken to change the PDEs into ODEs. √ = av w v = ayq′(η), u = axp′ (η), f p(η) +q(η) , (12) a −T o − T∞ = T ∞ . −C o − C ∞ = C ∞ , r(η) T η = z vf , s(η) C The differentiation is w.r.t η. The flow Eq. (1) of mass conversation is satisfied identically by employing Eq. (12). The Eqs. (2,3,5) and Eq. (11) for momentum, energy, and concentration will take the following form after using Eq. (12) with boundary conditions: 2 p′′′ (η) = H1 ∗ p′ (η) ∗ p′ (η) + q′ (η) − 2 ∗ δ ∗ q′ (η) + Z ∗ p′ (η) − ǫx ∗ r ∗ H4 + M 2 ∗ sin2 (α) ∗ p′ ∗ H5 ∗ K2 2 ′ q (η) = H1 ∗ q (η) p (η) + q (η) − 2 ∗ ∗ p′ (η) + Z ∗ q′ (η) − ǫy ∗ r ∗ H4 + M 2 ∗ sin2 (α) ∗ q′ ∗ H5 δ ′′′ ′ ′ −1 4 2 r ′′ = − 1 + ∗π Pr ∗ H2 ∗ r ′ p(η) + q(η) + r ′ ∗ s′ ∗ Nb + r ′ Nt 3 ∗ H3 2 2 2 2 + K2 ∗ Ec ∗ p′′ + q′′ + H5 ∗ M 2 ∗ sin2 (α) ∗ Ec ∗ p′ + q′ ′′ s = Sc ∗ Nt ∗ r ′′ − s ∗ Kc p+q ∗r + Nb ′ (13) ∗ K2 (14) (15) (16) The non-dimensional quantities H1 , H2 , H3 , H4 , H 5 , and K2 , are hybrid NPs relations (presented in Table 2), and , δ, Z, ǫ, M, Pr, π, Ec, Sc, Nt , τ , Nb , and Kc are defined as = ω∗ a ,δ Pr = y = x,Z = vf kf 2 Nb = Scientific Reports \| Vol:.(1234567890) (2024) 14:1207 \| = Gr ,M Rex 2 = σf B0 2 aρf , (ρCp)s DB vf , τ = (ρCp)f , (ρCp)s DB (Cw −C∞ ) 1 , (Tw −T∞ ) , Nt = T∞ (ρCp)f vf (ρCp)s DB (Tw −T∞ ) , Kc = Kar (ρCp)f vf ,π = uw Ec = (ρC p )f µhnf aρhnf ko , ǫ 4σ ∗ T∞ 3 k1 kf , Sc = https://doi.org/10.1038/s41598-024-51195-4 (17) 6 www.nature.com/scientificreports/ g ∗ (ρBt ) hnf Here Gr = (T − T∞ )x 3 , Rex = uvwf . vf 2 The modified boundary conditions are presented as follows: atη = 0, s = 1, r = 1, q′ = γ , q = 0, p′ = 1, p = 0, asη → ∞, s → 0, r → 0, q′ → 0, p′ → 0, (18) Here γ = ab presents the dimensionless stretching ratio. Engineering parameters of interest: There are the following most important quantities regarding to engineering perspective. (A) Skin friction: The significant surface skin friction along the x-axis and y-axis are Cf x , Cf y defined as: Cf x = τzy τzx , Cf y = ρf uw 2 ρf vw 2 The τzx and τzy indicate shear stress along the stretched wall x-axis and y-axis which is defined as: ∂v ∂w ∂u ∂w + + , τzy = µhnf τzx = µhnf ∂z ∂x z=0 ∂z ∂y z=0 (19) (20) The dimensionless form of Eq. (20) with the help of Eq. (16) is: (Rex )1/2 Cfx = µ hnf ′′ µhnf ′′ p (0), (Rex )1/2 Cfy = q (0), µf µf (B) Rate of heat and mass transfer: The HMT rates, expressed through Nusselt and Sherwood numbers are the following: By utilizing the temperature field, the thermal diffusion rate is characterized by the Nusselt number: xqw ∂T , qw = −khnf + (qr )w , Nux = kf (Tw − T∞ ) ∂z z=0 By using the similarity transformation, the transformed form of Eq. (22) is given below: 4 Nux = − B3 + π r′(0), 3 (21) (22) (23) By utilizing the concentration field, the mass transmission rate is characterized by the Sherwood number: ∂C xqm , qm = Dm Shx = (24) Dm (Cw − C∞ ) ∂z z=0 By using the similarity transformation, the transformed form of Eq. (24) is given below: Shx = −s′(0). (25) Numerical solution This section provides the numerical solution methodology as presented in Fig. 2 to get the solution of higherorder nondimensional ODEs. For this purpose, the bvp4c method is used to tackle the boundary value problem by transforming it into an initial value problem. The Fig. 3 validates the code of the present problem. For this purpose, newly defined variables are as follows: z3 ′ = p′′′ , z3 = p′′ , z2 = p′ , z1 = p, z6 ′ = q′′′ , z6 = q′′ , z5 = q′ , z4 = q, z8 ′ = r ′′ , z8 = r ′ , z7 = r, (26) z10 ′ = s′′ , z10 = s′ , z9 = s, The following form of equations is used in MATLAB to get the numerical solution. z3 ′ = H1 ∗ z1 ′ = z2 (27) z2 ′ = z3 (28) 2 z2 ∗ (z1 + z4 ) − [2 ∗ ∗ δ ∗ z5 ] + [Z ∗ z2 ] − [ǫx ∗ z7 ∗ H4 ] + M 2 ∗ z2 ∗ sin2 (α) ∗ H5 ∗ K2 (29) z4 ′ = z5 Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 (30) 7 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 2. Flow chart for the numerical solution. Figure 3. Validation of code by velocity profile p′ (η) and q′ (η). z5 ′ = z 6 z6 ′ = H1 ∗ 1 z5 2 ∗ (z1 + z4 ) − 2 ∗ ∗ ∗ z2 + [Z ∗ z5 ] − ǫy ∗ z7 ∗ H4 + M 2 ∗ sin2 (α) ∗ z5 ∗ H5 ∗ K2 δ (32) z7 ′ = z8 Scientific Reports \| Vol:.(1234567890) (2024) 14:1207 \| (31) https://doi.org/10.1038/s41598-024-51195-4 (33) 8 www.nature.com/scientificreports/ −1 4 ′ Pr ∗ H2 ∗ z8 (z1 + z4 ) + z8 ∗ z10 ∗ Nb + z8 2 Nt z8 = − 1 + ∗π 3 ∗ H3 + K2 ∗ Ec ∗ z3 2 + z6 2 + H5 ∗ M 2 ∗ sin2 (α) ∗ Ec ∗ z2 2 + z5 2 (34) z9 ′ = z10 (35) Nt ′ ∗ z8 ′ − (z10 ∗ Kc ) z10 = Sc ∗ ((z4 + z1 ) ∗ z8 ) + Nb (36) The transformed boundary conditions have been modified into the following form: atη = 0, z1 = 0, z2 = 1, z4 = 0, z5 = γ , z7 = 1, as η → ∞, z2 → 0, z5 → 0, z7 → 0. (37) Results and discussion The problem is formulated for HNF in the previous section. The authors obtained the results of the current problem separately for both NF and HNF and discussed them physically. The obtained influences of different constitutes by using the bvp4c algorithm at MATLAB are described in the following. Velocity profiles The influences of study parameters on velocities are discussed in this section. The influence of rotation on velocity profile p′(η) when Nb = 0.1, δ = 0.8, γ = Z = ǫ = π = Kc = Ec = 0.5, Nt = Le = M = 0.3, α = 45◦ is presented in Fig. 4a. The graph displays the velocity profiles p′(η), demonstrating a decrease in velocity as the rotation parameter increases for both NF and HNF cases. Initially, at = 0 and = 1, there is minimal alteration in the velocity profiles. However, a significant acceleration in the decay of these profiles is observed as the rotation parameter rises to = 2 and = 3. The main reason behind this is enhanced viscous dissipation that affects the velocity profile. Figure 4b represents the influence of the magnetic field on velo city q′(η)whenNb = 0.1, δ = 0.8, γ = Z = ǫ = π = Kc = Ec = 0.5, Nt = Le = = 0.3, α = 45◦ . The magnetic parameter has a great influence on the velocity profile. In the non-existence (M = 0) of the magnetic field, the momentum BL looks similar but when it upsurges between 1 ≤ M ≤ 3, a big change is noted due to the existing Lorentz force which decreases the fluid motion and consequently momentum BL decay. A wider momentum BL is noted for HNF as associated with NF. This is due to the generation of damping electromagnetic force which restricts the fluid’s motion and results in a gradual reduction in velocity profile. Figure 4c depicts the impact of the angle of inclination 0◦ ≤ α ≤ 90◦ on velocity profile p′(η) whenNb = 0.1, δ = 0.8, γ = Z = ǫ = π = Kc = Ec = 0.5, Nt = Le = = 0.3, M = 0.8 . The parameter at α = 0 represents the magnetic force parallel to the x − axis and varies from α = 0◦ to α = 90◦ i.e., parallel to the y-axis. As the inclination angle increases, the momentum BL decreases because Lorentz force increases gradually. The extended momentum BL is noted for HNF when α = 30◦ , 45◦ and 90◦ whereas NF has smooth BL. A minimum flow for NF is observed when the inclination angle is parallel to the y − axis because greater interaction between the magnetic field and fluid increases flow resistance. The mixed convection parameter 0 ≤ ǫ ≤ 30 has a direct relation with velocity profile p′(η) as presented in Fig. 4d whenNb = 0.1, δ = 0.8, γ = Z = π = Kc = Ec = 0.5, Nt = Le = = 0.3, α = 45◦ , M = 3 and a higher velocity profile is observed for NF as associated with the HNF. This is owing to the driving forces associated with convection which typically increases the fluid’s velocity. Figure 4e shows that porous medium parameter Z has a negligible decreasing influence on velocity profile p′(η) . whenNb = 0.1, δ = 0.8, γ = ǫ = π = Kc = Ec = 0.5, Nt = Le = = 0.3, α = 45◦ , M = 0.8 The porosity acts as a flow impediment, and the drag force significantly reduces the velocity of the fluid. The stretching ratio parameter γ represents the effects on velocity profile q′(η) in Fig. 4f whenNb = 0.1, δ = 0.8, = Z = ǫ = π = Kc = Ec = 0.5, Nt = Le = 0.3, α = 45◦ , M = 0.8. When the stretching rate along the x − axis increases as compared to the y-axis , the velocity momentum BL expands for both types of NFs. Temperature and concentration profiles The following Fig. 5a–h show the impression of different constraints on temperature and concentration profiles. The influence of magnetic parameters on temperature r(η) is represented in Fig. 5a whenNb = 0.1, δ = 0.8, γ = Z = ǫ = π = Kc = Ec = 0.5, Nt = Le = = 0.3, α = 45◦ a n d c a n b e seen that the temperature increases when magnetic strength increases. The justification for this is that Lorentz force reduces the fluid motion and as a result, a maximum HT and a higher thermal BL are noted for HNF as compared to NF. Figure 5b indicates the effect of rotation on the temperature (η) whenNb = 0.1, δ = 0.8, M = 0.3, Z = ǫ = π = Kc = Ec = 0.5, Nt = Le = = 0.3, α = 45◦ . W h e n t h e rotation of fluids increases the temperature profile shows the same behavior. Rotation increases temperature transmission by promoting fluid mixing, reducing boundary layer thickness, and enhancing convective heat transfer due to the formation of vortices and turbulence, leading to improved thermal. In this figure low but consistent HTR is noted for HNF. The influence of thermal radiation on the temperature r(η) is shown in Fig. 5c whenNb = 0.1, δ = 0.8, M = 03, γ = Z = ǫ = Kc = Ec = 0.5, Nt = Le = = 0.3, α = 45◦. In the absence of the radiation parameter, a higher temperature BL is detected for both NFs. Thermal Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 9 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 4. (a) Effect of on velocity p′(η). (b) Effect of M on velocity q′(η). (c) Effect of α on velocity p′(η). (d) Effect of ǫ on velocity q′(η). (e) Effect of Z on velocity p′(η). (f) Effect of on velocity q′(η). radiation typically decays temperature profiles by emitting heat energy from hotter regions to cooler surroundings, causing temperature gradients to diminish, resulting in a more uniform temperature distribution. The Eckert number characterizes the thermal energy conversion associated with the fluid’s motion. A higher Eckert number implies more kinetic energy, which promotes cooling, leading to a decrease in temperature gradients and a more uniform temperature profile. This phenomenon is presented in Fig. 5d whenNb = 0.1, δ = 0.8, γ = Z = ǫ = π = Kc = 0.5, Nt = Le = = 0.3, α = 45◦ , M = 03. Howe ver, we found that as viscous dissipation rises, the thermal BL gets thicker. The impact of mixed convection and CR is presented in Fig. 5e,f respectively. WhenNb = 0.1, M = 08, δ = 0.8, γ = Z = π = Kc = Ec = 0.5, Nt = Le = = 0.3, α = 45◦ the mixed convection and chemical reactions whenNb = 0.1, δ = 0.8, γ = Z = Scientific Reports \| Vol:.(1234567890) (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 10 www.nature.com/scientificreports/ Figure 5. (a) Effect of M on temperature r(η). (b) Effect of on temperature r(η). (c) Effect of π on temperature r (η). (d) Effect of Ec on temperature r(η). (e) Effect of ǫ on concentration s (η). (f) Effect of Kc on concentration s (η). (g) Effect of on concentration s (η). (h) Effect of Nt on concentration s (η). ǫ = π = Ec = 0.5, Nt = Le = = 0.3, M = 08, α = 45◦ decay concentration profiles due to enhanced fluid mixing from buoyancy and forced convection, leading to reduced concentration gradients and more uniform distribution. A higher concentration of BL is noted in the absence of mixed convection and CR for HNF as compared to NF which is presented in Fig. 5e,f for HNF. The rotation influence is represented in Fig. 5g whenNb = 0.1, δ = 0.8, γ = Z = ǫ = π = Kc = Ec = 0.5, Nt = Le = M = 0.3, α = 45◦ . The rotation and concentration profile have a direct relation. As the rotation parameter increases the mass Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 11 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 5. (continued) Z γ ǫ M α Cfx for NF Cfy for NF Cfx for HNF Cfy for HNF 0.0 0.7 0.5 0.3 0.8 45° −2.14850 −0.74840 −3.08458 −1.38589 1.0 −1.92663 −1.73975 −2.95226 −1.84510 2.0 −1.90891 −2.63017 −2.86364 −2.30971 −4.28186 −2.23697 −4.37705 −2.25723 20 −5.83925 −2.98470 −5.48274 −2.79401 30 −7.06607 −3.58458 −6.40189 −3.24491 −1.87550 −2.00135 −2.95067 −2.80315 1.0 −1.86189 −2.20911 −2.94398 −3.11017 1.1 −1.84833 −2.42367 −2.93726 −3.42038 −2.12530 −1.40129 −3.08560 −1.70111 01 −1.74097 −1.03913 −2.86990 −1.49433 02 −1.38247 −0.69217 −2.65904 −1.29093 −1.36554 −1.11913 −1.16233 −0.95291 50 −9.76931 −4.93298 −17.8269 −8.93220 10 −19.4546 −9.75085 −35.6205 −17.8196 0° −1.36554 −1.11913 −1.16233 −0.95291 30° −1.70734 −1.19708 −2.26901 −1.31224 45° −2.00639 −1.29060 −3.02022 −1.63860 60° −2.27259 −1.38630 −3.62387 −1.91633 90° −2.51397 −1.48003 −4.14240 −2.16088 0.5 0.5 0.5 0.5 0.5 10 0.7 0.7 0.7 0.7 0.5 0.9 0.5 0.5 0.5 0.3 0.3 0.0 0.3 0.3 0.8 0.8 0.8 00 0.8 45° 45° 45° 45° Table 5. Numerical results for skin frictions along x − axis Cfx and y − axisCfy when π = 0.7, Pr = 6.2, M = 0.8, Le = 0.6, Nt = 0.3, Ec = 0.5, Nb = 0.2 respectively. transfer rate also increases. Rotation increases the concentration profiles due to the centrifugal forces that drive fluid motion. The thermophoresis parameter effect on the concentration profile is shown in Fig. 5h whenNb = 0.1, δ = 0.8, γ = Z = ǫ = π = Kc = Ec = 0.5, M = 08, Le = = 0.3, α = 45◦ . A wider concentration of BL is observed for NF as compared to HNF when the thermophoresis parameter increases. This is due to the inclusion of single and double NPs inclusion in the host fluid. So, HNF shows a consistent mass transfer rate in Fig. 5h. Numerical outcomes The numerical outputs for skin frictions Cf x , Cf y , Nusselt number Nux , and Sherwood number Shx are presented in this section. Table 5 represents the obtained numerical results for skin frictions Cf x and Cf y for both nanofluids. A minimum skin friction Cf x is observed in the absence of rotation i.e. λ = 0 and increasing behavior is seen for increasing values of rotation parameter from = 1 to = 2, while an opposite behavior is observed for Cf y . Increased porosity from 10 ≤ Z ≤ 30 reduces skin friction in porous media because higher permeability allows fluid to flow more easily through the medium, reducing the resistance to motion and minimizing the friction between the fluid and the porous structure. The stretching ratio is defined by the rate along the y-axis to the Scientific Reports \| Vol:.(1234567890) (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 12 www.nature.com/scientificreports/ α π Pr M Kc Le Nt Ec Nb Nux for NF Shx for NF Nux for HNF Shx for HNF 0° 0.7 6.2 0.8 1.5 0.6 0.3 0.5 0.2 3.00058 1.72922 4.36870 1.74389 30° 2.74390 1.71586 3.04228 1.68995 45° 2.52236 1.70378 2.14219 1.65535 60° 2.32647 1.69307 1.42823 1.62959 90° 2.14992 1.68352 0.82732 1.60887 10.1829 1.72429 6.59213 1.66263 02 5.60675 1.68505 4.41799 1.64535 04 4.05846 1.67249 3.37044 1.63681 2.74899 1.70996 2.14219 1.65535 8.2 2.98656 1.71649 2.44237 1.66191 9.2 3.21141 1.72272 2.75913 1.66886 3.00058 1.72922 4.36870 1.74389 05 −2.2238 1.48799 −11.973 1.25456 10 −6.6810 1.33243 −26.824 0.903258 2.62898 0.58260 2.29389 0.504837 02 2.49564 2.16376 2.10901 2.123040 04 2.43024 4.10204 2.03810 4.07362 2.46683 2.51915 2.07947 2.41901 15 2.45546 2.75390 2.06659 2.64903 20 2.44728 2.94512 2.05751 2.83747 2.98666 1.64205 2.77205 1.61567 03 0.487884 1.66930 −0.2628 1.46950 06 0.060626 1.45236 −0.6053 1.16859 6.268410 1.80742 11.6602 1.86675 10 10.26151 1.91756 20.8553 2.07063 15 18.72051 2.13910 28.9276 2.24937 0.5 2.513160 1.79520 2.13120 1.71391 1.0 2.497920 1.94580 2.11298 1.80989 1.5 2.482801 2.09423 2.09489 1.90382 45° 45° 45° 45° 45° 45° 45° 45° 00 0.7 0.7 0.7 0.7 0.7 0.7 0.7 6.2 7.2 6.2 6.2 6.2 6.2 6.2 6.2 0.8 0.8 00 0.8 0.8 0.8 0.8 0.8 1.5 1.5 1.5 00 1.5 1.5 1.5 1.5 0.6 0.6 0.6 0.6 10 0.6 0.6 0.6 0.3 0.3 0.3 0.3 0.3 00 0.3 0.3 0.5 0.5 0.5 0.5 0.5 0.5 05 0.5 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Table 6. Numerical results for Nusselt number Nux and Sherwood number Shx when = 0.5, Z = 0.7, γ = 0.5, ǫ = 0.3, M = 0.8 respectively. stretching rate x-axis . Increased stretching ratio 0.9 ≤ γ ≤ 1.1 increases skin friction in the context of flow over a solid surface. This occurs because a higher stretching ratio leads to increased shear stress. Reduced skin friction is followed in the deprivation of mixed convection and magnetic field and increasing behavior is noted when it increases. The angle of inclination has a great influence on skin friction. When the magnetic field is parallel to the x-axis, a maximum skin friction Cf x is noted while it decays when the angle of inclination increases between the range 0 ≤ α ≤ 90. Minimum skin friction is noted when the magnetic field becomes parallel to the y − axis. Table 6 shows the influences of different constraints on Nusselt number Nux and Sherwood number Shx for both nanofluids. A higher HMT rate is noted when the angle of inclination for the magnetic field is parallel to the xaxis. Increasing radiation parameter 0 ≤ π ≤ 4 typically decreases the Nusselt and Sherwood numbers because radiative heat or mass transfer is a slower process compared to convection. As radiation becomes more significant, it diminishes the relative contribution of convection, leading to lower Nusselt and Sherwood numbers, indicating reduced HMTR and utmost Nusselt and Sherwood is seen in the absence of radiation, i.e. π = 0. Prandtl number 7.2 ≤ Pr ≤ 9.2 and magnetic field 0 ≤ M ≤ 15 have a direct relation and inverse relation with HMTR respectively because Pr implies that thermal or mass diffusion is rapid relative to momentum diffusion and the existence of magnetic field suppresses fluid movement, disrupting convection resulting in reducing HMTR. Increasing values of the CR parameter 0 ≤ Kc ≤ 4 and Lewis number 10 ≤ Le ≤ 20 decreases the Nusselt number while it increases the Sherwood number. CR consumes the temperature and concentration gradient and a higher Lewis number results in a more dominant effect on fluid behavior. Similar results are found for thermophoresis 0 ≤ Nt ≤ 6 and Brownian motion 0.5 ≤ Nb ≤ 1.5 factors corresponding to the Nusselt number and Sherwood number. As the viscous dissipation parameter increases between 5 ≤ Ec ≤ 15 the HMT rate increases in both cases of used nanofluids because increased Ec number indicates that the kinetic energy of the fluid is relatively higher than internal energy change. Conclusion In this present article, the authors discussed the heat and mass transfer rate of 3D rotating, incompressible NF and HNF fluid over the dual stretchable surface. The governing equations are transformed and tackled in MATLAB. We considered the inclined magnetic field, viscous dissipation, thermal radiation, and chemical reaction Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 13 Vol.:(0123456789) www.nature.com/scientificreports/ effects to provide a theoretical view of the study. The main findings that one needs to remember from this study are stated below: 1. Increasing the rotation, magnetic force, and porosity of the SS decays the velocity profile while it increases by a rise in mixed convection and stretching ratio parameters. 2. The temperature profile r(η) increases by an increase in rotation and magnetic field parameters. 3. The concentration profile decreases under the increasing influence of mixed convection parameters, and thermophoresis parameters whereas it increases by raising the value of the rotation parameter. 4. A maximum Nux = 3.00 and 4.36, Shx = 1.72 and 1.74, and minimum Cf x = −1.36 and −1.16, Cf y = −1.11 and −0.95 is noted when α = 00 i.e., angle of inclination is parallel to the x − axis, for NF and HNF. 5. By increasing Eckert number i.e., 5 ≤ Ec ≤ 10, the Nux = 18.7, 28.9, and Shx = 2.1, 2.2 is noted for NF and HNF respectively. 6. When the chemical reaction parameter increases i.e., 0 ≤ Kc ≤ 4, the Nusselt number 2.62 ≤ Nux ≤ 2.43 and 2.29 ≤ Nux ≤ 2.03 decreases, and Sherwood number 0.58 ≤ Shx ≤ 4.10 and 0.50 ≤ Shx ≤ 4.07 increases for NH and HNF respectively. 7. By changing the angle of inclination, the Nux performance is noted at 8% for NF and 33% for HNF which proves the high heat transfer rate efficiency of HNF. The strength of this study lies in its comprehensive analysis of the three-dimensional mixed convection flow of nanofluid over a dual stretching sheet, considering influential factors such as viscous dissipation, Joule heating, and solar radiation. The practical relevance to solar energy systems adds significance to the findings. However, limitations include potential simplifications in assumptions, applicability restricted to similar geometries, assumptions about material properties, and the influence of numerical solution techniques, emphasizing the need for careful interpretation of results in specific contexts. Data availability All data generated or analyzed during this study are included in this published article. Received: 29 November 2023; Accepted: 2 January 2024 References 1. Choi, S. U., & Eastman, J. A. Enhancing thermal conductivity of fluids with nanoparticles (No. ANL/MSD/CP-84938; CONF951135–29). Argonne National Lab.(ANL), Argonne, IL (United States) (1995). 2. Das, S., Jana, R. N. & Makinde, O. D. MHD boundary layer slip flow and heat transfer of nanofluid past a vertical stretching sheet with non-uniform heat generation/absorption. Int. J. Nanosci. 13(03), 1450019 (2014). 3. Reddy, P. S. & Sreedevi, P. Buongiorno’s model nanofluid natural convection inside a square cavity with thermal radiation. Chin. J. Phys. 72, 327–344 (2021). 4. Mehrizi, A. A., Besharati, F., Jahanian, O. & Hassanzadeh Afrouzi, H. Numerical investigation of conjugate heat transfer in a microchannel with a hydrophobic surface utilizing nanofluids under a magnetic field. Phys. Fluids 33(5), 1 (2021). 5. Jena, S., Mishra, S. R. & Pattnaik, P. K. Development in the heat transfer properties of nanofluid due to the interaction of inclined magnetic field and non-uniform heat source. J. Nanofluids 9(3), 143–151 (2020). 6. Parida, S. K. et al. Dynamics of dust particles in a conducting water-based kerosene nanomaterials: A computational approach. Int. J. Chem. Reactor Eng. 19(8), 787–797 (2021). 7. Pattnaik, P. K., Parida, S. K., Mishra, S. R., Abbas, M. A. & Bhatti, M. M. Analysis of metallic nanoparticles (Cu, Al2O3, and SWCNTs) on magnetohydrodynamics water-based nanofluid through a porous medium. J. Math. 2022, 1–12 (2022). 8. Pattnaik, P. K., Abbas, M. A., Mishra, S., Khan, S. U. & Bhatti, M. M. Free convective flow of hamilton-crosser model gold-water nanofluid through a channel with permeable moving walls. Combin. Chem. High Throughput Screen. 25(7), 1103–1114 (2022). 9. Sakiadis, B. C. Boundary-layer behavior on continuous solid surfaces: I. Boundary-layer equations for two-dimensional and axisymmetric flow. AIChE J. 7(1), 26–28 (1961). 10. Erickson, L. E., Fan, L. T. & Fox, V. G. Heat and mass transfer on moving continuous flat plate with suction or injection. Ind. Eng. Chem. Fund. 5(1), 19–25 (1966). 11. Baag, S., Mishra, S. R., Hoque, M. M. & Anika, N. N. Magnetohydrodynamic boundary layer flow over an exponentially stretching sheet past a porous medium with uniform heat source. J. Nanofluids 7(3), 570–576 (2018). 12. Nayak, B., Mishra, S. R. & Krishna, G. G. Chemical reaction effect of an axisymmetric flow over radially stretched sheet. Propul. Power Res. 8(1), 79–84 (2019). 13. Mishra, S. R., Hoque, M. M., Mohanty, B. & Anika, N. N. Heat transfer effect on MHD flow of a micropolar fluid through porous medium with uniform heat source and radiation. Nonlinear Eng. 8(1), 65–73 (2019). 14. Upreti, H., Mishra, S. R., Pandey, A. K. & Bartwal, P. Shape factor analysis in stagnation point flow of Casson nanofluid over a stretching/shrinking sheet using Cattaneo-Christov model. Num. Heat Transfer Part B Fund. 1, 1–17 (2023). 15. Seini, Y. I. & Makinde, O. D. MHD boundary layer flow due to exponential stretching surface with radiation and chemical reaction. Math. Probl. Eng. 1, 1 (2013). 16. Arshad, M. et al. Effect of inclined magnetic field on radiative heat and mass transfer in chemically reactive hybrid nanofluid flow due to dual stretching. Sci. Rep. 13(1), 7828 (2023). 17. Upreti, H., Mishra, S. R., Kumar Pandey, A., Joshi, N. & Joshi, B. P. Diversified role of fuzzified particle concentration on Casson gold-blood nanofluid flow through an elongating sheet for different shape nanoparticles. J. Taibah Univ. Sci. 17(1), 2254465 (2023). 18. Singh, K., Kumar, M. & Pandey, A. K. Melting and chemical reaction effects stagnation point flow of micropolar fluid over a stretchable porous medium in the presence of nonuniform heat source/sink. J. Porous Media 23(8), 1 (2020). 19. Pandey, A. K., Upreti, H., Joshi, N. & Uddin, Z. Effect of natural convection on 3D MHD flow of MoS2–GO/H2O via porous surface due to multiple slip mechanisms. J. Taibah Univ. Sci. 16(1), 749–762 (2022). 20. Pandey, A. K., Upreti, H. & Uddin, Z. Magnetic SWCNT–Ag/H2O nanofluid flow over cone with volumetric heat generation. Int. J. Mod. Phys. B 2350, 253 (2023). 21. Sreedevi, P. & Reddy, P. S. Combined influence of Brownian motion and thermophoresis on Maxwell three-dimensional nanofluid flow over stretching sheet with chemical reaction and thermal radiation. J. Porous Media 23(4), 1 (2020). Scientific Reports \| Vol:.(1234567890) (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 14 www.nature.com/scientificreports/ 22. Rao, M., Lefèvre, F., Khandekar, S. & Bonjour, J. Heat and mass transfer mechanisms of a self-sustained thermally driven oscillating liquid–vapour meniscus. Int. J. Heat Mass Transfer 86, 519–530 (2015). 23. Arshad, M. et al. Exploration of heat and mass transfer subjected to first order chemical reaction and thermal radiation: Comparative dynamics of nano, hybrid and tri-hybrid particles over dual stretching surface. Int. Commun. Heat Mass Transfer 146, 106916 (2023). 24. Arshad, M. et al. Magneto-hydrodynamic flow above exponentially stretchable surface with chemical reaction. Symmetry 14(8), 1688 (2022). 25. Mathur, P., Mishra, S. R., Pattnaik, P. K. & Dash, R. K. Characteristics of Darcy-Forchheimer drag coefficients and velocity slip on the flow of micropolar nanofluid. Heat Transfer 50(7), 6529–6547 (2021). 26. Upreti, H., Pandey, A. K., Joshi, N. & Makinde, O. D. Thermodynamics and heat transfer analysis of magnetized Casson hybrid nanofluid flow via a Riga plate with thermal radiation. J. Comput. Biophys. Chem. 22(03), 321–334 (2023). 27. Jayavel, P., Upreti, H., Tripathi, D., & Pandey, A. K. Irreversibility and heat transfer analysis in MHD Darcy-Forchheimer flow of Casson hybrid nanofluid flow through cone and wedge. Numer. Heat Transfer Part A Appl. 1–27 (2023). 28. Hassan, A. et al. Heat and mass transport analysis of MHD rotating hybrid nanofluids conveying silver and molybdenum di-sulfide nano-particles under effect of linear and non-linear radiation. Energies 15(17), 6269 (2022). 29. Hussain, A. et al. Computational investigation of the combined impact of nonlinear radiation and magnetic field on threedimensional rotational nanofluid flow across a stretchy surface. Processes 9(8), 1453 (2021). 30. Pattanaik, P. C., Mishra, S. R., Jena, S. & Pattnaik, P. K. Impact of radiative and dissipative heat on the Williamson nanofluid flow within a parallel channel due to thermal buoyancy. Proc. Inst. Mech. Eng. Part N J. Nanomater. Nanoeng. Nanosyst. 236(1–2), 3–18 (2022). 31. Mohanty, B., Mohanty, S., Mishra, S. R. & Pattnaik, P. K. Analysis of entropy on the peristaltic transport of micropolar nanofluid: A simulation obtained using approximate analytical technique. Eur. Phys. J. Plus 136, 1–19 (2021). 32. Gupta, T., Pandey, A. K., & Kumar, M. Effect of Thompson and Troian slip on CNT-Fe3O4/kerosene oil hybrid nanofluid flow over an exponential stretching sheet with Reynolds viscosity model. Mod. Phys. Lett. B, 2350209 (2023). 33. Gupta, T., Pandey, A. K., & Kumar, M. Numerical study for temperature-dependent viscosity based unsteady flow of GP-MoS2/ C2H6O2-H2O over a porous stretching sheet. Numer. Heat Transfer Part A Appl. 1–22 (2023). 34. Joshi, N., Upreti, H. & Pandey, A. K. MHD Darcy-Forchheimer Cu-Ag/H2O-C2H6O2 hybrid nanofluid flow via a porous stretching sheet with suction/blowing and viscous dissipation. Int. J. Comput. Methods Eng. Sci. Mech. 23(6), 527–535 (2022). 35. Upreti, H., Pandey, A. K., Kumar, M. & Makinde, O. D. Darcy-Forchheimer flow of CNTs-H2O nanofluid over a porous stretchable surface with Xue model. Int. J. Mod. Phys. B 37(02), 2350018 (2023). 36. Upadhyay, S., Upreti, H., & Pandey, A. K. Three-dimensional flow of hybrid nanofluid through Darcy-Forchheimer porous surface: A Legendre wavelet collocation approach. Num. Heat Transfer Part A Appl. 1–25 (2023). 37. Upreti, H., Pandey, A. K., Gupta, T., & Upadhyay, S. Exploring the nanoparticle’s shape effect on boundary layer flow of hybrid nanofluid over a thin needle with quadratic Boussinesq approximation: Legendre wavelet approach. J. Thermal Anal. Calorimetry, 1–18 (2023). 38. Pattnaik, P. K., Moapatra, D. K., & Mishra, S. R. Influence of velocity slip on the MHD flow of a micropolar fluid over a stretching surface. In Recent Trends in Applied Mathematics: Select Proceedings of AMSE 2019 (pp. 307–321) (Springer Singapore, 2021). 39. Arshad, M. & Hassan, A. A numerical study on the hybrid nanofluid flow between a permeable rotating system. Eur. Phys. J. Plus 137(10), 1126 (2022). 40. Heidary, H., Hosseini, R., Pirmohammadi, M. & Kermani, M. J. Numerical study of magnetic field effect on nano-fluid forced convection in a channel. J. Magn. Magn. Mater. 374, 11–17 (2015). 41. Sheikholeslami, M. & Rokni, H. B. Simulation of nanofluid heat transfer in presence of magnetic field: A review. Int. J. Heat Mass Transfer 115, 1203–1233 (2017). 42. Makinde, O. D., & Mishra, S. R. Chemically reacting MHD mixed convection variable viscosity Blasius flow embedded in a porous medium. In Defect and Diffusion Forum (Vol. 374, pp. 83–91) (Trans Tech Publications Ltd., 2017). 43. Nisar, K. S., Mohapatra, R., Mishra, S. R. & Reddy, M. G. Semi-analytical solution of MHD free convective Jeffrey fluid flow in the presence of heat source and chemical reaction. Ain Shams Eng. J. 12(1), 837–845 (2021). 44. Mishra, S., Mahanthesh, B., Mackolil, J. & Pattnaik, P. K. Nonlinear radiation and cross-diffusion effects on the micropolar nanoliquid flow past a stretching sheet with an exponential heat source. Heat Transfer 50(4), 3530–3546 (2021). 45. Mehrizi, A. A., Vazifeshenas, Y. & Domairry, G. New analysis of natural convection boundary layer flow on a horizontal plate with variable wall temperature. J. Theor. Appl. Mech. 50(4), 1001–1010 (2012). 46. Reddy, P. S., Sreedevi, P. & Chamkha, A. J. Heat and mass transfer flow of a nanofluid over an inclined plate under enhanced boundary conditions with magnetic field and thermal radiation. Heat Transfer Asian Res. 46(7), 815–839 (2017). 47. Nayak, M. K. et al. Thermo-fluidic significance of non Newtonian fluid with hybrid nanostructures. Case Stud. Therm. Eng. 26, 101092 (2021). 48. Hayat, T. & Nadeem, S. An improvement in heat transfer for rotating flow of hybrid nanofluid: a numerical study. Can. J. Phys. 96(12), 1420–1430 (2018). 49. Jedi, A. et al. Statistical modeling for nanofluid flow: A stretching sheet with thermophysical property data. Colloids Interfaces 4(1), 3 (2020). 50. Hussain, A. et al. Numerical simulation and thermal enhancement of multi-based nanofluid over an embrittled cone. Case Stud. Thermal Eng. 28, 101614 (2021). 51. Hussain, A. et al. Heat transport investigation of engine oil based rotating nanomaterial liquid flow in the existence of partial slip effect. Case Stud. Therm. Eng. 28, 101500 (2021). 52. Wang, C. Y. Stretching a surface in a rotating fluid. Zeitschrift für angewandte Mathematik und Physik ZAMP 39(2), 177–185 (1988). 53. Nazar, R., Amin, N. & Pop, I. Unsteady boundary layer flow due to a stretching surface in a rotating fluid. Mech. Res. Commun. 31(1), 121–128 (2004). Acknowledgements The authors are thankful to the Deanship of Scientific Research, King Khalid University, Abha, Saudi Arabia, for financially supporting this work through the General Research Project under Grant No: RGP.1/435/44. This study is supported via funding from Prince Sattam bin Abdulaziz University project number (PSAU/2023/R/1444). Author contributions A.M. Galal: Supervision, Funding, Software. F.M. Alharbi: Data curation. Methodology. M. Arshad: Conceptualization, Writing- Original draft preparation, Writing- Reviewing and Editing. M.M. Alam: Funding, Software, Validation. T. Abdeljawad: Visualization, Funding. Q.M. Al-Mdallal: Formal analysis, Investigation. Scientific Reports \| (2024) 14:1207 \| https://doi.org/10.1038/s41598-024-51195-4 15 Vol.:(0123456789) www.nature.com/scientificreports/ Dedication This manuscript is dedicated to author Mubashar Arshad on his 26th birthday. Competing interests The authors declare no competing interests. Additional information Correspondence and requests for materials should be addressed to M.A., T.A. or Q.M.A.-M. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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https://openalex.org/W4248272564	https://peerj.com/articles/2707v0.3/submission	English	null	Peer Review Crossref Testing 002	null	2,020	cc-by	23,343	Manuscript to be reviewed Manuscript to be reviewed Dominance relationships in a family pack of captive arctic wolves (Canis lupus arctos): the influence of competition for food age and sex Simona Cafazzo Corresp., 1, 2 , Martina Lazzaroni 1, 2 , Sarah Marshall-Pescini 1, 2 1 Wolf Science Centre, Ernstbrunn, Austria 2 Comparative Cognition, Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University of Vienna, University of Vienna, Vienna, Austria 1 Wolf Science Centre, Ernstbrunn, Austria 2 Comparative Cognition, Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University of Vienna, University of Vienna, Vienna, Austria Wolf Science Centre, Ernstbrunn, Austria 2 Comparative Cognition, Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University Austria Corresponding Author: Simona Cafazzo Email address: simona.cafazzo@gmail.com Corresponding Author: Simona Cafazzo Email address: simona.cafazzo@gmail.com Background. Dominance is one of the most pervasive concepts in the study of wolf social behaviour but recently its validity has been questioned. For some authors the bonds between members of wolf families are better described as parent-off-spring relationships and the concept of dominance should be used just to evaluate the social dynamics of non- familial captive pack members (e.g. Mech & Cluff, 2010). However, there is a dearth of studies investigating dominance relationships and its correlates in wolf family packs. Methods. Here, we applied a combination of the most commonly used quantitative methods to evaluate the dominance relationships in a captive family pack of 19 Arctic wolves. Results. We found a significant linear and completely transitive hierarchy based on the direction of submissive behaviours and found that dominance relationships were not influenced by the competitive contexts (feeding vs. non-feeding context). A significant linear hierarchy also emerges amongst siblings once the breeding pair (the two top- ranking individuals) is removed from analyses. Furthermore, results suggest that wolves may use greeting behaviour as a formal signal of subordination. Whereas older wolves were mostly dominant over younger ones, no clear effect of sex was found. However, frequency of agonistic (submissive, dominant and aggressive) behaviours was higher between female-female and male-male dyads than female-male dyads and sex-separated linear hierarchies showed a stronger linearity than the mixed one. Furthermore, dominance status was conveyed through different behavioural categories during intra- sexual and inter-sexual interactions. Discussion. Current results highlight the importance of applying a systematic methodology considering the individuals’ age and sex when evaluating the hierarchical structure of a social group. Manuscript to be reviewed 1 Dominance relationships in a family pack of captive arctic wolves (Canis lupus arctos): 2 influence of competition for food, age and sex 3 4 1,2Simona Cafazzo, 1,2Martina Lazzaroni, 1,2Sarah Marshall-Pescini 5 1 Wolf Science Centre, Ernstbrunn, Austria 6 2 Comparative Cognition, Messerli Research Institute, University of Veterinary Medicine 7 Vienna, Medical University of Vienna, University of Vienna, Vienna, Austria 8 9 Corrisponding Author: 10 Simona Cafazzo1,2 11 Nikolaus Heid-Straße 74/1, Stockerau, 2000, Austria 12 Email address: simona.cafazzo@gmail.com 13 13 PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Dominance relationships in a family pack of captive arctic wolves (Canis lupus arctos): the influence of competition for food age and sex Moreover, they confirm the validity of the concept of dominance relationships in describing the social bonds within a family pack of captive wolves PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 14 Abstract 15 Background. Dominance is one of the most pervasive concepts in the study of wolf social 16 behaviour but recently its validity has been questioned. For some authors the bonds between 17 members of wolf families are better described as parent-off-spring relationships and the concep 18 of dominance should be used just to evaluate the social dynamics of non-familial captive pack 19 members (e.g. Mech & Cluff, 2010). However, there is a dearth of studies investigating 20 dominance relationships and its correlates in wolf family packs. 21 Methods. Here, we applied a combination of the most commonly used quantitative methods to 22 evaluate the dominance relationships in a captive family pack of 19 Arctic wolves. 23 Results. We found a significant linear and completely transitive hierarchy based on the directio 24 of submissive behaviours and found that dominance relationships were not influenced by the 25 competitive contexts (feeding vs. non-feeding context). A significant linear hierarchy also 26 emerges amongst siblings once the breeding pair (the two top-ranking individuals) is removed 27 from analyses. Furthermore, results suggest that wolves may use greeting behaviour as a forma 28 signal of subordination. Whereas older wolves were mostly dominant over younger ones, no 29 clear effect of sex was found. However, frequency of agonistic (submissive, dominant and 30 aggressive) behaviours was higher between female-female and male-male dyads than female- 31 male dyads and sex-separated linear hierarchies showed a stronger linearity than the mixed one 32 Furthermore, dominance status was conveyed through different behavioural categories during 33 intra-sexual and inter-sexual interactions. 34 Discussion. Current results highlight the importance of applying a systematic methodology 35 considering the individuals’ age and sex when evaluating the hierarchical structure of a social 14 Abstract 15 Background. Dominance is one of the most pervasive concepts in the study of wolf social 16 behaviour but recently its validity has been questioned. For some authors the bonds between 17 members of wolf families are better described as parent-off-spring relationships and the concept 18 of dominance should be used just to evaluate the social dynamics of non-familial captive pack 19 members (e.g. Mech & Cluff, 2010). However, there is a dearth of studies investigating 20 dominance relationships and its correlates in wolf family packs. 14 Abstract 15 Background. Dominance is one of the most pervasive concepts in the study of wolf social 16 behaviour but recently its validity has been questioned. Manuscript to be reviewed For some authors the bonds between 17 members of wolf families are better described as parent-off-spring relationships and the concept 18 of dominance should be used just to evaluate the social dynamics of non-familial captive pack 19 members (e.g. Mech & Cluff, 2010). However, there is a dearth of studies investigating 20 dominance relationships and its correlates in wolf family packs. 14 Abstract 21 Methods. Here, we applied a combination of the most commonly used quantitative methods to 22 evaluate the dominance relationships in a captive family pack of 19 Arctic wolves. 21 Methods. Here, we applied a combination of the most commonly used quantitative methods to 22 evaluate the dominance relationships in a captive family pack of 19 Arctic wolves. 23 Results. We found a significant linear and completely transitive hierarchy based on the direction 24 of submissive behaviours and found that dominance relationships were not influenced by the 25 competitive contexts (feeding vs. non-feeding context). A significant linear hierarchy also 26 emerges amongst siblings once the breeding pair (the two top-ranking individuals) is removed 27 from analyses. Furthermore, results suggest that wolves may use greeting behaviour as a formal 28 signal of subordination. Whereas older wolves were mostly dominant over younger ones, no 29 clear effect of sex was found. However, frequency of agonistic (submissive, dominant and 30 aggressive) behaviours was higher between female-female and male-male dyads than female- 31 male dyads and sex-separated linear hierarchies showed a stronger linearity than the mixed one. 32 Furthermore, dominance status was conveyed through different behavioural categories during 33 intra-sexual and inter-sexual interactions. 34 Discussion. Current results highlight the importance of applying a systematic methodology 35 considering the individuals’ age and sex when evaluating the hierarchical structure of a social 34 Discussion. Current results highlight the importance of applying a systematic methodology 35 considering the individuals’ age and sex when evaluating the hierarchical structure of a social PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 36 group. Moreover, they confirm the validity of the concept of dominance relationships in 37 describing the social bonds within a family pack of captive wolves. 38 39 Introduction 40 In group-living animals the natural asymmetries among individuals in their ability to 41 prevail in competition may result in social dominance hierarchies. In general dominant- 42 subordinate relationships may be defined as long-term dyadic relationships characterised by an 43 asymmetric distribution of agonistic behaviours (Drew, 1993). In line with this definition, 44 dominance relationships are measured in terms of the degree of the unidirectionality of 45 behaviours exhibited within a dyad (directional consistency index, Van Hoof & Wensing, 1987) 46 a high unidirectionality, for example, would emerge if A frequently showed submissive 47 behaviours to B, but B was never observed showing a submissive behaviour to A. When 48 dominant-subordinate relationships characterize all or most dyads in a social group, then it may 49 be possible to describe the overall social structure of that group as a ‘linear dominance 50 hierarchy’. To fit the linear hierarchy model, all or most relationships between group members 51 have to be transitive (A > B > C > D), rather than circular (A > B and B > C but C > A), 52 (Landau, 1951; Appleby, 1983; Van Hoof & Wensing, 1987; de Vries, 1995; Shizuka & 53 McDonald, 2012; Shizuka & McDonald, 2014). However, a major limitation of the linearity 54 index is that it becomes negatively biased when some pairs of individuals fail to interact (null 36 group. Moreover, they confirm the validity of the concept of dominance relationships in 37 describing the social bonds within a family pack of captive wolves. 36 group. Moreover, they confirm the validity of the concept of dominance relationships in 37 describing the social bonds within a family pack of captive wolves. 36 group. Moreover, they confirm the validity of the concept of dominance relationships in 37 describing the social bonds within a family pack of captive wolves. 38 39 Introduction 40 In group-living animals the natural asymmetries among individuals in their ability to 41 prevail in competition may result in social dominance hierarchies. In general dominant- 42 subordinate relationships may be defined as long-term dyadic relationships characterised by an 43 asymmetric distribution of agonistic behaviours (Drew, 1993). Manuscript to be reviewed In line with this definition, 44 dominance relationships are measured in terms of the degree of the unidirectionality of 45 behaviours exhibited within a dyad (directional consistency index, Van Hoof & Wensing, 1987), 46 a high unidirectionality, for example, would emerge if A frequently showed submissive 47 behaviours to B, but B was never observed showing a submissive behaviour to A. When 48 dominant-subordinate relationships characterize all or most dyads in a social group, then it may 49 be possible to describe the overall social structure of that group as a ‘linear dominance 50 hierarchy’. To fit the linear hierarchy model, all or most relationships between group members 51 have to be transitive (A > B > C > D), rather than circular (A > B and B > C but C > A), 52 (Landau, 1951; Appleby, 1983; Van Hoof & Wensing, 1987; de Vries, 1995; Shizuka & 53 McDonald, 2012; Shizuka & McDonald, 2014). However, a major limitation of the linearity 54 index is that it becomes negatively biased when some pairs of individuals fail to interact (null 55 dyads; de Vries, 1995; Klass & Cords, 2011). Therefore, a low level of linearity may not 56 necessarily indicate the absence of transitivity but may be due to the high percentage of null 57 dyads. More recently, triangle transitivity (ttri), which determines the level of hierarchy 58 transitivity and is less sensitive to unknown relationship (Shizuka & McDonald, 2012; Shizuka PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 59 & McDonald, 2014), has been used as a successful alternative (e.g. Norscia & Palagi, 2015). 60 Triangle transitivity is based on the transitivity of dominance relations among sets of three 61 individuals that all interact with each other (Shizuka & McDonald, 2012). The method by 62 Shizuka & McDonald (2012) follows logic similar to that of de Vries (1995), but the procedure 63 is conducted without filling in zero dyads (i.e. unknown relationships) with randomized 64 dominance relations. In fact, filling in zero dyads artificially decreases the level of linearity 65 because it creates cyclic (and not transitive) triads, e.g., A dominates B, B dominates C, and C 66 dominates A (A >B >C >A). Triangle transitivity and linearity are equivalent when the 67 dominance relations of all dyads are known but since such complete observations are rare in 68 empirical studies (Shizuka & McDonald, 2012); using both measures may provide a better 69 assessment of the hierarchical organization of a group. 70 As clearly demonstrated by Shizuka & McDonald (2015), another factor affecting the 71 linearity is the interaction rates of the top-ranked individuals. In fact, in groups where alpha 72 individuals engaged in more contests, there were more double-dominant triads (where A 73 dominates both B and C and any relationships is detectable between B and C) and fewer pass- 74 along triads (where A dominates B, B dominates C but any relationships is detectable between A 75 and C), suggesting that top-ranking individuals may have a disproportionate influence on 76 dominance hierarchies and highlighting the necessity to control for that (Shi et al., 2009, 77 Modlmeier et al., 2014; Shizuka & McDonald, 2015). 78 When a significant degree of linearity/transitivity exists in a group, the rank order most 79 consistent with a linear hierarchy may be obtained via the binary dyadic dominance relationship 80 method (I&SI, de Vries, 1998), which allows the characterization of the relationship between two 81 individuals in a group. However, the dyadic relationship between individuals may very across PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 82 contexts since the capacity and motivation of animals to obtain access to specific resources (e.g. 83 feeding, mating privileges etc.) may vary considerably. Since the costs and benefits of winning a 84 given conflict may be context-dependent, a dyad member may be dominant in one context but 85 not in others (Hand, 1986) thereby influencing the overall agonistic rank position of members in 86 a pack. Such differences in rank relationships depending on context have indeed been reported 87 for several species ranging from chimpanzees to cats (Noë, de Waal, & Van Hooff, 1980; 88 Bonanni et al. 2007). 82 contexts since the capacity and motivation of animals to obtain access to specific resources (e.g. 83 feeding, mating privileges etc.) may vary considerably. Since the costs and benefits of winning a 84 given conflict may be context-dependent, a dyad member may be dominant in one context but 85 not in others (Hand, 1986) thereby influencing the overall agonistic rank position of members in 86 a pack. Such differences in rank relationships depending on context have indeed been reported 87 for several species ranging from chimpanzees to cats (Noë, de Waal, & Van Hooff, 1980; 88 Bonanni et al. 2007). 89 A hierarchy within a group can be described in terms of the asymmetry amongst 90 individuals in winning conflicts (agonistic dominance), however it can also be described in terms 91 of formal dominance (de Waal, 1989). The latter is characterized by the exchange of ritualized 92 signals and/or greeting rituals, the direction of which is independent of the social context. When 93 the agonistic dominance is accepted by the subordinates, dominance relationships are stable (i.e. 94 no reversals or intransitivity in the hierarchy emerge; e.g. Fournier & Festa-Bianchet, 1995), and 95 formal and agonistic dominance coincide. In this case, the exchange of hierarchical status 96 information may be conveyed mainly through formalized submissive signals, resulting in a low 97 frequency of overt aggressive conflicts (de Waal, 1989). 89 A hierarchy within a group can be described in terms of the asymmetry amongst 90 individuals in winning conflicts (agonistic dominance), however it can also be described in terms 91 of formal dominance (de Waal, 1989). The latter is characterized by the exchange of ritualized 92 signals and/or greeting rituals, the direction of which is independent of the social context. Manuscript to be reviewed When 93 the agonistic dominance is accepted by the subordinates, dominance relationships are stable (i.e. 94 no reversals or intransitivity in the hierarchy emerge; e.g. Fournier & Festa-Bianchet, 1995), and 95 formal and agonistic dominance coincide. In this case, the exchange of hierarchical status 96 information may be conveyed mainly through formalized submissive signals, resulting in a low 97 frequency of overt aggressive conflicts (de Waal, 1989). 98 Wolves are a particularly interesting case in which to investigate hierarchical relationships 99 since the applicability of this concept to this species has been suggested to depend largely on the 100 type of social structure of the pack (e.g. Block, 2002; Mech & Cluff, 2010). The typical wild wolf 101 social structure is based around a bonded male-female pair that raises pups communally. The 102 offspring of a bonded pair may forego dispersal and remain with their native pack and help raise 103 later litters (e.g. Mech, 1999, 2000; Packard, 2003). Traditionally, dominance relationships in 98 Wolves are a particularly interesting case in which to investigate hierarchical relationships 99 since the applicability of this concept to this species has been suggested to depend largely on the 100 type of social structure of the pack (e.g. Block, 2002; Mech & Cluff, 2010). The typical wild wolf 101 social structure is based around a bonded male-female pair that raises pups communally. The 102 offspring of a bonded pair may forego dispersal and remain with their native pack and help raise 103 later litters (e.g. Mech, 1999, 2000; Packard, 2003). Traditionally, dominance relationships in PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) 1 The model was originally called the "age-graded model" but since it entails also an effect on sex on dominance relationships for a major clarity we prefer refers to it as the "age-(sex)-graded model" Manuscript to be reviewed This model has been presented in one of two ways: either simply as separate linear 108 hierarchies for each sex influenced but not determined by age (Schenkel, 1967; Zimen, 1982) or 109 as male dominance over females within each age class (Rabb, Woolpy & Ginsburg, 1967; Fox, 110 1980; Zimen, 1982; Van Hooff & Wensing, 1987; Savage, 1988). 104 wild wolf packs have been described in terms of the "age-(sex)-graded model1" (Zimen, 1982; 105 Packard, 2003, pp 54) in that within these family groups, there is a natural dominance order in 106 which offspring submit to parents, and puppies submit to both parents and older siblings (Mech, 107 1999). This model has been presented in one of two ways: either simply as separate linear 108 hierarchies for each sex influenced but not determined by age (Schenkel, 1967; Zimen, 1982) or 109 as male dominance over females within each age class (Rabb, Woolpy & Ginsburg, 1967; Fox, 110 1980; Zimen, 1982; Van Hooff & Wensing, 1987; Savage, 1988). 104 wild wolf packs have been described in terms of the "age-(sex)-graded model1" (Zimen, 1982; 105 Packard, 2003, pp 54) in that within these family groups, there is a natural dominance order in 106 which offspring submit to parents, and puppies submit to both parents and older siblings (Mech, 107 1999). This model has been presented in one of two ways: either simply as separate linear 108 hierarchies for each sex influenced but not determined by age (Schenkel, 1967; Zimen, 1982) or 109 as male dominance over females within each age class (Rabb, Woolpy & Ginsburg, 1967; Fox, 110 1980; Zimen, 1982; Van Hooff & Wensing, 1987; Savage, 1988). 110 1980; Zimen, 1982; Van Hooff & Wensing, 1987; Savage, 1988). 111 In contrast to this traditional “age-(and sex)-graded” model, more recently several authors 112 claim that the relationships in the typical wolf family, are better described as parents-offspring- 113 pups relationships rather than as a pecking order dominated by an alpha male (alpha, beta down 114 to omega animals; e.g. Bloch, 2002; Fatjó et al., 2007; Mech & Cluff, 2010). Thus, a dominance 115 hierarchy within the typical ‘nuclear’ family pack could be alternatively viewed as just a 116 reflection of the age, sex and reproductive structure of the group (Mech, 1999). Manuscript to be reviewed 104 wild wolf packs have been described in terms of the "age-(sex)-graded model1" (Zimen, 1982; 105 Packard, 2003, pp 54) in that within these family groups, there is a natural dominance order in 106 which offspring submit to parents, and puppies submit to both parents and older siblings (Mech, 107 1999). This model has been presented in one of two ways: either simply as separate linear 108 hierarchies for each sex influenced but not determined by age (Schenkel, 1967; Zimen, 1982) or 109 as male dominance over females within each age class (Rabb, Woolpy & Ginsburg, 1967; Fox, 110 1980; Zimen, 1982; Van Hooff & Wensing, 1987; Savage, 1988). 111 In contrast to this traditional “age-(and sex)-graded” model, more recently several authors 112 claim that the relationships in the typical wolf family, are better described as parents-offspring- 113 pups relationships rather than as a pecking order dominated by an alpha male (alpha, beta down 114 to omega animals; e.g. Bloch, 2002; Fatjó et al., 2007; Mech & Cluff, 2010). Thus, a dominance 115 hierarchy within the typical ‘nuclear’ family pack could be alternatively viewed as just a 116 reflection of the age, sex and reproductive structure of the group (Mech, 1999). Although the 117 same authors suggest that traditional concepts of dominance hierarchy may still be useful for 118 evaluating the social dynamics of captive families composed of artificially assembled unrelated 119 individuals (Mech, 1999; Packard, 2003; Fatjó et al., 2007). 120 To date, although dominance relationships have been described in several packs of wild 121 wolves (e.g. Mech, 1999; Bloch, 2002; Peterson et al. 2002; Sands & Creel, 2004; Baan et al. 122 2014), a quantitative assessment and statistical validation of the existence of linear dominance 123 relationships has been carried out only in wolves living in captivity and mostly in packs 124 i ti f ith ‘ l f ili ’ i d f tifi i ll bl d l t d 104 wild wolf packs have been described in terms of the "age-(sex)-graded model1" (Zimen, 1982; 105 Packard, 2003, pp 54) in that within these family groups, there is a natural dominance order in 106 which offspring submit to parents, and puppies submit to both parents and older siblings (Mech, 107 1999). Manuscript to be reviewed Packard, 2003 for a review; Mazzini et al 2013) or ‘disrupted families’ i.e. in 126 which one or both of the original parents are missing (e.g. Packard, 2003 for a review; Cordoni 127 & Palagi, 2008). To our knowledge, only two studies systematically tested and confirmed the 128 existence of a linear hierarchy between pack members in typical wolf families ('nuclear families' 129 with parents and multiple generations of offspring and 'extended families' consisting of parents 130 plus one of more of their siblings, and their direct offspring) (Van Hoof & Wensing, 1987; 131 Romero, et al., 2014). 132 Formal signals of dominance or submission have been described in wild wolves and 133 captive wolves (Schenkel, 1947; Feddersen-Petersen, 2004), for example Van Hoof & Wensing, 134 (1987) found that postural displays (namely high posture and low posture) were exhibited 135 consistently from one partner to the other and were therefore suggested as indicators of formal 136 dominance. However, signals of formal dominance have so far been systematically tested only in 137 domestic dogs (Cafazzo et al., 2010; van der Borg et al., 2015). In particular, mouth licking 138 associated with tail wagging (so called ‘greeting’ behaviour) occurring often during greeting 139 ceremonies fulfilled the criteria of a formal signal of subordination in both free-ranging (Cafazzo 140 et al., 2010) and group housed dogs (van der Borg et al., 2015). 132 Formal signals of dominance or submission have been described in wild wolves and 133 captive wolves (Schenkel, 1947; Feddersen-Petersen, 2004), for example Van Hoof & Wensing, 134 (1987) found that postural displays (namely high posture and low posture) were exhibited 135 consistently from one partner to the other and were therefore suggested as indicators of formal 136 dominance. However, signals of formal dominance have so far been systematically tested only in 137 domestic dogs (Cafazzo et al., 2010; van der Borg et al., 2015). In particular, mouth licking 138 associated with tail wagging (so called ‘greeting’ behaviour) occurring often during greeting 139 ceremonies fulfilled the criteria of a formal signal of subordination in both free-ranging (Cafazzo 140 et al., 2010) and group housed dogs (van der Borg et al., 2015). 141 In previous studies, although a linear hierarchy clearly emerged within the pack, there 142 was no quantitative assessment of the validity of the “age-(sex)-graded’ model. Manuscript to be reviewed Although the 117 same authors suggest that traditional concepts of dominance hierarchy may still be useful for 118 evaluating the social dynamics of captive families composed of artificially assembled unrelated 119 individuals (Mech, 1999; Packard, 2003; Fatjó et al., 2007). 1 The model was originally called the "age-graded model" but since it entails also an effect on sex on dominance relationships for a major clarity we prefer refers to it as the "age-(sex)-graded model" PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 125 individuals (e.g. Packard, 2003 for a review; Mazzini et al 2013) or ‘disrupted families’ i.e. in 126 which one or both of the original parents are missing (e.g. Packard, 2003 for a review; Cordoni 127 & Palagi, 2008). To our knowledge, only two studies systematically tested and confirmed the 128 existence of a linear hierarchy between pack members in typical wolf families ('nuclear families' 129 with parents and multiple generations of offspring and 'extended families' consisting of parents 130 plus one of more of their siblings, and their direct offspring) (Van Hoof & Wensing, 1987; 131 Romero, et al., 2014). 132 Formal signals of dominance or submission have been described in wild wolves and 133 captive wolves (Schenkel, 1947; Feddersen-Petersen, 2004), for example Van Hoof & Wensing, 134 (1987) found that postural displays (namely high posture and low posture) were exhibited 135 consistently from one partner to the other and were therefore suggested as indicators of formal 136 dominance. However, signals of formal dominance have so far been systematically tested only in 137 domestic dogs (Cafazzo et al., 2010; van der Borg et al., 2015). In particular, mouth licking 138 associated with tail wagging (so called ‘greeting’ behaviour) occurring often during greeting 139 ceremonies fulfilled the criteria of a formal signal of subordination in both free-ranging (Cafazzo 140 et al., 2010) and group housed dogs (van der Borg et al., 2015). 141 In previous studies, although a linear hierarchy clearly emerged within the pack, there 142 was no quantitative assessment of the validity of the “age-(sex)-graded’ model. Indeed, although 143 female and male separate linear hierarchies have been commonly described qualitatively in 144 captive wolves (e.g. Schenkel, 1947; Zimen, 1982), results tend to go in the opposite direction to 125 individuals (e.g. Packard, 2003 for a review; Mazzini et al 2013) or ‘disrupted families’ i.e. in 126 which one or both of the original parents are missing (e.g. Packard, 2003 for a review; Cordoni 127 & Palagi, 2008). To our knowledge, only two studies systematically tested and confirmed the 128 existence of a linear hierarchy between pack members in typical wolf families ('nuclear families' 129 with parents and multiple generations of offspring and 'extended families' consisting of parents 130 plus one of more of their siblings, and their direct offspring) (Van Hoof & Wensing, 1987; 131 Romero, et al., 2014). 125 individuals (e.g. Manuscript to be reviewed Finally, to help address the 159 controversy regarding the validity of the dominance concept in wolves, we also aimed to assess 160 5) if the typical age-(and sex)-graded model could adequately describe the relationships of the 161 nuclear family pack of wolves observed. Manuscript to be reviewed Indeed, although 143 female and male separate linear hierarchies have been commonly described qualitatively in 144 captive wolves (e.g. Schenkel, 1947; Zimen, 1982), results tend to go in the opposite direction to 145 descriptions of social relationships between the sexes in wild wolves (e.g. Clark, 1971; Haber, 146 1977; Mech, 1999). However, no study has been carried out to assess the validity of this model 147 neither in the wild nor in captivity. Furthermore, despite having been observed in a number of 141 In previous studies, although a linear hierarchy clearly emerged within the pack, there 142 was no quantitative assessment of the validity of the “age-(sex)-graded’ model. Indeed, although 143 female and male separate linear hierarchies have been commonly described qualitatively in 144 captive wolves (e.g. Schenkel, 1947; Zimen, 1982), results tend to go in the opposite direction to 145 descriptions of social relationships between the sexes in wild wolves (e.g. Clark, 1971; Haber, 146 1977; Mech, 1999). However, no study has been carried out to assess the validity of this model 147 neither in the wild nor in captivity. Furthermore, despite having been observed in a number of PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 148 species, no study, to our knowledge has evaluated the potential effect of different competitive 149 contexts on dominance relationships in wolves. 148 species, no study, to our knowledge has evaluated the potential effect of different competitive 149 contexts on dominance relationships in wolves. 148 species, no study, to our knowledge has evaluated the potential effect of different competitive 149 contexts on dominance relationships in wolves. 150 Hence, in the current study we applied a combination of the most commonly used 151 quantitative methods to evaluate dominance relationships, in order to investigate 1) whether an 152 agonistic dominance hierarchy could describe the relationships between members of a captive 153 family pack of Arctic wolves and whether a hierarchical structure would remain consistent 154 (amongst siblings) when the top-ranking individuals (i.e. the breeding pair) were removed from 155 analyses (see Shizuku & McDonald 2015); 2) whether the hierarchy remained consistent in a 156 feeding and non-feeding context; 3) which behaviours may be the best indicators of dominance, 157 4) whether mouth licking associated to tail wagging (greeting, hereafter) can be considered a 158 formal signal of submission in wolves, as has been found in dogs. Finally, to help address the 159 controversy regarding the validity of the dominance concept in wolves, we also aimed to assess 160 5) if the typical age-(and sex)-graded model could adequately describe the relationships of the 161 nuclear family pack of wolves observed. 150 Hence, in the current study we applied a combination of the most commonly used 151 quantitative methods to evaluate dominance relationships, in order to investigate 1) whether an 152 agonistic dominance hierarchy could describe the relationships between members of a captive 153 family pack of Arctic wolves and whether a hierarchical structure would remain consistent 154 (amongst siblings) when the top-ranking individuals (i.e. the breeding pair) were removed from 155 analyses (see Shizuku & McDonald 2015); 2) whether the hierarchy remained consistent in a 156 feeding and non-feeding context; 3) which behaviours may be the best indicators of dominance, 157 4) whether mouth licking associated to tail wagging (greeting, hereafter) can be considered a 158 formal signal of submission in wolves, as has been found in dogs. Manuscript to be reviewed The pack was structured as a nuclear family with all members born into the 175 pack except for the breeding male and the two unrelated breeding females. The number of 176 individuals decreased to 14 by the end of the period of study (Table 1) because 6 wolves were 177 removed from the pack. Specifically, one adult breeding female was removed because she was 178 badly injured by the other breeding female during the reproductive season. Two adult and two 179 sub-adult wolves were sold to another zoo. Finally an adult male was removed because of 180 continuous mobbing episodes from the whole pack. 170 We studied a pack of captive arctic wolves (Canis lupus arctos) at the Olomouc Zoo 171 (Moravia, Czech Republic) during a period of 5 months (Jan 2014–May 2014). The pack was 172 composed of 20 individuals: 7 adult males, 8 adult females, 3 sub-adult males, and 8 sub-adult 173 females. Adults were defined as individuals older than two years and sub-adults as individuals 174 younger than 2 years. The pack was structured as a nuclear family with all members born into the 175 pack except for the breeding male and the two unrelated breeding females. The number of 176 individuals decreased to 14 by the end of the period of study (Table 1) because 6 wolves were 177 removed from the pack. Specifically, one adult breeding female was removed because she was 178 badly injured by the other breeding female during the reproductive season. Two adult and two 179 sub-adult wolves were sold to another zoo. Finally an adult male was removed because of 180 continuous mobbing episodes from the whole pack. 181 The pack was kept from February to March in an enclosure of approximately 7000 m2 182 located in a naturally hilly area equipped with trees, branches and dens. For the rest of the study 183 period the animals were restricted in a smaller enclosure of about 3000 m2. The animals were fed 184 with pieces of meat, which were put on a table of 2 m2, 4 or 5 times every week in the early 185 afternoon. Water was available ad libitum. No stereotypic or aberrant behaviours characterized 186 the study group. Manuscript to be reviewed 170 We studied a pack of captive arctic wolves (Canis lupus arctos) at the Olomouc Zoo 171 (Moravia, Czech Republic) during a period of 5 months (Jan 2014–May 2014). The pack was 172 composed of 20 individuals: 7 adult males, 8 adult females, 3 sub-adult males, and 8 sub-adul 173 females. Adults were defined as individuals older than two years and sub-adults as individua 174 younger than 2 years. The pack was structured as a nuclear family with all members born into 175 pack except for the breeding male and the two unrelated breeding females. The number of 176 individuals decreased to 14 by the end of the period of study (Table 1) because 6 wolves were 177 removed from the pack. Specifically, one adult breeding female was removed because she wa 178 badly injured by the other breeding female during the reproductive season. Two adult and two 179 sub-adult wolves were sold to another zoo. Finally an adult male was removed because of 180 continuous mobbing episodes from the whole pack. 181 The pack was kept from February to March in an enclosure of approximately 7000 m2 182 located in a naturally hilly area equipped with trees, branches and dens. For the rest of the stu 183 period the animals were restricted in a smaller enclosure of about 3000 m2. The animals were 184 with pieces of meat, which were put on a table of 2 m2, 4 or 5 times every week in the early 185 afternoon. Water was available ad libitum. No stereotypic or aberrant behaviours characterize 186 the study group. 187 Data collection 188 The pack was observed 6 days per week for 2-3 hours a day, either in the morning or th 189 afternoon (the afternoon period included feeding time). Before commencing systematic data 190 collection, the observer (M.L.) underwent a 7-month training period on wolf behaviour whilst 191 collecting data at the Wolf Science Centre (Ernstrbunn, Austria). Then she carried out some 170 We studied a pack of captive arctic wolves (Canis lupus arctos) at the Olomouc Zoo 171 (Moravia, Czech Republic) during a period of 5 months (Jan 2014–May 2014). The pack was 172 composed of 20 individuals: 7 adult males, 8 adult females, 3 sub-adult males, and 8 sub-adult 173 females. Adults were defined as individuals older than two years and sub-adults as individuals 174 younger than 2 years. 163 Ethics statement 164 The study was purely observational with no manipulation of animals. The relevant 165 committee, Tierversuchs-kommission am Bundesministerium für Wissenschaft und Forschung 166 (Austria) allows us running this research without special permissions regarding animals (wolves) 167 since this is not required in such socio-cognitive studies in Austria (Tierversuchsgesetz 2012– 168 TVG 2012). PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed 192 preliminary observations (50 h) in order to 1) identify all individuals belonging to the pack and 193 2) establish the data collection methods. 192 preliminary observations (50 h) in order to 1) identify all individuals belonging to the pack and 193 2) establish the data collection methods. 194 Wolf behaviour was observed in 2 different social contexts: in the presence of food and in 195 the absence of this source of competition. Data collection was carried out following Altmann’s 196 (1974) methods: the focal animal sampling method was used in the absence of sources of 197 competition, whereas the subgroup animal sampling method was used in the presence of food. 198 During focal-subgroup sessions all occurrences of agonistic and greeting interactions were 199 recorded (Table 2). The "ad libitum" sampling method (Altmann, 1974) was also used to record 200 all agonistic behavioural patterns as well as greetings occurring out of focal-subgroup sampling 201 sessions; we gathered 154h of observations, distributed over 63 days. 194 Wolf behaviour was observed in 2 different social contexts: in the presence of food and in 195 the absence of this source of competition. Data collection was carried out following Altmann’s 196 (1974) methods: the focal animal sampling method was used in the absence of sources of 197 competition, whereas the subgroup animal sampling method was used in the presence of food. 198 During focal-subgroup sessions all occurrences of agonistic and greeting interactions were 199 recorded (Table 2). The "ad libitum" sampling method (Altmann, 1974) was also used to record 200 all agonistic behavioural patterns as well as greetings occurring out of focal-subgroup sampling 201 sessions; we gathered 154h of observations, distributed over 63 days. 202 Each individual measure of all behaviour patterns was corrected for animal observation 203 time because the latter varied between individuals; since some animals were removed from the 204 pack earlier. 202 Each individual measure of all behaviour patterns was corrected for animal observation 203 time because the latter varied between individuals; since some animals were removed from the 204 pack earlier. Manuscript to be reviewed D t ll ti 188 The pack was observed 6 days per week for 2-3 hours a day, either in the morning or the 189 afternoon (the afternoon period included feeding time). Before commencing systematic data 190 collection, the observer (M.L.) underwent a 7-month training period on wolf behaviour whilst 191 collecting data at the Wolf Science Centre (Ernstrbunn, Austria). Then she carried out some 188 The pack was observed 6 days per week for 2-3 hours a day, either in the morning or the 189 afternoon (the afternoon period included feeding time). Before commencing systematic data 190 collection, the observer (M.L.) underwent a 7-month training period on wolf behaviour whilst 191 collecting data at the Wolf Science Centre (Ernstrbunn, Austria). Then she carried out some PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 214 The values of the Landau’s corrected linearity index h′, Triangle transitivity (ttri), and DCI 215 ranged from 0 to 1. Values of 0 indicate a complete absence of linearity and transitivity (i.e. no 216 hierarchy) and a complete bidirectionality; values of 1 indicate a perfect linear hierarchy, the 217 absence of circular triads and a complete unidirectionality. 218 When significant linearity was detected, dominance ranks were determined using the I&SI 219 method which minimizes inconsistencies and strengths of inconsistencies in dominance 220 relationships (de Vries, 1998). An inconsistency occurs when individual j dominates i, and j's 221 rank is lower than i's (de Vries, 1998). The rank difference between two individuals involved in 222 an inconsistency is the strength of that inconsistency (de Vries, 1998). 223 The dominance rank order obtained for each matrix was standardized by distributing ranks 224 evenly between the highest (+1) and the lowest (-1), with the median rank being scored as 0 225 (East & Hofer, 2001). Then, in order to test the effect of social context, age and gender, we 226 calculated the Spearman’s correlation coefficient between 1) I&SI rank orders in the two 227 competitive contexts, 2) between inter-sexual and intra-sexual hierarchies, and 3) between rank 228 orders and age. 229 To classify greeting as a formal indicator of subordination we assessed whether it fit 230 specific criteria: a) completely unidirectional (DCI =1), b) shown between most of the pack 231 members (no null dyads) and c) correlation with dominance relationships based on agonistic 232 behaviours (de Waal & Luttrell, 1985; de Waal, 1986; 1989; Preuschoft, 1999; Vervaecke, de 233 Vries, H. & Van Elsacker, 2000). 234 All correlation analyses were plotted and visually checked to ascertain that data was not 235 clustered . 214 The values of the Landau’s corrected linearity index h′, Triangle transitivity (ttri), and DCI 215 ranged from 0 to 1. Values of 0 indicate a complete absence of linearity and transitivity (i.e. no 216 hierarchy) and a complete bidirectionality; values of 1 indicate a perfect linear hierarchy, the 217 absence of circular triads and a complete unidirectionality. 214 The values of the Landau’s corrected linearity index h′, Triangle transitivity (ttri), and DCI 215 ranged from 0 to 1. Values of 0 indicate a complete absence of linearity and transitivity (i.e. 205 Agonistic dominance hierarchy and behavioural analyses 206 In order to determine the agonistic dominance hierarchy, the outcomes of aggressive, 207 submissive, and dominance dyadic interactions (see Table 2 for a description) were ranked in 3 208 different squared matrices with winners on one axis and losers on the other. This procedure was 209 applied to interactions in both social contexts (i.e. presence and absence of food), resulting in a 210 total of 6 matrices, 3 for each context. In the same way, in order to analyse the use of greeting as 211 a formal signal of subordination, all greeting interactions observed (Table 2) were ranked in a 212 squared matrix. For each matrix, linearity and transitivity (and their statistical significance), as 213 well as directional consistency, were calculated. 206 In order to determine the agonistic dominance hierarchy, the outcomes of aggressive, 206 In order to determine the agonistic dominance hierarchy, the outcomes of aggressive, 207 submissive, and dominance dyadic interactions (see Table 2 for a description) were ranked in 3 208 different squared matrices with winners on one axis and losers on the other. This procedure was 209 applied to interactions in both social contexts (i.e. presence and absence of food), resulting in a 210 total of 6 matrices, 3 for each context. In the same way, in order to analyse the use of greeting as 211 a formal signal of subordination, all greeting interactions observed (Table 2) were ranked in a 212 squared matrix. For each matrix, linearity and transitivity (and their statistical significance), as 213 well as directional consistency, were calculated. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed 236 Linearity, DCI and I&SI rank orders were calculated using Matman 1.1 (10.000 237 randomizations; Noldus Information Technology, Wageningen, The Netherlands; de Vries, Netto 238 & Hanegraaf, 1993). Spearman's rank correlations were calculated in STATISTICA 7.1 edition 239 (StatSoft Italy s.r.l. 2005). We calculated the proportion of transitive triangles relative to all 240 triangles (Pt), the triangle transitivity metric (ttri) and its statistical significance using the codes 241 provided in Shizuka & McDonald (2012); supplementary material; errata corrige: Shizuka & 242 McDonald, 2014; package ‘statnet’, Handcock et al., 2003) in the R programme version 3.2.3 (R 243 Development Core Team, 2011). The alpha male and female totalized just under half of our data 244 points in all three categories of behaviours considered (41.84% of dominance behaviours; 245 44.06% of aggressive behaviors; 51.46% of submissive behaviors received). Hence, taking into 246 account the potential disproportionate role of these individuals in affecting the linearity of the 247 hierarchy (as suggested by Shizuku & McDonald 2015) we re-ran the analyses described above 248 without these two animals. 236 Linearity, DCI and I&SI rank orders were calculated using Matman 1.1 (10.000 237 randomizations; Noldus Information Technology, Wageningen, The Netherlands; de Vries, Netto 238 & Hanegraaf, 1993). Spearman's rank correlations were calculated in STATISTICA 7.1 edition 239 (StatSoft Italy s.r.l. 2005). We calculated the proportion of transitive triangles relative to all 240 triangles (Pt), the triangle transitivity metric (ttri) and its statistical significance using the codes 241 provided in Shizuka & McDonald (2012); supplementary material; errata corrige: Shizuka & 242 McDonald, 2014; package ‘statnet’, Handcock et al., 2003) in the R programme version 3.2.3 (R 243 Development Core Team, 2011). The alpha male and female totalized just under half of our data 244 points in all three categories of behaviours considered (41.84% of dominance behaviours; 245 44.06% of aggressive behaviors; 51.46% of submissive behaviors received). Manuscript to be reviewed no 216 hierarchy) and a complete bidirectionality; values of 1 indicate a perfect linear hierarchy, the 217 absence of circular triads and a complete unidirectionality. 223 The dominance rank order obtained for each matrix was standardized by distributing ranks 224 evenly between the highest (+1) and the lowest (-1), with the median rank being scored as 0 225 (East & Hofer, 2001). Then, in order to test the effect of social context, age and gender, we 226 calculated the Spearman’s correlation coefficient between 1) I&SI rank orders in the two 227 competitive contexts, 2) between inter-sexual and intra-sexual hierarchies, and 3) between rank 228 orders and age. 223 The dominance rank order obtained for each matrix was standardized by distributing ranks 224 evenly between the highest (+1) and the lowest (-1), with the median rank being scored as 0 225 (East & Hofer, 2001). Then, in order to test the effect of social context, age and gender, we 226 calculated the Spearman’s correlation coefficient between 1) I&SI rank orders in the two 227 competitive contexts, 2) between inter-sexual and intra-sexual hierarchies, and 3) between rank 228 orders and age. 229 To classify greeting as a formal indicator of subordination we assessed whether it fit 230 specific criteria: a) completely unidirectional (DCI =1), b) shown between most of the pack 231 members (no null dyads) and c) correlation with dominance relationships based on agonistic 232 behaviours (de Waal & Luttrell, 1985; de Waal, 1986; 1989; Preuschoft, 1999; Vervaecke, de 233 Vries, H. & Van Elsacker, 2000). 229 To classify greeting as a formal indicator of subordination we assessed whether it fit 230 specific criteria: a) completely unidirectional (DCI =1), b) shown between most of the pack 231 members (no null dyads) and c) correlation with dominance relationships based on agonistic 232 behaviours (de Waal & Luttrell, 1985; de Waal, 1986; 1989; Preuschoft, 1999; Vervaecke, de 233 Vries, H. & Van Elsacker, 2000). 234 All correlation analyses were plotted and visually checked to ascertain that data was not 235 clustered . PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Hence, taking into 246 account the potential disproportionate role of these individuals in affecting the linearity of the 247 hierarchy (as suggested by Shizuku & McDonald 2015) we re-ran the analyses described above 249 In order to investigate whether the dyadic distribution of greeting was influenced by rank, 250 age and sex we ran a generalized linear mixed model (GLMM with a Poisson distribution) with 251 the frequency of greeting behaviour as the response factor and sex combination of the dyad 252 (female-female, male-male and female-male), age difference expressed in months and rank 253 relationship between actor and receiver as independent factors. The identity of the actor and the 254 dyad were entered as random factors. Finally, to investigate the effect of sex on the distribution 255 of agonistic behaviours we ran a generalized linear mixed model (GLMM with a Poisson 256 distribution) with the frequency of agonistic behaviour as the response factor and gender 257 combination of the dyad (female-female, male-male and female-male) as the independent factor. 258 The identity of both individuals in the dyad was entered as random factors. 249 In order to investigate whether the dyadic distribution of greeting was influenced by rank, 250 age and sex we ran a generalized linear mixed model (GLMM with a Poisson distribution) with 251 the frequency of greeting behaviour as the response factor and sex combination of the dyad 252 (female-female, male-male and female-male), age difference expressed in months and rank 253 relationship between actor and receiver as independent factors. The identity of the actor and the 254 dyad were entered as random factors. Finally, to investigate the effect of sex on the distribution 255 of agonistic behaviours we ran a generalized linear mixed model (GLMM with a Poisson 256 distribution) with the frequency of agonistic behaviour as the response factor and gender 257 combination of the dyad (female-female, male-male and female-male) as the independent factor. 258 The identity of both individuals in the dyad was entered as random factors. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be revie Manuscript to be reviewed 281 Aggressive interactions (N = 277) recorded in the presence of food showed a significant 282 level of linearity but a very low unidirectionality. Both dominance (N = 272 interactions) and 283 submissive behaviour (N = 214 interactions) also showed a significant but low level of linearity 284 and the highest values of unidirectionality. The rank orders based on aggressive behaviour was 285 correlated to those based on dominance and submissive behaviours (rs = 0.72, n = 19, p = 286 0.0005; rs = 0.68, n = 19, p = 0.001, respectively) but it resulted in a high number of 281 Aggressive interactions (N = 277) recorded in the presence of food showed a significant 282 level of linearity but a very low unidirectionality. Both dominance (N = 272 interactions) and 283 submissive behaviour (N = 214 interactions) also showed a significant but low level of linearity 284 and the highest values of unidirectionality. The rank orders based on aggressive behaviour was 285 correlated to those based on dominance and submissive behaviours (rs = 0.72, n = 19, p = 286 0.0005; rs = 0.68, n = 19, p = 0.001, respectively) but it resulted in a high number of 281 Aggressive interactions (N = 277) recorded in the presence of food showed a significant 282 level of linearity but a very low unidirectionality. Both dominance (N = 272 interactions) and 283 submissive behaviour (N = 214 interactions) also showed a significant but low level of linearity 284 and the highest values of unidirectionality. The rank orders based on aggressive behaviour was 285 correlated to those based on dominance and submissive behaviours (rs = 0.72, n = 19, p = 286 0.0005; rs = 0.68, n = 19, p = 0.001, respectively) but it resulted in a high number of 287 inconsistencies. The dominance rank order was correlated to the submissive rank order (rs = 288 0.75, n = 19, p = 0.0002) and both generated 1 inconsistency with a strength of 2 for submissive 289 behaviours and a strength of 4 for dominance behaviours. Manuscript to be reviewed 259 The generalized linear mixed models were calculated using the codes provided in lme4- 260 package (Bates et al., 2016), in R version 3.2.3 (R Development Core Team, 2011). 261 Results 262 1) Agonistic dominance relationships in the absence and presence of food 263 The directional consistency (DCI), linearity (h'), the number and strength of inconsistency 264 (I&SI), the proportion of transitive triangles relative to all triangles (Pt), and the triangle 265 transitivity metric (ttri) for each agonistic behavioural category in the two different contexts 266 analysed (in the presence of food and in its absence) are summarised in Table 3. 267 Agonistic dominance in the absence of food 268 Aggressive interactions (N = 388) did not show significant linearity; nevertheless, the 269 behaviour showed a good level of unidirectionality. A significant linear dominance hierarchy, 270 emerged based on the direction of both submissive behaviours (N = 336 interactions) and 271 dominance behaviour (N = 469 interactions). However, the linearity for both behavioural 272 categories was not high, although submissive and dominance behaviours were both highly 273 unidirectional. Based on these results we applied the I&SI method to both behavioural categories 274 to find the best rank order to fit the linear model. Rank order based on submissive behaviour was 275 positively correlated with the rank order based on dominance behaviour (rs = 0.85, n = 19, p = 276 0.0001) but the former resulted in an inconsistency of a lower strength than the latter. Finally, 277 both dominance and submissive behaviours showed a high value of triangle transitivity, 278 indicating that the low level of linearity was due to the high percentage of null dyads (52.05% 279 both) than to a real absence of transitivity. 280 Agonistic behaviour in the presence of food 259 The generalized linear mixed models were calculated using the codes provided in lme4- 260 package (Bates et al., 2016), in R version 3.2.3 (R Development Core Team, 2011). 278 indicating that the low level of linearity was due to the high percentage of null dyads (52.05% 279 both) than to a real absence of transitivity. 278 indicating that the low level of linearity was due to the high percentage of null dyads (52.05% 279 both) than to a real absence of transitivity. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewe Manuscript to be reviewed All the behavioural categories showed 290 a high triangle transitivity, with dominance behaviours showing the highest value, indicating 291 that, as in the absence of food, the low level of linearity was due to the high percentage of null 292 dyads (aggressive behaviour: 51.86%, submissive behaviour: 60.82%, dominance behaviour: 293 63.13%) rather than to a real absence of transitivity. 287 inconsistencies. The dominance rank order was correlated to the submissive rank order (rs = 288 0.75, n = 19, p = 0.0002) and both generated 1 inconsistency with a strength of 2 for submissive 289 behaviours and a strength of 4 for dominance behaviours. All the behavioural categories showed 290 a high triangle transitivity, with dominance behaviours showing the highest value, indicating 291 that, as in the absence of food, the low level of linearity was due to the high percentage of null 292 dyads (aggressive behaviour: 51.86%, submissive behaviour: 60.82%, dominance behaviour: 293 63.13%) rather than to a real absence of transitivity. 294 In sum, both dominance and submissive behaviours showed a significant linearity, higher 295 values of directionality and triangle transitivity, and a lower number of inconsistencies than 296 aggressive behaviours, resulting in the better measures of dominance relationships. 297 2) Comparison of agonistic dominance in the absence, and in the presence of food 298 The I&SI rank orders based on submissive behaviours in the two contexts were highly 299 correlated (rs = 0.81, n = 19, p = 0.00008). In the same way, the I&SI rank based on dominance 300 behaviours found in the absence of food was highly correlated with the rank based on dominance 301 behaviours found in the presence of food (rs = 0.71, n = 19, p = 0.0007). We could reasonably 302 assert that the slight differences in the rank orders were probably due to the quite high percentage 294 In sum, both dominance and submissive behaviours showed a significant linearity, higher 295 values of directionality and triangle transitivity, and a lower number of inconsistencies than 296 aggressive behaviours, resulting in the better measures of dominance relationships. 294 In sum, both dominance and submissive behaviours showed a significant linearity, higher 295 values of directionality and triangle transitivity, and a lower number of inconsistencies than 296 aggressive behaviours, resulting in the better measures of dominance relationships. Manuscript to be reviewed 303 of null dyads observed in each context. Therefore, dominance relationships between wolves do 304 not appear to be affected by the competitive contexts. 305 3) Which behavioural category is the best indicator of dominance relationships? 306 Considering that from the previous analyses a high correlation was found in the presence 307 and absence of food and in order to minimize the number of null dyads and hence obtain the 308 most reliable dominance hierarchy, the outcomes of all submissive dyadic interactions (N = 550) 309 and all dominance interactions (N = 741) were ranked in two different squared matrices. For both 310 submission and dominance interactions we found a high level of directionality, significant levels 311 of linearity and high triangle transitivity (Table 3). By re-ordering the two matrices following the 312 I&SI method we found two highly correlated rank orders (rs = 0.90, N = 19, p = 0.0001; Figure 313 1). The alpha male and alpha female were the highest in rank, although they exchanged positions 314 between the two hierarchies, with the alpha female being the highest in rank in hierarchy based 315 on submissive behaviours and the alpha male being the highest in rank in the hierarchy based on 316 dominance behaviours. Nevertheless, the matrix of dominance behaviour generated 2 317 inconsistencies with a total strength of 9. Conversely, the matrix of submissive behaviours 318 (Table 4) showed no inconsistencies and revealed the highest triangle transitivity. Therefore, we 303 of null dyads observed in each context. Therefore, dominance relationships between wolves do 304 not appear to be affected by the competitive contexts. 303 of null dyads observed in each context. Therefore, dominance relationships between wolves do 304 not appear to be affected by the competitive contexts. 303 of null dyads observed in each context. Therefore, dominance relationships between wolves do 304 not appear to be affected by the competitive contexts. 305 3) Which behavioural category is the best indicator of dominance relationships? 306 Considering that from the previous analyses a high correlation was found in the presence 307 and absence of food and in order to minimize the number of null dyads and hence obtain the 308 most reliable dominance hierarchy, the outcomes of all submissive dyadic interactions (N = 550) 309 and all dominance interactions (N = 741) were ranked in two different squared matrices. Manuscript to be reviewed 297 2) Comparison of agonistic dominance in the absence, and in the presence of food 298 The I&SI rank orders based on submissive behaviours in the two contexts were highly 299 correlated (rs = 0.81, n = 19, p = 0.00008). In the same way, the I&SI rank based on dominance 300 behaviours found in the absence of food was highly correlated with the rank based on dominance 301 behaviours found in the presence of food (rs = 0.71, n = 19, p = 0.0007). We could reasonably 302 assert that the slight differences in the rank orders were probably due to the quite high percentage PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed For both 310 submission and dominance interactions we found a high level of directionality, significant levels 311 of linearity and high triangle transitivity (Table 3). By re-ordering the two matrices following the 312 I&SI method we found two highly correlated rank orders (rs = 0.90, N = 19, p = 0.0001; Figure 313 1). The alpha male and alpha female were the highest in rank, although they exchanged positions 314 between the two hierarchies, with the alpha female being the highest in rank in hierarchy based 315 on submissive behaviours and the alpha male being the highest in rank in the hierarchy based on 316 dominance behaviours. Nevertheless, the matrix of dominance behaviour generated 2 317 inconsistencies with a total strength of 9. Conversely, the matrix of submissive behaviours 318 (Table 4) showed no inconsistencies and revealed the highest triangle transitivity. Therefore, we 319 conclude that submission can be considered the most reliable indicator of linear dominance 320 relationships in our pack. 321 Excluding the alpha male and female from the analyses, we still found a highly significant linear 322 hierarchy based on both submissive and dominance behaviours (Table 3). Although both 323 linearity and unidirectionality were lower than those obtained including the breeding pair in the 324 analyses, rank orders were precisely the same. The triangle transitivity of dominance behaviours 321 Excluding the alpha male and female from the analyses, we still found a highly significant linear 322 hierarchy based on both submissive and dominance behaviours (Table 3). Although both 323 linearity and unidirectionality were lower than those obtained including the breeding pair in the 324 analyses, rank orders were precisely the same. The triangle transitivity of dominance behaviours PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed 325 decreased while that of submissive behaviour remained the same, showing again a complete 326 transitivity. 327 4) Can greeting behaviour be considered a signal of formal submission? 328 Greeting interactions (N = 182) showed a significant but very low linearity index (ILT: h' = 329 0.34, p = 0.008). Alpha male and the alpha females displayed greetings towards each other. This 330 determined an incomplete, yet still very high, level of unidirectionality (DCI = 0.98). The 331 complete triangle transitivity (ttri = 1.000, p = 0.000) indicated that the low level of linearity was 332 likely due to high percentage of null dyads (66.08%) and not due to the absence of transitivity. 333 Although the linearity was statistically significant, due to the high percentage of null dyads, the 334 I&SI rank order based on greeting was considered to be unreliable. The individual frequency of 335 greetings displayed was not correlated to the I&SI agonistic rank (rs = -0.13, n = 19, p = 0.58) 336 but the latter was highly and negatively correlated with the frequency of greetings received (rs = 337 0.87, n = 19, p = 0.0001). In other words, the higher the wolves were in rank, the more greetings 338 they received. In particular, 60.99% of greetings were received by the breeding pair. At the 339 dyadic level, greeting was directed mainly from subordinates towards dominant individuals 340 (GLM: z = -7.51, p < 0.0001); it also occurred most often between partners with higher age 341 differences (GLM: z = 5.91, p < 0.0001), with younger wolves displaying the behaviour more 342 often towards the older ones. The sex combination of the dyad did not affect the occurrence of 343 greeting (GLM: z = 0.37, p = 0.71). Overall, greeting was observed only in relatively few dyads. 344 Excluding the breeding pair, in such dyads, greeting was completely unidirectional with 345 subordinate wolves showing this behaviour towards dominant ones. 346 5) Does the rank order in our pack conform to the ‘age-(sex)-graded’ model? 325 decreased while that of submissive behaviour remained the same, showing again a complete 326 transitivity. 325 decreased while that of submissive behaviour remained the same, showing again a complete 326 transitivity. 346 5) Does the rank order in our pack conform to the ‘age-(sex)-graded’ model? 346 5) Does the rank order in our pack conform to the ‘age-(sex)-graded’ model? Manuscript to be reviewed 347 The agonistic rank order was significantly but only mildly correlated with the age of the 348 wolves (rs = 0.51, n = 19, p = 0.02; Figure 2) while sex had no significant effect on dominance 349 rank (U = 34, Z = -0.90, p = 0.37). These results could mainly be due to some adult males who 350 ranked at the bottom of the hierarchy. In particular, two of them (Storto and Procione, 17th and 351 18th rank position, respectively) showed lower levels of interactions (mean ± sd of interaction: 352 3.94 ± 0.002) and spent less time close to other wolves (mean ± sd of the average dyadic 353 association index, Clutton-Brock, 1982: 0.004 ± 0.002) than all other pack members (mean ± sd 354 of interaction: 8.39 ± 3.60; mean ± sd of the average dyadic association index: 0.03 ± 0.01). 355 Therefore, they appeared to be peripheral individuals in the pack who probably would have 356 dispersed in the wild. Excluding these two wolves from analyses shows the correlation between 357 rank and age to be noticeably higher (rs = 0.80, n = 17, p = 0.0001), while sex still has no effect 358 (U = 18, Z = -1.73, p = 0.09). Overall, dominance relationships appeared to be influenced by age, 359 with older wolves being dominant over younger individuals, but not by sex. 347 The agonistic rank order was significantly but only mildly correlated with the age of the 348 wolves (rs = 0.51, n = 19, p = 0.02; Figure 2) while sex had no significant effect on dominance 349 rank (U = 34, Z = -0.90, p = 0.37). These results could mainly be due to some adult males who 350 ranked at the bottom of the hierarchy. In particular, two of them (Storto and Procione, 17th and 351 18th rank position, respectively) showed lower levels of interactions (mean ± sd of interaction: 352 3.94 ± 0.002) and spent less time close to other wolves (mean ± sd of the average dyadic 353 association index, Clutton-Brock, 1982: 0.004 ± 0.002) than all other pack members (mean ± sd 354 of interaction: 8.39 ± 3.60; mean ± sd of the average dyadic association index: 0.03 ± 0.01). 355 Therefore, they appeared to be peripheral individuals in the pack who probably would have 356 dispersed in the wild. Manuscript to be reviewed 346 5) Does the rank order in our pack conform to the ‘age-(sex)-graded’ model? PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed 370 Based on these results we analysed the intra-sexual hierarchical organization. The 371 directional consistency (DCI), linearity (h'), the number and strength of inconsistency (I and SI), 372 the proportion of transitive triangles relative to all triangles (Pt), and the triangle transitivity 373 metric (ttri) for each agonistic behavioural category in the two different contexts analysed (in the 374 presence of food and in its absence) are summarised in Table 5. Aggressive behaviour emerged 375 as the best measure of dominance relationships between females since it showed the highest 376 value of linearity, complete triangle transitivity, and a rank order without any inconsistencies. A 377 similar result was found for submissive behaviours between females, although it showed a low 378 linearity due to the relatively high percentage of null dyads (36.11 %). However, the aggressive 379 rank order was highly correlated to the submissive rank order (rs = 0.97, n = 9, p = 0.00002). The 380 best measure of dominance relationships between males was based on submissive behaviours, 381 which showed the highest values of directionality and linearity, complete triangle transitivity and 382 no inconsistencies in the rank order. In fact, dominance behaviour between males also appeared 370 Based on these results we analysed the intra-sexual hierarchical organization. The 371 directional consistency (DCI), linearity (h'), the number and strength of inconsistency (I and SI), 372 the proportion of transitive triangles relative to all triangles (Pt), and the triangle transitivity 373 metric (ttri) for each agonistic behavioural category in the two different contexts analysed (in the 374 presence of food and in its absence) are summarised in Table 5. Aggressive behaviour emerged 375 as the best measure of dominance relationships between females since it showed the highest 376 value of linearity, complete triangle transitivity, and a rank order without any inconsistencies. A 377 similar result was found for submissive behaviours between females, although it showed a low 378 linearity due to the relatively high percentage of null dyads (36.11 %). However, the aggressive 379 rank order was highly correlated to the submissive rank order (rs = 0.97, n = 9, p = 0.00002). The 380 best measure of dominance relationships between males was based on submissive behaviours, 381 which showed the highest values of directionality and linearity, complete triangle transitivity and 382 no inconsistencies in the rank order. Manuscript to be reviewed Excluding these two wolves from analyses shows the correlation between 357 rank and age to be noticeably higher (rs = 0.80, n = 17, p = 0.0001), while sex still has no effect 358 (U = 18, Z = -1.73, p = 0.09). Overall, dominance relationships appeared to be influenced by age, 359 with older wolves being dominant over younger individuals, but not by sex. 360 However, sex did affect the distribution of agonistic behaviours. The frequency of 361 submissive and aggressive behaviours were higher between female-female (FF) and male-male 362 (MM) dyads than female-male dyads (submissive behaviours, GLM: FF-FM z = -3.76, p = 363 0.0004; MM-FM z = 4.04, p = 0.0002; aggressive behaviours, GLM: FF-FM z = -5.19, p = 364 0.001; MM-FM z = 3.25, p = 0.003) but no differences emerged between male-male and female- 365 female dyads (submissive behaviours, GLM: FF-MM z = -0.06, p = 0.99; aggressive behaviours, 366 GLM: FF-MM z = -1.59, p = 0.24). Female-female dyads showed a frequency of dominance 367 behaviours higher than female-male dyads (GLM: FF-FM z = -5.20, p = 0.001) but no significant 368 difference was found between all other combinations (GLM: MM-FM z = 1.96, p = 0.11; FF- 369 MM z = -2.13, p = 0.07). 360 However, sex did affect the distribution of agonistic behaviours. The frequency of PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed In fact, dominance behaviour between males also appeared 383 to be a good measure of hierarchy and the relative rank order was highly correlated to that based 384 on submissive behaviours (rs = 0.94, n = 10, p = 0.00006). 381 which showed the highest values of directionality and linearity, complete triangle transitivity and 382 no inconsistencies in the rank order. In fact, dominance behaviour between males also appeared 383 to be a good measure of hierarchy and the relative rank order was highly correlated to that based 384 on submissive behaviours (rs = 0.94, n = 10, p = 0.00006). 385 The female rank orders based on both aggressive and submissive behaviours and the male 386 rank orders based on both submissive and dominance behaviours were highly correlated to the 387 rank order that both sexes had in the entire inter-sexual hierarchy based on submissive 388 behaviours (female aggressive behaviour: rs = 0.98, n = 9, p < 0.00001; female submissive 389 behaviour: rs = 0.98, n = 9, p < 0.00001; male submissive behaviour: rs = 0.93, n = 10, p = 390 0.0001; male dominance behaviour: rs = 0.99, n = 10, p < 0.00001). 391 Female rank orders were positively correlated to age (aggressive rank order: rs = 0.76, n = 385 The female rank orders based on both aggressive and submissive behaviours and the male 386 rank orders based on both submissive and dominance behaviours were highly correlated to the 387 rank order that both sexes had in the entire inter-sexual hierarchy based on submissive 388 behaviours (female aggressive behaviour: rs = 0.98, n = 9, p < 0.00001; female submissive 389 behaviour: rs = 0.98, n = 9, p < 0.00001; male submissive behaviour: rs = 0.93, n = 10, p = 390 0.0001; male dominance behaviour: rs = 0.99, n = 10, p < 0.00001). 391 Female rank orders were positively correlated to age (aggressive rank order: rs = 0.76, n = 392 9, p = 0.02; submissive rank order: rs = 0.83, n = 9, p = 0.005). For males, positive and 391 Female rank orders were positively correlated to age (aggressive rank order: rs = 0.76, n = 392 9, p = 0.02; submissive rank order: rs = 0.83, n = 9, p = 0.005). For males, positive and PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 393 significant correlations were found only when the two potentially dispersing adult males were 394 not considered (all males: submissive behaviour, rs = 0.38, n = 10, p = 0.28 and dominance 395 behaviour, rs = 0.37, n = 10, p = 0.29; without the two dispersing males: submissive behaviour, 396 rs = 0.78, n = 10, p = 0.002 and dominance behaviour, rs = 0.79, n = 10, p = 0.002). 397 Overall, although no sex effect emerged on the hierarchical rank order of the pack, both 398 males and females showed agonistic behaviours preferentially towards other males and females, 399 respectively. Sex-separate hierarchies showed higher linearity than the hierarchy including the 400 whole pack. Male hierarchical relationships appeared to be based on dominance and submissive 401 behaviours, while female hierarchal relationships appeared to be based on aggressive and 402 submissive behaviours. 393 significant correlations were found only when the two potentially dispersing adult males were 394 not considered (all males: submissive behaviour, rs = 0.38, n = 10, p = 0.28 and dominance 395 behaviour, rs = 0.37, n = 10, p = 0.29; without the two dispersing males: submissive behaviour, 396 rs = 0.78, n = 10, p = 0.002 and dominance behaviour, rs = 0.79, n = 10, p = 0.002). 397 Overall, although no sex effect emerged on the hierarchical rank order of the pack, both 398 males and females showed agonistic behaviours preferentially towards other males and females, 399 respectively. Sex-separate hierarchies showed higher linearity than the hierarchy including the 400 whole pack. Male hierarchical relationships appeared to be based on dominance and submissive 401 behaviours, while female hierarchal relationships appeared to be based on aggressive and 402 submissive behaviours. 404 Using linearity (de Vries, 1995), triangle transitivity (Shizuka & McDonald, 2014), matrix- 405 ranking procedures (MatMan; de Vries, 1998), and taking into account both the sex and ages of 406 the wolves in our pack, we found: 1) the existence of a clear linear hierarchy unaffected by the 407 competitive context and which remained solid also when the highest ranking individuals (the 408 breeding pair) were removed from analyses, 2) evidence suggesting the use of ‘greeting’ as a 409 formalised signal of subordination, and 3) partial support for the age-(and sex)-graded model. Manuscript to be reviewed 416 behaviours (e.g. crouch, passive and active submission, flee, etc.). The breeding pair was 417 involved in most of the interactions, as previously reported also in other captive and wild packs 418 (e.g. Van Hooff & Wensing, 1987¸ Mech, 1999), and although Shizuka & McDonald (2015) 419 point out that linearity of a hierarchy may be ‘skewed’ due to dominant individuals showing 420 more behaviours than the rest, this was not the case in our study. Indeed results showed that a 421 linear and completely transitive hierarchy based on submissive behaviours was still highly 422 significant when the two top-ranking individuals were removed from the analyses. These results 423 indicate that clear dominance relationships exist among all siblings and confirm submissive 424 behaviours as a more reliable indicator of hierarchical relationships in the pack than aggressive 425 and dominance behaviours. 416 behaviours (e.g. crouch, passive and active submission, flee, etc.). The breeding pair was 417 involved in most of the interactions, as previously reported also in other captive and wild packs 418 (e.g. Van Hooff & Wensing, 1987¸ Mech, 1999), and although Shizuka & McDonald (2015) 419 point out that linearity of a hierarchy may be ‘skewed’ due to dominant individuals showing 420 more behaviours than the rest, this was not the case in our study. Indeed results showed that a 421 linear and completely transitive hierarchy based on submissive behaviours was still highly 422 significant when the two top-ranking individuals were removed from the analyses. These results 423 indicate that clear dominance relationships exist among all siblings and confirm submissive 424 behaviours as a more reliable indicator of hierarchical relationships in the pack than aggressive 425 and dominance behaviours. 426 Although the common social structure in wild wolves is usually made up of the 427 reproducing parent pair and their offspring of the last two years, ranging from 2 to 15 individuals 428 (e.g. Bloch, 2002), families composed of several generations of up to 19-26 individuals (e.g. 429 Landau, 1993; White, 2001; VonHoldt, et al., 2008; Smith et al., 2011), have been described. 430 Hence our pack of Arctic wolves, composed of 5 generations and a total of 19 individuals, can be 431 considered representative of a multigenerational family pack of wolves. Manuscript to be reviewed 410 1) Agonistic dominance relationships in the presence and absence of food 411 The main result of the current study is that the relationship between family pack members 412 of the Arctic wolves studied were not randomly distributed but rather, showed a high linear, 413 transitive, and significant hierarchy, which remained constant across both the feeding and non- 414 feeding contexts. Furthermore, we found that the best indicator of dominance, which resulted in 415 the clearest hierarchical relationship between individuals, was the direction of submissive 411 The main result of the current study is that the relationship between family pack members 412 of the Arctic wolves studied were not randomly distributed but rather, showed a high linear, 413 transitive, and significant hierarchy, which remained constant across both the feeding and non- 414 feeding contexts. Furthermore, we found that the best indicator of dominance, which resulted in 415 the clearest hierarchical relationship between individuals, was the direction of submissive PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Therefore, the clear 432 presence of a linear hierarchy in our family pack of wolves goes against recent suggestions that 433 hierarchical relationships may only be adequate to describe atypical pack structures such as 434 disrupted families or forced packs of unrelated individuals (Mech & Cluff, 2010) and rather 435 supports the importance of this concept also in describing the relationship between wolves in a 436 multi generational family pack. 426 Although the common social structure in wild wolves is usually made up of the 427 reproducing parent pair and their offspring of the last two years, ranging from 2 to 15 individuals 428 (e.g. Bloch, 2002), families composed of several generations of up to 19-26 individuals (e.g. 429 Landau, 1993; White, 2001; VonHoldt, et al., 2008; Smith et al., 2011), have been described. 430 Hence our pack of Arctic wolves, composed of 5 generations and a total of 19 individuals, can be 431 considered representative of a multigenerational family pack of wolves. Therefore, the clear 432 presence of a linear hierarchy in our family pack of wolves goes against recent suggestions that 433 hierarchical relationships may only be adequate to describe atypical pack structures such as 434 disrupted families or forced packs of unrelated individuals (Mech & Cluff, 2010) and rather 435 supports the importance of this concept also in describing the relationship between wolves in a 436 multi generational family pack. 437 A further confirmation of the importance of dominance relationships in wolves comes from 438 our second finding, that such relationships remained constant across competitive and non- 437 A further confirmation of the importance of dominance relationships in wolves comes from 438 our second finding, that such relationships remained constant across competitive and non- 437 A further confirmation of the importance of dominance relationships in wolves comes from 438 our second finding, that such relationships remained constant across competitive and non- PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 439 competitive contexts. Indeed the dominance and submissive rank orders detected in the presence 440 of food were highly correlated to the respective rank orders detected in the absence of food. The 441 slight differences observed between the two contexts may be explained by the high percentage of 442 null dyads (Van Hoof & Wensing, 1987). Indeed, when adding together all submissive and 443 dominant interactions occurring in the two contexts, we found improved values of linearity and 444 unidirectionality for both hierarchies, further indicating that dominance relationships in our 445 family pack were not influenced by the competitive context. In some mammal species, it may be 446 reasonable to predict the existence of asymmetries in fighting abilities and resource value, 447 especially between different age-sex classes, leading to different rank orders in different 448 contexts. Indeed, food is considered a major determinant of the reproductive success of 449 individuals hence, in species where females play the main role in rearing pups food should have 450 a higher value for them than for males, leading to a female over male dominance hierarchy 451 during feeding competition but not in other contexts. This has been found to be the case in both 452 chimpanzees and cats, where females raise their infants largely with no male intervention (e.g. 453 chimpanzees, Noë, de Waal, & Van Hooff, 1980; domestic cats, Bonanni et al., 2007). However, 454 wolf packs rely on cooperation between all pack members in both rearing pups and providing 455 food; this may account for the consistency of dominance relationships and the absence of a 456 diverse effect of sex in the feeding and non-feeding context. 457 Submissive behaviours best fulfilled the criteria of agonistic dominance indicators since 439 competitive contexts. Indeed the dominance and submissive rank orders detected in the presence 440 of food were highly correlated to the respective rank orders detected in the absence of food. The 441 slight differences observed between the two contexts may be explained by the high percentage of 442 null dyads (Van Hoof & Wensing, 1987). Indeed, when adding together all submissive and 443 dominant interactions occurring in the two contexts, we found improved values of linearity and 444 unidirectionality for both hierarchies, further indicating that dominance relationships in our 445 family pack were not influenced by the competitive context. Manuscript to be reviewed In some mammal species, it may be 446 reasonable to predict the existence of asymmetries in fighting abilities and resource value, 447 especially between different age-sex classes, leading to different rank orders in different 448 contexts. Indeed, food is considered a major determinant of the reproductive success of 449 individuals hence, in species where females play the main role in rearing pups food should have 450 a higher value for them than for males, leading to a female over male dominance hierarchy 451 during feeding competition but not in other contexts. This has been found to be the case in both 452 chimpanzees and cats, where females raise their infants largely with no male intervention (e.g. 453 chimpanzees, Noë, de Waal, & Van Hooff, 1980; domestic cats, Bonanni et al., 2007). However, 454 wolf packs rely on cooperation between all pack members in both rearing pups and providing 455 food; this may account for the consistency of dominance relationships and the absence of a 456 diverse effect of sex in the feeding and non-feeding context. 457 S b i i b h i b t f lfill d th it i f i ti d i i di t i 457 Submissive behaviours best fulfilled the criteria of agonistic dominance indicators since 458 they showed a higher linearity, a complete transitivity, and rank orders with no inconsistencies. 459 The importance of submissive behaviours in establishing and maintaining dominance 460 relationships have been widely highlighted in primates (Rowell, 1974; de Wall & Luttrell, 1985) 461 but also in wolves (Schenkel, 1967; Mech, 1999). In our pack, subordinate individuals often 457 Submissive behaviours best fulfilled the criteria of agonistic dominance indicators since 458 they showed a higher linearity, a complete transitivity, and rank orders with no inconsistencies. 459 The importance of submissive behaviours in establishing and maintaining dominance 460 relationships have been widely highlighted in primates (Rowell, 1974; de Wall & Luttrell, 1985) 461 but also in wolves (Schenkel, 1967; Mech, 1999). In our pack, subordinate individuals often 457 Submissive behaviours best fulfilled the criteria of agonistic dominance indicators since 458 they showed a higher linearity, a complete transitivity, and rank orders with no inconsistencies. 459 The importance of submissive behaviours in establishing and maintaining dominance 460 relationships have been widely highlighted in primates (Rowell, 1974; de Wall & Luttrell, 1985) 461 but also in wolves (Schenkel, 1967; Mech, 1999). Manuscript to be reviewed In our pack, subordinate individuals often PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 462 determined the outcome of agonistic interactions by lowering themselves when being 463 approached by or when approaching dominant individuals, as described in wild wolf interactions 464 (Mech, 1999). Similar results have also been found in other captive family packs (Van Hooff & 465 Wensing, 1987; Romero et al., 2014). 466 In sum, to date the results suggest that submissive behaviours play a more relevant role 467 than dominance displays in terms of maintaining dominance relationships between all members 468 of a family pack, although further investigation should assess the importance of submissive 469 behaviours in promoting friendly relations and pack cohesion in wolf pack, as has been 470 suggested by some authors (e.g. Mech, 1999). 471 2) Greeting as formal indicator of submission 472 Overall, our results showed that greeting in Arctic wolves partially fulfilled the criteria of a 473 formal signal of submission, although it occurred only in a limited number of dyads, it was 474 almost completely unidirectional and it was exhibited in line with the agonistic dominance 475 hierarchy, in that it was displayed mainly by subordinate individuals towards dominant ones. The 476 main exception was the breeding pair, in which this behaviour was exchanged equally. This is 477 particularly interesting considering that their relative position in the hierarchy was also not 478 always fixed; the male appearing dominant over the female when calculating the rank based on 479 dominance displays and vice versa when basing the rank on submissive behaviours. This might 480 indicate a relaxed dominance relationship between breeding partners, with the females prevailing 481 in some situations and the male in others, as described in other captive family packs (Van Hooff 482 & Wensing, 1987) and wild packs (e.g. Mech, 1999). 483 The frequency of greeting behaviours however, did not correlate with the agonistic rank 484 making it an unreliable measure on which to base the ordering of pack members in a consistent 462 determined the outcome of agonistic interactions by lowering themselves when being 463 approached by or when approaching dominant individuals, as described in wild wolf interactions 464 (Mech, 1999). Similar results have also been found in other captive family packs (Van Hooff & 465 Wensing, 1987; Romero et al., 2014). 462 determined the outcome of agonistic interactions by lowering themselves when being 463 approached by or when approaching dominant individuals, as described in wild wolf interactions 464 (Mech, 1999). Manuscript to be reviewed Similar results have also been found in other captive family packs (Van Hooff & 465 Wensing, 1987; Romero et al., 2014). 466 In sum, to date the results suggest that submissive behaviours play a more relevant role 467 than dominance displays in terms of maintaining dominance relationships between all members 468 of a family pack, although further investigation should assess the importance of submissive 469 behaviours in promoting friendly relations and pack cohesion in wolf pack, as has been 470 suggested by some authors (e.g. Mech, 1999). 471 2) Greeting as formal indicator of submission 472 Overall, our results showed that greeting in Arctic wolves partially fulfilled the criteria of a 473 formal signal of submission, although it occurred only in a limited number of dyads, it was 474 almost completely unidirectional and it was exhibited in line with the agonistic dominance 475 hierarchy, in that it was displayed mainly by subordinate individuals towards dominant ones. The 476 main exception was the breeding pair, in which this behaviour was exchanged equally. This is 477 particularly interesting considering that their relative position in the hierarchy was also not 478 always fixed; the male appearing dominant over the female when calculating the rank based on 479 dominance displays and vice versa when basing the rank on submissive behaviours. This might 480 indicate a relaxed dominance relationship between breeding partners, with the females prevailing 481 in some situations and the male in others, as described in other captive family packs (Van Hooff 482 & Wensing, 1987) and wild packs (e.g. Mech, 1999). 483 The frequency of greeting behaviours however, did not correlate with the agonistic rank 484 making it an unreliable measure on which to base the ordering of pack members in a consistent 483 The frequency of greeting behaviours however, did not correlate with the agonistic rank 484 making it an unreliable measure on which to base the ordering of pack members in a consistent PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 485 linear hierarchy. This was most likely due to the numerous dyads in which no greeting 486 behaviours were observed. Indeed greeting in wolves is widely described to occur upon reunion 487 after a period of separation and before travelling and hunting (e.g. Mech, 1999; Peterson et al., 488 2002). This context is limited in captivity, which may explain why, for many dyads, we were 489 unable to observe this behaviour. Nevertheless, with the only exception of the breeding pair, who 490 exchanged greetings exclusively towards each other, in all other dyads the behaviour was 491 completely unidirectional, displayed mainly from subordinates towards the dominant wolves and 492 with all pack members showing this behaviour most often towards the dominant breeding pair. 493 This is consistent with studies in other canids, showing that the use of greeting is a signal of 494 acknowledgment of dominance status (Ethiopian wolves, Sillero-Zubiri, et al 1996; domestic 495 dog, Cafazzo et al., 2010), and with theories suggesting that the mouth licking behaviour that 496 occurs during greeting interactions may be derived from food begging behaviour displayed from 497 the offspring towards the breeding pair to elicit regurgitation (Schenkel, 1967; Mech, Wolf & 498 Packard, 1999). 499 3) The age-(sex)-graded model 500 In our pack we found an overall dominance hierarchy based on submissive behaviours in 485 linear hierarchy. This was most likely due to the numerous dyads in which no greeting 486 behaviours were observed. Indeed greeting in wolves is widely described to occur upon reunion 487 after a period of separation and before travelling and hunting (e.g. Mech, 1999; Peterson et al., 488 2002). This context is limited in captivity, which may explain why, for many dyads, we were 489 unable to observe this behaviour. Nevertheless, with the only exception of the breeding pair, who 490 exchanged greetings exclusively towards each other, in all other dyads the behaviour was 491 completely unidirectional, displayed mainly from subordinates towards the dominant wolves and 492 with all pack members showing this behaviour most often towards the dominant breeding pair. Manuscript to be reviewed 508 work both in captivity and in the wild never statistically tested the effect of sex and age. Our 509 results support the effect of age, showing largely that older siblings are dominant over younger 510 ones, but do not support a sex effect of males being dominant over females. 511 Interestingly however, when looking at the frequency of the three main behavioural 512 categories used to calculate dominance relationships in our pack, we found that female-male 513 agonistic interactions were fewer compared to intra-sexual (female-female and male-male) 514 agonistic interactions. In other words, although an overall hierarchy including animals of both 515 sexes was detected, agonistic displays were not so frequently expressed in interactions between 516 females and males. In fact, females showed agonistic behaviours preferentially towards other 517 females and males towards other males. Taking into account this differential pattern of 518 behaviours, we calculated sex-separate linear hierarchies, which, in both cases, showed stronger 519 linearity than the mixed hierarchy. Moreover, dominance relationships appeared to be expressed 520 making use of different behavioural categories in male’s and female’s hierarchies. The best 521 hierarchies (in terms of unidirectionlity, linearity, and transitivity) in females were based on 522 aggressive and submissive interactions, whereas in males, hierarchies based on submission and 523 dominance behaviours showed better indices. Taken together, results suggest that although the 524 pack as a whole shows a clear hierarchical organization, the structure of the hierarchy within 525 each sex is even clearer. Furthermore, it appears that females and males may use different ways 526 to communicate their reciprocal rank when interacting with members of their own sex. Van 527 Hooff & Wensing (1997) found similar results in a family pack of European wolves, where intra- 528 sexual relationships were characterized by a higher intensity of exchange of agonistic behaviour 529 than inter-sexual relationships. 508 work both in captivity and in the wild never statistically tested the effect of sex and age. Our 509 results support the effect of age, showing largely that older siblings are dominant over younger 510 ones, but do not support a sex effect of males being dominant over females. 511 Interestingly however, when looking at the frequency of the three main behavioural 512 categories used to calculate dominance relationships in our pack, we found that female-male 513 agonistic interactions were fewer compared to intra-sexual (female-female and male-male) 514 agonistic interactions. Manuscript to be reviewed 493 This is consistent with studies in other canids, showing that the use of greeting is a signal of 494 acknowledgment of dominance status (Ethiopian wolves, Sillero-Zubiri, et al 1996; domestic 495 dog, Cafazzo et al., 2010), and with theories suggesting that the mouth licking behaviour that 496 occurs during greeting interactions may be derived from food begging behaviour displayed from 497 the offspring towards the breeding pair to elicit regurgitation (Schenkel, 1967; Mech, Wolf & 498 Packard, 1999). 499 3) The age-(sex)-graded model 500 In our pack we found an overall dominance hierarchy based on submissive behaviours in 501 which males were not, on average, higher in rank than females, but older wolves were dominant 502 over younger ones (with the exception of two adult males who ranked at the bottom of the 503 hierarchy). In wild wolf packs, it is usually reported that all members submit to the breeding pair, 504 and the breeding female to the breeding male, with no clear dominance displays being observed 505 between offspring (Mech, 1999; Bloch, 2002). In captive studies, males are mostly described as 506 being dominant over females and older individuals over younger ones (e.g. Van Hooff & 507 Wensing, 1987; Romero et al., 2014). However, differently from the current study, previous 500 In our pack we found an overall dominance hierarchy based on submissive behaviours in 501 which males were not, on average, higher in rank than females, but older wolves were dominant 502 over younger ones (with the exception of two adult males who ranked at the bottom of the 503 hierarchy). In wild wolf packs, it is usually reported that all members submit to the breeding pair, 504 and the breeding female to the breeding male, with no clear dominance displays being observed 505 between offspring (Mech, 1999; Bloch, 2002). In captive studies, males are mostly described as 506 being dominant over females and older individuals over younger ones (e.g. Van Hooff & 507 Wensing, 1987; Romero et al., 2014). However, differently from the current study, previous PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed In other words, although an overall hierarchy including animals of both 515 sexes was detected, agonistic displays were not so frequently expressed in interactions between 516 females and males. In fact, females showed agonistic behaviours preferentially towards other 517 females and males towards other males. Taking into account this differential pattern of 518 behaviours, we calculated sex-separate linear hierarchies, which, in both cases, showed stronger 519 linearity than the mixed hierarchy. Moreover, dominance relationships appeared to be expressed 520 making use of different behavioural categories in male’s and female’s hierarchies. The best 521 hierarchies (in terms of unidirectionlity, linearity, and transitivity) in females were based on 522 aggressive and submissive interactions, whereas in males, hierarchies based on submission and 523 dominance behaviours showed better indices. Taken together, results suggest that although the 524 pack as a whole shows a clear hierarchical organization, the structure of the hierarchy within 525 each sex is even clearer. Furthermore, it appears that females and males may use different ways 526 to communicate their reciprocal rank when interacting with members of their own sex. Van 527 Hooff & Wensing (1997) found similar results in a family pack of European wolves, where intra- 528 sexual relationships were characterized by a higher intensity of exchange of agonistic behaviour 529 than inter-sexual relationships. 508 work both in captivity and in the wild never statistically tested the effect of sex and age. Our 509 results support the effect of age, showing largely that older siblings are dominant over younger 510 ones, but do not support a sex effect of males being dominant over females. 508 work both in captivity and in the wild never statistically tested the effect of sex and age. Our 509 results support the effect of age, showing largely that older siblings are dominant over younger 510 ones, but do not support a sex effect of males being dominant over females. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed 530 An unexpected result is that females, but not males, appeared to use aggression to 531 communicate their reciprocal status in interactions with other females. This result disagrees with 532 most of the studies on hierarchies outlining submission as the best measure of dominance 533 relationships (e.g. Rowel, 1974; Bernstein, 1981; Hand, 1986; Cafazzo et al., 2010). A potential 534 explanation is that in general, aggressive interactions were frequent between individuals of our 535 study pack, potentially due to the data being collected mostly during the breeding season, which 536 starts in January and lasts approximately until April. During this time the hierarchical structure of 537 the pack likely regulates breeding activity, and aggressive interactions may be used to more 538 forcefully maintain the status among individuals. Further studies are needed to ascertain whether 539 indeed the behaviours used to maintain the hierarchy are different during breeding and non- 540 breeding periods. 541 The greater linearity of the hierarchical organization and the differential patterns of 542 behaviours used to maintain it in males and females raises the question about the most 543 appropriate way to characterize the dominance relationships among members of a wolf pack. Are 544 sex-separate hierarchies a better model than all-member hierarchies to describe such 545 relationships? Several authors suggest that separate same-sex hierarchies best describe the social 546 structure of wolf packs (Schenkel, 1947; Rabb, Woolpy & Ginsburg, 1967; Zimen, 1975, 1978; 547 Derix et al. 1993; Derix & VanHooff, 1995). Nevertheless, Zimen highlighted the existence of an 548 overall hierarchy with males being dominant over females in each age class, which is also the 549 usual model reported in studies of wild wolves (Clark, 1971; Mech, 1999). Unfortunately, as 550 stated above, most of the previous studies carried out both in captivity and in the wild, did not 551 follow a systematic procedure aimed to statistically show the dominance relationships in the 552 pack, which makes a comparative assessment of results difficult. Based on current results, when 530 An unexpected result is that females, but not males, appeared to use aggression to 531 communicate their reciprocal status in interactions with other females. This result disagrees with 532 most of the studies on hierarchies outlining submission as the best measure of dominance 533 relationships (e.g. Rowel, 1974; Bernstein, 1981; Hand, 1986; Cafazzo et al., 2010). PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 553 considering the pack as a whole the hierarchical structure does not show males being dominant 554 over females, but given the stronger linearity indices of separate male and female hierarchies, it 555 would appear that status within sexes may carry an even greater weight than within the mixed 556 sex group. 557 A final point to consider is the validity of captive-based studies when attempting to 558 characterize the social structure of a wild species. It is undeniable that studies with wild animals 559 are preferable when exploring such topics, however it is perhaps interesting to note that in a 560 metanalyses involving 113 studies looking at dominance structures in 85 species (172 groups), 561 Shizuku and McDonald (2015) found that whether studies were conducted in the wild or in a 562 captive setting did not affect results. With such elusive species as wolves, partial reliance on 563 captive studies is probably unavoidable, however future research using the same methodologies 564 adopted here on wild animals would be particularly important to further our understanding of 565 wolves’ social behaviour. 557 A final point to consider is the validity of captive-based studies when attempting to 558 characterize the social structure of a wild species. It is undeniable that studies with wild animals 559 are preferable when exploring such topics, however it is perhaps interesting to note that in a 560 metanalyses involving 113 studies looking at dominance structures in 85 species (172 groups), 561 Shizuku and McDonald (2015) found that whether studies were conducted in the wild or in a 562 captive setting did not affect results. With such elusive species as wolves, partial reliance on 563 captive studies is probably unavoidable, however future research using the same methodologies 564 adopted here on wild animals would be particularly important to further our understanding of 565 wolves’ social behaviour. Manuscript to be reviewed A potential 534 explanation is that in general, aggressive interactions were frequent between individuals of our 535 study pack, potentially due to the data being collected mostly during the breeding season, which 536 starts in January and lasts approximately until April. During this time the hierarchical structure of 537 the pack likely regulates breeding activity, and aggressive interactions may be used to more 538 forcefully maintain the status among individuals. Further studies are needed to ascertain whether 539 indeed the behaviours used to maintain the hierarchy are different during breeding and non- 540 breeding periods. 530 An unexpected result is that females, but not males, appeared to use aggression to 531 communicate their reciprocal status in interactions with other females. This result disagrees with 532 most of the studies on hierarchies outlining submission as the best measure of dominance 533 relationships (e.g. Rowel, 1974; Bernstein, 1981; Hand, 1986; Cafazzo et al., 2010). A potential 534 explanation is that in general, aggressive interactions were frequent between individuals of our 535 study pack, potentially due to the data being collected mostly during the breeding season, which 536 starts in January and lasts approximately until April. During this time the hierarchical structure of 537 the pack likely regulates breeding activity, and aggressive interactions may be used to more 538 forcefully maintain the status among individuals. Further studies are needed to ascertain whether 539 indeed the behaviours used to maintain the hierarchy are different during breeding and non- 540 breeding periods. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 575 We are grateful to Dr. Jitka Vokurková, the director, and employees of Olomouc Zoo f 576 allowing us to run observations in their institution. Special thanks are due to Daizaburo Shizuk 577 for statistical advice and to Rachel Dale for language revision. We also thank two anonymou 578 referees for useful and constructive suggestions that improved the manuscript. 579 580 References 581 Altmann J. 1974. Observational study of behavior: sampling methods. Behaviour. 48:227–265. 582 Appleby MC. 1983. The probability of linearity in hierarchies. Anim Behav. 31:600–608. 583 Baan C, Bergmüller R, Smith DW & Molnar B. 2014. Conflict management in free-ranging 584 wolves, Canis lupus. Animal Behaviour, 90, 327–334. 585 http://doi.org/10.1016/j.anbehav.2014.01.033 586 Bates D, Maechler M, Bolker B, Walker S. 2015. Fitting Linear Mixed-Effects Models Using 587 lme4. Journal of Statistical Software, 67(1), 1-48. doi:10.18637/jss.v067.i01. 588 Bernstein IS. 1981. Dominance: the baby and the bathwater. Behav Brain Sci. 4:419–457. 589 Bloch G. 2002. Alpha-concept, dominance & leadership in wolf families. Wolf! Magazine Vol. 590 20, No.2 591 Bonanni R, Cafazzo S, Fantini C, Pontier D, Natoli E. 2007. Feeling order in an urban feral 592 domestic cat colony: relationship to dominance rank, sex and age. Anim Behav. 74:1369– 593 1379. 594 Cafazzo S, Valsecchi P, Bonanni R, Natoli E. 2010. Dominance in relation 586 to age, sex, and 595 competitive contexts in a group of free-ranging domestic dogs. Behav. Ecol. 21 (3), 443- 596 455. 575 We are grateful to Dr. Jitka Vokurková, the director, and employees of Olomouc Zoo for 576 allowing us to run observations in their institution. Special thanks are due to Daizaburo Shizuka 577 for statistical advice and to Rachel Dale for language revision. We also thank two anonymous 578 referees for useful and constructive suggestions that improved the manuscript. 581 Altmann J. 1974. Observational study of behavior: sampling methods. Behaviour. 48:227–265. 582 Appleby MC. 1983. The probability of linearity in hierarchies. Anim Behav. 31:600–608. 583 Baan C, Bergmüller R, Smith DW & Molnar B. 2014. Conflict management in free-ranging 584 wolves, Canis lupus. Animal Behaviour, 90, 327–334. 584 wolves, Canis lupus. Animal Behaviour, 90, 327–334. 584 wolves, Canis lupus. Animal Behaviour, 90, 327–334. 585 http://doi.org/10.1016/j.anbehav.2014.01.033 585 http://doi.org/10.1016/j.anbehav.2014.01.033 586 Bates D, Maechler M, Bolker B, Walker S. 2015. Fitting Linear Mixed-Effects Models Using 587 lme4. Journal of Statistical Software, 67(1), 1-48. doi:10.18637/jss.v067.i01. 586 Bates D, Maechler M, Bolker B, Walker S. 2015. 566 Conclusion 567 In conclusion, we emphasize the importance of applying a systematic methodology 568 including both age and sex in order to analyse dominance relationships between pack members. 569 Results clearly show that both within each sex and for the pack as a whole, dominance 570 relationships are a meaningful concept, which can be used to describe the structure of a multi- 571 generational family pack of captive wolves. Future studies analysing the potential effects of 572 dominance relationships on other aspects of the animal’s lives will likely help to further establish 573 the importance of this concept to describe the social lives of wolves. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed Fitting Linear Mixed-Effects Models Using , , , W S g U g 587 lme4. Journal of Statistical Software, 67(1), 1-48. doi:10.18637/jss.v067.i01. 587 lme4. Journal of Statistical Software, 67(1), 1-48. doi:10.18637/jss.v067.i01. 588 Bernstein IS. 1981. Dominance: the baby and the bathwater. Behav Brain Sci. 4:419–457. 589 Bloch G. 2002. Alpha-concept, dominance & leadership in wolf families. Wolf! Magazine Vol. 590 20, No.2 591 Bonanni R, Cafazzo S, Fantini C, Pontier D, Natoli E. 2007. Feeling order in an urban feral 592 d ti t l l ti hi t d i k d A i B h 74 1369 591 Bonanni R, Cafazzo S, Fantini C, Pontier D, Natoli E. 2007. Feeling order in an urban feral 591 Bonanni R, Cafazzo S, Fantini C, Pontier D, Natoli E. 2007. Feeling order in an urban feral 592 domestic cat colony: relationship to dominance rank, sex and age. Anim Behav. 74:1369– 593 1379. y p , g 593 1379. 594 Cafazzo S, Valsecchi P, Bonanni R, Natoli E. 2010. Dominance in relation 586 to age, sex, and 595 competitive contexts in a group of free-ranging domestic dogs. Behav. Ecol. 21 (3), 443- 596 455. 594 Cafazzo S, Valsecchi P, Bonanni R, Natoli E. 2010. Dominance in relation 586 to age, sex, and 595 competitive contexts in a group of free-ranging domestic dogs. Behav. Ecol. 21 (3), 443- 596 455. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be Manuscript to be reviewed 597 Clark KRF. 1971. Food habits and behavior of the tundra wolf on central Baffin island. Ph.D. 598 thesis, University of Toronto. Toronto, Ont. [Available from the National Library of 599 Canada, Ottawa, Ont.] 597 Clark KRF. 1971. Food habits and behavior of the tundra wolf on central Baffin island. Ph.D. 598 thesis, University of Toronto. Toronto, Ont. [Available from the National Library of 599 Canada, Ottawa, Ont.] 600 Derix R, Van Hooff J, Vries AD, Wensing J. 1993. Male and female mating competition in 601 wolves: female suppression vs. male intervention. Behaviour 127(1/2): 141–174. 602 Derix R. & VanHooff J. 1995. Male and female partner preferences in a captive wolf pack: 603 specificity vs. spread of sexual attention. Behavior. 1-2: 132-149. 604 de Vries H, Netto WJ, Hanegraaf PLH. 1993. MatMan: a program for the analysis of sociometr 605 matrices and behavioural transition matrices. Behaviour 125:157–175 DOI 606 10.1163/156853993X00218. 607 .de Vries H. 1995. An improved test of linearity in dominance hierarchies containing unknown 608 or tied relationships. Anim Behav. 50:1375–1389. 609 de Vries H. 1998. Finding a dominance order most consistent with linear hierarchy: a new 610 procedure and review. Anim Behav. 55:827–843. 611 d W l FBM & L tt ll LM 1985 Th f l hi h f h k A i ti ti 600 Derix R, Van Hooff J, Vries AD, Wensing J. 1993. Male and female mating competition in 601 wolves: female suppression vs. male intervention. Behaviour 127(1/2): 141–174. 602 Derix R. & VanHooff J. 1995. Male and female partner preferences in a captive wolf pack: 603 specificity vs. spread of sexual attention. Behavior. 1-2: 132-149. 604 de Vries H, Netto WJ, Hanegraaf PLH. 1993. MatMan: a program for the analysis of sociometric 604 de Vries H, Netto WJ, Hanegraaf PLH. 1993. MatMan: a program for the analysis of sociometric 605 matrices and behavioural transition matrices. Behaviour 125:157–175 DOI 605 matrices and behavioural transition matrices. Behaviour 125:157–175 DOI 606 10.1163/156853993X00218. 607 .de Vries H. 1995. An improved test of linearity in dominance hierarchies containing unknown 608 or tied relationships. Anim Behav. 50:1375–1389. 609 de Vries H. 1998. Finding a dominance order most consistent with linear hierarchy: a new 609 de Vries H. 1998. Finding a dominance order most consistent with linear hierarchy: a new 610 procedure and review. Anim Behav. 55:827–843. 609 de Vries H. 1998. Manuscript to be reviewed Finding a dominance order most consistent with linear hierarchy: a new 610 procedure and review. Anim Behav. 55:827–843. 610 procedure and review. Anim Behav. 55:827–843. 611 de Waal FBM & Luttrell LM. 1985.The formal hierarchy of rhesus monkeys: An investigation of h 615 b d h di l A J P i l 9 73 85 611 de Waal FBM & Luttrell LM. 1985.The formal hierarchy of rhesus monkeys: An investigation of 612 the 615 bared teeth display. Am. J. Primatol., 9,73-85. y y g 612 the 615 bared teeth display. Am. J. Primatol., 9,73-85. 612 the 615 bared teeth display. Am. J. Primatol., 9,73-85. 613 de Waal FBM. 1986. Deception in the natural communication of chimpazees. In RW Mitchell 614 &NS Thompson (Eds) Decption: Perspecti es on h man and nonh man deceit (pp 221 613 de Waal FBM. 1986. Deception in the natural communication of chimpazees. In RW Mitchell 614 &NS Thompson (Eds), Decption: Perspectives on human and nonhuman deceit (pp. 221- 615 244). Albany: SUNY Press 614 &NS Thompson (Eds), Decption: Perspectives on human and nonhuman deceit (pp. 221- 244) Alb SUNY P 616 de Waal FBM. 1989. Dominance ‘style’ and primate social organisation. In: Staden V, Foley 617 RA, editors. Comparative socioecology: the behavioural ecology of humans and other 618 mammals. Oxford: Blackwell Scientific. p. 243–263. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed 619 Drews C. 1993. The concept and definition of dominance in animal behaviour. Behaviour. 620 125:283–313. 621 East ML & Hofer H. 2001. Male spotted hyenas (Crocuta crocuta) queue for status in social 622 groups dominated by females. Behavioral Ecology, 12, 558–568. 621 East ML & Hofer H. 2001. Male spotted hyenas (Crocuta crocuta) queue for status in social 622 groups dominated by females. Behavioral Ecology, 12, 558–568. 622 groups dominated by females. Behavioral Ecology, 12, 558–568. 623 Fatjo J, Feddersen-Petersen D, Ruiz de la Torre JL, Amat M, Mets M, Braus B, Manteca X. 624 2007. Ambivalent signals during agonistic interactions in a captive wolf pack. Appl. Anim. 624 2007. Ambivalent signals during agonistic interactions in a captive wolf pack. Appl. Anim. 625 Behav. Sci 105, 274-283. 624 2007. Ambivalent signals during agonistic interactions in a captive wolf pack. Appl. Anim. 625 Behav. Sci 105, 274-283. 625 Behav. Sci 105, 274-283. 626 Feddersen-Petersen D. 2004. Hundepsychologie. Kosmos, Stuttgart, Germany, pp 113. 627 Fox MW. 1980. The soul of the wolf. Boston (MA): Little Brown. 628 Haber GC. 1977. Socio-ecological dynamics of wolves and prey in a subarctic ecosystem. Ph.D. 629 thesis, University of British Columbia. Vancouver. 630 Hand JL. 1986. Resolution of social conflicts: dominance, egalitarianism, spheres of dominance, 631 and game theory. Quart Rev Biol. 61(2):201–219. 631 and game theory. Quart Rev Biol. 61(2):201–219. 632 Handcock M, Hunter D, Butts C, Goodreau S, Krivitsky P, Bender-deMoll S & Morris M. 2015. 633 statnet: Software Tools for the Statistical Analysis of Network Data_. The Statnet Project 634 (<URL: http://www.statnet.org>). R package version 2015.11.0, <URL: CRAN.R- 635 project.org/package=statnet>. 636 Klass K & Cords M. 2011. Effect of unknown relationships on linearity, steepness and rank 637 ordering of dominance hierarchies: simulation studies based on data from wild monkeys. 638 BehaviouralProcesses 88:168–176 DOI 10.1016/j.beproc.2011.09.003. 639 Landau HG. 1951. On dominance relations and the structure of animal societies: I. Effect of 640 inherent characteristics. Bulletin of Mathematical Biophysics 13:1–19 DOI 641 10.1007/BF02478336. 632 Handcock M, Hunter D, Butts C, Goodreau S, Krivitsky P, Bender-deMoll S & Morris M. 2015. 632 Handcock M, Hunter D, Butts C, Goodreau S, Krivitsky P, Bender-deMoll S & Morris M. 2015. 633 statnet: Software Tools for the Statistical Analysis of Network Data_. The Statnet Project 634 (<URL: http://www.statnet.org>). R package version 2015.11.0, <URL: CRAN.R- 635 project.org/package=statnet>. 636 Klass K & Cords M. 2011. Manuscript to be reviewed Effect of unknown relationships on linearity, steepness and rank 637 ordering of dominance hierarchies: simulation studies based on data from wild monkeys. 638 BehaviouralProcesses 88:168–176 DOI 10.1016/j.beproc.2011.09.003. 636 Klass K & Cords M. 2011. Effect of unknown relationships on linearity, steepness and rank 637 ordering of dominance hierarchies: simulation studies based on data from wild monkeys. 638 BehaviouralProcesses 88:168–176 DOI 10.1016/j.beproc.2011.09.003. 639 Landau HG. 1951. On dominance relations and the structure of animal societies: I. Effect of 640 inherent characteristics. Bulletin of Mathematical Biophysics 13:1–19 DOI 641 10.1007/BF02478336. 639 Landau HG. 1951. On dominance relations and the structure of animal societies: I. Effect of 640 inherent characteristics. Bulletin of Mathematical Biophysics 13:1–19 DOI 641 10.1007/BF02478336. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 642 Landau D. 1993. Wolf: spirit of the wild. The Nature Company, Berkeley, California, USA. 643 Mazzini F, Townsend SW, Virányi Z, Range F. 2013. Wolf howling is mediated by relationship 644 quality rather than underlying emotional stress. Current Biology , 23 (17): 1677-1680. 645 Mech LD. 1999. Alpha status, dominance, and division of labor in wolf packs. Can J Zool. 646 77:1196–1203. 645 Mech LD. 1999. Alpha status, dominance, and division of labor in wolf packs. Can J Zool. 77 1196 1203 p , , p 646 77:1196–1203. 646 77:1196–1203. 647 Mech LD. 2000. Leadership in wolf, Canis lupus, packs. Canadian Field Naturalist, 114, 259- 648 263. 647 Mech LD. 2000. Leadership in wolf, Canis lupus, packs. Canadian Field Naturalist, 114, 259- 647 Mech LD. 2000. Leadership in wolf, Canis lupus, packs. Canadian Field Naturalist, 114, 259 648 263. 648 263. 649 Mech LD, Wolf PC, Packard JM. 1999. Regurgitative food transfer among wild wolves. Can J 649 Mech LD, Wolf PC, Packard JM. 1999. Regurgitative food transfer among wild wolves. Can J 650 Zool 77: 1192–1195. 650 Zool 77: 1192–1195. 651 Mech LD & Cluff HD. 2010. Prolonged intensive dominance behavior between grey wolves, 651 Mech LD & Cluff HD. 2010. Prolonged intensive dominance behavior between grey wolves, 652 Canis lupus. Canadian Field-Naturalist 124, 215-218. 652 Canis lupus. Canadian Field-Naturalist 124, 215-218. 652 Canis lupus. Canadian Field-Naturalist 124, 215-218. 653 Modlmeier AP, Keiser CN, Watters JV, Sih A, Pruitt JN. 2014 The keystone individual concept: 653 Modlmeier AP, Keiser CN, Watters JV, Sih A, Pruitt JN. 2014 The keystone individual concept: 654 an ecological and evolutionary overview. Animal Behaviour 89: 53–62. (DOI: 653 Modlmeier AP, Keiser CN, Watters JV, Sih A, Pruitt JN. 2014 The keystone individual concept: 654 an ecological and evolutionary overview. Animal Behaviour 89: 53–62. (DOI: 654 an ecological and evolutionary overview. Animal Behaviour 89: 53–62. (DOI: 654 an ecological and evolutionary overview. Animal Behaviour 89: 53–6 655 10.1016/j.anbehav.2013. 12.020) 656 Noë R, De Waal FBM, Van Hooff JARAM. 1980. Types of dominance in a chimpanzee colony. 657 Folia Primatol. 34:90–110. 658 Norscia i & Palagi E. 2015. The socio-matrix reloaded: from hierarchy to dominance profile in 659 wild lemurs. PeerJ 3:e729; DOI 10.7717/peerj.729 658 Norscia i & Palagi E. 2015. The socio-matrix reloaded: from hierarchy to dominance profile in ild l P J 3 729 DOI 10 7717/ j 729 659 wild lemurs. Manuscript to be reviewed PeerJ 3:e729; DOI 10.7717/peerj.729 660 Packard JM. 2003. Wolf behavior: reproductive, social and intelligent. In: Mech LD, Boitani L, 661 editors. Wolves: behavior, ecology, and conservation. Chicago and London: The 662 University of Chicago Press. p. 35–65. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 663 Peterson RO, Jacobs AK, Drummer TD, Mech LD, Smith DW. 2002. Leadership behaviour in 664 relation to dominance and reproductive status in gray wolves, Canis lupus. Can. J.Zool. 80, 665 1405-718 1412. 666 Preuschoft S. 1999. Are primates Behaviorists? Formal dominance, cognition and free floating i l J C P h l 113 91 95 666 Preuschoft S. 1999. Are primates Behaviorists? Formal dominance, cognition and free floating 667 rationales. J. Comp . Psychol. 113, 91-95. 667 rationales. J. Comp . Psychol. 113, 91-95. 668 R Development Core Team. 2011. R: a language and environment for statistical computing. Vi h R F d i f S i i l C i A il bl h // R 668 R Development Core Team. 2011. R: a language and environment for statistical computing. 668 R Development Core Team. 2011. R: a language and environment for statistical computing. 669 Vienna: the R Foundation for Statistical Computing. Available at http://www.R- 670 project.org/. 669 Vienna: the R Foundation for Statistical Computing. Available at http://www.R- 670 project.org/. 671 Rabb GB, Woolpy JH, Ginsburg BE. 1967. Social relationships in a group of captive wolves. 672 Am Zool. 7:305–312. 671 Rabb GB, Woolpy JH, Ginsburg BE. 1967. Social relationships in a group of captive wolves. 671 Rabb GB, Woolpy JH, Ginsburg BE. 1967. Social relationships in a group of captive wolves. 672 Am Zool. 7:305–312. 672 Am Zool. 7:305–312. 673 Romero T, Ito M, Saito A & Hasegawa T. 2014. Social Modulation of Contagious Yawning in 674 Wolves. PLoS ONE, 9(8), e105963. http://doi.org/10.1371/journal.pone.0105963 673 Romero T, Ito M, Saito A & Hasegawa T. 2014. Social Modulation of Contagious Yawning in 674 Wolves PLoS ONE 9(8) e105963 http://doi org/10 1371/journal pone 0105963 673 Romero T, Ito M, Saito A & Hasegawa T. 2014. Social Modulation of Contagious Yawning in 674 Wolves. PLoS ONE, 9(8), e105963. http://doi.org/10.1371/journal.pone.0105963 675 Rowell TE. 1974 The concept of social dominance. Behav. Biol. 11, 131–154. 676 (doi:10.1016/S0091- 6773(74)90289-2). 677 Sands J & Creel S. 2004. Social dominance, aggression and faecal glucocorticoid levels in a wild 678 population of wolves, Canis lupus. Animal. Behaviour 67, 387–39. 677 Sands J & Creel S. 2004. Social dominance, aggression and faecal glucocorticoid levels in a wild 678 population of wolves, Canis lupus. Animal. Behaviour 67, 387–39. 679 Savage C. 1988. Wolves. San Francisco (CA): Sierra Club Books. 680 Schenkel R. 1947. Ausdruckstudien an Wölfen. Behaviour 1, 81-129. Manuscript to be reviewed 681 Schenkel R. 1967. Submission: its features and function in the wolf and dg. Am Zool. 7:319– 682 329. 683 Shi A, Hanser SF, McHugh KA. 2009 Social network theory: new insights and issues for 684 behavioral ecologists. Behavioral Ecology and Sociobiology 63: 975–988. (DOI: 685 10.1007/s00265-009-0725-6) 681 Schenkel R. 1967. Submission: its features and function in the wolf and dg. Am Zool. 7:319– 682 329. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 686 Shizuka D & McDonald DB. 2012. A social network perspective on measurements of dominance 687 hierarchies. Animal Behaviour 83:925–934. (DOI 10.1016/j.anbehav.2012.01.011). 686 Shizuka D & McDonald DB. 2012. A social network perspective on measurements of dominance 687 hierarchies. Animal Behaviour 83:925–934. (DOI 10.1016/j.anbehav.2012.01.011). 688 Shizuka D & McDonald DB. 2014. Errata corrige to Shizuka D &McDonald DB (Animal 689 Behaviour, 83, 925-934). Animal Behaviour 87: 243. (DOI 688 Shizuka D & McDonald DB. 2014. Errata corrige to Shizuka D &McDonald DB (Animal 689 Behaviour, 83, 925-934). Animal Behaviour 87: 243. (DOI 689 Behaviour, 83, 925-934). Animal Behaviour 87: 243. (DOI 689 Behaviour, 83, 925-934). Animal Behaviour 87: 243. (DOI 690 10.1016/j.anbehav.2013.10.016). 691 Shizuka D & McDonald DB. 2015. The network motif architecture of dominance hierarchies. 692 J l f th R l S i t I t f 12: 105 (DOI: 10 1098/rsif 2015 0080) 691 Shizuka D & McDonald DB. 2015. The network motif architecture of dominance hierarchies. 692 Journal of the Royal Society Interface 12: 105. (DOI: 10.1098/rsif.2015.0080) 692 Journal of the Royal Society Interface 12: 105. (DOI: 10.1098/rsif.2015.0080) 693 Smith DW, Stahler DR, Stahler E, McIntyre R, Metz M, Irving J, Raymond R, Anton C, 694 Kindermann R & Bowersock N. 2012. Yellowstone Wolf Project: Annual Report, 2011. 695 National Park Service, Yellowstone Center for Resources, Yellowstone National Park, 696 Wyoming YCR-2012-01 695 National Park Service, Yellowstone Center for Resources, Yellowstone National Park, 696 Wyoming, YCR-2012-01. 697 StatSoft Italia srl 2005. STATISTICA (sistema software di analisi dei dati), versione 7.1. 698 www.statsoft.it. 699 van der Borg JAM, Schilder MBH, Vinke CM, de Vries H. 2015. Dominance in Domestic Dogs: 700 A Quantitative Analysis of Its Behavioural Measures. PLoS ONE 10(8): e0133978. 701 doi:10.1371/journal. pone.0133978. 702 Van Hooff JARAM & Wensing JAB. 1987. Dominance and its behavioural measures in a 703 captive wolf pack. In: Frank HW, editor. Man and Wolf. Dordrecht, Olanda (Netherlands): 704 Junk Publishers. p. 219–252. 702 Van Hooff JARAM & Wensing JAB. 1987. Dominance and its behavioural measures in a 703 captive wolf pack. In: Frank HW, editor. Man and Wolf. Dordrecht, Olanda (Netherlands): 702 Van Hooff JARAM & Wensing JAB. 1987. Dominance and its behavioural measures in a 703 captive wolf pack. In: Frank HW, editor. Man and Wolf. Dordrecht, Olanda (Netherlands): 704 Junk Publishers. p. 219–252. 703 captive wolf pack. In: Frank HW, editor. Man and Wolf. Manuscript to be reviewed Dordrecht, Olanda (Netherlands): 704 Junk Publishers p 219–252 703 captive wolf pack. In: Frank HW, editor. Man and Wolf. Dordrecht, Olanda (Netherlands): 704 Junk Publishers. p. 219–252. 705 Vervaecke, H., De Vries, H., Van Elsacker, L. 2000. Dominance and its behavioral measures in a 706 captive group of bonobos (Pan paniscus). International Journal of Primatology, 21(1): 47- 707 68. 705 Vervaecke, H., De Vries, H., Van Elsacker, L. 2000. Dominance and its behavioral measures in a 706 captive group of bonobos (Pan paniscus). International Journal of Primatology, 21(1): 47- 707 68. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed Manuscript to be reviewed 708 VonHoldt BM, Stahler DR, Smith DW, Earl DA, Pollinger JP, Wayne RK. 2008. The genealogy 709 and genetic viability of reintroduced Yellowstone grey wolves. Molecular Ecology, 17, 710 252–274. 711 Zimen E. 1975. Social dynamics of the wolf pack. In: Fox MW, editor. The wild Canids. New 712 York: Van Nostrand Reinhold. p. 336–362. 711 Zimen E. 1975. Social dynamics of the wolf pack. In: Fox MW, editor. The wild Canids. New 712 Y k V N t d R i h ld 336 362 711 Zimen E. 1975. Social dynamics of the wolf pack. In: Fox MW, editor. The wild Canids. New 712 York: Van Nostrand Reinhold. p. 336–362. 713 Zimen E. 1978. The wolf. Delacorte Press, New York. 714 Zimen E. 1982. A wolf pack sociogram. In: Harrington FH, Paquet PC, editors. Wolves of the 715 world: perspectives of behaviour, ecology and conservation. Park Ridge (NJ): Noyes 716 Publication. p. 282–322. 714 Zimen E. 1982. A wolf pack sociogram. In: Harrington FH, Paquet PC, editors. Wolves of the 715 world: perspectives of behaviour, ecology and conservation. Park Ridge (NJ): Noyes 717 White AB. 2001. Wild and captive wolf (Canis lupus) aggression in relation to territory 718 availability and pack size. Honors thesis - University of Colorado, Boulder. 719 PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 724 Name Sex Date of birth Age class Removed Returned Viki Female mar-04 Adult Beta* Female apr-07 Adult 09-feb Normale Female apr-12 Sub-adult 13-mar Uno Beta Female apr-12 Sub-adult Lacrima Female apr-12 Sub-adult Volpe Female apr-12 Sub-adult 22-apr 12-may Musocorto Female apr-12 Sub-adult Husky Female apr-13 Sub-adult 13-mar Sosia Female apr-13 Sub-adult Cane Female apr-12 Sub-adult Macchia Male mar-04 Adult Maschera Male apr-09 Adult 13-mar Sfregiato Male apr-09 Adult Storto Male apr-09 Adult 13-mar Procione Male apr-10 Adult 22-apr Secondo Male may-11 Adult Taglio Male apr-12 Sub-adult Musolungo Male mag-11 Adult Due Male apr-12 Sub-adult Zampa Male apr-12 Sub-adult 726 727 Table 2. Ethogram 727 Table 2. Ethogram 27 Table 2. Ethogram Behavioural categories Behavioural pattern Description Greeting behaviours Greeting To interact in a friendly and relaxed manner holding the ears back, showing much tail wagging and licking of the other’s mouth/muzzle. The subject however does not show crouching/lowered hindquarters nor is the tail tucked between the legs Dominance behaviours Stand tall Subject straightens up to full height, with a rigid posture and tail, may include raised hackles, ears erect and tail perpendicular or above the back Stand over To stand over another's body, with all four paws on the ground and the tail above the plane of the back. The receiver may have either the whole body or just the forepaws under the actors’ belly/side Paw on To place one or both forepaws on the other’s back Ride up To mount another one from behind or from the side, exhibiting a thrusting motion Head on The subject approaches another’s shoulder/back with the tail above the plane of the back and puts its head on it. Most of times formation looks like a capital “T” Muzzle bite To grab the muzzle of another subject softly Approach dominant To approach another subject within one body length for at least 5 seconds, with the tail perpendicular or above the plane of the back and the ears erect and pointed forward Submissive behaviours Crouch Lowering the head, sometimes bending the legs, arching the back, lowering the tail between the hind legs, and avoiding eye contact Passive submission To lie on the back showing the stomach and holding the tail between the legs. The ears are held back and close to the head and the subject raises a hind leg for inguinal presentation Active submission The subject has its tail tucked between the hind legs sometimes wagging it while he is in a crouched position (with hindquarters lowered) and may attempt to paw and lick the side of actors’/aggressor’s muzzle. The behaviour may include urination. Withdrawing The subject withdraws from another moving away slowly in the opposite direction, displaying a submissive posture. It occurs when a subject has been threatened or attacked by another, or a fight has taken place Flee To run away from another with tail tucked between the legs and body ducked. PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) 727 Table 2. Ethogram It occurs when a subject has been threatened or attacked by another, or after a fight Avoidance In response to another reducing the distance towards it, the subject moves away displaying a submissive posture. The subject may also look at the individual he is trying to avoid Approach submissive To slowly approach another within one body length remaining within that distance for at least 5 seconds. The approach is characterized by a ducked posture and tail between the legs. Subject can also be moving in a wavy line and in a hesitant (stop-start) manner Aggressive behaviours Threat Subject orients towards another performing one or more of the following: staring intently at, curling of the lips, baring of the canines, raising the hackles, snarling, growling, and barking, sometimes with the tail perpendicular or above the back Attack Running into or jumping onto another with tail, ears and sometimes hackles up, often with bites at the neck Knock down To strike another subject sharply with the chest or shoulder so that the other falls to the ground Pin To grab another at the neck or at the muzzle, forcing it down to the ground and holding it there Chase A subject runs after a conspecific, exhibiting threatening behaviours (see ‘threat’ above) Snapping To snap teeth into the air, noisily Bite Bite a conspecific, without inhibition, with enough pressure to cause potential injury 28 To interact in a friendly and relaxed manner holding the ears back, showing much tail wagging and licking of the other’s mouth/muzzle. The subject however does not show crouching/lowered hindquarters nor is the tail tucked between the legs Subject straightens up to full height, with a rigid posture and tail, may include raised hackles, ears erect and tail perpendicular or above the back To stand over another's body, with all four paws on the ground and the tail above the plane of the back. The receiver may have either the whole body or just the forepaws under the actors’ belly/side To mount another one from behind or from the side, exhibiting a thrusting motion The subject approaches another’s shoulder/back with the tail above the plane of the back and puts its head on it. Manuscript to be reviewed 729 Table 3. Summary of values of the directionality (directional consistency index, DCI), linearity h' and its 730 significance level, number and strength of inconsistencies (No. I and SI, respectively) for the I&SI rank orders, and 731 triangle transitivity (proportion of transitive triangles relative to all triangles Pt, triangle transitivity metric ttri, and 732 significance level) for all behavioural categories in the absence, and in the presence, of food and for all dominance 733 and submissive interactions regardless of the context. 729 Table 3. Summary of values of the directionality (directional consistency index, DCI), linearity h' and its 730 significance level, number and strength of inconsistencies (No. I and SI, respectively) for the I&SI rank orders, and 731 triangle transitivity (proportion of transitive triangles relative to all triangles Pt, triangle transitivity metric ttri, and 732 significance level) for all behavioural categories in the absence, and in the presence, of food and for all dominance 733 and submissive interactions regardless of the context. 729 Table 3. Summary of values of the directionality (directional consistency index, DCI), linearity h' and its 730 significance level, number and strength of inconsistencies (No. I and SI, respectively) for the I&SI rank orders, and 731 triangle transitivity (proportion of transitive triangles relative to all triangles Pt, triangle transitivity metric ttri, and 732 significance level) for all behavioural categories in the absence, and in the presence, of food and for all dominance 733 and submissive interactions regardless of the context. Agonistic behaviours displayed in the absence of food Directionality Linearity Inconsistency: No. I (and SI) Triangle transitivity Aggressive behaviour DCI =0.94 No (h'=0.25, p=0.08) - Pt=0.88, ttri=0.52,p=0.009 Dominance behaviour DCI =0.97 Yes (h'=0.38, p=0.002) 1 (3) Pt=0.99, ttri=0.97, p=0.000 Submissive behaviour DCI =0.98 Yes (h'=0.45, p=0.0001) 1 (2) Pt=0.99, ttri=0.97, p=0.000 Agonistic behaviours displayed in the presence of food Directionality Linearity Inconsistency: No. I (and SI) Triangle transitivity Aggressive behaviour DCI =0.85 Yes (h'=0.36, p=0.003) 4 (24) Pt=0.98, ttri=0.90,p=0.000 Dominance behaviour DCI =0.99 Yes (h'=0.33, p=0.006) 1 (4) Pt=1.00, ttri=1.000,p=0.000 Submissive behaviour DCI =0.99 Yes (h'=0.30, p=0.02) 1 (2) Pt=0.99, ttri=0.95,p=0.000 Agonistic behaviours displayed in both the absence and the presence of food Directionality Linearity Inconsistency: No. Manuscript to be reviewed I (and SI) Triangle transitivity All dominance behaviours DCI =0.97 Yes (h'=0.58, p=0.0001) 2 (9) Pt=0.992, ttri=0.970,p=0.000 All submissive behaviours DCI =0.97 Yes (h'=0.56, p=0.0001) 0 Pt=1.000, ttri=1.000,p=0.000 Agonistic behaviours displayed in both the absence and the presence of food without considering the alpha male and female Directionality Linearity Inconsistency: No. I (and SI) Triangle transitivity All dominance behaviours DCI =0.96 Yes (h'=0.44, p=0.0008) 2 (9) Pt=0.983, ttri=0.932,p=0.000 All submissive behaviours DCI =0.94 Yes (h'=0.40, p=0.005) 0 Pt=1.000, ttri=1.000,p=0.000 splayed in both the absence and the presence of food without considering the alpha male and female 735 727 Table 2. Ethogram Most of times formation looks like a capital “T” To grab the muzzle of another subject softly To approach another subject within one body length for at least 5 seconds, with the tail perpendicular or above the plane of the back and the ears erect and pointed forward p The subject has its tail tucked between the hind legs sometimes wagging it while he is in a crouched position (with hindquarters lowered) and may attempt to paw and lick the side of actors’/aggressor’s muzzle. The behaviour may include urination. gg y The subject withdraws from another moving away slowly in the opposite direction, displaying a submissive posture. It occurs when a subject has been threatened or attacked by another, or a fight has taken place To slowly approach another within one body length remaining within that distance for at least 5 seconds. The approach is characterized by a ducked posture and tail between the legs. Subject can also be moving in a wavy line and in a hesitant (stop-start) manner Subject orients towards another performing one or more of the following: staring intently at, curling of the lips, baring of the canines, raising the hackles, snarling, growling, and barking, sometimes with the tail perpendicular or above the back Running into or jumping onto another with tail, ears and sometimes hackles up, often with bites at the neck Bite a conspecific, without inhibition, with enough pressure to cause potential injury 728 PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 736 Table 4. Dominance relationships based on all submissive behavioural patterns recorded between wolves. 737 Bold type: males; italic type: females. The signallers, who are the performers of the submissive behaviours, are 738 listed in rows, whereas the recipients in columns. 4. Dominance relationships based on all submissive behavioural patterns recorded between wolves. 736 Table 4. Dominance relationships based on all submissive behavioural patterns recorded between wolves. 737 Bold type: males; italic type: females. The signallers, who are the performers of the submissive behaviours, are 738 listed in rows, whereas the recipients in columns. Manuscript to be reviewed vik mac mas sec sfr zam due unb vol lac nor can tag mul muc sos sto pro hus vik * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 mac 1 * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 mas 1 14 * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 sec 3 14 4 * 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 sfr 2 4 13 4 * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 zam 0 3 0 0 0 * 0 0 0 0 0 0 0 0 0 0 0 0 0 due 1 10 3 2 1 0 * 0 0 0 0 0 0 0 0 0 0 0 0 unb 48 6 1 0 0 0 1 * 0 0 0 0 0 0 0 0 0 0 0 vol 31 4 1 2 2 0 0 2 * 0 0 0 0 1 0 0 0 0 0 lac 27 3 1 2 1 0 0 1 9 * 0 0 0 1 0 0 0 0 0 nor 3 1 0 0 0 0 0 0 4 6 * 0 0 0 0 0 0 0 0 can 1 2 0 0 0 0 0 3 0 6 0 * 0 0 0 1 0 0 0 tag 2 13 3 0 7 1 1 0 0 0 0 0 * 0 0 0 1 0 0 mul 5 22 10 0 10 0 0 1 1 2 0 0 0 * 0 0 0 0 0 muc 11 13 0 2 2 0 1 1 8 16 1 42 1 1 * 2 0 0 0 sos 1 2 0 0 1 1 1 0 0 0 0 1 0 0 11 * 0 0 0 sto 4 4 3 0 1 0 0 1 2 1 2 0 4 0 2 0 * 0 0 pro 1 23 3 2 5 2 7 1 0 1 0 7 2 5 3 1 1 * 0 hus 2 1 4 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 * 9 740 PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) Manuscript to be reviewed 741 Table 5. Summary of values of the directionality (directional consistency index, DCI), linearity h' and its 742 significance level, number and strength of inconsistencies (No. I and SI, respectively) for the I&SI rank orders, and 743 triangle transitivity (proportion of transitive triangles relative to all trangles Pt, triangle transitivity metric ttri, and 744 significance level) for all behavioural categories for females and males separately. 741 Table 5. Summary of values of the directionality (directional consistency index, DCI), linearity h' and its 742 significance level, number and strength of inconsistencies (No. I and SI, respectively) for the I&SI rank orders, and 743 triangle transitivity (proportion of transitive triangles relative to all trangles Pt, triangle transitivity metric ttri, and 744 significance level) for all behavioural categories for females and males separately. Agonistic behaviours displayed between females Directionality Linearity Inconsistency: No. I (and SI) Triangle transitivity Female aggressive behaviour DCI =0.92 Yes (h'=0.78, p=0.004) 0 Pt=1.000, ttri=1.000,p=0.008 Female dominance behaviour DCI =0.98 Yes (h'=0.74, p=0.007) 1 (2) Pt=0.973, ttri=0.892,p=0.004 Female submissive behaviour DCI =0.98 Yes (h'=0.59, p=0.048) 0 Pt=1.000, ttri=1.000,p=0.008 Agonistic behaviours displayed between males Directionality Linearity Inconsistency: No. I (and SI) Triangle transitivity Male aggressive behaviour DCI =0.90 Yes (h'=0.61, p=0.015) 1 (4) Pt=0.972, ttri=0.889,p=0.002 Male dominance behaviour DCI =0.96 Yes (h'=0.78, p=0.0009) 0 Pt=1.000, ttri=1.000,p=0.001 Male submissive behaviour DCI =0.97 Yes (h'=0.81, p=0.0002) 0 Pt=1.000, ttri=1.000,p=0.001 745 746 PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) 747 Figure 1. 48 The relation between the rank order based on all dominance behaviours and the rank order based 49 on all submissive behaviours. 748 The relation between the rank order based on all dominance behaviours an 748 The relation between the rank order based on all dominance behaviours and the rank order based 749 on all submissive behaviours. 749 on all submissive behaviours. 750 751 750 752 PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016) 47 Figure 1. Manuscript to be reviewed Manuscript to be reviewed 53 Figure 2. 753 Figure 2. elation between the agonistic rank order based on submission and the age of wolves 754 The relation between the agonistic rank order based on submission and the age of wolves. 755 755 755 PeerJ reviewing PDF \| (2016:06:11561:2:0:NEW 7 Oct 2016)
https://openalex.org/W2948991595	https://europepmc.org/articles/pmc6603983?pdf=render	English	null	Metabolic Outcomes in Southern Italian Preadolescents Residing Near an Industrial Complex: The Role of Residential Location and Socioeconomic Status	International journal of environmental research and public health/International journal of environmental research and public health	2,019	cc-by	10,220	International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Article Metabolic Outcomes in Southern Italian Preadolescents Residing Near an Industrial Complex: The Role of Residential Location and Socioeconomic Status Esha Bansal 1,2, Donatella Placidi 1,* , Shaye Carver 1,3, Stefano Renzetti 4,5 , Augusto Giorgino 6, Giuseppa Cagna 1, Silvia Zoni 1, Chiara Fedrighi 1, Miriana Montemurro 1,6, Manuela Oppini 1, Michele Conversano 6, Stefano Guazzetti 1,7, Robert O. Wright 2, Donald Smith 8, Luz Claudio 2 and Roberto G. Lucchini 1,2 1 Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia, 25123 Brescia, Italy; esha.bansal@icahn.mssm.edu (E.B.); carversh@bc.edu (S.C.); giuseppa.cagna@unibs.it (G.C.); silvia.zoni@unibs.it (S.Z.); chiara.fedrighi@unibs.it (C.F.); miriana.montemurro@unibs.it (M.M.); manuela.oppini@unibs.it (M.O.); stefano.guazzetti@ausl.re.it (S.G.);roberto.lucchini@unibs.it (R.G.L.) g 2 Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; robert.wright@mssm.edu (R.O.W.); luz.claudio@mssm.edu (L.C.) 2 Department of Environmental Medicine and Public Health, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; robert.wright@mssm.edu (R.O.W.); luz.claudio@mssm.edu (L.C.) 3 Department of Biology, Morrissey College of Arts and Sciences, Boston College, Chestnut Hill, PA 02467, USA g ( ) ( ) 3 Department of Biology, Morrissey College of Arts and Sciences, Boston College, Chestnut Hill, PA 02467, USA 4 Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy; stefano.renzetti@unibs.it 5 Department of Clinical Sciences and Community Health, University of Milan, 20122 Milan, It 6 6 Department of Prevention, Local Health Authority of Taranto, 74121 Taranto, Italy; augusto.giorgino@asl.taranto.it (A.G.); michele.conversano@asl.taranto.it (M.C.) g g g 7 Local Health Authority of Reggio Emilia, 42122 Reggio Emilia, Italy y gg gg y 8 Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, CA 95064, USA; drsmith@ucsc.edu y gg gg y 8 Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, S t C CA 95064 USA d ith@ d * Correspondence: donatella.placidi@unibs.it; Tel.: +39-030-399-6834 1. Introduction Exposure to industrial pollutants is a serious environmental health problem, particularly as it aﬀects children. Even low-level toxicity from contaminated air, water, and food sources has been implicated in pediatric pathologies of high morbidity and mortality, such as obesity, organ damage, and neurological dysfunction [1–3]. Many of the pediatric metabolic conditions linked to environmental exposure have been studied and found to be associated with speciﬁc sociodemographic conditions. This is particularly true for the Mediterranean region of Europe, where childhood obesity is increasing at alarming rates [4–7]. In Italy, 22.2% of children at the age of 8–9 years are overweight, and 17.4% are obese. These rates have worsened substantially over the past decades, particularly for girls, yet remain higher in males relative to females and in Southern relative to Northern Italy [4,7,8]. A recent study of 15,035 Italian schoolchildren aged 11–15 years found 21.8% of boys and 13.3% of girls to be overweight or obese in 2014 [9]. These ﬁndings agree with prior literature, which associates childhood obesity with low socioeconomic status (SES) and low maternal education level [5,8–10]. Childhood obesity has also been linked to cognitive conditions, including ADHD, risky behavior, and antisocial behavior [11,12]. However, such health conditions have not been as well described in the context of multiple sociodemographic and anthropometric variables, or in the setting of industrial activity. Much remains to be understood about the role of sociodemographic and lifestyle factors in mediating harmful pediatric health eﬀects linked to environmental exposure. Numerous chemicals present in industrial sites—including heavy metals, polychlorinated biphenyls, dioxins, and bisphenol A—are considered obesogens because they have been shown to alter adipose tissue formation and neuroendocrine regulation of appetite [13]. Exposure to industrial chemicals may also increase obesity indirectly by promoting impulsive behaviors that interfere with appetite control [12]. While children are far more sensitive than adults to such toxins, relatively little is known about the diﬀerential eﬀects of each chemical based on child characteristics such as sex and body size [14]. To our knowledge, there is also scarce currently published work that evaluates the metabolic outcomes of children living near industrial sites. This knowledge gap could limit the investigation of environmental contributors to metabolic syndromes. In addition to body size metrics, metabolic pathologies have been described in relation to early-life environmental exposure. Received: 13 May 2019; Accepted: 6 June 2019; Published: 8 June 2019 Abstract: Evidence suggests that environmental exposures and socioeconomic factors may interact to produce metabolic changes in children. We assessed the inﬂuence of residential location and socioeconomic status (SES) on pediatric body mass index (BMI) Z-score and fasting blood glucose (FBG) concentration. Participants included 214 children aged 6–11 years who live near a large industrial complex in Taranto, Italy. Participants were grouped into residential zones based on the distance between their home address and the complex periphery (Zone 1: 0.000–4.999 km, Zone 2: 5.000–9.999 km, Zone 3: 10.000–15.000 km). BMI Z-scores were calculated via World Health Organization (WHO) pediatric reference curves. FBG was obtained via venous blood sampling. Closer residential location to the industrial complex on the order of 5.000 km was signiﬁcantly associated with worsened metabolic outcomes, particularly in female children. Zone 1 participants had higher BMI-adjusted FBG than Zone 2 and 3 participants (p < 0.05 versus Zone 2; p < 0.01 versus Zone 3). SES did not signiﬁcantly inﬂuence BMI-adjusted FBG. Moreover, BMI Z-scores indicated high rates of overweight (22.0%) and obesity (22.9%) in the cohort. BMI Z-score was not signiﬁcantly associated with SES or residential zone but was negatively associated with maternal education level (p < 0.05). These results oﬀer new evidence that residing near industrial activity may predict adverse eﬀects on child metabolic health. Int. J. Environ. Res. Public Health 2019, 16, 2036; doi:10.3390/ijerph16112036 www.mdpi.com/journal/ijerph 2 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 Keywords: children; socioeconomic status; residential location; industrial; air pollution; body mass index; BMI; blood glucose; obesity Keywords: children; socioeconomic status; residential location; industrial; air pollution; body mass index; BMI; blood glucose; obesity 2.1. Study Design and Population A cross-sectional study was carried out with a cohort of 214 primary school children aged 6–11 years, recruited from 12 public primary schools in the Taranto municipality. Among the 432 children who received parental consent to participate in this study, full or partial data were collected from the 312 children that satisfied the inclusion and exclusion criteria. Of the 312 eligible participants, 214 participants (68.6%) provided sufficient data to be included in the analyses of this paper. The cohort of 214 participants included in these analyses did not differ significantly from the 98 participants whose data were not used, on any analyzed characteristic. A more detailed breakdown of enrollment data is available upon request. Inclusion Criteria: To participate in the study: (1) children must have been born and raised continuously in one of the target study areas in the province of Taranto, Italy; and (2) the pregnancy of the child’s mother must have been carried out within the participant’s current area of residence at the time of recruitment. Exclusion Criteria: Subjects were excluded from the study for having one or more of the following conditions: history of total parenteral nutrition; history of neurodegenerative disease; current use of pharmaceuticals aﬀecting the nervous system or with known neuropsychological side eﬀects; inadequately corrected visual defects; deﬁcits in hand and/or ﬁnger function; and/or presence of a neurological, neuropsychiatric, hematological, metabolic, endocrine, kidney, or bile tract pathology. Children excluded due to pathologies were referred to the pediatric care service of the Azienda Sanitaria Locale (ASL) of Taranto. Because participants were below 12 years of age, informed written consent for child participation and for anonymous data collection was obtained from both parents of each participant. Children were enrolled in this study from March 2015 to May 2016. This study was conducted in accordance with the Declaration of Helsinki. On September 11, 2014, the study was reviewed and approved by the Independent Medical Ethics Committee of the ASL of Brindisi, the approval body responsible for health research conducted in the Province of Taranto (ID number: 142/14). 1. Introduction For instance, the ambient air pollutants ozone and sulfate have been characterized as triggers of Type I diabetes in early childhood [15]. Literature indicates that arsenic, a heavy metal compound produced by steel plants, contributes to both obesity and prediabetic insulin resistance, an important step in the pathogenesis of Type II diabetes [16]. This study was conducted in the areas surrounding an industrial complex that includes the largest steel plant in the European Union (Taranto, Italy). The industrial activities of this complex are known to produce substances that aﬀect child obesity and metabolism. The objective of this study is to describe the metabolic health eﬀects of industrial site proximity on school-aged children, a population segment that has not been well studied in this context. In particular, this paper aims to: (1) describe trends in body mass index (BMI) Z-score and fasting blood glucose (FBG) concentration among a cohort of urban children aged 6–11 years residing near the industrial complex; and (2) relate these ﬁndings to children’s residential distance from the industrial complex and SES. Our approach combines bodily measurements and physiological data with sociodemographic and geographic information. In this way, the study oﬀers a deeper analysis of the relationship between metabolic outcomes and environmental exposure than is typically possible in pediatric population studies about industrial activity. The authors Int. J. Environ. Res. Public Health 2019, 16, 2036 3 of 16 intend that these analyses will be considered when designing future health interventions for children with environmental exposure to industrial emissions. intend that these analyses will be considered when designing future health interventions for children with environmental exposure to industrial emissions. 2.3. Division of Residential Location into Residential Zones 2 3 Division of Residential Location into Residential Zones To categorize residential location, the Taranto municipality was divided geographically into three concentric residential zones (1, 2, 3) of increasing radial distance from the industrial complex. The industrial complex was deﬁned as a polygonal region in which processes of steel preproduction (including iron ore extraction), production, and reﬁnery were performed regularly (Figure 1). 2.3. Division of Residential Location into Residential Zones To categorize residential location, the Taranto municipality was divided geographically into three concentric residential zones (1, 2, 3) of increasing radial distance from the industrial complex. The industrial complex was defined as a polygonal region in which processes of steel preproduction (including iron ore extraction), production, and refinery were performed regularly (Figure 1) Figure 1. Geographic description of the residential zones (circles) used to define participant proximity to the industrial complex (polygon). Figure 1. Geographic description of the residential zones (circles) used to deﬁne participant proximity to the industrial complex (polygon). Figure 1. Geographic description of the residential zones (circles) used to define participant proximity to the industrial complex (polygon). Figure 1. Geographic description of the residential zones (circles) used to deﬁne participant proximity to the industrial complex (polygon). First, geolocalization of participants’ reported residence and school of attendance was performed using the World Geodetic System (WGS84). Thereafter, residential distance from the industrial complex was calculated for each participant as the straight-line distance between his/her home address and the closest point of the polygon denoting the perimeter of the complex. The perimeter, rather than center, of the industrial complex was used in distance measurement due to the presence of accumulated metal dusts at the industrial complex borders; wind forces have been kno n to resuspend these dusts and transport them airborne across the city First, geolocalization of participants’ reported residence and school of attendance was performed using the World Geodetic System (WGS84). Thereafter, residential distance from the industrial complex was calculated for each participant as the straight-line distance between his/her home address and the closest point of the polygon denoting the perimeter of the complex. The perimeter, rather than center, of the industrial complex was used in distance measurement due to the presence of accumulated metal dusts at the industrial complex borders; wind forces have been known to resuspend these dusts and transport them airborne across the city. known to resuspend these dusts and transport them airborne across the city. 2.2. Study Design and Enrollment Subject enrollment was conducted in primary schools. Within each of the three geographic zones delineated in the study, four public primary schools were selected, for a total of 12 schools (Table 1). These schools were chosen in order to accurately represent the environmental eﬀects of industrial complex emissions within the Province of Taranto. To this eﬀect, maps of average PM10 concentration at ground level in the Province of Taranto, created by the Puglia Regional Agency for Prevention and Protection of the Environment (ARPA Puglia) in 2010, informed school selection. After verifying children’s registration forms and eligibility for the study, participants were enrolled, and the cohort was balanced for zone, school, age, and gender. Data were anonymized by assigning each participant a numerical identiﬁcation code. 4 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 R di l Di t f Table 1. Description of primary school enrollment sites by residential zone, distance from industrial complex, and neighborhood. Residential Zone Radial Distance from Industrial Complex (km) Neighborhoods Primary Schools 1 0.000–4.999 Tamburi, Città Vecchia, Borgo Vico, Deledda, Giusti, XXV Luglio 2 5.000–9.999 Italia Montegranaro, Tre Carrare Battisti, Solito Corvisea, Salinella, Paolo VI, Comune di Statte Alﬁeri, Viola, Pertini, Giovanni XXIII 3 10.000–15.000 Talsano-San Vito-Lama Frascolla, Salvemini, Sciascia, De Amicis f d l d l Zone Industrial Complex (km) Neighborhoods Primary Schools 1 0.000–4.999 Tamburi, Città Vecchia, Borgo Vico, Deledda, Giu XXV Luglio 2 5.000–9.999 Italia Montegranaro, Tre Carrare Battisti, Solito Corvisea, Salinella, Paolo VI, Comune di Statte Alfieri, Viola, Perti Giovanni XXIII 3 10.000–15.000 Talsano-San Vito-Lama Frascolla, Salvemin Sciascia, De Amic Table 1. Description of primary school enrollment sites by residential zone, distance from industrial complex, and neighborhood. Zone Industrial Complex (km) Neighborhoods Primary Sch 2.3. Division of Residential Location into Residential Zones 2 3 Division of Residential Location into Residential Zones 2.6. Determination of BMI Z-Score A clinical determination of underweight, healthy weight, overweight, or obese was made for each participant based on parent-reported data, using the World Health Organization (WHO) BMI-for-age reference charts for girls and boys. Based on these charts, participants with calculated BMI Z-scores below or equal to −2 standard deviations (SD) for their age and gender were classiﬁed as underweight. Participants with Z-scores from −2 SD up to and including +1 SD were classiﬁed as healthy weight. Z-scores from +1 SD up to and including +2 SD were considered overweight, and those greater than +2 SD were classiﬁed as obese [20,21]. To corroborate the accuracy of Z-score results, clinical weight classiﬁcation via BMI was also conducted based on BMI percentile, using the Children’s BMI Tool for Schools of the United States Centers for Disease Control and Prevention (CDC; values not used for analysis). All correlations of BMI percentile and BMI-adjusted fasting blood glucose with other covariates remained at the same level of statistical signiﬁcance using this approach. The distribution of participants into weight classiﬁcations was also comparable (6.5% underweight, 52.8% healthy weight, 20.1% overweight, 20.6% obese). 2.7. Determination of SES SES was classiﬁed for each participating child as low, medium, or high, according to the validated protocol described by Cesana et al [22]. The following characteristics of both parents were used as inputs to determine the SES classiﬁcation of each child: age, years of formal education, occupation, and work-related stress perception. 2.5. Collection of BMI Z-Score, SES, and Lifestyle Data 2.5. Collection of BMI Z-Score, SES, and Lifestyle Data A written questionnaire adapted from the Elementary Home Observation for Measurement of the Environment (HOME) Inventory [17] was administered to the parent(s) of participants to obtain self-reported child data. For determination of BMI Z-score, these included participant birthdate, sex, height, and weight. Parental assessments of child height and weight have been described as reasonably accurate for this age group, particularly when classifying large cohorts into obese and non-obese categories [18,19]. Similarly, information on residential address, parental occupation, and parental education level was collected from parents via the Elementary HOME Inventory. Participant lifestyle data, including weekly hours of sports activity, weekly hours of outdoor play, and total weekly hours of physical activity, were also provided by parents in this manner. The questionnaire was completed by the parent(s) at the primary school of the child, during the evaluation of the child by study personnel. 2.4. Collection and Analysis of Fasting Blood Glucose Data Fasting blood glucose measurements were collected at participants’ primary schools by trained medical and nursing personnel of the Taranto ASL Department of Prevention. Venous whole blood was collected from participating children in the morning hours after fasting overnight, with at least one parent present. Blood specimens were drawn in heparinized tubes and promptly transported to the analytical laboratories of the Hospital of Taranto for immediate analysis. 2.3. Division of Residential Location into Residential Zones 2 3 Division of Residential Location into Residential Zones Subsequently, the distance from the industrial complex perimeter to the farthest participant residential location was measured and divided by three, to form three levels of geographical distance (zones). Residential distance from the industrial complex was categorized as follows: less than 5.000 km (Zone 1), between 5.000 km and 9.999 km (Zone 2), or between 10.000 km and 15.000 km (Zone 3). Int. J. Environ. Res. Public Health 2019, 16, 2036 5 of 16 3.1. Sociodemographic and Physical Characteristics of the Cohort The main sociodemographic and physical characteristics of participating children (n = 214) are summarized in Table 2. The three residential zones had similar numbers of participants (n = 62 for Zone 1; n = 77 for Zone 2; n = 75 for Zone 3). The cohorts were also well-balanced by sex (n = 114 or 53.3% female; n = 100 or 46.7% male). Zone 1 had more females than males, while Zones 2 and 3 had nearly equal numbers of males and females (62.9% female in Zone 1; 49.4% female in Zone 2; 49.3% female in Zone 3). The mean age of all study participants was 8.6 years, and the age distribution was quite consistent for all zones and sexes (8.9 ± 1.6 years for Zone 1; 8.2 ± 1.4 years for Zone 2; 8.6 ± 1.5 years for Zone 3; 8.5 ± 1.5 years for all females; 8.6 ± 1.5 years for all males). Table 2. Sociodemographic and physical characteristics of the study cohort (mean (± SD) or n (%)) in totality, by residential zone, and by sex (BMI = Body mass index; SES = socio-economic status; FBG = fasting blood glucose). Table 2. Sociodemographic and physical characteristics of the study cohort (mean (± SD) or n (%)) in totality, by residential zone, and by sex (BMI = Body mass index; SES = socio-economic status; FBG = fasting blood glucose). 2.8. Statistical Analysis The cohort was ﬁrst described using standard statistics (mean and standard deviation for continuous variables; frequency, and percentage for discrete variables) to explore the characteristics of each residential zone and group. Generalized additive models were used to conﬁrm a linear relationship between each independent variable—residential zone and SES—and each metabolic dependent variable of interest [23]. Linear modeling was then performed to explore these relationships quantitatively. Diﬀerent residential proximities to the industrial complex were represented and compared via residential zone, 6 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 the categorical variable described above (Zone 2 vs. Zone 1; Zone 3 vs. Zone 1). SES was expressed and compared in terms of the above-described categorized indices (High vs. Low; Medium vs. Low). We also performed a mediation analysis to test the role of BMI Z-score as a mediator in the association of SES and residential-to-industrial-complex distance with FBG. The 95% conﬁdence intervals were calculated for each regression. Diﬀerences between groups were considered statistically signiﬁcant at p < 0.05 for a two-tailed hypothesis test. All statistical analyses, tables, and graphs were performed and generated with R 3.5.2 [24]. 3.1. Sociodemographic and Physical Characteristics of the Cohort Measured Characteristic Total (n = 214) Zone 1 (n = 62) Zone 2 (n = 77) Zone 3 (n = 75) Females (n = 114) Males (n = 100) Sex (F) 114 (53.3%) 39 (62.9%) 38 (49.4%) 37 (49.3%) Age (years) 8.6 (±1.5) 8.9 (±1.6) 8.2 (±1.4) 8.6 (±1.5) 8.5 (±1.5) 8.6 (±1.5) Weight (kg) 33.1 (±10.1) 35.4 (±10.9) 31.5 (±9.4) 33.0 (±10.0) 31.8 (±9.0) 34.7 (±11.2) Height (cm) 133.6 (±11.5) 134.5 (±13.5) 132.3 (±10.6) 134.0 (±10.8) 132.9 (±11.2) 134.3 (±11.9) BMI Z-Score 0.8 (±1.5) 1.1 (±1.5) 0.62 (±1.5) 0.66 (±1.6) 0.56 (±1.3) 1.0 (±1.7) Underweight 9 (4.2%) 2 (3.2%) 5 (6.5%) 2 (2.7%) 5 (4.4%) 4 (4%) Healthy Weight 109 (50.9%) 27 (43.6%) 39 (50.6%) 43 (57.3%) 64 (56.1%) 45 (45%) Overweight 47 (22.0%) 16 (25.8%) 20 (26.0%) 11 (14.7%) 32 (28.1%) 15 (15%) Obese 49 (22.9%) 17 (27.4%) 13 (16.9%) 19 (25.3%) 13 (11.4%) 36 (36%) SES Low 91 (42.5%) 41 (66.1%) 26 (33.8%) 24 (32.0%) 48 (42.1%) 43 (43%) Medium 70 (32.7%) 16 (25.8%) 25 (32.5%) 29 (38.7%) 41 (36.0%) 29 (29%) High 53 (24.8%) 5 (8.1%) 26 (33.8%) 22 (29.3%) 25 (21.9%) 28 (28%) FBG (mg/dL) 1 85.5 (±6.6) 87.8 (±6.3) 85.5 (±7.1) 83.5 (±5.6) 85.4 (±6.8) 85.6 (±6.3) 1 For FBG data, n = 212 for cohort (Zone 1: n = 62; Zone 2: n = 76; Zone 3: n = 74; females: n = 113; males: n = 99). Across the three residential zones and both sexes, the distribution of height was quite uniform (133.6 ±11.5 cm for all participants). Weight was slightly higher in Zone 1 relative to Zones 2 and 3 (35.4 ± 10.9 kg for Zone 1; 31.5 ± 9.4 kg for Zone 2; 33.0 ± 10.0 kg for Zone 3), as well as in males relative to females (31.8 ± 9.0 kg for females; 34.7 ± 11.2 kg for males). g g In general, the BMI classiﬁcation of participating children showed a disproportionately high prevalence of overweight and obesity (4.2% underweight, 50.9% healthy weight, 22.0% overweight, 22.9% obese). This trend towards overweight and obesity was especially strong among participants in Zone 1 (25.8% overweight, 27.4% obese) and among male participants (15% overweight, 36% obese), as over 50% of participants in these two groups were overweight or obese. Across the three residential zones and both sexes, the distribution of height was quite uniform (133.6 ±11.5 cm for all participants). Weight was slightly higher in Zone 1 relative to Zones 2 and 3 (35.4 ± 10.9 kg for Zone 1; 31.5 ± 9.4 kg for Zone 2; 33.0 ± 10.0 kg for Zone 3), as well as in males relative to females (31.8 ± 9.0 kg for females; 34.7 ± 11.2 kg for males). 3.1. Sociodemographic and Physical Characteristics of the Cohort Mean BMI Z-score for the cohort was 0.8 ± 1.5 (1.1 ± 1.5 for Zone 1; 0.62 ± 1.5 for Zone 2; 0.66 ± 1.6 for Zone 3; 0.56 ± 1.3 for females; 1.0 ± 1.7 for males). In general, the BMI classiﬁcation of participating children showed a disproportionately high prevalence of overweight and obesity (4.2% underweight, 50.9% healthy weight, 22.0% overweight, 22.9% obese). This trend towards overweight and obesity was especially strong among participants in Zone 1 (25.8% overweight, 27.4% obese) and among male participants (15% overweight, 36% obese), as over 50% of participants in these two groups were overweight or obese. Mean BMI Z-score for the cohort was 0.8 ± 1.5 (1.1 ± 1.5 for Zone 1; 0.62 ± 1.5 for Zone 2; 0.66 ± 1.6 for Zone 3; 0.56 ± 1.3 for females; 1.0 ± 1.7 for males). 7 of 16 7 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 Study participants tended towards low SES (42.5% low SES; 32.7% medium SES; 24.8% high SES). This tendency occurred because Zone 1 participants were disproportionately concentrated in the low SES index (66.1% low SES, 25.8% medium SES, 8.1% high SES). By contrast, Zone 2 and Zone 3 were relatively well-balanced by SES index (Zone 2: 33.8% low SES, 32.5% medium SES, 33.8% high SES; Zone 3: 32.0% low SES, 38.7% medium SES, 29.3% high SES). Further, males and females had SES distributions, with nearly equal portions of each sex in the low SES index, a slightly greater portion of females in the medium SES index, and a slightly greater portion of males in the high SES index (females: 42.1% low SES; 36.0% medium SES; 21.9% high SES; males: 43% low SES, 29% medium SES; 28% high SES). g Fasting blood glucose values were within the age-appropriate range of normality for all subjects [25], with a cohort average of 85.5 ± 6.6 mg/dL, and are described in detail in Section 3.2. 3.2. FBG by Sociodemographic Factors Tables 3–5 present regression coefficients and 95% confidence intervals for the association of sociodemographic factors with BMI Z-score and FBG (Table 3 describes all participants, Table 4 describes female participants only, and Table 5 describes male participants only). When analyzing FBG, the relationship between FBG and BMI Z-Score was taken into account. Overall, Zone 1 participants had significantly higher FBG compared to Zone 2 and Zone 3 participants (p < 0.05 vs. Zone 2; p < 0.001 vs. Zone 3; Table 3). When considering only female participants; Zone 1 continued to show significantly elevated FBG relative to Zone 3 (p < 0.001), with a higher magnitude of association (βAll participants = −4.6; βFemales = −6.3). The difference in FBG between Zones 1 and 2 dropped below the threshold of significance for female participants only, yet the magnitude of association increased relative to the full cohort (βAll participants = −2.5; βFemales = −3.0; Table 4). When only male participants were analyzed, none of the differences in FBG by residential zone remained statistically significant. For males only, FBG comparisons by residential zone showed lower magnitudes of association than in the full cohort (Table 5). Low SES participants did not show statistically significant differences in FBG relative to medium SES or high SES participants, neither for any particular sex nor for the cohort as a whole. Table 3. All participants (n = 214): Linear regression coeﬃcients and 95% conﬁdence intervals for associations of (i) residential zone; (ii) socioeconomic status (SES) index; and (iii) maternal education level to body mass index (BMI) Z-score and BMI-adjusted fasting blood glucose (FBG) (mg/dL). Sociodemographic Variable BMI Z-Score FBG Adjusted by BMI Z-Score BMI Z-Score 0.337 (−0.254, 0.928) Zone 2 vs. Zone 1 −0.223 −2.518 * (−0.755, 0.309) (−4.813, −0.224) Zone 3 vs. Zone 1 −0.052 −4.638 *** (−0.606, 0.501) (−7.020, −2.255) SES: Medium vs. Low −0.23 1.453 (−0.729, 0.269) (−0.699, 3.606) SES: High vs. Low 0.199 1.71 (−0.699, 1.096) (−2.157, 5.576) Maternal Education: 13 years vs. 5–8 years −0.539 * (−1.056, 0.023) −0.203 (−2.450, 2.044) Maternal Education: 16+ years vs. 5–8 years −1.031 * (−2.014, −0.048) −0.761 (−5.037, 3.514) Constant 1.347 * 87.036 * (0.934, 1.760) (85.089, 88.983) Legend: * p < 0.05; *** p < 0.001. Sociodemographic Variable BMI Z-Score FBG Adjusted by BMI Z-Score Legend: * p < 0.05; * p < 0.001. Int. J. Environ. Res. 3.2. FBG by Sociodemographic Factors Public Health 2019, 16, 2036 8 of 16 Table 4. Female participants (n = 114): Linear regression coeﬃcients and 95% conﬁdence intervals for associations of (i) residential zone; (ii) socio-economic status (SES) index; and (iii) maternal education level to body mass index (BMI) Z-score and BMI-adjusted fasting blood glucose (FBG). Sociodemographic Variable BMI Z-Score FBG Adjusted by BMI Z-Score BMI Z-Score 0.537 (−0.412, 1.485) Zone 2 vs. Zone 1 −0.011 −3.033 (−0.629, 0.608) (−6.080, 0.015) Zone 3 vs. Zone 1 0.015 −6.312 * (−0.676, 0.706) (−9.717, −2.907) SES: Medium vs. Low −0.209 0.659 (−0.825, 0.408) (−2.383, 3.702) SES: High vs. Low −0.002 3.623 (−1.406, 1.402) (−3.291, 10.536) Maternal Education: 13 years vs. 5–8 years −0.664 * (−1.279, −0.048) 1.037 (−2.059, 4.132) Maternal Education: 16+ years vs. 5–8 years −0.537 (−2.015, 0.942) −2.899 (−10.199, 4.401) Constant 0.998 * 87.319 * (0.527, 1.469) (84.813, 89.825) Legend: * p < 0.05; * p < 0.001. Table 5. Male participants (n = 100): Linear regression coeﬃcients and 95% conﬁdence intervals for associations of (i) residential zone; (ii) socio-economic status (SES) index; and (iii) maternal education level to body mass index (BMI) Z-score and BMI-adjusted fasting blood glucose (FBG). Sociodemographic Variable BMI Z-Score FBG Adjusted by BMI Z-Score BMI Z-Score 0.265 (−0.536, 1.066) Zone 2 vs. Zone 1 −0.664 −1.213 (−1.617, 0.289) (-4.918, 2.491) Zone 3 vs. Zone 1 −0.458 −1.659 (−1.414, 0.498) (−5.354, 2.036) SES: Medium vs. Low −0.104 2.994 (−0.945, 0.737) (−0.241, 6.229) SES: High vs. Low 0.196 1.303 (−1.090, 1.483) (−3.649, 6.255) Maternal Education: 13 years vs. 5–8 years −0.462 (−1.344, 0.419) −1.959 (−5.369, 1.452) Maternal Education: 16+ years vs. 5–8 years −1.311 (−2.788, 0.166) 0.196 (−5.582, 5.974) Constant 1.923 * 85.993 * (1.173, 2.674) (82.721, 89.266) Legend: * p < 0.001. BMI Z-score showed a positive association with FBG but was not statistically signiﬁcant for any sex or for the cohort as a whole. The same analysis was performed using categorical BMI, and results were very similar to those previously described. Examining FBG results by clinical BMI subgroup (with all participants included), we found an almost signiﬁcant diﬀerence in FBG between healthy weight Int. J. Environ. Res. Public Health 2019, 16, 2036 9 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 9 of 16 subjects and obese subjects, with the former category showing a lower average FBG concentration (β = −2.0; CI = −4.2, 0.3). 3.3. BMI Z-Scores and Clinical Subgroups by Sociodemographic Factors Results on the impact of sociodemographic factors on BMI Z-score are shown in Tables 3–5. No statistically signiﬁcant relationship was found between residential zone and BMI Z-score for any sex or for the cohort as a whole. BMI Z-score was also not signiﬁcantly diﬀerent across SES categories for males, females, or the entire cohort. Across the cohort, participants whose mothers had completed high school education (13 years of schooling) had signiﬁcantly lower BMI Z-scores than those whose mothers were formally educated up to middle or primary school (5–8 years) (p < 0.05; Table 3). The same was true for participants whose mothers had a university-level education or higher (16+ years in total) with respect to those whose mothers completed up to middle or primary school (p < 0.05; Table 3). Likewise, female children whose mothers completed high school had signiﬁcantly lower BMI Z-scores than female children whose mothers completed only 5–8 years of formal education (p < 0.05; Table 4). However, the relationship between BMI Z-score and maternal education level was no longer signiﬁcant when comparing female children whose mothers had university-level education or higher with female children whose mothers had only middle or primary school education (Table 4). For male children, no comparison by maternal education level showed a signiﬁcant diﬀerence in BMI Z-score (Table 5). Furthermore, no signiﬁcant association with BMI Z-score or FBG was found for weekly hours of sports activity, weekly hours of outdoor play, or total weekly hours of physical activity (p > 0.05 for all comparisons). Similarly, no signiﬁcant diﬀerence between residential zones was found for any of the lifestyle variables (p > 0.05 for all comparisons). Low SES participants had signiﬁcantly lower reported weekly hours of outdoor play than high SES participants (p < 0.05), although all other comparisons of lifestyle outcomes by SES were statistically nonsigniﬁcant. 3.2. FBG by Sociodemographic Factors Additionally, in our analyses, BMI Z-score was not a mediator of the relationships between FBG and either residential zone or SES (results not shown). When FBG was not corrected for BMI Z-score, similarly signiﬁcant associations at the same p-value thresholds were found between these residential zones and SES indices. 4. Discussion This study describes the BMI values and FBG levels of a cohort of 214 Taranto schoolchildren aged 6–11 years, in relation to residential distance from a nearby industrial complex and SES. Our results showed that residential proximity to the complex was associated with high BMI-adjusted FBG. This eﬀect of residential location on BMI-adjusted FBG was stronger in female children than in male children. Residential proximity to the industrial complex was not associated with BMI Z-score, and SES was not associated with BMI Z-score or FBG. Additionally, higher maternal education level was associated with lower BMI Z-score. Both BMI Z-score and the resultant clinical subgroups (underweight, healthy weight, overweight, obese) showed positive but nonsigniﬁcant associations to FBG. 4.1. Fasting Blood Glucose Concentration by Sociodemographic Factors Despite no signiﬁcant diﬀerence in BMI Z-score makeup across SES and residential groups, FBG was higher in children who lived closer to the industrial complex. At the same time, SES and BMI Z-score were not associated with FBG. Taken together, these results show that residential location with respect to the industrial complex inﬂuenced children’s FBG levels; they suggest that the presence of the industrial complex plays a role in the corporeally elevated fasting blood glucose of children in the surrounding area. While consistent with preliminary literature on the child health outcomes and hyperglycemia in the setting of industrial metal exposure [26–29], this association has not been described previously. More thorough investigation of the relationship between residential proximity to industrial activity and increase in pediatric FBG is highly merited. 10 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 As residential proximity to the industrial complex increased, female children experienced substantially more signiﬁcant increases in FBG than male children. This male–female disparity was strongly apparent in the Zone 1 vs. Zone 3 comparison. The Zone 1 vs. Zone 2 comparison also suggested that females may be more vulnerable to increased FBG based on closer residential location to the industrial complex, yet the reduced sample size of the single-sex analyses limited the detection of statistically signiﬁcant diﬀerences between boys and girls at this level. These ﬁndings concur with prior literature that describes female children as potentially more vulnerable than male children to the health consequences of environmental exposure [30,31]. For instance, a study of 6730 Chinese children aged 3–7 years found that higher concentrations of particulate matter, sulfur dioxide, and nitrogen dioxides in ambient air were signiﬁcantly associated with increased respiratory symptoms in girls, but not in boys [31]. However, since there is no clear consensus about the eﬀects of sex on pollution sensitivity, nor about the reasons for which pollution sensitivity may diﬀer between boys and girls, the role of sex in the relationship between FBG and residential proximity to the industrial complex should be studied further. In addition, further analysis is required to understand the mechanisms of this eﬀect. For instance, FBG levels could be mediated in several ways by environmental exposure to obesogenic and metabolically disruptive steel production byproducts. 4.1. Fasting Blood Glucose Concentration by Sociodemographic Factors Potential pathways for this mechanism include ambient air or water contamination, a greater frequency of contact with persons directly exposed to toxins from the industrial complex, or behavior modiﬁcation due to negative perceptions of the industrial complex. Considering the lack of a statistically signiﬁcant BMI Z-score gradient with respect to residential proximity to the industrial complex, the latter possibility seems less probable. Moreover, these data suggest a geographic threshold for the negative eﬀects of residing near an industrial complex. While Zone 1 had signiﬁcantly worse outcomes than Zones 2 and 3 with regard to FBG levels, diﬀerences between Zones 2 and 3 were not statistically signiﬁcant. This ﬁnding indicates that a categorical interpretation of distance may be eﬀective for identifying groups most impacted by industrial activity. The positive association of BMI Z-score with FBG and the nearly signiﬁcant diﬀerence in FBG between healthy weight and obese participants may be relevant to preventative health measures. A 2017 study of obese, nondiabetic children aged 8–15 years in the United States found that proximity to air with nitrogen dioxide and particulate matter contaminants decreased insulin sensitivity and increased Type II diabetes risk at early adulthood [32]. The interaction between BMI status, FBG, and environmental exposure suggested by our study and others should be further explored, in order to inform clinical and public health management of metabolic disease risks in childhood and early adulthood. 4.2. BMI Z-Scores and Clinical Subgroups by Sociodemographic Factors As a whole, the BMI Z-score makeup of participating children was congruent with that described by prior studies of age-matched Italian cohorts; 44.9% of children were overweight or obese, and higher obesity and overweight/obesity rates were found in male children than in female children [4–10]. In our study, overweight or obese status were not linked to any particular socioeconomic or residential condition in a statistically signiﬁcant way. This contrasts with prior literature, in which the presence of overweight/obesity in children was linked to economic disadvantage [33–35] and poor neighborhood conditions [36,37]. However, the comparatively smaller size of the Taranto cohort may have limited the statistical power of our study to capture SES inﬂuences. The association of higher maternal education level with lower pediatric BMI Z-score, and the lack thereof with BMI-adjusted FBG, agrees with previous literature [5,8]. In adults, formal education level has been shown to aﬀect health literacy and, in turn, health outcomes [38]. Given the central role of the maternal ﬁgure in controlling child diet in Southern Italian society, the inﬂuence of maternal education level on child BMI was considered worthy of investigation. Notably, maternal education was lowest 11 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 in Zone 1, the area closest to the plant and with the lowest overall SES. This diﬀerence in maternal education by zone would likely have been more apparent with a larger sample size. Similarly, the weakening of the association between maternal educational level and BMI Z-score when the cohort was disaggregated by sex most likely results from the reduction in sample size (n = 214 total participants; n = 114 females, n = 100 males). In 2019, a population-based study of 45,000 Italian adolescents recorded signiﬁcantly lower overweight/obesity rates for both males and females as maternal education increased [39]. In our study, however, the stronger relationship between maternal education and BMI Z-score for female children relative to male children is nonetheless noteworthy. This observation merits deeper investigation, particularly in light of the more intense association of residential location to BMI-adjusted FBG for girls. Additionally, the association between maternal education level and child BMI Z-score supports the notion that obesity is mediated by the interaction of multiple diﬀerent environmental conditions, rather than a single predominant factor. 4.3. Implications This study found that closer residential location to a nearby industrial complex on the order of 5.000 km was signiﬁcantly associated with worsened metabolic outcomes. This result is consistent with past studies conducted by our research group in Brescia Province (Italy), in which greater outdoor dust Manganese concentrations were observed within 2.000 km of ferromanganese alloy plants [43]. SES (low, medium, or high) was not signiﬁcantly associated with any studied change in metabolic variables. Therefore, these ﬁndings support the notion that residential area may be a more eﬀective starting point to reach populations experiencing the negative health eﬀects of industrial exposure [44,45]. Furthermore, the FBG consequences of residing near the industrial complex were more pronounced in female children than in male children. This indicates that young girls may be more susceptible to the health eﬀects of industrial exposure, and that their potentially elevated health risks from exposure merit special attention. 4.2. BMI Z-Scores and Clinical Subgroups by Sociodemographic Factors A genetic modeling study of 925 pediatric twin pairs found that children living in obesogenic homes (deﬁned by quantitative measurement of the food, physical activity, and media inﬂuences present in the household) displayed greater BMI heritability than those in non-obesogenic homes [40]. Such literature suggests that the home environment in early life may play an important role in modifying genetic propensities for obesity. In this regard, the lack of association between BMI Z-score and weekly hours of sports activity, weekly hours of outdoor play, or total weekly hours of physical activity in this study is notable. This result suggests that, while an important component of obesity prevention, physical activity alone may be insuﬃcient to alter obesity prevalence on a population level. This insight concords with past literature and is an important consideration for public health policies and programs addressing obesity [41,42]. Finally, the SES index used in our analyses was derived from the parental occupation and education level. As a result, it may have been diﬀerently informative about parental awareness of, and ability to aﬀord, healthy lifestyle options when compared to the SES classiﬁcations used by other studies [5,33–35]. For instance, the idea that parental occupation may not be related to childhood overweight and/or obesity is corroborated by prior work [5]. Finally, it is possible that obesogenic factors originating from the industrial complex are ubiquitous in the local environment and aﬀect the entire cohort, altering expected patterns in overweight/obesity [5,36,37]. 4.4. Strengths, Limitations, and Future Directions This study describes the interrelationships between two important environmental factors (SES and residential location) and the metabolic health outcomes of a unique, sizable, and robust pediatric cohort. While previous publications have focused on urban children aged 6–11 years or populations residing near industrial sites, few studies have explored both conditions simultaneously. The quantity and diversity of available data allowed us to study the metabolic results of BMI and FBG together. Int. J. Environ. Res. Public Health 2019, 16, 2036 12 of 16 For this reason, a fuller understanding of how SES and residential proximity to the industrial complex inﬂuence metabolic health could be obtained. This cohort was also balanced across the variables of age, sex, SES, and residential proximity to the industrial complex, oﬀering a useful system to study these multiple relationships. However, this study was not without limitations. BMI inputs of height and weight were based on parental perceptions and not measured according to a standardized protocol, allowing for subjective bias that is diﬃcult to quantify. Furthermore, the sample size of this cohort did not provide enough statistical power to analyze severely obese participants in a separate BMI category. Severe obesity is deﬁned as a BMI percentile value over 1.2 times that of the 95th percentile for the child’s age and gender according to the WHO reference curve and predicts distinctly elevated cardio-metabolic risk [46]. Another important consideration regards the approach by which height, weight, age, and sex data are converted into pediatric BMI measurements and cutoﬀs for underweight, healthy weight, overweight, and obese. The WHO standards used in this paper are considered most appropriate for measuring obesity and overweight at the population level, given their higher sensitivity relative to other systems. Additionally, the use of Z-scores was determined to be more statistically meaningful for our analytical purposes than the percentile values generated by the CDC tool [20,21,46]. At the same time, Italian national standards for pediatric BMI measurement and classiﬁcation have also been developed previously. While more speciﬁc than the WHO standards used herein for identifying children at high cardiovascular risk, these standards are considered less useful for cohort studies due to their lower sensitivity and tendency to underestimate rates of overweight and obesity in larger samples [47]. References 1. Hanna-Attisha, M.; LaChance, J.; Sadler, R.C.; Champney Schnepp, A. Elevated blood lead levels in children associated with the Flint drinking water crisis: A spatial analysis of risk and public health response. Am. J. Pub. Health 2016, 106, 283–290. [CrossRef] [PubMed] 1. Hanna-Attisha, M.; LaChance, J.; Sadler, R.C.; Champney Schnepp, A. Elevated blood lead levels in children associated with the Flint drinking water crisis: A spatial analysis of risk and public health response. Am. J. Pub. Health 2016, 106, 283–290. [CrossRef] [PubMed] 2. Hong, S.B.; Im, M.H.; Kim, J.W.; Park, E.J.; Shin, M.S.; Kim, B.N.; Yoo, H.J.; Cho, I.H.; Bhang, S.Y.; Hong, Y.C.; et al. Environmental lead exposure and attention deficit/ hyperactivity disorder symptom domains in a community sample of South Korean school-age children. Environ. Health Perspect. 2015, 123, 271–276. [CrossRef] [PubMed] 2. Hong, S.B.; Im, M.H.; Kim, J.W.; Park, E.J.; Shin, M.S.; Kim, B.N.; Yoo, H.J.; Cho, I.H.; Bhang, S.Y.; Hong, Y.C.; et al. Environmental lead exposure and attention deficit/ hyperactivity disorder symptom domains in a community sample of South Korean school-age children. Environ. Health Perspect. 2015, 123, 271–276. [CrossRef] [PubMed] 3. Jerrett, M.; McConnell, R.; Wolch, J.; Chang, R.; Lam, C.; Dunton, G.; Gilliland, F.; Lurmann, F.; Islam, T.; Berhane, K. Traﬃc-related air pollution and obesity formation in children: A longitudinal, multilevel analysis. Environ. Health 2014, 13. [CrossRef] [PubMed] 3. Jerrett, M.; McConnell, R.; Wolch, J.; Chang, R.; Lam, C.; Dunton, G.; Gilliland, F.; Lurmann, F.; Islam, T.; Berhane, K. Traﬃc-related air pollution and obesity formation in children: A longitudinal, multilevel analysis. Environ. Health 2014, 13. [CrossRef] [PubMed] 4. Lombardo, F.L.; Spinelli, A.; Lazzeri, G.; Lamberti, A.; Mazzarella, G.; Nardone, P.; Pilato, V.; Buoncristiano, M.; Caroli, M. Severe obesity prevalence in 8-to 9-year-old Italian children: A large population-based study. Eur. J. Clin. Nutr. 2015, 69, 603–608. [CrossRef] [PubMed] 4. Lombardo, F.L.; Spinelli, A.; Lazzeri, G.; Lamberti, A.; Mazzarella, G.; Nardone, P.; Pilato, V.; Buoncristiano, M.; Caroli, M. Severe obesity prevalence in 8-to 9-year-old Italian children: A large population-based study. Eur. J. Clin. Nutr. 2015, 69, 603–608. [CrossRef] [PubMed] 5. Grassi, T.; De Donno, A.; Bagordo, F.; Serio, F.; Piscitelli, P.; Ceretti, E.; Zani, C.; Viola, G.; Villarini, M.; Moretti, M.; et al. Socio-Economic and environmental factors associated with overweight and obesity in children aged 6–8 years living in ﬁve Italian cities (the MAPEC_LIFE cohort). Int. J. Environ. Res. Public Health 2016, 13, 1002. [CrossRef] [PubMed] 5. 4.4. Strengths, Limitations, and Future Directions Public Health 2019, 16, 2036 13 of 16 13 of 16 studies have demonstrated a link between obesity and elevated serum creatinine levels in children above 10 years of age, with implications for renal function [51]. In a prior cohort study of 396 Italian children aged 5–11 years, urine creatinine levels were directly correlated with measurements of urban benzene pollution surrounding the home environment [52]. Therefore, more detailed and multifactorial analyses are required to elucidate the relationships of residential location and SES with these variables. 5. Conclusions Our study found that greater residential proximity to an industrial complex was associated with signiﬁcant increases in FBG concentration among a cohort of primary school children in Taranto, Italy. BMI Z-scores were not signiﬁcantly diﬀerent by residential zone, and neither BMI Z-score nor FBG was signiﬁcantly associated with SES. Moreover, the relationship between decreased residential distance from the industrial complex and increased FBG was particularly strong among female children. Taken together, these results provide additional evidence that living close to industrial activity can negatively aﬀect the metabolic health of children aged 6–11 years. These data also support residential location as a fundamental consideration for reaching the pediatric populations most acutely impacted by industrial exposure. They corroborate the need for customized interventions that address potential diﬀerences in the health needs and risk proﬁles of boys and girls. As a result, this study provides useful evidence for the design of public health policies and interventions for children living in the setting of industrial activity. Author Contributions: Conceptualization: E.B., D.P., S.C., R.O.W., D.S., L.C., and R.G.L.; Data curation: S.R. and S.G.; Formal analysis: S.R. and S.G.; Funding acquisition: R.G.L.; Investigation: A.G., G.C., S.Z., C.F., M.M., M.O., and M.C.; Methodology: E.B. and D.P.; Project administration: D.P.; Resources: R.G.L.; Software, S.R.; Supervision: D.P. and R.G.L.; Validation: S.R.; Visualization: E.B. and S.R.; Writing—original draft: E.B.; Writing—review and editing: E.B., D.P., R.O.W., D.S., L.C. and R.G.L. Funding: This research was funded by the Italian Ministry of Health (grant number CCM 2013: “Biomonitoring and toxicity of pollutants in the territory of Taranto, Italy”) and by the National Institute of Environmental Health Sciences (grant numbers R01ES019222 and R56ES019222). The APC was funded by the Department of Environmental Medicine and Public Health of the Icahn School of Medicine at Mount Sinai. Acknowledgments: We thank the Mount Sinai International Exchange Program for Minority Students funded by the National Institute of Minority Health and Health Disparities (T37 MD001452) and the International Training and Research Program in Environmental and Occupational Health funded by the Fogarty International Center (Fogarty International Center TW00640). Conﬂicts of Interest: The authors declare no conﬂict of interest. 4.4. Strengths, Limitations, and Future Directions The analysis and interpretation of pediatric BMI values in this study is advantageous for the sensitivity and broad applicability of the study, although other methods to ensure compatibility with national and regional tendencies in body composition should also be considered. Participants’ residential distance from the plant was analyzed as a categorical variable in order to represent the neighborhood structure of the Taranto municipality. In this way, the study design attempted to maintain consistency with health policies and interventions, which are most often organized on a discrete zone-by-zone or neighborhood-by-neighborhood basis. At the same time, two potential limitations of this approach exist. First, the similarity and contrast of environmental exposure proﬁles within and across the 5.0 km zones, respectively, have not been experimentally veriﬁed. Second, therefore, multiple equally reasonable zone delineations might have emerged when clustering participant residences by distance from the industrial complex. However, when the same correlations were run using distance as a continuous variable (measured in kilometers from the perimeter of the industrial complex), nearly identical correlations were obtained. This suggests that categorical measures of distance employed herein are an appropriate unit of analysis in the context of this study. These results would also be stronger if combined with quantitative measures of industrial complex-associated pollution in the studied residential areas and/or with biomarkers of exposure. Therefore, future studies from our group will aim to measure industrial particles and byproducts in ambient air, soil, and water of the three residential zones, as well as participants’ physiological uptake of these substances [48]. By specifying the relationship between residential location and environmental/physiological exposure to industrial activities, these data will better contextualize the results described herein. Additionally, expanding this cohort to include a control population far removed from the industrial complex and accounting for variation in participants’ dietary and lifestyle behaviors will permit a more potent interpretation of these results [49]. More broadly, sociodemographic and biological factors beyond the scope of this study such as home environment and diet may play relevant obesogenic roles in interaction with SES and residential proximity to the industrial complex [50]. Other biological variables should also be incorporated into future studies. For instance, blood creatinine levels were collected for all participants in this cohort. These data were not analyzed in relation herein, because biological and environmental mediators of blood creatinine levels are vast. However, these data merit further investigation. In fact, prior Int. J. Environ. Res. References Grassi, T.; De Donno, A.; Bagordo, F.; Serio, F.; Piscitelli, P.; Ceretti, E.; Zani, C.; Viola, G.; Villarini, M.; Moretti, M.; et al. Socio-Economic and environmental factors associated with overweight and obesity in children aged 6–8 years living in ﬁve Italian cities (the MAPEC_LIFE cohort). Int. J. Environ. Res. Public Health 2016, 13, 1002. [CrossRef] [PubMed] 14 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 6. Olaya, B.; Moneta, M.V.; Pez, O.; Bitfoi, A.; Carta, M.G.; Eke, C.; Goelitz, D.; Keyes, K.M.; Kuijpers, R.; Lesinskiene, S.; et al. Country-level and individual correlates of overweight and obesity among primary school children: A cross-sectional study in seven European countries. Bmc Pub. Health 2015, 15, 475. [CrossRef] 7. Silano, M.; Agostoni, C.; Fattore, G. Italy’s unsolved childhood obesity crisis. Arch. Dis Child. 20 202–203. [CrossRef] 8. Parrino, C.; Vinciguerra, F.; La Spina, N.; Romeo, L.; Tumminia, A.; Baratta, R.; Squatrito, S.; Vigneri, R.; Frittitta, L. Inﬂuence of early-life and parental factors on childhood overweight and obesity. J. Endocrinol. Investig. 2016, 39, 1315–1321. [CrossRef] 9. Lazzeri, G.; Dalmasso, P.; Berchialla, P.; Borraccino, A.; Charrier, L.; Giacchi, M.V.; Simi, R.; Lenzi, M.; Vieno, A.; Lemma, P.; et al. Trends in adolescent overweight prevalence in Italy according to socioeconomic position. Ann. Ist. Super Sanità. 2017, 53, 283–290. 10. Lazzeri, G.; Pammolli, A.; Simi, R.; Pilato, V.; Giacchi, M.V. BMI from nutritional surveillance of 8-9 years old children in Tuscany (Italy). J. Prev. Med. Hyg. 2011, 52, 181–185. 11. Cortese, S.; Moreira-Maia, C.R.; St. Fleur, D.; Morcillo-Peñalver, C.; Rohde, L.A.; Faraone, S.V. Association between ADHD and obesity: A systematic review and meta-analysis. Am. J. Psychiatry 2015, 173, 34–43. [CrossRef] [PubMed] 12. Sfera, A.; Osorio, C.; Inderias, L.A.; Parker, V.; Price, A.I.; Cummings, M. The obesity–impulsivity axis: Potential metabolic interventions in chronic psychiatric patients. Front. Psychiatry 2017, 13, 8–20. [CrossRef] [PubMed] 13. Janesick, A.S.; Blumberg, B. Obesogens: An emerging threat to public health. Am. J. Obs. Gynecol. 2016, 214, 559–565. [CrossRef] [PubMed] 14. Landrigan, P.J.; Goldman, L.R. Children’s vulnerability to toxic chemicals: A challenge and opportunity to strengthen health and environmental policy. Health Aﬀ. 2011, 30, 842–850. [CrossRef] [PubMed] 15. Lanzinger, S.; Rosenbauer, J.; Sugiri, D.; Schikowski, T.; Treiber, B.; Klee, D.; Holl, R.W. Impact of long-term air pollution exposure on metabolic control in children and adolescents with type 1 diabetes: Results from the DPV registry. Diabetologia 2018, 1–8. [CrossRef] [PubMed] 16. References Pizzino, G.; Irrera, N.; Bitto, A.; Pallio, G.; Mannino, F.; Arcoraci, V.; Aliquò, F.; Minutoli, L.; de Ponte, C.; D’andrea, P.; et al. Cadmium-induced oxidative stress impairs glycemic control in adolescents. Oxid. Med. Cell Longev. 2017, 2017, 6341671. [CrossRef] 29. Dunea, D.; Iordache, S.; Liu, H.Y.; Bøhler, T.; Pohoata, A.; Radulescu, C. Quantifying the impact of PM2.5 and associated heavy metals on respiratory health of children near metallurgical facilities. Environ. Sci. Poll. Res. 2016, 23, 15395–15406. [CrossRef] 30. Dong, G.H.; Chen, T.; Liu, M.M.; Wang, D.; Ma, Y.N.; Ren, W.H.; Lee, Y.L.; Zhao, Y.D.; He, Q.C. Gender diﬀerences and eﬀect of air pollution on asthma in children with and without allergic predisposition: Northeast Chinese children health study. PLoS ONE 2017, 6, e22470. [CrossRef] 31. Liu, M.M.; Wang, D.; Zhao, Y.; Liu, Y.Q.; Huang, M.M.; Liu, Y.; Sun, J.; Ren, W.H.; Zhao, Y.D.; He, Q.C.; et al. Eﬀects of outdoor and indoor air pollution on respiratory health of Chinese children from 50 kindergartens. J. Epidemiol. 2013, 23, 280–287. [CrossRef] 32. Alderete, T.L.; Habre, R.; Toledo-Corral, C.M.; Berhane, K.; Chen, Z.; Lurmann, F.W.; Weigensberg, M.J.; Goran, M.I.; Gilliland, F.D. Longitudinal associations between ambient air pollution with insulin sensitive, beat-cell function, and adiposity in Los Angeles Latino children. Diabetes 2017, 66, 1989–1996. [CrossRef] 33. Frederick, C.B.; Snellman, K.; Putnam, R.D. Increasing socioeconomic disparities in adolescent obesity. PNAS 2014, 111, 1338–1342. [CrossRef] 34. Chung, A.; Backholer, K.; Wong, E.; Palermo, C.; Keating, C.; Peeters, A. Trends in child and adolescent obesity prevalence in economically advanced countries according to socioeconomic position: A systematic review. Obes. Rev. 2016, 17, 276–295. [CrossRef] 35. Walsh, B.; Cullinan, J. Decomposing socioeconomic inequalities in childhood obesity: Evidence from Ireland. Econ. Hum. Biol. 2015, 16, 60–72. [CrossRef] 36. Suglia, S.F.; Shelton, R.C.; Hsiao, A.; Wang, Y.C.; Rundle, A.; Link, B.G. Why the neighborhood social environment is critical in obesity prevention. J. Urban. Health 2016, 93, 206–212. [CrossRef] 37. Sullivan, S.M.; Peters, E.S.; Trapido, E.J.; Oral, E.; Scribner, R.A.; Rung, A.L. Neighborhood environment measurements and anthropometric indicators of obesity: Results from the Women and Their Children’s Health (WaTCH) study. Environ. Behav. 2018, 50, 1032–1055. [CrossRef] 38. Van der Heide, I.; Wang, J.; Droomers, M.; Spreeuwenberg, P.; Rademakers, J.; Uiters, E. The relationship between health, education, and health literacy: Results from the Dutch adult literacy and life skills survey. J. Health Commun. 2013, 18, 172–184. [CrossRef] 39. References Farkhondeh, T.; Samarghandian, S.; Azimi-Nezhad, M. The role of arsenic in obesity and diabetes. J. Cell Physiol. 2019. [CrossRef] 17. Bradley, R.H.; Caldwell, B.M.; Rock, S.L.; Hamrick, H.M.; Harris, P. Home Observation for Measurement of the Environment: Development of a Home Inventory for use with families having children 6 to 10 years old. Contemp. Educ. Psychol. 1988, 13, 58–71. [CrossRef] 18. Weden, M.M.; Brownell, P.B.; Rendall, M.S.; Lau, C.; Fernandes, M.; Nazarov, Z. Parent-reported height and weight as sources of bias in survey estimates of childhood obesity. Am. J. Epidemiol. 2013, 178, 461–473. [CrossRef] 19. Garcia-Marcos, L.; Valverde-Molina, J.; Sanchez-Solis, M.; Soriano-Pérez, M.J.; Baeza-Alcaraz, A.; Martinez-Torres, A.; Perez-Fernandez, V.; Guillen-Perez, J.J. Validity of parent-reported height and weight for deﬁning obesity among asthmatic and nonasthmatic schoolchildren. Int. Arch. Allergy Immunol. 2006, 139, 139–145. [CrossRef] 20. Butte, N.F.; Garza, C.; De Onis, M. Feasibility of international growth standards for school-aged children and adolescents. J. Nutr. 2007, 137, 153–157. [CrossRef] 21. De Onis, M.; Lobstein, T. Deﬁning obesity risk in childhood population: Which cut-oﬀs should we use? Int. J. Ped. Obes. 2010, 5, 458–460. [CrossRef] [PubMed] 22. Cesana, G.C.; Ferrario, M.; De, G.V.; Sega, R.; Grieco, A. Evaluation of the socioeconomic status in epidemiological surveys: Hypotheses of research in the Brianza area MONICA project. Med. Lav. 1995, 86, 16–26. 23. Wood, S.N. Fast stable restricted maximum likelihood and marginal likelihood estimation of semiparametric generalized linear models. J. R Stat. Soc. Ser. B Stat. Methodol. 2011, 73, 3–36. [CrossRef] 24. Team, R. Core. A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2013. 25. Rudesill, C.L.; Henderson, R.A. Normal blood sugar values in children. Am. J. Dis. Child. 1941, 61, 108–115. [CrossRef] 26. González-Villalva, A.; Colín-Barenque, L.; Bizarro-Nevares, P.; Rojas-Lemus, M.; Rodríguez-Lara, V.; García-Pelaez, I.; Ustarroz-Cano, M.; López-Valdez, N.; Albarrán-Alonso, J.C.; Fortoul, T.I. Pollution by metals: Is there a relationship in glycemic control? Environ. Toxicol. Pharm. 2016, 46, 337–343. [CrossRef] 15 of 16 15 of 16 Int. J. Environ. Res. Public Health 2019, 16, 2036 27. Neisi, A.; Goudarzi, G.; Akbar Babaei, A.; Vosoughi, M.; Hashemzadeh, H.; Naimabadi, A.; Mohammadi, M.J.; Hashemzadeh, B. Study of heavy metal levels in indoor dust and their health risk assessment in children of Ahvaz city, Iran. Toxin. Rev. 2016, 35, 16–23. [CrossRef] 28. References Lauria, L.; Spinelli, A.; Buoncristiano, M.; Nardone, P. Decline of childhood overweight and obesity in Italy from 2008 to 2016: Results from 5 rounds of the population-based surveillance system. Bmc Public Health 2019, 19, 618. [CrossRef] 40. Schrempft, S.; van Jaarsveld, C.H.; Fisher, A.; Herle, M.; Smith, A.D.; Fildes, A.; Llewellyn, C.H. Variation in the Heritability of Child Body Mass Index by Obesogenic Home Environment. Jama Pediatr. 2018. Advance online publication. [CrossRef] 41. Faith, M.S.; Epstein, L.H. Healthy homes and obesogenic genes in young children: Rigorous behavioral theory and measurement and the detection of gene-environment interactions. Jama Pediatr. 2018, 172, 1121–1122. [CrossRef] 42. National Institutes of Health, National Heart, Lung, and Blood Institute. Clinical guidelines on the identiﬁcation, evaluation, and treatment of overweight and obesity in adults: The evidence report. Obes. Res. 1998, 6, 51S–210S. 43. Lucas, E.L.; Bertrand, P.; Guazzetti, S.; Donna, F.; Peli, M.; Jursa, T.P.; Lucchini, R.; Smith, D.R. Impact of ferromanganese alloy plants on household dust manganese levels: Implications for childhood exposure. Environ. Res. 2015, 138, 279–290. [CrossRef] 44. Diez Roux, A.V.; Mair, C. Neighborhoods and health. Ann. N. Y. Acad. Sci. 2010, 1186, 125–145. [CrossRef] 44. Diez Roux, A.V.; Mair, C. Neighborhoods and health. Ann. N. Y. Acad. Sci. 2010, 1186, 125–145. [CrossRef] 45. Christian, H.; Zubrick, S.R.; Foster, S.; Giles-Corti, B.; Bull, F.; Wood, L.; Knuiman, M.; Brinkman, S.; Houghton, S.; Boruﬀ, B. The inﬂuence of the neighborhood physical environment on early child health and 44. Diez Roux, A.V.; Mair, C. Neighborhoods and health. Ann. N. Y. Acad. Sci. 2010, 1186, 125–145. [CrossRef] 45. Christian, H.; Zubrick, S.R.; Foster, S.; Giles-Corti, B.; Bull, F.; Wood, L.; Knuiman, M.; Brinkman, S.; Houghton, S.; Boruﬀ, B. The inﬂuence of the neighborhood physical environment on early child health and development: A review and call for research. Health Place 2015, 33, 25–36. [CrossRef] Int. J. Environ. Res. Public Health 2019, 16, 2036 16 of 16 16 of 16 46. Barlow, S.E.; Expert Committee. Expert committee recommendations regarding the prevention, assessment, and treatment of child and adolescent overweight and obesity: Summary report. Pediatrics 2007, 120, S164–S192. [CrossRef] 7. Società Italiana di Pediatria. Società Italiana di Endocrinologia e Diabetologia; Consensus su diagnosi, trattam e prevenzione dell’obesità del bambino e dell’adolescente: Rome, Italy, 2017. 48. Pavilonis, B.T.; Lioy, P.J.; Guazzetti, S.; Bostick, B.C.; Donna, F.; Peli, M.; Zimmerman, N.J.; Bertrand, P.; Lucas, E.; Georgopoulos, P.G. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Manganese concentrations in soil and settled dust in an area with historic ferroalloy production. J. Expo. Sci. Environ. Epidemiol. 2015, 25, 443–450. [CrossRef] 49. Lucchini, R.; Placidi, D.; Cagna, G.; Fedrighi, C.; Oppini, M.; Peli, M.; Zoni, S. Manganese and developmental neurotoxicity. In Neurotoxicity of Metals; Aschner, M., Costa, L.G., Eds.; Springer International Publishing: Cham, Switzerland, 2017; pp. 13–34. 50. Katzmarzyk, P.T.; Barreira, T.V.; Broyles, S.T.; Champagne, C.M.; Chaput, J.P.; Fogelholm, M.; Hu, G.; Johnson, W.D.; Kuriyan, R.; Kurpad, A.; et al. Relationship between lifestyle behaviors and obesity in children ages 9–11: Results from a 12-country study. Obesity 2015, 23, 1696–1702. [CrossRef] 51. Li, X.M.; Ma, Y.T.; Xie, X.; Yang, Y.N.; Zheng, Y.Y. Relationship between serum creatinine and obesity in children in Xinjiang, China. Genet. Mol. Res. 2014, 13, 2409–2416. [CrossRef] 52. Protano, C.; Andreoli, R.; Manini, P.; Vitali, M. Urinary trans, trans-muconic acid and S-phenylmercapturic acid are indicative of exposure to urban benzene pollution during childhood. Sci. Total Environ. 2012, 435–436, 115–123. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4391827234	https://sciendo.com/pdf/10.2478/amns-2024-0135	English	null	The application of information fusion technology in the cultivation of talents in fine arts majors	Applied mathematics and nonlinear sciences	2,024	cc-by	10,240	Submission Info Communicated by Z. Sabir Received December 18, 2023 Accepted December 23, 2023 Available online January 31, 2024 Communicated by Z. Sabir Received December 18, 2023 Accepted December 23, 2023 Available online January 31, 2024 Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 The application of information fusion technology in the cultivation of talents in fine arts majors The application of information fusion technology in the cultivation of talents in fine arts majors Zuochuan Wang1, 2,† 1. Nanjing University of the Arts, Nanjing, Jiangsu, 210013, China. 2. Suke Jiangong, Yancheng, Jiangsu, 224000, China. 1. Nanjing University of the Arts, Nanjing, Jiangsu, 210013, China. Abstract In this paper, we use information fusion technology to describe the problem formalization of art major talent cultivation and obtain the similarity coefficients and probability matrices of art major talent cultivation by calculating the ability level of art majors. On this basis, the art major talent cultivation path is proposed from two aspects: the planning of the art major cultivation path and the recommendation of a personalized art major academic talent cultivation path. The data set is selected, the experimental environment is determined, and the data analysis software is used to analyze the example of the ant colony optimization algorithm-based talent cultivation path planning for fine arts majors and the CNN-based talent cultivation path recommendation for fine arts majors. The results show that the teaching effect of mastering knowledge points is poor, and the duration has a more obvious change (598s), which confirms that the ant colony optimization algorithm can optimize the teaching duration of video knowledge points according to the situation of talent cultivation path of fine arts majors. CNN, in solving the problem of recommending the talent cultivation path of fine arts majors, compares with the genetic algorithm GA (0.662) and the original ant colony algorithm ACO (0.665); the most personalized learning path recommended is not only more suitable for the user’s learning style, but also in line with the actual learning sequence, and thus can help users efficiently achieve their career goals. rds: Information fusion technology; CNN; Ant colony optimization algorithm; Similarity; Fine arts major 010 codes: 94A08 †Corresponding author. Email address: wdchydq@163.com ISSN 2444-8656 https://doi.org/10.2478/amns-2024-0135 © 2023 Zuochuan Wang, published by Sciendo. This work is licensed under the Creative Commons Attribution alone 4.0 License. ISSN 2444-8656 https://doi.org/10.2478/amns-2024-0135 ISSN 2444-8656 https://doi.org/10.2478/amns-2024-0135 ISSN 2444-8656 1. Nanjing University of the Arts, Nanjing, Jiangsu, 210013, China. 2. Suke Jiangong, Yancheng, Jiangsu, 224000, China. 1 Introduction Local undergraduate colleges and universities are accustomed to formulating their talent cultivation programs based on the talent cultivation mode of professional art colleges and setting up professional directions and curriculum teaching according to the idea of cultivating specialized art talents [1-2]. At present, local colleges and universities specializing in fine arts have the old talent training mode, the convergence of talent training pathways, and the fuzzy positioning of talent training goals [3-4]. The knowledge and skills learned in school do not match with the ability required by the job after work, which directly leads to the difficult employment of graduates [5]. However, the social demand for high-level specialized personnel also presents a “labor shortage”, resulting in “on the one hand, the market demand for professionals is far from being able to meet, on the other hand, college graduates face ‘hard to get a job’ the Huge pressure, society ‘labor shortage’ and college graduates ‘difficult to employ’ phenomenon coexist” [6-7]. At present, there are a series of problems in the training of talents in the fine arts program in domestic universities, resulting in the long-term development being affected, and the graduates of the fine arts program are unable to meet the status quo of social positions and talent needs [8]. Under the new situation that emphasizes “talent is competitiveness”, how colleges and universities, especially local colleges and universities, can combine the characteristics of fine arts majors and the characteristics of the institutions and construct a set of effective application-oriented talent cultivation modes is a problem that practitioners of fine arts education need to pay attention to [9-10]. Under the background of big data technology, accelerating the reform of innovative talent cultivation mode of fine arts majors, optimizing the content of talent cultivation, and perfecting the talent evaluation system, we need to respond to the social demand for the intersection of disciplines and emphasize the in-depth fusion of modern information technology, such as artificial intelligence, and fine arts majors as well as the talent cultivation mechanism guided by application-oriented talents. Literature [11] suggests that virtual continuing professional education programs are excellent learning platforms for art professionals that can provide evidence-based knowledge to a global audience and help them gain knowledge, even in a distance learning environment. Art professionals can access excellent resource tools for knowledge at the fastest speed. ISSN 2444-8656 https://doi.org/10.2478/amns-2024-0135 Zuochuan Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 2 1 Introduction Literature [12] analyzes the differences between sculpture professional artists and students in the process of sculpture art creation, aiming to understand those factors that have the greatest impact on sculpture art creation and then to derive the mechanism of influence on sculpture art creation, which is a guide for the enhancement of the students’ sculpture art creation ability. Literature [13] explains the theory of life education in the form of the professional framework of fine arts and tries to let students use the knowledge of art theory to solve social and life problems, which is conducive to student’s development of good artistic literacy and values. The aim of Literature [14] is to promote the development of contemporary art discourse by studying black women who make impassioned art statements in American museums using the direct address strategy. Literature [15] selected 16 female art therapy students to participate in eight simulated art therapy sessions and divided the 16 research subjects into two groups to study the mechanism of the art therapy program’s effect on the student’s professional development as well as their career planning by means of simulation experiments. Literature [16] distributed questionnaires to find out the similarities and connections between art therapists and psychotherapists, as well as art therapists’ views on artworks and exhibitions, and to give the reasons why it is difficult for art therapists to be accepted by people. Literature [17] collected research data through unstructured, open-ended interviews in an attempt to understand students’ perceptions of the art practice course in the Master of Arts in Art Education program at the University of Roehampton, London, as well as exploring the link between theoretical knowledge of art teaching and art practice. Literature [18] improves the creative and practical skills of art students by maintaining an active art/design practice; therefore, the art and design practice course plays a crucial role in the path of developing artists. The application of information fusion technology in the cultivation of talents in fine arts majors 3 Literature [19] explores the way painting is taught and assessed in schools in the UK by means of a survey and demonstrates that incorporating the art history elements of the college art syllabus is into art practice to further improves students’ aesthetic skills. 1 Introduction Literature [19] explores the way painting is taught and assessed in schools in the UK by means of a survey and demonstrates that incorporating the art history elements of the college art syllabus is into art practice to further improves students’ aesthetic skills. In this paper, firstly, from the perspective of information fusion technology, we formally describe the problem of art major talent cultivation in the era of information fusion technology and obtain the art major ability level, similarity coefficient and probability matrix through the calculation of art major ability level based on information fusion. Secondly, on the basis of the ability level and similarity degree of fine arts majors, the talent cultivation path of fine arts majors based on information fusion technology is proposed from the four aspects of current cognitive state annotation of fine arts majors’ learners, target learning state annotation, group learning path planning, and personalized fine arts majors’ talent cultivation path recommendation. Finally, the experimental environment and dataset are determined, and through the empirical analysis of the ant colony optimization algorithm-based talent cultivation path planning for fine arts majors, the empirical analysis of the convolution-based talent cultivation path recommendation for fine arts majors, and the correlation analysis of the talent cultivation mode for fine arts majors in the era of information fusion technology, the effective feasibility of this paper’s research results are confirmed, and it can provide guidance value for the cultivation of fine arts majors in colleges and universities in the era of information fusion. Guiding value. 2.1.2 Calculation of professional competence level in fine arts based on information fusion Aiming at a certain knowledge and corresponding skill point j Kt of students majoring in fine arts, combining multiple learning result data E and learning behavior data , the calculation formula of each sub-ability i in each professional ability  (for example, 5 represents information acquisition ability and 8 represents professional attitude) is shown in formula (1). 1 n i j ij j w B  = = (1) (1) Where, ( 1,2, , ) j w j n = is the weighting coefficient of the j nd learning outcome data je and ij B is the value of the professional competency level of the i th professional competency in Fine Arts achieved in the j th learning activity as shown in Equation (2). Where, ( 1,2, , ) j w j n = is the weighting coefficient of the j nd learning outcome data je and ij B is the value of the professional competency level of the i th professional competency in Fine Arts achieved in the j th learning activity as shown in Equation (2). ( ) , ( 1,2, , ; 1,2, , ) ij j j B F e i m j n  = = = (2) (2) where ( ) , j j F e  is a calculated function of the level of professional competence in fine arts that can be achieved in that learning activity using a single learning outcome and learning behavior data combined. 2.1.1 Formal description of the problem In the era of information integration, in order to improve the quality of training of fine arts professionals, it is necessary to abandon the previous education concept of pan-fine arts professionals and change the existing teaching methods. In the training of fine arts professionals, we need to reflect on the integration of production and education as an educational concept, in the integration of production and education concept, we need to highlight the integration of the relationship between the school and the industry, the school in the training of fine arts professionals, we need to take the industry as the object of service, for the industry to deliver the fine arts professionals it needs, and this requires the school to optimize the existing professional setup, the full use of information integration technology Understand the actual needs of the industry, on this basis to strengthen the cooperation between schools and enterprises, so that the school can carry out the combination of production, learning and research, so as to realize the synergistic cultivation of art professionals, and then make the school and the enterprise can really achieve a win-win situation. Learning outcome data for students majoring in Fine Arts in higher education is defined as   1 2 , , , n E e e e = , where, ie is the outcome of the i th learning of a certain knowledge and corresponding skill point j Kt . Learning behavior data is defined as   1 2 , , , n   = , where i is the learning behavior data of the i th learning of a certain knowledge and corresponding skill point j Kt . Student Learning Competency is defined as   1 2 , , m   = , where i is the professional competency for a knowledge and corresponding skill point j Kt . Zuochuan Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 4 2.1.3 Calculation of similarity coefficients The collegiate art studies major group is defined as   1 2 , , , n Stu s s s = , and the corresponding art major competencies are defined as   1 2 , , , n X x x x = , where, ix represents the learner is art major competency data.   1 2 , , , k   = represents the k base clusters of the n learner occupational college art major competency data set, where   1 2 , , , k j j j j n C C C = is the j th base cluster, j i C is the i th cluster in j , and j n is the number of base classes in k . The set of all clusters in the cluster integration  is denoted as   1 2 , , , cn C C C C = , where i C is the i th cluster and cn is the total number of clusters in  . The similarity between two clusters is measured by considering the intersection size and concatenation size of the clusters through Jaccard’s coefficient as shown in Equation (3). ( ) , i j i j i j C C Jaccard C C C C  =  (3) (3) where  is the intersection of two clusters,  is the concatenation of two clusters, and \| \| is the number of learners in a cluster. The Jaccard coefficient is used as a similarity measure between the clusters to construct an initial cluster similarity graph, with each cluster as a graph node, as shown in Equation (4). where  is the intersection of two clusters,  is the concatenation of two clusters, and \| \| is the number of learners in a cluster. The Jaccard coefficient is used as a similarity measure between the clusters to construct an initial cluster similarity graph, with each cluster as a graph node, as shown in Equation (4). { , }  = G V (4) (4) (4) where V  = is the set of nodes and  is the set of edges in Fig. G. The weight , i j C C V of an edge between two nodes is calculated as in equation (5). 2.1.3 Calculation of similarity coefficients where V  = is the set of nodes and  is the set of edges in Fig. G. The weight , i j C C V of an edge between two nodes is calculated as in equation (5). The application of information fusion technology in the cultivation of talents in fine arts majors 5 ( ) , ij i j e Jaccard C C = (5) (5) (5) The similarity calculation, which incorporates multidimensional information, is a dynamic process that switches from one node to one of its neighbors at each step with a certain probability, and the probabilistic transfer probability matrix  ij N N P p  = is computed in Equation (6) as follows. The similarity calculation, which incorporates multidimensional information, is a dynamic process that switches from one node to one of its neighbors at each step with a certain probability, and the probabilistic transfer probability matrix  ij N N P p  = is computed in Equation (6) as follows. , if 0, if k i ij ik ij C C e i j e p i j     =   =   (6) (6) where ij p is the random probability that the learner transitions from node i C to node j C in one step, which is proportional to the edge weights between them. Based on the single-step transfer probability matrix, we can obtain the multi-step transfer probability matrix   ( ) ( ) t t ij N N P p  = , as shown in Equation (7). ( ) ( 1) , if 1 , if 1 t t P t P P p t − =  =     (7) (7) where ( )t ij p denotes the random probability that a learner majoring in Fine Arts transitions from node i C to node j C in t steps. By capturing the multidimensional information in the graph G and using the random trajectories at different step sizes to refine the cluster similarity, the new similarity matrix between all clusters in  is shown in Equation (8). ( ) (1: ) (1: ) : : , t t ij i j Z Sim p p = (8) (8) 2.2 Talent cultivation path for fine arts majors based on information fusion technology Information fusion technology can also be called data fusion technology, and data fusion technology utilizes a variety of computer algorithms to associate, correlate and synthesize data and information from single and multiple information sources. On the basis of the ability level and similarity of the art studies major, the information fusion technology-based art studies major talent training path recommendation process is proposed as shown in Fig. 1 from the two aspects of talent training path planning for art studies major learners and personalized art studies major academic talent training path recommendation. Information fusion technology is employed to categorize major learners in the subject knowledge map based on their current cognitive state of the art. Talent cultivation path planning for fine arts majors, according to the current cognitive state of fine arts majors and the target learning state, the improved ant colony optimization algorithm is used to plan the talent cultivation path planning for fine arts majors. Personalized learning path recommendation, according to the learner characteristics, using the improved convolutional neural network to recommend personalized art major talent training paths for different art major learners to meet their needs. Zuochuan Wang. 2.2 Talent cultivation path for fine arts majors based on information fusion technology Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 6 Learner Profile Basic Characteristic Attributes Learning Behavioral Attributes Learning Outcome Attributes Learning Objectives Knowledge Learning Objectives Problem-solving objectives Competency Development Objectives Disciplinary knowledge mapping Learning path planning for group learners Group Knowledge Learning Pathway 1 3 5 7 9 10 1 2 3 4 8 10 1 2 4 6 8 10 K K K K K K K K K K K K K K K K K K → → → → → → → → → → → → → → → Group Problem Solving Pathways 1 2 3 4 5 10 1 3 4 7 0 10 1 4 7 8 9 10 Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q → → → → → → → → → → → → → → → Pathways to group capacity formation 1 2 4 7 9 10 1 3 6 8 9 10 1 3 4 6 8 10 A A A A A A A A A A A A A A A A A A → → → → → → → → → → → → → → → Current cognitive status of group students i1 i2 in o1 o2 on Hidden Layer Extrinsic Layer Cohort Student Goal Perception Status 1 2 10 1 2 10 1 2 10 K K K Q Q Q A A A → → → → → → → → → Group Knowledge Learning Pathway 1 3 5 7 9 10 1 2 3 4 5 10 1 2 4 7 9 10 K K K K K K Q Q Q Q Q Q A A A A A A → → → → → → → → → → → → → → → Figure 1. The teaching of artistic learning is the recommended process of the path recommendation Disciplinary knowledge mapping Figure 1. The teaching of artistic learning is the recommended process of the path recommendation 2.2.1 Talent cultivation path planning for fine arts majors Using the improved ant colony optimization algorithm to mine the optimal path in the candidate set of paths for talent cultivation of fine arts majors, the ant colony optimization algorithm is a heuristic search algorithm for the shortest path, and its basic idea is that ants release a special substance pheromone in the process of searching for food, and when more ants pass through a certain path, the greater the pheromone concentration of the path, and for the shorter paths, the higher the residual concentration of pheromone will be, so that there is a greater probability of attracting more ants to choose that path. For the shorter path, the higher the residual concentration of pheromone will be, so that there is a greater probability of attracting more ants to choose that path, and under the action of positive feedback mechanism, it will eventually lead to the whole ant colony finding the shortest path. The ant colony algorithm has the problems of low search efficiency, long search time, and easy to fall into the local optimum, while Dijkstra’s algorithm has a low time complexity in the case of fewer nodes, and it can search for the optimal solution quickly. For this reason, this paper adopts the improved ant colony algorithm (Dijkstra and ant colony algorithm combined) to carry out the path planning of talent cultivation for fine arts majors. 1) Dijkstra algorithm solution Numbering the entities in the set of learning paths as 1 2 3 , , e e e  gives the set of entity vertices   1 2 3 , , , E e e e = . Numbering all backward and forward relationships in the set of learning paths as 1 2 3 , , v v v  gives the set of backward and forward edges   1 2 3 , , V v v v = . Numbering the entities in the set of learning paths as 1 2 3 , , e e e  gives the set of entity vertices   1 2 3 , , , E e e e = . Numbering all backward and forward relationships in the set of learning paths as 1 2 3 , , v v v  gives the set of backward and forward edges   1 2 3 , , V v v v = . 2.2.1 Talent cultivation path planning for fine arts majors (4) Repeat steps 2 and 3 to find the shortest path length from 0v to the end point iv in set V , and record the set k E where the shortest path set kv is located. 2) Ant colony optimization algorithm fine arts professional training path planning and the maximum number of iterations and other parameters are initialized. Due to the existence of before-and-after relationship between entities, ants searching for paths will bring different time and cost overheads due to different path combinations, for this reason, this study introduces delay arrays and cost arrays. 2) Ant colony optimization algorithm fine arts professional training path planning and the maximum number of iterations and other parameters are initialized. Due to the existence of before-and-after relationship between entities, ants searching for paths will bring different time and cost overheads due to different path combinations, for this reason, this study introduces delay arrays and cost arrays. (1) Define the ant colony search node array [ ][ ] vv i j , which consists of the start point, the end point and the set k K where the set kv of shortest paths in Dijkstra’s algorithm is located. (2) Perform the ant colony search, starting from the starting point 0v , and calculate the delay array [ ][ ] delay i j and cost array [ ][ ] cost i j between the node where the ant colony is located and the reachable nodes. 2.2.1 Talent cultivation path planning for fine arts majors (3) Based on [ ][ ] delay i j and [ ][ ] cost i j , the next node of the colony is selected and the transfer probability of a single ant moving from node i to node j is calculated as shown in (9): (3) Based on [ ][ ] delay i j and [ ][ ] cost i j , the next node of the colony is selected and the transfer probability of a single ant moving from node i to node j is calculated as shown in (9): feasible domains ( ) ( ) , , ( ) ( ) ( ) , O e 0 th r ij ij k ij ij ij t t i j P t t t            =    (9) (9) Where, ( ) ij t  represents the pheromone concentration between nodes i and j on the ant colony search path, and ( ) ij t  is the heuristic information indicating the degree of transfer from node i to node j expectation. Where, ( ) ij t  represents the pheromone concentration between nodes i and j on the ant colony search path, and ( ) ij t  is the heuristic information indicating the degree of transfer from node i to node j expectation. 2.2.1 Talent cultivation path planning for fine arts majors The application of information fusion technology in the cultivation of talents in fine arts majors 7 7 7 (1) Define sets M , P ,  0 , [ ] [0][ ], 0,1, , 1 M v P i Arcs i i n = = =  −, where N is the set of vertices of the shortest path and P is the length of the shortest path from the start point 0v to the end point iv . (1) Define sets M , P ,  0 , [ ] [0][ ], 0,1, , 1 M v P i Arcs i i n = = =  −, where N is the set of vertices of the shortest path and P is the length of the shortest path from the start point 0v to the end point iv . (1) Define sets M , P ,  0 , [ ] [0][ ], 0,1, , 1 M v P i Arcs i i n = = =  −, where N is the set of vertices of the shortest path and P is the length of the shortest path from the start point 0v to the end point iv . (2) Select jv such that   [ ] min [ ]\| , 0,1, , 1 i P j P i v M N i n =  − =  −; (3) Modify the shortest path length from 0v to any node kv on set M N − , or [ ] [ ] [ ][ ] P k P j Arcs j k = + if [ ] [ ] [ ][ ] P k P j Arcs j k  + ; (3) Modify the shortest path length from 0v to any node kv on set M N − , or [ ] [ ] [ ][ ] P k P j Arcs j k = + if [ ] [ ] [ ][ ] P k P j Arcs j k  + ; (4) Repeat steps 2 and 3 to find the shortest path length from 0v to the end point iv in set V , and record the set k E where the shortest path set kv is located. 2.2.1 Talent cultivation path planning for fine arts majors (4) After each ant search, the pheromone concentration on the path needs to be updated, and the pheromone update formula is shown in (10-12): (4) After each ant search, the pheromone concentration on the path needs to be updated, and the pheromone update formula is shown in (10-12): ( 1) ( ) ( , 1) ij ij ij t t t t    + = +  + (10) 1 ( , 1) ( , 1) n k ij ij k t t t t   =  + =  +  (11) ( 1) ( ) ( , 1) ij ij ij t t t t    + = +  + (10) ( 1) ( 1) n k    ( 1) ( ) ( , 1) ij ij ij t t t t    + = +  + (10) (10) 1 ( , 1) ( , 1) n k ij ij k t t t t   =  + =  +  (11) (11) Zuochuan Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 8 8 Ants through p / , ( ) 0, Ant without assing k k ij k ij k ij F S t    =   (12) (12) Where, F denotes the pheromone intensity factor, which represents the total amount of pheromone released by an ant searching once. k S denotes the total length of the path traveled by the k rd ant during the search. (5) Repeat steps 2, 3, and 4 for the next generation of search until all ants complete the search to obtain the optimal population learning path. (5) Repeat steps 2, 3, and 4 for the next generation of search until all ants complete the search to obtain the optimal population learning path. 2.2.2 Recommendation of personalized art major talent cultivation paths The personalized art major talent training path recommendation is a learning sequence based on the analysis of learning behaviors and learning results of each art major learner, which provides learning contents and learning activities that meet their learning styles, cognitive levels, and learning styles, and is self-paced and controlled by the learners. Based on the group learning path planned above, the basic structure of the neural network for the personalized learning path is trained using an improved convolutional neural network (CNN) to generate a personalized art professional talent cultivation path [20]. 1) Input layer, the input layer in this study is the group learning path. 2) Convolutional layer, the function of the convolutional layer is to extract features from the input data, and the convolutional kernel calculation formula is shown in (13): 2) Convolutional layer, the function of the convolutional layer is to extract features from the input data, and the convolutional kernel calculation formula is shown in (13): ( ) 1 1 1 0 0 1 1 1 ( , ) ( , ) , ( , ) l l l l K f f l l k k k x y Z i j Z w i j b Z s i x s j y w x y b + + + = = =   =  +     = + + +    (13) (13) The summation part of Eq. is equivalent to solving a cross-correlation, where b is the deviation, l Z and 1 l Z + denote the convolutional inputs and outputs of layer 1 l + , ( , ) Z i j corresponds to the pixels of the feature map, K is the number of channels of the feature map, and f , 0s , and p are the parameters of the convolutional layer corresponding to the convolutional kernel size, the convolutional step size, and the number of filler layers. The summation part of Eq. 2.2.2 Recommendation of personalized art major talent cultivation paths is equivalent to solving a cross-correlation, where b is the deviation, l Z and 1 l Z + denote the convolutional inputs and outputs of layer 1 l + , ( , ) Z i j corresponds to the pixels of the feature map, K is the number of channels of the feature map, and f , 0s , and p are the parameters of the convolutional layer corresponding to the convolutional kernel size, the convolutional step size, and the number of filler layers. 3) Normalization layer, the normalization layer is after the convolutional layer, before entering the next layer, the input of the previous layer will be normalized, which can speed up the network training, effective and prevent the gradient disappearance. Its calculation formula is shown in (14-17): 3) Normalization layer, the normalization layer is after the convolutional layer, before entering the next layer, the input of the previous layer will be normalized, which can speed up the network training, effective and prevent the gradient disappearance. Its calculation formula is shown in (14-17): 1 1 n i i X n  = =  (14) ( ) 2 2 1 1 n i i X n   = = −  (15) 2 i i X Y    − = + (16) (14) (15) (16) The application of information fusion technology in the cultivation of talents in fine arts majors The application of information fusion technology in the cultivation of talents in fine arts majors The application of information fusion technology in the cultivation of talents in fine arts majors 9 i i Y Y   = + (17) (17) Where  represents the mean, X represents the original data, n represents the number of samples, 2  is the variance,  represents the constant, and  &  are the learning parameters. Where  represents the mean, X represents the original data, n represents the number of samples, 2  is the variance,  represents the constant, and  &  are the learning parameters. Where  represents the mean, X represents the original data, n represents the number of samples, 2  is the variance,  represents the constant, and  &  are the learning parameters. 2.2.2 Recommendation of personalized art major talent cultivation paths 4) Pooling layer, the function of the pooling layer in this study is to convert the results of a single point in the feature map into the statistics of its neighboring regions. In this study, Lp pooling model is used and its basic representation formula is shown in (18): ( ) 1 0 0 1 1 ( , ) , f f p p l l k k x y A i j A s i x s j y = =   = + +    (18) (18) 5) Fully-connected layer, the fully-connected layer is located in the last part of the implicit layer of the convolutional neural network and transmits signals to other fully-connected layers. 5) Fully-connected layer, the fully-connected layer is located in the last part of the implicit layer of the convolutional neural network and transmits signals to other fully-connected layers. 6) Output layer: the output layer in this study uses the classical logistic regression model to generate personalized learning paths. 6) Output layer: the output layer in this study uses the classical logistic regression model to generate personalized learning paths. 3.1.1 Experimental environment In order to facilitate the subsequent empirical analysis of the ant colony optimization algorithm-based talent cultivation path planning for fine arts majors proposed in this paper, the experiments in this paper have selected the appropriate hardware and software environments, and the experimental hardware and software environments are shown in Table 1. Table 1. Experimental hardware and hardware environment Experimental environment Parameter Operating system Windows 10 x64 Processor Intel(R) Core(TM) i7-4210U Main frequency 2.6GHz Running memory 32GB Hard disk space 512GB Solid disk Experimental platform MATLAB R2018b Visual tool Pajek32 5.09 3.1.2 Data sets The experimental dataset of this paper comes from a variety of behavioral data generated in the talent cultivation process of fine arts majors at Shanghai Art College. The selected course is “Career Planning for Fine Arts Majors”, which contains 200 video knowledge points, and the experiments are selected from the fine arts majors in 2019 intermediate, 2020 junior, and 2021 senior level, which covers the learner’s learning behavioral log information on each video knowledge point, interaction behavior log information and test information. Zuochuan Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 10 3.1.3 Results of Talent Cultivation Path Planning for Fine Arts Majors According to the level of competence and similarity of the art studies major that integrates multiple factors obtained above, the time series data generated during the learning process of the beginner in 2020, the intermediate in 2019 and the advanced in 2021 are analyzed by using the ant colony optimization algorithm to solve the degree of mastery of the knowledge points, the degree of interaction of the knowledge points, the degree of achievement of the conceptual interactions, and the teaching effect of the learners of different levels. Figure 2 shows the optimization results of the talent cultivation path of fine arts majors in 2020, Figure 3 shows the optimization results of the talent cultivation path of fine arts majors in 2021, and Figure 4 shows the optimization results of the talent cultivation path of fine arts majors in 2019. The knowledge node color is related to the number of times learners learn in the planning of the talent cultivation path for fine arts majors, with purple knowledge nodes indicating one occurrence in the planned talent cultivation path for fine arts majors, orange knowledge nodes indicating two occurrences, and blue knowledge nodes indicating three and more occurrences. The paths planned for beginner learners are relatively shorter and simpler; due to the lower cognitive level of this type of learner, each knowledge point may take longer to master, and learning simpler paths in the same learning time is more conducive to improving the conceptual interaction of knowledge points reached by learners of this level. The paths planned for advanced learners are longer and more complex, and this type of learner may be able to master a knowledge point in a relatively short period, but the learners’ conceptual interaction attainment of the knowledge point decreases over time, so they need to constantly review the relevant knowledge points they have learned before to strengthen their understanding of the current learning knowledge point, which is conducive to improving the quality and level of talent cultivation of the university’s fine arts program. 3.1.3 Results of Talent Cultivation Path Planning for Fine Arts Majors In 2021, the optimization result of the training path of the professional talents 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 60 59 58 57 56 55 54 53 52 51 50 49 48 47 46 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 125 124 123 122 121 120 119 93 91 81 80 79 78 77 76 126 127 128 129 130 114 115 116 117 118 105 106 107 82 83 100 99 98 97 96 95 94 92 90 89 88 87 86 85 84 101 102 103 104 108 109 110 111 112 113 131 132 133 134 135 150 149 148 147 146 145 144 143 142 141 140 139 138 137 136 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 180 179 178 177 176 175 174 173 172 171 170 169 168 167 166 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 207 206 205 204 203 202 201 200 199 198 197 196 Figure 3. In 2021, the optimization result of the training path of the professional talents Figure 3. In 2021, the optimization result of the training path of the professional talents Figure 3. 3.1.3 Results of Talent Cultivation Path Planning for Fine Arts Majors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 60 59 58 57 56 55 54 53 52 51 50 49 48 47 46 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 125 124 123 122 121 120 119 93 91 81 80 79 78 77 76 126 127 128 129 130 114 115 116 117 118 105 106 107 82 83 100 99 98 97 96 95 94 92 90 89 88 87 86 85 84 101 102 103 104 108 109 110 111 112 113 131 132 133 134 135 150 149 148 147 146 145 144 143 142 141 140 139 138 137 136 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 180 179 178 177 176 175 174 173 172 171 170 169 168 167 166 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 207 206 205 204 203 202 201 200 199 198 197 196 Figure 2. The optimization result of the training path of the professional talents of art science in 2020 Figure 2. The optimization result of the training path of the professional talents of art science in 2020 Figure 2. 3.1.3 Results of Talent Cultivation Path Planning for Fine Arts Majors In 2021, the optimization result of the training path of the professional talents 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 44 43 42 41 40 39 36 37 36 35 34 33 18 17 16 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 74 73 72 71 70 69 68 67 66 65 64 63 62 61 60 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 107 106 105 104 103 102 101 100 99 96 97 93 92 91 90 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 178 176 175 174 173 172 171 170 129 128 127 126 125 124 123 179 180 181 182 183 184 185 186 187 188 189 190 191 19 20 94 96 95 32 31 30 29 28 27 26 25 24 23 22 21 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 159 158 157 156 155 154 153 152 151 150 149 148 147 146 145 160 161 162 163 164 165 166 167 168 169 177 192 193 194 195 207 206 205 204 203 202 201 200 199 198 197 196 Figure 4. In 2019, the optimization result of the professional talents training path is optimized Figure 4. In 2019, the optimization result of the professional talents training path is optimized 3.1.3 Results of Talent Cultivation Path Planning for Fine Arts Majors The optimization result of the training path of the professional talents of art science in 2020 The application of information fusion technology in the cultivation of talents in fine arts majors 11 11 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 60 59 58 57 56 55 54 53 52 51 50 49 48 47 46 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 125 124 123 122 121 120 119 93 91 81 80 79 78 77 76 126 127 128 129 130 114 115 116 117 118 105 106 107 82 83 100 99 98 97 96 95 94 92 90 89 88 87 86 85 84 101 102 103 104 108 109 110 111 112 113 131 132 133 134 135 150 149 148 147 146 145 144 143 142 141 140 139 138 137 136 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 180 179 178 177 176 175 174 173 172 171 170 169 168 167 166 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 207 206 205 204 203 202 201 200 199 198 197 196 Figure 3. 3.1.4 Results of the optimization of teaching hours in the talent cultivation path of fine arts majors The data on teaching hours generated in the process of cultivating talents in art studies for three categories of students, namely, beginners in 2020, intermediate in 2019 and advanced in 2021, were analyzed, and the allocation of hours was adjusted based on the teaching effect of the learners and used to optimize the design of the teaching process, while the teaching hours remained unchanged. When the teaching effect of the knowledge point is poor, a longer teaching time is planned for the learner to give the learner sufficient time to understand and master the knowledge point, and when the teaching effect of the knowledge point is good, it means that the learner understands and applies Zuochuan Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 12 the knowledge point better, and the teaching time can be reduced appropriately. The ant colony optimization algorithm is used to optimize teaching duration, and the comparison between the optimized duration and the original duration for each level is depicted in Fig. 5, where (a), (b), and (c) is the comparison of the duration of the beginner learner, the comparison of the duration of the intermediate learner and the comparison of the duration of the advanced learner, respectively. The teaching of mastering the knowledge point is less effective, and there is a more significant change in the length of time (598s). The teaching duration for the same knowledge point after optimizing the 3 levels is evidently different, with 3430s, 2481s, and 2358s, respectively. It confirms that the ACO algorithm can optimize the teaching duration of video knowledge points according to the situation of the talent cultivation path of fine arts majors in order to meet the learning needs of the learners, improve the learning efficiency of the learners, and provide a basis for teachers to carry out the design of the teaching duration of the knowledge point, and to improve the Fine arts professional talent cultivation efficiency. (a) Primary learner time contrast (b) Intermediate learners’ time contrast (c) Senior learner time contrast (a) Primary learner time contrast (a) Primary learner time contrast (b) Intermediate learners’ time contrast (c) Senior learner time contrast Figure 5. The comparison of the length of the teaching and the original length of the class 3.2.1 Feasibility Analysis of Talent Training Path Recommendation In order to verify the feasibility of CNN-based talent cultivation path recommendation for fine arts majors, this paper looks at the two aspects of user satisfaction and the degree of matching between the path and the user’s learning style. In this experiment, the initial population number is set to be 5, the number of nodes is set to be 10, and the maximum number of iterations is set to be 50. Taking The application of information fusion technology in the cultivation of talents in fine arts majors 13 13 user 2 as an example, whose learning style is (-3, 1, -5, 7), the CNN-based fine arts majors proposed by this paper Talent cultivation path recommended in this paper after 50 iterations. The similarity between the talent cultivation path for fine arts majors and the learning style of user 2 is shown in Table 2, and the experimental results of the three methods for four types of users when the number of iterations is from 1 to 50 are shown in Fig. 6, in which (a)~(d) are users 1-4, respectively. It can be clearly seen that the three methods will continuously derive learning paths with higher user satisfaction with the increase in the number of iterations and will be gradually stabilized after reaching a certain height. Gradually stabilizes. It can be seen that all three methods are capable of planning a talent cultivation path for users who specialize in fine arts with different learning styles. However, from the experimental results of all four users, it can be seen that through 50 iterations, the matching degree (0.75) of the CNN-based art professional talent cultivation path recommendation method proposed in this paper is better than the other two (0.703), (0.687), that is, the CNN-based art professional talent cultivation path method proposed in this paper can obtain users more in line with the user’s 2 learning style. The CNN-based art major talent cultivation path recommendation research plays an important auxiliary function in art major talent cultivation so that students in the art major category can carry out independent learning and online learning. 3.2.1 Feasibility Analysis of Talent Training Path Recommendation Furthermore, online assessment can be carried out with the help of computers to comprehensively improve the quality of talent cultivation of fine arts majors, give full play to the advantages of the application of information fusion technology in teaching and learning, and provide students of fine arts majors with a more flexible, interactive and open learning mode. Table 2. The professional talent training path is similar to the learning style of student 2 Method The art students cultivate the path nodes Matching degree 1 2 3 4 5 6 7 8 9 10 This method 0.73 0.76 0.79 0.81 0.78 0.73 0.75 0.71 0.73 0.71 0.75 GA 0.77 0.65 0.64 0.55 0.71 0.68 0.75 0.81 0.78 0.69 0.703 ACO 0.73 0.65 0.72 0.81 0.83 0.63 0.65 0.56 0.61 0.68 0.687 (a) User 1 :(1,-1,0,-3) (b) User 2 :(-3,1,-5,7) (a) User 1 :(1,-1,0,-3) (b) User 2 :(-3,1,-5,7) Zuochuan Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 14 (c) User 1 :(7,9,-11,-9) (d) User 2 :(-11,-3,7,0) Figure 6. The number of iterations from 1to 50 is the result of four kinds of users (c) User 1 :(7,9,-11,-9) (d) User 2 :(-11,-3,7,0) Figure 6. The number of iterations from 1to 50 is the result of four kinds of users 3.2.2 Effectiveness Analysis of Talent Cultivation Path Recommendation Conduct experiments based on 760 candidate learning materials in the computer field, 450 candidate learning materials in the art field, and all 1210 candidate learning materials, respectively. Set the initial population number as 5 and the number of nodes as 10, and take user 2 as an example to plan the optimal talent cultivation path for the art major through 1-50 iterations. The matching degree of learning paths and user learning styles with different numbers of candidate resources is shown in Table 3, and the experimental results of the three methods for user 2 at three candidate learning resources are shown in Fig. 7, in which (a)~(c) the candidate learning materials are 450, 760, and 1,210, respectively, and even in the case of the different numbers of candidate learning resources, the CNN-based talent cultivation path recommendation of the fine arts majors proposed in this paper can be optimized after After 50 iterations, it is able to effectively construct a talent cultivation path for users that meets their learning styles and actual learning sequences and is able to obtain a higher user similarity than the GA and ACO algorithms. In order to further verify that the cultivation path derived from CNN is more suitable for the user’s learning style than GA and ACO algorithms, this paper adopts the Euclidean-based similarity calculation method to calculate the similarity between the optimal learning paths derived from the three methods and the user’s learning style under different candidate resources. It can be seen that the CNN proposed in this paper can bring greater user satisfaction to users with different learning styles than the genetic algorithm GA (0.662) and the original ant colony algorithm ACO (0.665) in solving the problem of recommending paths for cultivating professionals in the field of fine arts, and the most personalized learning paths recommended in this paper not only suit the learning styles of the users better but also conform to the actual learning sequence, which can help users realize their learning styles efficiently. The most personalized learning paths recommended are not only more suitable for users’ learning styles but also conform to the actual learning sequence, which can help users realize their career goals efficiently. We will continue to explore the training programs for art majors that can meet the requirements of industrial development and continuously deliver high-quality, applied and innovative art majors to society. 3.2.2 Effectiveness Analysis of Talent Cultivation Path Recommendation The application of information fusion technology in the cultivation of talents in fine arts majors 15 15 Table 3. The number of different candidate resources is matched by the user learning style N Method The art students cultivate the path nodes Matching degree 1 2 3 4 5 6 7 8 9 10 760 This method 0.71 0.88 0.71 0.83 0.81 0.55 0.56 0.72 0.82 0.61 0.72 GA 0.61 0.62 0.71 0.77 0.74 0.55 0.58 0.62 0.73 0.69 0.662 ACO 0.64 0.66 0.77 0.61 0.74 0.55 0.68 0.73 0.65 0.62 0.665 450 This method 0.83 0.83 0.87 0.85 0.81 0.71 0.55 0.54 0.57 0.81 0.737 GA 0.88 0.52 0.73 0.71 0.66 0.89 0.67 0.64 0.66 0.47 0.683 ACO 0.66 0.65 0.62 0.75 0.68 0.75 0.68 0.64 0.85 0.51 0.679 (a) 450 (b) 760 (c) 1210 Figure 7. Three methods of candidate learning resources are the results of the experiment of user 2 (a) 450 (c) 1210 (b) 760 ure 7. Three methods of candidate learning resources are the results of the experiment of user 2 3 Analysis of the Relevance of Talent Cultivation Mode for Fine Arts Majors Pearson correlation coefficient analysis is used to study the correlation between overall satisfaction and six items, namely, personnel training objectives, curriculum system, faculty, teaching system, training evaluation, and implicit curriculum construction, Pearson correlation coefficient is used to indicate the degree of correlation between the variables, which can present the degree of correlation and the direction of the effect between the variables. When the absolute value of the correlation coefficient is larger, the higher the degree of correlation between the variables, and the positive and negative signs indicate whether the direction of change between the variables is the same. A correlation coefficient greater than 0.7 indicates that the variables are strongly correlated, a correlation coefficient between 0.3 and 0.7 indicates that the variables are moderately correlated, and a correlation coefficient less than 0.3 indicates that the variables are weakly correlated. The results of the correlation analysis of the talent cultivation mode of fine arts majors in the era of information integration are shown in Figure 8. The correlation coefficient between the overall satisfaction of the talent cultivation mode of fine arts majors in the era of information fusion and the talent cultivation goal is 0.752, so there is a significant positive correlation between the overall satisfaction and the talent cultivation goal. The application of information fusion technology in the cultivation of fine arts majors can improve the students’ internationalized fine arts professional level while providing endogenous power for the development of fine arts in colleges and universities. Zuochuan Wang. Applied Mathematics and Nonlinear Sciences, 9(1) (2024) 1-17 16 Figure 8. Analysis of the relationship between the training mode of the art and the training of the art Figure 8. Analysis of the relationship between the training mode of the art and the training of the art Figure 8. Analysis of the relationship between the training mode of the art and the training of the art References [1] Dikiy, Igor, S., Dikaya, Liudmila, & A., et al. (2016). Neurophysiological correlates of artistic creativity of art representatives with different professional levels. International Journal of Psychophysiology. [2] Webb, A. K., & Fitzjohn, J. (2016). Poetry, music, writing and painting; developing the artistic talents of adults with cystic fibrosis - sciencedirect. Paediatric Respiratory Reviews, 17, 39-41. [3] Mariana, M., Sattin, Vickitoriana, K.,. A., & Silva, et al. (2018). Use of a garment as an alternative to body painting in equine musculoskeletal anatomy teaching. Journal of Veterinary Medical Education, 45(1), 119-125. [4] Finn, G. M., & White, P. M. (2012). The role of colour in knowledge retention: a body‐painting study. The FASEB Journal, 26(S1), 529.5-529.5. [5] Kong, X. (2014). Xu beihong’s artistic influence on current university education teaching of chinese painting art. International Journal of Technology, Management(011), 000. [6] Sui, G. (2018). Reverse ekphrasis: teaching poetry-inspired chinese brush painting workshops - in english. International Journal of Art & Design Education, 38. [7] Elkoshi, R. (2019). Tone painting and painting tones: a follow-up study of listeners’ audiovisual responses to beethoven’sthunder storm:. International Journal of Music Education, 37(3), 476-492. [8] Tara, & Chittenden. (2013). Body-building: a female student’s use of the transitional spaces of a painting degree course to explore her sexual desirability and aesthetics as a ‘grotesque’ female body. International Journal of Art & Design Education, 32(1), 55-67. [9] Lohr, V. I. (2013). Diversity in landscape plantings: broader understanding and more teaching needed. Horttechnology, 23(1), 126-129. [10] You, J. (2021). Retraction note to: distribution of earthquake activity in mountain area based on big data and teaching of landscape design. Arabian Journal of Geosciences, 14(22), 1-1. [11] Nijs, M., Morroll, D., Lynch, C., Levett, S., Fleming, S., & Chin, R., et al. (2021). P–786 virtual continual professional education programs in art in time of sars-cov–2: do they deliver?. Human Reproduction (Supplement_1), Supplement_1. [12] Puppe, L., Jossberger, H., & Gruber, H. (2020). Creation processes of professional artists and art students in sculpting. Empirical Studies of the Arts(11), 027623742094271. [13] Meyer, M., & Wood, L. (2020). My living theory of a professional framework for art education. Action Research(4), 147675032097101. [14] Williams, S. S. (2016). Speaking Out of Turn: Race, Gender, and Direct Address in American Art Museums. (Doctoral dissertation, University of Southern California.). [15] Eyal-Cohen, D., Regev, D., Snir, S., & Bat-Or, M. (2020). 4 Conclusion In the era of information fusion, it is increasingly difficult to meet the requirements of the development of the past talent training requirements of fine arts majors, and this paper makes full use of information fusion technology to integrate the on-campus and off-campus faculty and training path. On the basis of information fusion technology, the proposed training path of fine arts majors based on E-GPPE-C model is designed to make fine arts majors always meet the requirements of industrial development in training. The following conclusions are drawn: 1) The teaching duration after optimization of the three levels of primary study, intermediate and advanced, is obviously different, respectively 33430s, 2481s and 2358s, which confirms that the ACO optimization algorithm can optimize the teaching duration of video knowledge points according to the situation of the cultivation path of fine arts majors, and comprehensively improve the quality of the cultivation of fine arts majors in colleges and universities, so as to make the cultivation of fine arts majors can be truly in line with the industry. 2) Through 50 iterations, the matching degree (0.75) of the CNN-based talent cultivation path recommendation method for art majors proposed in this paper is better than the other two (0.703) and (0.687). It can bring greater user satisfaction to users with different learning styles, and the most personalized learning path recommended is not only more suitable for the user’s learning style but also has a very important impact on promoting the cultivation of talents in fine arts majors, promoting the increase of employment rate, and stabilizing social relations. 3) The correlation coefficient between the overall satisfaction of the training mode of fine arts majors in the era of information fusion and the talent cultivation goal is 0.752, which is statistically significant at the level of 0.01. Therefore, there is a significant positive correlation between overall satisfaction and the talent cultivation goal. Making use of the advantages of information fusion technology in education and learning and providing Fine Arts students with a more flexible, interactive, and open approach to talent development. The application of information fusion technology in the cultivation of talents in fine arts majors 17 17 References Developing the professional identity of art therapy students as reflected in art therapy simulation sessions. The Arts in Psychotherapy, 71(3), 101706. [16] Jue, & Juliet. (2017). The extent of engagement in art making and exhibition by art therapy practitioners and students. Arts in Psychotherapy, 55, 32-39. [17] Hall, J. (2010). Making art, teaching art, learning art: exploring the concept of the artist teacher. International Journal of Art & Design Education, 29(2), 103-110. [18] Budge, & Kylie. (2016). Learning to be: the modelling of art and design practice in university art and design teaching. International Journal of Art & Design Education, 35(2), 243-258. [19] Owen, C. (2020). Through a glass darkly: the teaching and assessment of drawing skills in the uk post‐16 art & design curriculum. International Journal of Art & Design Education, 39(2). [20] Saravanapriya, M., Senthilkumar, R., & Saktheeswaran, J. (2022). Multi-label convolution neural network for personalized news recommendation based on social media mining. Journal of Scientific & Industrial Research.
https://openalex.org/W2119069124	https://sajhivmed.org.za/index.php/hivmed/article/download/108/173	English	null	From the Executive	Southern African journal of HIV medicine	2,012	cc-by	339	F Conradie President F Conradie President Southern African HIV Clinicians Society Johannesburg fconradie@witshealth.co.za President Southern African HIV Clinicians Society Johannesburg fconradie@witshealth.co.za MESSAGE MESSAGE MESSAGE MESSAGE MESSAGE From the Executive M F 'The times they are a-changing.' The South African National AIDS Council (SANAC) reconvened at an impressive inaugural meeting in Pietermaritzburg on 4 October 2012, attended by the Minister of Health, Dr Aaron Motsaeledi, and Deputy President Kgalema Motlanthe. SANAC announced that the incidence of mother-to-child transmission of HIV was down and life expectancy was up – all very good news. However, in my opinion, the most important development is that fixed- dose combinations are now going to be part of the national tender. While I think that this will improve adherence, more importantly, we will not be faced with stock-out of a single drug. The impact of Register for the conference as soon as you can. The line-up of speakers is impressive, and we have very interesting debates planned. Skills building will be provided in many areas, and conference attendees will have the chance to rub shoulders with the academics. More than that, the conference will present an opportunity to meet a number of brave and hardworking healthcare providers from the region. I encourage you to share your experiences and learn. patients having received two out of three of their drugs will be felt in the years to come. The Southern African HIV Clinicians Society has been invited to be a plenary member of SANAC, so we will continue our role as the voice of reason to the Department of Health, to ensure that the goals of the National Strategic Plan are achieved. This is not the time to let up in any way – an AIDS-free generation is within our grasp. Lastly, if you have not yet made travel arrangements to attend the conference of the Southern African HIV Clinicians Society in Cape Town from 25 to 28 November 2012, you should get onto it as soon as possible. 160 SAJHIVMED November 2012, Vol. 13 No. 4
W4361025176.txt	https://www.degruyter.com/document/doi/10.1515/9783839462225-016/pdf	de		Das Dölitzer Schlösschen	transcript Verlag eBooks	2,023	cc-by-sa	5,144	Das Dölitzer Schlösschen Geschichte und Narrativ Georg-Felix Sedlmeyer Etwa fünf Kilometer südlich des Leipziger Stadtzentrums liegt die ehemalige Schlossanlage im Stadtteil Dölitz-Dösen. Das einstige Rittergut, vom Flüsschen Mühlpleiße umflossen, lässt sich bis ins 13. Jahrhundert zurückverfolgen. Es erfuhr einen Ausbau im Stil der Renaissance,1 erlitt eine Beschädigung im Dreißigjährigen Krieg, wurde wieder instandgesetzt und bei einem Besitzübergang 1636 zur vierflügeligen Anlage ausgebaut.2 Am 16. und 18. Oktober 1813 wurde die Schlossanlage Schauplatz von Kampf‌handlungen der Völkerschlacht bei Leipzig und erlangte damit eine weitere geschichtliche Auf‌ladung.3 Die Stadt erwarb die Schlossanlage 1927 und richtete dort ab 1931 eine Freiluftschulungsstätte ein.4 So galt das Schloss früher bzw. gilt sein Ort heute als Erinnerungsstätte des Dreißigjährigen Kriegs und der Völkerschlacht bei Leipzig.5 Spricht man heute vom Dölitzer Schlösschen, so sind im Wesentlichen nur dessen verbliebene Bauteile in Form eines Torhauses und einer Gartenanlage gemeint. Das Hauptgebäude ist in den Jahren von 1944 bis 1947 beschädigt, geplündert und abgebrochen worden. Die Ereignisse sind unterschiedlich überliefert. In dem vom Krieg verschonten separaten Torhaus 6 wird über die Baugeschichte des Anwesens informiert: »In der Nacht vom 20. zum 21. Februar 1944 wird das Schloss durch eine Fliegerbombe schwer beschädigt und nach einem tödlichen Unfall 1947 gesprengt.« 7 Ähnlich knapp, sogar widersprüchlich geben andere 1 So die Angaben auf der Informationstafel im Torhaus Dölitz. 2 Cornelius Gurlitt: Amthauptmannschaft Leipzig (Leipzig Land). Beschreibende Darstellung der älteren Bau- und Kunstdenkmäler des Königreichs Sachsen. Bd. 16. Dresden: Meinhold 1894, S. 11–12. 3 Gurlitt 1894 (wie Anm. 2), S. 12. 4 Die Freiluftschulungsstätte wurde für den planmäßigen Volksschulunterricht während der Sommermonate genutzt. Stadtarchiv Leipzig (StadtAL): Kapitelakten (Kap.) 19, Nr. 155 Bh. 3. Bd. 4: Fragebogen des Landesdenkmalpflegers in Sachsen vom 28. September 1943. 5 Siehe weiter unten und vgl. Anm. 1. 6 Das Torhaus wurde ab den 1950er Jahren saniert und beherbergt heute ein Zinnfigurenmuseum. Vgl. Horst Immisch: Dölitz. Zur Geschichte eines Leipziger Vorortes. Leipzig: Pro Leipzig 2002, S. 31–38. 7 Vgl. Immisch 2002 (wie Anm. 6). 228 Georg-F elix Sedlmeyer Autoren das Ende der Baugeschichte wieder.8 Der Lokalhistoriker Horst Immisch berichtete 2002 etwas ausführlicher, indem er auf fehlende Baustoffe zur Instandsetzung, den witterungsbedingten Verfall und die Plünderung von Brennmaterialien durch die Bevölkerung hinwies.9 Das vorhandene Narrativ zum Verlust des Bauwerks ist damit kurzgehalten und lässt viel Raum für eine individuelle und freie Interpretation, obwohl es ein wichtiger Teil der regionalen Architekturgeschichte ist.10 Doch wer verhandelte und entschied aus welchen Gründen über die Zukunftsfähigkeit des Objekts? Wer plädierte nun für die Sprengung des Schlosses und warum, wer erkannte welche Werte in der Ruine und wer trug letzten Endes die Entscheidungsgewalt? Darüber hinaus kann gefragt werden, ob die Objektgeschichte mit dessen Abbruch tatsächlich endet oder ob durch die Zuweisung von Erinnerungswerten die Schlossgeschichte fortgeschrieben werden sollte und muss – auch weil lokale Aktivistinnen gegenwärtig an eine Rekonstruktion denken. Spätestens die Schrift des Architekturhistorikers Erwin Schleichs zur »zweite[n] Zerstörung Münchens« von 1978 machte auf den Verlust von Baudenkmälern nach dem Zweiten Weltkrieg und im Kontext des Wiederauf‌baus aufmerksam.11 Die Bauhistoriker Hartwig Beseler und Niels Gutschow zeigten 1988 die Dimensionen der Verluste an westdeutschen Baudenkmälern auf. Auch der Abbruch von rettbaren Schlossbauten wie etwa das Braunschweiger Residenzschloss wurden thematisiert.12 Im selben Rahmen wies der Politikwissenschaftler Klaus von Beyme 1988 ebenfalls auf die Tragweite von »Vorentscheidungen durch Ruinenpolitik« hin, die vielerlei bauliche Verluste einleiten konnte.13 An anderen Stellen wurde belegt, dass man Kriegsbeschädigungen als Chance zur Umgestaltung ganzer Quartiere sah, wobei mancherorts kulturgeschichtlich wertvolle Bausubstanz bereitwillig einer neuen Planung geopfert wurde.14 Dazu muss angemerkt werden, dass Schlossbauten, meist aufgrund von Größe und Lage, hohe Ansprüche an Nutzungskonzepte stellen. Für das Dölitzer Schlösschen 8 Vgl. Immisch, 2002 (wie Anm. 6), S. 31–38; Götz Eckardt, Horst Drescher, Ludwig Deiters: Schicksale deutscher Baudenkmale im Zweiten Weltkrieg. Eine Dokumentation der Schäden und Totalverluste auf dem Gebiet der neuen Bundesländer. Bd. 2: Bezirke Halle, Leipzig, Dresden, Karl-Marx-Stadt (Chemnitz), Erfurt, Gera, Suhl. Wiesbaden: Panorama-Verlag 2000; vgl. auch Wikipedia: Torhaus Dölitz, https://de.wikipedia.org/wiki/Torhaus_Dölitz (aufgerufen am 16.05.2022). 9 Immisch 2002 (wie Anm. 6). 10 Vgl. Andrew Herscher: Violence Taking Place. T‌he Architecture of the Kosovo Conflict. Stanford: Stanford University Press 2010. Herscher macht darauf aufmerksam, dass das existenzielle Ende von Bauwerken Teil ihrer Geschichte ist. 11 Erwin Schleich: Die zweite Zerstörung Münchens. Neue Schriftenreihe des Stadtarchivs München. Bd. 100. Stuttgart: Steinkopf 1978. 12 Hartwig Beseler, Niels Gutschow: Kriegsschicksale Deutscher Architektur. 2 Bde. Neumünster: Wachholtz 1988, S. XVII. 13 Klaus von Beyme: Der Wiederauf‌bau. Architektur und Städtebaupolitik in beiden deutschen Staaten. München: Piper 1987, S.101–110. 14 Jörn Düwel, Niels Gutschow, Freie Akademie der Künste (Hg.): A Blessing in Disguise. War and Town Planning in Europe, 1940–1945. Berlin: DOM Publishers 2013. Das Dölitzer Schlösschen. Geschichte und Narrativ schienen zudem Abbrüche bei Beschädigungen aus ideologischen Gründen denkbar, da man vergangenen Herrschaftsstrukturen kein neues Denkmal setzen wollte.15 All dies trifft hier aber nicht zu, wie im Folgenden noch belegt wird. Im Fall Dölitz wird wie an vielen anderen Orten auch ausgespart, warum und wie es zur Niederlegung und nicht zu einer Sicherung und Instandsetzung eines (lokal-) geschichtlich bedeutsamen Bauwerks kam. Handlungsabläufe zum materiellen Ende von Bauwerken wurden bislang noch zu wenig beschrieben; das gilt insbesondere für die zeitlich kurzen Phasen nach Kriegsbeschädigungen und vor Wiederauf‌bauausführungen am Ende des Zweiten Weltkriegs. Dieser Beitrag möchte einen Lückenschluss für die hier angesprochene Zeitspanne versuchen, um am konkreten Beispiel anhand von Schadenskarten, -listen und Korrespondenzen unterschiedlicher beteiligter Ämter die variierenden Wahrnehmungen des beschädigten Schlosses zwischen Staat, Stadt und Bürgerinnen nachzuzeichnen. Die Verhandlungen über und der Umgang mit dem Schloss können als beispielhaft für die Schicksale zahlreicher kulturhistorisch wichtiger Bauten im Kontext des Zweiten Weltkriegs gesehen werden. Die Schadensfeststellungen Für einen Überblick zur historischen Situation und für einen Zugang zu einstigen Bewertungs- und Verhandlungsdiskursen dienen als Ausgangspunkt damalige Schadensbewertungen und Schadenserfassungen. Aus den Akten geht dazu hervor, dass der allgemein für Leipzig als verheerend geltende Luftangriff am 4. Dezember 1943 das Stadtplanungsamt veranlasste, bis Anfang Januar 1944 eine Prüfung des aktuellen Zustands aller Denkmäler der Stadt vorzunehmen, die nach dem Ortsgesetz über die Pflege des Stadtbilds von 1929 gelistet waren. Das Dölitzer Schloss wurde hierin schon genannt und dementsprechend geprüft.16 Es wurde als »nicht betroffen« verzeichnet.17 Ein handschriftlich hinzugefügtes »T« weist aber auf »total zerstört« bis spätestens zum 24. Juni 1946 hin.18 In einem Nachtrag zur Liste über den Zustand der Denkmäler lassen sich Schäden an weiteren Bauten zum Fliegerangriff vom 20. Februar 1944 festmachen. Hier erscheint das Schloss nun mit der wenig aussagekräftigen Beschreibung: »teils total zerstört, teils in verschiedenem Grade beschädigt«.19 Beigefügte Wertungen, die auf eine Instandsetzungs- oder Abbruchmaßnahme abhängig 15 Vgl. Beseler, Gutschow 1988, S. XVII (wie Anm. 12). 16 StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4: Schreiben des Stadtplanungsamtes an den Landesdenkmalpfleger vom 8. Januar 1944. 17 StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4, Anlage A. 18 StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4, Vermerk vom 24. Juni 1946. Gemeint ist der Zeitpunkt der Erfassung, welcher den Bauzustand beschreibt. Neben Kriegsbeschädigungen bis Kriegsende können hier auch Abbrüche, Einsturz oder Rückbau nach Kriegsende gemeint sein. 19 StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4, Addendum zu Anlage A, Schäden beim Fliegerangriff vom 20. Februar 1944. 229 230 Georg-F elix Sedlmeyer vom ermittelten Schaden hindeuten, wurden nicht gefunden. Damit hatte das Stadtplanungsamt den Schaden vorerst neutral zur Kenntnis genommen. Im Aktenverkehr 20 zur Leipziger Denkmalpflege findet sich eine undatierte Fotografie, die einen bildlichen Eindruck von dem Zustand im Jahr 1944 vermittelt (Abb. 1).21 Der Leipziger Stadtbaurat Baumeister kommentierte dazu, er habe die fotografische Aufnahme nach dem 20. Februar 1944 anfertigen lassen und der Bombeneinschlag sei von der Wasserseite aus erfolgt: »Die Vorderseite mit dem Eingang und dem Vorhofe sowie die Seite nach dem Parke zu bleiben im [W]esentlichen unbeschädigt.« 22 Die Ruine ist demnach als Schlossbau noch deutlich zu erkennen. Dennoch erscheinen dem Foto nach Material- und Personalaufwand für eine statische und substanzielle Sicherung des Baukörpers erheblich, insbesondere in Anbetracht der Kriegsbedingungen. Eine weitere Notiz zur Lädierung des Bauwerks findet sich in zwei verschiedenen Schadenskarten. Es handelt sich an erster Stelle um einen Organisationsplan der Luftschutzstellen (LS-Stellen) und Luftschutzreviere (Abb. 2), der auch Schadensstellen nach unterschiedlichen Merkmalen verzeichnet. An zweiter Stelle wird eine Schadenskarte des LS-Reviers 11 c zum Abschnitt Dölitz-Dösen (Abb. 3) wiedergegeben, welche Kriegseinwirkungen in drei verschiedenen Schadensgraden inklusive eines Datums markiert. Anhand von beiden Karten lässt sich die Einwirkung am Schloss der Kategorie »schwerer Schaden« zuordnen.23 Eine weiterführende Wertung der Beeinträchtigung hinsichtlich eines darauf‌folgenden Umgangs mit dem Bauwerk lassen die Karten alleine nicht zu. Die vorangegangene Schadensdokumentation vor Ort hatten sogenannte Revierarchitekten im Einvernehmen mit dem Stadtplanungsamt und LS-Stellen übernommen.24 Unklar muss an diesem Punkt bleiben, nach welchen Maßstäben oder unter welchem Blickwinkel die kriegsbeschädigten Bauten betrachtet wurden. Denkbar wäre eine Schadensbewertung unter dem Gesichtspunkt des voraussichtlichen Aufwands einer etwaigen Instandsetzung. Weitere Schadenskarten, die den Stadtteil Dölitz abbilden, sind im Auf‌trag von Albert Speers »Arbeitsstab für Wiederauf‌bauplanung« gezeichnet worden. Hier wurden reichsweit Schadenskarten mit dem Stand zum 1. Mai 1944 erstellt, 20 StadtAL, Stadtverordnetenversammlung und des Rats der Stadt Leipzig (StVuR) 15408: Schreiben der Abteilung der Sofortmaßnahmen vom 30. Januar 1945. Eine Aktenablage zum Schloss beim Stadtplanungsamt soll bei dem Luftangriff am 6. April 1945 verloren gegangen sein. Dies geht aus einer handschriftlichen Notiz auf dem Schreiben hervor. Eine Recherche zu den Bauakten des Schlosses fand nicht statt. 21 StadtAL, StVuR 15408: Aktenvermerk mit Abbildung des Schloss Dölitz ohne Datum. 22 StadtAL, StVuR 15408: Auszug aus Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. 23 StadtAL, StVuR 13486: Legende auf Planblatt 11 a. 24 Die Revierarchitekten waren in Leipzig über die Polizei- und LS-Stellen organisiert; insgesamt gab es 29 LS-Reviere mit Unterteilungen für das Stadtgebiet Leipzig. Der Stadtteil Dölitz-Dösen befand sich im LS-Revier 11 c. Der Revierarchitekt stellte die durch »Feindeinwirkung« entstandenen Schäden an Baulichkeiten fest und arbeitete zur Begrenzung von Folgeschäden mit der »Abteilung für Sofortmaßnahmen« zusammen. Vgl. Deutsche Fotothek, DF_DK_0006495: Übersichtskarte der LS-Reviere in Leipzig, nach 1943. Das Dölitzer Schlösschen. Geschichte und Narrativ Abb. 1: Schloss Dölitz nach der Beschädigung, Foto: 1944. so auch für Leipzig.25 Diese Karten wurden bei städtischen Ämtern vorgezeichnet und anschließend durch den Arbeitsstab vereinheitlicht.26 Der bauliche Verlust am Dölitzer Schlösschen war zum Zeitpunkt der Kartenerstellung schon eingetreten und spätestens ab dem 14. April 1944 der Stadtverwaltung bekannt.27 Jedoch wurde die Kriegseinwirkung am Schloss nicht in diese Karten aufgenommen, obwohl sie schwere bis totale Schäden verzeichneten. Dieses Fehlen kann 25 Zu Leipzig finden sich zwei Karten in den Carleton University Archives, Ottawa (German destruction maps, Leipzig); ausführlicher dazu Werner Durth: Der programmierte Auf‌bau: Speers ›Arbeitsstab zum Wiederauf‌bau bombenzerstörter Städte‹. In: Bauwelt 48 (1984), S. 378–390. 26 Für Augsburg beschrieben bei Georg-Felix Sedlmeyer: Augsburg. Die Funktion der Kriegsschadenserfassung in der Wiederauf‌bauplanung. In: Lisa Marie Selitz, Sophie Stackmann (Hg.): Wertzuschreibungen und Planungslogiken in historischen Stadträumen. Neue Beiträge zur städtebaulichen Denkmalpflege. Bamberg: University of Bamberg Press 2018, S. 13–53, hier S. 27–29. 27 Vgl. StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4: Schreiben des Vermietungsamtes 5. Bezirk an das Stadtplanungsamt vom 14. April 1944. 231 232 Georg-F elix Sedlmeyer Abb. 2: Kartenausschnitt, Ortsteil Dölitz, Organisationsplan der LS-Stellen Leipzig mit Schadenseintragungen nach 1943. Abb. 3: Kartenausschnitt, Plan 11c des Revierarchitekten, Schadens dokumentation 29. März 1944. Das Dölitzer Schlösschen. Geschichte und Narrativ Abb. 4: Kartenausschnitt, Schadenskarte des ›Arbeitsstabes für Wiederauf‌bau planung‹ mit Stand zum 1. Mai 1944. damit erklärt werden, dass sich der Arbeitsstab lediglich für stadtplanerische Belange interessierte.28 Da in der Anlage weder Wohnungen verloren gegangen waren noch städtebauliche Veränderungen erstrebenswert schienen, war das Schloss städtebaulich nicht von Interesse. Die vorweg unternommene Schadensdokumentation wurde von dem Tief‌bauamt 29 für weiterführende Zwecke neu bewertet, das dem Dölitzer Schlösschen keine städtebauliche Relevanz attestierte. Für die Forschung zu Schadenskarten, insbesondere zum Kartenbestand des Arbeitsstabs, ließe sich hieraus ableiten, dass die Angaben der Karten des Arbeitsstabs keine absolute Übersicht über alle tatsächlich entstandenen Schäden oder Schadensstellen der Stadt wiedergeben, sondern subjektiv nur für den Stab relevante Inhalte anzeigen (Abb. 4). 28 Uta Hohn: Die Zerstörung deutscher Städte im Zweiten Weltkrieg. Regionale Unterschiede in der Bilanz der Wohnungstotalschäden und Folgen des Luftkrieges unter bevölkerungsgeographischem Aspekt. Dortmund: Dortmunder Vertrieb für Bau- und Planungsliteratur 1991, S. 36. 29 Vgl. Kartensignatur, Carleton University Archives (German destruction maps, Leipzig). 233 234 Georg-F elix Sedlmeyer Unterschiedliche Interessen: Die Verhandlungen um das beschädigte Schloss Bewertung in Abhängigkeit zur Nutzung: Vermietungsamt und Stadtplanungsamt Amtsinterne Korrespondenzen geben eine Diskussion zu erkennen, welche die Zerstörung des Schlosses nachzeichnen und nachfolgend unterschiedliche Bewertungen der Ruine aufzeigen. Das städtische Vermietungsamt erhielt als Eigentümer des Schlosses 30 unmittelbar nach dem Schadensfall einen Zustandsbericht vom Stadtplanungsamt einschließlich eines Hinweises auf dessen Bedeutung als Baudenkmal.31 In einer Rückantwort vom 14. April 1944 lässt sich darin erstmals eine wertende Beschreibung des Schadens ausmachen: Bei dem Fliegerangriff am 20.2.44 ist am Schloß Dölitz etwa 8 m hinter der Rückfront eine große Sprengbombe niedergegangen, die das Mauerwerk der Umfassungswände an der Südwestecke weggerissen und alles übrige Mauerwerk, Zwischenwände, Fußböden, [ . . . ] usw. stark beschädigt hat. Ein Brand ist nicht ausgebrochen. Wiederauf‌bau ist sehr fraglich. Der Revierarchitekt hat hiervon Kenntnis. Das Gebäude steht unter Heimatschutz und ich bitte um Kenntnisn[ahme] und weitere Veranlassung.32 Obwohl das Schloss noch Ende 1943 als »kriegswichtig« 33 eingestuft worden war, wurde durch das Vermietungsamt die Option einer Instandsetzung und somit der Substanzsicherung erstmals infrage gestellt. Das Amt betrachtete – so lässt sich vermuten – die Liegenschaft aus einer wirtschaftlich-rationalen Perspektive hinsichtlich der Nutzung. Im Kontext des Kriegs und der damit zusammenhängenden wirtschaftlichen Situation scheint man Nutzen oder Bedeutung des Baus gegen Arbeitsaufwand und Kosten abzuwägen, wobei es als für eigene Zwecke nicht mehr instandsetzungsfähig eingeschätzt wird. Das Vermietungsamt scheint sich dahingehend mit einer fachlichen Expertise – nachstehend zitiert – beim Stadtplanungsamt über eine weitere Behandlung der Liegenschaft absichern zu wollen. Das Stadtplanungsamt konstatierte dazu am 19. April 1944: Da die Bombe im Park hinter dem Schlosse einschlug, ist der Hauptbau an der Westseite durch Luftdruckwirkung zu 2/3 total und zu 1/3 schwer beschädigt worden. Soweit noch etwas vom Mauerwerk bestehen blieb, ist es samt den Gewölben 30 StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4: Fragebogen des Landesdenkmalpflegers in Sachsen vom 28. September 1943. 31 Vgl. StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. Der Zustandsbericht konnte nicht ermittelt werden. 32 StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4: Schreiben des Vermietungsamtes 5. Bezirk an das Stadtplanungsamt vom 14. April 1944. 33 Nutzung durch die Nationalsozialistische Volkswohlfahrt; StadtAL: Kap. 19, Nr. 155 Bh. 3. Bd. 4: Fragebogen des Landesdenkmalpflegers in Sachsen vom 28. September 1943. Antwortschreiben des Stadtplanungsamtes auf der Rückseite vom 6. Oktober 1943. Das Dölitzer Schlösschen. Geschichte und Narrativ und Decken so gesprengt und gerissen, daß nur ein völliger Neubau in Frage käme. Das Giebeldach einschl. des Turmes ist zwar stark beschädigt, aber in der Gesamtform noch erhalten. Der südliche Anbau ist auch total zerstört. Dagegen haben die beiden nach Osten gerichteten Seitenflügel und die Vorhalle samt dem Innenhof mit den Holzgalerien weniger gelitten. Im Ganzen gesehen sind diese Schäden viel zu umfangreich, um sie in den Rahmen der Sofortmaßnahmen bringen zu können.34 Im Schreiben des Vermietungsamts vom 14. April lassen sich Zweifel an einer Sicherung für eine Weiternutzung des Schlosses erkennen, welche das Stadtplanungsamt am 19. April bestätigte. Es beschrieb die Schäden als so schwerwiegend, dass nur ein Neubau sinnvoll wäre. Die Bewertung vom 19. April war an der vorhergehenden Nutzung orientiert; dies wird deutlich, da auf die historische Bedeutung der Anlage nicht weiter eingegangen und zusätzlich auf ältere bauliche Mängel verwiesen wurde: Auch müßte mit einer Wiederinstandsetzung des Schlosses über die Behebung der Fliegerschäden hinaus eine gründliche Sanierung des ganzen Gebäudes verbunden werden. Vor allem müßten die Fundamente und Keller gegen die aus den Pleiße-Armen aufsteigende Feuchtigkeit isoliert werden, um die Räume in allen Geschossen für die Schule voll ausnutzbar zu machen. Aus diesen Gründen bleibt z. Zt. nichts übrig, als das Schloß im jetzigen Zustand zu belassen und die Frage des Wiederauf‌baues und der Instandsetzung bis nach Beendigung des Krieges zurückzustellen.35 Positive Wertungen hinsichtlich einer Bauwerkssicherung erfolgten nicht. Maßnahmen zur Notsicherung wurden – im Gegenteil – sogar als unrealistisch abgetan, was auf die kriegswirtschaftlichen Bedingungen zurückgeführt werden darf. Eine Ablehnung von Sicherungsmaßnahmen bedingte allerdings einen weiteren Verfall der noch jungen Ruine durch Witterungseinflüsse. Dieser Tatsache dürfte sich das Stadtplanungsamt bewusst gewesen sein; doch zu diesem Zeitpunkt wird keine Nutzungsperspektive bestanden haben. Weder vom Stadtplanungsamt noch vom Vermietungsamt wurde der Ruine eine Zukunftsfähigkeit zugestanden. Für den Eigentümer wäre ein Abbruch angesichts der Hinweise des Stadtplanungsamts nun eine wirtschaftlich-logische Konsequenz gewesen. Das Stadtplanungsamt hingegen sprach sich dafür aus, die Entscheidung erst nach Kriegsende zu treffen. Vermutlich erhoffte man für die Nachkriegszeit zumindest, bessere wirtschaftliche oder zumindest klarere Bedingungen hinsichtlich eines Wiederauf‌baus, einer neuen Nutzung oder einer endgültigen Zweckbestimmung vorzufinden. 34 StadtAL, Kap. 19, Nr. 155 Bh. 3. Bd. 4: Antwortschreiben des Stadtplanungsamtes vom 19.04.1944. Der Begriff »Sofortmaßnahmen« bezieht sich auf bauliche Maßnahmen zu Notsicherung und für provisorische Nutzung, ausgeführt durch Bautrupps der Abteilung für Sofortmaßnahmen. 35 StadtAL, Kap. 19, Nr. 155 Bh. 3. Bd. 4: Antwortschreiben des Stadtplanungsamtes vom 19.04.1944. 235 236 Georg-F elix Sedlmeyer Neubewertung als Erinnerungsstätte: Stadtplanungsamt und Abteilung für Sofortmaßnahmen In der zuvor angesprochenen Vertagung der Entscheidung über den weiteren Umgang kann auch der Wunsch nach Erhaltung des Bauwerks erkannt werden, eben wegen der vorher unausgesprochenen kulturgeschichtlichen Bedeutung des Orts. Es sei an dieser Stelle darauf hingewiesen, dass rechtlich die Möglichkeit bestanden hätte, Sicherungen einzuleiten. Die Reichsgesetzgebung billigte »Maßnahmen zur Beseitigung von Bombenschäden« sowie »Sicherungsarbeiten an beschädigten Baudenkmälern«.36 Demnach konnten substanzielle Sicherungen eingeleitet werden, »wenn von dem zuständigen amtlichen Denkmalpfleger das Vorhaben und sein Umfang als unabweisbar dringlich bescheinigt« würde.37 Eine solche Bescheinigung geht jedoch aus den Leipziger Akten nicht hervor. Selbst wenn eine Sicherung beantragt worden wäre, hätte das Problem eines Mangels an Baumaterialien und Arbeitskräften fortbestanden. Für die Kriegszeit lassen sich dementsprechend keine Sicherungsarbeiten belegen, womit einem weiteren Verfall Vorschub geleistet wurde. Der geschichtlichen Bedeutung der Schlossanlage schien man sich im Stadtplanungsamt stets bewusst gewesen zu sein. Ein Schreiben des Amts, hier in der Funktion der Bauberatungsstelle, vom 30. Januar 1945 an die Leitung der Abteilung für Sofortmaßnahmen belegt dies anschaulich: Wie ich von Anfang an dargelegt habe, muß das Schloß nämlich in seiner ganzen historischen Anlage als Erinnerungsstück an die Völkerschlacht erhalten bleiben, soweit es möglich ist. Wenn die Schloßanlage leider nicht mehr völlig wiederhergestellt werden kann, so muß man sich eben mit einer teilweise [sic!] Ruine zufrieden geben, wie ja auch viele andere historische Denkmäler nur in mehr oder weniger zerstörtem Zustand bestehen. Zur Sicherung der Ruine wie der erhaltenen Gebäudeteile werden Baustoffe vom alten Bestand (Dachziegel und Mauersteine alten Formates, [ . . . ] usw.) von hohem Wert sein.38 Gegenüber der Leitung der Abteilung für Sofortmaßnahmen wurde knapp ein Jahr später (und im Gegensatz zum Briefwechsel mit dem Vermietungsamt) die Wertigkeit der Schlossanlage als Denkmal mit der Erinnerungsfunktion an einen Kriegsschauplatz hervorgehoben. Ob hier die damalige Kriegspropaganda einbezogen wurde, bliebe an anderer Stelle noch zu erörtern. Die Würdigung des Bauwerks als Zeugnis der Spätrenaissance tritt nicht auf. Auch die vorherige Nutzung als Freiluftschulungsstätte bzw. die erst kurz zuvor als »kriegswichtig« attestierte Nutzung scheinen keinen Einfluss zu nehmen. In der Korrespondenz ging es offensichtlich nicht um die Bewertung im Sinne einer ehemaligen Nutzung, sondern um den tatsächlichen Substanzerhalt für den Zweck 36 Runderlass des Reichsministeriums des Innern vom 18. November 1942. 37 Runderlass 1942 (wie Anm. 36), Abs. 6. 38 StadtAL, StVuR 15408: Schreiben des Stadtplanungsamts an die Sofortmaßnahme, Abt. Lenkung vom 30. Januar 1945. Das Dölitzer Schlösschen. Geschichte und Narrativ einer zukünftigen Nutzung als Erinnerungsstätte – wie oben erwähnt auch als »teilweise Ruine«. Das Stadtplanungsamt versuchte damit, der Abteilung für Sofortmaßnahmen zu verdeutlichen, wie wichtig es sei, die noch vorhandene Bausubstanz weitestmöglich am Ort zu erhalten. Das schien notwendig, da die Abteilung für Sofortmaßnahmen Baumaterialien von Schadensstellen entfernen lassen durfte, um sie an anderen Baustellen einzusetzen.39 Die Forderung nach Substanzerhalt wurde auch weiterhin unterstrichen, da in diesem Schreiben nun doch eine spätere Instandsetzung erwogen wurde. Für Anfang 1945 lässt sich konstatieren, dass das Stadtplanungsamt eine Bedeutsamkeit der Ruine als mögliche Erinnerungsstätte erkannt hatte und nun den Schutz der verbliebenen Baustrukturen forderte. Dieser Forderung nach Erhaltung sollten aber andere Interessen entgegengestellt werden – nämlich von privater Seite. In demselben Schreiben an die Abteilung für Sofortmaßnahmen wurde beschrieben, inwieweit die in der Ruine vorhandenen Baumaterialien doch für Zwecke Dritter abgegeben werden können: Gegen die Überlassung der Heizkörper und -rohre sowie des Küchenherdes aus den zerstörten Räumen des Schlosses an den Bewerber bestehen keine Bedenken. Das Aufräumen der Schadensstelle von Einsturzmaterialien ist aber nur soweit unbedenklich, als das Material später nicht zur Instandsetzung des Schlosses selbst gebraucht wird. [ . . . ] Auf keinen Fall darf das Schloß, wie es der Bewerber beabsichtigt, auf Abbruch vergeben werden. Ich bitte, bei der Dölitzer Schloßanlage die spätere Erhaltung, wenn auch als teilweise Ruine, im Auge zu behalten. Soweit Baumaterialien und Bauteile entnommen sind, die wir für eine spätere Konservierungsarbeit selbst benötigen, sind sie für uns sicherzustellen.40 Aus diesem Schriftverkehr geht hervor, dass auch die Interessen von Privatpersonen mit in die Verhandlungen um die baulichen Strukturen und deren Erhalt eingingen. Offenbar lag eine private Anfrage an die Abteilung für Sofortmaßnahmen zur Nutzung vorhandener Baumaterialien vor. Das Gesuch des »Bewerbers« lag weder in der Nutzung noch in der Wiederinstandsetzung der Schlossanlage; zudem wurden an dieser Stelle keine Erinnerungswerte als Argument angeführt, sondern lediglich das reine Interesse an vorhandenem Baumaterial. Angenommen werden darf, dass der »Bewerber« sich in einer Notlage befand und versuchte, sein eigenes beschädigtes Anwesen wiederaufzubauen oder instand zu setzen. Er forderte aus einem banalen, aber verständlichen Grund den Abbruch der Schlossruine, dem vonseiten des Stadtplanungsamts aber entschieden widersprochen wurde.41 So fand am 3. Februar 1945 eine Ortsbegehung des Schlosses statt, an der Stadtbauräte, Vertreter des Vermietungsamts und der Revierarchitekt teilnahmen. Konsens unter den Teilnehmenden der Ortsbegehung 39 Runderlass des RMI vom 18. November 1942; Runderlass des GB-Bau vom 16. September 1943, Nr. GB 8544/43 VIII. 40 StadtAL, StVuR 15408: Schreiben des Stadtplanungsamtes an die Sofortmaßnahme, Abt. Lenkung vom 30. Januar 1945. 41 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. 237 238 Georg-F elix Sedlmeyer war die Erhaltung der Schlossanlage, die Sicherstellung von wertvollen oder historischen Baumaterialien sowie weitere Entscheidungen nach Kriegsende. Zu einer beabsichtigten Beratung mit dem sächsischen Landesdenkmalpfleger in Dresden ist es vor Kriegsende nicht mehr gekommen.42 Später wurde zu der Ortsbegehung am 3. Februar 1945 notiert, dass der »Bewerber« um die Baumaterialien, »der es nicht unterließ, das Schloß weiterhin für seine eigenen Bauten in der Gärtnerei auszubeuten, strengste Anweisung [erhielt], eine weitere Ausplünderung zu unterlassen.« 43 Die Entfernung von Baumaterialien aus der Ruine wurde mit dem Erkennen einer zukünftigen Nutzungsmöglichkeit untersagt, daher wurde die Erhaltung der Schlossruine gefordert. Wie ferner aus den beiden zitierten Schreiben des Stadtplanungsamts hervorgeht, bewertete das Amt die Zukunftsfähigkeiten der Schlossanlage auf zwei Ebenen. Die erste Ebene bezog sich im April 1944 auf die Nutzung der Anlage in ihrer bisherigen Form vor der Beschädigung. Dabei ist anzunehmen, dass das Amt die Ruine unter wirtschaftlichen Gesichtspunkten zur Wiederinstandsetzung betrachtete und den Bau gegenüber dem Eigentümer mehr oder weniger aufgab. Die Aussicht auf Weiternutzung wäre nur mit einem Abbruch und einem darauf‌folgenden Neubau erstrebenswert gewesen. Auf einer zweiten Ebene (mit dem Erkennen einer möglichen zukünftigen und geänderten Nutzung Anfang 1945) bewertete das Stadtplanungsamt die Ruine hinsichtlich ihrer kulturellen Bedeutung neu. Hierbei wurde der verbliebenen Baustruktur eine Zukunftsfähigkeit eben als Erinnerungsstätte und auch als teilweise Ruine zugesprochen. Es wurde gegenüber der Abteilung für Sofortmaßnahmen und dem privaten Bewerber dementsprechend eine Konservierung gefordert, »soweit es möglich ist«.44 Die Bausubstanz dürfe nur vom Ort entfernt werden, wenn sie keine historische oder bautechnische Wertigkeit besaß. Bei den beiden unterschiedlichen Stellungnahmen des Stadtplanungsamts soll nun auf den zeitlichen Abstand eingegangen werden. Im Jahr 1944 stand gegenüber dem Vermietungsamt die frühere Nutzung des Objekts im Vordergrund, welche für die Zukunft nicht mehr erkannt wurde; im Jahr 1945 schien das Stadtplanungsamt in dem Schloss eine Perspektive als Erinnerungsstätte zu sehen, welche sich vorher noch nicht belegen lässt. Vermutlich unterstützte der durch den Kriegsverlauf weiter fortschreitende Verlust an Kulturgütern diese unterschiedliche Wertung. Die Option zur Sicherung der Ruine als Erinnerungsstätte schien das Stadtplanungsamt mit Blick auf den Zeitpunkt der Luftkriegsbeschädigung damit erst spät anzugehen. 42 StadtAL, StVuR 15408: Aktennotiz zum Schreiben des Stadtplanungsamtes an die Sofortmaßnahme, Abt. Lenkung vom 30. Januar 1945. 43 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. 44 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. Das Dölitzer Schlösschen. Geschichte und Narrativ Weitere Zerstörung und eine erneute Bewertung: Bürger, Hochbauamt und Stadtplanungsamt Weitere Akten zum Fall sind erst nach Kriegsende wieder auszumachen. Am 26. Juni 1945 wurde mit einer Bitte um den gegenwärtigen Sachstand der Fall des Dölitzer Schlösschens erneut aufgegriffen. Unmittelbar darauf, am 5. Juli 1945, erfolgte nochmals eine Ortsbegehung mit Vertretern des Stadtrats, des Stadtbaurats, unter anderem mit dem für den Leipziger Wiederauf‌bau bedeutsamen Stadtbaurat Walther Beyer, des stadtgeschichtlichen Museums, der Baupolizei, des Kulturamts, des Schulamts und dem Revierarchitekten. Die Vielzahl der anwesenden Amtsmänner zeigt bereits, welche Bedeutung der Schlossruine innerhalb der Stadtverwaltung beigemessen wurde. Der ebenfalls anwesende Stadtbaurat Baumeister legte im Nachhinein dar, welche Werte das Stadtplanungsamt in der Schlossruine erkannt hatte: Ich habe sofort dem Vermietungsamt einen Bericht von dem Zustand gegeben mit dem Hinweis, daß die ganze Gebäudeanlage als Baudenkmal des 16. und 17. Jahrhunderts und Erinnerungsstück an die Völkerschlacht von 1813 und auch an diesen Krieg, wenn auch als teilweise Ruine erhalten bleiben müsse. Ich habe mich auch mit dem Rev. Architekten [ . . . ] ins Benehmen gesetzt und ihm ausführlich dargelegt, daß zur Sicherung des Bestandes später die alten Baumaterialien und Bauteile dringend benötigt würden [ . . . ].45 Nach Kriegsende wurden von dem Stadtbaurat die Denkmalwerte der Schlossanlage aufgezählt, die nun dringlich für eine Erhaltung – auch als Ruine – gesprochen hätten. Der Stadtbaurat plädierte somit für die Zukunftsfähigkeit der verbliebenen Bausubstanz und fügte im Nachgang sogar eine weitere Bedeutungsebene als Erinnerungsort für den soeben erst beendeten Krieg hinzu. Es zeigt sich damit, wer die Deutungshoheit über die Zukunft der Ruine beanspruchte. Dass diese aber nicht in den Händen der zuvor genannten Amtsmänner lag, belegt der weitere Teil des Protokolls: Mehrere Bürger*innen des Ortsteils wären demnach für die »völlige Zerstörung und Plünderung« verantwortlich zu machen.46 Sie hätten der Ruine Baumaterialien entnommen, wobei ein Mann von herabstürzenden Trümmern erschlagen wurde. Ein polizeiliches Eingreifen sei aber abgelehnt worden, da zu diesem Zeitpunkt amerikanische Truppen die Stadt eingenommen hätten. Der bereits genannte »Bewerber« gab an, »er habe nicht widerrechtlich gehandelt, denn er habe vom städt. Hochbauamt die schriftliche Ermächtigung erhalten, außer anderem 15.000 Mauersteine aus dem Schloßbau für seine Zwecke herauszuholen«.47 Das Protokoll kommentiert dazu: 45 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. 46 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. 47 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. 239 240 Georg-F elix Sedlmeyer Abb. 5: Schloss Dölitz nach der Ausbeutung von Baumaterialien, Foto: 1945. Es erregte von allen Teilnehmern der Ortsbesichtigung Verwunderung, daß eine Abteilung der Stadtverwaltung, obwohl ihr bekannt sein mußte, daß das Schloß unbedingt zu erhalten sei, die Genehmigung erteilte, eine so beträchtliche Anzahl von Mauersteinen aus dem Schloß herauszunehmen. [ . . . ] Jedenfalls muß nach übereinstimmender Ansicht aller Teilnehmer an der Ortsbesichtigung das Schloß als Baudenkmal nunmehr aufgegeben werden und nur die Eingangspartie [ . . . ] wird von dem einstigen Herrenhause zeugen.48 Nach der Ortsbegehung im Juli 1945 wurde eine weitere Fotografie angefertigt. Das Schloss zeigt sich in sichtlich zurückgebauter Form, wobei die Baustoffentnahme zum Einsturz des Dachs führte.49 Die erwogene Instandsetzung oder Erhaltung als teilweise Ruine im Sinne eines Erinnerungsorts wurde nach der letzten Begehung verworfen. Somit findet sich hier die Erklärung, wieso der Schlossbau in der zuvor genannten Liste über den Zustand der Baudenkmäler im Nachhinein als totalbeschädigt nachmarkiert wurde (Abb. 5). 48 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. 49 StadtAL, StVuR 15408: Protokoll der Ortsbegehung am Schloss Dölitz vom 6. Juli 1945. Das Dölitzer Schlösschen. Geschichte und Narrativ Einordnung Die Stadtverwaltung war mit mehreren Ämtern der Bauverwaltung mit dem Objekt betraut. Nutzungen, Werte, aber auch Schäden waren ihr bekannt gewesen. Es lässt sich festhalten, dass für das erste Jahr nach der Beschädigung des Schlosses keine Erhaltungsforderung aufgestellt wurde, was wahrscheinlich in der kriegswirtschaftlichen Mangelsituation begründet war. Erst mit der auf‌kommenden Perspektive zur Nutzung der Ruine als Erinnerungsstätte versuchte das Stadtplanungsamt, diese aktiv zu schützen. Es erwog gar eine Wiederinstandsetzung des Schlosses, obwohl sich die schwierige wirtschaftliche Situation bis unmittelbar nach Kriegsende kaum verändert haben dürfte. Es darf angenommen werden, dass die bis dahin weiter fortgeschrittenen Verluste an Kulturgütern die städtischen Organe zu einer vehementeren Schutzforderung bewogen. Dennoch bleibt fraglich, warum das Leipziger Hochbauamt entgegen bestehender städtischer Schutzforderungen dem »Bewerber« Baumaterialien in einer derartigen Höhe zur Entnahme freigab, welche die schwerwiegende Beschädigung des Objekts erst hervorrief. Schlussendlich bediente sich die kriegsleidende Bevölkerung an den Baustoffen. Einen Schutz der Ruine konnten oder wollten die exekutiven Organe des zusammenbrechenden NS-Staats nicht mehr leisten. Dabei wäre interessant zu wissen, ob eine zügige Instandsetzung als Erinnerungsstätte, zumindest aber eine provisorische Sicherung, eine Plünderung der Baumaterialien seitens der Bevölkerung verhindert hätte. Insgesamt wäre ein Schloss als mahnende Kriegsruine eine Seltenheit gewesen, sind es doch wiederholt innerstädtische Kirchen, denen diese Funktion zugesprochen wurde. Die Diskussionen um den Wiederauf‌bau oder den Abbruch von Schlössern nach 1945 sind bereits als widersprüchlich für innerstädtische Prestigebauten bekannt.50 So sollte mit diesem Beitrag ein weiterer Einblick in die Debatte gegeben werden, da diese noch nicht abgeschlossen scheint, wenn auch bis heute noch neue Nutzungen für bereits abgegangene Schlösser gesucht werden. Die Ereignisse um das Dölitzer Schlösschen zum Kriegsende dürften symptomatisch für vielerlei weniger beachtete, aber kulturgeschichtlich wertvolle Bauten sein. Das Ausplündern einer Ruine nach Baumaterialien ist dabei kein singuläres Phänomen unterlassener Absperrarbeiten, sondern wurde auch institutionell zur Baumaterialgewinnung betrieben. Die Weiterverwendung von Baumaterial – vor allem aus Burgen, Schlössern oder Herrschaftshäusern – ist historisch gesehen kein Novum. Die Weiterverwendung von Trümmern oder Trümmermaterial ist bekannt, insgesamt aber für den Zweiten Weltkrieg noch recht wenig erforscht. In diesem Kontext bleiben vielfach auch die Diskussionen um den Umgang mit Ruinen unbekannt. 50 Vgl. Beseler, Gutschow 1988, S. XVIII–XIX (wie Anm. 12). 241 242 Georg-F elix Sedlmeyer Abstract T‌he Manor House of Dölitz History and Narrative In the final phase of the Second World War, and before the first measures of reconstruction, the future viability of war-related ruins was negotiated all across Germany. T‌his was also the case for the Dölitzer Schlösschen (the manor house of Dölitz) near Leipzig. Depending on the divergent cultural and material values of those involved, former structures were to be either rebuilt, repaired, and reinterpreted, or abandoned and demolished. Damage maps and lists, as well as correspondence, reveal who had what particular interest in the ruins. T‌he documents show hitherto unknown decisions, actors, and valorisations in a short but significant period of time that decisively shaped Dölitzer Schlösschen’s further history.
https://openalex.org/W2081978128	https://www.app.pan.pl/article/item/article/item/app20080057.html?pdf=39	English	null	A New Genus of Lungfish from the Givetian (Middle Devonian) of Central Australia	Acta Palaeontologica Polonica	2,009	cc-by	358	A new genus of lungfish from the Givetian (Middle Devonian) of central Australia Alice M. Clement Acta Palaeontologica Polonica 54 (4), 2009: 615-626 doi: http://dx.doi.org/10.4202/app.2008.0057 Alice M. Clement Acta Palaeontologica Polonica 54 (4), 2009: 615-626 doi: http://dx.doi.org/10.4202 Acta Palaeontologica Polonica 54 (4), 2009: 615-626 doi: http://dx.doi.org/10.4202/app.2008.0057 A new Dipterus−like lungfish, Harajicadipterus youngi, is described from the Givetian (Middle Devonian) Harajica Sandstone Member of central Australia. The material is comprised of five specimens representing the skull roof, orbital bones, tooth plates, operculo−gular bones, a partial pectoral girdle, centra and scales. Harajicadipterus can be distinguished from other dipnoans by its long postorbital cheek, broad B bone, lack of contact between E and C bones, and radiating tooth rows with some denticles evident between the rows. Results of a cladistic analysis of 81 characters for 33 dipnoan taxa resolved Harajicadipterus below the holodontid clade but as more derived than Dipterus and the chirodipterid clade. words: Dipnoi, phylogeny, Devonian, Givetian, Harajica Formation, Australia. Key words: Dipnoi, phylogeny, Devonian, Givetian, Harajica Formation, Australia. Alice M. Clement [aclement@museum.vic.gov.au], Museum Victoria, PO Box 666, Melbourne, Victoria, 3001, Australia; Research School of Earth Sciences, Australian National University, Canberra, Australian Capital Territory, 0200, Australia. Alice M. Clement [aclement@museum.vic.gov.au], Museum Victoria, PO Box 666, Melbourne, Victoria, 3001, Australia; Research School of Earth Sciences, Australian National University, Canberra, Australian Capital Territory, 0200, Australia. This is an open-access article distributed under the terms of the Creative Commons This is an open-access article distributed under the terms of the Creative Commons Attribution License (for details please see creativecommons.org), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This is an open access article distributed under the terms of the Creative Commons Attribution License (for details please see creativecommons.org), which permits unrest distribution, and reproduction in any medium, provided the original author and source a ion License (for details please see creativecommons.org), which permits unrestricted us i d d i i di id d h i i l h d di tion, and reproduction in any medium, provided the original author and source are credit Full text (1,142.2 kB) Full text (1,142.2 kB)
https://openalex.org/W2900292961	https://europepmc.org/articles/pmc6221338?pdf=render	English	null	Lack of serological and molecular evidence of arbovirus infections in bats from Brazil	PloS one	2,018	cc-by	6,262	RESEARCH ARTICLE Editor: Naomi Forrester, Keele University Faculty of Natural Sciences, UNITED KINGDOM Copyright: © 2018 Bittar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was financially supported by FAPESP (São Paulo Research Foundation - http:// www.fapesp.br/) grant number 2015/09704-6, received by RRGM and by CNPq (National Council for Scientific and Technological Development - http://cnpq.br/) grant number 165802/2015-4, received by CB. Lack of serological and molecular evidence of arbovirus infections in bats from Brazil Cı´ntia Bittar1☯, Rafael R. G. MachadoID1☯, Manuela T. Comelis2, Larissa M. Bueno2, Eliana Morielle-Versute2, Matheus R. Beguelini2,3, Renato P. de Souza4, Maurı´cio L. Nogueira5, Paula Rahal1 Cı´ntia Bittar1☯, Rafael R. G. MachadoID1☯, Manuela T. Comelis2, Larissa M. Bueno2, Eliana Morielle-Versute2, Matheus R. Beguelini2,3, Renato P. de Souza4, Maurı´cio L. Nogueira5, Paula Rahal1 1 Laborato´rio de Estudos Genoˆmicos, Instituto de Biociências, Letras e Ciências Exatas, UNESP— Universidade Estadual Paulista “Ju´lio de Mesquita Filho”, São Jose´ do Rio Preto, São Paulo, Brazil, 2 Laborato´rio de Chiroptera, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Ju´lio de Mesquita Filho”, São Jose´ do Rio Preto, São Paulo, Brazil, 3 Centro das Ciências Biolo´gicas e da Sau´de, UFOB—Universidade Federal do Oeste da Bahia, Barreiras, Bahia, Brazil, 4 Nu´cleo de Doenc¸as de Transmissão Vetorial, Instituto Adolfo Lutz, São Paulo, Brazil, 5 Laborato´rio de Pesquisa em Virologia, FAMERP-Faculdade de Medicina de São Jose´ do Rio Preto, São Jose´ do Rio Preto, São Paulo, Brazil a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. * rafarahalmachado@gmail.com ☯These authors contributed equally to this work. * rafarahalmachado@gmail.com Abstract OPEN ACCESS Citation: Bittar C, Machado RRG, Comelis MT, Bueno LM, Morielle-Versute E, Beguelini MR, et al. (2018) Lack of serological and molecular evidence of arbovirus infections in bats from Brazil. PLoS ONE 13(11): e0207010. https://doi.org/10.1371/ journal.pone.0207010 OPEN ACCESS Viruses are important agents of emerging zoonoses and are a substantial public health issue. Among emerging viruses, an important group are arboviruses, which are character- ized by being maintained in nature in cycles involving hematophagous arthropod vectors and a wide range of vertebrate hosts. Recently, bats have received increasing attention as an important source for the emergence of zoonoses and as possible viral reservoirs. Among the arboviruses, there are many representatives of the genera Flavivirus and Alphavirus, which are responsible for important epidemics such as Dengue virus, Zika virus and Chikun- gunya virus. Due to the importance of analyzing potential viral reservoirs for zoonosis control and expanding our knowledge of bat viruses, this study aimed to investigate the presence of viruses of the Alphavirus and Flavivirus genera in bats. We analyzed serum, liver, lungs and intestine from 103 bats sampled in northeast and southern Brazil via Nested-PCR and the hemagglutination inhibition test. All samples tested in this study were negative for arbovi- ruses, suggesting that no active or past infection was present in the captured bats. These data indicate that the bats examined herein probably do not constitute a reservoir for these viruses in the studied areas. Further studies are needed to clarify the role of bats as reser- voirs and sources of infection of these viral zoonoses. Citation: Bittar C, Machado RRG, Comelis MT, Bueno LM, Morielle-Versute E, Beguelini MR, et al. (2018) Lack of serological and molecular evidence of arbovirus infections in bats from Brazil. PLoS ONE 13(11): e0207010. https://doi.org/10.1371/ journal.pone.0207010 Editor: Naomi Forrester, Keele University Faculty of Natural Sciences, UNITED KINGDOM Received: May 16, 2018 Accepted: October 23, 2018 Published: November 7, 2018 Editor: Naomi Forrester, Keele University Faculty of Natural Sciences, UNITED KINGDOM Received: May 16, 2018 Accepted: October 23, 2018 Published: November 7, 2018 Editor: Naomi Forrester, Keele University Faculty of Natural Sciences, UNITED KINGDOM Ethics statement The animals were collected under environmental licenses from the Brazilian Institute of Environment and Natural Renewable Resources (IBAMA), SISBIO n˚: 21707–1 (Process: 02027.001957 / 2006–02); 46190–1 and handled according to the Guide for the Care and Use of Laboratory Animals of the Institute for Laboratory Animal Resources Commission on Life Sciences National Research Council [21]. In addition, it should be noted that this project was approved by the Ethics Committee on the Use of Animals of the Institute of Biosciences, Let- ters and Exact Sciences (CEUA / IBILCE—Protocol 135/2016). Study areas Sampling was performed between 2014 and 2017 at ten different sites in urban and peri-urban areas of two cities from two different states of Brazil: São Jose´ do Rio Preto, state of São Paulo (SP) and Barreiras, state of Bahia (BA). The complete geographical coordinates address and ecological characteristics of all sampling sites are available as supplementary information (S1 Table). Additionally, climatological characteristics (temperature and rainfall) are given in S1 Fig (“A” for São Jose´ do Rio Preto—SP and “B” for Barreiras-BA). Investigation of arbovirus in bats Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Bats are mammals belonging to the order Chiroptera [3], and they are considered to be one of the most abundant, diverse and geographically distributed vertebrates in the world [4]. In Brazil, there is a great diversity of bats, with approximately 179 species (10 of which are endemic) and 68 subspecies belonging to 68 genera documented [5,6]. They present a broad geographic distribution, being able to fly long distances and often coming into direct or indi- rect contact with humans [3]. Recently, these vertebrates have received increasing attention as an important source for the emergence of zoonoses and possibly as viral reservoirs [7–9]. The infection of bats by arboviruses has long been reported by several authors [8,10–14]. Although the possibility of them acting as reservoirs has been raised, it is not clear yet if they play this role in the ecological cycle [15,16]. Being a large highly populated tropical country with one third of its territory covered by forests, Brazil presents ideal conditions for the existence of many arboviruses [17]. More than 200 arboviruses have been isolated in the country, and approximately 40 of these viruses cause diseases in humans [17,18]. The country presents a constant risk of emergence and re-emer- gence of arboviruses due to the existence of densely populated cities infested by mosquitoes of the genera Culex and Aedes, which are important vectors for arboviruses [1,7,19,20]. Focusing on the importance of the analysis of potential sources of zoonoses and viral reser- voirs for the control of emerging viruses, the aim of this study was to investigate arbovirus infections, of the Alphavirus and Flavivirus genera, in bats from southeast and northeast Brazil. In addition, we aim to provide relevant information that may contribute to the epidemiologi- cal surveillance of diseases of great public health impact. Introduction Among emerging viruses, an important group are arboviruses (arthropod-borne viruses), which are characterized by being maintained in nature in cycles involving hematophagous arthropod vectors and a wide range of hosts [1,2]. These hosts are often vertebrates, especially mammals and birds. 1 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 Samples Bats were collected using mist nets, and the sex and species of the animals were determined [22–24]. Following euthanasia, which was performed by subcutaneous anesthesia with 80 mg/ kg ketamine (Dopalen-Vertebrands, Paulı´nia, SP, Brazil) and 20 mg/kg xylazine (Rompun- Bayer S.A., São Paulo, SP, Brazil), the liver, intestines and lungs from each specimen were removed and stored at -150˚C. Blood was also collected, and serum was separated and stored at -150˚C. Finally, the animals were fixed in 10% formaldehyde for 24 hours and deposited in PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 2 / 10 Investigation of arbovirus in bats 70% alcohol in the Chiroptera Collection of the Department of Zoology and Botany—IBILCE / UNESP, where they are available for taxonomic studies. 70% alcohol in the Chiroptera Collection of the Department of Zoology and Botany—IBILCE / UNESP, where they are available for taxonomic studies. Endogenous control amplification After cDNA synthesis, in order to check the RNA quality, PCR was performed for the endoge- nous control, β-actin, as described previously [25]. Molecular detection of Alphavirus and Flavivirus Detection of viral RNA in the tissue samples was carried out by Nested-PCR assay. We designed a set of primers targeting the nsp4 region of Alphavirus based on 23 viruses from this genus (S1 File). To test for Flavivirus, we used PCR primers external to Flav100F and Flav200R, published by Maher-Sturgess et al., 2008 [26], for the Nested-PCR reaction, which targets the NS5 region. The sets of primers used for each reaction is described in Table 1. Addi- tional information on primer design and molecular tests are available in S1 and S2 Files. The PCR reaction was carried out using Long PCR Enzyme Mix (Thermo Fisher Scientific, Wal- tham, MA, USA). Amplification conditions were 3 min at 94˚C, 35 cycles at 94˚C for 1 min, annealing at 50˚C (for Alphavirus) and 48˚C (for Flavivirus) for 45 seconds, and extension at 72˚C for 1 min followed by a final extension at 72˚C for 10 minutes. The Nested-PCR reaction was performed as described above except for the annealing temperature, 45˚C (for Alphavirus) and 42˚C (for Flavivirus). Samples were resolved in 1% agarose gels. The expected sizes of the amplicons were 803 bp for Flavivirus and 913 bp for Alphavirus. RNA extraction and cDNA synthesis The RNA of the lungs, intestines and liver was extracted in order to test for the presence of Alphavirus and Flavivirus RNA. Tissues were homogenized in a Turrax-MA102 (Marconi, Pir- acicaba, SP, Brazil), and pellets were used for RNA extractions in TRIzol (Thermo Fisher Sci- entific, Waltham, MA, USA), according to manufacturer’s instructions. Finally, the RNA was resuspended in 100 μl of water treated with DEPC (Sigma Aldrich, St. Louis, Missouri, USA) and stored at -150˚C. Quantification of samples was performed on the NanoDrop 2000 spec- trophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Nucleic acids extracted from the organs of individual bats were subjected to cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Alphavirus and Flavivirus antibody detection The presence of antibodies for Alphavirus and Flavivirus in the serum samples were tested by hemagglutination inhibition (HI), as described previously [27]. Additional information about Table 1. Sequences 5’-3’ of Alphavirus and Flavivirus specific primers used for Nested-PCR reactions. PCR reaction Nested-PCR reaction Name Sequence 5’-3’ Name Sequence 5’-3’ Alphavirus α.nsP4.PF TAYYTDGAYATGGTIGABGG α.nsP4.NF TGYAAYGTIACICARATGMG α.nsP4.PR GGYTTICCIADYTTVAAIAG α.nsP4.NR AAICCICCRCARAARTAIGG Flavivirus F.NS5P-F ACIMTBITSTGTGACATHGGIGA Flav100F AAYTCIACICAIGARATGTAY F.NS5P-R GTBAHIGTGTTIARGGCRTAIGT Flav200R CCIARCCACATRWACCA Flav100F and Flav200R primers were published by Maher-Sturgess et al., 2008 [26]. https://doi.org/10.1371/journal.pone.0207010.t001 Table 1. Sequences 5’-3’ of Alphavirus and Flavivirus specific primers used for Nested-PCR reactions. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 3 / 10 Investigation of arbovirus in bats the antigens and the positive controls used in the HI, are given in S2 Table. The tests were per- formed by the Adolfo Lutz Institute–SP. the antigens and the positive controls used in the HI, are given in S2 Table. The tests were per- formed by the Adolfo Lutz Institute–SP. Results A total of 39 animals were collected in Barreiras-BA and 64 in the region of São Jose´ do Rio Preto-SP, totaling 103 bats. These were distributed in four families: Molossidae, Phyllostomi- dae, Emballonuridae and Vespertilionidae (Fig 1 and Table 2). The diversity of species and the amount of male/female per species are reported in Table 2. The livers and intestines were col- lected from 28 specimens from Barreiras–BA and only the livers from 11 specimens. From the region of São Jose´ do Rio Preto-SP liver, lungs and intestines were collected from all bats. Detailed information on species, sex, organs tested, site of sampling and month of collection for each specimen are presented in S3 Table. The necropsies did not show any morphological changes that indicated a pathological condition. Detailed information on species, sex, organs tested, site of sampling and month of collection for each specimen are presented in S3 Table. The necropsies did not show any morphological changes that indicated a pathological condition. RNA from all tissue samples was extracted and quantified. The quality of the RNA was con- firmed by the amplification of the endogenous gene, β-actin, from all tested samples. The presence of Alphavirus and Flavivirus RNA was investigated by Nested-PCR in all available tissue samples. The results revealed that none of the 103 samples were infected by Alphavirus and Flavivirus at the time they were collected. We also tested for serological evidence of previous infections by viruses from this groups using HI. A total of 73 serum samples (46 from São Jose´ do Rio Preto–SP and 27 from Bar- reiras–BA) were tested for anti-DENV-1, anti-DENV-2, anti-DENV-3, anti-DENV-4, anti- Fig 1. Locations of bat collections and the families found in each sampling site. On the left is the map of Latin America, highlighting the two Brazilian states, Bahia (in blue) and São Paulo (in green) in which bats were collected. At greater magnification, the collection sites within the regions of São Jose´ do Rio Preto–SP and Barreiras–BA are shown. The families of bats collected are represented by colored circles. The image was designed using QGIS 2.18.15 software (http://www.qgis.org/en/ site/about/index.html). Fig 1. Locations of bat collections and the families found in each sampling site. On the left is the map of Latin America, highlighting the two Brazilian states, Bahia (in blue) and São Paulo (in green) in which bats were collected. Results At greater magnification, the collection sites within the regions of São Jose´ do Rio Preto–SP and Barreiras–BA are shown. The families of bats collected are represented by colored circles. The image was designed using QGIS 2.18.15 software (http://www.qgis.org/en/ site/about/index.html). https://doi.org/10.1371/journal.pone.0207010.g001 https://doi.org/10.1371/journal.pone.0207010.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 4 / 10 Family Species Number of specimens Male Female Molossidae Eumops glaucinus - 1 Molossops temminckii 1 - Molossus molossus 6 18 Molossus rufus 14 4 Vespertilionidae Eptesicus furinalis 1 2 Eptesicus sp - 1 Myotis nigricans 1 - Phyllostomidae Artibeus lituratus 17 5 Artibeus planirostris 9 3 Carollia perspicillata 1 - Desmodus rotundus 1 - Diphylla ecaudata 2 - Glossophaga soricina 2 1 Phyllostomus discolor 3 2 Phyllostomus hastatus 1 - Platyrrhinus lineatus 1 - Sturnira lilium 1 - Vampyressa pussilla - 1 Emballonuridae Peropteryx leucoptera 1 - https://doi.org/10.1371/journal.pone.0207010.t002 MAYV, anti-WEEV, anti-EEEV, anti-YFV, anti-SLEV, anti-ROCV, anti-ILHV, anti-MUCV and anti-IGUV antibodies. No HI was detected in any of the bats’ serum or in the negative serum used as a control, indicating no previous infection by any of these viruses. Di i Table 2. Diversity of the species collected in relation to the sex of each specimen. Family Species Number of specimens Male Female Molossidae Eumops glaucinus - 1 Molossops temminckii 1 - Molossus molossus 6 18 Molossus rufus 14 4 Vespertilionidae Eptesicus furinalis 1 2 Eptesicus sp - 1 Myotis nigricans 1 - Phyllostomidae Artibeus lituratus 17 5 Artibeus planirostris 9 3 Carollia perspicillata 1 - Desmodus rotundus 1 - Diphylla ecaudata 2 - Glossophaga soricina 2 1 Phyllostomus discolor 3 2 Phyllostomus hastatus 1 - Platyrrhinus lineatus 1 - Sturnira lilium 1 - Vampyressa pussilla - 1 Emballonuridae Peropteryx leucoptera 1 - https://doi.org/10.1371/journal.pone.0207010.t002 MAYV anti-WEEV anti-EEEV anti-YFV anti-SLEV anti-ROCV anti-ILHV anti-MUCV Table 2. Diversity of the species collected in relation to the sex of each specimen. Family Species Number of specimens Male Female Molossidae Eumops glaucinus - 1 Molossops temminckii 1 - Molossus molossus 6 18 Molossus rufus 14 4 Vespertilionidae Eptesicus furinalis 1 2 Eptesicus sp - 1 Myotis nigricans 1 - Phyllostomidae Artibeus lituratus 17 5 Artibeus planirostris 9 3 Carollia perspicillata 1 - Desmodus rotundus 1 - Diphylla ecaudata 2 - Glossophaga soricina 2 1 Phyllostomus discolor 3 2 Phyllostomus hastatus 1 - Platyrrhinus lineatus 1 - Sturnira lilium 1 - Vampyressa pussilla - 1 Emballonuridae Peropteryx leucoptera 1 - https://doi.org/10.1371/journal.pone.0207010.t002 Table 2. Diversity of the species collected in relation to the sex of each specimen. MAYV, anti-WEEV, anti-EEEV, anti-YFV, anti-SLEV, anti-ROCV, anti-ILHV, anti-MUCV and anti-IGUV antibodies. No HI was detected in any of the bats’ serum or in the negative serum used as a control, indicating no previous infection by any of these viruses. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 Investigation of arbovirus in bats MAYV, anti-WEEV, anti-EEEV, anti-YFV, anti-SLEV, anti-ROCV, anti-ILHV, anti-MUCV and anti-IGUV antibodies. No HI was detected in any of the bats’ serum or in the negative serum used as a control, indicating no previous infection by any of these viruses. Discussion The role of bats in the transmission and ecology of arboviruses, such as Flavivirus and Alpha- virus, is not fully elucidated; however, several studies over the past 40 years have demonstrated that bats are susceptible to infection by viruses of these genera. For example, evidence of nucleic acids and antibodies from the four DENV serotypes has been reported in bats in Cen- tral and South America [28]. In addition, CHIKV was detected in bats from Asia [29]. In this study, we analyzed the presence of RNA from Alphavirus and Flavivirus and antibodies from viruses from these genera in animals collected in São Jose´ do Rio Preto (São Paulo State) and Barreiras (Bahia State). The first city is in the northwest region of São Paulo state and com- prises an area with a high incidence of arbovirus infection in humans. Several studies have shown that this is a region of YFV transmission [30,31], is hyper-endemic for DENV [32–34], has confirmed cases of MAYV [35,36], Saint Louis encephalitis virus (SLEV) [34,37] and ZIKV [38–40]. Additionally, some cases of DENV intra-serotype co-infection [32] and co- infection with other Flaviviruses [34,41] have been reported. In the Barreiras region, epidemio- logical reports from 2014 to 2017 also show the occurrence of DENV, CHIKV, ZIKV and YFV [42–44]. In addition, all animals were collected in densely populated urban or peri-urban areas where the main vector of these arboviruses, Aedes aegypti, has extensive circulation. Our results suggest that no active or past infections by the arboviruses in this study were present in th t d b t Th f t i di t th t lth h th b t ll t d i l t t ith Table 2. Diversity of the species collected in relation to the sex of each specimen. Discussion The role of bats in the transmission and ecology of arboviruses, such as Flavivirus and Alpha- virus, is not fully elucidated; however, several studies over the past 40 years have demonstrated that bats are susceptible to infection by viruses of these genera. For example, evidence of nucleic acids and antibodies from the four DENV serotypes has been reported in bats in Cen- tral and South America [28]. In addition, CHIKV was detected in bats from Asia [29]. In this study, we analyzed the presence of RNA from Alphavirus and Flavivirus and antibodies from viruses from these genera in animals collected in São Jose´ do Rio Preto (São Paulo State) and Barreiras (Bahia State). The first city is in the northwest region of São Paulo state and com- prises an area with a high incidence of arbovirus infection in humans. Several studies have shown that this is a region of YFV transmission [30,31], is hyper-endemic for DENV [32–34], has confirmed cases of MAYV [35,36], Saint Louis encephalitis virus (SLEV) [34,37] and ZIKV [38–40]. Additionally, some cases of DENV intra-serotype co-infection [32] and co- infection with other Flaviviruses [34,41] have been reported. In the Barreiras region, epidemio- logical reports from 2014 to 2017 also show the occurrence of DENV, CHIKV, ZIKV and YFV [42–44]. In addition, all animals were collected in densely populated urban or peri-urban areas where the main vector of these arboviruses, Aedes aegypti, has extensive circulation. Our results suggest that no active or past infections by the arboviruses in this study were present in the captured bats. These facts indicate that although the bats collected are in close contact with these viruses, that they are not being infected and that they probably do not constitute a reser- voir in the studied regions. 5 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 Investigation of arbovirus in bats Our study sampled bats from the Molossidae, Vespertilionidae, Phyllostomidae and Embal- lonuridae families. The few previous studies that sought to identify arboviruses in these bat families show that Molossidae and Phyllostomidae have the highest incidence of arbovirus infection. As an example of Molossidae infection, St. Louis encephalitis virus (SLEV) was iso- lated from Tadarida brasiliensis mexicana in Texas [45]. Additionally, in east Africa, antibod- ies reactive to ZIKV were detected at a high seroprevalence in Mops condylurus using the indirect hemagglutination test (HAI) [10]. Discussion In the Phyllostomidae family, there is a single work that detected the viral RNA of DENV-1 in Carollia perspicillata in French Guiana [46]. Addi- tionally, specific antibodies against West Nile Virus (WNV), SLEV and DENV 1–4 were detected by the plaque reduction neutralization test (PRNT) in three bat species from Mexico: Glossophaga soricina, Artibeus jamaicensis and Artibeus lituratus, with a Flavivirus antibody prevalence of 33%, 24%, and 9%, respectively [47]. For the Emballonuridae and Vespertilioni- dae families, there are no records of viral RNA or antibodies against the Flavivirus and Alpha- virus genera [46]. Even with the absence of studies that reported the lack of active and/or past arbovirus infec- tion in bats, some studies with DENV corroborate the results obtained in this work. In a study carried out in Mexico, which investigated the presence of the 4 serotypes of DENV in serum, lung and liver of 240 bats, no active or past infection in these animals was found [48]. Addi- tionally, other studies with DENV, demonstrate that some serotypes of this virus did not repli- cate efficiently in cell lines derived from neotropical bat species and indicate that some species are incapable of sustaining Dengue virus replication and are unlikely to act as reservoirs for this virus [49–51]. Moreover, a recent work showed that bats sampled from households in Costa Rican urban environments do not sustain DENV amplification, since they do not sup- port sufficient virus replication. These findings excluded them as potential hosts or reservoirs with no role in the transmission cycle and more likely are functioning as epidemiological dead-end hosts for this virus [52]. To date, few studies have aimed to identify arbovirus in bats, even with all the recognition of the importance of these animals in the emergence of zoonotic viruses. Some studies have demonstrated that ecological, behavioral and phylogenetic characteristics can influence and diversify the immunological response to viral infections in different species of bats [53,54]. For example, some evidence suggests that large colonies and higher species richness were signifi- cantly positively associated with European Bat lyssavirus 1 (EBLV-1) seroprevalence [54]. Additionally, they observed that EBLV-1 seroprevalence in bats from the Vespertilionidae and Rhinolophidae families were different. This difference is likely due to differences between bat species in the immune response and the lifespan of immunity to a virus infection [53,55]. Discussion Therefore, it is more likely to find seropositive bats in species with long lifespan immunity [55]. However, no study aimed to investigate differences in immunological responses between bat species to arbovirus infection. Even though there is no serological and molecular evidence of arbovirus infections in Bra- zilian bats from the studied regions, we emphasize the importance of continuing studies in other locations in order to evaluate the importance of bats as arbovirus reservoirs and to deter- mine if these animals are an important part of its enzootic cycle. Acknowledgments The authors would also like to thank all members of the Laboratory of Genomics Studies (IBILCE-UNESP, BR) for their helpful suggestions and discussions. This work was financially supported by FAPESP (São Paulo Research Foundation) grant number 2015/09704-6 and by CNPq (National Council for Scientific and Technological Development) grant number 165802/2015-4. Supporting information S1 Fig. Climatological characteristics of the sampling sites. A. Average monthly rainfall and temperature in the São Jose´ do Rio Preto region, from 2014 to 2017. The data were provided by the Integrated agrometeorological information center (CIIAGRO-Brazil: http://www. ciiagro.sp.gov.br). B. Average monthly rainfall and temperatures in the Barreiras region, from 6 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 Investigation of arbovirus in bats www.inmet.gov.br). (TIF) S1 Table. Sampling sites information. Additional information, geographic coordinates and ecological characteristics of sampling sites. The ecological characteristics were obtained by the Brazilian company of agricultural research—Ministry of Agriculture, Livestock and Supply (EMBRAPA-Brazil: https://www.embrapa.br/) and by the Forestry Institute—Sao Paulo’s State Government—Secretariat of Environment (IF-Brazil: http://iflorestal.sp.gov.br/). (XLSX) S2 Table. HI Antigens and Positive Controls. Description of the antigens (strain, virus and sample) and the positive controls used in HI. The positive controls were pools of mouse hyper- immune sera for each subgroup of arboviruses (Pool A—Alphavirus and B—Flavivirus). (XLSX) S3 Table. Characteristics of bats collected. Characteristics of bats collected (identification number—ID, family, species, sex, type of sample collected of each specimen and sampling site and month/year of collection). (XLSX) S1 File. Alphavirus primers design and molecular tests. (PDF) S2 File. Flavivirus primers design and molecular tests. (PDF) Author Contributions Conceptualization: Cı´ntia Bittar, Paula Rahal. Data curation: Cı´ntia Bittar, Rafael R. G. Machado. Formal analysis: Cı´ntia Bittar, Rafael R. G. Machado, Paula Rahal. Funding acquisition: Maurı´cio L. Nogueira, Paula Rahal. Investigation: Cı´ntia Bittar, Rafael R. G. Machado. Methodology: Cı´ntia Bittar, Rafael R. G. Machado, Manuela T. Comelis, Larissa M. Bueno, Eliana Morielle-Versute, Matheus R. Beguelini, Renato P. de Souza. Project administration: Cı´ntia Bittar, Rafael R. G. Machado. Resources: Paula Rahal. Supervision: Maurı´cio L. Nogueira, Paula Rahal. Validation: Cı´ntia Bittar, Rafael R. G. Machado. Conceptualization: Cı´ntia Bittar, Paula Rahal. Data curation: Cı´ntia Bittar, Rafael R. G. Machado. Data curation: Cı´ntia Bittar, Rafael R. G. Machado. Formal analysis: Cı´ntia Bittar, Rafael R. G. Machado, Paula Rahal. Funding acquisition: Maurı´cio L. Nogueira, Paula Rahal. Investigation: Cı´ntia Bittar, Rafael R. G. Machado. Methodology: Cı´ntia Bittar, Rafael R. G. Machado, Manuela T. Comelis, Larissa M. Bueno, Eliana Morielle-Versute, Matheus R. Beguelini, Renato P. de Souza. Project administration: Cı´ntia Bittar, Rafael R. G. Machado. Project administration: Cı´ntia Bittar, Rafael R. G. Machado. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 References 1. Weaver SC, Barrett ADT. Transmission cycles, host range, evolution and emergence of arboviral dis- ease. Nat Rev Microbiol. 2004; 2: 789–801. https://doi.org/10.1038/nrmicro1006 PMID: 15378043 2. Coffey LL, Vasilakis N, Brault AC, Powers AM, Tripet F, Weaver SC. Arbovirus evolution in vivo is con- strained by host alternation. Proc Natl Acad Sci U S A. 2008; 105: 6970–6975. https://doi.org/10.1073/ pnas.0712130105 PMID: 18458341 3. Hill JE, Smith JD. Bats: a natural history. London: British Museum (Natural History); 1984. 4. Feldhamer GA, Drickamer LC, Vessey SH, Merritt JF, Krajewski C. Mammalogy: Adaptation, Diversity, Ecology. 4th ed. Baltimore: Johns Hopkins University Press; 2015. 5. Nogueira MR, Lima IP De, Moratelli R, Cunha V, Gregorin R, Peracchi AL. Checklist of Brazilian bats, with comments on original records. J species List Distrib. 2014; 10: 808–821. https://doi.org/10.15560/ 10.4.808 6. Gregorim R. Cata´logo Taxonoˆmico da Fauna do Brasil. In: Nogueira MR, Isaac P. Lima, editors. Boletim da Sociedade Brasileira para o Estudo de Quiro´pteros. 2nd ed. Rio de Janeiro; 2016. p. 28. 7. Olival KJ, Hosseini PR, Zambrana-Torrelio C, Ross N, Bogich TL, Daszak P. Host and viral traits predict zoonotic spillover from mammals. Nature. Nature Publishing Group; 2017; 546: 646–650. https://doi. org/10.1038/nature22975 PMID: 28636590 8. Calisher CH, Childs JE, Field HE, Holmes K V., Schountz T. Bats: Important reservoir hosts of emerging viruses. Clin Microbiol Rev. 2006; 19: 531–545. https://doi.org/10.1128/CMR.00017-06 PMID: 16847084 9. Wang L-F, Cowled C. Bats and Viruses: A New Frontier of Emerging Infectious Diseases. Hoboken, New Jersey: Wiley-Blackwell; 2015. 10. Shephard RC WM. Studies on viruses in east African bats (Chiroptera). 1. Hemagglutination inhibition and circulation of arboviruses. Zoonoses Res. 1964; 3: 125–139. PMID: 5895899 11. Thompson NN, Auguste AJ, Travassos da Rosa APA, Carrington CVF, Blitvich BJ, Chadee DD, et al. Seroepidemiology of selected alphaviruses and flaviviruses in bats in Trinidad. Zoonoses Public Health. 2015; 62: 53–60. https://doi.org/10.1111/zph.12118 PMID: 24751420 12. Luis AD, Hayman DTS, O’Shea TJ, Cryan PM, Gilbert AT, Pulliam JRC, et al. A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? Proc Biol Sci. 2013; 280: 20122753. https:// doi.org/10.1098/rspb.2012.2753 PMID: 23378666 13. Williams MC, Simpson DI SR. Studies on viruses in East African bats. (Chiroptera). 2. Virus isolation. Zoonoses Res. 1964; 3: 141–53. PMID: 5895900 14. Williams MC, Simpson DIH, Shepherd RC. Bats and Arboviruses in East Africa. Nature. 1964; 203: 670. 15. Kuno G, Chang GJJG-JJ, Kuno G, Chang GJJG-JJ. Project administration: Cı´ntia Bittar, Rafael R. G. Machado. Resources: Paula Rahal. Supervision: Maurı´cio L. Nogueira, Paula Rahal. Validation: Cı´ntia Bittar, Rafael R. G. Machado. 7 / 10 7 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 Investigation of arbovirus in bats Visualization: Cı´ntia Bittar, Rafael R. G. Machado. Visualization: Cı´ntia Bittar, Rafael R. G. Machado. Visualization: Cı´ntia Bittar, Rafael R. G. Machado. Writing – original draft: Cı´ntia Bittar, Rafael R. G. Machado. Writing – original draft: Cı´ntia Bittar, Rafael R. G. Machado. Writing – review & editing: Cı´ntia Bittar, Rafael R. G. Machado, Paula Rahal. References Epidemiol Infect. 2008; 136: 1678–1683. https://doi. org/10.1017/S0950268808000460 PMID: 18325131 29. Dash a. P, Bhatia R, Sunyoto T, Mourya DT. Emerging and re-emerging arboviral diseases in Southeast Asia. J Vector Borne Dis. 2013; 50: 77–84. PMID: 23995308 30. Moreno ES, Rocco IM, Bergo ES, Brasil RA, Siciliano MM, Suzuki A, et al. Reemergência de febre amarela: Detecc¸ão de transmissão no estado de São Paulo, Brasil, 2008. Rev Soc Bras Med Trop. 2011; 44: 290–296. https://doi.org/10.1590/S0037-86822011005000041 PMID: 21739073 31. Saad LDC, Barata RB, Saad LDC, Barata RB. Surtos de febre amarela no estado de São Paulo, 2000– 2010. Epidemiol e Servic¸os Sau´de. 2016; 25: 531–540. https://doi.org/10.5123/S1679- 49742016000300009 PMID: 27869924 32. Colombo TE, Vedovello D, Mondini A, Reis AFN, Cury AAF, Oliveira FH de, et al. Co-infecc¸ão por vı´rus dengue, sorotipos 1 e 4, em paciente de cidade de porte me´dio no Brasil. Rev Inst Med Trop Sao Paulo. 2013; 55: 275–281. https://doi.org/10.1590/S0036-46652013000400009 33. Colombo TE, Vedovello D, Pacca-Mazaro CC, Mondini A, Arau´jo JP, Cabrera E, et al. Dengue virus surveillance: Detection of DENV-4 in the city of São Jose´ do Rio Preto, SP, Brazil. Acta Trop. Elsevier B.V.; 2016; 164: 84–89. https://doi.org/10.1016/j.actatropica.2016.09.004 PMID: 27609639 34. Mondini A, Bronzoni RV de M, Cardeal ILS, Santos TMIL dos, La´zaro E, Nunes SHP, et al. Simulta- neous infection by DENV-3 and SLEV in Brazil. J Clin Virol. 2007; 40: 84–86. https://doi.org/10.1016/j. jcv.2007.06.007 PMID: 17658293 35. Mota MTO, Vedovello D, Estofolete C, Malossi CD, Arau´jo JP, Nogueira ML. Complete Genome Sequence of Mayaro Virus Imported from the Amazon Basin to São Paulo State, Brazil. Genome Announc. 2015; 3: e01341–15. https://doi.org/10.1128/genomeA.01341-15 PMID: 26607884 36. Estofolete CF, Mota MTO, Vedovello D, de Go´ngora DVN, Maia IL, Nogueira ML. Mayaro fever in an HIV-infected patient suspected of having chikungunya fever. Rev Soc Bras Med Trop. 2016; 49: 648– 652. https://doi.org/10.1590/0037-8682-0093-2016 PMID: 27812665 37. Mondini A, Cardeal ILS, La´zaro E, Nunes SH, Moreira CC, Rahal P, et al. Saint Louis encephalitis virus, Brazil. Emerg Infect Dis. 2007; 13: 176–178. https://doi.org/10.3201/eid1301.060905 PMID: 17370543 38. Colombo TE, Estofolete CF, Reis AFN, da Silva NS, Aguiar ML, Cabrera EMS, et al. Clinical, laboratory and virological data from suspected ZIKV patients in an endemic arbovirus area. J Clin Virol. Elsevier; 2017; 96: 20–25. https://doi.org/10.1016/j.jcv.2017.09.002 PMID: 28918127 39. Fernanda Estofolete C, Terzian ACB, Parreira R, Esteves A, Hardman L, Greque GV, et al. References Biological Transmission of Arboviruses: Reexami- nation of and New Insights into Components, Mechanisms, and Unique Traits as Well as Their Evolu- tionary Trends Biological Transmission of Arboviruses: Reexamination of and New Insights into Components, M. Clin Microbiol Rev. 2005; 18: 608–637. https://doi.org/10.1128/CMR.18.4.608-637. 2005 PMID: 16223950 16. Kuno G, Mackenzie JS, Junglen S, Huba´lek Z, Plyusnin A, Gubler DJ. Vertebrate reservoirs of arbovi- ruses: Myth, synonym of amplifier, or reality? Viruses. 2017; 9: 1–28. https://doi.org/10.3390/v9070185 PMID: 28703771 17. Figueiredo LTM. Emergent arboviruses in Brazil. Rev Soc Bras Med Trop. 2007; 40: 224–229. https:// doi.org/10.1590/S0037-86822007000200016 PMID: 17568894 18. Casseb ADR, Casseb LMN, Silva SP Da, Vasconcelos PFDC. Arbovı´rus: Importante Zoonose Na Ama- zoˆnia Brasileira. Veterina´ria e Zootec. 2013; 20: 391–403. Available: http://www.fmvz.unesp.br/rvz/ index.php/rvz/article/view/191 19. Woolhouse MEJ, Gowtage-Sequeria S. Host range and emerging and reemerging infectious diseases. Emerg Infect Dis. 2005; 11: 1842–1847. https://doi.org/10.3201/eid1112.050997 PMID: 16485468 20. Kuno G, Chang GJ, Tsuchiya KR, Karabatsos N, Cropp CB. Phylogeny of the genus Flavivirus. J Virol. 1998; 72: 73–83. PMID: 9420202 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 8 / 10 Investigation of arbovirus in bats 21. Institute for Laboratory Animal Resources Comission on Life Sciences National Research Council. Guide for the Care and Use of Laboratory Animals [Internet]. 8th ed. Washington, D.C.: The National Academies Press; 2011. https://doi.org/10.1163/1573-3912_islam_DUM_3825 22. Vizotto LD, Taddei VA. Chave para determinac¸ão de quiro´pteros brasileiros. São Jose´ do Rio Preto, SP: Gra´fica Francal; 1973. 23. Miranda JMD, Bernardi IP, Passos FC. A new species of Eptesicus (Mammalia: Chiroptera: Vespertilio- nidae) from the Atlantic Forest, Brazil. Zootaxa. 2006; 1383: 57–68. 24. Laval RK. A revision of the neotropical bats of the genus Myotis. Sci Bull Nat Hist Museum Los Angeles Cty. 1973; 15: 1–53. 25. Spandidos A, Wang X, Wang H, Seed B. PrimerBank: A resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res. 2009; 38: 792–799. https:// doi.org/10.1093/nar/gkp1005 PMID: 19906719 26. Maher-Sturgess SL, Forrester NL, Wayper PJ, Gould E a, Hall R a, Barnard RT, et al. Universal primers that amplify RNA from all three flavivirus subgroups. Virol J. 2008; 5: 16. https://doi.org/10.1186/1743- 422X-5-16 PMID: 18218114 27. Clarke D, Casals J. Techniques for hemagglutination and hemagglutination-inhibition with arthropod- borne viruses. Am J Trop Med Hyg. 1958; 7: 561–573. PMID: 13571577 28. Aguilar-Setie´n a, Romero-Almaraz ML, Sa´nchez-Herna´ndez C, Figueroa R, Jua´rez-Palma LP, Garcı´a- Flores MM, et al. Dengue virus in Mexican bats. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 References Clinical and laboratory profile of Zika virus infection in dengue suspected patients: A case series. J Clin Virol. Else- vier B.V.; 2016; 81: 25–30. https://doi.org/10.1016/j.jcv.2016.05.012 PMID: 27289428 40. Nogueira ML, Rocha Nery Ju´nior NR, Estofolete CF, Bernardes Terzian AC, de Freitas Guimarães G, Zini N, et al. Adverse birth outcomes associated with Zika virus exposure during pregnancy in São Jose´ do Rio Preto, Brazil [Internet]. Clinical Microbiology and Infection. European Society of Clinical Microbi- ology and Infectious Diseases; 2017. https://doi.org/10.1016/j.cmi.2017.11.004 PMID: 29133154 41. Terzian ACB, Mondini A, de Moraes Bronzoni RV, Drumond BP, Ferro BP, Cabrera EMS, et al. Detec- tion of Saint Louis Encephalitis Virus in Dengue-Suspected Cases During a Dengue 3 Outbreak. Vec- tor-Borne Zoonotic Dis. 2011; 11: 291–300. https://doi.org/10.1089/vbz.2009.0200 PMID: 20645866 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 9 / 10 Investigation of arbovirus in bats 42. Costa IMP, Calado DC, Costa IMP, Calado DC. Incidência dos casos de dengue (2007–2013) e distri- buic¸ão sazonal de culicı´deos (2012–2013) em Barreiras, Bahia. Epidemiol e Servic¸os Sau´de. 2016; 25: 735–744. https://doi.org/10.5123/S1679-49742016000400007 PMID: 27869967 43. Bahia S da S do E da. Boletim Epidemiolo´gico da Febre Amarela. 2017; 3. Available: http://www.saude. mg.gov.br/component/gmg/story/9282-informe-epidemiologico-da-febre-amarela-12-04 44. Sau´de S de V em S−M da. Boletim Epidemiolo´gico. 2017;48: 1–10. 44. Sau´de S de V em S−M da. Boletim Epidemiolo´gico. 2017;48: 1–10. 45. Sulkin SE, Sims RA, Allen R. Isolation of St. Louis Encephalitis Virus from Bats (Tadarida b. mexicana) in Texas. Science (80-). 1966;Vol. 152: 223–225. 46. Chen L, Liu B, Yang J, Jin Q. DBatVir: The database of bat-associated viruses. Database. 2014; 2014: 1–7. https://doi.org/10.1093/database/bau021 PMID: 24647629 47. Machain-williams C, Lo´pez-uribe M, Talavera-aguilar L, Vera-escalante L, Puerto-manzano F, Ulloa A, et al. Serologic Evidence of Flavivirus Infection in Bats in the Yucatan Peninsula of Mexico. J Wildl Dis. 2014; 49: 1–8. https://doi.org/10.7589/2012-12-318.Serologic 48. Cabrera-Romo S, Max Ramirez C, Recio-To´toro B, Tolentino-Chi J, Lanz H, del A´ ngel RM, et al. No Evi- dence of Dengue Virus Infections in Several Species of Bats Captured in Central and Southern Mexico. Zoonoses Public Health. 2016; 63: 579–583. https://doi.org/10.1111/zph.12276 PMID: 27357156 49. Moreira-Soto A, Soto-Garita C, Corrales-Aguilar E. Neotropical primary bat cell lines show restricted dengue virus replication. Comp Immunol Microbiol Infect Dis. Elsevier Ltd; 2017; 50: 101–105. https:// doi.org/10.1016/j.cimid.2016.12.004 PMID: 28131369 50. Perea-Martı´nez L, Moreno-Sandoval HN, Moreno-Altamirano MM, Salas-Rojas M, Garcı´a-Flores MM, Are´chiga-Ceballos N, et al. Experimental infection of Artibeus intermedius bats with serotype-2 dengue virus. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207010 November 7, 2018 References Comp Immunol Microbiol Infect Dis. Elsevier Ltd; 2013; 36: 193–198. https://doi.org/10.1016/j. cimid.2012.12.002 PMID: 23312108 51. Cabrera-Romo S, Recio-Totoro B, Alcala´ AC, Lanz H, Del Angel RM, Sa´nchez-Cordero V, et al. Experi- mental inoculation of artibeus jamaicensis bats with dengue virus serotypes 1 or 4 showed no evidence of sustained replication. Am J Trop Med Hyg. 2014; 91: 1227–1234. https://doi.org/10.4269/ajtmh.14- 0361 PMID: 25311698 52. Vicente-Santos A, Moreira-Soto A, Soto-Garita C, Chaverri LG, Chaves A, Drexler JF, et al. Neotropical bats that co-habit with humans function as dead-end hosts for dengue virus. PLoS Negl Trop Dis. 2017; 11: 1–18. https://doi.org/10.1371/journal.pntd.0005537 PMID: 28545090 53. Lo´pez-Roig M, Bourhy H, Lavenir R, Serra-Cobo J. Seroprevalence dynamics of European bat Lyssa- virus type 1 in a multispecies bat colony. Viruses. 2014; 6: 3386–3399. https://doi.org/10.3390/ v6093386 PMID: 25192547 54. Serra-Cobo J, Lo´pez-Roig M, Seguı´ M, Sa´nchez LP, Nadal J, Borra´s M, et al. Ecological Factors Asso- ciated with European Bat Lyssavirus Seroprevalence in Spanish Bats. PLoS One. 2013; 8. https://doi. org/10.1371/journal.pone.0064467 PMID: 23700480 55. Serra-Cobo J, Lopez-Roig M. Bats and Emerging Infections: An Ecological and Virological Puzzle. In: Giuseppe R, Ippolito G, editors. Emerging and Re-emerging Viral Infections: Advances in Microbiology, Infectious Diseases and Public Health. 2017. pp. 35–49. 10 / 10
W3090346229.txt	https://link.springer.com/content/pdf/10.1007/s42597-020-00037-x.pdf	de		Strategien von Frieden und Erneuerung in Belfast	Zeitschrift für Friedens- und Konfliktforschung	2,020	cc-by	10,561	Z Friedens und Konflforsch (2020) 9:233–254 https://doi.org/10.1007/s42597-020-00037-x AUFSATZ Strategien von Frieden und Erneuerung in Belfast Der nordirische Unionismus auf der Suche nach einem Platz im Friedensprozess Felix Schiedlowski Eingegangen: 11. Dezember 2019 / Überarbeitet: 7. September 2020 / Angenommen: 12. September 2020 / Online publiziert: 1. Oktober 2020 © Der/die Autor(en) 2020 Zusammenfassung Mehr als zwanzig Jahre nach dem Abschluss des Karfreitagsabkommens steckt der nordirische Friedensprozess in einer Sackgasse. Während der Einfluss paramilitärischer Gruppen massiv schrumpfte, stagniert die politische und gesellschaftliche Annäherung zwischen den britisch-protestantischen Unionisten und den irisch-katholischen Nationalisten. Dieser Artikel identifiziert daher einen Friedensprozess der zwei Geschwindigkeiten, welcher nirgendwo so offen zu Tage tritt wie in Belfast. In der Hauptstadt hat sich die Form der politischen Auseinandersetzung partiell verschoben, Faktoren wie Geschichte, Identität oder Kultur sind unlängst zur Ware geworden. Insbesondere der Unionismus hat Schwierigkeiten, mit dieser Entwicklung Schritt zu halten. Während der Unionismus landläufig mit der konfliktträchtigen Vergangenheit des Landes assoziiert wird, inszeniert sich insbesondere das Stadtzentrum von Belfast als aufstrebend und befriedet. Für die unionistische Gemeinschaft der Stadt stellt sich jedoch die Frage, wie sie an diesem Wandel Anteil nehmen kann und muss. Dieser Prozess ist zugleich eine Suche nach einem Platz im nordirischen Friedensprozess an sich. Schlüsselwörter Friedensprozess · Urbanität · Kulturerbe · Belfast · Nordirland Strategies of peace and renewal in Belfast Northern Irish Unionism and the search for a place in the peace process Abstract More than twenty years after the signing of the Good Friday Agreement, the Northern Irish peace process seems to be locked in a stalemate. While the influence of paramilitary groups decreased massively, the political and social relationship F. Schiedlowski () Seminar für Ethnologie, Martin-Luther-Universität Halle-Wittenberg, Reichardtstraße 11, 06114 Halle, Deutschland E-Mail: felix.schiedlowski@ethnologie.uni-halle.de K 234 F. Schiedlowski between the British-Protestant unionist and the Irish-Catholic nationalist community remains tensed. Therefore, this article identifies a Janus-faced peace process which becomes openly visible in Belfast. In the capital city, factors such as history, identity, or culture have become commodities. The unionist community, in particular, has difficulties to keep pace with this trend. While unionism tends to be critical of the developments post-1998, some corners of Belfast appear to be a built manifestation of a positive peace process. For the city’s unionist community, however, this opens up the question as to how it can participate in this change. This process is also a search for a place in the Northern Ireland peace process in itself. Keywords Peace process · Urbanity · Cultural heritage · Belfast · Northern Ireland 1 Einleitung Belfast galt lange Zeit als Epizentrum des Nordirlandkonflikts, doch mit dem Karfreitagsabkommen von 1998 erlebt die Stadt einen bemerkenswerten Aufschwung. Mit diesem Wandel, vom Sinnbild einer schmutzigen Vergangenheit (Theroux 1983 (1999): 73 f) zur angesagten Partystadt (Lonely Planet o. J.), symbolisiert Belfast die Erfolgsgeschichte des nordirischen Friedensprozesses. Mit dem massiven Rückgang der Gewalt einher ging die politische Zusammenarbeit zwischen ehemals verfeindeten britisch-protestantischen Unionisten und irisch-katholischen Nationalisten. Mit Hilfe lokaler und internationaler Investitionen entstand eine post conflict Infrastruktur bestehend aus Hotels, Besucherzentren und landmark buildings. Doch zugleich ist der Friedensprozess vor Rückschlägen nicht gefeit. Noch immer existieren paramilitärische Strukturen (Gray et al. 2018, S. 106–112). Und während die politische Zusammenarbeit zwischen Unionisten und Nationalisten regelmäßig von Krisen erschüttert wird (Nagle 2017), ist die Polarisierung zwischen beiden Bevölkerungsgruppen kaum weniger geworden (Jarman 2016, S. 144). Der nordirische Friedensprozess ist geprägt von diesem Widerspruch, welcher sich nirgendwo so gut beobachten lässt wie in Belfast. Dieser Artikel tritt jenem Paradoxon mittels einer räumlich-kulturell verorteten Studie zum Unionismus in Belfast entgegen. Die Parallelität und Gleichzeitig eines gelungenen und eines stagnierenden Friedensprozesses ist in Belfast auch in den Stadtraum eingeschrieben. Zentral geht es um die Frage: Wie verorten sich unionistische Akteure von Belfast in diesem widersprüchlichen Friedensnarrativ? Ziel ist es, durch ethnologische Perspektiven auf Prozesse der urbanen Erneuerung einen alternativen Blick auf den nordirischen Friedensprozess zu eröffnen. Auf lokaler Ebene werden Begriffe wie Frieden und urbaner Wandel geschaffen und verhandelt, so auch in den mehrheitlich protestantischen Wohnvierteln Sandy Row und OstBelfast, welche in diesem Artikel im Fokus stehen. Primärdaten aus Interviews und ethnographischen Beschreibungen lassen dabei erkennen, wie in beiden Stadträumen Strategien der Abschottung und Anpassung geprobt werden, um Anschluss an K Strategien von Frieden und Erneuerung in Belfast 235 dominante Repräsentationsmuster von einem befriedeten Belfast zu finden.1 Dafür ist es zu Beginn nötig, das Gewebe der unionistischen Bedeutungswelt in Belfast, genauer in Pitt Park, zu verorten. 2 Pitt Park: Unionistische Identität in einem neuen Belfast Pitt Park, gelegen an der unteren Newtownards Road im Osten von Belfast, wirkt im Vergleich zu einer klassischen Grünanlage eher unscheinbar: Eine Rasenfläche in der Größe eines Fußballplatzes, ein paar Bäume, einige Bänke und ein Kinderspielplatz, mehr nicht. Dennoch lädt dieser Ort zum Verweilen ein, wenn man auf der Suche nach dem protestantisch-britischen Belfast ist. Pitt Park ist symbolisch aufgeladen, vereint auf engstem Raum jene Mythen und Geschichten, welche die Identität von Nordirlands Unionisten prägen: Das Gedenken an den Ersten Weltkrieg ist hier allgegenwärtig. Im November eines jeden Jahres, rund um den Remembrance Day, entsteht auf der Grünfläche des Parks ein Meer aus kleinen Holzkreuzen, versehen mit dutzenden Poppies – stilisierte rote Mohnblüten als Zeichen des Erinnerns an die Männer aus Ost-Belfast, welche in beiden Weltkriegen für King and Country gefallen sind. Als 1914 der erste jener Kriege ausbrach, waren viele der Männer bereits an den Waffen organisiert, als freiwillige Mitglieder der 1912 gegründeten Ulster2 Volunteer Force (UVF). In der protestantischen Miliz fanden sich tausende britische Iren zusammen, welche bereit waren, gegen eine immer stärker werdende irische Nationalbewegung zu kämpfen. Schließlich unterband der Erste Weltkrieg dieses Vorhaben, und aus der UVF wurde die 36th Division, eine offizielle Einheit der britischen Armee. Der Kampf um das Vaterland fand von nun an auf den Feldern Frankreichs statt. Den Ulstermen an der Waffe ist in Pitt Park ein Wandgemälde gewidmet, For Valour – für Tapferkeit, steht es in großen Lettern an einer Mauer geschrieben. Nie soll die Opfergabe dieser Männer vergessen werden, nie soll vergessen werden, wie loyal man der britischen Nation sein kann. Aufrichtigkeit, Hingabe und Stolz sind auch die Werte, die einem anderen Ereignis jener Zeit zugeschrieben werden. Vor der Rasenfläche des Parks wird mit den Skulpturen von drei Arbeitern – sie tragen Westen und Ballonmützen, sind dargestellt mit ernstem Blick und schnellem Schritt, auf dem Weg in die Werft von Harland & Wolff – an den Bau der Titanic erinnert. To the memory of men who built 1 Die Daten für diesen Artikel wurden zwischen Juli und September 2017 im Rahmen einer ethnologischen Feldforschung erhoben. Hierfür wurden vorrangig qualitative Interviews (elf formelle, zahlreiche informelle) und teilnehmende Beobachtungen im Stadtraum von Belfast durchgeführt. Ausschnitte der Forschungsergebnisse wurden im Oktober 2019 auf der Jahreskonferenz der Assoziation Peace and Conflict Studies in Anthropology in Belfast vorgestellt. 2 Die irische Insel ist in vier historische Provinzen eingeteilt: Leinster im Osten, Munster im Süden, Connacht im Westen und Ulster im Norden. Die Provinzen sind wiederum in 32 Grafschaften aufgegliedert. Während Leinster, Munster und Connacht vollständig zum Hoheitsgebiet der Republik Irland gehören, ist Ulster geteilt: Sechs Grafschaften bilden den nordirischen Staat, drei Grafschaften sind Teil der Republik. Viele Unionisten verwenden Ulster als einen historischen Namen für Nordirland sowie als Ausdruck ihrer irisch-britischen Identität. K 236 F. Schiedlowski giants steht es neben den 2012 errichteten Figuren geschrieben, 100 Jahre nachdem das größte Schiff seiner Zeit Belfast verließ und wenig später mit dem Untergang im Atlantik zum Mythos wurde. Lange genug ist dieses Unglück nun her, so dass der Stolz über die Leistung der zumeist protestantischen Yardmen stärker wiegt als das unheilvolle Ende der Titanic. Die Schiffsbautradition der Stadt begann Mitte des 19. Jahrhunderts und war lange eine Erfolgsgeschichte (Lynch 2012, S. 41), von der noch heute zwei riesige, gelbe Kräne im Hafen der Stadt zeugen. In den 1970er Jahren gebaut, thronen sie im Hintergrund über Pitt Park, von fast überall in Belfast sind sie zu sehen. Die Kräne der Werft wurden zum Wahrzeichen der Stadt, zu einem Monument der protestantischen Arbeitsethik. Gegenüber von Pitt Park, auf der anderen Seite der Newtownards Road, steht die Westbourne Church, in Erinnerung an die Arbeiter aus der Gegend auch Shipyard Church genannt. Unter dem Dach der presbyterianischen Kirche, dem mitgliederstärksten Zweig des protestantischen Glaubens in Nordirland, wird Religion zum Motor der Tüchtigkeit. Auch politisch hat die Westbourne Church eine stolze Geschichte zu erzählen. Im Jahr 1912 rief die unionistische Führungselite Nordirlands die protestantische Bevölkerung dazu auf, mit ihrer Unterschrift den Verlust des britischen Irlands an die Anhänger der irischen Unabhängigkeit zu verhindern. Am 28. September 1912, dem Anlass entsprechend Ulster Day getauft, fanden sich tausende Unionisten in dutzenden öffentlichen Einrichtungen des Landes zusammen, um den Ulster Covenant zu unterzeichnen: Das förmlich verbriefte und stolz ausgerufene Versprechen, mit aller Macht und mit allen Mitteln der britischen Krone treu zu bleiben. Die Westbourne Church war einer der Orte, an welchen sich die protestantischen Männer (und ihre Frauen, welche eine ähnliche Erklärung with their menfolk unterschrieben) in großer Zahl einfanden. Heute wird diese Geschichte beständig weitererzählt, von der Kirchengemeinde unter der Leitung von Pfarrer Mervyn Gibson. Gibson ist gleichzeitig hochrangiges Mitglied der Bruderschaft des Oranier-Ordens, welcher die Einheit bildet zwischen dem protestantischen Glauben und der britischen Identität in Nordirland. Während der marching season in den Sommermonaten macht der Orden mit zahlreichen Paraden auf sich aufmerksam, es ist das vielleicht bekannteste Element protestantischer Identität in Nordirland. Der Paradentradition ist dann auch eine weitere Wandinstallation in Pitt Park gewidmet, auf einem asphaltierten Areal neben der Grünanlage, angebracht an der Längsseite eines Schiffscontainers.3 Darauf zu sehen sind drei Männer in Reih und Glied, abgebildet in historischen Gewändern der UVF. Sie sind in Bewegung dargestellt, als Teilnehmer einer Parade, spielen die pipes and drums, die Flöten und Trommeln als klassische Begleittöne solcher Märsche (Law 1998, S. 52–54). Die Figuren sind größer als der Container, im Drang des Marschierens scheinen sie auszubrechen 3 Nordirland ist berühmt für unzählige Wandgemälde oder -installationen: Sowohl innerhalb der protestantischen wie auch innerhalb der katholischen Gemeinschaft werden sie genutzt, um politische Botschaften in die Öffentlichkeit zu tragen. Im frühen 20. Jahrhundert wurden die so genannten murals zunächst zu einer frühen Form protestantischer Popkultur, ehe sie auch von der katholischen Bevölkerung übernommen wurden. Mit dem Ausbruch des Nordirlandkonflikts in den 1960er Jahren wurden sie zum Träger politischer Botschaften, seit den 1990er Jahren ziehen sie zunehmend das Interesse von Touristen an und werden auch von unpolitischen Akteuren gestaltet (Jarman 1998). K Strategien von Frieden und Erneuerung in Belfast 237 aus dem Wandbild. Neben ihnen, in weißer Schrift auf gelb-rotem Grund, steht geschrieben: Culture Threatens No one. 2.1 Protestantisches Kulturerbe – doppelter Boden Doch Culture Threatens No one bedeutet mehr als ein oft beschworener protestantischer Stolz. In Nordirland, wo das Gegeneinander zwischen britischen Protestanten und irischen Katholiken nach wie vor das politische und gesellschaftliche Leben definiert, wird auch der Kulturbegriff4 in diesen Kategorien gedacht. Der nordirische Konflikt hat sich in den vergangenen 20 Jahren von einer militärischen zu einer kulturellen Auseinandersetzung gewandelt (siehe Bryan 2000; Jarman 1997; Parr 2017). Kultur kann eben doch eine Gefahr sein für die einen, oder selbst in Gefahr geraten, meinen die anderen. Kultur kann gesellschaftlicher Kitt sein, ist aber oft spaltender Pfahl, kann gleichermaßen gewichtiges Kapital sein oder erdrückende Last. Kultur wird zum Indikator, wie gut sich die beiden politischen Traditionen mit dem Friedensprozess arrangieren können. In den vieldeutigen und gegensätzlichen Bedeutungen, die insbesondere dem protestantischen Kulturerbe zugeschrieben sind, spiegelt sich der gegenwärtige Zustand der Tradition des Unionismus. Den Erinnerungsorten von Pitt Park sind noch weitere Narrative zugeschrieben, viele davon bereiten der unionistischen Gemeinschaft ein ernsthaftes Imageproblem. Der Unionismus, so scheint es, stand und steht das ein oder andere Mal auf der falschen Seite der Geschichte. Mit der Teilung der irischen Insel im Jahr 1921 wurde der Grundstein gelegt für einen Konflikt, welcher bis heute nicht überwunden ist.5 Aus den 26 irischen Grafschaften, welche den von Großbritannien unabhängigen irischen Freistaat begründeten, wurde „ein katholischer Staat für ein katholisches Volk“, wie es Regierungschef Éamon de Valera im Jahr 1934 formulierte. Dem setzte Nordirlands Premierminister James Craig entgegen, die sechs Grafschaften der Provinz Ulster, welche das britische Nordirland formten, hätten ein „protestantisches Volk und ein protestantisches Parlament“ (Edwards 2011, S. 16). Beide Bekundungen gingen 4 „Anthropologists get remarkably nervous when they discuss culture“ – mit dieser Feststellung behält Adam Kuper (1999, S. 226) bis heute recht. Einst von zentraler Bedeutung in der Ethnologie, ist der Kulturbegriff im Zuge von Neuausrichtungen des Faches in die Kritik geraten (siehe Clifford und Marcus 1986; Ortner 1999). Dieser ethnologische Streit soll hier jedoch keine Rolle spielen. Stattdessen wird Kultur als emische Kategorie verstanden: Um diesen Kulturbegriff, wie er von lokalen Akteuren genutzt wird, von einer theoretischen Definition abzugrenzen, wird Kultur in dieser Arbeit kursiv geschrieben. 5 Die bis heute gültige konstitutionelle Aufteilung der irischen Insel in einem britischen Norden und einen unabhängigen Süden geht auf das Jahr 1921 zurück. Die Frage nach dem Ursprung der irischen Frage hat jedoch deutlich tiefere Wurzeln. Austausch zwischen Irland und Britannien findet bereits seit dem 5. Jahrhundert v. Chr. statt, mit dem 12. Jahrhundert n. Chr. waren beide Inseln in einer frühen Form staatlicher Kooperation miteinander verbunden (Maurer 2013, S. 13, 36 f). Die Plantation of Ulster (1603–1685) legte schließlich den Grundstein für einen protestantisch geprägten Norden Irlands (Bardon 1992, S. 124–128). Nachdem dutzende englische Versuche scheiterten, dem Protestantismus eine Machtbasis im katholischen Irland zu verschaffen, setzte König James I auf schottische Siedler. Zwar missglückte schlussendlich auch dieser Versuch, die irische Insel komplett unter britische Kontrolle zu bringen. Im nördlich gelegenen Ulster jedoch, wo sich aufgrund der geografischen Nähe zu Schottland die meisten Siedler niederließen, entstanden größere protestantische Gemeinschaften. Ulster wurde von da an zur Bastion britisch-protestantischer Loyalität in Irland. K 238 F. Schiedlowski jeweils zulasten der katholischen Minderheit im Norden und der protestantischen Minderheit im Süden. Beide Gruppen waren im Idealbild der neuen Staaten nicht vorgesehen. Doch während das Schicksal der deutlich kleineren protestantischen Bevölkerung im Süden Irlands von Auswanderung, Resignation oder Anpassung geprägt war (Bury 2017), gipfelten die Forderungen der nordirischen Katholiken nach Gleichberechtigung lautstark in den Bürgerrechtsbewegungen der 1960er Jahre. Der nordirische Staat war unwillig und später unfähig, Ungleichbehandlungen bei der Arbeits- und Wohnungsvergabe sowie bei der politischen Partizipation zu beheben. Bürgerrechtsdemonstrationen mündeten zunehmend in Ausschreitungen, mobilisierten mehr und mehr katholische Unterstützer und protestantische Widersacher (Bardon 1992, S. 643–655). Ende der 1960er Jahre entwickelte sich eine Spirale der Gewalt, welche weder durch zaghafte politische Reformen noch durch den Einsatz der britischen Armee gestoppt werden konnte. Schließlich schlitterte Nordirland in einen fast 30 Jahre währenden Konflikt, welcher knapp 3600 Todesopfer forderte und tiefe gesellschaftliche Narben hinterlässt (Downs et al. 2009, S. 894). In diesem historischen Kontext wurde eine wiederbelebte UVF zu einer der tödlichsten paramilitärischen Akteure des Nordirlandkonflikts. Dabei machte man sich den traditionsreichen und respektierten Namen aus dem frühen 20. Jahrhundert zu nutze. Heute hat sich die UVF, wie andere lokale Paramilitärs, neue Tätigkeitsfelder gesichert, viele davon im Bereich der organisierten Kriminalität (Basra und Neumann 2018). Der Osten von Belfast, rund um Pitt Park, gilt als ein Hoheitsgebiet der Organisation, dargestellt durch Flaggen oder, als bildliche Vergegenwärtigung eines eigentlich beendeten Konflikts, Wandbilder von vermummten, bewaffneten Volunteers, dazu in großen Lettern die Erläuterung: We seek nothing but the elementary right implanted in every man: The right if you are attacked, to defend yourself. Auf der Suche nach einer Aufgabe im heutigen Nordirland dient der Name der historischen UVF den Paramilitärs als Anker. Eine UVF im Weltkrieg, eine andere UVF im Nordirlandkonflikt aktiv. Die eine geehrt, die andere gefürchtet. Oder, wie es die moderne UVF formulieren würde: In beiden Fällen waren es mutige Männer, die getan haben, was getan werden musste. Während die Bombardements der IRA in manchen Kreisen als legitimier Freiheitskampf interpretiert werden und sogar von Popgrößen wie John Lennon honoriert wurden (Brown et al. 2001, S. 69), gilt das Werk der Loyalisten durchweg als Frevel. Im Gegensatz zu ihnen haben die Kämpfer der IRA ihren Platz im Frieden gefunden. Als Sinn Féin Mitte der 1980er Jahre überraschende Wahlerfolge feierte, wandelte sich deren Strategie von einer militärischen in eine politische. Die Kampagne der IRA wurde langsam abgewickelt, aus IRA volunteers wurden einflussreiche Sinn Féin Politiker (Belfast News Letter 3. Juni 2011). Den Loyalisten hingegen fehlen bis heute einflussreiche Partner. Auf der parteipolitischen Ebene gilt die kleine, lediglich auf kommunaler Ebene vertretene Progressive Unionist Party als politischer Arm der UVF (McAuley 2004, S. 527 f). An der Basis, in den ehemaligen Hoheitsgebieten der protestantischen Paramilitärs, fungiert die Vereinigung Action for Community Transformation als wohltätige Einrichtung und zivile Nachfolgeorganisation der UVF (Edwards 2017, S. 312 f, 333). Dennoch, so der Historiker Tony Novosel, sehen die Unionisten der Mittelklasse die Loyalisten der UVF häufig als „embarrassment and not worthy of study or serious consideration because of their involvement in violence“ (2013, S. 5). Loyalismus in K Strategien von Frieden und Erneuerung in Belfast 239 Zeiten des Friedens bedeutet dem Journalisten Malachi O’Doherty zu Folge: „All that is required of them is that they be silent“ (2015, S. 32). Der Nordirlandkonflikt erschütterte auch die stolze protestantische Schiffsbautradition. In den 1970er und 1980er Jahren neigte sich die goldene Ära der Schwerindustrie überall in Westeuropa ihrem Ende zu, in Belfast wurde dieser Prozess noch verstärkt durch den alltäglichen Terror in der Stadt (O’Connell 2012, S. 290–296). Während Belfast zum Schauplatz eines blutigen konfessionellen Konflikts wurde, brach der Stadt die wichtigste Industrie weg: Das britische Königreich hat seine Vormachtstellung auf den Weltmeeren verloren, der Hafen in Belfast war nicht eingestellt auf den Bedarf an immer größer werdenden Schiffen, die Konkurrenz aus Asien und Ost-Europa wurde übermächtig (Lynch 2012, S. 50–59). Vom Aus der Schiffsindustrie war fast ausschließlich die protestantische Arbeiterklasse betroffen, nur wenige Katholiken waren in diesem Sektor angestellt. Immerhin hat der Mythos der Titanic überlebt. Kein Schiff verkörpert den Stolz der Yardmen aus Ost-Belfast so sehr wie der Luxusliner. Es war ein Schiffsbautriumph, zwischen 1909 und 1912 erschaffen in der Werft von Harland & Wolff. Wenig später, nach dem kolossalen Unglück auf dem Atlantik, wurde das Schiff zunächst zum Symbol für das Unheil der Moderne sowie für die Grenzen des technologischen Fortschritts (Neill 2011, S. 71 f). Aus Stolz wurde Schmach, welche sich schnell in eine gesellschaftliche Erinnerungslücke verwandelte. Doch mit James Camerons Leinwanderfolg aus dem Jahr 1997 kam die Titanic zurück nach Belfast (Hodson 2019, S. 232). Das Schiff entwickelte sich zu einer Chance. In Nordirland stritten Parteien, Paramilitärs und die Zivilgesellschaft um und für einen politischen Frieden, Belfast wollte nicht länger Europas Hauptstadt des Terrors sein, sehnte sich nach Normalität und sah in der Titanic das Potential eines gänzlich unpolitischen Alleinstellungsmerkmals. Das Ship of Dreams wurde zu einem gewaltigen Infrastrukturprogramm für die Stadt, ein Teil des verwaisten Hafengeländes auf Queen’s Island wurde in ein Titanic Quarter umgewandelt. Im Jahr 2012, 100 Jahre nach dem Unglück, eröffnete hier das neue Wahrzeichen der Stadt, ein interaktives Museum mit dem Namen Titanic Belfast. Die in das Schiff gesetzten Hoffnungen gingen auf, Belfast lockt nunmehr als Titanic Town Touristen aus aller Welt an. Doch die Suche nach einem unpolitischen Symbol geht zu Lasten der unionistischen Geschichte: Selten wird vom Platz des Schiffes im kulturellen Gedächtnis der Protestanten gesprochen, von der Titanic als Sinnbild britisch-protestantischer Ingenieurskunst. Der Mythos vom Schiffbau hat in Belfast überlebt, ist in Form der Titanic gar zum größten Exportschlager der Stadt geworden. Doch dass der neue Stolz ein Stück protestantische Kultur darstellt, geht dabei allgemein unter (Hodson 2019, S. 237–238). Im Gegensatz dazu steht die Politik und Tradition der presbyterianischen Kirche allzu sehr im Fokus und verkörpert für viele den Habitus des nordirischen Unionismus. Mit der fundamentalen Ablehnung der gleichgeschlechtlichen Ehe steht die Glaubensgemeinschaft zunehmend auf Kriegsfuß mit weiten Teilen der nordirischen Bevölkerung. Insbesondere im urbanen Kontext von Belfast zeichnet sich eine starke Opposition gegen die Position der presbyterianischen Kirche ab. Eine Umfrage aus dem Jahr 2015 zeigt, dass 68 % der nordirischen Bevölkerung schon lange auf die Einführung der gleichgeschlechtlichen Ehe drängte, welche erst seit Januar 2020 K 240 F. Schiedlowski gesetzlich verankert ist. Doch verteilt man die Zahlen auf die politischen Parteien, zeigt sich das Dilemma des Unionismus. Während die irisch-katholischen Parteien Sinn Féin (zu 80 %) und die Social Democratic and Labour Party (zu 61 %), ebenso wie die überkonfessionelle Alliance Party (zu 79 %), geeint für die gleichgeschlechtliche Ehe stritten, ist der Unionismus gespalten: Die beiden großen Parteien, die Democratic Unionist Party (DUP, zu 45 %) und die Ulster Unionist Party (zu 49 %), weisen deutlich geringere Zustimmungsraten auf (Cromie, 6. Juli 2015). In der Debatte um die gleichgeschlechtliche Ehe steht der Unionsmus isoliert und ohne Verbündete da. Mit der presbyterianischen Kirche eng verbunden ist der Oranier-Orden, wie das Beispiel von Orangeman Gibson als Pfarrer der Shipyard Church illustriert. Die Paradentradition der Bruderschaft ist zum Sinnbild des nordirischen Kulturkampfs geworden. Darf der Oranier-Orden, auf der Grundlage seiner altbewährten Marschrouten, auch weiterhin Stadtviertel passieren, welche heute vornehmlich katholisch bewohnt sind? Die Bruderschaft beruft sich auf ihr right to march, das Grundrecht auf Demonstrationsfreiheit. Katholische Anwohnerinitiativen hingegen verstehen diese Forderung häufig als Provokation. Für sie ist der Oranier-Orden das historische Sinnbild protestantischer Fremdherrschaft über die Katholiken. Der Paradenkonflikt symbolisiert die nordirische Gretchenfrage: Ist der Boden, auf dem Unionisten und Nationalisten gemeinsam leben, nun ein britischer oder ein irischer Boden? Das Marschieren oder Blockieren ist dabei ein Versuch, diese Frage öffentlichkeitswirksam zu beantworten (Bryan 2000). Nicht selten endet der Streit um eine Parade in handfester Gewalt und tagelangen Unruhen, zuletzt in den Jahren 2013 und 2015. Es sind solche Bilder, die ein düsteres Bild der protestantischen Paradentradition zeichnen. Es spricht vieles dafür, Nordirlands Unionisten eine Krise der Identität zu attestieren. Seit der Teilung der irischen Insel im Jahr 1921 verteidigen die Unionisten ihre politische und kulturelle Überzeugung von einem britischen Nordirland. Im Ringen um den richtigen Weg liegt dabei ein schier unerschütterlicher Stolz und die ständige Angst vor dem Ausverkauf der eigenen kulturellen Identität eng beieinander. Die Geschichten aus Pitt Park illustrieren das Dilemma des Unionismus: Der vielfach zitierte Stolz jener Tradition ist nicht universell, steht aus anderen Perspektiven für bedrückende Überreste des Nordirlandkonflikts, für fortschrittsfeindliche Positionen, für einfältige und bornierte Überzeugungen. Alte Ideale der protestantischen Ethik gelten vielen Unionisten noch immer als vorbildlich. Die Verpflichtung gegenüber der Kirche und der Krone, harte Arbeit und Disziplin, der individuelle und kritische Geist. Doch diese Taktik stößt an ihre Grenzen. Generell leidet der Unionismus unter dem Stigma der Kulturlosigkeit. Fernab des Oranier-Ordens, so die immer wieder durchschimmernde Ansicht von Politikern und Journalisten, habe der Unionismus kaum kulturelles Kapital anzubieten. Während die irische Nationalbewegung seit jeher mit kulturellen Elementen wie der irisch-gälischen Sprache, traditional Irish music oder Gaelic sporting Games in Verbindung gebracht wird, fehlt den Unionisten eine solche Beziehung (McCoy und O’Reilly 2003, S. 156; Vallely 2008, S. 249). Der Vorwurf der unionistischen Kulturlosigkeit ist dabei, wie es der Politikwissenschaftler Connal Parr aufzeigt, Teil der politischen Debatte: Der Unionismus, so das Argument vieler irisch-katholischer Na- K Strategien von Frieden und Erneuerung in Belfast 241 tionalisten, sei auf der Illusion von einem britischen Nordirland aufgebaut, und die Identitätskrise mit all ihren kulturpolitischen Ursachen sei die logische Konsequenz aus diesem Trugschluss (2017, S. 12–19). Diese Darstellung mag misslich sein, doch wirklich gekümmert hat sie einen Großteil der Unionisten bislang nicht. Schließlich, folgen wir dem Argument der protestantischen Ethik, wussten die Unionisten um ihre Stärken. Der Pomp und die Zurschaustellung sei den Katholiken überlassen, mit besserer PR gewinnt man vielleicht Sympathien, jedoch nicht die Deutungshoheit über die irische Frage. Doch der Friedensprozess von 1998 bringt eine neue Form der politischen Auseinandersetzung mit sich, welche diese defensive Position des Unionismus ins Wanken bringt. 2.2 Frieden in Belfast: doppelte Geschwindigkeit Im imposanten Rathaus von Belfast ist seit 2017 auch eine Dauerausstellung zur lokalen Geschichte untergebracht. Zentral gelegen, ist das Rathaus oft die erste Anlaufstelle für Gäste der Stadt, welche sich nun über den Wandel von Belfast im Spiegel der Zeit informieren können, vom ruhigen Dorf zur „vibrant, innovative, attractive city it is today“ (Love Belfast, 15. Mai 2017). So ist fast alles zu sehen, was Belfast ausmacht, von berühmten Söhnen und Töchtern der Stadt über den Aufund Abstieg als Industriemetropole bis hin zum Belfast slang. Auffallend wenig Aufmerksamkeit wird jedoch der jüngeren Geschichte gewidmet. Erklärungen und Einordnungen zum Nordirlandkonflikt bleiben aus. Stattdessen ist dieser Periode der Geschichte ein Reflection Space gewidmet. Zitate von Opfern des Konflikts stehen an den Wänden eines kleinen, mit weißem Licht gefluteten Raums geschrieben. Die wenigen Quadratmeter vermitteln einen Eindruck vom Leid dieser Zeit und den damit verbundenen, anhaltenden Schmerzen. Doch Fragen zur Schuld werden nicht gestellt, Täter werden nicht genannt. Voraussetzung für das Entstehen der Ausstellung war es, dass die beiden dominanten politischen Traditionen sich angemessen repräsentiert sahen. Geht es um das Erbe des Konflikts und um Fragen von Schuld und Verantwortung, war ein Raum der Ruhe der Minimalkonsens. Oder, wie es einer meiner Forschungspartner ausdrückte: „They couldn’t agree on anything“. Das Karfreitagsabkommen von 1998 beendete die heiße Phase des Konflikts. Die Auseinandersetzungen dieser Zeit sind Geschichte, gut aufgehoben in einem Reflection Space und taugen nicht als Zukunftsvisionen für Nordirland. Darüber sind sich die meisten protestantischen und katholischen Akteure einig, doch viel weiter reicht der Konsens nicht. Weder das Friedensabkommen noch die seither verstrichene Zeit haben erreicht, dass Misstrauen und Zorn zwischen beiden Seiten verschwinden. Auch scheitert man daran, ein universelles Narrativ für die Vergangenheit zu etablieren, Fragen von Schuld und Verantwortung bleiben unbeantwortet, Definitionen von Opfern und Tätern umkämpft. Das Karfreitagsabkommen skizzierte vielmehr, manchmal vage und manchmal konkret, wie ein friedliches Zusammenleben in Nordirland aussehen sollte. Das Jahr 1998 markiert einen Meilenstein für den hiesigen Friedensprozess, der jedoch als dessen Anfang, nicht als Endpunkt zu verstehen ist. Wandel basierend auf Vertrauen einst verfeindeter politischer Fraktionen oder Bevölkerungsgruppen wird auf der Grundlage politischer Abkommen ermöglicht, braucht aber deutlich mehr Zeit und nicht selten auch mehr Mühe (Brewer 2010, K 242 F. Schiedlowski S. 15). Für Nordirland bedeutet das, dass sich die nach wie vor existente Polarisierung zwischen Katholiken und Protestanten nicht aufgelöst hat. Das gilt auch für Belfast: Viele Bereiche des alltäglichen Lebens sind vom Gegensatz zwischen britisch-protestantisch und irisch-katholisch gezeichnet. Ein Großteil der Grundschulen sind protestantisch oder katholisch, integrated education bleibt die Ausnahme. Auch, weil working class districts überwiegend als homogen britische oder irische Viertel markiert sind. Während sich Belfast bereits zu Zeiten der industriellen Revolution in vornehmlich irische und britische Stadtviertel unterteilen ließ, verstärkte sich dieser Trend mit dem Ausbruch des nordirischen Konflikts. Die Angst vor und das Erleben von ethnisch begründeter Gewalt trieb die Menschen in homogene Nachbarschaften (Doherty und Poole 1997, S. 532 f). Und wo unterschiedlich geprägte Stadtviertel aneinandergrenzen, ist die Trennung auch baulich markiert, durch Hauptverkehrsstraßen, Sackgassen, Brachland, Zäune oder Mauern (Belfast Interface Project 2017, S. 7). Um der Gewalt an den konfessionellen Grenzen in der Stadt Herr zu werden, wurden provisorische Barrieren errichtet. Aus den Barrieren der späten 1960er Jahre wurden Mauern, und über die Jahrzehnte wurden es immer mehr. Nur die wenigsten konnten wieder abgerissen werden, die meisten wuchsen in die Höhe, viele davon auch nach dem Karfreitagsabkommen von 1998. Die sogenannten Friedensmauern machen einen sozialen Friedensprozess an diesen Orten fast unmöglich, stattdessen definieren sie Orte als politische und ökonomische Brennpunkte. Doch allzu oft erfüllen sie eben auch noch ihren Zweck. Sie sichern den brüchigen Frieden an diesen Orten, und einige Nachbarschaften fürchten sich vor einem Rückbau der Barrieren (Wilson 2016, S. 64–66). Hinter den Friedensmauern ringen paramilitärische Gruppen noch immer mit Gewalt, Einschüchterungen und kriminellen Geschäften um die Kontrolle über die oft abgehängten Viertel (Nolan 23. April 2018). Friedensmauern sind zu einem Symbol verkommen für die Spaltung der nordirischen Gesellschaft und für die Tücken des Friedensprozesses. Und währenddessen – als Folge von traumatischen Erlebnissen aus dem Nordirlandkonflikt, der ständigen Angst vor Gewalt, ökonomischer sowie sozialer Isolation – weist Nordirland die höchste Selbstmordrate des Vereinigten Königreichs auf, zudem fanden 45 % der zwischen 1965 und 2012 verzeichneten Suizide nach 1998 statt (McKee 25. August 2017). In einigen Teilen der Stadt bleibt der Frieden somit ein unerfülltes Versprechen. Doch Städte tragen verschiedene, oft gegensätzliche Repräsentationen mit sich (Shields 1996, S. 235, 245). Belfast kann nach wie vor als das Sinnbild einer polarisierten Stadt gelesen werden, doch parallel gilt die Stadt auch als befriedet, aufstrebend und angesagt. Mit dem Friedensprozess der 1990er Jahre ging die von katholischen und protestantischen Paramilitärs ausgeübte Gewalt spürbar zurück. Im Jahr 2007 schließlich zog die britische Armee ihre Soldaten ab, beendete ihren über 30 Jahre währenden Einsatz im Nordirlandkonflikt. Das Stadtzentrum rund um die prachtvolle City Hall ist nicht länger ein militärisch gesicherter Bereich, sondern das kommerzielle und kulturelle Herz von Belfast. Diverse Kaufhäuser aus viktorianischer Zeit, etliche Zweckbauten der 1970er und 1980er Jahre, sowie moderne Glasfassaden der frühen 2000er Jahre bilden die urbane Kulisse für Einheimische und Gäste. Während der Konflikt also keinesfalls komplett aus Belfast verschwand, blühten insbesondere das Stadtzentrum und die daran angrenzenden Viertel auf (siehe: Komarova und O’Dowd 2013; Murtagh 2018; Shirlow 2006). K Strategien von Frieden und Erneuerung in Belfast 243 Doch sind diese aufstrebenden und kommodifizierten Stadtnarrative vereinbar mit dem unionistischen Identitätskorsett, wie es sich in Pitt Park präsentiert? Die Altlasten des Nordirlandkonflikts sind nicht beräumt. Nationalisten drängen auf ein vereinigtes Irland, Unionisten wehren sich. Um in diesem ständigen Abnutzungskampf zu überleben, so der Politikwissenschaftler Arthur Aughey, entwickelte der Unionismus eine „Kultur des Fatalismus“, welche sich aus einem fragilem ,Wir sind immer noch da‘ – Optimismus speist (2000, S. 185 f). Die Zukunft gilt als prekär, Misstrauen und Unsicherheit bestimmen den unionistischen Handlungsspielraum. Den nordirischen Friedensprozess entwickelt sich somit in zwei Geschwindigkeiten. Nirgendwo sonst wird dieser Dualismus so deutlich wie in der Hauptstadt Belfast. Die doppeldeutige unionistische Bedeutungswelt, wie sie in Pitt Park manifestiert ist, verbindet die Gemeinschaft mit den Idealen des alten, konfliktträchtigen Belfasts, mit einem unperfekten Frieden. In den Stadtvierteln Sandy Row und Ost-Belfast ist man schließlich gezwungen, dieser Dialekt zu begegnen. 3 Sandy Row Das unscheinbare Wohnviertel Sandy Row, eine protestantische Enklave, grenzt direkt an die Innenstadt von Belfast. Bislang hat das Areal kaum vom Aufschwung der Stadt profitiert. Während der Stadtkern immer weiter an Sandy Row heranrückt, machen die hiesigen Gassen einen geschundenen Eindruck. Der geplante Umbau des angrenzenden Bahnhofs an der Great Victoria Street könnte das Schicksal des Viertels endgültig besiegeln, fürchten Anwohner. Ein Bauvorhaben zieht besonders viel Protest auf sich: Im Zuge der Umbaumaßnahmen soll die unscheinbare aber historische Boyne Bridge abgerissen werden. Dagegen machen die Boyne Bridge Defenders mobil. Noch endet Belfasts Innenstadt dort, wo Sandy Row beginnt. Doch der Aufschwung der Stadt stellt diese alten urbanen Grenzen in Frage. Während Hotels, Restaurants und Geschäfte immer mehr Platz brauchen, vegetiert Sandy Row vor sich hin. Es scheint nur eine Frage der Zeit, bis sich der kapitalistische Trend auch in diesen Teil der Stadt sichert. Sandy Row wäre lukratives Bauland für Hotels, Bars und teure Wohnungen in bester Lage. Rings herum entsteht bereits viel Neues, doch das kleine Arbeiterviertel scheint sich dem Trend zu verwehren. Einfache Häuser und graue Gassen, zwischendrin Brachland, ein Community Club, ein kleines Ladengeschäft, eine Kneipe, ein Gemeindehaus des Oranier-Ordens und diverse Wandbilder: Manche zieren die Queen oder Nordirlands Fußballer, andere die Wappen loyalistischer Paramilitärs: Nicht selten wird Sandy Row als ein place apart bezeichnet, der nicht recht reinpassen will in seine moderne Umwelt. Doch nun könnte ein Infrastrukturprojekt dieses Image aufbrechen. Der in die Jahre gekommene Bahnhof Great Victoria Street soll zu einem Transport Hub umgebaut werden, welcher den Ansprüchen einer expandieren Stadt Rechnung trägt. Gleichzeitig soll rings um den neuen Hauptbahnhof auch ein neues Stadtviertel, Wavers Cross, entstehen. Laut Translink, dem nordirischen Nahverkehrsbetrieb und Auftraggeber des Projekts, ermöglicht der Transport Hub Besuchern „an excellent first impression of a confident capital city“ (2017, S. 1). K 244 F. Schiedlowski Doch endet dieser tolle erste Eindruck nicht vielleicht in just dem Moment, in dem sich der Blick auf Sandy Row richtet? Was bedeutet die Vision von Wavers Cross für das schmucklose Arbeiterviertel? Anwohner haben wenig Vertrauen. Sie kämpfen um den Erhalt ihres Viertels, konkret um die unscheinbare aber historisch bedeutende Boyne Bridge. In Sandy Row wird Kultur zur letzten Patrone. Auf den folgenden Seiten wird deutlich, dass der Streit um die Boyne Bridge auch die Geschichte eines Friedensprozesses ist, der sich für die Bewohner von Sandy Row zu einem gebrochenen Versprechen entwickelte. 3.1 „I don’t trust anyone“: Streit um die Boyne Bridge Vielleicht zwanzig Personen haben sich an einem Samstagvormittag im August vor dem Rathaus von Belfast versammelt.6 Im Gewusel der Wochenendbesorgungen geht die Gruppe fast unter, lediglich die Uniformen von drei Polizisten sowie dutzende Union Jacks und Banner (Loyal Peoples Protest) machen deutlich, dass es sich hier um eine Demonstration für den Erhalt der Boyne Bridge handelt. Ich geselle mich dazu, und sofort nimmt mich ein älterer Herr in Beschlag. Besagte Brücke, so erklärt es mir der Herr, wurde in seiner heutigen Form im Jahr 1936 errichtet, ihr Ursprung soll jedoch auf das 15. Jahrhundert zurück gehen. Als Nord-Süd-Achse war die Boyne Bridge lange Zeit eine bedeutende Verkehrsader in Belfast, Mächtige aller Couleur sollen die Brücke überquert haben (BBC 9. August 2017; Save the Boyne Bridge 2020). Heute passieren Züge von oder nach Great Victoria Street die Brücke, welche wie eine profane Straße daherkommt. Auch ihre strategische Bedeutung hat die Boyne Bridge längst eingebüßt, wird der NordSüd-Verkehr doch heute über den nahe gelegenen Westlink geleitet. Doch auf diese Punkte kommt der Herr nicht zu sprechen, schnell verschiebt sich sein Fokus, weg von der Brücke, hin zu den Verantwortlichen des Projekts. Translink will die Brücke in eine Straße umwandeln und mit künstlerischen Elementen an ihre historische Bedeutung erinnern. Doch für die Demonstranten wäre das kein annehmbarer Kompromiss. Die Brücke dürfe für den Transport Hub nicht abgerissen werden, im Gegenteil, ihr Wert soll durch eine umfassende Renovierung hervorgehoben werden. Mehr als 3000 Unterschriften haben sie bereits für den Erhalt der Boyne Bridge gesammelt, das könne Translink nicht einfach ignorieren. Ich frage ihn, ob sich sein Protest auch gegen den Transport Hub und das neue Stadtviertel Wavers Cross an sich richtet. Er sei jedenfalls nicht davon überzeugt, dass Sandy Row von dem Unterfangen profitieren würde, seine Erklärungen mir gegenüber werden nun emotionaler. Verbittert verweist er auf frühere Versprechen, welche den Bewohnern von Sandy Row gemacht wurden. Ihre Unterstützung für Bauprojekte rings um Sandy Row herum sollte mit dringend benötigten Investitionen im Viertel belohnt werden. Der Bau eines Hotels sollte mit dem Bau neuer Wohnungen einhergehen, stattdessen entstand ein Parkhaus. Dementsprechend misstrauisch beäugen die Demonstranten das Translink-Projekt: „I don’t trust anyone“, versichert mir der Herr. Der Protest wirkt bizarr und doch nachvollziehbar. Sandy Row, soviel ist sicher, hat Modernisierungsbedarf. Das Bauvorhaben von Translink wird die direkte Nach6 26. August 2017. K Strategien von Frieden und Erneuerung in Belfast 245 barschaft des Viertels herausputzen. Davon könnte auch Sandy Row profitieren, geht mit städtebaulichen Investitionen in Millionenhöhe doch stets der Wunsch einher, kulturhistorische Orte aufzuwerten. Doch das Argument, Kultur zu retten kann nur dort greifen, wo Kultur auch als solche anerkannt wird. Stadträume sind dabei stets mit Bedeutungen aufgeladen, gelten als Träger von lokalen Werten und Interessen (Kuper 1972, S. 421). In diesem Kontext gilt Sandy Row vielen als protestantische Enklave, schützenswertes Kulturerbe ist hier nicht die erste Assoziation. Die misstrauischen Reaktionen auf das Bauprojekt rund um Sandy Row spiegeln auch eine unionistische Geisteshaltung gegenüber den aus ihrer Sicht oft faulen Versprechen des Friedensprozesses, von welchen wir im folgenden Abschnitt mehr hören werden. Für den älteren Herren von der Demonstration ist die Sache jedenfalls klar: „I am not prepared to sell my history to anyone!“, ist seine trotzige Reaktion auf den Wandel der Stadt. 3.2 „We believed we were save“: Vom Frieden enttäuscht An einem Freitagvormittag im September7 bin ich mit Oliver8 verabredet, einige Tage zuvor habe ich den Mann im mittleren Alter bei einer Führung zum Erbe des Ersten Weltkriegs in Ost-Belfast getroffen. Zunächst bin ich von der Hoffnung getrieben, dank Oliver mehr über die Bedeutung des Great War für den Unionismus zu erfahren. Dazu kommt es nicht, unser geplanter Besuch des Somme Heritage Centres muss ausfallen, das Museum bleibt freitags geschlossen, unsere Ausfahrt wird nicht belohnt. Doch bereits einige Stunden zuvor treffen wir uns in Olivers Haus in Sandy Row, wir trinken Tee und unterhalten uns, über sein Viertel, die Boyne Bride, den nordirischen Frieden. Oliver will mir seine Leidenschaft, das Gedenken an den Ersten Weltkrieg, näherbringen. Dazu lotst er mich auf seine Facebookseite, auf welcher er dutzende historische Fotos und Dokumente teilt und hochlädt. Beim Überfliegen seiner Inhalte erblicke ich auch politische Inhalte, zu den meisten schweigt Oliver. Auf einen Beitrag von Save the Boyne Bridge kommt er jedoch zu sprechen: Oliver erläutert mir den Hintergrund des Brückendisputs und bekennt, dass er sich selbst für den Erhalt der Boyne Bridge engagiert. Den Abriss hält er, wenig überraschend, für unnötig. Schnell, kommt Oliver auf größere Sachverhalte zu sprechen. Er ruft die einstige Größe von Sandy Row in Erinnerung und erläutert, wie das Viertel mit der Expansion des Stadtzentrums immer kleiner wurde. Er spricht davon, wie den Bewohnern von Sandy Row seit Jahren neue, dringend benötigte Häuser verwehrt werden, weil die Finanzkräfte kein Interesse an diesem Viertel hätten. Ich kann dem Eindruck von Oliver wenig entgegensetzen. Obwohl das Viertel nicht groß ist – die Sandy Row, Herzstück und Namensgeber, von welcher kleine Gassen abgehen, misst keinen Kilometer – ist der Kontrast zur direkten Umgebung gewaltig. In Sandy Row vertreibt ein Geschäft diverse Paraden-Utensilien, über Lautsprecher wird die Straße mit protestantischer Marschmusik beschallt. Und als bräuchten Olivers Worte noch weitere Beweise, ist eine Brachfläche mitten auf der 7 1. September 2017. 8 Name geändert. K 246 F. Schiedlowski Sandy Row drapiert wie eine Kulisse: Statt der ersehnten Wohnhäuser befinden sich hier nur haufenweise leere Dosen und Flaschen als Überbleibsel der Oranierparade vom 12. Juli, dahinter ein Altkleidercontainer, das alles vor einer angrenzenden Gibelwand und einem großen Banner mit der Losung British and Proud. Nach einer Tasse Tee verlassen wir Sandy Row, Oliver will mich jemandem vorstellen, seinem Bekannten Jack9, Pfarrer und Gründer einer Community Church im Niemandsland zwischen der katholischen Falls Road und der protestantischen Shankill Road. Die Kirche hat mit einem sakralen Gebäude wenig gemein, erinnert eher an eine große Turnhalle. Doch für Jack, der in etwa Olivers Alter teilt, ist die Community Church vielmehr ein Ort, um die Nachbarn aus der Shankill Road und der Falls Road zusammen zu bringen, der Glaube an Gott ist dabei nachrangig. Jack ist mit seiner Arbeit ein Advokat des Friedens, und doch entwickelt sich dieses Gespräch zwischen ihm und Oliver zu einer Abrechnung mit dem, was man in Nordirland Friedensprozess nennt. Jack gibt uns einen Kaffee aus, später einen zweiten, dazu bestellt er einige süße Teilchen. Jack erzählt zunächst seine Geschichte: Er stammt aus Ballymurphy, einer Gegend im Westen von Belfast, seine Familie musste zu Beginn des Nordirlandkonflikts jedoch in die Shankill Road umziehen, ihre protestantische Konfession drohte ihnen in ihrem Heimatort zum Verhängnis zu werden. In den 1960er Jahren schließlich war Jack selbst involviert in die täglichen Straßenkämpfe, beteiligte sich an Angriffen auf Katholiken. Er teilt diese Geschichte mit Oliver, auch er war in die alltägliche Gewalt involviert, verbüßte seine Taten im Gefängnis. Jack hat sich später schließlich dem Ulster Defence Regiment der Britischen Armee angeschlossen und ist seit 1982 verbunden mit dieser Kirche. Die persönlichen Einlassungen von Jack bewegen schließlich auch Oliver, eine Geschichte aus dieser Zeit mit uns zu teilen. Auf seiner damaligen Arbeitsstätte kannte er einen jungen Katholiken flüchtig. Eines Tages wurde der junge Mann erschossen, Paramilitärs hatten ihn verwechselt. Oliver war in der Nähe, der junge Mann starb in seinen Armen. Die Geschichte ist kurz, jäh ist ihr Ende. Beide machen deutlich, wie sehr sie den heutigen Frieden schätzen, und doch teilen sie eine tiefe Wut. Dabei haben beide Männer für den Friedensvertrag von 1998 gestimmt, Oliver gar aus tiefer Überzeugung. Den Tür-zu-Tür-Wahlkämpfern der DUP, damals Gegner des Karfreitagsabkommens, hat er vor dem Hintergrund der andauernden Gewalt und mit Blick auf seine Kinder und Enkelkinder gesagt: „Don’t ask me to keep this going“. Jack und seine Frau wollten zunächst mit Nein stimmen: „We wanted peace but not at this price“. Doch nach einem Wahlkampfauftritt von Tony Blair in Belfast und den dort gemachten (und später, so Jack, gebrochenen) Versprechen, katholischen Paramilitärs keine Zugeständnisse zu machen, haben sie im Vertrauen auf ihren Premierminister und gläubigen Christen Blair mit Ja gestimmt.10 Heute 9 Name geändert. 10 Ein Friedensprozess ohne die Einbeziehung von Sinn Féin war nicht möglich, gleichzeitig versuchte Tony Blair damals zu versichern, dass der IRA gegenüber keine unmoralischen Versprechen gemacht werden (Brown und McKittrick, 26. Juni 1997). Kritiker jedoch verloren schnell das Vertrauen in derlei Ankündigungen. Der versprochene Entwaffnungsprozess dauerte bis 2005 an, IRA-Anschläge gingen zunächst weiter. Dennoch wurde Sinn Féin als politischer Arm der Paramilitärs die Teilnahme an den Friedensgesprächen gewährt. Im Jahr 2015 wurde schließlich bekannt, dass die Blair-Regierung dutzende On K Strategien von Frieden und Erneuerung in Belfast 247 sagt Jack: „If I would turn the time back I would vote no“. Für beide sind die Ziele des Karfreitagsabkommens zu einer Illusion verkommen. Wir machen eine kleine Tour durch die Kirche, ich sehe den Kinderspielbereich, die Veranstaltungshalle sowie den Fußballplatz. Anschließend kehren wir zurück, zum zweiten Kaffee. Oliver und Jack sind mittlerweile auf das Problem der unionistischen PR zu sprechen gekommen, oder wie es Oliver formuliert: „The unionist PR is not existing“. Beide waren gegen die Einführung der gleichgeschlechtlichen Ehe, doch Oliver stellt fest: „If we are not progressive the other side will see us as dinosaurs“, worauf Jack entgegnet: „I am not a dinosaur. I have an opinion. That opinion should not make me a dinosaur“. „We are not good in telling our story“ sagt Jack schließlich, und da sind sich beide einig, doch woran liegt das? Die Unionisten, so Jack, hätten stets gedacht: „We believed we were save. [...] We won two world wars. But we were wrong“. Jack und Oliver leben im Frieden, sie wollen keine Rückkehr der Gewalt. Doch ihr Verdruss gilt den politischen Prozessen hinter dem Karfreitagsabkommen. Ihnen zu Folge müssen die Unionisten bis heute einen hohen Preis zahlen: Die Aufgabe alter Privilegien, die Neuverhandlung öffentlicher Symbole, die gesellschaftliche Rehabilitierung von Kämpfern der IRA. Stadträume wie Sandy Row bilden vor diesem Hintergrund Rückzugsorte in eine alte Welt. Hier akzeptiert man das Schweigen der Waffen, will aber darüber hinaus wenig mit dem heutigen Friedensprozess zu tun haben. 3.3 Abschottung als Taktik In Sandy Row wird die unscheinbare Boyne Bridge aufgewertet und umgedeutet zu einem politischen Symbol für die unionistische Unzufriedenheit mit dem Friedensprozess. Der Stadtraum wird zum Träger einer manifesten und unüberhörbaren Botschaft (Kuper 1972, S. 258). Der Protest illustriert den symbolischen und politischen Charakter, welchen Stadträume einnehmen können. Die Stadt ist fluide und formbar, ein Metatext mit parallelen und widersprüchlichen Botschaften (Shields 1996, S. 234, 245). Oliver und die Boyne Bridge Protestor widersetzen sich dem Narrativ vom modernen Belfast, welches eng verzahnt ist mit dem Narrativ vom befriedeten Belfast, suchen zugleich nach eigenen Anknüpfungspunkten. Sandy Row ist dieser Hinsicht entkoppelt von der nach kapitalistischem Vorbild modernisierten Innenstadt von Belfast, und das in ökonomischer, infrastruktureller und ideeller Hinsicht. Das Leben in geschröpften Städten und Vierteln verlangt von dessen Bewohnern, ihr gebautes Umfeld ständig neu zu denken und zu erfinden (Mah 2012, S. 175). In Sandy Row vollzieht sich dieser Wandel durch das Bewahren des Alten. Der Kampf um die Boyne Bridge soll unionistisches Kulturgut retten und zugleich die Brandmauer zu einem Belfast markieren, welches, anders als Sandy Row, von den Versprechen des Friedens profitierte. Noch hält die Opposition gegen Verdrängung und Gentrifizierung. Der mit der Brücke verbundene Streit lässt sich von dem the run letters versendete. Darin wurde flüchtigen Paramilitärs verbindlich Straffreiheit zugesichert. Nach dem Bekanntwerden der Briefe platzen juristische Prozesse gegen Ex-IRA-Mitglieder. Tony Blair verteidigte die Briefe als notwendiges Mittel, um Sinn Féin und die IRA zum Frieden zu bewegen (McDonald und Perraudin, 13. Januar 2015). K 248 F. Schiedlowski Bauwerk loslösen und als Taktik auf andere Kulturgüter übertragen. Kultureller Wettbewerb bedeutet in Sandy Row Widerstand und Abschottung. Doch es bleibt fraglich, wie viel Kultur sich noch zur Waffe schmieden lässt, wie lange diese Taktik Erfolg haben wird. Auch in Ost-Belfast steht man vor der Aufgabe, Verbindungen zum neuen Belfast aufzubauen. In der Ausgangslage ähneln sich beide Viertel, doch die Herangehensweise ist dort eine gänzlich andere. 4 Ost-Belfast Ost-Belfast galt einst als Heimat der hiesigen Schwerindustrie und protestantischer Paramilitärs. Bis heute zehrt man von dieser Geschichte, welche, so wird es im folgenden Kapitel deutlich, stets auch in der Gegenwart erlebt und auf die Zukunft projiziert wird. Anders als Sandy Row ist der mehrheitlich protestantische Osten fernab der Innenstadt, getrennt durch den Fluss Lagen. In dieser Abgeschiedenheit versucht man nun, die Geschichten von industriellem Niedergang und politischer Gewalt umzuschreiben, um so ein modernes und auf den städtischen Wandel abgestimmtes Ost-Belfast zu gestalten. Aslan der Löwe gilt als Star im Figurenensemble der Chroniken von Narnia, dem Märchen und Kinderbuchklassiker des in Ost-Belfast geborenen Autors C. S. Lewis. Aslan ist eine mystische Erscheinung, allwissend und barmherzig, er besitzt die Fähigkeit in anderen Körpern zu leben und mit Menschen zu sprechen. Lewis, selbst gläubiger Christ, versteckte biblische Parallelen in seinen Märchen, in welchen Aslan die Fleischwerdung Jesu darstellt. Auch im heutigen Ost-Belfast ist von der Inkarnation des prächtigen Löwen etwas zu spüren: Dem 2016 eröffneten Eastside Visitor Centre auf der Newtownards Road ist der C. S. Lewis Square angeschlossen. In dem Figurenpark, bestehend aus metallischen Nachbildungen von Charakteren der Narnia-Saga, bildet der auf einem Hügel thronende Aslan das Herz der Anlage. Wo Sandy Row dem Wandel der Stadt skeptisch gegenüber steht, hat ihn Ost-Belfast zu einem gewissen Teil bereits vollzogen. Der Untergang der Schiffsindustrie war ein schwerer Schlag für Ökonomie und Seele des Viertels, als Heimat der loyalistischen UVF erlebte der Osten eine besondere Bindung zum Nordirlandkonflikt und von den positiven Entwicklungen in der Innenstadt profitierte man schon allein deshalb nur zum Teil, weil der Fluss Lagan den Osten in mancher Hinsicht zu einem abgeschiedenen Teil der Stadt macht. Wie in keinem anderen protestantischen Viertel lernte man in Ost-Belfast daher schnell, notwendige Veränderungen selbst voranzutreiben. So ist die eingangs zitierte, hoffnungsfrohe Botschaft Aslans Teil der Geschichte von Ost-Belfast. Orte wie der C. S. Lewis Square geben Auskunft darüber, mit welchen Antworten dem Wandel von Belfast begegnet wird. Das Zusammenspiel zwischen urbanen Veränderungen und unionistischem Agieren präsentiert sich in diesem Kapitel in einer neuen, weil mutigen und fortschrittsfreundlichen Form. Das aktive Erinnern an eine andere Geschichte von Ost-Belfast ist somit auch als Versuch zu lesen, Anschluss zu finden an das Narrativ vom befriedeten Belfast. K Strategien von Frieden und Erneuerung in Belfast 249 4.1 „Belfast is a transformed space“: Führungen durch das Kulturerbe des Ostens „It is once again a place we can be proud of“ meint Tourguide Laura ganz am Ende der knapp dreistündigen Yardmen Walking Tour, quer durch den industriellen Nachlass der hiesigen Schiffsindustrie.11 Es ist ein sonniger Nachmittag im August, allein der lausig kalte Wind lässt keine Sommergefühle aufkommen. Die 22-köpfige Gruppe wirkt nach dem langen Fußmarsch erschöpft, doch mit ihren Abschlussworten gewinnt Laura alle Aufmerksamkeit zurück. Die Tour begann und endete am Eastside Visitor Centre, die Backsteinfassade des Gebäudes verbindet die Nostalgie lokaler Industriegeschichte mit den Vorzügen zeitgenössischer Architektur, hier in Form von vielen Fenstern und Glaselementen. Das Besucherzentrum ist nicht besonders groß, trotzdem bildet das Gebäude, gemeinsam mit den Metallskulpturen des C. S. Lewis Square und einem kleinen Nachbarschaftsgarten, einen Dreh- und Angelpunkt im sozialen Leben des Ostens. Dieses Areal und der damit verbundene Tourismus, so Tourguide Laura abschließend, schaffe etwas lang ersehntes: „Giving us a sense of pride in what we once had“. Stunden zuvor, als das Sonnenlicht tatsächlich mit vergleichsweise sommerlichen Temperaturen daherkam, beginnt die Tour „into the footsteps of the Yardmen“, wie Laura es formuliert, später spricht sie von „men who built giants“. Die Yardmen Tour ist eine Erzählung über die Größe und Bedeutung des Industriestadtteils OstBelfast, weit über den Schiffsbau hinaus, von der Leinen-Produktion bis zum besten italienischen Eis weit und breit. Die Industrialisierung veränderte diesen Teil von Belfast grundlegend. Die schweren Arbeitsbedingungen in den Werften, der ökonomische Verfall, heftige Zerstörungen durch Bombenangriffe der deutschen Luftwaffe im Zweiten Weltkrieg, schließlich der Nordirlandkonflikt und dessen Nachwirkungen: „I don’t want to give the impression that it was glorious“, sagt Laura über die Lebensbedingungen der Arbeiterschaft, sinnbildlich gilt diese Aussage wohl für das gesamte Viertel. Doch am Ende sind es die positiven Entwicklungen, welche die Tour maßgeblich prägen. Laura betont das stolze Erbe der Yardmen, darüber hinaus lobt sie die Entwicklungen der letzten Jahre, den Frieden und den Tourismus. Tourguide Laura trifft den Geist ihrer Gruppe, wenn sie schließlich resümiert: „Belfast is a transformed space“. Dieser Wandel soll auch auf Häuserwänden in den Straßen rund um das Visitor Centre sichtbar werden. Dutzende Wandgemälde entstanden in den letzten Jahren und sollen Botschaften von Frieden und Aufbruch vermitteln: Looking to a Better Future steht es an einem Haus geschrieben, Dream, Seek, Educate, Achieve! an einem anderen. In direkter Nachbarschaft finden sich prominente Fassaden, an welchen sich die UVF ihren Anteil am Straßenbild sichert: The Ulster Conflict Is About Nationality. This We Shall Maintain ist eine Botschaft. An anderer Stelle ist es eine schwarz gestrichene Giebelwand, in ihrer Mitte ein weißer Kreis, die Losung We Are The Pilgrims, Master; We Shall Go Always A Little Further sowie drei mit Maschinenpistolen und Handgranaten ausgerüstete, vermummte Loyalisten. Ost-Belfast erlebt die Gleichzeitigkeit von bewaffneten Geistern der Vergangenheit, bunten Frie11 8. August 2017. K 250 F. Schiedlowski densbilder und dem Kulturerbe einer lokalen Fußballikone. Sie alle streiten um ihr symbolisches Überleben im Frieden. 4.2 „It’s a lovely part of Belfast“: Heimat von George Best „Has anyone ever met someone from the Best family?“ fragt Tourguide Peter, ein Herr im besten Alter und im Shirt des nordirischen Fußballverbands, in die zehnköpfige Teilnehmerrunde.12 Peter akzeptiert das kollektive Nein aus der Gruppe, doch eine Bekanntschaft mit dem Fußballtalent von Manchester United oder seiner Familie hätte ihn vermutlich auch nicht tiefer überrascht. Auf der George Best Bus Tour erzählt Peter nicht vorrangig die Geschichte einer Sportikone, sondern die eines East Belfast Boys, welcher den Stolz der hiesigen Fußballwelt verkörperte und dessen früher Tod im Jahr 2005 noch immer eine Zäsur darstellt. Seine fußballerische Heimat fand Best bei Manchester United, für die nordirische Nationalmannschaft hingegen spielte er nur selten, auch aufgrund seines exzessiven Lebensstils, für den Best im Laufe der Jahre ähnlich berühmt wurde wie für sein sportliches Talent.13 Diese Schattenseite aus dem Leben von George Best wird von Peter jedoch so gut es geht ausgeblendet, Fragen und Kommentare dazu hält er klein. Über Best, so hält es Peter seit jeher, wird nur mit größtem Respekt und Anstand gesprochen. Wir halten im Burren Way in Outer East Belfast, die Gruppe entert das alte Reihenhaus der Familie Best, hier wuchs die Ikone auf. „This is living history, you can book it for 70 Pound a night“ sagt Peter, heute ist das nach Vorbild der 1970er Jahre eingerichtete Haus im Besitz der örtlichen Tourismusbehörde. Jeder von uns bekommt eine Tasse Tee, während Peter voller Stolz ein von Best gezeichnetes Trikot präsentiert, er zeigt zudem Zeitungen herum, welche nach seinem Tod erschienen – Remember me for my Football, titelte der Belfast Telegraph. Auf einem Platz ganz in der Nähe lernte Best das Fußballspielen, ein Wandgemälde erinnert hier an ihn. Noch heute, so Peter, leben Katholiken und Protestanten in diesem Wohnviertel zusammen: „It’s a lovely part of Belfast“. „I feel very proud of it. A very, very sad occasion. But what a legacy“: Peter kommt auf die Beerdigung von Best zu sprechen. Zu Tausenden säumten die Menschen von Ost-Belfast und darüber hinaus die Straßen um ihrem Belfast Boy die letzte Ehre zu erweisen. Roselawn Cemetery, Grabstätte von George Best, markiert dann auch den letzten Punkt der Tour. Mit Zustimmung seiner Familie ist dieser Ort der Öffentlichkeit zugänglich, Fans bringen Blumen, Fotos und Fanschals zu dem ansonsten schlichten Grab. „I couldn’t come here for ten years. I couldn’t face it“, bringt Peter seine Anteilnahme zum Ausdruck. 12 Tour, 11. August 2017. 13 Bests Alkoholsucht war kein Geheimnis. Über viele Jahre war es der Öffentlichkeit möglich, Bests sportliche, gesundheitliche und familiäre Probleme mitzuerleben. Wenige Monate vor seinem Tod attestierte ihm die BBC: „Arguably the most naturally gifted footballer of his generation, Best is famous for squandering his skill and failing to achieve his full potential“ (14. Juni 2005). K Strategien von Frieden und Erneuerung in Belfast 251 4.3 Kulturerbe als Ausweg Der Zustand relativen Friedens gibt Ost-Belfast die Möglichkeit, Kulturerbe fernab des Nordirlandkonflikts zu produzieren. Die Beispiele der Yardmen-Tradition oder des Gedenkens an George Best machen deutlich, dass Rückgriffe auf die Vergangenheit für die Kulturerbeproduktion zwingend notwendig sind. Um aus Fragmenten lokaler Geschichte jedoch einen identitäts- und sinnstiftenden Faktor zu machen, müssen Teile dieser Geschichte im Hier und Jetzt neu definiert, konstruiert und zu einem einenden Gebilde modelliert werden (Kirschenblatt-Gimblett 1995, S. 370). Darüber hinaus verrät das aus der Vergangenheit entlehnte und in der Gegenwart nutzbar gemachte Kulturerbe auch etwas über die Zukunft: Im Konstruieren einer stolzen Vergangenheit spiegeln sich nicht selten Wünsche und Vorstellungen. Ist die Vergangenheit und Gegenwart geordnet und von lästigem Ballast bereinigt, kann sie als Inspiration für kommende Entwicklungen dienen (Bryant und Knight 2019, S. 196, 200). Führungen durch das Kulturerbe von Ost-Belfast spielen nicht allein im Gestern, sie appellieren viel mehr daran, lokalen Stolz wiederzuentdecken und in die Stadt zu tragen. Die Ethnologen Jean und John Comaroff sprechen in solchen Fällen von empowerment, wenn sie die Suche nach einem kommodifizierbaren und in vielerlei Hinsicht gewinnbringenden Alleinstellungsmerkmal beschreiben (2009, S. 15). Weltweit vonstattengehende Prozesse der Kulturerbeproduktion, inklusive der damit einhergehenden ökonomischen und politischen Erfolge, werden auch in Ost-Belfast als Alternativen, „als vorgestellte Leben“ wahrgenommen und eröffnen die Möglichkeit, auch die eigene Zukunft in diese Richtung zu imaginieren (Appadurai 1996, S. 53). 5 Schluss Ziel dieses Artikels war es, zu erkennen, wie sich unionistische Akteure von Belfast in einem Friedensprozess zweier Geschwindigkeiten positionieren, wie er in Nordirlands Hauptstadt auftritt. Das Bild der befriedeten Stadt hat die Assoziationen zu Terror und Konflikt nicht verdrängt, sondern ergänzt. Im Zustand von Belfast lässt sich somit die Parallelität eines erfolgreichen und eines stagnierenden Friedensprozesses erkennen. Für Unionisten, welche sich durch vermeintliche falsche Versprechen und der Omnipräsenz vom Albtraum eines vereinigten Irlands als Verlierer des Friedensprozesses erachten, bedeutet das eine Assoziation mit dem konfliktträchtigen Belfast. Für Akteure wie Oliver und Jack in Sandy Row oder Laura und Peter in Ost-Belfast gilt es, sich von diesem Narrativ zu lösen und Anschluss zu finden an das aufstrebende Belfast. In beiden Stadträumen lassen sich kreative Antworten auf den Wandel von Belfast und die Hürden des Friedensprozesses erkennen, in Form von kulturpolitischem Widerstand oder Anpassung. Diese Antworten und Strategien werden im öffentlichen Raum geschaffen und in Stellung gebracht gegenüber den neuen politischen Deutungswelten vom boomenden Belfast. Das Verhalten gegenüber diesem urbanen Wandel kann auch als Suche nach einem Platz im hiesigen Friedensprozess verstanden werden. Egal ob die Strategie Abschottung oder An- K 252 F. Schiedlowski passung lautet, es lässt sich in beiden Fallstudien erkennen, dass urbane Friedenskonsolidierung durch den Zugriff auf neue Stadtnarrative passiert. Dieser Artikel macht mit seiner ethnographischen Herangehensweise deutlich, dass übergeordnete Prozesse wie Frieden und Konflikt vor Ort konstruiert, negiert und gelebt werden. Der Blick auf Sandy Row und Ost-Belfast ermöglicht somit einen alternativen Blick auf regionale Entwicklungen des nordirischen Friedensprozesses. In beiden protestantischen Stadtvierteln bestimmt der Umgang mit der lokalen Vergangenheit über gegenwärtige Verortungen unionistischer Akteure im Friedensprozess. Es ist davon auszugehen, dass der Wandel von Belfast zum Ideal einer post conflict city fortgeschrieben wird. Diese Entwicklung betrifft verlangt von den handelnden Akteuren vor Ort Antworten auf Prozesse urbaner Erneuerung, auf Friedensnarrative, vielleicht gar auf den konstitutionellen Status des Landes. Nordirland feiert im Jahr 2021 sein hundertjähriges Bestehen. Doch Nordirland als Herz und Heimathafen des Unionismus steht zur Diskussion. Umfragen deuten an, dass sich eine knappe Mehrheit der Nordiren im Zuge des britischen EU-Austritts für ein vereinigtes Irland aussprechen könnte. Die Verlässlichkeit der Daten ist dabei von nachrangiger Bedeutung, viel mehr ist die Irland-Frage im Zentrum der politischen Auseinandersetzung angekommen. All diese Debatten spiegeln sich im Stadtraum, hier konstituieren sich daraufhin Strategien und Antworten. Die in Belfast anzutreffende Gleichzeitigkeit von erfüllten und unerfüllten Friedensversprechen, von urbanem Aufstieg und Verfall, von unionistischem Stolz sowie der Angst vor einem vereinigten Irland ist hier kein Widerspruch, sondern Zustand der gelebten Alltagswelt. Funding Open Access funding enabled and organized by Projekt DEAL. Open Access Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenz veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/ licenses/by/4.0/deed.de. Literatur Appadurai, Arjun. 1996. Modernity at Large. Cultural Dimensions of Globalization. Minneapolis: University of Minnesota Press. Aughey, Arthur. 2000. Learning from ‘the Leopard. In Aspects of the belfast agreement, Hrsg. Rick Wilford, 184–201. Oxford: University of Oxford Press. Bardon, Jonathan. 1992. A history of ulster. Belfast: Blackstaff Press. Basra, Rajan, und Peter R. Neumann. 2018. The crime-terror nexus in the United Kingdom and Ireland. London.: International Centre for the Study of Radicalisation. BBC. 2005. Best. Decline of the golden boy. http://news.bbc.co.uk/2/hi/uk_news/4090840.stm; (Erstellt: 14. Juni 2005). Zugegriffen: 17. Aug. 2020. K Strategien von Frieden und Erneuerung in Belfast 253 BBC. 2017. Protest staged to ‘save Belfast’s Boyne bridge. http://www.bbc.com/news/uk-northernireland-40874628; (Erstellt: 9. Aug. 2017). Zugegriffen: 17. Aug. 2020. Belfast Interface Project. 2017. Interfacebarriers, peacelines and defensive architecture. http://www. belfastinterfaceproject.org/publication/interface-barriers-peacelines-and-defensive-architecture. Zugegriffen: 17. Aug. 2020. Belfast News Letter. 2011. Sinn Fein members with IRA links. http://www.newsletter.co.uk/news/sinnfein-members-with-ira-links-1-2738798 (Erstellt: 3. Juni 2011). Zugegriffen: 12. Dez. 2018. Belfast, Love. 2017. A new £1.3 million visitor attraction at Belfast city hall. http://lovebelfast.co.uk/ 43878-2/; (Erstellt: 15. Mai 2017). Zugegriffen: 17. Aug. 2020. Brewer, John D. 2010. Peace processes. A sociological approach. Cambridge: Polity Press. Brown, Kris, Gordon Gillespie, Allan Leonhard, und Yvonne Murphy. 2001. Troubled images. Posters and image of the Northern Ireland conflict. Belfast: Linen Hall Library. Brown, Colin, und David McKittrick. 1997. Blair’s promise of peace to Ulster’s young. Unionists give lukewarm response to new timetable, in: The Independent. http://www.independent.co.uk/news/ blairs-promise-of-peace-to-ulsters-young-1257986.html (Erstellt: 26. Juni 1997). Zugegriffen: 17. Aug. 2020. Bryan, Dominic. 2000. Orange parades. The politics of ritual, tradition and control. London: Pluto Press. Bryant, Rebecca, und Daniel M. Knight. 2019. The anthropology of the future. Cambridge: Cambridge University Press. Bury, Robin. 2017. Buried lives. The protestants of Southern Ireland. Dublin: The History Press Ireland. Clifford, James, und George Marcus (Hrsg.). 1986. Writing culture. The poetics and politics of ethnography. Berkeley: University of California Press. Comaroff, Jean, und John Comaroff. 2009. Ethnicity. Chicago: University of Chicago Press. Cromie, Claire. 2015. Gay marriage now has overwhelming support in Northern Ireland—poll, in: Belfast Telegraph. http://www.belfasttelegraph.co.uk/news/northern-ireland/gay-marriage-now-hasoverwhelming-support-in-northern-ireland-poll-31355428.html; (Erstellt: 6. Juli 2015). Zugegriffen: 17. Aug. 2020. Doherty, Paul, und Michael A. Poole. 1997. Ethnic residential segregation in Belfast, Northern Ireland, 1971–1991. Geographical Review 87(4):520–536. Downs, Joni, Victor Messev, und Peter Shirlow. 2009. The geography of conflict and death in Belfast, Northern Ireland. Annals of the Association of American Geographers 99(5):893–903. Edwards, Aaron. 2011. The northern Ireland troubles. Operation banner 1969–2007. Oxford: Osprey Publishing. Edwards, Aaron. 2017. UVF. Behind the mask. Newbridge: Merrion Press. Gray, Ann Marie, Hamilton Jennifer, Kelly Gráinne, Brendan Lynn, Martin Melaugh, und Gillian Robinson. 2018. Northern Ireland peace monitoring report. Bd. 5. Belfast: Community Relations Council. Hodson, Pete. 2019. Titanic struggle. Memory, heritage and shipyard deindustrialization in Belfast. History Workshop Journal 87:224–249. Jarman, Neil. 1997. Material conflicts. Parades and visual displays in Northern Ireland. Oxford: Routledge. Jarman, Neil. 1998. Painting landscapes. The place of murals in the symbolic construction of urban space. In Symbols in Northern Ireland, Hrsg. Anthony D. Buckley, 81–98. Belfast: Institute of Irish Studies, Queen’s University Belfast. Jarman, Neil. 2016. The challenge of peace building and conflict transformation. A case study of northern Ireland. Kyiv-Mohyla Law and Politics Journal 2:129–146. Kirschenblatt-Gimblett, Barbara. 1995. Theorizing heritage. Ethnomusicology 39(3):367–380. Komarova, Milena, und Liam O’Dowd. 2013. Three narratives in search of a city. Researching Belfast’s ‘post-conflict’ transition. City 17(4):526–546. Kuper, Adam. 1999. Culture. The anthropologists’ account. Cambridge: Harvard University Press. Kuper, Hilda. 1972. The language of sites in the politics of space. American Anthropologist 74(3):411–425. Law, Gary. 1998. The cultural traditions dictionary. Belfast: Blackstaff Press. Lonely Planet (o. J.). Welcome to Belfast. http://www.lonelyplanet.com/ireland/northern-ireland/belfast. Zugegriffen: 17. Aug. 2020. Lynch, John. 2012. Belfast built ships. Stroud: The History Press. Mah, Alice. 2012. Industrial ruination, community, and place. Landscapes and legacies of urban decline. Toronto: University of Toronto Press. Maurer, Michael. 2013. Geschichte Irlands, 3. Aufl., Stuttgart: Reclam. McAuley, James W. 2004. ‘Just fighting to survive’. Loyalist paramilitary politics and the progressive unionist party. Terrorism and Political Violence 16(3):522–543. K 254 F. Schiedlowski McCoy, Gordon, und Camille O’Reilly. 2003. Essentializing ulster? The ulster-scots language movement. In Language and tradition in Ireland. Continuities and displacements, Hrsg. Colin Ireland, Maria Tymoczko, 156–171. Amherst: University of Massachusetts Press. McDonald, Henry, und Frances Perraudin. 2015. Blair: Without IRA Letters, Peace Process would have Collapsed. Giving Evidence to MPs, Former Prime Minister Defends His Role in Allowing the OnThe-Run Scheme in 1999, in: The Guardian. http://www.theguardian.com/politics/2015/jan/13/tonyblair-defends-on-the-run-letters-ira-northern-ireland; (Erstellt: 13. Jan. 2015). Zugegriffen: 17. Aug. 2020. McKee, Lyra. 2017. The suicide of the ceasefire babies, in: the open university. http://www.open.edu/ openlearn/health-sports-psychology/mental-health/the-suicide-the-ceasefire-babies?in_menu=730 993; (Erstellt: 25. Aug. 2017). Zugegriffen: 17. Aug. 2020. Murtagh, Brendan. 2018. Contested space, peacebuilding and the post-conflict city. Parliamentary Affairs 71:438–460. Nagle, John. 2017. Between conflict and peace. An analysis of the complex consequences of the good friday agreement. Parliamentary Affairs 71:395–416. Neill, William J.V. 2011. The debasing of myth. The privatization of titanic memory in designing the ‘postconflict’ city. Journal of Urban Design 16(1):67–86. Nolan, Paul. 2018. The crucial peace. Killings in Northern Ireland since the good friday agreement, in: the detail. http://www.thedetail.tv/articles/the-cruel-peace-killings-in-northern-ireland-since-the-goodfriday-agreement; (Erstellt: 23. Apr. 2018). Zugegriffen: 17. Aug. 2020. Novosel, Tony. 2013. Northern Ireland’s lost opportunity. The frustrated promise of political loyalism. London: Pluto Press. O’Connell, Sean. 2012. An age of conservative modernity, 1914–1968. In Belfast 400. People, place and history, Hrsg. S.J. Connolly, 271–315. Liverpool: Liverpool University Press. O’Doherty, Malachi. 2015. Lost in translation. Loyalism and the media. In The contested identities of ulster protestants, Hrsg. Thomas Burges, Mulvenna, und Gareth, 31–38. London: Palgrave Macmillan. Ortner, Sherry B. 1999. The fate of ‘culture’. Geertz and beyond. Berkeley: University of California Press. Parr, Connal. 2017. Inventing the myth. Political passions and the ulster protestant imagination. Oxford: Oxford University Press. Save the Boyne Bridge. Front page. http://www.boynebridge.co.uk/435567839;. Zugegriffen: 17. Aug. 2020. Shields, Rob. 1996. A guide to urban representation and what to do about it. Alternative traditions of urban theory. In Re-Presenting the City. Ethnicity, Capital and Culture in the 21st-Century Metropolis, Hrsg. Anthony D. King, 227–252. New York: New York University Press. Shirlow, Peter. 2006. Belfast. The ‘post-conflict’ city. Space and Polity 10(2):99–107. Theroux, Paul. 1983. The kingdom by the sea. In 1999: the belfast anthology, Hrsg. Patricia Craig, 73–74. Belfast: Blackstaff Press. Translink. 2017. Belfast transport hub and weavers cross. Community update August 2017. http://www. translink.co.uk/Translink-Footer/the-hub. Zugegriffen: 20. Dez. 2018. Vallely, Finton. 2008. Scenting the paper rose. The ulster-scots quest for music as identity. In Irish protestant identities, Hrsg. Mervyn Busteed, Frank Neal, und Jonathan Tonge, 247–256. Manchester: Manchester University Press. Wilson, Robin. 2016. Northern Ireland peace monitoring report. Schriftenreihe des Northern Ireland Community Relations Council, Bd. 4. Belfast: Community Relations Council. Felix Schiedlowski (M.A.) ist Doktorand und wissenschaftlicher Mitarbeiter am Seminar für Ethnologie der Martin-Luther-Universität Halle-Wittenberg. K
https://openalex.org/W4313271240	https://zenodo.org/records/7491868/files/2472-Article%20Text-7276-1-10-20221209.pdf	English	null	Bloom’s taxonomy for the digital age student in a rural African context	Eureka, Social and Humanities./Eureka, Social and Humanities	2,022	cc-by	5,476	Abstract Bloom’s Taxonomy serves as an important guide for teachers in building their student’s cognition from low to high order thinking. This taxonomy has been widely implemented in face-to-face settings worldwide. With the increased use of technology and blended and online learning on the rise, there is a need for teachers to warm up to digital learning. Teachers must be innovative in us ing technology to improve the quality of education and its delivery. Bloom’s Digital Taxonomy serves as an updated original Bloom’s Taxonomy for designing technology-rich instruction for quality outcomes. This qualitative study explores the use of technology and digital tools to facilitate student learning experiences and outcomes in the modern era. The secondary data collected and analyzed was gathered from existing sources of information. The study recommends that for teachers to be proficient and achieve the best outcomes and add incentives for the students, there is a need to follow the utilization of Bloom’s Digital Taxonomy and consolidate innovation, following the application will get students closer to accomplishing their learning objectives. Teachers need support to prevent forestall disdain and advancement weariness. To conclude the Bloom Digital Taxonomy is well practised in classrooms, and teachers can rest assured that the students they produce will make a difference in this world through the skills they have accumulated during their schooling years. Keywords: bloom taxonomy, digital technology, blooms digital taxonomy, rural learners, African educatio Original Research Article: full paper (2022), «EUREKA: Social and Humanities» Number 6 BLOOM’S TAXONOMY FOR THE DIGITAL AGE STUDENT IN A RURAL AFRICAN CONTEXT Morakinyo Akintolu1 akintl@unisa.ac.za Ntandokamezi Dlamini Department of Curriculum and Instructional Studies2 Moeketsi Letseka1 1UNESCO Chair on Open Distance Learning2 2College of Education University of South Africa 11-14 Winnie Madikizela-Mandela Building, Pretoria, South Africa Corresponding author Original Research Article: full paper Original Research Article: full paper (2022), «EUREKA: Social and Humanities» Number 6 BLOOM’S TAXONOMY FOR THE DIGITAL AGE STUDENT IN A RURAL AFRICAN CONTEXT Ntandokamezi Dlamini Department of Curriculum and Instructional Studies2 1UNESCO Chair on Open Distance Learning2 2College of Education University of South Africa 11-14 Winnie Madikizela-Mandela Building, Pretoria, South Africa Corresponding author (2022), «EUREKA: Social and Humanities» Number 6 Original Research Article: full paper mary school, begins too late in life to meet the issue. Education should be encouraged early and often, both inside and outside of the formal schooling system, according to the Science of Brain Development (year). Programs that include prenatal health and early childhood development are good examples of informal education before children start schooling. The author stresses that qual ity instruction is critical in the early years to provide pupils with the core reading and numeracy skills for lifelong learning. mary school, begins too late in life to meet the issue. Education should be encouraged early and often, both inside and outside of the formal schooling system, according to the Science of Brain Development (year). Programs that include prenatal health and early childhood development are good examples of informal education before children start schooling. The author stresses that qual ity instruction is critical in the early years to provide pupils with the core reading and numeracy skills for lifelong learning. To bridge the gap between teachers and students at an early age for sustainable pedagogical delivery Benjamin Bloom developed a learning theory, known as Bloom’s Taxonomy, which pro vides a framework for developing strong learning objectives for children [2]. Bloom’s Taxonomy serves as a toolbox that teachers and students can use to classify and organise learning objectives. However, when it was created in 1956 eLearning did not exist; it was intended for in-class style teaching training. Therefore, the thrust of this article is how can this learning theory, Bloom’s Tax onomy be adapted to create strong learning objectives through digital technology for children in a rural classroom setting in this era. In recent times, studies have indicated that today’s students, regardless of demographics, have shown an interest in digital learning opportunities, and the range of Web or mobile technology that enables collaboration, innovation, and individual exploration is incredible [5–7]. According to [6], providing students with authentic learning experiences is a critical component of digital learning. An ongoing debate is how teachers and all other stakeholders provide meaningful learn ing experiences for students using intrinsically motivating tools. However, the debate of such needs to provide teachers with an innovative way of integrating digital technology that can encourage higher-order critical thinking skills and creativity among children/ students. (2022), «EUREKA: Social and Humanities» Number 6 In closing the gap, this study seeks to provide a guide for teachers on how to use technology and digital tools to facilitate the student learning experience and outcomes using the framework of Bloom’s Revised Taxonomy, most especially in the rural African context. 1. Introduction “Success provides confidence, security, a sense of well-being, the ability to contribute at a greater level, hope and leadership,” [1]. Every parent wants their child to be successful in life and one way that has been identified as key to ensuring success is education. Parents, therefore, take their children to school to get an education. The people are responsible for ensuring that the dreams and wishes of the parents for their children to achieve success are put in the hands of the teach ers. Teachers are given the responsibility to provide learning experiences for the students, which ensure that the dreams of the parents come true. The education they provide must be increasingly concerned about the whole development of all individuals or learners, and it is the responsibility of the school management, including teachers, to seek learning conditions that will enable individ uals to reach the highest level of learning possible [2]. According to the Sustainable development Goals (SDGs) United Nations Educational, Scientific and Cultural Organization’s (UNESCO’s) agenda for 2030, the task of education is to offer children tools and strategies for access to knowl edge, criteria for interpreting, abilities, and knowledge for living and growing in their life contexts. The SDG goal four states, ‘to ensure inclusive and equitable quality education and promote lifelong learning opportunities for all both rural and urban environments [3]. Noting the importance of education for children, King [4] posits that Early learning skills pave the way for a lifetime of learning. The traditional concept of education, which begins in pri 39 Social Sciences (2022), «EUREKA: Social and Humanities» Number 6 1. 1. Conceptualization of terms Bloom’s Taxonomy: Bloom’s taxonomy is a set of three hierarchical models, used to cate gorize educational learning objectives according to their complexity and specificity. The models categorize learning objectives into three categories: cognitive, affective, and sensory/psychomotor. However, the focus of this paper is on the cognitive domain. Digital/Technology: Technology is the application of scientific knowledge to the practical aims of human life, which promote the use of gadgets, devices, and equipment as well as online network [8]. In other words, technology creates an opportunity for students to learn using online platforms. In this study, technology is a form of learning that requires teaching to be modernised and flexible using various technological tools. Digital Bloom’s Taxonomy: It is a form of selecting an appropriate digital tool by the teach er based on the kind of learning experiences the students need to acquire. Children/Students: The word children vary from place to place [9], according to the [10], refers the word child to anyone under 18 years unless, under the law applicable to the child, majorly attained earlier. Merriam-Webster dictionary defines a child as a young person, especially between infancy and puberty. However, a report, conducted by [11], affirmed that there are some 18 years old individuals who are still children because of a lack of awareness and experience in life, making them incapable of taking the right actions or decisions. For this study, the word students and chil dren are used interchangeably. Rural Area: Rural area varies according to location [12] as well as Akintolu and [8]. Rural areas in developed countries are distinct from those in developing or underdeveloped countries, the majority of which are African. This is due to the characteristics that distinguish each of these regions. For instance, [13] affirm that these are locations that are largely characterized by underde velopment, high rate of poverty, illiteracy, untarred roads, poor electricity supply, and poor internet connectivity among others. In the context of this study, rurality implies areas within the African continent that are characterized by high levels of illiteracy, unemployment, poverty, and untarred and poor road networks. 40 Social Sciences Original Research Article: full paper (2022), «EUREKA: Social and Humanities» Number 6 (2022), «EUREKA: Social and Humanities» Number 6 Rural African context: These are specific strategic locations within and around African settlements that lack development, high levels of illiteracy, poverty, and poor road networks. 1. 2. Rationale of the study The purpose of this study is to explore the use of technology and digital tools to facilitate student learning experiences and outcomes in this era. The original Bloom’s taxonomy served as an excellent tool for guiding teachers in the 50s going forward. The revision, done by Krathwolh and his team, enabled teachers to implement Bloom’s Taxonomy in the 90s. The Covid-19 pandemic has pushed many teaching and learning institutions to online learning after closure to control the spread of the virus. While some teaching and learning institutions are back to face-to-face, others are still fully online. Even those who are back have adopted the blended learning method. Blended learning is a term, coined in the late 1990s [14], which refers to combining face-to-face and computer-medi ated learning [15]. Bloom’s Taxonomy has generally been used in face-to-face situations; this study explores how it can be made relevant in today’s classrooms. This study attempts to show teachers how to embed digital technology in Bloom’s Taxonomy to facilitate students learning experiences and outcomes. This study expands upon the skills, associated with each level of bloom’s taxonomy as technology becomes a more ingrained essential part of learning. 2. Material and Methods This study employed a qualitative approach with an exploratory research design. The broad aim of this study is to systematically review relevant kinds of literature that present an insight into the bloom digital taxonomy and its relevance to education, centred on digital-age students in a rural context. The input of data review is generated by the combination of secondary data from multiple discussions relating to the subject matter. The databases consulted are google scholar, articles on springer link and Taylor and Francis as well as data and reports from international organisations. Fig. 1. Bloom’s Taxonomy revision [17] Knowledge is the basis of the six cognitive processes: remember, understand, apply, an alyze, evaluate, and create. The authors of the new framework also identified different types of knowledge used in cognition: factual knowledge, conceptual knowledge, procedural knowledge, and metacognitive knowledge. The lower-order thinking skills remain at the base of the pyramid with the higher-order skills at the pinnacle. (2022), «EUREKA: Social and Humanities» Number 6 (2022), «EUREKA: Social and Humanities» Number 6 Original Research Article: full paper Original Research Article: full paper Fig. 1. Bloom’s Taxonomy revision [17] 3. 1. An overview of bloom’s taxonomy The cognitive domain of Bloom’s Taxonomy is the most popular one, hence this study’s focus. It assumes that learning should be structured from easy to difficult in the following 6 step levels, arranged from the least to the most difficult level: 1) knowledge; 2) comprehension; 3) application; 4) analysis; 5) synthesis; 6) evaluation. The original Bloom’s Taxonomy served educators well, but a group deemed it important to revise it such that it is more user-friendly. This classification of human cognition, which in cludes thinking, learning and understanding, is a good tool to guide instructional activities as well as assessment in the classroom. These levels are organized in a hierarchy, the bottom three lev els (knowledge, comprehension and application) being the most basic levels of cognition, referred to as lower-order thinking, and the top three levels (analysis, synthesis, evaluation) being higher-or der thinking skills. Teachers use this taxonomy to structure the learning process and guide students from low to high levels of thinking in their learning experiences. A group of researchers, led by David Krathworl and Lorin Anderson, saw a need to revise this taxonomy in the late 90s to make it more relevant and easily usable by 21st-century teachers in their classrooms. The major change they came with was changing the nouns in the hierarchy to verbs. The reasoning was to bring an action to the hierarchy, since learning is an active process rather than a passive one [16]. The nouns were then replaced by action words, i. e. remember, understand, apply, analyse, evaluate and create. They also re-arranged the top two levels, synthesis and evaluation to start with synthesising and then creating. The changes are clearer in the Fig. 1, taken from David Krathwolh. 41 Social Sciences Social Sciences (2022), «EUREKA: Social and Humanities» Number 6 The fourth level is analysed. On the fourth level, we learn to analyse. This involves exam ining and breaking down information into components, determining how the parts relate to one another and finding evidence to support generalizations. We study the lemon flesh, examine the skin and look at the levels of vitamins. We conclude that we can eat everything inside, while the skin tastes bitter and contains traces of toxic pesticides. It ought not to be consumed. The fifth level is evaluation. On this level, we analyse, critique and compare. For example, to evaluate our lemon as a good source of vitamins, we compare it to other sources, such as oranges, grapes and supplements. In this case, we consider the following properties: vitamin levels, afford ability, taste, and packaging waste. If we evaluate our thoughts critically and without bias, we learn where the lemons score high and where others score higher. The sixth and final level is created. Now after we have learned, understood, applied, ana lyzed and evaluated, we are ready to create. As we now really understand lemons, also in compari son to similar things, we can formulate a plan to create our natural lemonade. It is now easy to come up with a cute shop design, a good name sweet lemon and a good slogan natural healthy yummy. So, to design learning activities for children using digital bloom taxonomy, it is important to put all six levels into consideration. As educators and administrators, we need to design content that will engage and provoke critical thinking and problem-solving skills in these learners by putting different alternative approaches into consideration as highlighted in the example of lemonade above. Original Research Article: full paper Original Research Article: full paper Creating Blogging, Building, Animating, Adapting, Collaborating, Composing, Directing, Devising, Podcasting, Wiki Building, Writing, Filming, Programming, Simulating, Role-Playing, Solving, Remixing, Facilitating Designing a Presentation (Prezi or YouTube Channel), Negotiating and Leading 3. 3. Bloom’s Taxonomy Power Verbs Remembering Copying, Defining, Finding, Locating, Quoting, Listening, Googling, Repeating, Retriev ing, Outlining, Highlighting, Memorizing, Networking, Searching, Identifying, Selecting, Tabulat ing, Duplicating, Matching, Curating & Bookmarking and Bullet-pointing. Understanding Copying, Defining, Finding, Locating, Quoting, Listening, Googling, Repeating, Retriev ing, Outlining, Highlighting, Memorizing, Networking, Searching, Identifying, Selecting, Tabulat ing, Duplicating, Matching, Curating & Bookmarking and Bullet-pointing. Understanding Annotating, Tweeting, Associating, Tagging (tagging your curriculum for example), Sum marizing, Relating, Categorizing, Paraphrasing, Predicting, Comparing, Contrasting, Comment ing, Journaling, Interpreting, Grouping, Inferring, Estimating, Extending, Gathering, Exemplify ing and Expressing. Applying Acting out, Articulate, Reenact, Loading, Choosing, Determining, Displaying, Revising Search Keywords, Executing, Examining, Implementing, Sketching, Experimenting, Hacking, In terviewing, Painting, Preparing, Playing, Integrating, Presenting and Charting. Analyzing Calculating, Categorizing (e. g., web content, search results, etc.), Breaking Down, Cor relating, Deconstructing, Strategic Hyperlinking, Supporting (e. g., a cause), Mind-Mapping, Or ganizing, Appraising, Advertising, Dividing, Deducing, Distinguishing, Illustrating, Questioning, Structuring, Integrating, Attributing, Estimating and Explaining. Analyzing Calculating, Categorizing (e. g., web content, search results, etc.), Breaking Down, Cor relating, Deconstructing, Strategic Hyperlinking, Supporting (e. g., a cause), Mind-Mapping, Or ganizing, Appraising, Advertising, Dividing, Deducing, Distinguishing, Illustrating, Questioning, Structuring, Integrating, Attributing, Estimating and Explaining. Evaluating Arguing & Debating, Validating, Testing, Scoring, Assessing, Criticizing, Commenting, Iterating or Pivoting (e. g., a startup or app), Defending, Detecting, Experimenting, Grading, Hy pothesizing, Judging, Moderating, Posting, Predicting, Rating, Reflecting, Reviewing (e. g., a ser vice or platform) and Editorializing. Original Research Article: full paper 3. 2. Level of Bloom’s Taxonomy The first level is remembered. On the first level, we learn to remember. At this level, there is just rote memorization and recollection of facts without much understanding. For example, if we learn about lemons, we want to remember their name, shape, colour, and size and that they are sour. Once we memorized these essentially meaningless facts, we move to the second level of learning. The second level is understood. On the second level, we begin to decode information and learn that a lemon is yellow when it is ripe to eat, and if we take a bite, it is super sour. Furthermore, we also understand that lemons love sunshine and that they contain lots of vitamin C, which is a great natural antioxidant that keeps us healthy. Hence, as we understand a lemon, we can work with it. The third level is applied. On the third level, we apply what we know. We have understood that while lemons are sour, they are also a great provider of vitamin C. To apply this knowledge in a meaningful way. We could boil a lemon in hot water and add some honey. Then serve this hot lemon to our sick sister, who needs treatment. 42 42 Social Sciences (2022), «EUREKA: Social and Humanities» Number 6 other skills. A good understanding of Bloom’s Taxonomy would drive teaching and learning to impact other very important skills, such as critical thinking skills, problem-solving, experiential learning and interpersonal relationship among others. other skills. A good understanding of Bloom’s Taxonomy would drive teaching and learning to impact other very important skills, such as critical thinking skills, problem-solving, experiential learning and interpersonal relationship among others. Creating Creating Canva design. Create online interactive posters Video editing. Create and share online video lessons and explanations YouTube: Upload, edit and share videos Story creation Show me Whiteboard 3. 5. Exploring bloom’s digital taxonomy in the classroom It is important, that teachers create the best experiences for the students and remain relevant, thus, equipping them with skills that will enable them to be marketable and ready for the work place. The integration of the use of technology into Bloom’s Taxonomy creates what is now known as Bloom’s Digital Taxonomy. The purpose of Bloom’s Digital Taxonomy is to help educators use technology and digital tools to facilitate student learning experiences and outcomes (Fig. 2). Fig. 2. Bloom’s Digital Taxonomy activities. Adapted from [18] Fig. 2. Bloom’s Digital Taxonomy activities. Adapted from [18] 44 Creating Canva design. Create online interactive posters Video editing. Create and share online video lessons and explanations YouTube: Upload, edit and share videos Story creation Show me Whiteboard Evaluating Voice thread: Good tools for online discussions Protagonize: publish and collaborate online Google Docs: Create, edit, share, and collaborate on online docs, forms, drawings and spreadsheets Google Classroom Edmodo Zoom Good drive And canva design Analyzing Mindomo: create and share online mind maps Poll Everywhere: Create and share polls/ surveys to collect information Collaborative Classroom: Online classroom discussions Vokiet avatars do the talking for you Evaluating Voice thread: Good tools for online discussions Protagonize: publish and collaborate online Social Sciences 3. 4. Why bloom’s taxonomy Bloom’s Taxonomy helps provoke critical thinking and problem-solving in learners. In ad dition, it elicits innate talents in learners. The learning methodology in a typical African learning space limits learning outcome to the acquisition of knowledge (cognitive skills), and hardly any 43 Social Sciences Original Research Article: full paper (2022), «EUREKA: Social and Humanities» Number 6 Analyzing 44 3. 5. Why bloom’s digital taxonomy should be integrated The integration of technology into classrooms is always praised and teachers are encouraged to integrate it into their lessons. However, it is not always the case; some teachers still do not. There are many reasons for the teachers to appear resisting. Some teachers are digital immigrants [19], also known as BBTs (born before technology), they, therefore, fear being a laughingstock in class, since the younger generation is born into technology and it surrounds most of their lives like the internet, television, computers, emails etc. People are classified according to the time they were born when it comes to technology. [19] describes the classes of people as baby boomers (1940–1959), gener ation X (1960–1979), generation Y (1980–2000) and generation Z (after 2000). Prensky further groups them according to their levels of exposure to technology at an early age; he states that baby boomers and generation Y people are digital immigrants (born before the widespread adoption of digital technology), while generation Y are digital natives (born after the widespread adoption of digital technology). The older generation was exposed to digital technologies later in life, while the younger one is born into them. The older generation is still in schools, teaching the younger ones. It, therefore, becomes very important that they are aware of the era they are teaching in and the kind of students they are teaching, since they are very different from them. They must provide them with an education that is relevant to the times they live in. Times have changed, and so have people and technology. Technology keeps advancing and so should the people; [19] calls this ‘singularity”. It, therefore, becomes important that the gradu ates produced are ready for the workplace and meet the requirements of the job market. It remains the responsibility of the teachers to equip students with all the skills they require to survive in the job market. It is essential now more than before, that student is equipped with digital skills as well as 21st-century skills, identified as critical and creative thinking, problem-solving and communi cation ones. Digital Natives think differently. Unlike digital immigrants, digital natives are more com fortable with new technologies and are less scared of making mistakes [20]. This change in the new generations calls for a change in the way they are taught so that learning takes place. 3. 5. Why bloom’s digital taxonomy should be integrated Teachers cannot stick to the old ways of teaching when teaching the new generation because digital natives learn and communicate in a different way than digital immigrants. Digital natives have a shorter attention span than the traditional way of learning. Bloom’s Digital Taxonomy encourages interaction and creates motivation. Generally, chil dren learn better when they have an interest in something. If they are not engaged in the activity, they can easily get bored and lose interest, and not learn as a result. Deep learning is what Benja min Bloom intended with his taxonomy, and what the Education Department and parents envisage from the students. Making the students interested in the lesson makes even the job of the teacher easy, they get to learn the high-order skills, fostered through the top three levels of Bloom’s Digital Taxonomy. 44 Original Research Article: full paper Original Research Article: full paper (2022), «EUREKA: Social and Humanities» Number 6 (2022), «EUREKA: Social and Humanities» Number 6 Story bird: Write online stories Google map Google slide Simple mind 3. 8. Limitation and suggestions for further study This study is only limited to a systematic review of secondary data from existing pieces of literature. Hence, further study in this regard is suggested through an intervention using digital tools adopting Bloom’s Digital Taxonomy using a quantitative method, which can be used to test the students’ attitude, academic performance and achievement. Also, further study can explore the use of experimental research by having experiments and control groups. 3. 7. How schools can support the use of bloom’s digital technology The provision of gadgets and connectivity – Schools need to invest in the provision of gad gets for teachers and learners. Gadgets can be in the form of laptops and tablets. However, the South African President, Mr Cycil Ramaphosa in his state of the nation address announced that every South African learner will get a tablet [24]. The Minister of Basic Education, Mrs Angie Motshekga affirmed the president’s words and added that the distribution would start in 2020 in multigrade, multiphase and rural schools. According to the plan by the Minister, all learners should have received their tablets by the year 2025. The aim is to enable all learners to access teaching and learning support materials digitally. In the meantime, teachers can work with what they have, allowing students to access computer labs even for non-ICT subjects. Training-It can be challenging and overwhelming for teachers to learn to use technology, es pecially those who are less tech-savvy. Training is very important when one introduces something new. As mentioned above, some of the teachers in schools are digital immigrants, but that does not mean that they cannot learn to use technology. Some are even using it even though they started later in their lives. The key is to give them support in the form of training. Digital immigrants need to be trained before they can be able to use digital technology in their classrooms. They need support from the schools and the Department of Education to achieve this. Training can be in the form of workshops. For the workshops to be effective, they must be ongoing so that if the teachers encounter challenges, they have people assisting them. This can help them feel at ease with the use of technology. 3. 6. How to bloom’s digital taxonomy can be integrated Teachers must find ways to incorporate different teaching strategies to ensure that they cater for the kind of students they are teaching. However, this does not mean that they abandon traditional teaching strategies. The best way would be to use both traditional and modern teaching strategies to produce students who are ready to take on the world. Traditional teaching strategies are more on passive involvement of students, while modern learning strategies call for the involve ment of students in the lesson. For the effective use of Bloom’s digital taxonomy, active learning strategies must be used; teachers must involve in student teaching. Since the younger generation has a short attention span, they will get bored of being passive in the classroom, which could result in the lesson being ineffective. Involving learners actively in the lesson has benefits, they learn to 45 45 Social Sciences (2022), «EUREKA: Social and Humanities» Number 6 (2022), «EUREKA: Social and Humanities» Number 6 Original Research Article: full paper take responsibility for their learning, they get a deeper understanding of the lesson, and their read ing, writing and thinking skills are developed (….). [21] developed the Cone of Learning, which shows that students can remember 70–90 % of what was learnt if they were actively involved in the lesson. Furthermore, [22] states that active participation in the classroom is enjoyed by the students compared to passive. The active involvement of students does not only accommodate the digital natives, but it is also true since students assimilate and process information differently. The VARK model classifies students as visual, aural, read/write and kinesthetic [23]. This model supports the involvement of students when teaching and knowing their needs, so that the teacher is in a better position of assisting them. take responsibility for their learning, they get a deeper understanding of the lesson, and their read ing, writing and thinking skills are developed (….). [21] developed the Cone of Learning, which shows that students can remember 70–90 % of what was learnt if they were actively involved in the lesson. Furthermore, [22] states that active participation in the classroom is enjoyed by the students compared to passive. The active involvement of students does not only accommodate the digital natives, but it is also true since students assimilate and process information differently. The VARK model classifies students as visual, aural, read/write and kinesthetic [23]. This model supports the involvement of students when teaching and knowing their needs, so that the teacher is in a better position of assisting them. Benefits of adopting bloom’s digital taxonomy in education: Benefits of adopting bloom’s digital taxonomy in education: – restructuring of teaching approaches to make it in form of learners centred approach; – it encourages immediate feedback; – it inspires the learners the possibility to actively participate; – improves digital competency; – makes learning fun (gamification); – increases engagement. Social Sciences (2022), «EUREKA: Social and Humanities» Number 6 Original Research Article: full paper References [1] Cardone, G. (2011). Success Is Important! Huffpost. Available at: https://www.huffpost.com/entry/importance-of-suc cess_b_837924#:~:text=Success%20provides%20confidence%2C%20security%2C%20a,entire%20civilizations%20 cease%20to%20survive [1] Cardone, G. (2011). Success Is Important! Huffpost. Available at: https://www.huffpost.com/entry/importance-of-suc cess_b_837924#:~:text=Success%20provides%20confidence%2C%20security%2C%20a,entire%20civilizations%20 cease%20to%20survive [2] Bloom, B. S. (1956). Taxonomy of educational objectives. Vol. I: The cognitive domain. New York: David M [3] Goal 4: Ensure inclusive and equitable quality education and promote lifelong learning opportunities for all. United Nations Statistics Division Development Data and Outreach Branch (2017). New York: UNESCO. Available at: https://unstats.un.org/ sdgs/report/2017/goal-04/ [4] King, E. (2011). Education is Fundamental to development and growth. Education for Global Development. Available at: https://blogs.worldbank.org/education/education-is-fundamental-to-development-and-growth [5] Adelore, O., Akintolu, M. (2016). Effects of Mobile Technology on Adult Learners ‘Achievement in Literacy Programmes in Ibadan. Nigeria Journal of Social Work Education, 15, 187–199. [6] Lightle, K. (2011). More than just the technology. Science Scope, 34 (9), 6–9. [7] Makoe, M. (2011). Academics going mobile: New roles for new technologies. Progressio, 33 (2), 174–188. [8] Akintolu, M., Uleanya, C. (2021). Ensuring Sustainable Development Goal in Rural Africa through Adult Literacy Programme: A Case Study of Technology Usage in Developing Nations. Universal Journal of Educational Research, 9 (4), 713–719. doi: https://doi.org/10.13189/ujer.2021.090401 [9] The African Child Policy Forum (2013) Definition of the Child: The International/Regional Legal Framework. Available at: http:// www.africanchildforum.org/clr/Harmonisation%20of%20Laws%20in%20Africa/other-documents-harmonisation_1_en.pdf [10] Convention on the Rights of the Child (1989). Office of the United Nations High Commissioner for Human Rights (OHCHR) Geneva: OHCHR. Available at: https://www.ohchr.org/EN/ProfessionalInterest/Pages/CRC.aspx [11] UNICEF Annual Report 2020 (2020). UNICEF. [12] Flora, C. B., Flora, J. L., Gasteyer, S. P. (2018). Rural communities: Legacy and change. Routledge. doi: https://doi. org/10.4324/9780429494697 [13] Uleanya, C., Yu, K. (2019). Resilience: Solution to learning challenges amongst rural African undergraduates. Gender and Behaviour, 17 (2), 12921–12935. [14] Lalima, Dr., Lata Dangwal, K. (2017). Blended Learning: An Innovative Approach. Universal Journal of Educational Re search, 5 (1), 129–136. doi: https://doi.org/10.13189/ujer.2017.050116 [15] Graham, C. R. (2006). Blended learning systems. The handbook of blended learning: Global perspectives, local designs, 1, 3–21. [16] Vygotsky, L. S., Cole, M. (1978). Mind in society: Development of higher psychological processes. Harvard university press, 174. doi: https://doi.org/10.2307/j.ctvjf9vz4 [16] Vygotsky, L. S., Cole, M. (1978). Mind in society: Development of higher psychological processes. Harvard university press, 174. doi: https://doi.org/10.2307/j.ctvjf9vz4 [17] Krathwohl, D. R. (2002). A Revision of Bloom’s Taxonomy: An Overview, Theory into Practice, 41 (4), 212–218. doi: http:// doi.org/10.1207/s15430421tip4104_2 [18] Sneed, O. (2016). Integrating technology with Bloom’s Taxonomy. How to cite: Akintolu, M., Dlamini, N., Letseka, M. (2022). Bloom’s taxonomy for the digital age student in a rural African con text. EUREKA: Social and Humanities, 6, 39–47. doi: http://doi.org/10.21303/2504-5571.2022.002472 4. Conclusion For professional educators to achieve the best results and add value for the learners, there is a need to follow the application of Bloom’s Taxonomy and incorporate = technology, follow ing the above application will get students closer to achieving their learning goals. Teachers need support to prevent resentment and innovation fatigue. With this taxonomy well practised in class rooms, teachers can rest assured that the students they produce will make a difference in this world through the skills they have accumulated during their schooling years. 46 46 Social Sciences (2022), «EUREKA: Social and Humanities» Number 6 Original Research Article: full paper Original Research Article: full paper (2022), «EUREKA: Social and Humanities» Number 6 Arizona State University Teach Online. Available at: https:// teachonline.asu.edu/2016/05/integrating-technology-blooms-taxonomy/#comments [19] Prensky, M. (2001). Digital Natives, Digital Immigrants Part 2: Do They Really Think Differently? On the Horizon, 9 (6), 1–6. doi: https://doi.org/10.1108/10748120110424843 [20] Traynor, L. (2019). Exploratory study on the adoption of Financial Technology among Digital Natives and Digital Immigrants in Ireland. Dublin. [21] Dale, E. (1969). Audio-Visual Methods in Teaching. Holt, Rinehart & Winston, New York: Dryden Press, 10 [22] Močinić, S. N. (2012). Active teaching strategies in higher education. Metodički obzori: časopis za odgojno-obrazovnu teoriju i praksu, 7 (15), 97–105. doi: https://doi.org/10.32728/mo.07.2.2012.08 [23] Prithishkumar, I., Michael, S. (2014). Understanding your student: Using the VARK model. Journal of Postgraduate Medicine, 60 (2), 183–186. doi: https://doi.org/10.4103/0022-3859.132337 [24] Government is giving every South African learner a table – here’s who is getting theirs first (2019). Business Tech. Received date 11.10.2022 Received date 11.10.2022 Accepted date 24.11.2022 Accepted date 24.11.2022 Published date 30.11.2022 How to cite: Akintolu, M., Dlamini, N., Letseka, M. (2022). Bloom’s taxonomy for the digital age student in a rural African con text. EUREKA: Social and Humanities, 6, 39–47. doi: http://doi.org/10.21303/2504-5571.2022.002472 47 Social Sciences
https://openalex.org/W4224237022	https://revistas.unjbg.edu.pe/index.php/eyn/article/download/1349/1506, https://dialnet.unirioja.es/descarga/articulo/8617573.pdf	es		Gestión de riesgos en la estrategia de transición a un centro de servicios compartidos: percepción de los colaboradores de una empresa transnacional	Economía y negocios	2,022	cc-by	5,068	Economía & Negocios, Vol. 4 Núm.1, abril-septiembre, pp. 29-40 (2022) ISSN-e 2708-6062 ©Universidad Nacional Jorge Basadre Grohmann, Fondo Editorial Universitario. Tacna, Perú. DOI: https://doi.org/10.33326/27086062.2022.1.1349 ARTÍCULO ORIGINAL Gestión de riesgos en la estrategia de transición a un centro de servicios compartidos: percepción de los colaboradores de una empresa transnacional Risk management in the transition strategy to a shared services center: perception of the collaborators of a transnational company Juan Heriberto Robledo Martínez Universidad Autónoma de San Luis Potosí San Luis Potosí, México Autor de correspondencia: herirobledo@hotmail.com https://orcid.org/0000-0002-0945-5488 Rosalba Martínez Hernández Universidad Autónoma de San Luis Potosí San Luis Potosí, México rosalba.martínez@uaslp.mx https://orcid.org/0000-0003-3721-6094 [Presentado: 21/02/2022, Aceptado: 20/03/2022] RESUMEN Los centros de servicios compartidos son un modelo de negocio que permite eficientar operaciones, ubicándose geográficamente en lugares estratégicos. Entender la visión global de una organización respecto a los riesgos en la transición de un centro de servicio compartido, conocer la forma en que se ha desarrollado la implementación del proceso de gestión de riesgos de acuerdo con las normas internacionales y analizar la forma en que se integra la administración estratégica a los procesos de gestión de riesgos, son los objetivos de este trabajo. Para ello, se recogió la perspectiva de los colaboradores del CoE mediante una encuesta. Los resultados revelan que el concepto de los riesgos y su gestión son ampliamente conocidos en la organización y se cuenta con un ambiente de control que permite mitigar la mayoría de los riesgos que se presentan, dado que la organización está alineada a las directrices emitidas por el Committee of Sponsoring Organizations of the Treadway Commission (COSO) e ISO-31000 mediante la adaptación de un marco propio y una plataforma robusta como parte de su estrategia de gestión de riesgos. La principal contribución de este trabajo es evidenciar la forma en que una empresa líder en la industria llevó a cabo una gestión de riesgos en la transición de las operaciones de sus países al centro de servicios compartidos. Palabras clave: Centros de servicios compartidos, gestión de riesgos empresariales, estrategia, ISO-31000 y COSO. ABSTRACT Shared service centers are a business model that makes operations more efficient, locating geographically in strategic places. Understand the global vision of an organization regarding the risks in the transition of a shared service center, know the way in which the implementation of the risk management process has been developed in accordance with international standards and analyze the way in which it is integrates strategic management to risk management processes, are the objectives of this work. For this, the perspective of the CoE collaborators was collected through a survey. The results reveal that the concept of risks and their management are widely known in the organization and there is a control environment that allows mitigating most of the risks that arise, since the organization is aligned with the guidelines issued by the COSO and ISO-31000 by adapting its own framework and robust platform as part of its risk management strategy. The main contribution of this work is to show the way in which a leading company in the industry carried out risk management in the transition of the operations of its countries to the shared services center. Keywords: Shared service center, enterprise risk management, strategy, ISO-3100 y COSO. Este artículo es publicado por la Revista Economía & Negocios de la Universidad Nacional Jorge Basadre Grohmann. Este es un artículo de acceso abierto, distribuido bajo los términos de la Licencia Creative Commons Reconocimiento 4.0 Internacional. (https://creativecommons.org/licenses/by/4.0/deed.es). 29 Robledo et al. INTRODUCCIÓN Las empresas se ven sometidas a distintas presiones que las obligan a mejorar los niveles de servicios, reducir costos e incrementar sus controles y mantener su desempeño. Para resolver esta situación, han recurrido desde hace varios años al esquema de los servicios compartidos (SC), el cual es un modelo operativo en el que una entidad se especializa en proporcionar un servicio altamente transaccional para diferentes unidades de negocio (UN) con la finalidad de reducir costos, consolidar funciones administrativas y evitar la duplicidad de esfuerzos entre varias UN (Galaz, Yamazaki y Ruiz, S. C., 2013). Hoy en día, los centros de servicios compartidos (CSC) son vitales para toda estrategia comercial. Los CSC tienen una larga historia de desarrollo, evolucionaron de proveedores de actividades contables transaccionales y altamente repetitivas a organizaciones multifuncionales con un enfoque global y funciones adicionales, y ahora están cambiando hacia los servicios comerciales globales (Fane, 2020), lo que implica la integración completa de las actividades comerciales no esenciales en una organización de servicios consistente e independiente, con un enfoque específico en los procesos de extremo a extremo. Ahora bien, se espera que se lleven a cabo no solo actividades transaccionales, sino también intensivas en conocimiento y altamente sofisticadas para crear más valor en el negocio (Pricewaterhouse Coopers, 2019). La finalidad inicial de emplear los Centros de Servicios Compartidos (CSC) fue reducir costos, pero en los últimos años, los CSC han pasado de ser un ‘‘proveedor de lo que piden’’ a un generador de valor comercial tangible, especialmente cuando los CSC están presenciando una mayor penetración en funciones estratégicas y de gran interacción, como soporte al cliente, ventas, marketing y adquisiciones (Deloitte, 2019). Estas tendencias también se han soportado claramente en la literatura (Herráiz et al., 2016). Ante el interés por ubicar los CSC en países como Costa Rica y México, como una estrategia de ubicación de las empresas (Deloitte, 2019) son evidentes las oportunidades, pero también los desafíos en los CSC recolocados en los países señalados, al proveer servicios para la región de América. Existe un amplio abanico de métodos, herramientas y técnicas orientadas al desarrollo de mejores prácticas en el tema de los CSC. Sin embargo, la ejecución no siempre se lleva a cabo como se sugiere en las mejores prácticas consideradas en la fase de planeación, lo que implica que la organización se vuelva vulnerable a ciertos riesgos en la fase de ejecución. Si bien los procesos de gestión de riesgos son observables en algunas empresas, el conocimiento es escaso respecto a la implementación de un proceso de gestión de riesgos empresariales (ERM). La mayoría de estudios sobre ERM se han llevado a cabo en otras partes del mundo (Ahmad, Ng, y McManus, 2014). En definitiva, tanto en el campo de la práctica profesional (Galaz, Yamazaki, Ruiz Urquiza, S.C., 2013; Deloitte, 2019; Fane, 2020; Pricewaterhouse Coopers, 2019), como en el campo académico se han desarrollado numerosos estudios sobre los CSC (Bangemann, 2005; Herráiz López, 2016; Marciniak, 2013; Mikson, Fielt, Bandara y Gable, 2013; Passerini, 2008; Petkovic y Lazarevic, 2012; Schulman, Harmer, Dunleavy, y Lusk, 1999; San Martín, 2002; Ulbrich, 2006; Wirtz, Tuzovic, y Ehret, 2015; Zamorano, 2014); sin embargo, hasta el momento no se ha identificado con los recursos de los que se dispone, o alguna investigación que aborde el tema de servicios compartidos vinculado a la gestión de riesgos y gestión estratégica. Por tanto, un estudio que analice la relación entre estos aspectos cobra Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) 30 Gestión de riesgos en la estrategia de transición a un centro de servicios compartidos: percepción de los colaboradores de una empresa transnacional relevancia, resulta necesario y útil tanto para la comunidad científica como para los profesionales. Así se justifica la conveniencia de analizar los procesos de gestión de riesgos en el contexto de los Centros de Servicios Compartidos alineados a la estrategia de una empresa con presencia global. Por tanto, el objetivo de este trabajo es realizar un análisis de los procesos de gestión de riesgos y administración estratégica bajo las directrices de COSO e ISO-31000 en la transición de un Centro de Servicios Compartidos (CSC) de otros países a México, a través del método de la encuesta. Para ello, es necesario considerar como punto de partida el significado y evolución de los CSC hasta llegar a los servicios globales de negocio Global Business Services (GBS) así como del concepto de gestión de riesgos de conformidad con directrices como la Sarbaness Oxley, COSO 2004 y 2017 e ISO 31000 vinculado a la gestión estratégica (Apartado II). A continuación, se describe la metodología empleada (Apartado III), en donde se identifica a la empresa transnacional objeto de estudio y la estrategia de negocio con el objetivo de traspasar las operaciones de Contraloría y Planeación Financiera de 12 plantas productivas de los países del continente americano al Centro de Experiencia (CoE) situado en San Luis Potosí y se describe la técnica de recolección de datos utilizada. Finalmente se presenta (en el apartado IV de resultados), la percepción de los colaboradores de la empresa objeto de estudio sobre la visión y gestión del riesgo e integración de los procesos de gestión de riesgos a la estrategia del negocio, así como las conclusiones (Apartado V). MATERIALES Y MÉTODOS Los materiales y métodos utilizados en estudios relacionados al tema de gestión de riesgos, han sido estudios de caso desarrollados mediante cuestionarios y entrevistas (Beck et al., 2018; GonzálezFajardo et al., 2018). Para efectos de este trabajo, la empresa sujeta de estudio, es una corporación transnacional de capital extranjero, cuya sede se encuentra en Zúrich (Suiza), cuyos mayores negocios son los de tecnologías en generación de energía eléctrica y en automatización industrial. Actualmente, opera en 100 países y emplea alrededor de 135 mil personas en todo el mundo. El giro del negocio está relacionado con las industrias digitales vinculados originalmente a cuatro negocios enfocados en el cliente a nivel mundial: 1) Electrificación (Electrification-EL), 2) Automatización Industrial (Industrial Automation-IA), 3) Motorización (Motion-MO), y 4) Robótica y Automatización Discreta (Robotics and Discrete Automation-RA), todos ellos respaldados por la plataforma digital propia de la empresa (ABB, 2016). La empresa cuenta con cinco oficinas centrales en el mundo, las cuales incluyen esta modalidad de servicios compartidos y se encuentran en Xiamen (China), Bangalore (India), Cracovia (Polonia), Tallin (Estonia) y San Luis Potosí (México). La oficina central de San Luis Potosí (México) cuenta con 418 empleados en donde se encuentra el Centro de Experiencia en Planeación y Controlaría (Center of Expertise-CoE), el departamento de Servicios de Negocios Globales (GBS), compras, recursos humanos, Sistemas de Información (Information Systems, IS), digitalización de negocios, y logística (Fleet) (ABB, 2016). En este contexto y como parte de la estrategia de negocio del corporativo de la empresa en Suiza, se decidió traspasar las operaciones relacionadas con las áreas de Contraloría y Planeación Financiera de las plantas productivas de los países del continente americano (Canadá, Estados Unidos, México, Panamá, Colombia, Ecuador, Perú, Paraguay, Uruguay, Argentina, Chile y Brasil) al Centro de Experiencia (CoE) situado en San Luis Potosí (México). Esto con la finalidad de ejecutar las actividades de 31 Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) Robledo et al. análisis como son: revisión de costos, participación en el presupuesto anual, revisión gastos mensuales real contra presupuesto, atender requerimientos de auditoría interna y externa, registros contables manuales, cumplimiento con los controles internos establecidos, análisis de cuentas contables, análisis y contrataciones de coberturas para ventas y compras, conciliaciones de cuentas contables, además de actividades no transaccionales para agregar valor al negocio con la experiencia de sus colaboradores, como la digitalización que mediante la herramienta Power BI permite a los usuarios de la información contar con la información financiera al momento que se está generando de una manera más dinámica y rápida (ABB, 2016). La empresa programó y desarrolló un plan de transición de los diversos procesos de negocio de las divisiones de cada país al CoE, para asegurar el éxito de esta transición y también mitigar los riesgos inherentes en esta etapa de transición. El proceso de transición incluyó un primer contacto con las divisiones de los negocios para la adecuada planeación de entrega-recepción de las actividades, permitiéndole al negocio identificar y preparar las actividades a entregar. En una siguiente etapa, se realizaron una o más visitas al país de origen para el comienzo de la transferencia y explicación de las actividades, esto dependió de la complejidad y barreras que surgieron en la primera visita. La siguiente etapa consistió en el control y ejecución “ownership” de las actividades transferidas para iniciar en una etapa posterior la “armonización” de las mismas. Una vez logrando tal armonización, se decide llegar a la etapa de ir en vivo (Go-live) en donde finalmente el CoE se vuelve autónomo e independiente ejecutando las actividades transferidas. Una vez lograda la etapa de ir en vivo (Go-live) el CoE comienza la fase de estabilización, para lograr identificar las lecciones aprendidas, conseguir mejorar el trabajo en equipo, mejorar las habilidades de comunicación en el CoE local y globalmente. Posteriormente, se comienza una fase de estandarización en los procesos y actividades, implementar esfuerzos de digitalización, logrando tener eficiencias en los procesos, implementar indicadores de desempeño “KPI” (Key Performance Indicators) y llevar a cabo encuestas de satisfacción del cliente para medir la calidad de servicio. Para mejorar la calidad de servicio, se llevó a cabo una iniciativa llamada plan táctico de mejora “TIP” (Tactical Improvement Plan) en donde se definieron proyectos que ayudaron a lograr tal iniciativa. Como toda investigación que se apoya en técnicas o instrumentos específicos para la obtención de datos (Mendoza et al., 2018), en este trabajo se utilizó como técnica de recolección de datos, un cuestionario y análisis de resultados mediante estadística descriptiva con el objetivo de recoger la percepción de los colaboradores en una empresa transnacional, respecto al proceso de gestión de riesgos y su integración a la estrategia. El cuestionario diseñado se estructuró en tres secciones: Visión de riesgo (1), proceso de gestión de riesgo (2), e integración de la estrategia en la gestión de riesgo (3) y tiempo (4) con 12 preguntas en total, considerando entre ellas la escala Likert para su diseño. La población objeto de estudio son los colaboradores (analistas, especialistas y líderes de equipos) ejecutores y responsables de los procesos y actividades de Planeación Financiera y Contraloría que laboran en el Centro de Experiencia (CoE) de una empresa de capital extranjero. Así la población del CoE se conformó por el total de los colaboradores que en ese momento prestaban servicio, siendo 58 funcionarios. El cuestionario se aplicó al personal que en su momento estaba presente en las instalaciones del CoE y a todos los niveles jerárquicos analistas, especialistas y líderes. Se obtuvieron 44 cuestionarios completos y una tasa de respuesta de 76 % (Tabla 1). El cuestionario se aplicó en 3 semanas correspondientes al mes de enero del año 2020. Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) 32 Gestión de riesgos en la estrategia de transición a un centro de servicios compartidos: percepción de los colaboradores de una empresa transnacional Tabla 1 Distribución de la muestra por género y área. Área Planeación financiera Contraloría de fabrica Entidad legal Contraloría de proyectos Contraloría funcional Total por género Hombres Mujeres Total 11 3 14 9 6 15 0 1 1 1 4 5 5 4 9 26 18 44 59 % 41 % RESULTADOS Visión de riesgo El concepto de riesgo es usado como un valor esperado, una distribución de probabilidad, como incertidumbre o como un evento. En este contexto, se cuestionó al personal su familiaridad con el concepto de riesgo, el 50 % está familiarizado, el 43 % está muy familiarizado, el 7 % es indiferente respecto al concepto de riesgo. Estos porcentajes demuestran el grado de conocimiento y experiencia de los colaboradores del departamento CoE respecto del concepto de riesgo. Referente al ambiente de control, la gran mayoría de los encuestados (66 % +16 %) consideran que dicho ambiente de control que se está experimentando en el CoE es el adecuado, el 11 % informan que no existe un adecuado ambiente de control y el 7 % tienen una opinión neutral. Los resultados reflejan que la gran mayoría de los colaboradores identifican que existe un ambiente de control adecuado en el CoE. Cuando se asignan tareas nuevas, el 77 % de los encuestados se consideran capaces de identificar inmediatamente los riesgos en dichas tareas, el 14 % mostraron indiferencia a esta situación y el 9 % no identificaron riesgos. Este resultado puede ser atribuido al grado de experiencia de los colaboradores para identificar los riesgos mencionados. Referente al riesgo en la información financiera del negocio, la mitad de los encuestados (36 % + 14 %) contestaron que, si existía riesgo por la inadecuada gestión del riesgo en el CoE, una cuarta parte (23 % + 2 %) respondieron que no percibieron tal riesgo y la otra cuarta parte (25 %) les fue indiferente esta percepción (Figura 1). De este modo, se vuelve de suma importancia rediseñar la estrategia de gestión de riesgos basados más tal vez en controles que mitiguen riesgos financieros. 33 Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) Robledo et al. Figura 1 Resultados de la encuesta de la sección de visión de riesgo Proceso de gestión de riesgo empresarial Respecto a la identificación de riesgos que representaban un peligro para el éxito de la transición de conocimiento del país de origen al CoE, se identificó que más de la mitad de los encuestados (43 % + 14 %) lograron identificar estos riesgos, una quinta parte (18 % + 2 %) no percibieron estos riesgos y el 23 % les fue indiferente a tal situación. Lo que se traduce en que la organización pudiera diseñar e implementar algunos controles mayormente en la etapa de transición para garantizar el éxito de esta transición. En relación con dicha identificación de los riesgos, se preguntó si lo comunicaron a las instancias adecuadas. Más de la mitad de los encuestados (48 % + 23 %) respondieron que si lo comunicaron, el 11 % contestaron que no lo hicieron y casi una quinta parte (18 %) les fue indiferente. Estos porcentajes reflejan una adecuada reacción de los colaboradores al comunicar tales riesgos con los líderes de equipos para evitar un impacto mayor. Respecto si los controles sobre los procesos críticos han sido implementados para mitigar riesgos potenciales, casi tres cuartas partes (52 % + 16 %) de los encuestados consideraron que si se implementaron dichos controles, una minoría (11 %) respondieron que no se implementaron y una quinta parte (20 %) no lograron identificar tal situación. Estos porcentajes indican la adecuada gestión de riesgos que la empresa ABB lleva cabo para mitigar los riesgos que implica un proceso de transición de los procesos de negocio mencionados. Sobre la pregunta si los encuestados consideran que todavía existen riesgos que no han sido mitigados en los diferentes procesos de negocio, la mitad (48 % + 2 %) consideran que todavía existen riesgos, una cuarta parte (16 % + 9 %) consideran que ya no existen riesgos y la otra cuarta parte (25 %) no mostraron conocimiento del tema. Estos resultados reflejan que las acciones llevadas a cabo representan una oportunidad para realizar mejoras en los procesos correspondientes para mitigar los riesgos identificados. Respecto a la segregación de funciones como parte de una mitigación de riesgos, tres cuartas partes de los encuestados (57 % + 18 %) manifestaron que existe una adecuada segregación de funciones, el 11 % respondieron que no existe tales controles y el 14 % desconocen el tema (Figura 2). Estos Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) 34 Gestión de riesgos en la estrategia de transición a un centro de servicios compartidos: percepción de los colaboradores de una empresa transnacional porcentajes indican que la empresa ABB dentro de un contexto de gestión de riesgos, ejecuta una adecuada segregación de funciones. Figura 2 Resultados de la encuesta de la sección de proceso de gestión de riesgos Integración de la estrategia en la gestión de riesgos Sobre la mitigación de riesgos, casi tres cuartas partes de los encuestados (64 % + 9 %) consideran que los riesgos están mayormente mitigados en las actividades que ejecutan, tan solo el 7 % consideran que no están mitigados y el 20 % informan desconocer el tema. Estos resultados denotan que la organización dentro de su estrategia de la gestión de riesgos ha conseguido mitigarlos. Referente a la estrategia en la gestión de riesgos, la gran mayoría de los encuestados (73 % + 9 %) respondieron que el CoE llevó acciones adecuadas para mitigar los riesgos en forma exitosa, una mínima parte (7 % + 2 %) informan que no han sido así, y el 9 % denotan desconocimiento o indiferencia del tema. Respecto a la capacitación para un entendimiento de la gestión del riesgo, casi la mitad de los encuestados (34 % + 11 %) respondieron que el CoE los capacitó para lograr un entendimiento pleno de la gestión de riesgo, casi una cuarta parte (20 % + 2 %) informan que no ha sucedido tal capacitación y el 32 % expresan indiferencia o desconocimiento (Figura 3). Estos resultados denotan que la organización dentro de su estrategia de gestión de riesgos ha realizado esfuerzos por capacitar a sus colaboradores, a fin de lograr que tengan los conocimientos necesarios para identificar riesgos y tomar las adecuadas acciones para su mitigación, pero hay un área de oportunidad referente a la capacitación porque existe indiferencia o desconocimiento de una tercera parte de los participantes. 35 Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) Robledo et al. Figura 3 Resultados de la encuesta de la sección en la Estrategia de Integración de Gestión de Riesgos Ante la pregunta “¿Cuánto tiempo te tomó tener el control total de las actividades que estás desempeñando?” Los encuestados respondieron de la siguiente manera: al 34 % le tomó 6 meses, el 25 % indicó que le tomó 9 meses, el 20 % comentó que le tomó 3 meses, el 14 % respondió que le tomó otro tiempo (no indicado en la encuesta) y el 7 % comentó que le tomó 12 meses (Figura 4). Cabe mencionar que, en la opción de otro tiempo, la mayoría de los casos es personal nuevo que aún están en la etapa de transferencia del conocimiento. Es importante resaltar que en promedio el 18 % de los encuestados respondieron a la opción de ni estar de acuerdo o estar de acuerdo, lo que pudiera indicar indiferencia o desconocimiento del tema sujeto de investigación. Figura 4 Resultados de la encuesta en la sección del tiempo en tener el control de las actividades DISCUSIÓN Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) 36 Gestión de riesgos en la estrategia de transición a un centro de servicios compartidos: percepción de los colaboradores de una empresa transnacional DISCUSIÓN Derivado de los resultados obtenidos, se identifica para la empresa objeto de estudio, el cumplimiento en lo establecido en las directrices COSO e ISO-31000 y que una administración estratégica alineada permite mitigar los riesgos en la transición de un CSC de una empresa transnacional después de ser evaluados. Pareciera algo muy sencillo, sin embargo, los resultados encontrados por González-Fajardo et al. (2018) en un estudio realizado a ocho empresas constructoras en el sureste mexicano, evidencian la dificultad de adoptar adecuadamente la gestión de riesgos, pues solo una empresa observada entendió los beneficios de ERM en todos los niveles de la organización. Lo que indica la necesidad de habilitar al personal responsable de estos procesos en las organizaciones. Si bien las empresas que GonzálezFajardo et al. (2018) analizan, obtuvieron resultados positivos en los componentes de ambiente interno y establecimiento de objetivos, correspondientes a los niveles iniciales de ERM; en los componentes esenciales de ERM, como la evaluación de riesgos y respuesta al riesgo, presentaron puntajes más bajos. Finalmente, los resultados encontrados en este trabajo realizado para una compañía multinacional, se suman a los trabajos que buscan mejorar en los procesos de gestión de riesgos, consistentes con los resultados obtenidos por Beck et al. (2018) en el sector salud, obtenidos mediante entrevistas con 15 directores de riesgos (8 de Estados Unidos y 7 de Brasil) en donde se confirma que la adopción de ERM para las organizaciones ha cobrado impulso y se ha convertido en una prioridad, y que la demanda de orientación respecto a la fase de evaluación sobre el riesgo es común entre los administradores de riesgos en el sector salud. En este contexto, cabe subrayar las oportunidades de investigación respecto al diseño, implementación y formalización de los procesos de gestión de riesgos, en distintos sectores y niveles de las organizaciones a nivel global y sobre todo en países de contextos como el latinoamericano. CONCLUSIONES Entender la visión global de una organización respecto a los riesgos en la transición de un centro de servicio compartido, conocer la forma en que se ha desarrollado la implementación del proceso de gestión de riesgos de acuerdo con las normas internacionales y analizar la forma en que se integra la administración estratégica a los procesos de gestión de riesgos, son los objetivos de este trabajo. Para ello, se recogió la perspectiva de los colabores del CoE mediante una encuesta. Los resultados indican que la visión global de esta organización respecto al significado de riesgos corresponde a una empresa de primer mundo, ya que se identificó que el personal que labora en el CoE está mayormente familiarizado con el concepto de riesgo y logran identificar los riesgos en las tareas que ejecutan, esto demuestra el grado de experiencia y conocimiento en el personal respecto al concepto de riesgo. También, se identifica que existe un adecuado ambiente de control interno con el que se logra tener una mayor confianza en la gestión de riesgos. En cuestión del riesgo en la información financiera es importante rediseñar la estrategia de la gestión de riesgos a una que se base en los controles que mitiguen estos riesgos financieros. Por otro lado, respecto al proceso de la gestión de riesgo empresarial se identificó un área de oportunidad en el diseño e implementación de controles en la transición del conocimiento para garantizar el éxito de 37 Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) Robledo et al. esta. Por el contrario, existió una adecuada comunicación de los colaboradores de los riesgos que estos identificaron en esta etapa. Adicionalmente, la empresa cuenta con una adecuada gestión de riesgos para que estos sean mitigados en este proceso de transición. Y, por último, ABB cuenta con una adecuada segregación de funciones. En materia de integración de la estrategia en la gestión de riesgos, se identificó que en ABB los riesgos están mayormente mitigados; de igual forma, el CoE llevó a cabo acciones para mitigar riesgos en la fase de transición como parte de esta estrategia. Además, la organización dentro de su estrategia de gestión de riesgos realizó esfuerzos por capacitar a sus colaboradores con la finalidad de tener los conocimientos necesarios para identificar riesgos y tomar las adecuadas acciones para su mitigación, aunque también implica un área de oportunidad referente a la capacitación porque existe indiferencia o desconocimiento de una tercera parte de los colaboradores. Como una línea de investigación futura, es posible profundizar sobre las propuestas presentadas en este trabajo relacionadas a robustecer el tema de control interno, reforzar las capacitaciones al personal en materia de control interno, crear conciencia intercultural para un mejor entendimiento de las necesidades de los clientes en otros países. Finalmente, cabe señalar como limitación a este trabajo de tesis, el tamaño de muestra restringida a los colaboradores de una empresa caso de estudio, de ahí que una oportunidad para investigaciones futuras sea ampliar el tamaño de muestra y el número de empresas. REFERENCIAS ABB. (2016). ABBA: Nueva etapa de creación de valor. https://new.abb.com/news/es/detail/36909/ abb-nueva-etapa-de-creacion-de-valor Ahmad, S., Ng, C., y McManus, L. A. (2014). Enterprise risk management (ERM) implementation: Some empirical evidence from large Australian companies. Procedia-Social and Behavioral Sciences, 164, 541-547. https://doi.org/10.1016/j.sbspro.2014.11.144 Bangemann, T. O. (2005). Shared Services in Finance and Accounting. (1st ed.). Routledge. https://doi. org/10.4324/9781315243269 Beck Da Silva Etges, A. P., Grenon, V., Siqueira de Souza, J., Kliemann Neto, F. J., y Felix, E. A. (2018). ERM for Health Care Organizations: An Economic Enterprise Risk Management Innovation Program. Value in Health Regional Issues, 17, 102-108. https://doi.org/10.1016/j.vhri.2018.03.008 Committee of Sponsoring Organizations of the Treadway Commission (2017). Enterprise Risk Management - Integrating with Strategy and Performance. COSO - Executive Summary. https://www.coso.org/Shared%20Documents/2017-COSO-ERM-Integrating-with-StrategyandPerformance-Executive-Summary.pdf Deloitte (2019). Global Shared Services Survey Report Executive Summary. https://www2.deloitte. com/content/dam/Deloitte/us/Documents/process-andoperations/2019-global-shared-servicessurvey-results.pdf Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) 38 Gestión de riesgos en la estrategia de transición a un centro de servicios compartidos: percepción de los colaboradores de una empresa transnacional Fane, S. (28 de junio de 2020). A Definition for Global Business Services. Sharespace. https://sharespace. digital/blog/definition-global-business-services Galaz, Yamazaki, Ruiz Urquiza, S.C. (2013). ¿Qué son los Servicios Compartidos? Diferentes negocios, un solo soporte. https://www2.deloitte.com/content/dam/Deloitte/pe/Documents/processandoperations/shared-services.pdf González-Fajardo, J., Solis-Carcaño, R., y Be-García, J. (2018). ERM in Construction Companies in the Mexican Southeast. International Journal of Construction Engineering and Management, 7(4), 153-161. http://article.sapub.org/10.5923.j.ijcem.20180704.03.html Herráiz López, M. A. (2016). Los centros de servicios compartidos: Razones, Pros y Contras en el ámbito financiero. [Tesis de maestría, Universidad Politécnica de Madrid]. Archivo Digital UPM. https://oa.upm.es/42656/1/TFM_ANTONIA_HERRAIZ_LOPEZ.pdf Marciniak, R. (2013). Measuring Service Satisfaction in Shared Service Organizations. Procedia Social and Behavioral Sciences, 81, 217-223. https://doi.org/10.1016/j.sbspro.2013.06.416 Mikson, S., Fielt, E., Bandara, W., y Gable, G. (2013). Towards a typology of structural arrangements for shared services: evidence from the higher education sector. Electronic Markets, 23(2), 149162. https://doi.org/10.1007/s12525-012-0116-0 Passerini, F. (2008). From internal service provider to strategic partner: An interview with the head of Global Business Services at PyG. Entrevistado por M. Bloch, y E. C. Lempres. McKinsey Digital. https://www.mckinsey.com/business-functions/mckinsey-digital/our-insights/frominternalservice-provider-to-strategic-partner-an-interview-with-the-head-of-global-businessservicesat-p-and-g Petkovic, M., y Lazarevic, S. (2012). Managing Interorganizational Relations: Design of Shared Services Centre. Belgrado: Management Journal for Theory and Practice Management, 64, 5567. http://managementstari.fon.bg.ac.rs/management/e_management_64_engleski_07.pdf Pricewaterhouse Coopers. (2019). PwC: Shared Services-Digitalise Your Services. https://www.pwc. de/de/prozessoptimierung/pwc-studie-shared-services.pdf San Martín, P. (2002). Servicios Compartidos (Shared Services) - Función Financiera que agrega valor. IBM Business Consulting Services - Financial Management Services. https://es.slideshare.net/ PEDROSANMARTIN/8-shared-services Schulman, D. S., Harmer, M. J., Dunleavy, J. R., y Lusk, J. S. (1999). Shared Services Adding Values to the Business Units. New York: John Wiley y Sons, Inc. Ulbrich, F. (2006). Improving shared service implementation: adopting lessons from the BPR movement. Business Process Management Journal, 12(02) 191-205. https://doi. org/10.1108/14637150610657530 39 Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) Robledo et al. Wirtz, J., Tuzovic, S., y Ehret, M. (2015). Global Business Services: Increasing Specialization and Integration of the World Economy as Drivers of Economic Growth. Journal of Service Management, 26(4),565-587. https://doi.org/10.1108/JOSM-01-2015-0024 Zamorano Velásquez, L. A. (2014). Centros de servicios compartidos y su evolución a Servicios Globales de Negocio. [Tesis de pregrado, Colegio de Estudios Superiores de AdministraciónCESA]. Repositorio CESA. http://hdl.handle.net/10726/1219 Economía & Negocios, Vol. 4 Núm.1, pp. 29-40 (2022) 40
https://openalex.org/W2257823342	https://europepmc.org/articles/pmc4491812?pdf=render	English	null	Supramolecular amplification of amyloid self-assembly by iodination	Nature communications	2,015	cc-by	9,390	ARTICLE Received 5 Mar 2015 \| Accepted 19 May 2015 \| Published 30 Jun 2015 Results Si l Single crystal X-ray structure of p-iodo-phenylalanine. The halogen bond21 is the strong, speciﬁc and directional interaction involving halogen atoms as electrophilic species22. Increasing in strength with halogen atom polarizability, halogen bonds are most efﬁcient with iodine substituents. This rather new interaction is virtually unexplored in the ﬁeld of amino acid and peptide self-assembly. For this reason, to evaluate the possibility of exploiting the halogen bond in the self-assembly of amyloid peptides/proteins, we have ﬁrst studied the single crystal X-ray structure of the amino acid p-I-Phe. Amyloid ﬁbrils are characterized by their cross-b-sheet structures whereby bundles of b-strands form highly ordered ﬁlaments that run perpendicular to the ﬁbre axis11,13. The stability of amyloid structures can be attributed to noncovalent interactions, namely hydrogen bonds, p–p stacking and hydrophobic interactions occurring between both side chain and backbone atoms, which hold the b-sheets and strands together11. On the basis of the assumption that noncovalent interactions are key to the formation of the amyloid structure, and since halogen bonds have been recently appreciated as new tools for supramolecular engineering14, we decided to study the impact of introducing halogen-bond donor groups, for example, iodine atoms, into an already known amyloidogenic motif to study the potential of halogen bonding in controlling and promoting amyloid self-assembly. The crystal structure of p-I-Phe (Supplementary Fig. 1 and Supplementary Table 1) highlights the amphoteric behaviour of the iodine atom, which functions as Lewis acid along the extension of the C–I bond and as Lewis base at the belt perpendicular to it (Supplementary Fig. 2a). In fact, the crystal packing of this iodinated amino acid is characterized by two strands of amino acids running along the b crystallographic axis and connected through hydrogen bonds (Supplementary Fig. 2b). These strands are then laterally assembled into two-dimensional supramolecular sheets thanks to type II iodine iodine contacts23, that is, halogen bonds (I I distance 3.7517(4) Å, I I I angle 89.66), occurring at the strand surface (Supplementary Fig. 2c). Importantly, no sign of p–p stacking is observed, while C–H p interactions involving the benzyl hydrogens contribute to aromatic side-chain ordering. Despite the fact that inferring structural information on the amyloid state from high-resolution X-ray diffraction analysis of small-molecule models is difﬁcult, we introduced the p-I-Phe residue into the amyloidogenic core sequence DFNKF24 (NH2-Asp-Phe-Asn-Lys- Phe-COOH, Fig. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 A 1 A myloid ﬁbrils are a class of nanomaterials that can be assembled from a wide variety of peptides and proteins and have an array of functional and pathological roles in nature1. In the latter, the self-assembly process can be triggered by peptide mutations and leads to the formation of insoluble ﬁbrillar aggregates that are associated with Alzheimer’s, Parkinson’s, Creutzfeldt–Jakob and Huntington’s diseases2–6. Amyloid ﬁbrils also perform a number of physiological roles including being constituents of protective envelopes of ﬁsh and insect eggs, essential amphipathic materials of fungi and bacteria and a vital constituent of spider silk7. This wide variety of physiological functions of amyloids has inspired many nanocomposites thanks to the robustness of these ﬁbrillar materials, which are comparable to dragline silk and collagen, as well as biomaterial applications like tissue engineering8–11. In this context, it is important that the amyloidogenic peptide/protein self-assembles in a tunable and controlled manner to provide ﬁbrils of well-deﬁned architecture12. of the obtained hydrogels. This has been explained by the signiﬁcant electronic effects of ﬂuorine and the subsequent perturbation of the energetics of the p–p stacking interactions, although hydrophobic and steric effects could not be discounted20. Conversely, there is only one report on the ﬁbrillar nanostructures given by a diiodinated dipeptide17 but, to the best of our knowledge, no detailed study on the effect of iodination on promoting/inhibiting the self-assembly of amyloidogenic peptides. Under the hypothesis that introducing an iodine atom on the p-position of the benzyl side chain of an aromatic amino acid may result in the formation of halogen bonding thus promoting ﬁbrils’ formation, we studied the structure of p-iodo-phenylalanine (p-I-Phe) and the self- assembly behaviour of a series of iodinated derivatives of a known amyloidogenic core sequence. Results Si l 1), with the purpose of exploiting similar Although many modiﬁcations of amyloidogenic sequences have been utilized to tune their self-assembly behaviour12, halogenation has rarely been pursued and only on single amino acids or on a dipeptide15–17. The advantage of a strategy based on the introduction of halogen atoms on amyloidogenic motifs lies in the fact that halogenation is a minimal structural modiﬁcation, which, on the other hand, may induce a large difference in the peptide supramolecular behaviour as a consequence of the rich variety of noncovalent interactions given by halogen atoms18. It has been shown previously that incorporation of single halogen substituents on the aromatic side chain of amino acids enhances their self-assembly into amyloid-like ﬁbrils that promote hydrogelation in aqueous solvents19. In particular, ﬂuorinated amino acids were found to function better than chlorinated and brominated analogues in terms of assembly kinetics and rigidity O O O O O OH O OH H H2N NH2 NH2 R2 R2 R1 R1 DFNKF H H H H I Br Br CI CI I I I DFNKF(I) DF(I)NKF DF(I)NKF(I) DF(Br)NKF(Br) DF(CI)NKF(CI) hCT15–19 NH2 – Asp – Phe – Asn – Lys – Phe – COOH 1 2 3 4 5 O N H H N N H N Figure 1 \| Molecular formulae of the peptides used in this study. The natural peptide DFNKF was modiﬁed by halogenation on the phenylalanine residues in the para position. Modiﬁcations are shown in the peptide sequence in brackets where F(I) denotes 4-iodophenylalanine, F(Br) denotes 4-bromophenylalanine and F(Cl) denotes 4-chlorophenylalanine. O O O O O OH O OH H H2N NH2 NH2 R2 R1 hCT15–19 NH2 – Asp – Phe – Asn – Lys – Phe – COOH 1 2 3 4 5 O N H H N N H N hCT15–19 Figure 1 \| Molecular formulae of the peptides used in this study. The natural peptide DFNKF was modiﬁed by halogenation on the phenylalanine residues in the para position. Modiﬁcations are shown in the peptide sequence in brackets where F(I) denotes 4-iodophenylalanine, F(Br) denotes 4-bromophenylalanine and F(Cl) denotes 4-chlorophenylalanine. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications Macmillan Publishers Limited All rights reserved NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 2 2 \| \| / \| / & 2015 Macmillan Publishers Limited. All rights reserved. Supramolecular ampliﬁcation of amyloid self-assembly by iodination Arianna Bertolani1, Lisa Pirrie1,2, Loic Stefan1, Nikolay Houbenov3, Johannes S. Haataja3, Luca Catalano1, Giancarlo Terraneo1, Gabriele Giancane4, Ludovico Valli4, Roberto Milani2, Olli Ikkala3, Giuseppe Resnati1 & Pierangelo Metrangolo1,2 Amyloid supramolecular assemblies have found widespread exploitation as ordered nanomaterials in a range of applications from materials science to biotechnology. New strategies are, however, required for understanding and promoting mature ﬁbril formation from simple monomer motifs through easy and scalable processes. Noncovalent interactions are key to forming and holding the amyloid structure together. On the other hand, the halogen bond has never been used purposefully to achieve control over amyloid self-assembly. Here we show that single atom replacement of hydrogen with iodine, a halogen-bond donor, in the human calcitonin-derived amyloidogenic fragment DFNKF results in a super-gelator peptide, which forms a strong and shape-persistent hydrogel at 30-fold lower concentration than the wild-type pentapeptide. This is remarkable for such a modest perturbation in structure. Iodination of aromatic amino acids may thus develop as a general strategy for the design of new hydrogels from unprotected peptides and without using organic solvents. 1 Laboratory of Nanostructured Fluorinated Materials (NFMLab), Department of Chemistry, Materials, and Chemical Engineering ‘Giulio Natta’, Politecnico di Milano, Via Mancinelli 7, Milano I-20131, Italy. 2 VTT-Technical Research Centre of Finland, PO Box 1000, Espoo FI-02044, Finland. 3 Department of Applied Physics, Aalto University, PO Box 15100, Espoo FI-02150, Finland. 4 Dipartimento Beni Culturali, Universita` del Salento, Lecce I-73100, Italy. Correspondence and requests for materials should be addressed to P.M. (email: pierangelo.metrangolo@polimi.it). NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications 1 1 & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 halogen bonds at the ﬁbril strand surface to promote ﬁbril–ﬁbril lamination effects. ﬂuorescence was observed upon mixing ThT with peptide solutions. This is well in accordance with the literature since previous studies have demonstrated that DFNKF does not bind this ﬁbril marker dye29. DFNKF is a short segment (residues 15–19) of the human calcitonin (hCT) hormone25, an amyloidogenic protein with 32 amino acids, whose amyloid formation is related to medullary carcinoma of the thyroid24. This particular pentapeptide is widely used for investigating the nanoscopic arrangement of a ﬁbril complex since DFNKF forms well-ordered ﬁbrils similar to the aggregates of hCT24,25. For this reason, we selected DFNKF as a simple model compound for ﬁbril formation and obtained its halogenated derivatives by introducing I, Br and Cl atoms at the p-position of the phenylalanine (Phe) benzene ring. To study the effect on ﬁbrillation of the introduction of halogen atoms on speciﬁc positions of the pentapeptide, we studied the 5-p-iodo- Phe derivative DFNKF(I), the 2-p-iodo-Phe derivative DF(I)NKF, the 2,5-bis-p-iodo-Phe derivative DF(I)NKF(I), as well as its bis- brominated and bis-chlorinated analogues DF(Br)NKF(Br) and DF(Cl)NKF(Cl), respectively (Fig. 1). Importantly, all of the studied peptides carry free amino (N) and carboxyl (C) termini. y Subsequently, a working concentration of 15 mM was chosen where all halogenated peptides formed clear and elastic gels within a reasonable time (r12 h). At this concentration, the speed of hydrogel formation followed the order DF(I)NK- F(I)4DF(I)NKFZDF(Br)NKF(Br)4DF(Cl)NKF(Cl)ZDFNKF(I), whereas DFNKF did not form a gel even after 30 days (Supplementary Table 3). The diiodinated peptide formed the most homogeneous and shape-persistent gel (Fig. 2b), whereas peptides DF(Cl)NKF(Cl) and DFNKF(I) showed phase separation of the ﬁbrous aggregates from the aqueous phase. The obtained hydrogels were all thermoreversible with gel–sol transition temperatures ranging from 65 C (DFNKF(I) to 119 C (DF(l)NKF(l); Fig. 2c). In particular, the observed transition temperatures followed the same order of the gel forming kinetics reported above. These results clearly show that, in general, halogenation of the DFNKF model peptide greatly promotes hydrogel formation, with the diiodinated peptide being the most efﬁcient. This efﬁciency decreases with the polarizability of the halogen atom, that is, DF(l)NKF(l)4DF(Br)NKF(Br)4 DF(CI)NKF(CI), and also depends on the speciﬁc position of the halogen atom in the peptide scaffold, that is, the peptide iodinated at the Phe in the second position is more efﬁcient than the one iodinated in the ﬁfth. & 2015 Macmillan Publishers Limited. All rights reserved. Hydrogelation properties of halogenated DFNKF derivatives. Hydrogelation is highly indicative of ﬁbrillation, therefore all peptides were in the ﬁrst instance assessed for their ability to form hydrogels26,27. All of the studied peptides were found to self- assemble into hydrogels over a minimum gelation concentration. Only the hydrogel of the wild-type peptide DFNKF has been previously reported, though of its N-acetylated form at the N terminus28. All the reported halogenated peptides are novel types of gelators and, importantly, formed gels (Fig. 2a and Supplementary Table 2) at concentrations much lower than that of their wild-type parent DFNKF, that is, 75 mM, with the super- gelator peptide DF(I)NKF that showed a minimum gelation concentration of o0.2% (2.5 mM; 30-fold lower than DFNKF). Characterization of the different halogenated peptide hydrogels was done by oscillatory rheology (ring-cast method) using the 15 mM concentration. At this low concentration, the wild-type peptide DFNKF only formed a viscous ﬂuid and was not studied further. The diiodinated peptide DF(I)NKF(I) was conﬁrmed to form the stiffest gel, which is reﬂected in its high elastic modulus (G04104 Pa and G044G00; Fig. 2d and Supplementary Fig. 3) and by the fact that the gel assumes the well-deﬁned shape of the mould (Fig. 2b). The G0 value for DF(I)NKF(I) is comparable to Attempts to study the kinetics of ﬁbril formation using Thioﬂavin T (ThT) were unsuccessful since no increase in 1 10 100 1,000 10,000 100,000 Storage modulus, G′ (Pa) Oscillation strain (%) Amplitude sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) 1 10 100 1,000 10,000 100,000 0.1 Storage modulus, G′ (Pa) Angular frequency (rad s–1) Frequency sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) Peptide DFNKF(I) DF(I)NKF DF(I)NKF(I) DF(Br)NKF(Br) DF(Cl)NKF(Cl) WT Halogenated Transition T (°C) 64.7–69.0 88.2–92.7 114.0–119.0 92.1–96.4 78.1–82.4 100 10 1 100 10 1 0.1 0.01 Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. (b) The hydrogel formed by DF(I)NKF(I) was the most shape-persistent of all gels. The gel was prepared using the ring-cast method at 15 mM concentration and imaged after 48 h. (c) Gel–sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. & 2015 Macmillan Publishers Limited. All rights reserved. (d) Rheological characterization of the gels by frequency sweep studies whereby the storage modulus (G0) was recorded as a function of angular frequency (o). The peptide DF(I)NKF(I) was determined to be the strongest gel with G04104 Pa. (e) Amplitude sweep studies of peptide hydrogels showing G0 as a function of oscillation strain (g). All halogenated hydrogels show similar linear viscoelastic region (LVR) proﬁles. Peptide hydrogels at a concentration of 15 mM were utilized for the rheological measurements after 48 h from preparation. For the loss moduli, see the Supplementary Fig. 3. WT Halogenated 1 10 100 1,000 10,000 100,000 Storage modulus, G′ (Pa) Oscillation strain (%) Amplitude sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) 100 10 1 0.1 0.01 1 10 100 1,000 10,000 100,000 0.1 Storage modulus, G′ (Pa) Angular frequency (rad s–1) Frequency sweep DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DFNKF(I) DF(Cl)NKF(Cl) 100 10 1 Storage modulus, G′ (Pa) ) a) Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. (b) The hydrogel formed by DF(I)NKF(I) was the most shape-persistent of all gels. The gel was prepared using the ring-cast method at 15 mM concentration and imaged after 48 h. (c) Gel–sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. (d) Rheological characterization of the gels by frequency sweep studies whereby the storage modulus (G0) was recorded as a function of angular frequency (o). The peptide DF(I)NKF(I) was determined to be the strongest gel with G04104 Pa. (e) Amplitude sweep studies of peptide hydrogels showing G0 as a function of oscillation strain (g). All halogenated hydrogels show similar linear viscoelastic region (LVR) proﬁles. Peptide hydrogels at a concentration of 15 mM were utilized for the rheological measurements after 48 h from preparation. For the loss moduli, see the Supplementary Fig. 3. Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. & 2015 Macmillan Publishers Limited. All rights reserved. Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon aging 12 h at RT After this period, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂuid. (b) The hydrogel formed by DF(I)NKF(I) was the most shape-persistent of all gels. The gel was prepared using the ring-cast method at 15 mM concentration and imaged after 48 h. (c) Gel–sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. (d) Rheological Figure 2 \| Characterization of hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials contain rogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions upon hydrogels formed by halogenated DFNKF derivatives (a) Photograph of vials containing 15 mM peptide solutions up d, all halogenated peptides formed clear hydrogels whereas the wild-type peptide DFNKF (WT) remained as a viscous ﬂ concentration and imaged after 48 h. (c) Gel sol transition temperatures of 15 mM halogenated peptide hydrogels formed in 48 h. (d) Rheological characterization of the gels by frequency sweep studies whereby the storage modulus (G0) was recorded as a function of angular frequency (o). The peptide DF(I)NKF(I) was determined to be the strongest gel with G04104 Pa. (e) Amplitude sweep studies of peptide hydrogels showing G0 as a function of oscillation strain (g). All halogenated hydrogels show similar linear viscoelastic region (LVR) proﬁles. Peptide hydrogels at a concentration of 15 mM were utilized for the rheological measurements after 48 h from preparation. For the loss moduli, see the Supplementary Fig. 3. 3 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 that of Ac-LIVAGD, a hexamer that forms one of the best performing hydrogels in terms of ease of hydrogel formation and strength30,31. Interestingly, the trend of G0 values parallels those of gel formation efﬁciency and thermal stability whereby gels of peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br) are the stiffest and DFNKF(I) and DF(Cl)NKF(Cl) formed the weakest ones. similar to those of the diiodinated peptide DF(I)NKF(I) (Fig. 4a and Supplementary Fig. 4a), which, in turn, had the most complex structures with twisted ribbon-like ﬁbrils tens of nanometre wide and micrometre long. & 2015 Macmillan Publishers Limited. All rights reserved. These results highlight the importance of the speciﬁc position of the iodine atom in the peptide structure and therefore the subsequent speciﬁc inter- actions, which result in different amyloid ﬁbril topographies. That the ﬁbrillation and twisting is not due to drying is excluded by high-resolution cryo-TEM and electron tomography (ET) studies. This is exempliﬁed for the peptide DF(I)NKF(I) corresponding to the strongest hydrogel. It shows ﬁbrils with lateral sizes of 20–25 nm and helical twisting with periodicities between 250 and 140 nm (Fig. 4h and Supplementary Fig. 4b). Such periodicities correspond to the different maturation steps of amyloid ﬁbrils as previously reported for b-lactoglobulin ﬁbrils33. ET allowed three-dimensional visualization of the twisting morphology of a DF(I)NKF(I) ﬁbril imaged in solution (Fig. 4i). Morphology and microarchitecture of the self-assembled ﬁbrillar networks constituting the halogenated hydrogels were evaluated by confocal microscopy (Fig. 3)32. The network of the hydrogel of DF(I)NKF(I) appeared to be very ﬁlamentous with micrometre-long ﬁbrils assembled in bundles with a helical sense, which propagate along the same direction. These micrometre- long ﬁbrils become fewer and thinner on going from DF(I)NKF(I) to DF(I)NKF and DF(Br)NKF(Br), and disappear in the hydrogels of DF(CI)NKF(CI) and DFNKF(I) where only entangled matrices of small ﬁbrils and aggregates were observed. These observations ﬁt well with the rheology data where the strongest gels result from the entanglement of the longest ﬁbrils. In agreement with the confocal microscopy study, the hydrogel formed by the peptide DF(I)NKF(I) showed an intertwined network of long ﬁbrils in its transmission electron microscopy (TEM) image (Fig. 3f). Amyloid structure of ﬁbrils of halogenated peptides. There are three criteria that deﬁne a protein aggregate as an amyloid ﬁbril: green birefringence upon staining with Congo Red, ﬁbrillar morphology and b-sheet secondary structure34. Therefore, Congo red staining was carried out for all the peptides (at the same 15 mM concentration) and strong green-gold birefringence was observed under polarized light, which is a reporter for long-range cross-b structure (Supplementary Fig. 5). The nanostructure of the halogenated ﬁbrils was revealed by atomic force microscopy (AFM) (Fig. 4). The peptides were ﬁrst imaged after incubation in dilute aqueous conditions for 9 days (see also Supplementary Fig. 4) and Fig. 4a–c highlight the most strongly ﬁbrillating peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br). While only small spherical aggregates were observed for the wild-type peptide DFNKF (Supplementary Fig. 4f), all of the halogenated peptides showed pronounced ﬁbrillar structures. & 2015 Macmillan Publishers Limited. All rights reserved. Large differences were, however, observed between the monoiodinated peptides DFNKF(I) and DF(I)NKF: Peptide DFNKF(I) (Supplementary Fig. 4e) showed only small protoﬁbrils and aggregates, whereas the peptide DF(I)NKF (Fig. 4b and Supplementary Fig. 4c) showed large twisted ﬁbrils Further evidence of amyloid nature was obtained by Fourier transform infrared (FT-IR) spectroscopy. The FT-IR spectra of both the solution and the gel state in D2O were recorded (Supplementary Fig. 6). A solution of the wild-type peptide DFNKF showed no signiﬁcant amide I’ band, however, using a concentration at which it forms a gel (75 mM), this band became visible. This peak at around 1,630 cm 1 can be attributed to the peptide bond carbonyl upon formation of the amyloid b-sheet structure and subsequent gelation35. The spectra of the gel state DF(Br)NKF(Br) DF(I)NKF(I) DF(I)NKF DF(Br)NKF(Br) DF(Cl)NKF(Cl) DFNKF(I) DF(I)NKF(I) Figure 3 \| Fibrillar morphologies of halogenated hydrogels. (a–e) Confocal microscopy images of peptide hydrogels at a concentration of 15 mM upon aging 48 h at RTand after staining with Rhodamine B (scale bar, 100 mm). Panels a, b and c show bundles of long twisted ﬁbrils belonging to hydrogels of peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br), respectively. Panels d and e show much smaller ﬁbrils of hydrogels of peptides DF(Cl)NKF(Cl) and DFNKF(I), which intertwine and form a matrix-type structure. (f) TEM image of the dried hydrogel of peptide DF(I)NKF(I) showing an intertwined network of long ﬁbrils (scale bars, 0.5 mm and 5 nm). DF(I)NKF(I) DF(I)NKF DF(I)NKF(I) DF(Cl)NKF(Cl) DFNKF(I) DFNKF(I) Figure 3 \| Fibrillar morphologies of halogenated hydrogels. (a–e) Confocal microscopy images of peptide hydrogels at a concentration of 15 mM upon aging 48 h at RTand after staining with Rhodamine B (scale bar, 100 mm). Panels a, b and c show bundles of long twisted ﬁbrils belonging to hydrogels of peptides DF(I)NKF(I), DF(I)NKF and DF(Br)NKF(Br), respectively. Panels d and e show much smaller ﬁbrils of hydrogels of peptides DF(Cl)NKF(Cl) and DFNKF(I), which intertwine and form a matrix-type structure. (f) TEM image of the dried hydrogel of peptide DF(I)NKF(I) showing an intertwined network of long ﬁbrils (scale bars, 0.5 mm and 5 nm). NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 4 & 2015 Macmillan Publishers Limited. All rights reserved. Electron tomography reconstructions were collected upon tilting of samples up to ±69 (scale bar, 25 nm). of the halogenated peptides showed this same amide I’ band consistent with the formation of amyloid ﬁbrillar species comprising b-sheet elements. These data conﬁrm that all the studied halogenated derivatives of DFNKF form ﬁbrils of the same amyloid nature to their wild-type parent. Interestingly, this 1,630 cm 1 peak is red-shifted in the spectra of DF(I)NKF(I) and DF(I)NKF, while it is not in the one of DFNKF(I) (Supplementary Fig. 6). This red-shift indicates a reduced electron density on the peptide bond and is consistent with the involvement of the carbonyl oxygen in halogen bonding36. others41. A direct correlation exists between the role of oxidative stress and protein ﬁbrillation42. However, how halogenation affects protein structure, folding and functioning is not yet known. To study to what extent a partial degree of halogenation may promote the ampliﬁed formation of amyloid ﬁbrils, we studied the co-assembly43 of the strongly self-assembling diiodinated peptide DF(I)NKF(I) with the wild-type peptide DFNKF. Mixing DFNKF and DF(I)NKF(I) at concentrations at which normally neither form gels alone (Fig. 5a), 15 and 0.75 mM (5%), respectively, resulted in a mixed hydrogel within 18 h. Characterization of the physical properties of the mixed hydrogel was performed by oscillatory rheology (ring-cast method) where the mixed hydrogel was found to be weaker than the hydrogel containing only DF(I)NKF(I) (Fig. 5b versus Fig. 2d). It was also observed that increasing the amount of the strong gelator DF(I)NKF(I) led to an increase in the elastic modulus value, whereas the gel containing 10% was stronger than the one containing 5% of DF(I)NKF(I). Interestingly, circular dichroism (CD) analysis of the mixed hydrogels showed the same structural features observed in the CD spectra of the pure iodinated peptides (see Supplementary Figs 8 and 9). Raman spectroscopy is a powerful tool to investigate the occurrence of the halogen bond37. For this reason, we followed the changes experienced by the C–I stretching band at around 167 cm 1 upon formation of the gel. Interestingly, compared with the bulk powders of the starting materials, in the dried gels of the iodinated peptides DF(I)NKF(I) and DF(I)NKF, this band shifted to lower frequency (see Supplementary Fig. 7). This shift is perfectly consistent with the weakening of the C–I bond as a consequence of its involvement in halogen bonding upon formation of the hydrogels37. & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 0 0 4 0.4 0.8 1.2 μm 8 nm 0 0 0 0.1 0.2 0.3 0.4 0.5 0.6 4 0.4 0.8 1.2 μm μm Helical turn Half-pitch helical cut 8 nm 0 1 3 2 nm 0 0 4 0.4 0.8 25 nm 25 nm 250 nm 20 nm 20 nm 255 nm 150 nm 150 nm 1.2 μm 8 nm 0 0 4 0.4 0.8 1.2 μm 8 12 nm Figure 4 \| Nanoﬁbrillation of the halogenated pentapeptides. AFM image close-up insets of (a) peptide DF(I)NKF(I), (b) peptide DF(I)NKF, and (c) peptide DF(Br)NKF(Br) evaporated on mica substrates after 9 days incubation in aqueous solutions (scale bar, 1 mm). (d–f) Height proﬁles of ﬁbrils from a, b and c (cross-section lines highlighted in white). (g) Cross-sectional analysis on the top of a ﬁbril segment in a showing a helicoidal proﬁle along the longitudinal direction of the ﬁbrils (highlighted in black). (h) Cryo-TEM images of the dried hydrogel of DF(I)NKF, showing a network of long ﬁbrils (scale bar, 200 nm). (i) In situ electron tomography of half-pitch helicoidal peptide DF(I)NKF(I) vitriﬁed from aqueous solution. The twisted ﬁbril morphology is highlighted by arrows. Electron tomography reconstructions were collected upon tilting of samples up to ±69 (scale bar, 25 nm). 0 0 4 0.4 0.8 1.2 μm 8 nm 0 0 4 0.4 0.8 1.2 μm 8 12 nm 0 0.1 0.2 0.3 0.4 0.5 0.6 μm μm Helical turn Half-pitch helical cut 0 1 3 2 nm 0 0.1 0.2 0.3 0.4 0.5 0.6 μm 0 1 3 2 nm 25 nm 20 nm 255 nm 150 nm 25 nm 250 nm 20 nm 150 nm Figure 4 \| Nanoﬁbrillation of the halogenated pentapeptides. AFM image close-up insets of (a) peptide DF(I)NKF(I), (b) peptide DF(I)NKF, and (c) peptide DF(Br)NKF(Br) evaporated on mica substrates after 9 days incubation in aqueous solutions (scale bar, 1 mm). (d–f) Height proﬁles of ﬁbrils from a, b and c (cross-section lines highlighted in white). (g) Cross-sectional analysis on the top of a ﬁbril segment in a showing a helicoidal proﬁle along the longitudinal direction of the ﬁbrils (highlighted in black). (h) Cryo-TEM images of the dried hydrogel of DF(I)NKF, showing a network of long ﬁbrils (scale bar, 200 nm). (i) In situ electron tomography of half-pitch helicoidal peptide DF(I)NKF(I) vitriﬁed from aqueous solution. The twisted ﬁbril morphology is highlighted by arrows. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 (d) AFM of a sample of the mixed hydrogel containing 15 mM DFNKF and 1.5 mM DF(I)NKF(I) showed larger and more entangled ﬁbrils (scale bar, 400 nm). Figure 5 \| Mixed hydrogels formed by co-assembly of DFNKF and DF(I)NKF(I). (a) Photograph of vials after 48 h containing (I) 15 mM DFNKF, which did not form hydrogel; (II) 1.5 mM DF(I)NKF(I), which did not form hydrogel and some precipitation was observed; (III) 15 mM DFNKF:0.15 mM DF(I)NKF(I), which did not form hydrogel; (IV) 15 mM DFNKF:0.75 mM DF(I)NKF(I), which formed hydrogel within 48 h; (V) 15 mM DFNKF:1.5 mM DF(I)NKF(I), which formed hydrogel within 48 h. (b) Rheological characterization of mixed hydrogels by frequency sweep studies whereby the storage concentration of 15 mM after 48 h were utilized for the rheological measurements. (c) AFM of a 15 mM solution of wild-type peptide DFNKF showed small ﬁbrils and aggregates unable to network sufﬁciently to form a hydrogel. (d) AFM of a sample of the mixed hydrogel containing 15 mM DFNKF and 1.5 mM DF(I)NKF(I) showed larger and more entangled ﬁbrils (scale bar, 400 nm). Figure 6 \| Potential halogen bonds involving p-iodo-phenylalanine. IR and Raman spectroscopies are more consistent with the occurrence of type a interaction, however, other interactions reported cannot be discounted. (a) Orthogonal hydrogen and halogen bonds involving the carbonyl oxygen atom of the peptide bond36. (b) Type II iodine iodine contacts23. (c) Iodine p interactions49. (d) Triangular iodine synthon50. too small to crosslink and form a network (Fig. 5c). On the addition of 10% DF(I)NKF(I), the resulting ﬁbrils were much longer and intertwined to form a ﬁbrous network resulting in the hydrogelation of the wild-type peptide DFNKF solution (Fig. 5d and Supplementary Fig. 10a). The weakness of the resulting mixed hydrogel compared with that of DF(I)NKF(I) alone can be explained by the relative size and network formation of the ﬁbrils (Supplementary Fig. 10b). This result may have important implications in a biological setting since only small amounts of brominated or chlorinated peptides may be required to amplify the supramolecular self-assembly of an amyloid ﬁbril. Role of halogen bonding in hydrogels’ self-assembly. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 1 100 10,000 0.1 1 10 100 Storage/loss moduli (Pa) Angular frequency (rad s–1) Frequency sweep 10% DF(I)NKF(I) G′ 10% DF(I)NKF(I) G″ 5% DF(I)NKF(I) G′ 5% DF(I)NKF(I) G″ 15 mM DFNKF 1.5 mM DF(I)NKF(I) 15 mM DFNKF 1 100 10,000 0.1 1 10 100 Storage/loss moduli (Pa) Angular frequency (rad s–1) Frequency sweep 10% DF(I)NKF(I) G′ 10% DF(I)NKF(I) G″ 5% DF(I)NKF(I) G′ 5% DF(I)NKF(I) G″ II III IV V I 15 mM DFNKF 1.5 mM DF(I)NKF(I) Figure 5 \| Mixed hydrogels formed by co-assembly of DFNKF and DF(I)NKF(I). (a) Photograph of vials after 48 h containing (I) 15 mM DFNKF, which did not form hydrogel; (II) 1.5 mM DF(I)NKF(I), which did not form hydrogel and some precipitation was observed; (III) 15 mM DFNKF:0.15 mM DF(I)NKF(I), which did not form hydrogel; (IV) 15 mM DFNKF:0.75 mM DF(I)NKF(I), which formed hydrogel within 48 h; (V) 15 mM DFNKF:1.5 mM DF(I)NKF(I), which formed hydrogel within 48 h. (b) Rheological characterization of mixed hydrogels by frequency sweep studies whereby the storage concentration of 15 mM after 48 h were utilized for the rheological measurements. (c) AFM of a 15 mM solution of wild-type peptide DFNKF showed small ﬁbrils and aggregates unable to network sufﬁciently to form a hydrogel. (d) AFM of a sample of the mixed hydrogel containing 15 mM DFNKF and 1.5 mM DF(I)NKF(I) showed larger and more entangled ﬁbrils (scale bar, 400 nm). 1 100 10,000 0.1 1 10 100 Storage/loss moduli (Pa) Angular frequency (rad s–1) Frequency sweep II III IV V I 10% DF(I)NKF(I) G′ 10% DF(I)NKF(I 5% DF(I)NKF(I) G′ 5% DF(I)NKF(I) 15 mM DFNKF 1.5 mM DF(I)NKF(I) 15 mM DFNKF Figure 5 \| Mixed hydrogels formed by co-assembly of DFNKF and DF(I)NKF(I). (a) Photograph of vials after 48 h containing (I) 15 mM DFNKF, which did not form hydrogel; (II) 1.5 mM DF(I)NKF(I), which did not form hydrogel and some precipitation was observed; (III) 15 mM DFNKF:0.15 mM DF(I)NKF(I), which did not form hydrogel; (IV) 15 mM DFNKF:0.75 mM DF(I)NKF(I), which formed hydrogel within 48 h; (V) 15 mM DFNKF:1.5 mM DF(I)NKF(I), which formed hydrogel within 48 h. (b) Rheological characterization of mixed hydrogels by frequency sweep studies whereby the storage concentration of 15 mM after 48 h were utilized for the rheological measurements. (c) AFM of a 15 mM solution of wild-type peptide DFNKF showed small ﬁbrils and aggregates unable to network sufﬁciently to form a hydrogel. & 2015 Macmillan Publishers Limited. All rights reserved. p p pp y g The resulting ﬁbril morphologies in the mixed hydrogels were studied by AFM (Fig. 5c,d). A 15 mM sample of the wild-type peptide DFNKF showed only a mixture of intertwined aggregates and small ﬁbrils, which is consistent with the observation that no gelation is observed at this concentration since the ﬁbrils appear Co-assembly of wild-type peptide with diiodinated analogue. Bromination and chlorination of proteins in vivo has been related to a series of oxidative stress-related diseases such as cystic ﬁbrosis38, atherosclerotic intima39, sepsis40 and asthma, among 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Methods R t C Congo red staining. All the samples were monitored for green birefringence using an Olympus BX50 polarizing microscope with a SensiCam PCO camera used to display and enhance images. An 80% ethanol solution saturated with NaCl and Congo red was freshly prepared before each measurement. A piece of each peptide hydrogel was placed on a glass microscope, allowed to air dry and then stained with Congo red solution. Subsequently, excess Congo red solution was blotted off the slide and the samples were analysed using both bright and polarized light. Reagents. Congo red, Rhodamine B, NaCl and D2O were purchased from Sigma- Aldrich and used without further puriﬁcation. Peptides with conﬁrmed amino acid analysis (purity Z98%), were purchased from Biopeptek (Malvern, USA). The integrity of all peptides was conﬁrmed by ion spray mass spectrometry and the purity was determined by reverse phase high-pressure liquid chromatography. Hydrogel preparation. Hydrogels were prepared by dissolving each peptide (15 mM for halogenated peptides and 75 mM for the wild-type peptide DFNKF) in deionized water (18.2 Mo cm) or in D2O (Z99.9 atom % deuterium). The glass vials containing the 500 ml solutions were sealed, sonicated for 20 s, heated using a heat gun until complete dissolution of the peptides, before slow cooling to room temperature (RT) All the samples were stored at RT for 48 h before analysis. Infrared spectroscopy (FT-IR). Infrared spectra were recorded at RT using a Nicolet iS50 FT-IR spectrometer equipped with a DTGS detector. Peptides were analysed as solutions (after heating at 100 C to break any pre-formed ﬁbrils) or gels at 15 mM in D2O (for DFNKF 75 mM concentration was used). Spectra represent an average of 64 scans recorded in a single-beam mode with a 4 cm 1 resolution and corrected for the background. The second-derivative analyses of the spectra were performed using the Nicolet FTIR software, Omnic 9.0, with a 13-point and third polynomial order Savitzky and Golay function. Second- derivative spectra generated negative bands as compared with the original spectra, thus for comparison all the second-derivative spectra were multiplied by 1. Preparation of peptide solutions. Peptide solutions (40 mM) were freshly prepared in deionized water (18.2 Mo cm), sonicated for 20 s, and gently warmed to reach 90 C before ﬁltration through a 0.22 mm Millipore ﬁlter. The peptide solutions were stored in sealed vials at RT for varying time points before analysis. X-ray crystallography. Discussion In summary, we have demonstrated that iodination of a short amyloidogenic core sequence strongly promotes its ﬁbril formation ability and affects the structure of formed ﬁbrils. In particular, the 2-p-iodo-Phe derivative DF(I)NKF was demonstrated to be 30-fold more efﬁcient in forming hydrogels than the wild-type pentapeptide DFNKF. This is remarkable for such a modest perturbation in structure. Preliminary results indicate that the strong effect of iodination on peptide self- assembly is general and not limited to the core sequence reported here. In fact, iodination of the amyloidogenic core sequence KLVFF (residues 16–20 of Ab) as well as of the full-length hCT similarly promotes ﬁbril formation efﬁciency and self-assembly (data not shown). Iodination of aromatic amino acids may thus develop as a general strategy for the design of new hydrogels starting from unprotected peptides and without the use of organic solvents44,45. Rheology. Rheology experiments were performed using a TA instrument ARG2 Rheometer. A 20 mm stainless steel, parallel-plate geometry was used with a gap distance of 1,000 mm. Oscillatory frequency sweep studies were performed for a range of 0.1–100 rad s 1, using a 0.5% strain. Oscillatory amplitude sweep studies were conducted from 0.01 to 100% strain with an angular frequency of 1 rad s 1. The ring-cast method was used for hydrogel preparation at a peptide concentration of 15 mM. The peptide solution was sonicated for 20 s in a sealed glass vial before heating to 90 C to afford complete dissolution of the peptide. After cooling, the solutions were transferred into ring casts of 22 mm diameter and placed in tightly sealed tissue-culture dishes for 48 h. All the measurements were repeated a minimum of three times. Circular dichroism spectroscopy. All the CD experiments were carried out in deionized water (18.2 MO cm) in a 0.01 mm detachable quartz cuvette, using a JASCO J-815 CD spectrometer. Acquisitions were performed between 190 and 300 nm with a 0.1 nm data pitch, 1 nm bandwidth, 100 nm min 1 scanning speed and 1 s response time. All the spectra are an average of 10 scans and were corrected from a reference solution, comprising deionized water (18.2 MO.cm) alone. Discussion Raw data (y, in millidegree) were subsequently converted to mean residue ellipticity ([y] in deg cm2 dmol 1) for the sake of comparison, in accordance with the following formulae: The results reported in this paper are relevant in the ﬁeld of the programmed synthesis of amyloid supramolecular assemblies as well as in the context of amyloid-dependent diseases. In the former ﬁeld, we have demonstrated that single atom mutations of amyloidogenic core sequences by replacing hydrogen with iodine atoms greatly promote ﬁbril formation and self-assembly. Methods promoting the formation of mature ﬁbrils are advantageous for use, among others, as scaffolds for tissue engineering, controlled drug release, surgical reconstruction applications, microﬂuidic devices, biosensors and bioswitches7–11. As far as amyloid-dependent diseases are concerned, our results suggest that oxidative stress-induced halogenation of proteins might potentially be the triggering point for the transformation of a natively folded protein into a halogen- bonded and ﬁbrillar malfunctioning form. Research in this direction is currently being carried out in our laboratories and will be reported elsewhere. y½ ¼ y 10lcðn 1Þ ; ð1Þ ð1Þ where y is the observed ellipticity in millidegree, c is the concentration of the sample in mol l 1, (n 1) is the number of peptide bonds and l is the path length of the cuvette in centimetre. Confocal microscopy. Hydrogels were imaged using a Zeiss LSM 710 microscope with a He/Ne laser (lex ¼ 543 nm). The ﬂuorescent dye, Rhodamine B, was incorporated into an aged hydrogel (48 h) scaffold by the addition of 10 ml of the dye solution (0.1% w/v). Following complete absorption of the dye, the sample was excited at 543 nm and emitted light recorded using the E570LP emission ﬁlter. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 DF(Br)NKF(Br) and DF(Cl)NKF(Cl) are both more hydrophobic than DF(I)NKF (see Supplementary Table 6), which instead forms the second stiffest gel. Moreover, the two monoiodinated peptides, despite having similar hydrophobicity, display com- pletely different properties. For the same reasons discussed above, electrostatic and hydrophobic interactions also should not play an important role in the interﬁbrillar association observed in the halogenated gels. non-hydrogen atoms were reﬁned anisotropically and hydrogen atoms were ﬁxed geometrically and reﬁned isotropically (CCDC deposition number: 1049844). non-hydrogen atoms were reﬁned anisotropically and hydrogen atoms were ﬁxed geometrically and reﬁned isotropically (CCDC deposition number: 1049844). Mixed gels preparation. Mixed hydrogels were prepared using the same method as previously described. Brieﬂy, peptides were weighed into a glass vial and MilliQ water added. The glass vial was sealed and sonicated for 20 s before heating with a heat gun until complete solubilization of the peptides. The vials were allowed then to cool slowly to RT and stored at RT for 7 days before analysis. Thermal stability. The vials containing the hydrogels at 15 mM were inverted and ﬁxed at the bottom of an oil bath with stirring. The temperature was kept at 25 C for 10 min to equilibrate the system and then gradually increased from 25 to 140 C (at 1 C min 1). The temperature at which the gels break is reported as a range: the initial temperature corresponds to the fall of the ﬁrst drop and the ﬁnal one to the complete breakdown of the gel. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 Although no direct structural evidence of halogen bonding between our peptide monomers has been obtained, yet, the reported results, IR and Raman in particular, provided corroborative evidence that iodinated peptides DF(I)NKF and DF(I)NKF(I) potentially use halogen bonds to efﬁciently self-assemble into mature amyloid ﬁbrils. In fact, the particular position of the iodinated residue in the peptide sequence greatly inﬂuences the efﬁciency of its self- assembly process, thus suggesting the involvement of the iodine atom in a speciﬁc noncovalent interaction (Fig. 6a), rather than nonspeciﬁc hydrophobic and/or steric effects. This is further supported by considerations of halogen atom polarizability since the dibrominated and dichlorinated derivatives DF(Br)NKF(Br) and DF(Cl)NKF(Cl) showed less tendency to form ﬁbrils than their diiodinated analogue DF(I)NKF(I). This result allows interactions involving halogen atoms as hydrogen bond acceptors to be ruled out as Cl and Br would give stronger interactions than I, which was not observed. Electrostatic interactions involving the ionizable side chains of the peptides, Asp and Lys, may also be ruled out as the formation of these halogenated hydrogels is largely pH-independent (see Supplementary Table 4). As a Figure 6 \| Potential halogen bonds involving p-iodo-phenylalanine. IR and Figure 6 \| Potential halogen bonds involving p-iodo-phenylalanine. IR and Raman spectroscopies are more consistent with the occurrence of type a interaction, however, other interactions reported cannot be discounted. (a) Orthogonal hydrogen and halogen bonds involving the carbonyl oxygen atom of the peptide bond36. (b) Type II iodine iodine contacts23. (c) Iodine p interactions49. (d) Triangular iodine synthon50. conﬁrmation of the minor role played by electrostatic attraction in the self-assembly of the reported halogenated peptides, increasing the ionic strength of the peptide solutions resulted in accelerated formation of the hydrogels (see Supplementary Table 5). It is also unlikely that aromatic p–p interactions are playing an important role in the self-assembly of the halogenated hydrogels, since this would not explain the polarizability depen- dence that we observed. Finally, hydrophobic interactions could not justify the observed trends in hydrogel properties because NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 6 & 2015 Macmillan Publishers Limited. All rights reserved. References 41. Wu, W. et al. Eosinophils generate brominating oxidants in allergen-induced asthma. J. Clin. Invest. 105, 1455–1463 (2000). 10. Mankar, S., Anoop, A., Sen, S. & Maji, S. K. Nanomaterials: amyloids reﬂect their brighter side. Nano Rev. 2, 6032–6043 (2011). 42. Mazzulli, J. R., Hodara, R., Lind, S. & Ischiropoulos, H. Oxidative stress and protein deposition diseases in protein misfolding, aggregation, and conformational diseases. Prot. Rev. 4, 123–133 (2006). g Cherny, I. & Gazit, E. Amyloids: not only pathological agents 11. Cherny, I. & Gazit, E. Amyloids: not only pathological agents but also ordered nanomaterials. Angew. Chem. Int. Ed. 47, 4062–4069 (2008). conformational diseases. Prot. Rev. 4, 123–133 (2006). 43. Ryan, D. M., Doran, T. M. & Nilsson, B. L. Complementary p-p interactions induce multicomponent coassembly into functional ﬁbrils. Langmuir. 27, 11145–11156 (2011). g 12. Grifﬁth Jones, O. & Mezzenga, R. Inhibiting, promoting, and preserving stability of functional protein ﬁbrils. Soft Matter 8, 876–895 (2012). stability of functional protein ﬁbrils. Soft Matter 8, 876–895 (20 13. Petkova, A. T. et al. A structural model for Alzheimer’s b-amyloid ﬁbrils based on experimental constraints from solid state NMR. Proc. Natl Acad. Sci. USA 99, 16742–16747 (2002). 44. Gazit, E. Searching sequence space. Nat. Chem. 7, 14–15 (2015). 45. Frederix, P. W. J. M. et al. Exploring the sequence space for (tri-)peptide self- assembly to design and discover new hydrogels. Nat. Chem. 7, 30–37 (2015). 14. Metrangolo, P. & Resnati, G. Halogen versus hydrogen. Science 321, 918–919 (2008). 46. Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. Sect. A 64, 112–122 (2008). 15. Ryan, D. M., Anderson, S. B., Senguen, F. T., Youngman, R. E. & Nilsson, B. L. Self-assembly and hydrogelation promoted by F5-phenylalanine. Soft Matter 6, 475–479 (2010). 47. Macrae, C. F. et al. Mercury CSD 2.0 - new features for the visualization and investigation of crystal structures. J. Appl. Cryst. 41, 466–470 (2008). 48. Engelhardt, P. Electron tomography of chromosome structure. Encyclopedia of analytical chemistry (2006). 16. Wang, Y., Zhang, Z., Xu, L., Li, X. & Chen, H. Hydrogels of halogenated Fmoc-short peptides for potential application in tissue engineering. Colloids Surf. B Biointerfaces 104, 163–168 (2013). 49. Sirimulla, S., Bailey, J. B., Vegesna, R. & Narayan, M. Halogen interactions in protein–ligand complexes: implications of halogen bonding for rational drug design. J. Chem. Inf. Model. 53, 2781–2791 (2013). 17. Reches, M. & Gazit, E. References 35. Shivu, B. et al. Distinct b-sheet structure in protein aggregates determined by ATR-FTIR spectroscopy. Biochemistry 52, 5176–5183 (2013). 1. Knowles, T. P. J. & Buehler, M. J. Nanomechanics of functional and pathological amyloid materials. Nat. Nanotechnol. 6, 469–479 (2011). p py y 36. Vasylyeva, V. et al. Orthogonal halogen and hydrogen bonds involving a peptide bond model Cryst Eng Comm 16 8102 8105 (2014) 36. Vasylyeva, V. et al. Orthogonal halogen and hydrogen bonds involving a peptide bond model. Cryst. Eng. Comm. 16, 8102–8105 (2014). 2. Selkoe, D. J. Alzheimer’s disease: genes, proteins, and therapy. Physiol. Rev. 81, 741–766 (2001). peptide bond model. Cryst. Eng. Comm. 16, 8102–8105 (2014 37. Messina, M. T. et al. Infrared and Raman analyses of the halogen-bonded non-covalent adducts formed by a-$-diiodoperﬂuoroalkanes with DABCO 37. Messina, M. T. et al. Infrared and Raman analyses of the halogen-bonded non-covalent adducts formed by a-$-diiodoperﬂuoroalkanes with DABCO and other electron donors. J. Mol. Struct. 524, 87–94 (2000). 3. Dobson, C. M. Protein folding and misfolding. Nature 426, 884–890 (2003). 4. Selkoe, D. J. Folding proteins in fatal ways. Nature 426, 900–904 (2003). 3. Dobson, C. M. Protein folding and misfolding. Nature 426, 884–890 (2003). 4. Selkoe, D. J. Folding proteins in fatal ways. Nature 426, 900–904 (2003). and other electron donors. J. Mol. Struct. 524, 87–94 (2000) 5. Chiti, F. & Dobson, C. M. Protein misfolding, functional amyloid and human disease. Annu. Rev. Biochem. 75, 333–366 (2006). 38. Vliet, V. D. et al. Myeloperoxidase and protein oxidation in cystic ﬁbrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L537–L546 (2000). 6. Kreplak, L. & Aebi, U. From the polymorphism of amyloid ﬁbrils to their assembly mechanism and cytotoxicity. Adv. Protein Chem. 73, 217–233 (2006). 39. Hazen, S. L. 3-chlorotyrosine, a speciﬁc marker of myeloperoxidase? Catalyzed oxidation is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 99, 2075–2075 (1997). 7. Hauser, C. A. E., Maurer-Stroh, S. & Martins, I. C. Amyloid-based nanosensors and nanodevices. Chem. Soc. Rev. 43, 5326–5345 (2014). 40. Gaut, J. P. Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis. Proc. Nat Acad. Sci. USA 98, 11961–11961 (2001). 8. Adamcik, J. & Mezzenga, R. Fibrils from a polymer physics perspective. Macromolecules 45, 1137–1150 (2012). 9. Li, C. & Mezzenga, R. The interplay between carbon nanomaterials and amyloid ﬁbrils in bio-nanotechnology. Nanoscale 5, 6207–6218 (2013). References Designed aromatic homo-dipeptides: formation of ordered nanostructures and potential nanotechnological applications. Phys. Biol. 3, S10–S19 (2006). g f 50. Lu, Y. et al. Triangular halogen trimers. A DFT study of the structure, cooperativity, and vibrational properties. J. Phys. Chem. A 109, 11956–11961 (2005). 18. Metrangolo, P., Pilati, T. & Resnati, G. Halogen bonding and other noncovalent interactions involving halogens: a terminology issue. Cryst. Eng. Comm. 8, 946–947 (2006). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 and 340 cm 1. Obtained data were subsequently normalized, for the sake of comparison, and plotted using Origin Pro 8. and 340 cm 1. Obtained data were subsequently normalized, for the sake of comparison, and plotted using Origin Pro 8. 20. Ryan, D. M., Anderson, S. B. & Nilsson, B. L. The inﬂuence of side-chain halogenation on the self-assembly and hydrogelation of Fmoc-phenylalanine derivatives. Soft Matter 6, 3220–3231 (2010). f 21. Desiraju, G. R. et al. Deﬁnition of the halogen bond (IUPAC Recommendations 2013). Pure Appl. Chem. 85, 1711–1713 (2013). Atomic force microscopy. Peptide solution (10 ml, 40 mM) or diluted gel was deposited onto freshly cleaved mica surface and air dried. AFM characterizations were performed on a Veeco Dimension 5,000 Scanning Probe Microscope with a Nanoscope V controller (Digital Instruments, Inc.). All the samples were prepared on mica substrate and measured without treatment. Al-coated silicon AFM tips (NSC 15/AIBS, MikroMasch, Estonia) with a tip radius of 10 nm were used to probe the surface proﬁles of the ﬁlms. Tapping-mode AFM imaging was used according to well-established procedures. All the images were post-treated with NanoScope Analysis 1.5 Software. pp 22. Metrangolo, P. & Resnati, G. Tracing iodine. Nat. Chem. 3, 260 (2011). 23. Metrangolo, P. & Resnati, G. Type II halogen halogen contacts are halogen bonds. IUCrJ 1, 5–7 (2014). 24. Berger, G., Berger, N., Guillaud, M. H., Trouillas, J. & Vauzelle, J. L. Calcitonin-like immunoreactivity of amyloid ﬁbrils in medullary thyroid carcinomas. An immunoelectron microscope study. Virchows Arch. A Pathol. Anat. Histopathol. 412, 543–551 (1988). 25. Arvinte, T., Cudd, A. & Drake, A. F. The structure and mechanism of formation of human calcitonin ﬁbrils. J. Biol. Chem. 268, 6415–6422 (1993). Transmission electron microscopy. High-resolution transmission electron microscopy imaging was carried out using JEM-3200Fsc ﬁeld emission microscope (JEOL) operated at 300 kV in bright-ﬁeld mode with Omega-type Zero-loss energy ﬁlter. The images were acquired with ULTRASCAN 4,000 CCD camera (GATAN) and with GATAN DIGITAL MICROGRAPH software, while the specimen temperature was maintained at 187 C. Dried TEM and ET samples were prepared by placing 4 ml of 40 mM DF(I)NKF(I) solution on 200 mesh carbon only grid (CFT200-Cu) and the excess removed with ﬁlter paper. 26. Steed, J. W. Supramolecular gel chemistry: developments over the last decade. Chem. Commun. 47, 1379–1383 (2011). 27. Meazza, L. et al. ARTICLE Halogen-bonding-triggered supramolecular gel formation. Nat. Chem. 5, 42–47 (2013). 28. Lakshmanana, A. et al. Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis. Proc. Natl Acad. Sci. USA 110, 519–524 (2013). 29. Shtainfeld, A., Sheynis, T. & Jelinek, R. Speciﬁc mutations alter ﬁbrillation kinetics, ﬁber morphologies, and membrane interactions of pentapeptides derived from human calcitonin. Biochemistry 49, 5299–5307 (2010). Electron tomography. For image alignment purposes, the TEM grids were dipped in 11-mercapto-1-undecanol ligand-coated gold particle solution (d ¼ 3–10 nm) before sample deposition. ET tilt series were acquired with the SERIALEM- software package between tilt angles of ±69. Prealignment of tilt image series was done with IMOD and the ﬁne alignment and cropping with JPEGANIM software package48. The images were binned twice to reduce noise and computation time. Maximum entropy method reconstruction scheme was carried out with MEM software package48 on Linux cluster with regularization parameter value of d ¼ 5.0 10 2. Data visualization, volumetric graphics and analyses were performed with the UCSF CHIMERA package. The tomogram was ﬁltered with CHIMERA’s Gaussian ﬁlter with 1.5 voxel width. 30. Hauser, C. A. E. et al. Natural tri- to hexapeptides self-assemble in water to amyloid b-type ﬁber aggregates by unexpected a-helical intermediate 30. Hauser, C. A. E. et al. Natural tri- to hexapeptides self-assemble in water to amyloid b-type ﬁber aggregates by unexpected a-helical intermediate structures. Proc. Natl Acad. Sci. USA 108, 1361–1366 (2011). structures. Proc. Natl Acad. Sci. USA 108, 1361–1366 (2011 31. Mishra, A. et al. Ultrasmall natural peptides self-assemble to strong temperature-resistant helical ﬁbers in scaffolds suitable for tissue engineerin Nano Today 6, 232–239 (2011). 32. Smith, D. K. In Supramolecular Chemistry: From Molecules to Nanomaterials (John Wiley and Sons, Inc., 1999). y 33. Adamcik, J. et al. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nat. Nanotechnol. 5, 423–428 (2010). 34. Nilsson, M. R. Techniques to study amyloid ﬁbril formation in vitro. Methods 34, 151–160 (2004). Methods R t C The p-iodo-phenylalanine was suspended in water at RT and kept in an open vial under a hood. After 6 months, colourless crystalline needles appeared. Suitable crystals for XRD measurements were analysed without further manipulation. The crystals were measured using Mo-Ka radiation on a Bruker KAPPA APEX II diffractometer with a Bruker KRYOFLEX low tempera- ture device. The crystal structure was solved by direct method and reﬁned against F2 using SHELXL9746. Packing diagrams were generated using Mercury47. The Raman spectroscopy. Raman spectra were acquired at RT by using a Horiba Xplora MicroRaman instrument equipped with an Olympus BX-41 Microscope. An excitation wavelength of 785 nm was used. Laser power was attenuated by neutral density ﬁlters with a ﬁnal power density of B0.07 mW mm 2. The low wavenumber detection limit is 140 cm 1. Each spectrum was acquired with an exposure time of 5 s over 35 cycles. The raw data were ﬁrst corrected from the baseline, using the JASCO Nicolet FTIR software, Omnic 9.0, between 140 7 7 NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8574 Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ How to cite this article: Bertolani, A. et al. Supramolecular ampliﬁcation of amyloid self-assembly by iodination. Nat. Commun. 6:7574 doi: 10.1038/ncomms8574 (2015). Author contributions A.B. and L.P. performed most of the peptide self-assembly work. L.S. performed the IR spectroscopy and ionic strength-pH-dependence experiments. N.H. measured the samples by AFM. J.S.H. measured the samples by TEM. L.C. and G.T. performed the single crystal X-ray analysis. G.G. and L.V. performed the Raman spectroscopy. P.M., R.M., O.I., and G.R. conceived the experiments and contributed to results discussion. All the authors contributed to manuscript writing. Acknowledgements 19. Ryan, D. M., Doran, T. M., Anderson, S. B. & Nilsson, B. L. Effect of C-terminal modiﬁcation on the self-assembly and hydrogelation of ﬂuorinated Fmoc-Phe derivatives. Langmuir 27, 4029–4039 (2011). The European Research Council is acknowledged for the Starting Grant ERC-2012- The European Research Council is acknowledged for the Starting Grant ERC-2012- StG_20111012 FOLDHALO (Grant Agreement Number 307108) to P.M. Dr Claudia Piglia- celli (Politecnico di Milano) is acknowledged for help with the TEM experiment of Fig. 3f. The European Research Council is acknowledged for the Starting Grant ERC-2012- StG_20111012 FOLDHALO (Grant Agreement Number 307108) to P.M. Dr Claudia Piglia- celli (Politecnico di Milano) is acknowledged for help with the TEM experiment of Fig. 3f. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature. 8 & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7574 \| DOI: 10.1038/ncomms8574 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. Additional information Accession codes: The X-ray crystallographic coordinates for the structure reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number 1049844. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications naturecommunications
https://openalex.org/W3155551928	https://www.researchsquare.com/article/rs-193361/v1.pdf?c=1631868988000	English	null	Determination of Poultry Manure and Plant Residues Effects on Zn Bioavailable Fraction in Contaminated Soil via DGT Technique	Archives of environmental contamination and toxicology	2,021	cc-by	11,754	Determination of Poultry Manure and Plant Residues Effects on Zn Bioavailable Fraction in Contaminated Soil Via DGT Technique Amir Mohseni (  amir.mohseni65@gmail.com ) University of Tabriz https://orcid.org/0000-0002-2439-2972 Research Article Keywords: Chemical fraction, Clover residues, Sorghum residues, Organic amendment, Metal Posted Date: February 15th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-193361/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: February 15th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-193361/v1 DOI: https://doi.org/10.21203/rs.3.rs-193361/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Determination of poultry manure and plant residues effects on Zn bioavailable fraction in contaminated soil via DGT technique Amir Mohseni1. Saber Heidari2. Solmaz Bidast3. Bijan Raei1. Seyed Adel Moftakharzadeh4 1Department of Soil Science, University of Tabriz, Tabriz, Iran 2Faculty members of Soil and Water Research Department, South Kerman Agricultural and Natural Resources Research and Education Center, AREEO, Jiroft, Iran 3 Department of Soil Science, University of Zanjan, Zanjan, Iran 4Department of Animal Science, University of Tabriz, Tabriz, Iran Corresponding author: Amir Mohseni Department of Soil Science, University of Tabriz, Tabriz, Iran E-mail: Amir.mohseni65@gmail.com Mail address: 29 Bahman Blvd., Tabriz, Iran Postal Code: 5166616471 Phone number: +98-939-180-8604 Determination of poultry manure and plant residues effects on Zn bioavailable fraction in contaminated soil via DGT technique Amir Mohseni1. Saber Heidari2. Solmaz Bidast3. Bijan Raei1. Seyed Adel Moftakharzadeh4 Determination of poultry manure and plant residues effects on Zn bioavailable 1 fraction in contaminated soil via DGT technique 2 Determination of poultry manure and plant residues effects on Zn bioavailable 1 fraction in contaminated soil via DGT technique 2 3 Abstract 4 A greenhouse experiment was aimed at assessing the effects of poultry manure, sorghum, and clover 5 residues (0 and 15 g kg-1) on the zinc (Zn) bioavailable fraction in contaminated calcareous soil using two 6 chemical assay, diffusion gradient in thin films (DGT) and DTPA-TEA, and a bioassay with corn (Zea mase 7 L.). The results showed that poultry manure, clover, and sorghum residues application increased dissolved 8 organic carbon (DOC) by 53.6 and 36.1, and 9.2%, respectively, with respect to unamended soils, as well 9 as decreasing soil pH by 0.42, 0.26, and 0.06 units, respectively. These changes did result in increases of 10 Zn effective concentration (CE) and DTPA-Zn, and plant Zn concentration as a result of the increased 11 exchangeable form of Zn. In the sorghum residues-amended soils, a reverse trend was observed for CE-Zn 12 compared to the DTPA method. Correlation analyses revealed that unlike CE-Zn, DTPA-Zn had a positive 13 correlation significantly with organic fractions that can be considered as an equivalent to the fact that the 14 DTPA method had been overestimated Zn available to plants. The best correlations between corn metal 15 concentrations and soil metal bioavailability were for CE-Zn using DGT technique, which also provided the 16 best Zn bioavailability estimate. It is concluded that sorghum residues could be used to reduce the 17 phytotoxicity risk of Zn in calcareous contaminated soil, and DTPA method is the less robust indicator of 18 Zn bioavailability than DGT technique. 19 Keywords: Chemical fraction, Clover residues, Sorghum residues, Organic amendment, Metal 20 Abstract 4 A greenhouse experiment was aimed at assessing the effects of poultry manure, sorghum, and clover 5 residues (0 and 15 g kg-1) on the zinc (Zn) bioavailable fraction in contaminated calcareous soil using two 6 chemical assay, diffusion gradient in thin films (DGT) and DTPA-TEA, and a bioassay with corn (Zea mase 7 L.). The results showed that poultry manure, clover, and sorghum residues application increased dissolved 8 organic carbon (DOC) by 53.6 and 36.1, and 9.2%, respectively, with respect to unamended soils, as well 9 as decreasing soil pH by 0.42, 0.26, and 0.06 units, respectively. These changes did result in increases of 10 Zn effective concentration (CE) and DTPA-Zn, and plant Zn concentration as a result of the increased 11 exchangeable form of Zn. In the sorghum residues-amended soils, a reverse trend was observed for CE-Zn 12 compared to the DTPA method. Correlation analyses revealed that unlike CE-Zn, DTPA-Zn had a positive 13 correlation significantly with organic fractions that can be considered as an equivalent to the fact that the 14 DTPA method had been overestimated Zn available to plants. The best correlations between corn metal 15 concentrations and soil metal bioavailability were for CE-Zn using DGT technique, which also provided the 16 best Zn bioavailability estimate. It is concluded that sorghum residues could be used to reduce the 17 phytotoxicity risk of Zn in calcareous contaminated soil, and DTPA method is the less robust indicator of 18 Zn bioavailability than DGT technique. 19 Keywords: Chemical fraction, Clover residues, Sorghum residues, Organic amendment, Metal 20 I d i Metals present in different concentrations in all soils, most play a major role at relatively low levels for 22 biological life such as Zinc (Zn). Release of Zn to the soils occurs predominantly through industrial activities 23 such as smelting and mining (Chen et al. 2009). The focus on metal contamination has been aimed mainly 24 Metals present in different concentrations in all soils, most play a major role at relatively low levels for 22 biological life such as Zinc (Zn). Release of Zn to the soils occurs predominantly through industrial activities 23 such as smelting and mining (Chen et al. 2009). The focus on metal contamination has been aimed mainly 24 1 at preventing the accumulation of metal in the food chain and its toxic influences on plants and humans 25 (Alloway 2013). Abstract 4 Conversely, organic amendments have widely been used as a remediation method to reduce 26 the metals phytotoxicity and bioavailability in contaminated soils (Palansooriya et al. 2020). Organic 27 amendments affects the distribution of chemical forms of metals by modifying some soil properties 28 (Ashraf et al. 2019). The use of organic matter as a soil amendment is based on the hypothesis that metal 29 precipitation and sorption reactions induced by organic matter may result in an increase in metal retention 30 on the solid phase, and consequently a decrease in the metal availability (Nwachukwu and Pulford 2009), 31 while it is not always the case. Dodangeh et al. (2018) reported that the compost of municipal solid waste 32 affected the absorption properties of heavy metals through affecting the soil mineral fractions. This study 33 indicated that organic amendment increased soil absorption phases and reduce the metals availability. On 34 the other hand, Aziz et al. (2017) found that the application of farmyard manure to contaminated soils can 35 produce organo-metal complexes, which facilitate metal solubility and mobility. The organic amendment 36 efficacy at reducing the heavy metals bioavailability is mostly dependent on the type of organic amendment 37 and metal, as well as the potential measures of heavy metals. 38 at preventing the accumulation of metal in the food chain and its toxic influences on plants and humans 25 (Alloway 2013). Conversely, organic amendments have widely been used as a remediation method to reduce 26 the metals phytotoxicity and bioavailability in contaminated soils (Palansooriya et al. 2020). Organic 27 amendments affects the distribution of chemical forms of metals by modifying some soil properties 28 (Ashraf et al. 2019). The use of organic matter as a soil amendment is based on the hypothesis that metal 29 precipitation and sorption reactions induced by organic matter may result in an increase in metal retention 30 on the solid phase, and consequently a decrease in the metal availability (Nwachukwu and Pulford 2009), 31 while it is not always the case. Dodangeh et al. (2018) reported that the compost of municipal solid waste 32 affected the absorption properties of heavy metals through affecting the soil mineral fractions. This study 33 indicated that organic amendment increased soil absorption phases and reduce the metals availability. On 34 the other hand, Aziz et al. Abstract 4 DGT method is based on kinetic principles and can reflect the process of 52 resupply of heavy metals from the soil and the relationship between plant roots and soil. kinetic information 53 of heavy metal resupply process in soil can also be obtained using DGT induced fluxes (DIFS) model 54 (Harper et al 2000). A high correlation between the elements collected by DGT and their bioavailability in 55 plants has been concluded by various researchers (Degryse et al. 2009; Li et al. 2016). However, despite the 56 growing use of this method to evaluate metal availability in soil, insufficient rigorous work has established 57 the organic amendments effects on the validity of assay for metal bioavailability. Therefore, this experiment 58 was used to determine the organic residues effects on the validity of metal bioavailability assays in 59 contaminated calcareous soils. The overall purpose of this study was to evaluate measures of bioavailable 60 Zn across contrasting presence of organic residues in corn -planted soil by DTPA and DGT methods. 61 if the resupply and metals availability from the solid phase are kinetically limited or is not fast enough to 51 occur, uptake by plant is reduced. DGT method is based on kinetic principles and can reflect the process of 52 resupply of heavy metals from the soil and the relationship between plant roots and soil. kinetic information 53 of heavy metal resupply process in soil can also be obtained using DGT induced fluxes (DIFS) model 54 (Harper et al 2000). A high correlation between the elements collected by DGT and their bioavailability in 55 plants has been concluded by various researchers (Degryse et al. 2009; Li et al. 2016). However, despite the 56 growing use of this method to evaluate metal availability in soil, insufficient rigorous work has established 57 the organic amendments effects on the validity of assay for metal bioavailability. Therefore, this experiment 58 was used to determine the organic residues effects on the validity of metal bioavailability assays in 59 contaminated calcareous soils. The overall purpose of this study was to evaluate measures of bioavailable 60 Zn across contrasting presence of organic residues in corn -planted soil by DTPA and DGT methods. 61 Soil sampling and analysis 63 In this study, samples of contaminated soil (0-30 cm) were collected from zinc mine at 6 km in Zanjan 64 province, Iran. After sampling, soil samples were air-dried and sieved through a 2 mm sieve. Soil chemical 65 and physical analyses were done by a soil testing method, including total Zn concentration by acid digestion 66 (HNO3) (Sposito et al. 1982), Zn available concentration by DTPA-TEA solution (Lindsay and Norvell 67 1978), soil texture by hydrometer (Gee and Bauder 1986), Calcium carbonate equivalent (CCE) by titration 68 (Loeppert and Suarez 1996), exchange capacity using a methodology described by Chapman (1965), organic 69 carbon content by the Walkley-Black method (Nelson and Sommers 1996), pH and EC by pH meter (Mettler 70 Toledo, U.S.A.) and EC meter (4010 conductivity meter, Jenway Inc, England), respectively, in a 1: 5 soil- 71 to-water ratio (Thomas 1996; Rhoades et al. 1989). 72 Abstract 4 (2017) found that the application of farmyard manure to contaminated soils can 35 produce organo-metal complexes, which facilitate metal solubility and mobility. The organic amendment 36 efficacy at reducing the heavy metals bioavailability is mostly dependent on the type of organic amendment 37 and metal, as well as the potential measures of heavy metals. 38 In principle, measurement of total metal concentration is not generally considered to be an adequate estimate 39 for metal available fraction (McLaughlin et al., 2000). In contrast, some researches have shown that 40 chemical extractants such as buffer salts (NH4OAc), mineral acids (HCl, HNO3), and chelating agents 41 (EDTA, DTPA-TEA) may be suitable for the estimation of exchangeable metal fraction (Meers et al. 2007). 42 These extraction methods may provide an insight into the geochemical fractions of metals in the soil. 43 However, these methods do not consider the soil ability to sustain the metals concentration in soil solution 44 following the metals depletion by plant uptake (Zhang et al. 2001). 45 In principle, measurement of total metal concentration is not generally considered to be an adequate estimate 39 for metal available fraction (McLaughlin et al., 2000). In contrast, some researches have shown that 40 chemical extractants such as buffer salts (NH4OAc), mineral acids (HCl, HNO3), and chelating agents 41 (EDTA, DTPA-TEA) may be suitable for the estimation of exchangeable metal fraction (Meers et al. 2007). 42 These extraction methods may provide an insight into the geochemical fractions of metals in the soil. 43 However, these methods do not consider the soil ability to sustain the metals concentration in soil solution 44 following the metals depletion by plant uptake (Zhang et al. 2001). 45 The diffusive gradient in thin-film technique (DGT) was initially developed by Zhang and Davison (1995), 46 to measure in situ heavy metal concentration in soils, sediments, and aqueous solutions (Li et al. 2019). This 47 technique relies on the solutes accumulation onto the cation exchange resin after passing through the 48 diffusion hydrogel layer (Zhang et al. 1998). Since the mobility of heavy metals in the soil highly depends 49 on its resupply from the solid phase of the soil and their concentration in the soil solution (Nolan et al. 2005), 50 2 if the resupply and metals availability from the solid phase are kinetically limited or is not fast enough to 51 occur, uptake by plant is reduced. Organic amendment analysis 73 Organic amendments exerted in this study include poultry manure, sorghum, and white clover residues 74 which were ground and sieved using a 2 mm sieve. The sieved samples were then characterized for total Zn 75 Organic amendments exerted in this study include poultry manure, sorghum, and white clover residues 74 which were ground and sieved using a 2 mm sieve. The sieved samples were then characterized for total Zn 75 3 by wet digestion (Chen et al. 2001), EC and pH using a 1:5 sewage sludge to water ratio (Thomas 1996; 76 Rhoades 1996), dissolved organic carbon (DOC) using the methodology described by Mohseni et al. (2018), 77 organic carbon content by dichromate oxidation (Nelson and Sommers 1996), and total N by 78 the Kjeldahl method (Bremner and Mulvaney 1982). 79 Greenhouse experimental 80 To investigate the effect of organic amendments on Zn bioavailability, a factorial experiment was conducted 81 in a completely randomized block design with three replications. Treatments including: contaminated 82 calcareous soil, two levels of poultry manure, clover, and sorghum residues (0 and 15 g kg-1). Organic 83 amendments were mixed thoroughly in pot soil (three kg), wetted to field moisture capacity, and incubated 84 for 90 days at a constant temperature (25°C ± 2). After incubating, six seeds of corn (Zea mase L.), cultivar 85 704, were planted into each pot (except unamended soils). After a week, seedlings were thinned to three 86 plants per pot. Soil moisture was maintained at about field capacity, and pots irrigated every two days during 87 the growing season according to the control pot. All required nutrients except micronutrients were applied 88 according to soil exam and conventional fertilizer recommendations. The plants were grown under 14 hours 89 light /10 hours dark at temperature of 28°C ± 2. After eight weeks, the shoots and roots were harvested from 90 the soil, eluted with deionized water, and dried at 80 °C ± 5 to constant weight. Then, the samples were 91 milled and digested by wet oxidation (Lozano-Rodríguez et al. 1995). 92 After harvest, two potential measures of Zn bioavailability (DTPA-TEA extractant and DGT technique) 93 were used in each treatment. Concentration of DOC and pH value were also measured according to the 94 above-mentioned methods in the soil analysis section. In addition, sequential extraction procedure described 95 by Tessier et al. (1979) was used to determine the chemical distribution of Zn. This method separated Zn 96 chemical forms into the following five fractions: exchangeable Zn (EX-Zn), Zn-binding to carbonates 97 (CAR-Zn), organically bound Zn (OR-Zn), Zn-binding to oxides (OX-Zn), and residual Zn (RES-Zn) (Table 98 1). 99 100 101 After harvest, two potential measures of Zn bioavailability (DTPA-TEA extractant and DGT technique) 93 were used in each treatment. Concentration of DOC and pH value were also measured according to the 94 above-mentioned methods in the soil analysis section. In addition, sequential extraction procedure described 95 by Tessier et al. (1979) was used to determine the chemical distribution of Zn. This method separated Zn 96 chemical forms into the following five fractions: exchangeable Zn (EX-Zn), Zn-binding to carbonates 97 (CAR-Zn), organically bound Zn (OR-Zn), Zn-binding to oxides (OX-Zn), and residual Zn (RES-Zn) (Table 98 1). Greenhouse experimental 80 99 101 4 4 Table 1 sequential extraction procedure used in this study 102 Step Chemical form of Zn Extraction method 1 Exchangeable (EX-Zn) 8 ml of 1 M MgCl2 was added to 1 g soil and then shaken for two h 2 Carbonate-bound (CAR- Zn) 8 ml of 1 M NaOAc was added to the sediment and shaken for five h 3 oxide-bound (OX-Zn) 20 ml of NH2OH·HCl, 0.04 M was added to 25% w/v HOAc, the solution was mixed with sediment and then shaken for 0.5 h at 50 °C in a water bath. 4 Organically bound (OR- Zn) 5 ml of 30% m/v H2O2 + 3 ml of 0.02 M HNO3 was mixed with the sediment and shaken for one h in boiling water bath. 3 ml of 30% m/v H2O2 was added to the mixture and shaken for three h at 85 °C. 5 ml of 3.2M NH4OAc + 20 mM deionized water was added to the mixture and shaken for three min. 5 Residual (RES-Zn) The sediment was mixed with 20 ml HCl:HNO3 (3:1 v/v), incubated for 16 h at 25 °C, and then heated to 130 °C by a hot plate. 103 DGT technique 104 DGTs were prepared by Heidari et al. (2016) and evaluated for measuring the bioavailability of heavy metal 105 using Mohseni et al. (2020). After each application, the DGTs were removed then separated and eluted with 106 deionized water. After that, resin gel was digested with HNO3 (2 M) for 24 hours and shaken about eight 107 hours. Finally, the concentrations of Zn in acid were measured. When DGT is placed below the soil surface, 108 ions (Zn) move from the diffusion layer and immobilized in the resin layer. As a result, a steady-state 109 gradient linear concentration created in the diffusion layer. The ion flux (F) in the diffusion layer towards 110 resin layer was determined according to Fick's first law (Eq1). As the settling time increases, the ions in the 111 soil solution gradually deplete at the diffusive layer-soil interface, which induces the resupply of Zn from 112 the solid phase of the soil. After the deployed time of DGT in the soil sampling, the resin layer retrieved 113 following 24-h exposure and its total mass can be measured. The ion flux can also be explained according 114 to Equation 2. Greenhouse experimental 80 Finally, the average ion concentration in the contact area between the soil and DGT, which 115 Table 1 sequential extraction procedure used in this study 102 Step Chemical form of Zn Extraction method 1 Exchangeable (EX-Zn) 8 ml of 1 M MgCl2 was added to 1 g soil and then shaken for two h 2 Carbonate-bound (CAR- Zn) 8 ml of 1 M NaOAc was added to the sediment and shaken for five h 3 oxide-bound (OX-Zn) 20 ml of NH2OH·HCl, 0.04 M was added to 25% w/v HOAc, the solution was mixed with sediment and then shaken for 0.5 h at 50 °C in a water bath. 4 Organically bound (OR- Zn) 5 ml of 30% m/v H2O2 + 3 ml of 0.02 M HNO3 was mixed with the sediment and shaken for one h in boiling water bath. 3 ml of 30% m/v H2O2 was added to the mixture and shaken for three h at 85 °C. 5 ml of 3.2M NH4OAc + 20 mM deionized water was added to the mixture and shaken for three min. 5 Residual (RES-Zn) The sediment was mixed with 20 ml HCl:HNO3 (3:1 v/v), incubated for 16 h at 25 °C, and then heated to 130 °C by a hot plate. 102 5 5 is the amount of ions absorbed by the resin layer, can be calculated according to Equation 3 (Sochaczewski 116 et al. 2007). 117 is the amount of ions absorbed by the resin layer, can be calculated according to Equation 3 (Sochaczewski 116 et al. 2007). 117 116 𝐹(𝑥 ‚ 𝑡) = 𝜙𝑑𝐷𝑑 𝐶𝑎(𝑥 ‚ 𝑡) ∆𝑔 , 0 < t < T, x (-r , r) (1) 𝑀= 𝜙𝑑𝐷𝑑 ∆𝑔∫ ∫𝐶𝑎(𝑥 ‚ 𝑡)𝑑𝑡 𝑑𝑥 𝑇 0 𝑟 −𝑟 (2) 𝐶𝐷𝐺𝑇= ∫ ∫𝐶𝑎(𝑥 ‚ 𝑡)𝑑𝑡 𝑑𝑥 𝑇 0 𝑟 −𝑟 𝑇 = 𝑀 ∆𝑔 𝜙𝑑 𝐷𝑑𝑇 (3) 18 (1) (2) 𝐶𝑎(𝑥 ‚ 𝑡)𝑑𝑡 𝑑𝑥 𝑇 = 𝑀 ∆𝑔 𝜙𝑑 𝐷𝑑𝑇 (3) (3) (3) 118 Where M (mg), Δg, ϕd, D, A, and t (s) are accumulated masses of metals in the resin membrane, diffusion 119 layer thickness, gel porosity in the diffusion layer, diffusion coefficients of Zn in the diffusion layer, the 120 effective area of the gel which exposure with soil (3.14 cm2), and deployment time in the soil, respectively. 121 The ion mass in the resin layer gel was calculated using the equation M=𝐶𝑒(𝑉𝑔𝑒𝑙+ 𝑉𝑎𝑐𝑖𝑑). Greenhouse experimental 80 Where M 122 calculated by the concentration of elements measured by ICP-OES after eluted with 2 M nitric acid (Ce), 123 Volume of nitric acid (five ml) (Vacid) and resin gel volume (Vgel) (Zhao et al. 2006). 124 118 Where M (mg), Δg, ϕd, D, A, and t (s) are accumulated masses of metals in the resin membrane, diffusion 119 layer thickness, gel porosity in the diffusion layer, diffusion coefficients of Zn in the diffusion layer, the 120 effective area of the gel which exposure with soil (3.14 cm2), and deployment time in the soil, respectively. 121 The ion mass in the resin layer gel was calculated using the equation M=𝐶𝑒(𝑉𝑔𝑒𝑙+ 𝑉𝑎𝑐𝑖𝑑). Where M 122 calculated by the concentration of elements measured by ICP-OES after eluted with 2 M nitric acid (Ce), 123 Volume of nitric acid (five ml) (Vacid) and resin gel volume (Vgel) (Zhao et al. 2006). 124 To further explanation the DGT measurements, the concentration of elements directly calculated by the 125 DGT (CDGT) was converted to the effective concentration (CE) using Equation 4. Effective concentration 126 (CE) is completely explained by Zhang et al. (Zhang et al. 2001). 127 CE = CDGT Rdiff (4) (4) Where Rdiff is depleted concentration at the diffusive layer-soil interface. A two-dimensional numerical 128 model of the soil-DGT system called 2D-DIFS was used to calculate Rdiff. Moreover, since the input 129 parameters of DIFS software to calculate Rdiff relies on the assumption that the movement of elements 130 towards DGT is only based on the ion diffusion, TC (soil response time) and Kd (distribution ratio) were determined at 131 the maximum (1010 s) and the minimum value (10-10 gr cm-3), respectively. In this case, Rdiff depends only 132 on water holding capacity (WHC) of soils. Resultantly, soils with high water holding capacity are higher 133 Rdiff than soils with lower water holding capacity (Sochaczewski et al. 2007). 134 6 Comparisons of treatments data were statistically analyzed based on ANOVA with SAS 9.1 statistical 136 software. Significance differences between the treatments were also determined according to LSD’s test (p 137 ˂ 0.05). 138 Results 139 The soil characteristics results showed that the total concentration of Zn in the soil was in range of 140 contaminated soil according to critical range presented by Kabata-Pendias and Pendias (1984), and the pH 141 value of soil was also in the alkaline range (Table 2). Greenhouse experimental 80 According to table 3, the lowest value of pH was observed in 159 soil amended with poultry manure, followed by clover and sorghum residues-amended soil. 160 unamended soils, respectively. Moreover, the addition of poultry manure and clover residue significantly 152 increased, and sorghum residues decreased the effective concentration of Zn (CE-Zn) measured by DGT 153 method. CE-Zn increased by 8.7% and 3.6% in poultry manure and clover residual-amended soils with 154 respect to the unamended soils, respectively. In contrast, a 29.5% reduction in CE-Zn was recorded in 155 residual sorghum-amended soil. The results indicated that the presence of organic amendments significantly 156 increased the concentration of DOC compared to unamended soils. The DOC concentration in the poultry 157 manure, clover, and sorghum residues-amended soils were increased by 53.6 and 36.1, and 9.2% in 158 comparison to unamended soils, respectively. According to table 3, the lowest value of pH was observed in 159 soil amended with poultry manure, followed by clover and sorghum residues-amended soil. 160 Table 3 Chemical characteristics of amended and unamended soils with organic amendments. 161 Treatment DTPA- Zn (mg kg-1) CE -Zn (μg L−1) DOC (mg l-1) pH Control (unamended) 212.35±5.05d 1187.24±11.70c 26.85±1.22d 7.64±0.02a Poultry manure-amended soils 244.17±4.77a 1285.20±9.01a 41.23±4.52a 7.22±0.03c Sorghum residues-amended soils 225.21±1.79c 837.33±7.36d 29.34±2.65c 7.58±0.04a Clover residues-amended soils 236.21±1.01b 1229.74±12.81b 36.52±3.41b 7.38±0.02b Note: Results are means ± standard deviations. Values with the different lower-case letters within each column are significantly 162 different at p < 0.05 according to LSD test. 163 Distribution of Zn chemical forms in soil 164 Table 4 presented the effect of organic amendments on the distribution of Zn chemical forms in soil. The 165 results showed that the application of organic amendments significantly (p˂0.05) caused a change in Zn 166 concentration in the exchangeable, carbonate, oxide, and organic fraction compared to the unamended soils. 167 Meanwhile, there were no differences in the residual form of Zn in the organic-amended soils. The results 168 exhibited that poultry manure and clover residue increased the exchangeable form of Zn by 37.7 and 8.9%, 169 Table 3 Chemical characteristics of amended and unamended soils with organic amendments. Greenhouse experimental 80 The findings of organic amendments characteristics 142 used in this study revealed that the highest of Zn concentration was in poultry manure, clover, and sorghum 143 residues, respectively. In respect of carbon to nitrogen ratio (C:N), sorghum residues and poultry manure 144 showed the highest and the lowest values, respectively (Table 2). 145 The soil characteristics results showed that the total concentration of Zn in the soil was in range of 140 contaminated soil according to critical range presented by Kabata-Pendias and Pendias (1984), and the pH 141 value of soil was also in the alkaline range (Table 2). The findings of organic amendments characteristics 142 used in this study revealed that the highest of Zn concentration was in poultry manure, clover, and sorghum 143 residues, respectively. In respect of carbon to nitrogen ratio (C:N), sorghum residues and poultry manure 144 showed the highest and the lowest values, respectively (Table 2). 145 Table 2 Physicochemical characteristics of the studied soil and organic amendments. 146 Table 2 Physicochemical characteristics of the studied soil and organic amendments. Table 2 Physicochemical characteristics of the studied soil and organic amendments. 146 Property Soil Poultry manure Sorghum residues Clover residues Total Zn (mg kg-1) 976.45 32.97 8.78 10.97 DTPA-Zn (mg kg-1) 203.43 - - - EC (dS m-1) 0.80 11.54 7.17 5.48 pH 7.77 7.43 7.36 7.25 Organic carbon (g kg-1) 8.43 256.96 484.17 391.56 DOC (mg l-1) 20.53 33.45 24.69 31.36 Texture Clay loam - - - CEC (cmolc kg1) 16.58 - - - Total N (g kg-1) - 23.64 16.21 32.46 C/N - 10.87 29.86 12.06 147 7 7 unamended soils, respectively. Moreover, the addition of poultry manure and clover residue significantly 152 increased, and sorghum residues decreased the effective concentration of Zn (CE-Zn) measured by DGT 153 method. CE-Zn increased by 8.7% and 3.6% in poultry manure and clover residual-amended soils with 154 respect to the unamended soils, respectively. In contrast, a 29.5% reduction in CE-Zn was recorded in 155 residual sorghum-amended soil. The results indicated that the presence of organic amendments significantly 156 increased the concentration of DOC compared to unamended soils. The DOC concentration in the poultry 157 manure, clover, and sorghum residues-amended soils were increased by 53.6 and 36.1, and 9.2% in 158 comparison to unamended soils, respectively. Greenhouse experimental 80 161 Treatment DTPA- Zn (mg kg-1) CE -Zn (μg L−1) DOC (mg l-1) pH Control (unamended) 212.35±5.05d 1187.24±11.70c 26.85±1.22d 7.64±0.02a Poultry manure-amended soils 244.17±4.77a 1285.20±9.01a 41.23±4.52a 7.22±0.03c Sorghum residues-amended soils 225.21±1.79c 837.33±7.36d 29.34±2.65c 7.58±0.04a Clover residues-amended soils 236.21±1.01b 1229.74±12.81b 36.52±3.41b 7.38±0.02b Note: Results are means ± standard deviations. Values with the different lower-case letters within each column are significantly 162 different at p < 0.05 according to LSD test. 163 Table 3 Chemical characteristics of amended and unamended soils with organic amendments. 161 Distribution of Zn chemical forms in soil 164 Table 4 presented the effect of organic amendments on the distribution of Zn chemical forms in soil. The 165 results showed that the application of organic amendments significantly (p˂0.05) caused a change in Zn 166 concentration in the exchangeable, carbonate, oxide, and organic fraction compared to the unamended soils. 167 Meanwhile, there were no differences in the residual form of Zn in the organic-amended soils. The results 168 exhibited that poultry manure and clover residue increased the exchangeable form of Zn by 37.7 and 8.9%, 169 and sorghum residue reduced the exchangeable form of Zn by 25% compared to unamended soils, 170 respectively. In the carbonate form, a considerable 8.6, 3.2, and 6.3% reduction in Zn concentration was 171 Table 4 presented the effect of organic amendments on the distribution of Zn chemical forms in soil. The 165 results showed that the application of organic amendments significantly (p˂0.05) caused a change in Zn 166 concentration in the exchangeable, carbonate, oxide, and organic fraction compared to the unamended soils. 167 Meanwhile, there were no differences in the residual form of Zn in the organic-amended soils. The results 168 exhibited that poultry manure and clover residue increased the exchangeable form of Zn by 37.7 and 8.9%, 169 and sorghum residue reduced the exchangeable form of Zn by 25% compared to unamended soils, 170 respectively. In the carbonate form, a considerable 8.6, 3.2, and 6.3% reduction in Zn concentration was 171 8 9 recorded in soils amended with poultry manure, sorghum residues, and clover residue in comparison to the 172 unamended soil, respectively. 173 The effect of organic amendments on the oxide form of Zn was also statistically significant (p˂0.05). The 174 highest and lowest decreases were related to poultry manure (6.39%) and sorghum residue-amended soils 175 (1.13%) compared to the unamended soils, respectively. Besides, results suggested a significant increase in 176 Zn concentration in the organic fraction of amended soils compared to unamended soils. Overall, soils 177 amended with poultry manure, sorghum residue, and clover residue showed a 23.6, 60.7, and 39.7% increase 178 in the organic fraction of Zn with respect to unamended soil, respectively. 179 Table 4 Chemical fractions of Zn in amended and unamended soils with organic amendments. Distribution of Zn chemical forms in soil 164 180 Treatment EX CAR OX OR RES (mg kg-1) Control (unamended) 64.62±1.62c 195.66±3.85a 282.24±3.01a 49.31±1.62d 386.72±3.36a Poultry manure-amended soils 88.98±3.35a 178.66±2.20d 264.20±0.95c 60.97±3.39c 389.14±3.05a Sorghum residues-amended soils 48.35±1.45d 189.74±1.86b 279.03±4.03a 79.27±2.41a 384.22±5.99a Clover residues-amended soils 70.38±4.44b 183.41±2.84c 269.36±1.93b 68.89±3.53b 385.03±4.95a Note: Results are means ± standard deviations. Values with the different lower-case letters within each column are significantly 181 different at p < 0.05 according to LSD test. 182 Plant Zn concentration and yield 183 As shown in Table 5, the addition of organic amendments did result in significant changes in the corn Zn 184 concentration, shoot Zn content (uptake), and the plant dry weight (p˂0.05). The lowest plant Zn 185 concentration and uptake were observed in sorghum residue-amended soils, followed by unamended soil, 186 clover residue-amended soil, and poultry manure-amended soil. Overall, shoot and root Zn concentration, 187 and shoot Zn uptake were decreased in the plants grown in soils amended with sorghum residue by 36.5, 188 21.6, and 8.5% in comparison to unamended soils, respectively. Conversely, shoot and root Zn 189 concentration, and shoot Zn uptake were increased by 19.9, 4.9, and 33.8% in plants grown in poultry 190 manure-amended soil, and 11.6, 2.1, and 28.9% in plants grown in clover residue-amended soils with respect 191 to unamended soils, respectively. Moreover, the presence of organic amendments resulted in a 43.9 and 192 40.8% increase in plant shoot and root dry weight grown in sorghum residue-amended soil, and 15.5 and 193 recorded in soils amended with poultry manure, sorghum residues, and clover residue in comparison to the 172 unamended soil, respectively. 173 The effect of organic amendments on the oxide form of Zn was also statistically significant (p˂0.05). The 174 highest and lowest decreases were related to poultry manure (6.39%) and sorghum residue-amended soils 175 (1.13%) compared to the unamended soils, respectively. Besides, results suggested a significant increase in 176 Zn concentration in the organic fraction of amended soils compared to unamended soils. Overall, soils 177 amended with poultry manure, sorghum residue, and clover residue showed a 23.6, 60.7, and 39.7% increase 178 in the organic fraction of Zn with respect to unamended soil, respectively. 179 Table 4 Chemical fractions of Zn in amended and unamended soils with organic amendments. Distribution of Zn chemical forms in soil 164 180 Treatment EX CAR OX OR RES (mg kg-1) Control (unamended) 64.62±1.62c 195.66±3.85a 282.24±3.01a 49.31±1.62d 386.72±3.36a Poultry manure-amended soils 88.98±3.35a 178.66±2.20d 264.20±0.95c 60.97±3.39c 389.14±3.05a Sorghum residues-amended soils 48.35±1.45d 189.74±1.86b 279.03±4.03a 79.27±2.41a 384.22±5.99a Clover residues-amended soils 70.38±4.44b 183.41±2.84c 269.36±1.93b 68.89±3.53b 385.03±4.95a Note: Results are means ± standard deviations. Values with the different lower-case letters within each column are significantly 181 different at p < 0.05 according to LSD test. 182 9 16% increase in plant shoot and root dry weight grown in clover residue-amended soil compared to 194 unamended soils, respectively. In the case of poultry manure-amended soil, a significant increase was only 195 observed in shoot dry weight (13.4%). 196 Table 5 Mean of dry matter yield, Zn uptake, and Zn concentration in corn grown in amended and unamended soils with organic 197 amendments. 198 Treatment Zn-Shoot (mg kg-1) Zn-Root (mg kg-1) Zn-Shoot uptake (μg pot-1) Shoot dry weight Root dry weight Control (unamended) 205.46±4.01c 356.14±3.66c 2806.70±34.45c 13.65±1.01c 3.57±0.35c Poultry manure-amended soils 246.49±2.74a 373.84±4.32a 3757.94±19.16a 15.48±1.08b 3.91±0.44bc Sorghum residues-amended soils 130.34±2.52d 278.96±4.70d 2567.38±36.18d 19.65±2.11a 5.03±0.41a Clover residues-amended soils 229.35±2.73b 363.84±4.15b 3619.14±10.07b 15.77±1.03b 4.25±0.32b Note: Results are means ± standard deviations. Values with the different lower-case letters within each column are significantly 199 different at p < 0.05 according to LSD test. 200 Correlation between chemical forms of Zn and soil extraction methods 201 The correlation betwean different chemical forms of Zn in amended soils and Zn concentration measured 202 by DTPA and DGT methods represented in table 6. In all amended soils, the organic and carbonate forms 203 of Zn had the highest correlation with DTPA-extractable Zn with respect to CE-Zn. While, CE-Zn gave the 204 higher correlation with the exchangeable and oxide forms rather than DTPA-extractable Zn. On the other 205 hand, except for DTPA-Zn, which showed a positively significant correlation with organic fraction, CE-Zn 206 revealed a negatively significant correlation with organic forms of Zn. Furthermore, there were no 207 significant correlations between the residual forms of Zn and soil extraction methods in amended soils. 208 Table 6 Correlations (Pearson coefficients) between Zn fractions in amended soils with organic amendments and two measures of 209 Zn availability. Distribution of Zn chemical forms in soil 164 210 Fraction Poultry manure Sorghum residues Clover residues DTPA-Zn CE-Zn DTPA-Zn CE-Zn DTPA-Zn CE-Zn EX 0.78 0.85 0.81 0.89** 0.76* 0.86 CAR -0.81 -0.69ns -0.78* -0.71* -0.78* -0.72* OX -0.79* -0.81** -0.76* -0.84** -0.60ns -0.77* OR 0.89** -0.78* 0.86** -0.77* 0.86** -0.73* RES 0.58ns 0.51ns 0.44ns 0.59ns 0.41ns 0.43ns Note: * and ** p-values were significant at 0.01 and 0.05 levels, respectively. ns: Not significant. 211 16% increase in plant shoot and root dry weight grown in clover residue-amended soil compared to 194 unamended soils, respectively. In the case of poultry manure-amended soil, a significant increase was only 195 observed in shoot dry weight (13.4%). 196 16% increase in plant shoot and root dry weight grown in clover residue-amended soil compared to 194 unamended soils, respectively. In the case of poultry manure-amended soil, a significant increase was only 195 observed in shoot dry weight (13.4%). 196 Table 5 Mean of dry matter yield, Zn uptake, and Zn concentration in corn grown in amended and unamended soils with organic 197 amendments. 198 Treatment Zn-Shoot (mg kg-1) Zn-Root (mg kg-1) Zn-Shoot uptake (μg pot-1) Shoot dry weight Root dry weight Control (unamended) 205.46±4.01c 356.14±3.66c 2806.70±34.45c 13.65±1.01c 3.57±0.35c Poultry manure-amended soils 246.49±2.74a 373.84±4.32a 3757.94±19.16a 15.48±1.08b 3.91±0.44bc Sorghum residues-amended soils 130.34±2.52d 278.96±4.70d 2567.38±36.18d 19.65±2.11a 5.03±0.41a Clover residues-amended soils 229.35±2.73b 363.84±4.15b 3619.14±10.07b 15.77±1.03b 4.25±0.32b Note: Results are means ± standard deviations. Values with the different lower-case letters within each column are significantly 199 different at p < 0 05 according to LSD test 200 matter yield, Zn uptake, and Zn concentration in corn grown in amended and unamended soils with organic The correlation betwean different chemical forms of Zn in amended soils and Zn concentration measured 202 by DTPA and DGT methods represented in table 6. In all amended soils, the organic and carbonate forms 203 of Zn had the highest correlation with DTPA-extractable Zn with respect to CE-Zn. While, CE-Zn gave the 204 higher correlation with the exchangeable and oxide forms rather than DTPA-extractable Zn. On the other 205 hand, except for DTPA-Zn, which showed a positively significant correlation with organic fraction, CE-Zn 206 revealed a negatively significant correlation with organic forms of Zn. Furthermore, there were no 207 significant correlations between the residual forms of Zn and soil extraction methods in amended soils. Distribution of Zn chemical forms in soil 164 208 Table 6 Correlations (Pearson coefficients) between Zn fractions in amended soils with organic amendments and two measures of 209 Zn availability. 210 Fraction Poultry manure Sorghum residues Clover residues DTPA-Zn CE-Zn DTPA-Zn CE-Zn DTPA-Zn CE-Zn EX 0.78* 0.85 0.81 0.89** 0.76* 0.86 CAR -0.81 -0.69ns -0.78* -0.71* -0.78* -0.72* OX -0.79* -0.81** -0.76* -0.84** -0.60ns -0.77* OR 0.89** -0.78* 0.86** -0.77* 0.86** -0.73* RES 0.58ns 0.51ns 0.44ns 0.59ns 0.41ns 0.43ns Note: * and p-values were significant at 0.01 and 0.05 levels, respectively. ns: Not significant. 211 10 Estimations of Zn availability 212 The relationship between Zn concentration in soil and different plant tissues was investigated by linear 213 correlations (Figure 1). Relationships determined between pooled Zn concentrations of shoot and two 214 measures of Zn availability taken from pooled soil results were significant (P < 0.05). 215 Comparisons of the determination coefficients for tow assays of bioavailability and plant Zn concentrations 216 and uptakes, revealed that CE-Zn gave the most robust relationship for plant and soil data overall, closely 217 Comparisons of the determination coefficients for tow assays of bioavailability and plant Zn concentrations 216 and uptakes, revealed that CE-Zn gave the most robust relationship for plant and soil data overall, closely 217 followed by DTPA extractable Zn. 218 Comparisons of the determination coefficients for tow assays of bioavailability and plant Zn concentrations 216 and uptakes, revealed that CE-Zn gave the most robust relationship for plant and soil data overall, closely 217 followed by DTPA extractable Zn. 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 Fig. 1 Relationships determined between Zn concentration in plant tissues grown in amended and unamended soils and soil 240 extractable Zn. 241 242 0 200 400 600 800 1000 1200 0 300 600 90012001500 Zn shoot uptak (μg pot-1) CE-Zn (μg L-1) 0 100 200 300 400 500 0 300 600 90012001500 Zn root concentration (mg kg-1) CE-Zn (μg L-1) 0 50 100 150 200 250 0 300 600 90012001500 Zn shoot concentration (mg kg-1) CE-Zn (μg L-1) 0 200 400 600 800 1000 0 50 100 150 200 250 Zn shoot uptake (μg pot-1) DTPA-Zn (mg kg-1) 0 100 200 300 400 500 0 50 100 150 200 250 Zn root concentration (mg kg-1) DTPA-Zn (mg kg-1) 0 50 100 150 200 250 0 50 100 150 200 250 Zn shoot concentration (mg kg-1) DTPA-Zn (mg kg-1) r2=0.82 p < 0.01 r2=0.71 p < 0.05 r2=0.61 p < 0.05 r 2=0.97 p < 0.01 r 2=0.84 p < 0.01 r 2=0.91 p < 0.01 239 Fig. 1 Relationships determined between Zn concentration in plant tissues grown in amended and unamended soils and soil 240 extractable Zn. 241 242 Discussion 243 The addition of poultry manure, sorghum, and clover residues significantly increased the concentration of 244 DOC in soil solution as well as decreasing the amount of soil pH. Microbial degradation of soil organic 245 amendments and root exudates were the likely factor that resulted in these differences. Organic amendments 246 and root exudates provide microorganisms with a valuable source of carbon and nitrogen, which results in 247 considerable microbial activity (Khoshgoftarmanesh et al. 2018). Amending soils with poultry manure and 248 The addition of poultry manure, sorghum, and clover residues significantly increased the concentration of 244 DOC in soil solution as well as decreasing the amount of soil pH. Microbial degradation of soil organic 245 amendments and root exudates were the likely factor that resulted in these differences. Organic amendments 246 and root exudates provide microorganisms with a valuable source of carbon and nitrogen, which results in 247 considerable microbial activity (Khoshgoftarmanesh et al. 2018). Amending soils with poultry manure and 248 11 plant residues provide additional DOC to the soil solution. The presence of DOC, particularly humic acids, 249 may be the main factor contributing to the increased CE-Zn and DTPA-extractable Zn (except for CE-Zn in 250 sorghum residues- amended soil) through decreased pH and formation soluble organo-Zn. Increases of metal 251 bioavailability as a result of decreased pH can occur in different ways, including the acidic groups contained 252 in DOC such as hydroxyl functional groups and carboxyl (Adeleke et al. 2017), and the production of CO2 253 from root respiration and microbial activity, decrease pH, thereby increasing exchangeable metal form (Yao 254 et al. 2020). The increase in exchangeable form of Zn may be linked to decreased oxide fractions of Zn 255 which is likely due to the excretion of Zn2+/OH- to balance the internal charge variation as a result of 256 increased H+ in the soil solution. Another possible cause for increased Zn exchangeable form, is the presence 257 of decreased amounts of carbonate form due to decreased pH (Alloway 2013). Montalvo et al. (2016) found 258 that the increased concentration of DTPA-Zn in calcareous soil was related to the dissolution of zinc 259 carbonate minerals (ZnCO3) as a result of decreased pH in soil solution. Egene et al. (2018) observed that 260 municipal waste compost increased CE-Zn, which may be attributed to the significant decrease in soil pH. Discussion 243 (2018) observed that 260 municipal waste compost increased CE-Zn, which may be attributed to the significant decrease in soil pH. 261 The effects of these parameters present in organic amendments on CE-Zn and DTPA-Zn extractability 262 subsequently influenced the increase of corn Zn concentration. While dry matter biomass was not strongly 263 influenced by increased Zn concentration, which in turn was likely affected by the presence of enhanced 264 soil physical condition and increased concentration of macronutrients provided by organic amendments. 265 plant residues provide additional DOC to the soil solution. The presence of DOC, particularly humic acids, 249 may be the main factor contributing to the increased CE-Zn and DTPA-extractable Zn (except for CE-Zn in 250 sorghum residues- amended soil) through decreased pH and formation soluble organo-Zn. Increases of metal 251 bioavailability as a result of decreased pH can occur in different ways, including the acidic groups contained 252 in DOC such as hydroxyl functional groups and carboxyl (Adeleke et al. 2017), and the production of CO2 253 from root respiration and microbial activity, decrease pH, thereby increasing exchangeable metal form (Yao 254 et al. 2020). The increase in exchangeable form of Zn may be linked to decreased oxide fractions of Zn 255 which is likely due to the excretion of Zn2+/OH- to balance the internal charge variation as a result of 256 increased H+ in the soil solution. Another possible cause for increased Zn exchangeable form, is the presence 257 of decreased amounts of carbonate form due to decreased pH (Alloway 2013). Montalvo et al. (2016) found 258 that the increased concentration of DTPA-Zn in calcareous soil was related to the dissolution of zinc 259 carbonate minerals (ZnCO3) as a result of decreased pH in soil solution. Egene et al. (2018) observed that 260 municipal waste compost increased CE-Zn, which may be attributed to the significant decrease in soil pH. 261 The effects of these parameters present in organic amendments on CE-Zn and DTPA-Zn extractability 262 subsequently influenced the increase of corn Zn concentration. While dry matter biomass was not strongly 263 influenced by increased Zn concentration, which in turn was likely affected by the presence of enhanced 264 soil physical condition and increased concentration of macronutrients provided by organic amendments. Discussion 243 261 The effects of these parameters present in organic amendments on CE-Zn and DTPA-Zn extractability 262 subsequently influenced the increase of corn Zn concentration. While dry matter biomass was not strongly 263 influenced by increased Zn concentration, which in turn was likely affected by the presence of enhanced 264 soil physical condition and increased concentration of macronutrients provided by organic amendments. 265 Another factor that may have affected the increased dry matter production, is likely the formation of 266 phytochelatin-Zn complexes into plant cells as the primary mechanism contributing to the decrease in metals 267 mobility in plant tissues and several studies have linked the mobility of heavy metals in plants' tissues to the 268 synthesis of non-structural carbohydrates and phytoclates in the plants tissues (Chand et al. 2012; Sripriya 269 et al. 2016). 270 Moreover, unlike poultry manure and clover residues-amended soils, the addition of sorghum residues 271 resulted in a decrease in plant Zn concentration, which may be attributed to the significant decrease in CE- 272 Zn in the soil as a result of the conversion of exchangeable fraction into an organic fraction with lower 273 plant residues provide additional DOC to the soil solution. The presence of DOC, particularly humic acids, 249 may be the main factor contributing to the increased CE-Zn and DTPA-extractable Zn (except for CE-Zn in 250 sorghum residues- amended soil) through decreased pH and formation soluble organo-Zn. Increases of metal 251 bioavailability as a result of decreased pH can occur in different ways, including the acidic groups contained 252 in DOC such as hydroxyl functional groups and carboxyl (Adeleke et al. 2017), and the production of CO2 253 from root respiration and microbial activity, decrease pH, thereby increasing exchangeable metal form (Yao 254 et al. 2020). The increase in exchangeable form of Zn may be linked to decreased oxide fractions of Zn 255 which is likely due to the excretion of Zn2+/OH- to balance the internal charge variation as a result of 256 increased H+ in the soil solution. Another possible cause for increased Zn exchangeable form, is the presence 257 of decreased amounts of carbonate form due to decreased pH (Alloway 2013). Montalvo et al. (2016) found 258 that the increased concentration of DTPA-Zn in calcareous soil was related to the dissolution of zinc 259 carbonate minerals (ZnCO3) as a result of decreased pH in soil solution. Egene et al. Discussion 243 Conversely, despite the reduction of the exchangeable form of Zn, the concentration of 280 DTPA-Zn in the sorghum residues-amended soil increased with respect to CE-Zn. 281 to an increase in the organic fraction of Cu. The use of organic matter in the soil plays a key role on the 275 absorption of heavy metals. Molecular-scale spectroscopic studies have shown that these metals form strong 276 bonds with functional groups of organic matter such as carboxylic, phenolic, and thiols (Degryse et al. 277 2009). Therefore, an increased organic fraction of Zn in sorghum residues-amended soil may directly be 278 attributed to the higher carbon to nitrogen ratio (C:N) value of sorghum residue than poultry manure and 279 clover residues. Conversely, despite the reduction of the exchangeable form of Zn, the concentration of 280 DTPA-Zn in the sorghum residues-amended soil increased with respect to CE-Zn. 281 The reason that chelating agents such as DTPA-TEA might not be a good predictor of Zn bioavailability is 282 that not all chemical fractions of metals are thought to be available for plants, and it has been revealed that 283 free ion and labile complexes of metals are available for uptake, while a large amount of Zn organic form 284 extracted by this extractant was relatively unavailable for plant and it appears that this extractant 285 overestimated the phytoavailability (Soriano-Disla et al. 2010). The positive (DTPA-Zn) and negative (CE- 286 Zn) significant correlation obtained between organic fraction and two potential measures of Zn 287 bioavailability partly confirmed that DTPA might be extracted some of the Zn organic forms which was not 288 available for the plant. However, in poultry manure and clover residues-amended soils, due to the increase 289 in Zn exchangeable forms, it was impossible to distinguish whether the increased DTPA-extractable Zn was 290 related to the exchangeable fraction or the organic fraction. 291 Although DTPA extractant has been successfully used to measure deficiencies of metals in soils, the use of 292 this method to assess phytotoxic concentrations of metals have been less so, with plant responses typically 293 poorly correlated with DPTA (Karami et al. 2009; Zhang et al. 2010) which may be attributed to lack of 294 metals concentration measurement partitioned to the solid phase. Discussion 243 281 The reason that chelating agents such as DTPA-TEA might not be a good predictor of Zn bioavailability is 282 that not all chemical fractions of metals are thought to be available for plants, and it has been revealed that 283 free ion and labile complexes of metals are available for uptake, while a large amount of Zn organic form 284 extracted by this extractant was relatively unavailable for plant and it appears that this extractant 285 overestimated the phytoavailability (Soriano-Disla et al. 2010). The positive (DTPA-Zn) and negative (CE- 286 Zn) significant correlation obtained between organic fraction and two potential measures of Zn 287 bioavailability partly confirmed that DTPA might be extracted some of the Zn organic forms which was not 288 available for the plant. However, in poultry manure and clover residues-amended soils, due to the increase 289 in Zn exchangeable forms, it was impossible to distinguish whether the increased DTPA-extractable Zn was 290 related to the exchangeable fraction or the organic fraction. 291 Although DTPA extractant has been successfully used to measure deficiencies of metals in soils, the use of 292 to an increase in the organic fraction of Cu. The use of organic matter in the soil plays a key role on the 275 absorption of heavy metals. Molecular-scale spectroscopic studies have shown that these metals form strong 276 bonds with functional groups of organic matter such as carboxylic, phenolic, and thiols (Degryse et al. 277 2009). Therefore, an increased organic fraction of Zn in sorghum residues-amended soil may directly be 278 attributed to the higher carbon to nitrogen ratio (C:N) value of sorghum residue than poultry manure and 279 clover residues. Conversely, despite the reduction of the exchangeable form of Zn, the concentration of 280 DTPA-Zn in the sorghum residues-amended soil increased with respect to CE-Zn. 281 to an increase in the organic fraction of Cu. The use of organic matter in the soil plays a key role on the 275 absorption of heavy metals. Molecular-scale spectroscopic studies have shown that these metals form strong 276 bonds with functional groups of organic matter such as carboxylic, phenolic, and thiols (Degryse et al. 277 2009). Therefore, an increased organic fraction of Zn in sorghum residues-amended soil may directly be 278 attributed to the higher carbon to nitrogen ratio (C:N) value of sorghum residue than poultry manure and 279 clover residues. Discussion 243 265 Another factor that may have affected the increased dry matter production, is likely the formation of 266 Another factor that may have affected the increased dry matter production, is likely the formation of 266 phytochelatin-Zn complexes into plant cells as the primary mechanism contributing to the decrease in metals 267 mobility in plant tissues and several studies have linked the mobility of heavy metals in plants' tissues to the 268 synthesis of non-structural carbohydrates and phytoclates in the plants tissues (Chand et al. 2012; Sripriya 269 et al. 2016). 270 Moreover, unlike poultry manure and clover residues-amended soils, the addition of sorghum residues 271 resulted in a decrease in plant Zn concentration, which may be attributed to the significant decrease in CE- 272 Zn in the soil as a result of the conversion of exchangeable fraction into an organic fraction with lower 273 mobility. Garrido Reyeset et al. (2013) found that decreased CE-Cu in biosolids-amended soil was related 274 Moreover, unlike poultry manure and clover residues-amended soils, the addition of sorghum residues 271 resulted in a decrease in plant Zn concentration, which may be attributed to the significant decrease in CE- 272 Zn in the soil as a result of the conversion of exchangeable fraction into an organic fraction with lower 273 mobility. Garrido Reyeset et al. (2013) found that decreased CE-Cu in biosolids-amended soil was related 274 12 to an increase in the organic fraction of Cu. The use of organic matter in the soil plays a key role on the 275 absorption of heavy metals. Molecular-scale spectroscopic studies have shown that these metals form strong 276 bonds with functional groups of organic matter such as carboxylic, phenolic, and thiols (Degryse et al. 277 2009). Therefore, an increased organic fraction of Zn in sorghum residues-amended soil may directly be 278 attributed to the higher carbon to nitrogen ratio (C:N) value of sorghum residue than poultry manure and 279 clover residues. Conversely, despite the reduction of the exchangeable form of Zn, the concentration of 280 DTPA-Zn in the sorghum residues-amended soil increased with respect to CE-Zn. Discussion 243 While, in the case of DGT method, the 295 replenishment capacity of the solid phase is considered as a fundamental variable that resupply the amount 296 depleted in soil solution (Mason et al. 2010). 297 Analytically quantifying the potential measure of metals bioavailability by DGT technique has only been 298 attempted in a few studies, but in general, results from these predictions have not included calcareous soils 299 treated with the organic amendments. A study by Soriano-Disla et al. (2010) found that the correlation for 300 Analytically quantifying the potential measure of metals bioavailability by DGT technique has only been 298 attempted in a few studies, but in general, results from these predictions have not included calcareous soils 299 treated with the organic amendments. A study by Soriano-Disla et al. (2010) found that the correlation for 300 Analytically quantifying the potential measure of metals bioavailability by DGT technique has only been 298 attempted in a few studies, but in general, results from these predictions have not included calcareous soils 299 treated with the organic amendments. A study by Soriano-Disla et al. (2010) found that the correlation for 300 13 CE-Zn and sorghum root Zn concentration grown in was higher (r2 = 0.91), than DTPA-Zn concentration 301 (r2 = 0.89). Another successful relationship between Zn concentration of the shoot and CE-Zn (r2 = 0.76) 302 was also obtained in peppermint grown in sewage sludge-treated soils, but a less robust relationship was 303 found with Zn concentrations in the shoot and DTPA-Zn (r2 = 0.62) (Mohseni et al. 2020). 304 Conclusions 305 CE-Zn and sorghum root Zn concentration grown in was higher (r2 = 0.91), than DTPA-Zn concentration 301 (r2 = 0.89). Another successful relationship between Zn concentration of the shoot and CE-Zn (r2 = 0.76) 302 was also obtained in peppermint grown in sewage sludge-treated soils, but a less robust relationship was 303 found with Zn concentrations in the shoot and DTPA-Zn (r2 = 0.62**) (Mohseni et al. 2020). 304 Conclusions 305 This study showed that the organic amendment efficacy at reducing the bioavailability of the metal was 306 mostly dependent on the type of organic amendment and the potential measures of metal bioavailability for 307 plants. Unlike poultry manure and clover residues, sorghum residues significantly accounted for the most 308 reduction in Zn bioavailability as a result of higher carbon to nitrogen ratio. Discussion 243 DTPA method was poorly 309 correlated with plant metal concentrations, which may be associated with the extraction of the unavailable 310 form of Zn, especially Zn organic form. In contrast, the DGT technique had strong potential as a measure 311 of Zn bioavailability in amended soils. Thus the theory that DGT technique imitates important processes 312 involved in plant metal uptake makes it more reliable for estimating the bioavailability of Zn. 313 Declarations 314 Funding 315 This research did not receive any specific grant from funding agencies in the public, commercial, or not- 316 for-profit sectors. 317 Competing interests 318 The authors declare that they have no competing interests. 319 Availability of data and materials 320 The datasets used and/or analyzed during the current study are available from the corresponding author on 321 reasonable request. 322 Code availability 323 Not applicable 324 Acknowledgement 325 This study showed that the organic amendment efficacy at reducing the bioavailability of the metal was 306 mostly dependent on the type of organic amendment and the potential measures of metal bioavailability for 307 plants. Unlike poultry manure and clover residues, sorghum residues significantly accounted for the most 308 reduction in Zn bioavailability as a result of higher carbon to nitrogen ratio. DTPA method was poorly 309 correlated with plant metal concentrations, which may be associated with the extraction of the unavailable 310 form of Zn, especially Zn organic form. In contrast, the DGT technique had strong potential as a measure 311 of Zn bioavailability in amended soils. Thus the theory that DGT technique imitates important processes 312 involved in plant metal uptake makes it more reliable for estimating the bioavailability of Zn. 313 14 The authors appreciate the Agricultural Research, Education and Extension Organization, Tehran, Iran for 326 technical support of this study. 327 The authors appreciate the Agricultural Research, Education and Extension Organization, Tehran, Iran for 326 technical support of this study. 327 References 328 References 328 Adeleke, R., Nwangburuka, C., & Oboirien, B. (2017). Origins, roles and fate of organic acids in soils: A review. 329 South African Journal of Botany, 108, 393-406. 330 Alloway, B. J. (2013). Sources of heavy metals and metalloids in soils. In Heavy metals in soils (pp. 11-50): Springer. 331 Ashraf, S., Ali, Q., Zahir, Z. A., Ashraf, S., & Asghar, H. N. (2019). Phytoremediation: Environmentally sustainable 332 way for reclamation of heavy metal polluted soils. Ecotoxicology and Environmental Safety, 174, 714-727. 333 Aziz, M. A., Ahmad, H. R., Corwin, D. L., Sabir, M., Hakeem, K. R., & Öztürk, M. (2017). Influence of farmyard 334 manure on retention and availability of nickel, zinc and lead in metal-contaminated calcareous loam soils. 335 Journal of Environmental Engineering and Landscape Management, 25(3), 289-296. 336 Chand, S., Pandey, A., & Patra, D. (2012). Influence of nickel and lead applied in combination with vermicompost on 337 Chand, S., Pandey, A., & Patra, D. (2012). Influence of nickel and lead applied in combina 337 growth and accumulation of heavy metals by Mentha arvensis Linn. cv.‘Kosi’. 338 growth and accumulation of heavy metals by Mentha arvensis Linn. cv.‘Kosi’. 338 Chen, M., & Ma, L. Q. (2001). Comparison of three aqua regia digestion methods for twenty Florida soils. Soil science 339 society of America Journal, 65(2), 491-499. 340 Chen, Z., Setagawa, M., Kang, Y., Sakurai, K., Aikawa, Y., & Iwasaki, K. (2009). Zinc and cadmium uptake from a 341 metalliferous soil by a mixed culture of Athyrium yokoscense and Arabis flagellosa. Soil Science and Plant 342 Nutrition, 55(2), 315-324. 343 Chen, Z., Setagawa, M., Kang, Y., Sakurai, K., Aikawa, Y., & Iwasaki, K. (2009). Zinc and cadmium uptake from a 341 metalliferous soil by a mixed culture of Athyrium yokoscense and Arabis flagellosa. Soil Science and Plant 342 Nutrition, 55(2), 315-324. 343 Degryse, F., Smolders, E., Zhang, H., & Davison, W. (2009). Predicting availability of mineral elements to plants with 344 the DGT technique: a review of experimental data and interpretation by modelling. Environmental Chemistry, 345 6(3), 198-218. 346 Dodangeh, H., Rahimi, G., Fallah, M., & Ebrahimi, E. (2018). Investigation of heavy metal uptake by three types of 347 ornamental plants as affected by application of organic and chemical fertilizers in contaminated soils. 348 Environmental Earth Sciences, 77(12), 473. 349 Egene, C. E., Van Poucke, R., Ok, Y. S., Meers, E., & Tack, F. (2018). References 328 Impact of organic amendments (biochar, 350 compost and peat) on Cd and Zn mobility and solubility in contaminated soil of the Campine region after 351 three years. Science of the Total Environment, 626, 195-202. 352 Egene, C. E., Van Poucke, R., Ok, Y. S., Meers, E., & Tack, F. (2018). Impact of organic amendments (biochar, 350 compost and peat) on Cd and Zn mobility and solubility in contaminated soil of the Campine region after 351 15 Garrido Reyes, T., Mendoza Crisosto, J., & Velásquez Vergara, Y. (2013). Extractability of copper and application of 353 diffusive gradients in thin films: metal availability in contaminated soil by biosolids. Journal of the Brazilian 354 Chemical Society, 24(9), 1442-1450. 355 Gee, G. W., & Bauder, W. (1986). Particle-size analysis. In D. L. Sparks (Ed.), Methods of soil analysis. Part 1: 356 Physical and mineralogical methods (pp. 383–409). Madison: Soil Science Society of America. 357 Harper, M. P., Davison, W., & Tych, W. (2000). DIFS—a modelling and simulation tool for DGT induced trace metal 358 remobilisation in sediments and soils. Environmental Modelling & Software, 15(1), 55-66. 359 Heidari, S., Reyhanitabar, A., Oustan, S., & Olad, A. (2016). A new method of preparing gel for DGT technique and 360 application to the soil phosphorus availability test. Communications in Soil Science and Plant Analysis, 361 47(10), 1239-1251. 362 Kabata-Pendias, A., & Pendias, H. (1984). Trace elements in the soil and plants. Boca Rat 363 Kabata-Pendias, A., & Pendias, H. (1984). Trace elements in the soil and plants. Boca Raton, FL: CRC Press. 363 Karami, M., Afyuni, M., Rezainejad, Y., & Schulin, R. (2009). Heavy metal uptake by wh 364 Karami, M., Afyuni, M., Rezainejad, Y., & Schulin, R. (2009). Heavy metal uptake by wheat from a sewage sludge- 364 amended calcareous soil. Nutrient Cycling in Agroecosystems, 83(1), 51-61. 365 amended calcareous soil. Nutrient Cycling in Agroecosystems, 83(1), 51-61. 365 amended calcareous soil. Nutrient Cycling in Agroecosystems, 83(1), 51-61. 365 Khoshgoftarmanesh, A. H., Afyuni, M., Norouzi, M., Ghiasi, S., & Schulin, R. (2018). Fractionation and 366 g , , y , , , , , , , ( ) bioavailability of zinc (Zn) in the rhizosphere of two wheat cultivars with different Zn deficiency tolerance. 367 Geoderma, 309, 1-6. 368 Li, C., Ding, S., Yang, L., Wang, Y., Ren, M., Chen, M., et al. (2019). References 328 Diffusive gradients in thin films: devices, 369 Li, C., Ding, S., Yang, L., Wang, Y., Ren, M., Chen, M., et al. (2019). Diffusive gradients in thin films: devices, 369 materials and applications. Environmental Chemistry Letters, 17(2), 801-831. 370 materials and applications. Environmental Chemistry Letters, 17(2), 801-831. 370 materials and applications. Environmental Chemistry Letters, 17(2), 801-831. 370 Li, Z., Jia, M., Wu, L., Christie, P., & Luo, Y. (2016). Changes in metal availability, desorption kinetics and speciation 371 in contaminated soils during repeated phytoextraction with the Zn/Cd hyperaccumulator Sedum 372 plumbizincicola. Environmental pollution, 209, 123-131. 373 Lindsay, W. L., & Norvell, W. (1978). Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil 374 Science Society of America Journal 42(3) 421–428 375 Lindsay, W. L., & Norvell, W. (1978). Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil 374 Science Society of America Journal, 42(3),421–428. 375 Science Society of America Journal, 42(3),421–428. 375 Loeppert, R. H., & Suarez, D. L. (1996). Carbonate and gypsum. In D. L. Sparks (Ed.), Methods of soil analysis. Part 376 3: Chemical methods (pp. 437–474). Madison: Soil Science Society of America. 377 16 Lozano-Rodríguez, E., Luguera, M., Lucena, J., & Carpena-Ruiz, R. (1995). Evaluation of two different acid digestion 378 methods in closed systems for trace element determinations in plants. Quimica Analitica-Bellaterra-, 14, 27- 379 27. 380 Lozano-Rodríguez, E., Luguera, M., Lucena, J., & Carpena-Ruiz, R. (1995). Evaluation of two different acid digestion 378 methods in closed systems for trace element determinations in plants. Quimica Analitica-Bellaterra-, 14, 27- 379 27. 380 McLaughlin, M. J., Hamon, R., McLaren, R., Speir, T., & Rogers, S. (2000). A bioavailability-based rationale for 381 controlling metal and metalloid contamination of agricultural land in Australia and New Zealand. Soil 382 Research, 38(6), 1037-1086. 383 Meers, E., Du Laing, G., Unamuno, V., Ruttens, A., Vangronsveld, J., Tack, F., et al. (2007). Comparison of cadmium 384 extractability from soils by commonly used single extraction protocols. Geoderma, 141(3-4), 247-259. 385 Mohseni, A., Reyhanitabar, A., Najafi, N., Oustan, S., & Bazargan, K. (2018). Kinetics of DTPA extraction of Zn, Pb, 386 and Cd from contaminated calcareous soils amended with sewage sludge. Arabian Journal of Geosciences, 387 11(14), 384. 388 Mohseni, A., Reyhanitabar, A., Najafi, N., Oustan, S., & Bazargan, K. (2020). Effects of sludge on heavy metals 389 release from peppermint-planted soils during time as assessed by DGT technique. ew formulations and calibrations. Soil Science Society of America Journal, 53(2), 433-439. References 328 Archives of Agronomy and 390 Soil Science. 391 Nelson, D. W., & Sommers, L. E. (1996). Total carbon, organic carbon and organic matter. In D. L. Sparks (Ed.), 392 Methods of soil analysis. Part 3: Chemical methods (pp. 961–1010). Madison: Soil Science Society of 393 America. 394 Nelson, D. W., & Sommers, L. E. (1996). Total carbon, organic carbon and organic matter. In D. L. Sparks (Ed.), 392 Methods of soil analysis. Part 3: Chemical methods (pp. 961–1010). Madison: Soil Science Society of 393 America. 394 Nolan, A. L., Zhang, H., & McLaughlin, M. J. (2005). Prediction of zinc, cadmium, lead, and copper availability to 395 wheat in contaminated soils using chemical speciation, diffusive gradients in thin films, extraction, and 396 isotopic dilution techniques. Journal of environmental quality, 34(2), 496-507. 397 Nwachukwu, O. I., & Pulford, I. (2009). Soil metal immobilization and ryegrass uptake of lead, copper and zinc as 398 affected by application of organic materials as soil amendments in a short‐term greenhouse trial. Soil use and 399 management, 25(2), 159-167. 400 Palansooriya, K. N., Shaheen, S. M., Chen, S. S., Tsang, D. C., Hashimoto, Y., Hou, D., et al. (2020). Soil amendments 401 for immobilization of potentially toxic elements in contaminated soils: a critical review. Environment 402 international, 134, 105046. 403 Rhoades, J. D., Manteghi, N. A., Shouse, P. J., & Alves, W. J. (1989). Soil electrical conductivity and soil salinity: 404 New formulations and calibrations. Soil Science Society of America Journal, 53(2), 433-439. 405 Nolan, A. L., Zhang, H., & McLaughlin, M. J. (2005). Prediction of zinc, cadmium, lead, and copper availability to 395 wheat in contaminated soils using chemical speciation, diffusive gradients in thin films, extraction, and 396 isotopic dilution techniques. Journal of environmental quality, 34(2), 496-507. 397 Palansooriya, K. N., Shaheen, S. M., Chen, S. S., Tsang, D. C., Hashimoto, Y., Hou, D., et al. (2020). Soil amendments 401 for immobilization of potentially toxic elements in contaminated soils: a critical review. Environment 402 international, 134, 105046. 403 17 Sposito, G., Lund, L., & Chang, A. (1982). Trace metal chemistry in arid‐zone field soils amended with sewage sludge: 406 I. Fractionation of Ni, Cu, Zn, Cd, and Pb in solid phases. Soil Science Society of America Journal, 46(2), 407 260–264. 408 Tessier, A., Campbell, P. G., & Bisson, M. (1979). Sequential extraction procedure for the speciation of particulate 409 trace metals. Analytical chemistry, 51(7), 844-851. References 328 410 Thomas, G. W. (1996). Soil pH and soil acidity. In D. L. Sparks (Ed.), Methods of soil analysis. Part 3: Chemical 411 Thomas, G. W. (1996). Soil pH and soil acidity. In D. L. Sparks (Ed.), Methods of soil analysis. Part 3: Chemical 411 methods (pp. 475–490). Madison: Soil Science Society of America. 412 methods (pp. 475–490). Madison: Soil Science Society of America. 412 Yao, B., Hu, Q., Zhang, G., Yi, Y., Xiao, M., & Wen, D. (2020). Effects of Elevated CO2 Concentration and Nitrogen 413 Addition on Soil Respiration in a Cd-Contaminated Experimental Forest Microcosm. Forests, 11(3), 260. 414 Yao, B., Hu, Q., Zhang, G., Yi, Y., Xiao, M., & Wen, D. (2020). Effects of Elevated CO2 C 413 Q g ( ) g Addition on Soil Respiration in a Cd-Contaminated Experimental Forest Microcosm. Forests, 11(3), 260. 414 Addition on Soil Respiration in a Cd-Contaminated Experimental Forest Microcosm. Forests, 11(3), 260. 414 Addition on Soil Respiration in a Cd-Contaminated Experimental Forest Microco 414 Zhang, H., & Davison, W. (1995). Performance characteristics of diffusion gradients in thin films for the in situ 415 measurement of trace metals in aqueous solution. Analytical chemistry, 67(19), 3391-3400. 416 Zhang, H., Davison, W., Tye, A. M., Crout, N. M., & Young, S. D. (2006). Kinetics of zinc and cadmium release in 417 freshly contaminated soils. Environmental Toxicology and Chemistry: An International Journal, 25(3), 664- 418 670. 419 Zhang, M.-K., Liu, Z.-Y., & Wang, H. (2010). Use of single extraction methods to predict bioavailability of heavy 420 metals in polluted soils to rice. Communications in Soil Science and Plant Analysis, 41(7), 820-831. 421 Zhao, F. J., Rooney, C. P., Zhang, H., & McGrath, S. P. (2006). Comparison of soil solution speciation and diffusive 422 18 Figures Figures Figures Figure 1 Relationships determined between Zn concentration in plant tissues grown in amended and unamen soils and soil extractable Zn. Figure 1 Relationships determined between Zn concentration in plant tissues grown in amended and unamended soils and soil extractable Zn. Relationships determined between Zn concentration in plant tissues grown in amended and unamended soils and soil extractable Zn.
https://openalex.org/W3211016258	https://www.frontiersin.org/articles/10.3389/fendo.2021.732456/pdf	English	null	PACAP–PAC1 Signaling Regulates Serotonin 2A Receptor Internalization	Frontiers in endocrinology	2,021	cc-by	8,915	ORIGINAL RESEARCH published: 25 October 2021 doi: 10.3389/fendo.2021.732456 PACAP–PAC1 Signaling Regulates Serotonin 2A Receptor Internalization Atsuko Hayata-Takano 1,2†, Yusuke Shintani 1†, Keita Moriguchi 1, Naoki Encho 1, Kohei Kitagawa 1, Takanobu Nakazawa 1,3 and Hitoshi Hashimoto 1,2,4,5,6 1 Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan, 2 Molecular Research Center for Children’s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Suita, Japan, 3 Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Japan, 4 Division of Bioscience, Institute for Datability Science, Osaka University, Suita, Japan, 5 Transdimensional Life Imaging Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Japan, 6 Department of Molecular Pharmaceutical Science, Graduate School of Medicine, Osaka University, Suita, Japan Keywords: pituitary adenylate cyclase-activating polypeptide (PACAP), internalization, hallucination, b-arrestin, G protein-coupled receptor (GPCR), serotonin 2A receptor (5-HT2A) Reviewed by: Reviewed by: Lee E. Eiden, National Institutes of Health (NIH), United States Alessandro Castorina, University of Technology Sydney, Australia Correspondence: Hitoshi Hashimoto hasimoto@phs.osaka-u.ac.jp Atsuko Hayata-Takano a-hayata@phs.osaka-u.ac.jp †These authors have contributed Correspondence: Hitoshi Hashimoto hasimoto@phs.osaka-u.ac.jp Atsuko Hayata-Takano a-hayata@phs.osaka-u.ac.jp †These authors have contributed equally to this work and share ﬁrst authorship Correspondence: Hitoshi Hashimoto hasimoto@phs.osaka-u.ac.jp Atsuko Hayata-Takano a-hayata@phs.osaka-u.ac.jp †These authors have contributed equally to this work and share ﬁrst authorship Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Received: 29 June 2021 Accepted: 27 September 2021 Published: 25 October 2021 Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Received: 29 June 2021 Accepted: 27 September 2021 Published: 25 October 2021 Received: 29 June 2021 Accepted: 27 September 2021 Published: 25 October 2021 Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) display psychomotor abnormalities, most of which are ameliorated by atypical antipsychotics with serotonin(5-HT)2Areceptor(5-HT2A)antagonism.HeterozygousPacapmutantmiceshowa signiﬁcantly higher hallucinogenic response than wild-type mice to a 5-HT2A agonist. Endogenous PACAP may, therefore, affect 5-HT2A signaling; however, the underlying neurobiological mechanism for this remains unclear. Here, we examined whether PACAP modulates 5-HT2A signaling by addressing cellular protein localization. PACAP induced an increase in internalization of 5-HT2A but not 5-HT1A, 5-HT2C, dopamine D2 receptors or metabotropic glutamate receptor 2 in HEK293T cells. This PACAP action was inhibited by protein kinase C inhibitors, b-arrestin2 silencing, the PACAP receptor PAC1 antagonist PACAP6-38, and PAC1 silencing. In addition, the levels of endogenous 5-HT2A were decreased on the cell surface of primary cultured cortical neurons after PACAP stimulation and were increased in frontal cortex cell membranes of Pacap−/−mice. Finally, intracerebroventricular PACAP administration suppressed 5-HT2A agonist-induced head twitch responses in mice. These results suggest that PACAP–PAC1 signaling increases 5-HT2A internalization resulting in attenuation of 5-HT2A-mediated signaling, although further study is necessary to determine the relationship between behavioral abnormalities in Pacap−/−mice and PACAP-induced 5-HT2A internalization. Edited by: Edited by: Hubert Vaudry, Universite´ de Rouen, France Vector Construction The vector, pFN21A (HaloTag technology, Promega, Madison, WI, USA), encoding the secretory IL-6 signal peptide fused to the N-terminus of Halo-tag was a gift from Dr. Nagase (Kazusa DNA Research Institute). To generate the Halo-PAC1 construct, the hop1 splicing variant of a human PAC1 cDNA was subcloned into the pFN21A vector at SgfI and PmeI restriction sites as described previously (4). Human 5-HT1A, 5-HT2A, D2 and mGlu2 cDNAs were obtained from the Kazusa Collection of Flexi ORF Clones (Kazusa DNA Research Institute, Chiba, Japan). These clones were also subcloned into the pFN21A vector at SgfI and PmeI restriction sites. 5-HT2A has beenimplicated inmany psychiatric disorders, such as schizophrenia and affective disorders (13). Clinical studies have indicated that impaired 5-HT2A signaling plays a major role in schizophrenic episodes (14). Almost all currently available atypical antipsychotic drugs possess antagonistic effects against D2 and 5- HT2A (15).Cellular internalizationis knownto play a criticalrolein the regulation of 5-HT2A functions (16, 17). 5-HT, dopamine, DOI and clozapineinduce 5-HT2A internalizationand recycling, and the signaling processes through which each ligand induces its effect are differentially regulated (17). In addition, different classes of G- protein-coupled receptors (GPCRs) can form heteromeric complexes that potentially contribute to the regulation of receptor internalization or alteration of pharmacological signaling properties (18, 19). 5-HT2A/metabotropic glutamate receptor 2 (mGlu2) and 5-HT2A/D2 form heteromeric complexes that induce unique hallucinogen-speciﬁc signaling (20–23). Thus, the signaling pathways involved in 5-HT2A function are complicated, and the precise signaling pathways responsible for hallucinogenic and therapeutic effects remain unclear. Receptor Internalization in HEK293T Cells Receptor internalization was quantitatively assessed using HaloTag technology (Promega) as described previously (4). HEK293T cells were maintained in Dulbecco’s modiﬁed Eagle’s medium (DMEM, 5919, Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum. The cells were transfected with Halo-expressing vector and labeled with the cell-impermeable Alexa Fluor 488 ligand (Promega) in Opti-MEM for 15 min at 37°C. Each inhibitor or antagonist pretreatment was for 30 min. The cells were then treated with 1 µM PACAP, 5-HT or saline, washed with phosphate-buffered saline and ﬁxed in 4% paraformaldehyde. Cells were imaged using an FV1000D confocal microscope (Olympus, Tokyo, Japan) in sequential mode and membrane protein internalization was quantiﬁed using ImageJ software (NIH, MD, USA). Vector Construction To assess the internalization ratio, we deﬁned the shape of a whole-cell (region of interest, ROI, A) and its cytoplasmic region (ROI B) by reducing the size by 5–10 pixels and then determining the ﬂuorescence in both ROIs. The internalization ratio (%) was deﬁned by dividing the amount of luminescence in ROI B by that in ROI A. Our previous studies indicated that there are no signiﬁcant differences in 5-HT content in the cortex and striatum or in 5- HT2A protein levels in the somatosensory cortex between PACAP mutant and wild-type mice (5, 11, 24). Therefore, here, we examined the effect of PACAP signaling on 5-HT2A internalization and revealed that the PACAP–PAC1 signaling pathway regulates 5-HT2A internalization in a protein kinase C (PKC)- and b-arrestin2-dependent manner. These results further suggest the existence of functional crosstalk between PACAP and 5-HT2A-mediated signaling pathways in the brain. Drugs PACAP (PACAP-38, 4221-v), PACAP6-38 (4286-v) and VIP (4110-v) were purchased from Peptide Institute (Osaka, Japan). D-sphingosine (S7049), H89 (B1427) and 5-HT hydrochloride (H9523) were purchased from Sigma-Aldrich (St Louis, MO, USA). PD98059 (513000) was purchased from Calbiochem (CA, USA). H7 (BML-EI148) and HA1004 (BML-EI184) were purchased from ENZO Life Science (NY, USA). Citation: These ﬁndings indicate that 5-HT2A function may be involved in psychiatric conditions in which PACAP signaling is dysfunctional and that functional crosstalk may exist between PACAP and 5-HT2A signaling pathways. However, the underlying molecular mechanisms for this remain unclear. All animal care and handling procedures were performed in accordance with protocols approved by the Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences, Osaka University. All efforts were made to minimize the number of animals used. Citation: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that regulates a wide array of physiological responses, including emotion, cognition and motor function. It acts upon three G protein-coupled receptor subtypes: a PACAP-preferring receptor (PAC1) and two vasoactive intestinal polypeptide (VIP) receptors (VPAC1and VPAC2) (1, 2).PAC1signalingmediates cellular functions, such as transcriptional responses and cell survival, partly through its own internalization (3, 4). We previously reported that PACAP-deﬁcient (Pacap−/−) mice show behavioral Hayata-Takano A, Shintani Y, Moriguchi K, Encho N, Kitagawa K, Nakazawa T and Hashimoto H (2021) PACAP–PAC1 Signaling Regulates Serotonin 2A Receptor Internalization. Front. Endocrinol. 12:732456. doi: 10.3389/fendo.2021.732456 October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org Hayata-Takano et al. PACAP Serotonin 2A Crosstalk abnormalities such as locomotor hyperactivity in an open-ﬁeld, deﬁcits in prepulse inhibition (PPI) of the startle response, depression-like behavior and memory impairment (5–10). The hyperlocomotion and PPI deﬁcits in Pacap−/−mice were reversed by risperidone, an atypical antipsychotic drug with antagonism of serotonin (5-HT)2 receptors and dopamine D2 receptors (D2) (10). The depression-like behavior in Pacap−/−mice were ameliorated by risperidone and the selective 5-HT 2A receptor (5-HT2A) antagonist, ritanserin (7). In addition, Pacap−/−mice (7) and heterozygous mutant mice (Pacap+/−) (11) show exaggerated (±)-2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head- twitch responses compared with wild-type mice. Pacap−/−mice also have increased 5-HT content and 5-HT-immunoreactive cell counts inthe dorsalraphe(12) andslightly decreasedlevelsofthe 5- HT metabolite, 5-hydroxyindoleacetic acid, in the cortex and striatum (5). These ﬁndings indicate that 5-HT2A function may be involved in psychiatric conditions in which PACAP signaling is dysfunctional and that functional crosstalk may exist between PACAP and 5-HT2A signaling pathways. However, the underlying molecular mechanisms for this remain unclear. ICR background were obtained by crossing Pacap+/− heterozygous mice. abnormalities such as locomotor hyperactivity in an open-ﬁeld, deﬁcits in prepulse inhibition (PPI) of the startle response, depression-like behavior and memory impairment (5–10). The hyperlocomotion and PPI deﬁcits in Pacap−/−mice were reversed by risperidone, an atypical antipsychotic drug with antagonism of serotonin (5-HT)2 receptors and dopamine D2 receptors (D2) (10). The depression-like behavior in Pacap−/−mice were ameliorated by risperidone and the selective 5-HT 2A receptor (5-HT2A) antagonist, ritanserin (7). In addition, Pacap−/−mice (7) and heterozygous mutant mice (Pacap+/−) (11) show exaggerated (±)-2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head- twitch responses compared with wild-type mice. Pacap−/−mice also have increased 5-HT content and 5-HT-immunoreactive cell counts inthe dorsalraphe(12) andslightly decreasedlevelsofthe 5- HT metabolite, 5-hydroxyindoleacetic acid, in the cortex and striatum (5). Antibodies The following commercially available antibodies were used: rabbit polyclonal anti-PAC1 (ab54980, Abcam, Cambridge, UK), rabbit polyclonal anti-5-HT1A (ab44635, Abcam), rabbit polyclonal anti- 5-HT2A (ab16028, Abcam), rabbit polyclonal anti-D2 (ab21218, Abcam), rabbit polyclonal anti-mGlu2/3 (06-676, Millipore, Darmstadt, Germany), mouse monoclonal anti-b-actin (MAB1501, Millipore), mouse monoclonal anti-alpha 1 sodium potassium ATPase (ab7671, Abcam). Horseradish peroxidase- conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Cappel (Cochranville, PA, USA). Animals siRNA-mediated silencing of b-arrestins was performed exactly as described in our previous study (4). b-arrestin1 (6218S; Cell Signaling Technology, Danvers, MA, USA), b-arrestin2 (sc- 29743; Santa Cruz Biotechnology, Dallas, TX) or control ICR mice were purchased from Japan SLC (Shizuoka, Japan). Generation of Pacap−/−mice by gene targeting was reported previously (5). Pacap−/−mice and wild-type littermates on the October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org 2 Hayata-Takano et al. PACAP Serotonin 2A Crosstalk the bands of speciﬁc immune-complexes were analyzed using ImageJ software. siRNA (6568S; Cell Signaling Technology), each at 25 mM, were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. We conﬁrmed that the b-arrestin1 and b-arrestin2 siRNAs effectively decreased the respective b-arrestin levels to less than 35% in HEK293T cells in our previous study (4). Head Twitch Response and Intracerebroventricular Injections j Intracerebroventricular injections were performed as described previously (26). Head twitch responses were assessed as described previously (10). ICR mice were anesthetized and placed in a stereotaxic instrument (Narishige, Tokyo, Japan). A G-4 cannula (Eicom, Kyoto, Japan) was implanted, −0.4 mm posterior, 1.0 mm lateral, and 2.3 mm ventral from the bregma. After cannula implantation, each mouse was given 1 mg/kg buprenorphine (Sigma-Aldrich) to relieve pain and housed individually for at least 10 days before performing head-twitch experiments. Thirty minutes before DOI (Sigma-Aldrich) treatment, PACAP (10 pmol) was diluted in Ringer’s solution (1:100, Fuso Pharmaceutical Industries, Osaka, Japan) and a 3 ml volume was injected at an infusion rate of 1 ml/min using a microinjection pump (KD Scientiﬁc, MA, USA). For the pretreatment of the PAC1 antagonist, PACAP6-38 (100 pmol) were diluted and injected in the same way 30 min before PACAP treatment. The mice were individually placed in observation cages (19 × 10 × 11 cm) for a 30 min habituation period. They were then intraperitoneally injected with either saline or DOI, which were preparedjustbeforeuse,andrecordingsweremadeforadurationof 60 min. Scoring began immediately after injection by trained observers who were blind to the treatment. The head twitch response is a distinctive paroxysmal head-twitching behavior that iseasily distinguishedfromhead-bobbing,lateralmovementsofthe head and grooming. The intracerebroventricular injection was judged successful if the third ventricle was stained by Evans blue. Statistical Analysis Experimental data were analyzed using Student’s t-test, or one- way, two-way or two-way repeated measures analysis of variance (ANOVA). The Tukey-Kramer post hoc test was also performed after signiﬁcant main effects for interaction were observed. The criterion for statistical signiﬁcance was p < 0.05. Statistical analyses were performed using StatView software (version 5.0; SAS Institute, Cary, NC, USA). All experiments were performed in a blinded manner. The observers were blinded to the group of samples during the analyses by random numbering. Surface Biotinylation Assay and Membrane Protein Isolation A receptor biotinylation assay was performed using the Pierce cell surface protein isolation kit (Thermo Fisher Scientiﬁc, Waltham, MA, USA) as described previously (25). Primary cultures of cortical neurons were prepared as described previously (4). The surface proteins of mouse primary cultured cortical neurons at 14 days in vitro were biotinylated with EZ- Link Sulfo-NHS-SS-biotin for 30 min at 4°C. To collect the surface proteins, cells were lysed with lysis buffer and biotinylated proteins were precipitated with NeutrAvidin agarose. The collected surface proteins were analyzed by western blotting. Membrane protein isolation was performed using a plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN, USA) according to the manufacturer’s instructions. The collected membrane proteins were analyzed by western blotting. Western Blotting Collected surface proteins were suspended in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred electrophoretically onto polyvinylidene ﬂuoride membranes (Millipore). After blocking with 2% BSA in TBS buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), the membranes were incubated with an anti-PAC1 antibody (1:1,000 dilution), anti-5- HT1A antibody (1:1,000 dilution), anti-5-HT2A antibody (1:1,000 dilution), anti-D2 antibody (1:1,000 dilution), anti-mGlu2/3 antibody (1:1,000 dilution), anti-b-actin antibody (1:2000 dilution) or anti-alpha 1 sodium potassium ATPase antibody (1:1000 dilution) overnight at 4°C. After incubation with a horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution) or anti-mouse IgG (1:2,000 dilution) secondary antibody for 1 h at room temperature, proteins were detected by chemiluminescence and visualized with an ImageQuant LAS 4000 system (GE Healthcare, Little Chalfont, UK). For quantiﬁcation, PACAP-Induced Internalization of 5-HT2A in HEK293T Cells and 5-HT2A was below the detection limit of our RT-PCR analysis (Supplementary Figure 1A). Quantitative RT-PCR analysis showed thatPC12cellsandSH-SY5Ycellsexpressedrelativelyhigherlevelsof PAC1 mRNA as expected from the previous reports (27–29), and both our HEK293T cell cultures and the HEK293T cells provided by RIKENBRCCellBank(RCB2202;theNationalBio-ResourceProject of the MEXT/AMED, Japan) moderately expressed PAC1 mRNA at similar levels. In Hela cells, PAC1 expression was below the detection limit of our quantitative RT-PCR analysis (Supplementary Figure 1B). The nucleotide sequence of the cDNA fragment ampliﬁed from our HEK293T cell cultures was identical to that of the cDNA encoding the human PAC1 hop1 splice variant (NCBI Reference Sequence: NM_001199635.2). We then examined whether PACAP, maxadilan, a potent and speciﬁc PAC1 agonist (30), and VIP increase intracellular cyclic adenosine monophosphate (cAMP) levels in our HEK293T cell cultures and conﬁrmed that PACAP and maxadilan, both at ≥ 0.01 nM, signiﬁcantly increased intracellular cAMP levels, while VIP at higher concentrations (≥1 nM) increased intracellular cAMP levels (Supplementary Figure 1C). A B C FIGURE 1 \| PACAP induces internalization of 5-HT2A in HEK293T cells. (A) Representative images of HEK293T cells transfected with the indicated HaloTag receptors. The cells were labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (B) Quantiﬁcation of the indicated HaloTag receptor internalization. Values are the mean ± SEM of 40–64 cells obtained from three independent experiments. p < 0.01 vs. saline, Student’s t-test. (C) Time course of 5-HT2A and PAC1 internalization for 45 min after PACAP treatment. Values are the mean ± SEM of 23–69 cells obtained from three independent experiments. p < 0.01 vs. saline, two-way repeated-measures ANOVA followed by the Tukey-Kramer test. FIGURE 1 \| PACAP induces internalization of 5-HT2A in HEK293T cells. (A) Representative images of HEK293T cells transfected with the indicated HaloTag receptors. The cells were labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (B) Quantiﬁcation of the indicated HaloTag receptor internalization. Values are the mean ± SEM of 40–64 cells obtained from three independent experiments. p < 0.01 vs. saline, Student’s t-test. (C) Time course of 5-HT2A and PAC1 internalization for 45 min after PACAP treatment. Values are the mean ± SEM of 23–69 cells obtained from three independent experiments. p < 0.01 vs. PACAP-Induced Internalization of 5-HT2A in HEK293T Cells To examine whetherPACAP signalingmodulatesthe internalization of 5-HT2A and related GPCRs in HEK293T cells, we constructed membrane-speciﬁc Halo-tagged receptors for PAC1, 5-HT2A, 5- HT1A, 5-HT2c, D2 and mGlu2. As a ﬁrst step, we examined whether PAC1, VPAC1, VPAC2, and 5-HT2A mRNAs were expressed in HEK293T cells using reverse transcription (RT)-PCR analysis. In our HEK293T cell cultures, we detected the mRNA expression of PAC1 and VPAC1; however, the expression of VPAC2 October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org 3 PACAP Serotonin 2A Crosstalk Hayata-Takano et al. To detect receptor internalization, only cell surface GPCR- Halo proteins were labeled with the cell-impermeable Alexa Fluor 488 HaloTag ligand and the signal ratio of internalized GPCR vs. total GPCR was determined in each cell after 30 min of PACAP treatment. PACAP (1 µM) induced an increase in the internalization of 5-HT2A (saline, 10.64 ± 1.40; PACAP, 29.50 ± 2.07, p < 0.001, Student’s t-test) in HEK293T cells (Figures 1A, B). In accordance with previous reports (3, 4, 31, 32), PACAP also induced the internalization of PAC1 (saline, 9.52 ± 1.45; PACAP, 33.08 ± 0.56, p < 0.001, Student’s t-test) (Figures 1A, B). In contrast, PACAP did not affect the internalization of 5-HT1A (saline, 6.19 ± 0.61; PACAP, 7.18 ± 0.64, not signiﬁcant), 5-HT2c (saline, 49.35 ± 2.72; PACAP, 42.06 ± 2.09, not signiﬁcant), D2 (saline, 20.95 ± 1.93; PACAP, 17.87 ± 1.81, not signiﬁcant), or mGlu2 (saline, 11.64 ± 0.84; PACAP, 9.44 ± 0.95, not signiﬁcant) (Figures 1A, B). We also analyzed the time course of PACAP-induced internalization. The internalization ratios of 5-HT2A and PAC1 were similarly increased within 15 min after PACAP treatment and remained elevated for at least 45 min (two-way repeated-measures ANOVA; 5-HT2A, treatment effect, and 5-HT2A was below the detection limit of our RT-PCR analysis (Supplementary Figure 1A). Quantitative RT-PCR analysis showed thatPC12cellsandSH-SY5Ycellsexpressedrelativelyhigherlevelsof PAC1 mRNA as expected from the previous reports (27–29), and both our HEK293T cell cultures and the HEK293T cells provided by RIKENBRCCellBank(RCB2202;theNationalBio-ResourceProject of the MEXT/AMED, Japan) moderately expressed PAC1 mRNA at similar levels. In Hela cells, PAC1 expression was below the detection limit of our quantitative RT-PCR analysis (Supplementary Figure 1B). The nucleotide sequence of the cDNA fragment ampliﬁed from our HEK293T cell cultures was identical to that of the cDNA encoding the human PAC1 hop1 splice variant (NCBI Reference Sequence: NM_001199635.2). PACAP-Induced Internalization of 5-HT2A in HEK293T Cells saline, two-way repeated-measures ANOVA followed by the Tukey-Kramer test. October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org PACAP Serotonin 2A Crosstalk Hayata-Takano et al. HEK293T cells (4), b-arrestin2 siRNA, but not b-arrestin1 siRNA, blocked the PACAP-induced 5-HT2A internalization (two-way ANOVA, PACAP effect, F(1, 490) = 37.78, p < 0.001; silencing effect, F(2, 490) = 5.85, p = 0.0031; interaction, F(2, 490) = 7.61, p < 0.001) (Figures 3C, D). A negative control siRNA showed no effect on PACAP-induced 5-HT2A internalization (Figures 3C, D). F(1, 82) = 65.77, p < 0.001; time effect, F(3, 246) = 11.77, p < 0.001; interaction, F(3, 246) = 11.75, p < 0.001; PAC1, treatment effect, F(1, 54) = 96.14, p < 0.001; time effect, F(3, 162) = 19.41, p < 0.001; interaction, F(3, 162) = 18.66, p < 0.001) (Figure 1C). In accordance with previous reports (15, 16), 5-HT increased 5- HT2A internalization in a time-dependent manner, the pattern of which was similar to that of PACAP-induced 5-HT2A internalization (Supplementary Figure 2). PAC1 Mediates PACAP-Induced 5-HT2A Internalization To examine the subtypes of the three PACAP receptors (PAC1, VPAC1 and VPAC2) involved in PACAP-induced 5-HT2A internalization, we compared 5-HT2A internalization following administration of various doses of PACAP and VIP. PACAP (0.01, 0.1, and 1 µM) dose-dependently increased 5-HT2A internalization (one-way ANOVA, F(3, 321) = 29.44, p < 0.001), but VIP (0.01, 0.1, and 1 µM) did not (one-way ANOVA, F(3, 304) = 3.62, p = 0.054) (Figures 2A, B). Pretreatment with PACAP6-38, a PAC1 antagonist, signiﬁcantly inhibited the PACAP-induced 5- HT2A internalization (one-way ANOVA, F(2, 246) = 17.54, p < 0.001) (Figures 2C, D). In addition, shRNA-mediated PAC1 silencing in HEK293T cells, which effectively decreased PAC1 mRNA levels to less than 5% of normal levels, blocked the PACAP-induced 5-HT2A internalization (two-way ANOVA, PACAP effect, F(1, 156) = 79.51, p < 0.001; shRNA effect, F(1, 156) = 76.58, p < 0.001; interaction, F(1, 156) = 81.72, p < 0.001) (Supplementary Figure 3). Taken together these results indicate that PAC1 is involved in PACAP- induced 5-HT2A internalization. To conﬁrm the phenomenon of PACAP-induced 5-HT2A internalization in more neurologically relevant cells, we examined the effect of PACAP on the cell surface localization of endogenously expressed 5-HT2A in mouse primary cultured cortical neurons using a cell surface biotinylation assay. PACAP signiﬁcantly decreased the levels of cell-surface biotinylated 5- HT2A (saline, 1.00 ± 0.14; PACAP, 0.46 ± 0.10; p = 0.0077, Student’s t-test) (Figures 4A, B). As expected, cell-surface biotinylated PAC1 levels were also decreased by PACAP (saline, 1.00 ± 0.09; PACAP, 0.26 ± 0.055; p < 0.001, Student’s t-test) (Figures 4A, B). In contrast, levels of cell-surface biotinylated 5- HT1A (saline, 1.00 ± 0.17; PACAP, 1.50 ± 0.45; not signiﬁcant), D2 (saline, 1.00 ± 0.058; PACAP, 1.37 ± 0.22; not signiﬁcant) and mGlu2/3 (saline, 1.00 ± 0.10; PACAP, 0.68 ± 0.13; not signiﬁcant) were not affected by PACAP (Figures 4A, B). In addition, 5-HT2A levels in the membrane fraction of the frontal cortex were increased in Pacap–/– mice compared with wild-type mice (saline, 1.00 ± 0.10; PACAP, 1.64 ± 0.10; p = 0.002, Student’s t-test), although no signiﬁcant change was observed in total 5-HT2A protein levels between Pacap–/– and wild-type mice (saline, 1.00 ± 0.03; PACAP, 0.94 ± 0.03; not signiﬁcant, Student’s t-test) (Figures 5A, B). Intracerebroventricular PACAP Administration Ameliorates the Hallucinogenic Head Twitch Response Intracerebroventricular PACAP Administration Ameliorates the Hallucinogenic Head Twitch Response g We then addressed PACAP signaling involvement in 5-HT2A- dependent behavioral responses by examining the head twitch response, which is a characteristic head-shaking movement induced by a hallucinogenic drug through the stimulation of 5- HT2 receptors (33). DOI (1.0 mg/kg)-induced head twitch responses were signiﬁcantly fewer in mice administered PACAP (10 pmol) compared with vehicle control mice in the ﬁrst, third and fourth 10 min-bins of a 60-min observation period (Figure 5C). The numbers of head twitch responses induced by 0.3 and 1.0 mg/kg DOI during 60 min were signiﬁcantly lower in mice administered PACAP compared with vehicle control mice (two-way ANOVA, PACAP effect, F(1, 12) = 39.80, p < 0.001; dose effect, F(2, 12) = 50.90, p < 0.001; interaction, F(2, 12) = 11.03, p = 0.0019) (Figure 5D). In addition, we examined whether the inhibitory effect of PACAP on DOI- induced head twitch response is mediated by PAC1 by using the PAC1 antagonist PACAP6-38. Intracerebroventricular preadministration of PACAP6-38 (100 pmol) signiﬁcantly blocked the inhibitory effect of PACAP on DOI-induced head PKC Is Involved in PACAP-Induced 5-HT2A Internalization 2A We then addressed the signaling pathways involved in PACAP- induced 5-HT2A internalization. Pretreatment with the PKC inhibitor D-sphingosine (50 µM), but not the protein kinase A inhibitor H89 (20 µM), or the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (50 µM), blocked the PACAP- induced 5-HT2A internalization (two-way ANOVA, PACAP effect, F(1, 372) = 44.34, p < 0.001; inhibitor effect, F(3, 372) = 18.41, p < 0.001; interaction, F(3, 372) = 8.04, p < 0.001) (Figures 3A, B). Another PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) also signiﬁcantly blocked PACAP-induced 5-HT2A internalization, whereas HA1004, a structural analog of H7 and used as a control, did not signiﬁcantly inhibit the PACAP- induced 5-HT2A internalization (two-way ANOVA, PACAP effect, F(1, 448) = 33.37, p < 0.001; inhibitor effect, F(2, 448) = 8.98, p < 0.001; interaction, F(2, 448) = 4.53, p = 0.011) (Supplementary Figure 4). b-Arrestin2 Is Involved in PACAP-Induced 5-HT2A Internalization We recently reported that b-arrestin2, but not b-arrestin1, is involved in PACAP-induced internalization of PAC1 (4). We therefore examined whether b-arrestins are also involved in PACAP-induced 5-HT2A internalization. Although the b- arrestin1 and b-arrestin2 siRNAs effectively decreased respective b-arrestin levels to less than 35% of normal levels in October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org 5 Hayata-Takano et al. PACAP Serotonin 2A Crosstalk A B C D uces 5-HT2A internalization via PAC1 in HEK293T cells. (A) Representative images of HEK293T cells transfected with HaloTag 5-HT2A. The Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with the indicated concentrations of PACAP or VIP for m. (B) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 46–71 cells obtained from three independent experiments. e-way ANOVA followed by the Tukey-Kramer test. (C) Representative images of HEK293T cells transfected with 5-HT2A. The cells were ACAP6-38 or saline for 30 min, labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 100 nM min. Scale bar, 10 mm. (D) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 80–86 cells obtained from three s. p < 0.01, one-way ANOVA followed by the Tukey-Kramer test. A B B C C D D D FIGURE 2 \| PACAP induces 5-HT2A internalization via PAC1 in HEK293T cells. (A) Representative images of HEK293T cells transfected with HaloTag 5-HT2A. The cells were labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with the indicated concentrations of PACAP or VIP for 30 min. Scale bar, 10 mm. (B) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 46–71 cells obtained from three independent experiments. p < 0.01 vs. 0 mM, one-way ANOVA followed by the Tukey-Kramer test. (C) Representative images of HEK293T cells transfected with 5-HT2A. The cells were pretreated with 2 mM PACAP6-38 or saline for 30 min, labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 100 nM PACAP or saline for 30 min. Scale bar, 10 mm. (D) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 80–86 cells obtained from three independent experiments. p < 0.01, one-way ANOVA followed by the Tukey-Kramer test. October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org Hayata-Takano et al. b-Arrestin2 Is Involved in PACAP-Induced 5-HT2A Internalization PACAP Serotonin 2A Crosstalk A B C D FIGURE 3 \| Effect of kinase inhibitors or b-arrestin silencing on PACAP-induced 5-HT2A internalization in HEK293T cells. (A) Representative images of HEK293T cells transfected with 5-HT2A. The cells were pretreated with 50 mM D-sphingosine (PKC inhibitor), 20 mM H89 (protein kinase A inhibitor), 50 mM PD98059 (MEK inhibitor) or saline for 30 min, labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (B) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 34–51 cells obtained from three independent experiments. p < 0.01, two-way ANOVA followed by the Tukey-Kramer test. (C) Representative images of HEK293T cells cotransfected with 5-HT2A plus b-arrestin1 siRNA, b- arrestin2 siRNA or the negative control siRNA. The cells were labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (D) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 77–87 cells obtained from three independent experiments. p < 0.01, two-way ANOVA followed by the Tukey-Kramer test. A A B B C D GURE 3 \| Effect of kinase inhibitors or b-arrestin silencing on PACAP-induced 5-HT2A internalization in HEK293T cells. (A) Representative images of HEK293T C D FIGURE 3 \| Effect of kinase inhibitors or b-arrestin silencing on PACAP-induced 5-HT2A internalization in HEK293T cells. (A) Representative images of HEK293T cells transfected with 5-HT2A. The cells were pretreated with 50 mM D-sphingosine (PKC inhibitor), 20 mM H89 (protein kinase A inhibitor), 50 mM PD98059 (MEK inhibitor) or saline for 30 min, labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (B) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 34–51 cells obtained from three independent experiments. p < 0.01, two-way ANOVA followed by the Tukey-Kramer test. (C) Representative images of HEK293T cells cotransfected with 5-HT2A plus b-arrestin1 siRNA, b- arrestin2 siRNA or the negative control siRNA. The cells were labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (D) Quantiﬁcation of 5-HT2A internalization. b-Arrestin2 Is Involved in PACAP-Induced 5-HT2A Internalization Values are the mean ± SEM of 77–87 cells obtained from three independent experiments. p < 0.01, two-way ANOVA followed by the Tukey-Kramer test. FIGURE 3 \| Effect of kinase inhibitors or b-arrestin silencing on PACAP-induced 5-HT2A internalization in HEK293T cells. (A) Representative images of HEK293T cells transfected with 5-HT2A. The cells were pretreated with 50 mM D-sphingosine (PKC inhibitor), 20 mM H89 (protein kinase A inhibitor), 50 mM PD98059 (MEK inhibitor) or saline for 30 min, labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (B) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 34–51 cells obtained from three independent experiments. p < 0.01, two-way ANOVA followed by the Tukey-Kramer test. (C) Representative images of HEK293T cells cotransfected with 5-HT2A plus b-arrestin1 siRNA, b- arrestin2 siRNA or the negative control siRNA. The cells were labeled with Alexa Fluor 488 HaloTag membrane impermeable ligand for 15 min and then treated with 1 mM PACAP or saline for 30 min. Scale bar, 10 mm. (D) Quantiﬁcation of 5-HT2A internalization. Values are the mean ± SEM of 77–87 cells obtained from three independent experiments. p < 0.01, two-way ANOVA followed by the Tukey-Kramer test. October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org PACAP Serotonin 2A Crosstalk Hayata-Takano et al. A B FIGURE 4 \| PACAP signiﬁcantly decreases cell surface localization of 5-HT2A in primary cultured cortical neurons. (A) Representative immunoblots of cell surface biotinylated PAC1, 5-HT2A, 5-HT1A, D2, mGlu2/3 and alpha 1 sodium potassium ATPase (Na/K-ATPase) in primary cultured cortical neurons at 14 days in vitro treated with 1 mM PACAP or saline for 30 min. The band size is indicated for each blot. (B) Quantiﬁcation of cell surface levels of PAC1, 5-HT2A, 5-HT1A, D2 and mGlu2/3 normalized to the levels of Na/K-ATPase. Values are the mean ± SEM from three or four independent experiments. p < 0.01, p < 0.001 vs. saline, Student’s t-test. A B FIGURE 4 \| PACAP signiﬁcantly decreases cell surface localization of 5-HT2A in primary cultured cortical neurons. (A) Representative immunoblots of cell surface biotinylated PAC1, 5-HT2A, 5-HT1A, D2, mGlu2/3 and alpha 1 sodium potassium ATPase (Na/K-ATPase) in primary cultured cortical neurons at 14 days in vitro treated with 1 mM PACAP or saline for 30 min. b-Arrestin2 Is Involved in PACAP-Induced 5-HT2A Internalization The band size is indicated for each blot. (B) Quantiﬁcation of cell surface levels of PAC1, 5-HT2A, 5-HT1A, D2 and mGlu2/3 normalized to the levels of Na/K-ATPase. Values are the mean ± SEM from three or four independent experiments. p < 0.01, *p < 0.001 vs. saline, Student’s t-test. twitch response (one-way ANOVA, F(3, 12) = 47.77, p < 0.001) (Figure 5E). twitch response (one-way ANOVA, F(3, 12) = 47.77, p < 0.001) (Figure 5E). in mouse primary cultured cortical neurons and that 5-HT2A levels in the membrane fraction of the frontal cortex were increased in Pacap–/– mice compared with wild-type mice. Finally, we observed that intracerebroventricular administration of PACAP suppressed DOI-induced head twitch responses in mice. These results suggest that PACAP–PAC1 signaling increases 5-HT2A internalization, resulting in attenuation of 5-HT2A-meadiated signaling. DISCUSSION In the present study, we investigated the mechanisms underlying the relationship between PACAP and 5-HT2A signaling pathways. We found that PACAP time- and dose-dependently increased the internalization of 5-HT2A, but not 5-HT1A, 5-HT2c, D2 or mGlu2, in HEK293T cells and that the effect of PACAP was mediated by PAC1, PKC and b-arrestin2. In addition, we showed that PACAP decreased the cell surface levels of endogenously expressed 5-HT2A In the present study, we investigated the mechanisms underlying the relationship between PACAP and 5-HT2A signaling pathways. We found that PACAP time- and dose-dependently increased the internalization of 5-HT2A, but not 5-HT1A, 5-HT2c, D2 or mGlu2, in HEK293T cells and that the effect of PACAP was mediated by PAC1, PKC and b-arrestin2. In addition, we showed that PACAP decreased the cell surface levels of endogenously expressed 5-HT2A In the present study, it is still uncertain whether PACAP- induced 5-HT2A internalization can be a mechanism for behavioral abnormalities including hyperactivity, PPI deﬁcits, depressive-like behavior and memory impairment, reversal of the depressive-like behavior by the 5-HT2A antagonist ritanserin, and exaggerated DOI-induced hallucinogenic behaviors in Pacap–/– October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org 8 Hayata-Takano et al. PACAP Serotonin 2A Crosstalk A B D E C Increased 5-HT2A levels in the membrane fraction of the frontal cortex in Pacap–/– mice and PACAP-induced attenuation of DOI-induced head twitch A) Representative immunoblots of 5-HT2A in the cell membrane fraction (membrane) or total cell lysate (total) of the frontal cortex from wild-type (WT) or O) mice. As internal controls, Na/K-ATPase (membrane) and b-actin (total) were used. (B) Quantiﬁcation of 5-HT2A levels normalized to Na/K-ATPase or b-actin (total). Values are the mean ± SEM (n = 5). p < 0.01 vs. saline, Student’s t-test. (C, D) Mice intracerebroventricularly administered PACAP vehicle were treated with DOI and their head-twitch responses were counted. (C) Time course of DOI (1 mg/kg)-induced head twitch responses. (D) Head nses during 60 min in mice injected with the indicated doses of DOI. Values are the mean ± SEM (n = 3 per group). p < 0.05, p < 0.01 vs. vehicle, eated measures ANOVA (C) and two-way ANOVA (D) followed by the Tukey-Kramer test. (E) Effect of the PAC1 antagonist PACAP6-38 on the PACAP DOI-induced head twitch response. Thirty minutes before PACAP administration, PACAP6-38 (100 pmol) were preadministered intracerebroventricularly. he mean ± SEM (n = 4 per group). ETHICS STATEMENT This animal study was reviewed and approved by the Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences, Osaka University. We also examined the protein-protein interaction between PAC1 and 5-HT2A by co-immunoprecipitation using an anti-5- HT2A antibody; however, co-immunoprecipitation of PAC1 with 5-HT2A was not detected (data not shown). Therefore, it remains unclear how PACAP–PAC1 signaling induces 5-HT2A receptor internalization. We previously reported that PACAP–PAC1 signaling markedly reduces the association between DISC1 and DBZ in PC12 cells (44). DISC1 forms a protein complex of DISC1/Kalirin-7/PSD-95 (45). The Kalirin-7/PSD-95 complex is also directly associated with the 5-HT2A receptor and regulates 5- HT2A signaling and trafﬁcking in HEK293 cells (46, 47). In addition, we previously showed that b-arrestin2, but not b- arrestin1, was involved in PACAP-induced internalization of PAC1 (4). PACAP–PAC1 signaling may regulate 5-HT2A internalization through these adaptor proteins. DISCUSSION In addition, 5-HT2A expressed in the frontal cortex plays an important role in the pathophysiology and therapeutic effects of schizophrenia (12, 14). However, further analyses in other brain regions are needed, which will be investigated in our future work. the brain provides clues to elucidating the pathomechanisms of neurological and psychiatric disorders (53–55). The present study furthers understanding of PACAP–PAC1 signaling and shows that this pathway is a promising target for the development of neurotherapeutics. 5-HT2A internalizationisinvolvedindiverse signalingpathways dependingon different ligands. Recent studies indicate that 5-HT2A internalization signaling may be separated into hallucinogenic and antipsychotic speciﬁc pathways, because hallucinogenic and non- hallucinogenic 5-HT2A ligands induce distinct immediate early gene expression patterns (38–41). Hallucinogenic DOI-induced 5-HT2A internalization is independent on b-arrestins and antipsychotic clozapine-mediated internalization is independent on PKC (16, 42). Urs et al. (43) reported that b-arrestin-biased D2 ligands exert unique brain region-speciﬁc antipsychotic actions (43). The present observation that PACAP–PAC1 signaling regulates 5-HT2A internalization in a PKC- and b-arrestin2- dependent manner provides a new molecular mechanism for this peptidergic signaling that cross-talks with serotonergic signaling in the brain. FUNDING This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI, grant numbers JP16K08269 (AH-T), JP19K07121 (AH-T), JP20H00492 (HH), JP20H03429 (HH, AH-T), JP20K07736 (HH, AH-T), JP21K19335 (HH),MEXTKAKENHI,grantnumberJP18H05416(HH),AMED, grant numbers JP21dm0207117 (HH), and JP21am0101084 (HH), and a grant from the Takeda Science Foundation (HH). In the present study, we observed, in our HEK293T cell cultures, expression of PAC1 transcript, maxadilan-induced cAMP elevation, PACAP-induced 5-HT2A internalization as well as inhibition of the PACAP-induced 5-HT2A internalization by PACAP6-38 and shRNA-mediated PAC1 silencing. In addition, we observed that the HEK293T cells which was newly obtained from RIKEN BRC Cell Bank expressed PAC1 mRNA at a similar level with our HEK293T cell cultures used in the present 5-HT2A internalization study. However, previous studies have shown that HEK293T cells did not express PAC1 (3, 28, 48, 49) and therefore PAC1 was exogenously expressed to investigate the signal transduction system. In contrast, it was also reported that HEK293T cells expressed the PAC1 protein as observed by western blot analysis (50, 51). The reason for the disagreement in PAC1expressioninHEK293Tcellsiscurrentlyunknownbutmight be related with passage number and culture conditions. ACKNOWLEDGMENTS We are grateful to Dr. Atsuro Miyata at the Graduate School of Medical and Dental Sciences, Kagoshima University for his indispensable support. We are also grateful to the Center for Medical Research and Education, Graduate School of Medicine, Osaka University, for confocal microscopy analyses. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors. DISCUSSION p < 0.01 vs. vehicle, one-way ANOVA followed by the Tukey-Kramer test. A B D C A B B B C D D E E E FIGURE 5 \| Increased 5-HT2A levels in the membrane fraction of the frontal cortex in Pacap–/– mice and PACAP-induced attenuation of DOI-induced head twitch response. (A) Representative immunoblots of 5-HT2A in the cell membrane fraction (membrane) or total cell lysate (total) of the frontal cortex from wild-type (WT) or Pacap–/– (KO) mice. As internal controls, Na/K-ATPase (membrane) and b-actin (total) were used. (B) Quantiﬁcation of 5-HT2A levels normalized to Na/K-ATPase (membrane) or b-actin (total). Values are the mean ± SEM (n = 5). p < 0.01 vs. saline, Student’s t-test. (C, D) Mice intracerebroventricularly administered PACAP (10 pmol) or vehicle were treated with DOI and their head-twitch responses were counted. (C) Time course of DOI (1 mg/kg)-induced head twitch responses. (D) Head twitch responses during 60 min in mice injected with the indicated doses of DOI. Values are the mean ± SEM (n = 3 per group). p < 0.05, p < 0.01 vs. vehicle, two-way repeated measures ANOVA (C) and two-way ANOVA (D) followed by the Tukey-Kramer test. (E) Effect of the PAC1 antagonist PACAP6-38 on the PACAP inhibition of DOI-induced head twitch response. Thirty minutes before PACAP administration, PACAP6-38 (100 pmol) were preadministered intracerebroventricularly. Values are the mean ± SEM (n = 4 per group). p < 0.01 vs. vehicle, one-way ANOVA followed by the Tukey-Kramer test. mice. In order to address this, it is necessary to examine if increased cell surface expression of 5-HT2A in the frontal cortex (and possibly other brain regions as well) is relevant to behavioral impairments including exaggerated DOI-induced hallucinogenic behaviors and the effects of 5-HT2A antagonists on reversal of the impairments in Pacap–/–mice(5–10).Giventhatincreasedcellsurfaceexpressionof 5-HT2A leads to supersensitivity of the 5-HT2A-mediated 5-HT response, it isreasonable that 5-HT2Aantagonistseffectively reverse the behavioral impairments in Pacap–/– mice. The issue should also be addressed by examining whether 5-HT2A antagonists affect PAC1 and 5-HT2A interactions. We examined 5-HT2A levels in the membrane fraction of the frontal cortex in Pacap–/– mice, since both 5-HT2A, PACAP and PAC1 are expressed in this brain region (34–37), suggesting a October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org Frontiers in Endocrinology \| www.frontiersin.org 9 PACAP Serotonin 2A Crosstalk Hayata-Takano et al. potential colocalization of 5-HT2A and PAC1 in the frontal cortex. AUTHOR CONTRIBUTIONS AH-T: design, experimentation, statistics, visualization, and writing. YS: experimentation and statistics. KM: experimentation and statistics. NE: experimentation and statistics. KK: experimentation. TN: writing and supervision. HH: conception, writing, and supervision. All authors contributed to the article and approved the submitted version. Frontiers in Endocrinology \| www.frontiersin.org REFERENCES 18. Bulenger S, Marullo S, Bouvier M. Emerging Role of Homo- and Heterodimerization in G-Protein-Coupled Receptor Biosynthesis and Maturation. Trends Pharmacol Sci (2005) 26:131–7. doi: 10.1016/ j.tips.2005.01.004 1. Arimura A. Perspectives on Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in the Neuroendocrine, Endocrine, and Nervous Systems. Jpn J Physiol (1998) 48:301–31. doi: 10.2170/jjphysiol.48.301 1. Arimura A. Perspectives on Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in the Neuroendocrine, Endocrine, and Nervous Systems. Jpn J Physiol (1998) 48:301–31. doi: 10.2170/jjphysiol.48.301 19. Milligan G. G Protein-Coupled Receptor Hetero-Dimerization: Contribution to Pharmacology and Function. Br J Pharmacol (2009) 158:5–14. doi: 10.1111/ j.1476-5381.2009.00169.x 2. VaudryD,Falluel-MorelA,BourgaultS,BasilleM,BurelD,WurtzO,etal.Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years After the Discovery. Pharmacol Rev (2009) 61:283–357. doi: 10.1124/pr.109.001370 20. Gonzalez-Maeso J, Ang RL, Yuen T, Chan P, Weisstaub NV, Lopez-Gimenez JF, et al. Identiﬁcation of a Serotonin/Glutamate Receptor Complex Implicated in Psychosis. Nature (2008) 452:93–7. doi: 10.1038/nature06612 3. May V, Clason TA, Buttolph TR, Girard BM, Parsons RL. Calcium Inﬂux, But Not Intracellular Calcium Release, Supports PACAP-Mediated ERK Activation in HEK PAC1 Receptor Cells. J Mol Neurosci (2014) 54:342–50. doi: 10.1007/s12031-014-0300-0 21. Gonzalez-Maeso J, Sealfon SC. Psychedelics and Schizophrenia. Trends Neurosci (2009) 32:225–32. doi: 10.1016/j.tins.2008.12.005 22. Albizu L, Holloway T, Gonzalez-Maeso J, Sealfon SC. Functional Crosstalk and Heteromerization of Serotonin 5-HT2A and Dopamine D2 Receptors. Neuropharmacology (2011) 61:770–7. doi: 10.1016/j.neuropharm.2011.05.023 4. Shintani Y, Hayata-Takano A, Moriguchi K, Nakazawa T, Ago Y, Kasai A, et al. Beta-Arrestin1 and 2 Differentially Regulate PACAP-Induced PAC1 Receptor Signaling and Trafﬁcking. PLoS One (2018) 13:e0196946. doi: 10.1371/journal.pone.0196946 23. Fribourg M, Moreno JL, Holloway T, Provasi D, Baki L, Mahajan R, et al. Decoding the Signaling of a GPCR Heteromeric Complex Reveals a Unifying Mechanism of Action of Antipsychotic Drugs. Cell (2011) 147:1011–23. doi: 10.1016/j.cell.2011.09.055 5. Hashimoto H, Shintani N, Tanaka K, Mori W, Hirose M, Matsuda T, et al. Altered Psychomotor Behaviors in Mice Lacking Pituitary Adenylate Cyclase- Activating Polypeptide (PACAP). Proc Natl Acad Sci U S A (2001) 98:13355– 60. doi: 10.1073/pnas.231094498 24. Shibasaki Y, Hayata-Takano A, Hazama K, Nakazawa T, Shintani N, Kasai A. Atomoxetine Reverses Locomotor Hyperactivity, Impaired Novel Object Recognition, and Prepulse Inhibition Impairment in Mice Lacking Pituitary Adenylate Cyclase-Activating Polypeptide. Neuroscience (2015) 297:95–104. doi: 10.1016/j.neuroscience.2015.03.062 6. Tajiri M, Hayata-Takano A, Seiriki K, Ogata K, Hazama K, Shintani N, et al. REFERENCES Serotonin 5-HT(7) Receptor Blockade Reverses Behavioral Abnormalities in PACAP-Deﬁcient Mice and Receptor Activation Promotes Neurite Extension in Primary Embryonic Hippocampal Neurons: Therapeutic Implications for Psychiatric Disorders. J Mol Neurosci (2012) 48(3):473–81. doi: 10.1007/ s12031-012-9861-y 25. Hayata-Takano A, Kamo T, Kijima H, Seiriki K, Ogata K, Ago Y, et al. Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation. J Neurosci (2019) 39:4208–20. doi: 10.1523/JNEUROSCI.2468-18.2019 7. Hashimoto H, Hashimoto R, Shintani N, Tanaka K, Yamamoto A, Hatanaka M, et al. Depression-Like Behavior in the Forced Swimming Test in PACAP- Deﬁcient Mice: Amelioration by the Atypical Antipsychotic Risperidone. J Neurochem (2009) 110:595–602. doi: 10.1111/j.1471-4159.2009.06168.x 26. Kawaguchi C, Shintani N, Hayata-Takano A, Hatanaka M, Kuromi A, Nakamura R, et al. Lipocalin-Type Prostaglandin D Synthase Regulates Light-Induced Phase Advance of the Central Circadian Rhythm in Mice. Commun Biol (2020) 3:557. doi: 10.1038/s42003-020-01281-w 8. Gaszner B, Kormos V, Kozicz T, Hashimoto H, Reglodi D, Helyes Z. The Behavioral Phenotype of Pituitary Adenylate-Cyclase Activating Polypeptide- Deﬁcient Mice in Anxiety and Depression Tests is Accompanied by Blunted C- Fos Expression in the Bed Nucleus of the Stria Terminalis, Central Projecting Edinger-Westphal Nucleus, Ventral Lateral Septum, and Dorsal Raphe Nucleus. Neuroscience (2012) 202:283–99. doi: 10.1016/j.neuroscience.2011.11.046 27. Cavallaro S, D'Agata V, Guardabasso V, Travali S, Stivala F, Canonicon PL. Differentiation Induces Pituitary Adenylate Cyclase-Activating Polypeptide Receptor Expression in PC-12 Cells. Mol Pharmacol (1995) 48(1):56–62. 28. Hashimoto H, Hagihara N, Koga K, Yamamoto K, Shintani N, Tomimoto S, et al. Synergistic Induction of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Gene Expression by Nerve Growth Factior and PACAP in PC12 Cells. J Neurochem (2000) 74(2):501–7. doi: 10.1046/ j.1471-4159.2000.740501.x 9. Hattori S, Takao K, Tanda K, Toyama K, Shintani N, Baba A, et al. Comprehensive Behavioral Analysis of Pituitary Adenylate Cyclase- Activating Polypeptide (PACAP) Knockout Mice. Front Behav Neurosci (2012) 6:58. doi: 10.3389/fnbeh.2012.00058 29. Lutz EM, Ronaldson E, Shaw P, Johnson MS, Holland PJ, Mitchell R. Characterization of Novel Splice Variants of the PAC1 Receptor in Human Neuroblastoma Cells: Consequences for Signaling by VIP and PACAP. Mol Cell Neurosci (2006) 31(2):193–209. doi: 10.1016/j.mcn.2005.09.008 10. Hashimoto R, Hashimoto H, Shintani N, Chiba S, Hattori S, Okada T, et al. Pituitary Adenylate Cyclase-Activating Polypeptide Is Associated With Schizophrenia. Mol Psychiatry (2007) 12:1026–32. doi: 10.1038/sj.mp.4001982 11. Hazama K, Hayata-Takano A, Uetsuki K, Kasai A, Encho N, Shintani N, et al. REFERENCES Increased Behavioral and Neuronal Responses to a Hallucinogenic Drug in PACAP Heterozygous Mutant Mice. PLoS One (2014) 9:e89153. doi: 10.1371/ journal.pone.0089153 30. Uchida D, Tatsuno I, Tanaka T, Hirai A, Saito Y, Moro O, et al. Maxadilan is a Speciﬁc Agonist and its Deleted Peptide (M65) Is a Speciﬁc Antagonist for PACAP Type 1 Receptor. Ann N Y Acad Sci (1998) 865:253–8. doi: 10.1111/ j.1749-6632.1998.tb11185.x 12. Kormos V, Gaspar L, Kovacs LA, Farkas J, Gaszner T, Csernus V. Reduced Response to Chronic Mild Stress in PACAP Mutant Mice Is Associated With Blunted FosB Expression in Limbic Forebrain and Brain Stem Centers. Neurosci (2016) 330:335–58. doi: 10.1016/j.neuroscience.2016.06.004 31. Merriam LA, Baran CN, Girard BM, Hardwick JC, May V, Parsons RL. Pituitary Adenylate Cyclase 1 Receptor Internalization and Endosomal Signaling Mediate the Pituitary Adenylate Cyclase Activating Polypeptide- Induced Increase in Guinea Pig Cardiac Neuron Excitability. J Neurosci (2013) 33(10):4614–22. doi: 10.1523/JNEUROSCI.4999-12.2013 13. Norton N, Owen MJ. HTR2A: Association and Expression Atudies in Neuropsychiatric Genetics. Ann Med (2005) 37(2):121–9. doi: 10.1080/ 07853890510037347 32. May V, Buttolph TR, Girard BM, Clason TA, Parsons RL. PACAP-Induced ERK Activation in HEK Cells Expressing PAC1 Receptors Involves Both Receptor Internalization and PKC Signaling. Am J Physiol Cell Physiol (2014) 306(11):C1068–79. doi: 10.1152/ajpcell.00001.2014 14. Kang K, Huang XF, Wang Q, Deng C. Decreased Density of Serotonin 2A Receptors in the Superior Temporal Gyrus in Schizophrenia–A Postmortem Study. Prog Neuropsychopharmacol Biol Psychiatry (2009) 33:867–71. doi: 10.1016/j.pnpbp.2009.04.010 306(11):C1068–79. doi: 10.1152/ajpcell.00001.2014 33. Darmani NA, Shaddy J, Gerdes CF. Differential Ontogenesis of Three DOI- Induced Behaviors in Mice. Physiol Behav (1996) 60:1495–500. doi: 10.1016/ s0031-9384(96)00323-x 15. Meltzer HY, Li Z, Kaneda Y, Ichikawa J. Serotonin Receptors: Their Key Role in Drugs to Trear Schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry (2003) 27(7):1159–72. doi: 10.1016/j.pnpbp.2003.09.010 34. Weber ET, Andrade R. Htr2a Gene and 5-HT2A Receptor Expression in the Cerebral Cortex Studied Using Genetically Modiﬁed Mice. Front Neurosci (2010) 4:36. eCollection 2010. doi: 10.3389/fnins.2010.00036 16. Bhattacharyya S, Puri S, Miledi R, Panicker MM. Internalization and Recycling of 5-HT2A Receptors Activated by Serotonin and Protein Kinase C-Mediated Mechanisms. Proc Natl Acad Sci U S A (2002) 99:14470–5. doi: 10.1073/pnas.212517999 35. Hashimoto H, Nogi H, Mori K, Ohishi H, Shigemoto R, Yamamoto K, et al. Distribution of the mRNA for a Pituitary Adenylate Cyclase-Activating Polypeptide Receptor in the Rat Brain: An in Situ Hybridization Study. J Comp Neurol (1996) 371(4):567–77. doi: 10.1002/(SICI)1096-9861 (19960805)371:4<567::AID-CNE6>3.0.CO;2-2 17. SUPPLEMENTARY MATERIAL Serotonin syndrome is caused by adverse side effects of serotonergic drugs and is associated with increased serotoninergic activity (52). By indirectly antagonizing 5-HT2A function, PACAP signalingmay have the potential toameliorate serotonin syndrome. Accumulating evidence suggests that PACAP–PAC1 signaling in The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2021.732456/ full#supplementary-material October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org 10 PACAP Serotonin 2A Crosstalk Hayata-Takano et al. REFERENCES doi: 10.1073/pnas.0708862105 54. Ressler KJ, Mercer KB, Bradley B, Jovanovic T, Mahan A, Kerley K. Post- Traumatic Stress Disorder Is Associated With PACAP and the PAC1 Receptor. Nature (2011) 470(7335):492–7. doi: 10.1038/nature09856 43. Urs NM, Peterson SM, Caron MG. New Concepts in Dopamine D2 Receptor Biased Signaling and Implications for Schizophrenia Therapy. Biol Psychiatry (2017) 81:78–85. doi: 10.1016/j.biopsych.2016.10.011 55. Eiden LE, Goosens KA, Jacobson KA, Leggio L, Zhang L. Peptide-Liganded G Protein-Coupled Receptors as Neurotherapeutics. ACS Pharmacol Transl Sci (2020) 3(2):190–202. doi: 10.1021/acsptsci.0c00017 44. Hattori T, Baba K, Matsuzaki S, Honda A, Miyoshi K, Inoue K, et al. A Novel DISC1-Interacting Partner DISC1-Binding Zinc-Finger Protein: Implication in the Modulation of DISC1-Dependent Neurite Outgrowth. Mol Psychiatry (2007) 12:398–407. doi: 10.1038/sj.mp.4001945 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 45. Hayashi-Takagi A, Takaki M, Graziane N, Seshadri S, Murdoch H, Dunlop AJ, et al. Disrupted-In-Schizophrenia 1 (DISC1) Regulates Spines of the Glutamate Synapse via Rac1. Nat Neurosci (2010) 13:327–32. doi: 10.1038/nn.2487 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 46. Xia Z, Gray JA, Compton-Toth BA, Roth BL. A Direct Interaction of PSD-95 With 5-HT2A Serotonin Receptors Regulates Receptor Trafﬁcking and Signal Transduction. J Biol Chem (2003) 278:21901–8. doi: 10.1074/jbc.M301905200 47. Jones KA, Srivastava DP, Allen JA, Strachan RT, Roth BL, Penzes P. Rapid Modulation of Spine Morphology by the 5-HT2A Serotonin Receptor Through Kalirin-7 Signaling. Proc Natl Acad Sci U S A (2009) 106:19575– 80. doi: 10.1073/pnas.0905884106 Copyright © 2021 Hayata-Takano, Shintani, Moriguchi, Encho, Kitagawa, Nakazawa and Hashimoto. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 48. Emery AC, Eiden MV, Mustafa T, Eiden LE. REFERENCES Raote I, Bhattacharyya S, Panicker MM. Functional Selectivity in Serotonin Receptor 2A (5-HT2A) Endocytosis, Recycling, and Phosphorylation. Mol Pharmacol (2013) 83:42–50. doi: 10.1124/mol.112.078626 October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org 11 Hayata-Takano et al. PACAP Serotonin 2A Crosstalk Biological Activity at the PAC1 Receptor. Peptides (2016) 79:39–48. doi: 10.1016/j.peptides.2016.03.003 Biological Activity at the PAC1 Receptor. Peptides (2016) 79:39–48. doi: 10.1016/j.peptides.2016.03.003 36. Hannibal J. Pituitary Adenylate Cyclase-Activating Peptide in the Rat Central Nervous System: An Immunohistochemical and in Situ Hybridization Study. J Comp Neurol (2002) 453(4):389–417. doi: 10.1002/cne.10418 50. Yan Y, Zhou X, Pan Z, Ma J, Waschek JA, DiCicco-Bloom E. Pro- and Anti- Mitogenic Actions of Pituitary Adenylate Cyclase-Activating Polypeptide in Developing Cerebral Cortex: Potential Mediation by Developmental Switch of PAC1 Receptor mRNA Isoforms. J Neurosci (2013) 33(9):3865–78. doi: 10.1523/JNEUROSCI.1062-12.2013 37. Martelle SE, Cotella EM, Nawreen N, Chen C, Packard BA, Fitzgerald M, et al. Prefrontal Cortex PACAP Signaling: Organization and Role in Stress Regulation. Stress (2021) 24(2):196–205. doi: 10.1080/10253890.2021.1887849 38. González-Maeso J, Weisstaub NV, Zhou M, Chan P, Ivic L, Ang R, et al. Hallucinogens Recruit Speciﬁc Cortical 5-HT(2A) Receptor-Mediated Signaling Pathways to Affect Behavior. Neuron (2007) 53:439–52. doi: 10.1016/j.neuron. 2007.01.008 51. Egri P, Fekete C, Dénes Á ., Reglődi D, Hashimoto H, Fülöp BD, et al. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice. Endocrinology (2016) 157(6):2356–66. doi: 10.1210/en.2016-1043 39. Dong C, Ly C, Dunlap LE, Vargas MV, Sun J, Hwang IW. Psychedelic- Inspired Drug Discovery Using an Engineered Biosensor. Cell (2021) 184 (10):2779–92. doi: 10.1016/j.cell.2021.03.043 52. Sun-Edelstein C, Tepper SJ, Shapiro RE. Drug-Induced Serotonin Syndrome: A Review. Expert Opin Drug Saf (2008) 7(5):587–96. doi: 10.1517/ 14740338.7.5.587 40. Cameron LP, Tombari RJ, Lu J, Pell AJ, Hurley ZQ, Ehinger Y, et al. A Non- Hallucinogenic Psychedelic Analogue With Therapeutic Potential. Nature (2021) 589:474–9. doi: 10.1038/s41586-020-3008-z 53. Mabuchi T, Shintani N, Matsumura S, Okuda-Ashitaka E, Hashimoto H, Muratani T, et al. Pituitary Adenylate Cyclase-Activating Polypeptide Is Required for the Development of Spinal Sensitization and Induction of Neuropathic Pain. J Neurosci (2004) 24:7283–91. doi: 10.1523/JNEUROSCI. 0983-04.2004 41. López-Giménez JF, González-Maeso J. Hallucinogens and Serotonin 5-HT2A Receptor-Mediated Signaling Pathways. Curr Top Behav Neurosci (2018) 36:45–73. doi: 10.1007/7854_2017_478 42. Schmid RK, Bohn CL, Bohn LM. Agonist-Directed Signaling of the Serotonin 2A Receptor Depends on b-Arrestin-2 Interactions In Vivo. Proc Natl Acad Sci U S A (2008) 105:1079–84. October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org REFERENCES Rapgef2 Connects GPCR- Mediated cAMP Signals to ERK Activation in Neuronal and Endocrine Cells. Sci Signal (2013) 6(281):ra51. doi: 10.1126/scisignal.2003993 49. Emery AC, Alvarez RA, Abboud P, Xu W, Westover CD, Eiden MV, et al. C-Terminal Amidation of PACAP-38 and PACAP-27 Is Dispensable for October 2021 \| Volume 12 \| Article 732456 Frontiers in Endocrinology \| www.frontiersin.org 12
https://openalex.org/W4387378127	https://www.minbar.su/jour/article/download/1282/696	Russian	null	The Problem of Knowledge and Cognition in Islam	Minbar. Islamic studies/Minbar. Islamskie issledovaniâ	2,023	cc-by	6,505	А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 DOI 10.31162/2618-9569-2023-16-3-631-646 Original Pap УДК 297.1 Оригинальная стат А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 DOI 10.31162/2618-9569-2023-16-3-631-646 Original Pap УДК 297.1 Оригинальная стат А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 DOI 10.31162/2618-9569-2023-16-3-631-646 УДК 297.1 А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 Original Paper Оригинальная статья DOI 10.31162/2618-9569-2023-16-3-631-646 УДК 297.1 The Problem of Knowledge and Cognition in Islam A.A. Zakirov1а 1Center for Islamic Studies of Academy of Sciences of Tatarstan, Kazan, the Russian Federation аORCID: https://orcid.org/0000-0002-3745-4300, e-mail: aydarzak1@yandex.ru A.A. Zakirov1а 1Center for Islamic Studies of Academy of Sciences of Tatarstan, Kazan, the Russian Federation аORCID: https://orcid.org/0000-0002-3745-4300, e-mail: aydarzak1@yandex.ru Abstract: The article considers the epistemological teaching of Islamic religion, various types of knowledge and position of Islam regarding the matters of faith and reasoning. The author of the article answers the question about admissibility of examining exegesis as an object of cognition and an outcome of development of cogitative activity. The article off ers more ﬂ exible and liberal approach to the interpretation of notion of “ij tihad” and it is regarded as an attempt to revive the exploratory spirit of Islamic religion. The matter of faith is not introduced as an ultimate goal but as a mean of achieving success in religious life. “Is there any relation between the revelation of seventh century and emergence of empirical science?”, “Does the Quranic text foster the scientifi c research?”, “Is reality a rational comprehensible structure?”, – these are the questions the author seeks to provide the answers. Keywords: epistemology of Islam; faith and knowledge; religion and science; Islamic exegesis; Islamic theology; philosophy of Islam; postmodernism For citation: Zakirov A.A. The Problem of Knowledge and Cognition in Islam. Minbar. Islamic Studies. 2023;16(3):631–646. (In Russ.) DOI: 10.31162/2618-9569-2023-16- 3-631-646 Проблема знания и познания в исламе А.А. Закиров1а 1Центр исламоведческих исследований Академии наук Республики Татарстан, г. Казань, Российская Федерация аORCID: https://orcid.org/0000-0002-3745-4300, e-mail: aydarzak1@yandex.ru Резюме: В статье рассматриваются эпистемологическое учение ислама, виды знания и позиция ислама относительно вопроса веры и разума. Также автор ста- тьи предлагает ответ на вопрос о допустимости изучения экзегетики в качестве предмета познания и результата развития мыслительной деятельности. В статье предложен более гибкий и либеральный подход к интерпретации термина «идж- тихад», который рассматривается как стремление возродить исследовательский дух исламской религии. Вопрос знания представлен не как конечная цель, а как средство достижения успеха в религиозной жизни. Существует ли связь между кораническим откровением и возникновением эмпирической науки, стимулиру- ет ли коранический текст на научные изыскания, является ли реальность рацио- нально постижимой структурой – это те вопросы, на которые ищет ответы автор. Ключевые слова: эпистемология ислама; вера и знание; религия и наука; ис- ламская экзегетика; исламское богословие; философия ислама; постмодернизм Для цитирования: Закиров А.А. Проблема знания и познания в исламе. Minbar. Islamic Studies. 2023;16(3):631–646. DOI: 10.31162/2618-9569-2023-16-3-631-646 Резюме: В статье рассматриваются эпистемологическое учение ислама, виды знания и позиция ислама относительно вопроса веры и разума. Также автор ста- тьи предлагает ответ на вопрос о допустимости изучения экзегетики в качестве предмета познания и результата развития мыслительной деятельности. В статье предложен более гибкий и либеральный подход к интерпретации термина «идж- тихад», который рассматривается как стремление возродить исследовательский дух исламской религии. Вопрос знания представлен не как конечная цель, а как средство достижения успеха в религиозной жизни. Существует ли связь между кораническим откровением и возникновением эмпирической науки, стимулиру- ет ли коранический текст на научные изыскания, является ли реальность рацио- нально постижимой структурой – это те вопросы, на которые ищет ответы автор. Ключевые слова: эпистемология ислама; вера и знание; религия и наука; ис- ламская экзегетика; исламское богословие; философия ислама; постмодернизм Для цитирования: Закиров А.А. Проблема знания и познания в исламе. Minbar. Islamic Studies. 2023;16(3):631–646. DOI: 10.31162/2618-9569-2023-16-3-631-646 Контент доступен под лицензией Creative Commons Attribution 4.0 License. This work is licensed under a Creative Commons Attribution 4.0 License. 631 631 © А.А. Закиров, 2023 © Minbar. Islamic Studies, 2023 A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 Введение Эпистемология как область научного знания продолжает развиваться со времен возникновения философии наряду с такими направлениями, как этика, логика и метафизика, несмотря на то что термин «эпистемология» применяет- ся в современной науке не более пары сотен лет. В течение истории ее развития фокусом изучения становились различные аспекты. Древнегреческий философ Платон (427 до н. э. – 347 до н. э.) сформулировал определение понятия «зна- ние», описал его виды и характеристики, провел различение между «знанием» и «мнением». Он также разграничил «знание» относительного того, что существу- ет в реальном мире [1, с. 53–76]. Эпистемология И. Канта (1724–1804) была на- 632 © A.A. Zakirov, 2023 © Minbar. Islamic Studies, 2023 © A.A. Zakirov, 2023 © Minbar. Islamic Studies, 2023 А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 А.А. Закиров правлена на изучение условий возможностей человеческого познания, а Б. Рас- сел (1872–1970) делал попытки понять и оправдать современную науку с точки зрения чувственного опыта. На современном этапе развития эпистемология фор- мируется в рамках различных направлений как междисциплинарный комплекс научных дисциплин. Один из таких комплексов – это когнитивная наука, кото- рая объединяет исследования в сфере искусственного интеллекта, когнитивной психологии и лингвистики [2, с. 99–102]. правлена на изучение условий возможностей человеческого познания, а Б. Рас- сел (1872–1970) делал попытки понять и оправдать современную науку с точки зрения чувственного опыта. На современном этапе развития эпистемология фор- мируется в рамках различных направлений как междисциплинарный комплекс научных дисциплин. Один из таких комплексов – это когнитивная наука, кото- рая объединяет исследования в сфере искусственного интеллекта, когнитивной психологии и лингвистики [2, с. 99–102]. В современной философии эпистемология знания изучается в рамках двух направлений. В первом направлении большое внимание уделяется иррациональ- ным способам познания, лого и антропоцентризм подвергаются деконструкции (представители: Ф. Бекон (1561–1626), Дж. Локк (1632–1704), Д. Юм (1711– 1776), Дж. Беркли (1685–1753)). В другом направлении знание рассматривается как подлинно существующее и обоснованное мнение (представители: Р. Декарт (1596–1650), Б. Спиноза (1632–1677), Г. Лейбниц (1646–1716)). Основным предметом спора между ними был вопрос происхождения знания: это чувствен- ный опыт или деятельность разума? Вместе с тем следует упомянуть культурную легитимацию определенных видов знания в различные этапы развития истории. В европейской науке Нового времени опыт и эксперимент были основными источниками знания, в то время как в исламских странах первостепенное значение уделялось знанию, которое добывалось из недр традиции и толкования. Введение Рационализм и эмпиризм как кон- цептуальные направления эпистемологического знания в их зачаточном поло- жении были уделом небольшой группы людей, которые трудились при Домах мудрости. Однако со временем научные изыскания в исламском мире пришли в упадок в силу исторических обстоятельств, они не получили институциональ- ного развития, знание заключили в рамки сакральных текстов и их интерпре- тирующих субъектов, трансцендентальный субъективный опыт был заменен на объективно существующий, эмпирическое и рациональное восприятие как ин- струменты рационального познания, мысленные продукты, порождаемые разу- мом, стали служить субъективному ощущению и традиции, а их агенты начали доминировать в мусульманском общественном дискурсе. Эпистемологическое учение ислама В исламе вопрос «знания» поднимается в первых аятах суры «ал-‘Аляк», в начале ниспослания откровения Мухаммаду. В них отмечается, что человек со- 633 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) творен из куска плоти, ему было велено «читать и развиваться во имя Господа», который «научил его перу» и «поведал неизведанное». Муфассиры1 отмечают, что человек изначально появляется на свет без врожденного или встроенного умственного содержания, не обладая какими-либо сведениями об окружающем мире, в виде «tabula rasa», далее он познает окружающий мир посредством опы- та, чувственного восприятия и разума [3, с. 376]. творен из куска плоти, ему было велено «читать и развиваться во имя Господа», который «научил его перу» и «поведал неизведанное». Муфассиры1 отмечают, что человек изначально появляется на свет без врожденного или встроенного умственного содержания, не обладая какими-либо сведениями об окружающем мире, в виде «tabula rasa», далее он познает окружающий мир посредством опы- та, чувственного восприятия и разума [3, с. 376]. Согласно исламскому мировоззрению, Бог является творцом и первопри- чиной всего сущего и бытия, исходной точкой которого является «калимат Ал- лах» (слово Бога), следовательно, Он – единственный знаток истины вещей и источник знаний и наук. Человек получает знания посредством изучения «Книги сотворения», которая представлена тремя уровнями. Первый уровень – это зна- ния о «Книге происхождения, жизни и конечной судьбе сотворения» (аят аль- китаб). Второй – знания о «Книге человеческого общества» (аят аль-анфус), тре- тий – это знания о Вселенной (аят аль-каун) [4, p. 51–52]. Первый уровень представлен откровениями пророков, которые они полу- чали от Бога на протяжении жизни. С точки зрения ислама эти знания считаются достоверными, в исламе они представлены в виде свода Корана и сборников ха- дисов. В большинстве случаев положения коранических текстов и хадисов вза- имозаменяемы, дополняя друг друга, и относятся к категории шариатских зна- ний – «аль-‘улюм аль-наклиййа» (традиционные религиозные науки). Второй уровень знаний относится к изучению человека и общества. Цель – изучение предписанных Богом закономерностей функционирования человеческих сооб- ществ через пространство и время. В этих изысканиях человеческий интеллект, разум и органы чувств выступают основными инструментами научного позна- ния. Эти науки исламские ученые называют гуманитарными и рациональными науками. Третий уровень – это знания, полученные в результате изучения Все- ленной (аят аль-каун) или физических законов природы. Они представлены в виде закономерностей природных явлений и феноменов, а источниками этих данных выступают человеческий интеллект, чувства, систематизация, наблю- дение и опытно-экспериментальные работы. Эти науки называются естествен- ными. С точки зрения ислама они также относятся к рациональным наукам [5, p. 70]. 1 Муфассир – специалист по толкованию Корана, имеющий необходимые для этого навыки и знания. Эпистемологическое учение ислама Тем не менее, несмотря на четкое разделение религиозных и научных зна- ний, конфликт между их представителями оказался неизбежным. 1 Муфассир – специалист по толкованию Корана, имеющий необходимые для этого навыки и знания. 634 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) ISSN 2618-9569 (Print) 635 ISSN 2712-7990 (Online) А.А. Закиров р Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 Прежде чем изучить историю столкновения трансцендентального открове- ния с разумом, следует обозначить позицию ислама относительно вопроса веры, которая была руководством для мусульман. В Коране вера означает признание реальности и единства Аллаха и положения о том, что люди несут ответствен- ность за свою жизнь и деяния в День воскрешения. Единобожие является ядром исламского вероучения. С одной стороны, в исламе важное значение имеет вера, поклонение и послушание, а рациональные изыскания и дискурсивная мысль на- ходятся в подчинении веры и приобретают ценность в том случае, если они не противоречат основным постулатам веры. С другой стороны, Коран высоко оце- нивает потенциал человеческого интеллекта, призывая размышлять и развивать его. Вера в любом случае сохраняет приоритет над самостоятельным человече- ским разумом, наблюдает и ограничивает функции и полномочия интеллекта. Вопрос соотношения веры и разума стал одной из основных проблем в эпоху становления национальных государств и секуляризации многих сторон человеческой жизни. Исламские ученые подошли к решению этого вопроса с двух противоположных позиций – реформаторской и традиционной. Основ- ная проблема состояла в том, что исламские ученые игнорировали достижения гуманитарных и естественных наук, которые получили колоссальное развитие в последние несколько столетий. В исламском мире отсутствовали механизмы и площадки для обмена мнениями, и вследствие такой устоявшейся практики возникла вполне закономерная патовая ситуация: первая когорта уповала лишь на религиозные тексты, а вторая – надеялась только на социальный активизм первой и не нуждалась в их мнении. Осознавая положение вещей, современные исламские деятели предложили разграничить сферы эпистемологии и онтоло- гии и заново воссоздать эту утраченную связь. Вместо переноса богословской итерации из одной крайности в другую и обращения исключительно к религиоз- ным текстам они предложили всеобъемлющий подход, в котором рациональный аспект стал превалирующим. Именно этим положением мы можем объяснить попытку систематического и целостного осмысления современных неомодер- нистских тенденций, которые определяются как «прогрессивный ислам». Сов- ременный исследователь ислама А. Дудериджа определяет представителей этого движения как сообщество интеллектуалов, которые допускают определенные интерпретационные предположения эпистемологического, герменевтического и методологического характера в отношении осмысления традиции и ее первоис- точников [6, p. 2–3]. Характерным объединяющим качеством этого движения 635 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 является следование императивным нормам, выражающим теоретические и эти- ческие принципы ислама как универсальной религии [7, с. 55]. А.А. Закиров является следование императивным нормам, выражающим теоретические и эти- ческие принципы ислама как универсальной религии [7, с. 55]. р у р р [ ] В связи с этим возникают вполне целесообразные вопросы: может ли экзе- гетика быть предметом познания и развития мыслительной деятельности и пе- рестать быть исключительно богословской дисциплиной? Может ли она претен- довать на самостоятельный предмет и область изучения в рамках рационализма и быть одной из форм рефлексивного философского исследования? В разные исторические периоды ислама мы находим различные ответы на эти вопросы, и в этом отношении ислам предоставляет механизмы обновления религиозной мысли в каждом столетии. В достоверном хадисе пророка Мухаммада сказано о том, что Аллах будет посылать для этой общины каждые сто лет того, кто бу- дет возрождать для нее их религию [8, с. 688]. Обновителями религии в исламе называют «муджаддидами», а сам процесс обновления – «тадждидом». Концепт «тадждида» сыграл ключевую роль в современном исламском возрождении (ис- ламский ривайвелизм). Исламское возрождение – это общий термин, который включает в себя широкий спектр движений, зачастую с противоположными иде- ологическими установками (нетерпимыми или плюралистичными, научными или антинаучными, направленными на изменение себя или внешнего мира, апо- литичными или политическими, демократическими или авторитарными и т. д.). В общем, основные идеи ривайвелизма направлены на реконструкцию реально- го или воображаемого прошлого, подвергают сомнению современное состояние исламской мысли, нацелены на осознание эмоциональной близости к Богу и переживание личной встречи с ним. Их представители обвиняли современных мусульман в забвении религиозного учения ислама и в отступлении от ценно- стей «истинного ислама». В этом отношении представители исламского ривай- велизма не исключение, так как они предлагают доступ к «истинному посланию» и хотят восстановить его в первозданном виде [9, p. 30]. Даже так называемые «фундаменталисты» преследуют схожую цель, которая свидетельствует о слабой вере во внеконтекстуальную природу религии. Вместе с распространением идей исламского возрождения следует отме- тить особое место идеи «открытия врат иджтихада». Исламские ученые, бого- словы и законоведы на протяжении веков вели споры по поводу независимого суждения в религиозных делах, применяя термин «иджтихад». В религиозно- правовой терминологии «иджтихад» – это «труд, усердие в вынесении правовых предписаний и решении различных религиозных проблем и вопросов на осно- 636 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 вании аятов Корана, хадисов, кыяса и иджмы» [10, с. 266]. В исламе существует два подхода к иджтихаду. В первом подходе он представлен в узком смысле, в религиозно-правовом понимании. А.А. Закиров Под «иджтихадом» понимается процесс ре- лигиозно-правового суждения с целью определить дозволенность или запрет- ность какого-либо действия, которое не упоминается в основных источниках ислама. На самом деле этот подход часто применяется с целью ограничения права на независимое суждение среди мусульман и предоставления права пони- мания и разъяснения вопросов ислама исключительно мусульманским законо- ведам (факихам). Данный вид иджтихада противоречит свободному суждению, так как допускает его применение только в том случае, если рассматриваемый вопрос не упоминается в основных источниках ислама и ни один из средневеко- вых ученых не вынес решения по нему. В таком значении разум – это последний инструмент в понимании воли Бога. В более широком смысле под «иджтиха- дом» понимается процесс свободного суждения, рациональное размышление и поиск истины, в котором задействованы эпистемология науки, рационализм, человеческий опыт и критическое мышление. Обычно эту позицию отстаивают сторонники исламского модернизма и либерализма. Для модернистов иджти- хад – это дух изысканий и стремления ко всем формам знаний, не только рели- гиозных и правовых, и желание возродить и восстановить исследовательский дух ислама. Поэтому вопрос свободы и необходимости применения свободного суждения за пределами откровения был предметом спора на протяжении всей истории ислама. Следует отметить, что иджтихад периодически применялся во- преки духу откровения, который приводил к противоречиям. Исламские уче- ные считают, что иджтихад дал возможность мусульманам быть гибкими и пе- ренимать знания из других культур и цивилизаций. С точки зрения ислама ни один человек не обладает истиной, и верующий находится в постоянном поиске истины. Этот подход стал хорошим подспорьем для уважительного отношения к чужому мнению и признания своего мнения ошибочным, если в споре выясня- лось таковое. К сожалению, четыре-пять веков назад исламская цивилизация, в частности исламская мысль, начала терять былую гибкость и притягательность перед лицом достижений западной науки, заняла более консервативную пози- цию с целью сохранить традиционные ценности и институты. В итоге многие ис- ламские ученые стали отрицательно относиться к инновациям и чужому опыту, а в лучшем случае – настороженно [11, p. 2]. 637 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) A.A. Zakirov A.A. Zakirov Исламские богословы считают, что подчинение веры разуму может приве- сти к тому, что религия станет подотчетной философии. Они даже не допускают мысли о том, что откровение можно подвергнуть внешней философской оценке, исследовать религию на основании критериев, которые применяются при изуче- нии естественных наук. В конечном счете поздний исламский фидеизм приобре- тает схожие черты с христианским. Христианские фидеисты считают, что вера доминирует над разумом в вопросах веры, откровение является единственным источником просвещения верующего. Вера не нуждается ни в рациональной, ни в научной или в какой-либо внешней аргументации, она, наоборот, основывается на заверениях откровения, является судьей разума и его устремлений в вопросах веры [12, p. 81]. Следовательно, исторический конфликт между верой и разумом укоренен в отличных друг от друга эпистемологиях, которые предполагают наличие раз- личных рамок мышления и видов рациональности для исследования. Проблема знания у мусульман возникает не как вопрос секулярный и обособленный, а в большей степени как вопрос религиозный. Знание не является конечной целью, а лишь выступает средством достижения успеха в религиозной жизни, наподобие приобретения знаний естественных наук в наше время с целью заработка, контр- оля ресурсов и демонстрации власти. Коран обращается к верующим лишь с од- ним вопросом: «Во что следует уверовать верующему и как ему стоит поступать для того, чтобы он смог приобрести довольство Бога и войти в райские сады?». Такой узкий взгляд на религию неизбежно приводит к ограничению индивиду- ального разума и доступных возможностей для спекуляций. В результате этих ограничений мы получаем соразмерную зависимость от авторитетов мнений. Видимо, это положение стало причиной гибели зародившейся философии сре- ди мусульман. Были предприняты попытки развить религиозно-философскую мысль за пределами исламской ортодоксии, однако, как и в случае с естественны- ми науками, не были созданы институциональные условия для передачи резуль- татов этого опыта. Следующие поколения не имели непосредственного доступа к этим знаниям. Философия и наука впервые были внедрены культурно и институ- ционально в средневековых университетах Западной Европы и продолжают эти традиции по сей день. ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) 638 А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 Познаваемость Бога и Вселенной Мусульмане признают, что эпоха Просвещения оказала огромное влияние на исламскую цивилизацию. Развитие религиозной мысли вместе с распростра- нением и ростом популярности естественных наук обнажили некоторые пробле- мы традиционной веры. Мусульмане считают возможным сохранить аутентич- ное религиозное ядро откровения при условии, что эти проблемы будут приняты и рассмотрены с точки зрения разума. Об этом также свидетельствуют кораниче- ские нарративы о знамениях Бога в качестве проявлений божественного искусст- ва. Эти аяты внушают оптимизм на познаваемость Бога: истина завуалирована, потенциально выявляема и открывает себя усердному и умному наблюдателю – ищущему. Бог остается за завесой таинственности – Его воля совершенно ясна. Кораническое послание гласит, что человек наделен способностями различения истины ото лжи и приобретения знаний [13, с. 636]. Возникает вполне законо- мерный вопрос: существует ли какая-либо связь между возникновением эмпири- ческой науки и откровением Корана, которая побуждала большой интерес к из- учению проявлений Бога, признаваемых как различимые паттерны и физические законы природы? С точки зрения ислама божественное откровение не претенду- ет на соответствие научным критериям исследований и детализацию описанных в нем процессов и явлений. Более того, Коран не ставит целью обращение к пред- ставителям какого-то определенного поколения, в нашем случае – к современно- му. Цель Корана – вдохновление на изучение законов природы, следовательно, он прямо или косвенно способствовал к тому, чтобы природа изучалась как ав- тономная и независимая сущность. По сути, такую цель преследовала и наука. По этой причине заявления мусульман о том, что ислам дал стремительный им- пульс к развитию наблюдательной науки, имеет реальные основания [12, с. 237]. В этой связи возникает вопрос: каковы исходные положения Корана от- носительно научных исследований и каким образом коранический монотеизм стимулировал научные изыскания? Побуждает ли коранический текст на реф- лексивные размышления и непредвзятое изучение природы? Коранический на- рратив об определении Адама наместником Бога на земле вполне отвечает на этот вопрос. Согласно тексту Корана, Бог научил Адама всевозможным именам творений, а затем попросил ангелов назвать эти имена. Ангелы сказали, что они знают только то, чему научил их Бог [13, с. 36]. В данных аятах разъясняется, что человек наделен способностью запоминать, различать и классифицировать мате- 639 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 риальные предметы. Коранические откровения заверяют нас в том, что природа создавалась целенаправленно, следовательно, она постижима и научный поиск возможен. Познаваемость Бога и Вселенной К тому же следует отметить, что наука может возникнуть только в та- кой среде, где мир не воспринимается как иллюзия. риальные предметы. Коранические откровения заверяют нас в том, что природа создавалась целенаправленно, следовательно, она постижима и научный поиск возможен. К тому же следует отметить, что наука может возникнуть только в та- кой среде, где мир не воспринимается как иллюзия. 640 ISSN 2618-9569 (Print) Помимо метафизической поддержки в деле изучения постижимой и ау- тентичной реальности, монотеизм предложил возможность заниматься наукой тремя способами. Первый из них уникален для исламского монотеизма, осталь- ные – для западного монотеизма. В исламе осуждаются языческий политеизм и магические практики, которые имеют отношение к нему. В исламском праве кол- довство и черная магия строго запрещены [13, с. 46]. Во-первых, магия подвер- гается жесткой критике за то, что она вводит в заблуждение и является ложной религией. Она содержит псевдоритуалы, не ведет борьбу с невежеством своих последователей, следовательно, она не может быть мировоззренческой осно- вой для конструирования научных теорий. Во-вторых, язычество не побуждает к получению знаний о мире и рассматривает Вселенную как место состязания и поле битвы соперничающих божеств. Согласно учению ислама, политеизм яв- ляется источником хаоса на небесах и земле [13, с. 358], каждое божество дейст- вует в соответствии со своими интересами, следовательно, появление иерархии богов является неизбежной реальностью [13, с. 383]. Неорганизованность и не- стабильность – это сущностные характеристики политеизма, которые создают препятствия для рационального поиска. Выход из этой ситуации – монотеизм, избавляющий нас от иррациональных и непредсказуемых действий и хаоса язы- ческого пантеона. Верховная власть одного-единственного божества в природе гарантирует унификацию названий наблюдаемой Вселенной и создает объектив- ную основу для закономерностей, на которой базируется наука. В такой ситуации верующий в роли исследователя может планомерно изучить миропорядок, по- стижимый разумом. Наше понимание реальной действительности предполагает постоянную закономерность восприятия, которая в свою очередь основывается на предположении, что реальность – это рационально постижимая структура, до- ступная человеческому разуму. В монотеизме это предположение основывается на творческой и частично познаваемой воле Бога. В Коране многократно прохо- дит мысль о том, что мусульманам следует изучать физические законы природы. Усилия в этом направлении на протяжении веков привели к тому, что мусульмане стали передатчиками западу классической греческой учености. Однако и в этой ситуации сохранялась напряженность между ортодоксальными представителя- 640 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 ISSN 2618-9569 (Print) 641 ISSN 2712-7990 (Online) котором отсутствуют моральные критерии оценки и содержатся лишь формаль- ные процессуальные шаги для достижения цели, оправдывающей средства. котором отсутствуют моральные критерии оценки и содержатся лишь формаль- ные процессуальные шаги для достижения цели, оправдывающей средства. ми ислама и талантливыми и ми ислама и талантливыми интеллектуалами, которые были и мыслителями, и поэтами, и учеными-естествоиспытателями. В частности, ортодоксия запрещала хирургические вмешательства в человеческие тела и эксгумацию трупов. Религи- озное право также запрещало членовредительство, так как мусульмане убеждены в том, что они будут воскрешены физически. В общем, в исламе отсутствовала практика противодействия научным исследованиям как в средневековой церкви. Тем не менее в более поздние времена среди мусульман распространилось мнение о нежелательности занятия наукой. Они заявляли, что Коран представляет собой источник подлинной веры и заслуживает первоочередного изучения. Занятие философией и наукой требует более развитой религиозной мысли, допускаемой с одобрения откровения. Таким образом, изучение природы наделяло верующе- го инструментом для постижения, контроля и эксплуатации природных ресурсов в строго определенных моральных рамках и позволило ему развить необходи- мые технологии. Научный подход, санкционированный религией, предоставил возможность более активно накапливать опыт, который основывается на вере в закономерный и системный характер отношений в природе. Коран многократно заявляет, что природа содержит аллегорические указания на предустановленный порядок объективного мира и его смыслы. Если они указывают на присутствие и величие Бога, отношение к ним может быть определено не как субъект к объек- ту (как, например, к эмпирической науке), а как субъект к субъекту, так как мы имеем дело с проявлениями божественной сущности. Отношение субъект–субъ- ект объясняет причину того, почему довольно развитая исламская наука остава- лась свободной от скрытого скептицизма и оценивания – именно в этом кроется причина отличия западной науки с момента ее возникновения. Европейская на- ука изначально отказалась от рассмотрения природы как воплощения божест- венного присутствия в ней. Однако по мере изучения природы она утрачивала свою ценность. Ислам же прилагал большие усилия для сохранения этой свя- зи – изучение Вселенной должно было привести к большему пониманию смысла мироздания. Таким образом, Коран оказывал покровительство новым научным изысканиям, которые основывались на секулярной любознательности, а также призывал к поиску божественных замыслов в природе, которые уберегали науку от обесценивания. В итоге мы пришли к тому, что моральные нормы стали под- вергаться оценке со стороны инструментального разума: проводится техническая экспертиза без ценностных критериев, пренебрегается человеческая и моральная стоимость. Современный секулярный разум выиграл борьбу и создал алгоритм, в ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) 641 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 котором отсутствуют моральные критерии оценки и содержатся лишь формаль- ные процессуальные шаги для достижения цели, оправдывающей средства. Заключение Если мы определяем науку как стремление объяснить события и феномены физическими критериями, в этом случае она несовместима с теизмом, так как ее предпосылка изначально не содержит идеи вовлеченности Бога в процессы мироздания. Однако, если наука – это систематическое изучение природы, которое предполагает изучение фактов и данных в защиту или опровержение какой-либо выдвинутой теории, в этом слу- чае данное занятие совместимо с теизмом и было простимулировано исламом. Наука не занимается апологетикой религиозных постулатов, тем более телеоло- гией. С точки зрения науки Вселенная упорядочена, а ученые изучают сложные природные явления, их причинно-следственные связи, которые сформулирова- ны в натуралистических понятиях. Воздействие сверхъестественных сил на при- роду не принимается в качестве данных. То же самое принято и в гуманитарных науках, где ни один ученый не посмеет предположить, что демон оказал влияние на поведение серийного убийцы. Гипотезы, которые подразумевают вмешатель- ство сверхъестественных сил, не принимаются во внимание. В этой связи кора- нические нападки на магию и колдовство лишь усиливают позицию науки. ременной эмпирической науки и существует ли устойчивая институциональная связь между монотеизмом и наукой? Современные ученые заявляют, что суще- ствует объективная физическая реальность, которая постижима и упорядочена. Они предлагают полностью разграничить сферы религии и науки, воспринимая мир как самодостаточную детерминированную систему, и не нуждаются в идее Бога. Следовательно, ответ на вышеупомянутый вопрос зависит от того, что мы подразумеваем под наукой и ее конечными целями. Если мы определяем науку как стремление объяснить события и феномены физическими критериями, в этом случае она несовместима с теизмом, так как ее предпосылка изначально не содержит идеи вовлеченности Бога в процессы мироздания. Однако, если наука – это систематическое изучение природы, которое предполагает изучение фактов и данных в защиту или опровержение какой-либо выдвинутой теории, в этом слу- чае данное занятие совместимо с теизмом и было простимулировано исламом. Наука не занимается апологетикой религиозных постулатов, тем более телеоло- гией. С точки зрения науки Вселенная упорядочена, а ученые изучают сложные природные явления, их причинно-следственные связи, которые сформулирова- ны в натуралистических понятиях. Воздействие сверхъестественных сил на при- роду не принимается в качестве данных. То же самое принято и в гуманитарных науках, где ни один ученый не посмеет предположить, что демон оказал влияние на поведение серийного убийцы. Гипотезы, которые подразумевают вмешатель- ство сверхъестественных сил, не принимаются во внимание. В этой связи кора- нические нападки на магию и колдовство лишь усиливают позицию науки. Разрыв традиционной связи между нравственностью и религией не при- нес ущерба ни одной из сторон, а разрыв между наукой и религией причинил ущерб науке. Заключение Несмотря на то, что религия открыто не препятствует проведению науч- ных изысканий, будучи относительно гибкой в усвоении их результатов, она со скепсисом относится к утверждению о существовании первопричины возник- новения Вселенной. Средневековая религия утратила свою значимость в тот момент, когда ученые XVII в. отвергли формальную и заключительную пер- вопричины возникновения Вселенной. Древнегреческий философ Аристотель (384–322 гг. до н. э.) выделял четыре первопричины бытия вещей: материаль- ную (материя), формальную (суть и сущность), деятельную (активное деятель- ное начало) и целевую (предназначение) [14, с. 23]. Предполагалось, что целе- вая первопричина будет иметь глобальный характер в масштабах Вселенной, а формальная причина будет олицетворять замысел Творца. Верховное божество не принимало участия в мирских делах и пребывало вне времени. Аристотель и Платон были убеждены в истинности этих положений, и весь средневековый христианский мир придерживался этих идей. Мусульманские философы, кото- рые усвоили и развили идеи греческих философов, считали рационализм самой передовой формой религии и разработали более возвышенное представление о Боге по сравнению с кораническим Богом [15, с. 119]. Они нашли подтвержде- ние своих идей в кораническом тексте: «Господь наш – Тот, Кто создал все вещи, а затем указал [всему] путь» [13, с. 349]. А следующий аят служил доказатель- ством формальной первопричины сотворения: «Поистине, если Мы пожелаем [возникновения] чего-либо, Нам достаточно сказать: “Будь!”, и оно сбывается [творение возникает]» [13, с. 305]. Тем не менее в исламских странах научные изыскания пришли в упадок и не получили институционального развития – «ра- циональное божество» мусульманских философов погибло, несмотря на то что они не видели принципиальных противоречий между откровением и наукой, ве- рой и рациональностью [15, с. 120]. В большинстве исследований сущности религии утверждается, что она есть иррациональная и простейшая попытка контроля и умилостивления при- роды. Религия прочно сохраняет свое место и роль в жизни человека, несмотря на колоссальные достижения в современной науке. В этой связи возникает впол- не закономерный вопрос: был ли ислам частью зарождающегося процесса сов- 642 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 А.А. Закиров ременной эмпирической науки и существует ли устойчивая институциональная связь между монотеизмом и наукой? Современные ученые заявляют, что суще- ствует объективная физическая реальность, которая постижима и упорядочена. Они предлагают полностью разграничить сферы религии и науки, воспринимая мир как самодостаточную детерминированную систему, и не нуждаются в идее Бога. Следовательно, ответ на вышеупомянутый вопрос зависит от того, что мы подразумеваем под наукой и ее конечными целями. ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) Заключение Наука и технологии, которые не несут никакой ответственности перед трансцендентальным разумом, могут причинить необратимый вред че- ловечеству. В современную эпоху эти опасения в контексте глобального ядер- ного самоуничтожения и запрещенных технологий бактериологического и хи- мического оружия являются вполне обоснованными. Кораническое видение науки не получило должного развития, а восторжествовала философия эпохи Ренессанса в лице ее интеллектуального реформатора и философа естествен- ной науки Френсиса Бэкона (1561–1626), который приравнял знание к власти. Ф. Бэкон был убежден в том, что человек – властелин природы, признавая за природой статус второго священного писания в качестве открытой книги бо- жественного провидения. Он был очарован степенью достоверности резуль- 643 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 татов, получаемых опытным путем в природе – чистый разум гарантирует их правильную интерпретацию. Цель современной науки – это покорение при- роды и контроль над ее ресурсами, изучение ее предполагаемой пригодности к эксплуатации, а также получение выгоды и пользы. С точки зрения ислама эксплуатация природы с применением научных технических средств искажает ее истинную цель и предназначение. Пренебрежение религиозной телеологией природы приводит к отрицанию проявлений Бога, поэтому исследователь от- казывается от поиска более благородной цели, нежели ее эксплуатации. Приро- да нема, поэтому она нуждается в дополнительном откровении, а в секулярную эпоху у нее отсутствуют сакральные интерпретаторы. Религия уступила место секулярным интерпретаторам в лице искусства и науки, таким образом природа лишилась своей моральной значимости и ценности. Заполняя эту пустоту, Ко- ран заявляет, что только верующий осознает истинную ценность природы как места, где проявляется уникальное трансцендентное представительство Бога, где его деятельность становится постижимой разуму. Ведь в Коране сказано: «А на земле – знамения для людей убежденных, а также они [знамения] – в вас самих. Разве вы не видите?» [13, с. 555]. 8. Сунан Абу Дауда. Сборник хадисов. Казань: Издательство Иман; 2009. 888 с. 7. Мухетдинов Д.В. Исламский модернизм: эскиз обновленческой про- граммы. Ислам в современном мире. 2019;2:48–70. 1. Асмус В.Ф. Платон. М.: Мысль; 1975. 220 с. А.А. Закиров Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 Проблема знания и познания в исламе Minbar. Islamic Studies. 2023;16(3):631–646 9. Rippin A., Knappert J. Textual Sources for the Study of Islam. Manchester: Manchester University Press; 1986. 209 p. 10. Али-заде А.А. Исламский энциклопедический словарь. М.: Ансар; 2007. 920 с. 11. Smock D. Ijtihad: Reinterpreting Islamic Principles for the Twenty-fi rst Century. USIP Special Reports. №125. Washington: United States Institute of Peace; 2004. 8 p. 12. Akhtar Sh. The Quran and the Secular Mind. A philosophy of Islam. London, NY: Routledge; 2008. 400 p. 13. Калям Шариф. Перевод смыслов. Казань: Издательский дом «Хузур» – «Спокойствие»; 2020. 652 с. 14. Аристотель. Метафизика. Марков А.В. (пер. с древнегреч., вступитель- ная статья и комм.). М.: РИПОЛ классик; 2018. 384 с. 15. Армстронг К. История Бога. Семенов К. (пер.), Трилис В. и Доброволь- ский М. (ред.). К. – М.: София; 2004. 285 с. Литература 1. Асмус В.Ф. Платон. М.: Мысль; 1975. 220 с. 1. Асмус В.Ф. Платон. М.: Мысль; 1975. 220 с. 1. Асмус В.Ф. Платон. М.: Мысль; 1975. 220 с. 2. Никитина Е.А. Современная эпистемология: тенденции и направления развития. Социально-гуманитарные знания. 2010;1:91–103. 2. Никитина Е.А. Современная эпистемология: тенденции и направления развития. Социально-гуманитарные знания. 2010;1:91–103. 3. Коран: Пер. с араб. и коммент. М.–Н. Османова. М.: Ладомир, Восточная литература; 1995. 575 с. 3. Коран: Пер. с араб. и коммент. М.–Н. Османова. М.: Ладомир, Восточная литература; 1995. 575 с. 4. Faruqi I. Islamization of Knowledge: General Principles and Work Plan. Herndon: International Institute of Islamic Thought; 1987. 59 p. 4. Faruqi I. Islamization of Knowledge: General Principles and Work Plan. Herndon: International Institute of Islamic Thought; 1987. 59 p. 5. Lantong A. The Islamic Epistemology and its Implications for Education of Muslims in the Philippines. Advances in Social Science, Education and Humanities Research. Amsterdam: Atlantis Press; V. 125. 2018. P. 67–71. 5. Lantong A. The Islamic Epistemology and its Implications for Education of Muslims in the Philippines. Advances in Social Science, Education and Humanities Research. Amsterdam: Atlantis Press; V. 125. 2018. P. 67–71. 6. Duderij a A. The Imperatives of Progressive Islam. London: Routledge; 2017. 224 p. 6. Duderij a A. The Imperatives of Progressive Islam. London: Routledge; 2017. 224 p. 7. Мухетдинов Д.В. Исламский модернизм: эскиз обновленческой про- граммы. Ислам в современном мире. 2019;2:48–70. 8. Сунан Абу Дауда. Сборник хадисов. Казань: Издательство Иман; 2009. 888 с. 644 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) Информация об авторe Закиров Айдар Азатович, кандидат фи- лософских наук, старший научный со- трудник Центра исламоведческих ис- следований Академии наук РТ, Казань, Российская Федерация. Aydar A. Zakirov, Cand. Sci (Philosophy), Senior Research Fellow of the Center for Islamic Studies of Tatarstan Academy of Sciences, Kazan, the Russian Federation. Закиров Айдар Азатович, кандидат фи- лософских наук, старший научный со- трудник Центра исламоведческих ис- следований Академии наук РТ, Казань, Российская Федерация. Aydar A. Zakirov, Cand. Sci (Philosophy), Senior Research Fellow of the Center for Islamic Studies of Tatarstan Academy of Sciences, Kazan, the Russian Federation. A.A. Zakirov A.A. Zakirov The Problem of Knowledge and Cognition in Islam Minbar. Islamic Studies. 2023;16(3):631–646 8. Sunan Abu Dauda. Sbornik khadisov [Abu Daud’s Sunan. The Collection of hadiths]. Kazan: Iman Press; 2009. 888 p. (In Russian) 9. Rippin A., Knappert J. Textual Sources for the Study of Islam. Manchester: Manchester University Press; 1986. 209 p. 10. Ali-zade A.A. Islamskiy entsiklopedicheskiy slovar' [Islamic encyclopedic dictionary]. Мoscow: Ansar Press; 2007. 920 p. (In Russian) 11. Smock D. Ijtihad: Reinterpreting Islamic Principles for the Twenty-fi rst Century. USIP Special Reports. №125. Washington: United States Institute of Peace; 2004. 8 p. 12. Akhtar Sh. The Quran and the Secular Mind. A philosophy of Islam. London, New York: Routledge; 2008. 400 p. 13. Kalyam Sharif. Perevod smislov [Kalam Sharif. Interpretation of meanings]. Kazan: “Khuzur-Spokoystvie” Publishing House; 2020. 652 p. (In Russian) 14. Aristotel. Metafi zika [Metaphysics]. Markov A.V. (ancient Greek, introductory article and comments). Moscow: RIPOL Klassik Press; 2018. 384 p. (In Russian) 15. Armstrong K. Istoriya Boga [The History of the God]. Semenov K. (tr.), Trilis V. and Dobrovolskiy M. (eds). Kiev-Moscow: Sofi ya Press; 2004. 285 p. (In Russian) 15. Armstrong K. Istoriya Boga [The History of the God]. Semenov K. (tr.), Trilis V. and Dobrovolskiy M. (eds). Kiev-Moscow: Sofi ya Press; 2004. 285 p. (In Russian) References 1. Asmus V.F. Platon [Plato]. Moscow: Misl Press; 1975. 220 p. (In Russian) 2. Nikitina E. A. Sovremennaya epistemologiya: tendentsii i napravleniya razvitiya [The Modern Epistemology: tendencies and outlines for development]. Sotsial'no-gumanitarnye znaniya [Social and Humanitarian Knowledge]. 2010;1:91– 103. (In Russian) 2. Nikitina E. A. Sovremennaya epistemologiya: tendentsii i napravleniya razvitiya [The Modern Epistemology: tendencies and outlines for development]. Sotsial'no-gumanitarnye znaniya [Social and Humanitarian Knowledge]. 2010;1:91– 103. (In Russian) 3. Koran: Per. s arab. i komment. M.–N. Osmanova [Quran: Arabic translation and comments by M.N. Osmanov]. Moscow: Ladomir, Vostochnaya literature Press; 1993. 575 p. (In Russian) 3. Koran: Per. s arab. i komment. M.–N. Osmanova [Quran: Arabic translation and comments by M.N. Osmanov]. Moscow: Ladomir, Vostochnaya literature Press; 1993. 575 p. (In Russian) 4. Faruqi I. Islamization of Knowledge: General Principles and Work Plan. Herndon: International Institute of Islamic Thought; 1987. 59 p. 4. Faruqi I. Islamization of Knowledge: General Principles and Work Plan. Herndon: International Institute of Islamic Thought; 1987. 59 p. 5. Lantong A. The Islamic Epistemology and its Implications for Education of Muslims in the Philippines. Advances in Social Science, Education and Humanities Research. Amsterdam: Atlantis Press; V. 125. 2018, pp. 67–71. 5. Lantong A. The Islamic Epistemology and its Implications for Education of Muslims in the Philippines. Advances in Social Science, Education and Humanities Research. Amsterdam: Atlantis Press; V. 125. 2018, pp. 67–71. 6. Duderij a A. The Imperatives of Progressive Islam. London: Routledge; 2017. 224 p. 6. Duderij a A. The Imperatives of Progressive Islam. London: Routledge; 2017. 224 p. 7. Muhetdinov D.V. Islamskiy modernizm: eskiz obnovlencheskoy programmy [Islamic modernism: outlines of renewal programme]. Islam v sovremennom mire [Islam in the modern world]. 2019;2:48–70. (In Russian) 7. Muhetdinov D.V. Islamskiy modernizm: eskiz obnovlencheskoy programmy [Islamic modernism: outlines of renewal programme]. Islam v sovremennom mire [Islam in the modern world]. 2019;2:48–70. (In Russian) ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) 645 Раскрытие информации о конфликте интересов The author declares that there is no conﬂ ict of interest. Автор заявляет об отсутствии конфликта интересов. Информация о статье ф р Поступила в редакцию: 01 ноября 2022 Одобрена рецензентами: 20 августа 2023 Принята к публикации: 10 сентября 2023 ISSN 2618-9569 (Print) ISSN 2712-7990 (Online) 646
https://openalex.org/W2913909097	https://europepmc.org/articles/pmc6408386?pdf=render	English	null	Controllable synthesis of pomelo peel-based aerogel and its application in adsorption of oil/organic pollutants	Royal Society open science	2,019	cc-by	8,918	Research Cite this article: Shi G, Qian Y, Tan F, Cai W, Li Y, Cao Y. 2019 Controllable synthesis of pomelo peel-based aerogel and its application in adsorption of oil/organic pollutants. R. Soc. open sci. 6: 181823. royalsocietypublishing.org/journal/rsos Research Cite this article: Shi G, Qian Y, Tan F, Cai W, Li Y, Cao Y. 2019 Controllable synthesis of pomelo peel-based aerogel and its application in adsorption of oil/organic pollutants. R. Soc. open sci. 6: 181823. http://dx.doi.org/10.1098/rsos.181823 Received: 26 October 2018 Accepted: 3 January 2019 Subject Category: Chemistry Subject Areas: materials science Keywords: sponge aerogels, pomelo peel, silanization, oil-adsorption, recyclability Author for correspondence: Fengzhi Tan e-mail: fz_tan@126.com; tanfz@dlpu.edu.cn royalsocietypublishing.org/journal/rsos Research Cite this article: Shi G, Qian Y, Tan F, Cai W, Li Y, Cao Y. 2019 Controllable synthesis of pomelo peel-based aerogel and its application in adsorption of oil/organic pollutants. R. Soc. open sci. 6: 181823. http://dx.doi.org/10.1098/rsos.181823 Received: 26 October 2018 Accepted: 3 January 2019 Subject Category: Chemistry Subject Areas: materials science Keywords: sponge aerogels, pomelo peel, silanization, oil-adsorption, recyclability Author for correspondence: Fengzhi Tan e-mail: fz_tan@126.com; tanfz@dlpu.edu.cn royalsocietypublishing.org/journal/rsos Research Cite this article: Shi G, Qian Y, Tan F, Cai W, Li Y, Cao Y. 2019 Controllable synthesis of pomelo peel-based aerogel and its application in adsorption of oil/organic pollutants. R. Soc. open sci. 6: 181823. http://dx.doi.org/10.1098/rsos.181823 Received: 26 October 2018 Accepted: 3 January 2019 Subject Category: Chemistry Subject Areas: materials science Keywords: sponge aerogels, pomelo peel, silanization, oil-adsorption, recyclability Author for correspondence: Fengzhi Tan e-mail: fz_tan@126.com; tanfz@dlpu.edu.cn royalsocietypublishing.org/journal/rsos Guangyu Shi1, Yizhu Qian2, Fengzhi Tan1, Weijie Cai1, Yuan Li1 and Yafeng Cao1 http://dx.doi.org/10.1098/rsos.181823 1School of Light Industry and Chemical Engineering, Dalian polytechnic university, No. 1, Qinggongyuan, Ganjingzi District, Dalian 116034, People’s Republic of China 1School of Light Industry and Chemical Engineering, Dalian polytechnic g y g g, p y university, No. 1, Qinggongyuan, Ganjingzi District, Dalian 116034, People’s Republic of China Received: 26 October 2018 Accepted: 3 January 2019 Subject Category: Chemistry Subject Areas: materials science Keywords: sponge aerogels, pomelo peel, silanization, oil-adsorption, recyclability Author for correspondence: Fengzhi Tan e-mail: fz_tan@126.com; tanfz@dlpu.edu.cn 2Dalian No. 24 high school. No. 217, Jiefang Road, Zhongshan District, Dalian 116001, People’s Republic of China 2Dalian No. 24 high school. No. 217, Jiefang Road, Zhongshan District, Dalian 116001, People’s Republic of China 1School of Light Industry and Chemical Engineering, Dalian polytechnic university, No. 1, Qinggongyuan, Ganjingzi District, Dalian 116034, People’s Republic of China FT, 0000-0001-8715-2442 Oil/water separation is a field of high significance as it might efficiently resolve the contamination of industrial oily wastewater and other oil/water pollution. In this paper, an environmentally-friendly hydrophobic aerogel with high porosity and low density was successfully synthesized with renewable pomelo peels (PPs) as precursors. Typically, a series of sponge aerogels (HPSA-0, HPSA-1 and HPSA-2) were facilely prepared via high-speed dispersion, freeze-drying and silanization with methyltrimethoxysilane. Indeed, the physical properties of aerogel such as density and pore diameter could be tailored by different additives (filter paper fibre and polyvinyl alcohol). Hence, their physico-chemical properties including internal morphology and chemical structure were characterized in detail by Fourier transform infrared, Brunauer–Emmett–Teller, X-ray diffraction, scanning electron microscope, Thermal gravimetric analyzer (TG) etc. Moreover, the adsorption capacity was further determined and the results revealed that the PP-based aerogels presented excellent adsorption performance for a wide range of oil products and/ or organic solvents (crude oil 49.8 g g21, soya bean oil 62.3 g g21, chloroform 71.3 g g21 etc.). The corresponding cyclic tests showed the absorption capacity decreased slightly from 94.66% to 93.82% after 10 consecutive cycles, indicating a high recyclability. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 Nowadays, with the rapid development of the chemical industry and offshore exploitation, oily wastewater has become an extremely common pollutant, which can cause serious damage to the water environment and ecological systems [1,2]. In addition, the frequent oil leakages and organic pollutants spillage during marine transportation or crude oil production are potentially catastrophic to marine environments, and also a great waste of valuable natural resources [3]. To address the issues arising from oil spills, organic pollutants and industrial oily wastewater, various techniques have been developed for oily wastewater treatment, such as in situ burning, biodegradation, flotation, physical adsorption etc. Among these techniques, physical adsorption by porous materials has been proved to be very promising and efficient in the removal of oil/organic pollutants [4,5]. Many porous absorbent materials have been prepared and deeply investigated like activated carbon [6], polypropylene non-woven fabric [7], sponges [8], propylene foam [9] etc. However, such materials generally suffer from low capacities/output, high cost, disbiodegradation and lower separation selectivity because of the simultaneous adsorption of oil and water etc. [10,11]. Hence, it is critical to develop a novel type of sorbent which possesses the advantages of sorption capacity, selectivity, low cost, recyclability and environmental friendliness [12,13]. Cellulose is one of the abundant raw materials in nature. As a ‘young’ third generation of aerogel materials, cellulose-based aerogels seem to be one of the most fascinating natural oil sorbents after the appropriate modifications [14]. To date, various kinds of natural fibres have been reported as a precursor, such as kapok fibre [15], cotton [16], corn straw [17], banana peels [18], softwood [19] etc. It was accepted that the oil sorption capability of cellulose-based aerogel was attributed to its low density, high porosity and the three-dimensional network structure. In order to achieve excellent oil/water selectivity, most of the cellulose-based aerogels were further modified via esterification, nanocoating, carbonization, silanization and so on. Wang et al. fabricated a kind of superhydrophobic cellulose sponge for oil/water separation using a one-pot hydrothermal technique and subsequent hydrophobic modification with dodecanethiol. The as-prepared kapok fibre showed good sorption capacity up to 4–70 g g21 for oil and organic solvents [15]. Li et al. prepared a novel coconut peat powder-based magnetic sorbent for selective oil–water separation. The product was modified by low-surface-energy octadecylamine, and exhibited high recyclability, with a loss of less than 15.32% in oil absorption capacity after 11 absorption–desorption cycles [20]. Additionally, Lu et al. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 reported a superhydrophobic magnetic ethyl cellulose (EC) sponge with low density (less than 18 mg cm23) and high porosity (greater than 98%) [21]. Furthermore, the sponge showed excellent separation efficiency and good absorption capacity for oils and organic solvents (37–71 g g21). However, compared with natural fibre, to the best of our knowledge there has been little research on the aerogel-based citrus fruits peel. It was reported that the global production of citrus fruits amounts to over 100 million tons since 2010 [22,23]. A large amount of citrus waste was generated, which accounted for almost 50wt% of the fruit weight. Pomelo peel (PP), as an abundant pectin-rich fruit waste of Southeastern Asia, possessed ca 40 wt% of pomelo. Unfortunately, most of the PP was directly discarded in landfills, which resulted in resource waste and environmental pollution [24]. It should be noted that PP contained rich plant fibre, insoluble polysaccharides (e.g. cellulose, hemicellulose, pectin) and ligin [25]. This chemical composition rendered the PP a promising material for the cellulose-based aerogel. Furthermore, as a low-cost natural absorbent, PP presented high oil sorption capacity due to its porous structure, especially macropores (2–20 mm), which might provide sufficient space to adsorb oil and organic pollutants [24]. In this work, the PP derived hydrophobic porous aerogel was prepared by a facile and environmentally-friendly method [26,27]. The textural features of the investigated materials were determined and systemically discussed by various characterization techniques (Fourier transform infrared (FTIR), Brunauer–Emmett–Teller (BET), X-ray diffraction (XRD), scanning electron microscope (SEM), TG). More importantly, the as-prepared PP-based aerogel showed remarkably high recyclability (greater than 40 g g21, for diesel oil) even after 10 cycles, which made it an attractive candidate for use in efficient oil spill clean-up and water purification. Author for correspondence: This article has been edited by the Royal Society of Chemistry, including the commissioning, peer review process and editorial aspects up to the point of acceptance. Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare. c.4375658. Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9.figshare. & 2019 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. 1. Introduction 2 2.2. Pomelo peel pretreatment sos R. Soc. open sci. 6: 181823 A certain amount of grounded PP powder (80–100 meshes) was immersed into a solution of 4 wt% sodium hydroxide with a solid–liquid ratio of 1 : 15 and the mixture was kept at 408C for 6 h under magnetic stirring. After that, the product was filtered and thoroughly washed in deionized water until the filtrate was colourless and transparent (pH ¼ 7). The obtained material was dried at 608C for 24 h. 2.4. Preparation of hydrophobic sponge aerogels (HPSA-0, HPSA-1 and HPSA-2) In detail, the aerogel PSA-0 was placed in a glass bottle. An amount of 1.5 ml MTMS and 1.5 ml deionized water were inserted into a beaker, respectively. After that, two small beakers were placed inside the above-mentioned 250 ml bottle. Then, the glass bottle was tightly sealed and placed in an oven at 808C for 12 h. Finally, the silanized sample was placed in a vacuum oven at 608C for 24 h to remove the unreacted silane. The achieved samples were labelled as HPSA-0. The HPSA-1 and HPSA-2 were prepared following the same procedure as with HPSA-0. 2.3. Preparation of pomelo peel-based sponge aerogels (PSA-0, PSA-1 and PSA-2) Firstly, 1 g pretreated PP powder was added to the beaker containing 99 g H2O. The resulting mixture was continuously stirred at room temperature (25+ 18C) for 0.5 h using a high shear dispersing emulsifier. Then, the suspension was frozen for 24 h under 2188C, and subsequently freeze-dried at a condenser temperature of 2558C under vacuum for 24 h to obtain sponge sample PSA-0. The aerogel PSA-1 and PSA-2 were prepared using the same method as PSA-0. The pretreated PP powder was mixed with filter paper or PVA with a mass ratio of 7 : 3 to prepare sample PSA-1 and PSA-2, respectively. 2.1. Materials PP was provided by local WalMart as solid waste. The yellow skin of PP was chipped off, then the sponge residue was thoroughly washed with deionized water to remove all the dirty particles. After that, PP was grounded into granules after drying in an oven at 1008C for 10 h. The PP powder was stored in a glass desiccator for further use. Polyvinyl alcohol (PVA) and methyltrimethoxysilane (MTMS) were purchased from Aladdin Industrial Corporation. Ethanol, sodium hydroxide (NaOH), N,N- dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were purchased from the Kermel Chemical Reagent Co., Ltd (Tianjin, China). Diesel, crude oil and pump oil were supplied by local Sinopec Group (Dalian, China). Soya bean oil, peanut oil, sunflower oil etc. were purchased from a local market in Dalian, China. All chemicals were of analytical grade, and all chemicals were used without further purification. royalsocietypublishing. 3 3 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 2.7. Oil and organic solvent absorption capacity To assess the absorption capacity of the sponge aerogels, a series of oils and organic solvent were selected. An appropriate amount of oil or organic solvent was poured into a beaker, then the aerogel sample (initial weight was m0) was immersed in oil. The sample was taken out at intervals of 5 s and the oil located at material surface was carefully scraped with filter paper. The sample weight (m) was recorded after the test. This process was repeated several times until the aerogel sample reached adsorption equilibrium. The oil absorption capacity Q and oil absorption rate V of aerogels were quantified by formulas 1.3 and 1.4, respectively [31–33]. Q ¼ m m0 m0 , ð2:3Þ ð2:3Þ where Q, m and m0 represent the oil absorption capacity of the aerogel (g g21), the weight of aerogel after absorption (gram) and the weight of aerogel before absorption (gram). V ¼ Q t , V ¼ Q t , ð2:4Þ ð2:4Þ represents the oil absorption rate of samples (g s21). where V represents the oil absorption rate of samples (g s21). where V represents the oil absorption rate of samples (g s21). 2.6. Morphology characterization 4 The morphology of the samples was observed using a scanning electron microscope (SEM, JSM-6460LV) equipped with energy dispersive X-ray spectroscopy (EDS). The crystallinity of HPSA-0 (HPSA-1, HPSA- 2) was analysed by X-ray diffraction (XRD, SmartLab from Rigaku Co.). Water contact angles (WCAS) were measured by a contact angle system (KSV CM20, Finland) under ambient conditions. Fourier transform infrared (FTIR) spectroscopy spectra were recorded using a Bruker Tensor 27 spectrometer. And N2 adsorption–desorption experiments were carried out at 77 K using a SA 3100 apparature (Beckmann Coulter) to study the textural features of the samples (specific surface area, pore diameter/ volume). Thermal gravimetric analysis was conducted on a Thermo plus TG-8120 apparatus. Typically, 50 mg of the aerogel samples (HPSA-0, HPSA-1 and HPSA-2) were heated from 208C to 7008C at a rate of 108C min21 under a N2 flow 50 ml min21. 2.8. Reusability In order to evaluate the reusability of the aerogels, 10 consecutive cycles of absorption and regeneration were carried out. The saturated sample was immersed into 200 ml of anhydrous ethanol for 0.5 h at 708C. The oil dissolved in anhydrous ethanol completely, then it was separated from the obtained mixture by vacuum distillation. The anhydrous ethanol was recycled. 2.5. Density and porosity The apparent density of aerogel (r) was calculated based on the following equation: r ¼ m ð2pDHÞ=4 , ð2:1Þ ð2:1Þ r ¼ m ð2pDHÞ=4 , where r represents the density of aerogel (g cm23), m is the quality of the aerogel (gram), D is the diameter of cross section of aerogels (centimetre) and H is the height of the aerogel (centimetre). where r represents the density of aerogel (g cm23), m is the quality of the aerogel (gram), D is the diameter of cross section of aerogels (centimetre) and H is the height of the aerogel (centimetre). g g g Aerogel porosity was calculated by the aerogel apparent density r and skeleton density r0 using the following equation [28,29]: P ¼ 1 r r0 100%, ð2:2Þ ð2:2Þ where P is the porosity of aerogels (%), r is the apparent density of aerogel (g cm23) and r0 is the aerogel skeleton density (g cm23). where P is the porosity of aerogels (%), r is the apparent density of aerogel (g cm23) and r0 is the aerogel skeleton density (g cm23). Based on data in the literature, we used the density of cellulose (1.500 g cm23) and PVA (1.267 g cm23) as the solid density [30], so, r0(HPSA-0) ¼ 1.500 g cm23, r0(HPSA-1) ¼ 1.500 g cm23 and r0(HPSA-2) ¼ 1.430 g cm23 (the mass ratio of PP and PVA was 3 : 7). 3.1. Morphologies of the sponge aerogel The morphologies of the sponge aerogels before and after silanization were observed via SEM images. As shown in figure 1a–c, the aerogel surface was flaked with a small number of interspaces, and the macropores and micropores made up with interconnected PP powder particles and filter paper fibres or PVA were clearly observed in the aerogel cross section. These interspaces greatly enhanced the adsorption efficiency of aerogels. [34,35] Compared with raw PP (electronic supplementary material, figure S1), the aerogels showed a multilevel and an interconnected three-dimensional pore structure which was interweaved by flexible sheets. Noticeably, the results suggested that a large number of the tunnel structures were distributed in three aerogel samples, which made it easy for oil to enter the aerogels. In addition, the aerogels modified with MTMS (figure 1d,e) possessed an obviously layered structure, the surface shape was unchanged and the pore size was kept constant, which could reach up to 10–1000 mm. Unlike HPSA-1(PP/filter paper), a large amount of filamentous fibre structure in HPSA- 2(PP/PVA) was determined. Furthermore, as a water-soluble linear polymer, PVA presented its adhesive ability between PP powder particles, leading to the aerogel surface smoother than HPSA-1. However, it still possessed a layered structure and multi-level pore structure. (a) (c) (d) (b) (e) ( f ) Figure 1. SEM images of: (a) PSA-0, (b) PSA-1, (c) PSA-2, (d) HPSA-0, (e) HPSA-1 and (f ) HPSA-2. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181 5 (a) (c) (d) (b) (e) ( f ) Figure 1. SEM images of: (a) PSA-0, (b) PSA-1, (c) PSA-2, (d) HPSA-0, (e) HPSA-1 and (f ) HPSA-2. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 1 5 (a) (c) (d) (b) (e) ( f ) 5 (a) royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 5 (c) (b) (a) royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 (e) (d) ( f ) Figure 1. SEM images of: (a) PSA-0, (b) PSA-1, (c) PSA-2, (d) HPSA-0, (e) HPSA-1 and (f ) HPSA-2. Figure 1. SEM images of: (a) PSA-0, (b) PSA-1, (c) PSA-2, (d) HPSA-0, (e) HPSA-1 and (f ) HPSA-2. Table 1. Density, porosity and speciﬁc surface areas of pomelo peel-based sponge aerogels. ble 1. Density, porosity and speciﬁc surface areas of pomelo peel-based sponge aerogels. 3.1. Morphologies of the sponge aerogel aerogel r (g cm23) r0 (g cm23) porosity (%) BET surface area (m2 g21) PSA-0 0.0306 1.500 98.0 11.6 PSA-1 0.0214 1.500 98.6 16.0 PSA-2 0.0204 1.430 98.6 7.0 The density, porosity and specific surface areas of PP-based sponge aerogels were listed in table 1. It revealed that the lowest density was ca 0.0204 g cm23 and its porosity could be as high as 98.6%. All aerogel samples exhibited ultralight and highly porous (approx. 98%) features. The specific surface area was measured using the BET method. Among three aerogel samples, the surface area of HSPA-1 was larger than that of the others, probably due to the loose and porous structure of filter paper fibre. This result indicated that filter paper fibre was favourable for the formation of the multi-stage pore structure of aerogel. On the other hand, the microstructure of aerogel could be adjusted by the different additives, which might present a facile method of controlling material structure [36]. g p g Nitrogen adsorption–desorption tests have also been conducted to determine textural properties of the HPSA samples. Figure 2 shows the nitrogen adsorption–desorption isotherms for HPSA aerogels prepared with a different doping material. It could be observed that the HPSA-2 presented a small hysteresis loop at relative pressure from 0.4 to 0.6, indicating the mesoporous structure of the HPSA-2 sample. Similarly, the pore types of HPSA-0 samples were mainly mesoporous, which was in line with the results of pore size distribution curve (figure 2d,e). 3.2. Chemical component of aerogels 6: 181823 6 0 –2 0 2 4 6 adsorbed volume (cm3 g–1) 8 10 HPSA-0 adsorption curve desorption curve 12 14 0.2 0.4 relative pressure (P/P0) 0.6 0.8 1.0 (a) adsorbed volume (cm3 g–1) adsorbed volume (cm3 g–1) (d) 0 1 2 3 4 5 6 7 adsorbed volume (cm3 g–1) 0 relative pressure (P/P0) 0 0.2 0.4 0.6 0.8 1.0 HPSA-2 adsorption curve desorption curve (c) 0 0.002 0.004 0.006 0.008 0.010 0.012 dV/dD 0 5 10 0 pore diameter (nm) 15 20 25 30 HPSA-0 (d) os R. Soc. open sci. 6: 181823 dV/dD 0 0.005 0.010 0.015 0.020 dV/dD 0 pore diameter (nm) 0 5 10 15 20 HPSA-1 (e) 0 ( f ) 0 0.002 0.004 0.006 0.008 0.010 0.012 0.014 dV/dD pore diameter (nm) 0 5 10 15 20 25 HPSA-2 ( f ) Figure 2. The N2 adsorption–desorption isotherms of: (a) HPSA-0, (b) HPSA-1, (c) HPSA-2; the pore size distribution curves of: (d) HPSA-0, (e) HPSA-1 and (f ) HPSA-2. Table 2 shows the element component of raw PP, PSA-1, PSA-2 and silanized modified aerogel (HPSA-1, HPSA-2). In fact, there was no silicon element in the raw PP and alkali pretreatment PP, and silicon element was detected in the sponge aerogel (1.89 wt% and 1.02 wt%). The above results showed that sponge aerogel samples were successfully modified by MTMS during the fabrication of hydrophobic aerogel. It was known that crystallinity played an important role in the mechanical properties of aerogels. Figure 4 shows the XRD patterns of the modified aerogel samples (HPSA-0, HPSA-1 and HPSA-2) [30]. The results showed that all the samples presented a peak at around 2u ¼ 238 (002 peak) [35]. It could be seen from the XRD pattern that the HPSA-0 and HPSA-1 had an intense diffraction peak at 2u ¼ 22.608. This peak corresponded to the graphitic carbon structure of raw PP. The peak for HPSA-2 became narrow and stronger, suggesting that the crystallinity of HPSA-2 was higher than other samples. 3.2. Chemical component of aerogels FTIR and EDX analysis were used to identify the surface chemical component of aerogel samples, and the results are shown in figure 3 and table 2, respectively. As shown in figure 3, the dominant peaks in the 34 271 036 and 902 cm21 were ascribed to the stretching vibrations of –OH, C–O–C [28] and b-glycosidic linkage, respectively. It indicated that the surfaces of the raw PP were rich in hydroxyl, carbonyl, carboxyl and aromatic groups [37]. Additionally, the bands at 750 (d (C2H)) and 2980 cm21 (n (C2H)) were assigned to the vibration’s characteristic of the CH3 in silane. Besides, Si2CH3 bending vibration located at 1250–1390 cm21 was also noted. The characteristic peaks of Si2O2Si bonds in the siloxane were overlapped by the C2O bonds. The results revealed that MTMS could covalently form a strong cross-linked Si-O-Si monolayer coating under a modified reaction condition [38]. After silanized modification, the 2OH groups were displaced by O-Si(CH3)3 groups from MTMS resulting in the hydrophobic property of the aerogels [29]. 0 –2 0 0 1 2 3 4 5 6 7 0 0 0 0.002 0.004 0.005 0.010 0.015 0.020 0.006 0.008 0.010 0.012 0 0.002 0.004 0.006 0.008 0.010 0.012 0.014 2 2 4 4 6 6 adsorbed volume (cm3 g–1) adsorbed volume (cm3 g–1) dV/dD dV/dD dV/dD adsorbed volume (cm3 g–1) 8 8 10 10 HPSA-0 adsorption curve desorption curve 12 14 0.2 0.4 relative pressure (P/P0) relative pressure (P/P0) 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 5 10 relative pressure (P/P0) pore diameter (nm) pore diameter (nm) pore diameter (nm) 15 20 0 5 10 15 20 25 0 5 10 15 20 25 30 HPSA-1 HPSA-0 HPSA-2 HPSA-1 HPSA-2 adsorption curve desorption curve (e) ( f ) (b) (a) (c) (d) Figure 2. The N2 adsorption–desorption isotherms of: (a) HPSA-0, (b) HPSA-1, (c) HPSA-2; the pore size distribution curves of: (d) HPSA-0, (e) HPSA-1 and (f ) HPSA-2. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 6 0 2 4 6 adsorbed volume (cm3 g–1) 8 10 0 0.2 0.4 0.6 0.8 1.0 relative pressure (P/P0) HPSA-1 (b) 6 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 3.3. Thermal properties of aerogels This result indicated that the higher temperature of thermal decomposition was attributed to the removal of lignin from the fibres and the addition of filter paper in aerogel. However, the thermal stability of HPSA-2 was lower in comparison with the others, probably because of the decomposition of PVA. According to the literature, linear PVA would be degraded and produce low molecular weight products when the temperature was greater than 2648C [39]. The thermal stability of PVA can be improved mainly by increasing its degree of alcoholysis, thereby the PVA with 99% alcoholysis was selected as the starting material. clearly observed that only a little weight loss occurred in the range of 25–1008C owing to the evaporation of the adsorbed water. The main weight loss started at 227, 225 and 2128C for HPSA-0, HPSA-1 and HPSA-2, respectively in TGA. The degradation process reached its maximum at 313, 314 and 2988C, respectively as displayed in DTG. It is worth mentioning that the thermal stability of HPSA-0 was similar to HPSA-1. This result indicated that the higher temperature of thermal decomposition was attributed to the removal of lignin from the fibres and the addition of filter paper in aerogel. However, the thermal stability of HPSA-2 was lower in comparison with the others, probably because of the decomposition of PVA. According to the literature, linear PVA would be degraded and produce low molecular weight products when the temperature was greater than 2648C [39]. The thermal stability of PVA can be improved mainly by increasing its degree of alcoholysis, thereby the PVA with 99% alcoholysis was selected as the starting material. 3.3. Thermal properties of aerogels Considering the heat insulation application, thermal stability of aerogels is of great importance [36]. The results of thermogravimetry (TGA) and derivative thermogravimetry (DTG) are shown in figure 5. It was (a) (b) (c) wavenumbers (cm–1) 4000 3500 3000 2500 2000 1500 1000 500 wavenumbers (cm–1) 4000 3500 3000 2500 2000 1500 1000 500 wavenumbers (cm–1) 4000 3500 3000 2500 2000 PSA-0 PSA-1 PSA-2 HPSA-0 HPSA-1 HPSA-2 PP 625 1061 1630 890 1289 1626 1316 2905 1500 1000 500 Figure 3. FTIR spectra of (a) PSA-0 and HPSA-0, (b) PSA-1 and HPSA-1, (c) PSA-2 and HPSA-2. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 7 (a) (b) wavenumbers (cm–1) 4000 3500 3000 2500 2000 1500 1000 500 wavenumbers (cm–1) 4000 3500 3000 2500 2000 PSA-0 PSA-1 HPSA-0 HPSA-1 PP 625 1061 1630 890 1289 2905 1500 1000 500 royalsocietypublishing.org/journal/rsos R. 7 (a) wavenumbers (cm–1) 4000 3500 3000 2500 2000 PSA-0 HPSA-0 PP 625 1061 1630 2905 1500 1000 500 (b) wavenumbers (cm–1) 4000 3500 3000 2500 2000 1500 1000 500 PSA-1 HPSA-1 890 1289 royalsocietypublishing.org/journal/rsos 7 7 (b) royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 wavenumbers (cm–1) (c) wavenumbers (cm–1) 4000 3500 3000 2500 2000 1500 1000 500 PSA-2 HPSA-2 1626 1316 Figure 3. FTIR spectra of (a) PSA-0 and HPSA-0, (b) PSA-1 and HPSA-1, (c) PSA-2 and HPSA-2. (c) Figure 3. FTIR spectra of (a) PSA-0 and HPSA-0, (b) PSA-1 and HPSA-1, (c) PSA-2 and HPSA-2. Figure 3. FTIR spectra of (a) PSA-0 and HPSA-0, (b) PSA-1 and HPSA-1, (c) PSA-2 and HPSA-2. Table 2. EDS of PP, PP (pretreatment), PSA-1, HPSA-1, PSA-2 and HPSA-2. element PP PP (pretreatment) PSA-1 HPSA-1 PSA-2 HPSA-2 C (wt%) 63.87 62.82 54.99 57.37 47.8 51.44 O (wt%) 26.6 36.22 42.72 39.05 48.55 44.31 S (wt%) 0.44 0.48 0 0 0.21 0.2 Si (wt%) 0 0 0 1.89 0 1.02 clearly observed that only a little weight loss occurred in the range of 25–1008C owing to the evaporation of the adsorbed water. The main weight loss started at 227, 225 and 2128C for HPSA-0, HPSA-1 and HPSA-2, respectively in TGA. The degradation process reached its maximum at 313, 314 and 2988C, respectively as displayed in DTG. It is worth mentioning that the thermal stability of HPSA-0 was similar to HPSA-1. 3.4. Wettability Desirable oil/water selectivity is a critical property for oil absorbents [40,41]. To determine the effect of silanized modification on the hydrophobicity of aerogel, WCA was measured to check the 10 0 1000 2000 3000 intensity 4000 5000 HPSA-2 HPSA-1 HPSA-0 20 30 40 2q (°) hydrophobicity. When the contact angle was higher than 908, the material was classified as hydrophobic [42]. As expected, the MTMS-modified aerogels became hydrophobic after silylation. Noticeably, water formed droplets on the aerogel surface and maintained its spherical shape with a contact angle of 132.758 (figure 6). This result could be ascribed to successful silanizing modification. As shown in figure 7a, water (coloured with methylene blue) formed spherical droplets on the surface of HPSA-1, while kerosene (coloured with Sudan 3) was rapidly and completely absorbed. This revealed that the treatment of the sample with silanizing reagent showed the characteristics of hydrophobicity 100 0 20 40 60 weight (%) deriv. weight (%/°C) 80 100 227 212 298 225 314 313 0 0.8 0.6 0.4 0.2 1.0 200 300 400 temperature (°C) 500 600 100 200 300 400 temperature (°C) 500 600 HPSA-2 HPSA-1 HPSA-0 (b) (a) Figure 5. TGA (a) and DTG (b) thermograms of HPSA-0, HPSA-1 and HPSA-2. 10 0 1000 2000 3000 intensity 4000 5000 HPSA-2 HPSA-1 HPSA-0 20 30 40 2q (°) Figure 4. XRD patterns of HPSA-0, HPSA-1 and HPSA-2. WCA = 104.61° WCA = 113.49° WCA = 132.75° (a) (c) (b) Figure 6. Water contact angle of the (a) HPSA-0, (b) HPSA-1 and (c) HPSA-2. y yp g g/j / p 8 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 8 Figure 4. XRD patterns of HPSA-0, HPSA-1 and HPSA-2. 100 0 20 40 60 weight (%) deriv. weight (%/°C) 80 100 227 212 298 225 314 313 0 0.8 0.6 0.4 0.2 1.0 200 300 400 temperature (°C) 500 600 100 200 300 400 temperature (°C) 500 600 HPSA-2 HPSA-1 HPSA-0 (b) (a) Figure 5. TGA (a) and DTG (b) thermograms of HPSA-0, HPSA-1 and HPSA-2. 100 0 20 40 60 weight (%) deriv. weight (%/°C) 80 100 227 212 298 225 314 313 0 0.8 0.6 0.4 0.2 1.0 200 300 400 temperature (°C) 500 600 100 200 300 400 temperature (°C) 500 600 HPSA-2 HPSA-1 HPSA-0 (b) (a) Figure 5. TGA (a) and DTG (b) thermograms of HPSA-0, HPSA-1 and HPSA-2. deriv. 3.4. Wettability (a) Absorbed behaviour of the HPSA-1 (water and oil were coloured by methylene blue and Sudan 3, respectively), (b) behaviour of HPSA-2 in water. (b) (a) (a) (c) (d) (b) Figure 8. (a–d) Oil absorption process of the modified aerogel HPSA-2. R. Soc. open sci. 6: 181823 (b) (a) (c) (d) HPSA-2. (d) (c) Figure 8. (a–d) Oil absorption process of the modified aerogel HPSA-2. 3.4. Wettability weight (%/°C) 298 314 313 0 0.8 0.6 0.4 0.2 1.0 100 200 300 400 temperature (°C) 500 600 (b) Figure 5. TGA (a) and DTG (b) thermograms of HPSA-0, HPSA-1 and HPSA-2. Figure 5. TGA (a) and DTG (b) thermograms of HPSA-0, HPSA-1 and HPSA-2. WCA = 104.61° WCA = 113.49° WCA = 132.75° (a) (c) (b) Figure 6. Water contact angle of the (a) HPSA-0, (b) HPSA-1 and (c) HPSA-2. WCA = 132.75° (c) WCA = 104.61° WCA = 113.49° (a) (c (b) Figure 6. Water contact angle of the (a) HPSA-0, (b) HPSA-1 and (c) HPSA-2. WCA = 113.49° (b) HPSA (b) HPSA d ( ) HPSA (b) Figure 6. Water contact angle of the (a) HPSA-0, (b) HPSA-1 and (c) HPSA-2. hydrophobicity. When the contact angle was higher than 908, the material was classified as hydrophobic [42]. As expected, the MTMS-modified aerogels became hydrophobic after silylation. Noticeably, water formed droplets on the aerogel surface and maintained its spherical shape with a contact angle of 132.758 (figure 6). This result could be ascribed to successful silanizing modification. hydrophobicity. When the contact angle was higher than 908, the material was classified as hydrophobic [42]. As expected, the MTMS-modified aerogels became hydrophobic after silylation. Noticeably, water formed droplets on the aerogel surface and maintained its spherical shape with a contact angle of 132.758 (figure 6). This result could be ascribed to successful silanizing modification. As shown in figure 7a, water (coloured with methylene blue) formed spherical droplets on the surface of HPSA-1, while kerosene (coloured with Sudan 3) was rapidly and completely absorbed. This revealed that the treatment of the sample with silanizing reagent showed the characteristics of hydrophobicity [43]. In figure 7b, the lumpy sponge aerogels suspended on the water surface also elucidated the hydrophobicity features of the modified sponge aerogels. The excellent hydrophobic performance of modified aerogels could be used in effective oil/water separation. MTMS-modified PP-based aerogel, due to its low density, high porosity and surface hydrophobicity, may be a promising candidate for the elimination of oils and organic pollutants. water oil (a) (b) Figure 7. (a) Absorbed behaviour of the HPSA-1 (water and oil were coloured by methylene blue and Sudan 3, respectively), (b) behaviour of HPSA-2 in water. royalsocietypublishing.org/journal/rsos 9 (b) water oil (a) (b) (a) royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 Figure 7. 3.5. Absorption capacity Oil leakages lead to serious pollution of the water environment, and the treatment of oil and organic pollutants from water has attracted considerable academic and commercial interest [44]. Sorption processes of aerogels occurred because of their micro and microporous structures, and also the inter- and intra-fibre interactions of the aerogel [45]. The PP-based aerogels possessed a hierarchical pore structure, as already discussed by SEM, BET analysis, so the aerogel can absorb oil rapidly. As shown in figure 8a–d, the kerosene (stained with Sudan 3 dye) was put into the beaker containing water, and then a modified sponge aerogel HPSA-2 was added into the beaker. The dyed kerosene was absorbed by the modified aerogel within 1 min, then the water in the beaker was clean. The result indicated that the modified samples possessed excellent adsorption capacity for oils and organic pollutants. p p p p y g p Figure 9a–c introduces the adsorption capacity of HPSA-0, HPSA-1 and HPSA-2 samples for some commonly used oils/organic solvents (crude oil, diesel oil, kerosene, engine oil, DMF, DMSO, ethanol, n-hexane etc.). The oil’s adherence to the aerogel’s surface happens mainly due to the intramolecular interaction and van der Waals forces [46]. The different sorption capacity was attributed to the density, the molecular dimension, the surface tension and hydrophobicity of the organic solvents and oils [41]. As could be clearly observed, the adsorption capacity of HPSA-1 and HPSA-2 for various oils/organic solvents was better than that of HPSA-0. It was ascribed to a satisfied network structure that was formed by filter paper fibres (PVA) and PP closed interaction. Furthermore, PVA will provide royalsocietypublishing.org/journal/rsos R. Soc. open sci. 3.5. Absorption capacity 6: 181823 10 0 10 20 30 crude oil diesel oil soya bean oil pump oil methylbenzene DMSO peanut oil sunflower oil pumpkin seed oil motor oil corn germ oil kerosene ethyl alcohol methyl alcohol ispropyl alcohol n-hexane chloroform DMF crude oil diesel oil soya bean oil pump oil methylbenzene DMSO peanut oil sunflower oil pumpkin seed oil motor oil corn germ oil kerosene ethyl alcohol methyl alcohol isopropyl alcohol n-hexane chloroform DMF crude oil diesel oil soya bean oil pump oil methylbenzene DMSO peanut oil sunflower oil pumpkin seed oil motor oil corn germ oil kerosene ethyl alcohol methyl alcohol ispropyl alcohol n-hexane chloroform DMF 40 50 60 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 absorption capacity (g g–1) absorption capacity (g g–1) absorption capacity (g g–1) absorption capacity (g g–1) 70 80 –5 –10 0 10 20 30 40 50 60 70 0 5 10 15 time (s) 20 25 30 40 35 HPSA-2 HPSA-1 HPSA-0 HPSA-2 HPSA-1 HPSA-0 (b) (a) (c) (d) Figure 9. Absorption capacities for various oils and organic solvents (a) HPSA-0, (b) HPSA-1 and (c) HPSA-2. (d) Absorption rate curves of the HPSA-0, HPSA-1 and HPSA-2. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 10 crude oil diesel oil soya bean oil pump oil methylbenzene DMSO peanut oil sunflower oil pumpkin seed oil motor oil corn germ oil kerosene ethyl alcohol methyl alcohol ispropyl alcohol n-hexane chloroform DMF 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 absorption capacity (g g–1) HPSA-1 (b) 10 0 10 20 30 crude oil diesel oil soya bean oil pump oil methylbenzene DMSO peanut oil sunflower oil pumpkin seed oil motor oil corn germ oil kerosene ethyl alcohol methyl alcohol ispropyl alcohol n-hexane chloroform DMF 40 50 60 0 10 20 30 40 50 60 absorption capacity (g g–1) HPSA-0 (a) rsos R. Soc. open sci. 3.5. Absorption capacity 6: 181823 absorption capacity (g g–1) absorption capacity (g g–1) p p y (g g ) –5 –10 0 10 20 30 40 50 60 70 0 5 10 15 time (s) 20 25 30 40 35 HPSA-2 HPSA-1 HPSA-0 (d) crude oil diesel oil soya bean oil pump oil methylbenzene DMSO peanut oil sunflower oil pumpkin seed oil motor oil corn germ oil kerosene ethyl alcohol methyl alcohol isopropyl alcohol n-hexane chloroform DMF 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 absorption capacity (g g–1) 70 80 HPSA-2 (c) (c) (d) Figure 9. Absorption capacities for various oils and organic solvents (a) HPSA-0, (b) HPSA-1 and (c) HPSA-2. (d) Absorption rate curves of the HPSA-0, HPSA-1 and HPSA-2. Table 3. The adsorption capacity and adsorption rate of sponge aerogels on soya bean oil. Table 3. The adsorption capacity and adsorption rate of sponge aerogels on soya bean oil. Table 3. The adsorption capacity and adsorption rate of sponge aerogels on soya bean oil. additive mass ratios of additive-pomelo peel absorption capacity (g g21) absorption rate (g s21) ﬁlter paper ﬁbre 2 : 8 53.4 3.56 3 : 7 56.8 3.79 6 : 4 52.4 3.49 5 : 5 45.0 3.00 PVA 2 : 8 61.0 4.07 3 : 7 62.6 4.18 4 : 6 51.0 3.40 5 : 5 51.1 3.41 good properties as toughness and flexibility for composite aerogel. Herein, the absorption capacity of HPSA-2 for chloroform and DMSO reached as high as 71.3 g g21, 51.5 g g21, respectively. In addition, the adsorption capacity of the HPSA-1 sample for pumpkin seed oil and crude oil was 50.5 g g21 and 49.2 g g21, respectively. In conclusion, the adsorption capacity of HPSA-1 and HPSA-2 for most oils and organic solvents was promising and attractive. good properties as toughness and flexibility for composite aerogel. Herein, the absorption capacity of HPSA-2 for chloroform and DMSO reached as high as 71.3 g g21, 51.5 g g21, respectively. In addition, the adsorption capacity of the HPSA-1 sample for pumpkin seed oil and crude oil was 50.5 g g21 and 49.2 g g21, respectively. In conclusion, the adsorption capacity of HPSA-1 and HPSA-2 for most oils and organic solvents was promising and attractive. Figure 9d shows the absorption rate of HPSA-0, HPSA-1 and HPSA-2 samples for soya bean oil. 3.5. Absorption capacity Reusability of the (a) HPSA-0; (b) HPSA-1; (c) HPSA-2. adsorption capacity (g g–1) diesel oil HPSA-1 0 1 2 3 4 5 6 7 8 9 10 11 0 10 20 30 40 50 (b) adsorption capacity (g g–1) diesel oil HPSA-2 0 1 2 3 4 5 cycle number 6 7 8 9 10 11 0 10 20 30 40 50 60 (c) Figure 10. Reusability of the (a) HPSA-0; (b) HPSA-1; (c) HPSA-2. As could be seen from table 3, the synthesized sponge aerogels presented the highest adsorption capacity and/or adsorption rate when the mixed mass ratio was 3:7 regardless of whether PVA or filter paper was used. Compared with the oil absorption capacity of HPSA-0, it was concluded that the filter paper fibre or PVA could significantly increase the aerogel absorption capacity. The additives were favourable to forming a more flexible three-dimensional porous structure. 3.5. Absorption capacity In all cases, the adsorption rate of the three samples reaching saturation was below 20 s. Noticeably, the time for the HPSA-2 sample was as short as 15 s. This illustrated that the adsorption rate of the as-prepared sponge aerogel in this work was quite fast for oil phase and organic solvent. And both the adsorption capacity and adsorption rates greatly depended on the types of oils and organic solvents. could be seen from table 3, the synthesized sponge aerogels presented the highest adsorption ty and/or adsorption rate when the mixed mass ratio was 3:7 regardless of whether PVA or paper was used. Compared with the oil absorption capacity of HPSA-0, it was concluded that 0 1 2 3 4 5 adsorption capacity (g g–1) adsorption capacity (g g–1) adsorption capacity (g g–1) diesel oil HPSA-0 diesel oil HPSA-1 diesel oil HPSA-2 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 cycle number 6 7 8 9 10 11 0 10 20 30 40 50 0 10 20 30 40 50 60 10 5 0 20 30 40 15 25 35 45 (a) (b) (c) 10. Reusability of the (a) HPSA-0; (b) HPSA-1; (c) HPSA-2. 0 1 2 3 4 5 adsorption capacity (g g–1) diesel oil HPSA-0 6 7 8 9 10 11 10 5 0 20 30 40 15 25 35 45 (a) 11 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 11 As could be seen from table 3, the synthesized sponge aerogels presented the highest adsorption capacity and/or adsorption rate when the mixed mass ratio was 3:7 regardless of whether PVA or filter paper was used. Compared with the oil absorption capacity of HPSA-0, it was concluded that the filter paper fibre or PVA could significantly increase the aerogel absorption capacity. The additives were favourable to forming a more flexible three-dimensional porous structure. 0 1 2 3 4 5 adsorpti adsorption capacity (g g–1) adsorption capacity (g g–1) diesel oil HPSA-1 diesel oil HPSA-2 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 cycle number 6 7 8 9 10 11 0 10 20 30 40 50 0 10 20 30 40 50 60 10 5 0 15 (b) (c) Figure 10. References review. Chem. Eng. J. 300, 98–118. (doi:10. 1016/j.cej.2016.04.098) 1. Sabir S. 2015 Approach of cost-effective adsorbents for oil removal from oily water. Crit. Rev. Environ. Sci. Technol. 45, 1916–1945. (doi:10.1080/10643389.2014.1001143) activated carbon: a comparative study. Chem. Eng. J. 108, 179–185. (doi:10.1016/j.cej.2005. 01.016) activated carbon: a comparative study. Chem. Eng. J. 108, 179–185. (doi:10.1016/j.cej.2005. 01.016) activated carbon: a comparative study. Chem. Eng. J. 108, 179–185. (doi:10.1016/j.cej.2005. 01.016) review. Chem. Eng. J. 300, 98–118. (doi:10. 1016/j.cej.2016.04.098) 12 Pan Y Shi K Peng C Wang W Liu Z Ji X 2014 12. 12. Pan Y, Shi K, Peng C, Wang W, Liu Z, Ji X. 2014 Evaluation of hydrophobic polyvinyl-alcohol formaldehyde sponges as absorbents for oil spill. ACS Appl. Mater. Interfaces 6, 8651–8659. (doi:10.1021/am5014634) 7. Ren Y, Guo J, Lu Q, Xu D, Qin J, Yan F. 2018 Polypropylene nonwoven fabric@ poly(ionic liquid)s for switchable oil/water separation, dye absorption and antibacterial applications. ChemSusChem. 11, 1092–1098. (doi:10.1002/ cssc.201702320) 2. Nicholas TC, Christian A, Per TL, Lars W. 2012 Ultra porous nanocellulose aerogels as separation medium for mixtures of oil/water liquids. Cellulose 19, 401–410. (doi:10.1007/ s10570-011-9629-5) 13. Laitinen O, Suopaja¨rvi T, O¨sterberg M, Liimatainen H. 2017 Hydrophobic, superabsorbing aerogels from choline chloride- based deep eutectic solvent pretreated and silylated cellulose nanofibrils for selective oil removal. ACS Appl. Mater. Interfaces 9, 25 029–25 037. (doi:10.1021/acsami.7b06304) 3. Li J, Wan C, Lu Y, Sun Q. 2014 Fabrication of cellulose aerogel from wheat straw with strong absorptive capacity. Front. Agric. Sci. Eng. 1, 46–52 (doi:10.15302/J-FASE- 2014004) 3. Li J, Wan C, Lu Y, Sun Q. 2014 Fabrication of cellulose aerogel from wheat straw with strong absorptive capacity. Front. Agric. Sci. Eng. 1, 46–52 (doi:10.15302/J-FASE- 2014004) 8. Choi SJ, Kwon TH, Im H, Moon DI, Baek DJ, Seol ML, Duarte JP, Choi YK. 2011 A polydimethylsiloxane (PDMS) sponge for the selective absorption of oil from water. ACS Appl. Mater. Interfaces 3, 4552–4556. (doi:10.1021/ am201352w) 14. Karatum O, Steiner SA, Griffin JS, Shi W, Plata DL. 2016 Flexible, mechanically durable aerogel composites for oil capture and recovery. ACS Appl. Mater. Interfaces 8, 215–224. (doi:10. 1021/acsami.5b08439) 4. Zheng Q, Cai Z, Gong S. 2014 Green synthesis of polyvinyl alcohol (PVA)–cellulose nanofibril (CNF) hybrid aerogels and their use as superabsorbents. J. Mater. Chem. A 2, 3110–3118 (doi:10.1039/C3TA14642A) 9. 9. Liu H, Kang Y. 2018 Superhydrophobic and superoleophilic modified EPDM foam rubber fabricated by a facile approach for ail/water separation. Appl. Surf. Sci. 4. Conclusion To sum up, the sponge aerogels were synthesized from the waste PP using a simple and feasible method. The aerogels were characterized by FTIR, SEM, XRD and BET. The results indicated that HPSAs aerogels had an interconnected three-dimensional porous morphology. In order to render the ultralight and porous sponge aerogel hydrophobic, we exposed it to vapour phase deposition of MTMS. After modification, the contact angle at the water/sponge aerogel boundary remained high (WCA ¼ 132.758), indicating a strong hydrophobic character. In addition, the HPSAs aerogels exhibited a promising adsorption capacity (for chloroform, up to 70 g g21), and the as-prepared sponge aerogels could be recycled 10 times, with the adsorption capacity maintained at 94.92%. It is worth mentioning that the main advantage of the investigated material was its simplicity, low cost and environmental- friendliness. This will play a positive role in the development of highly efficient adsorbents for different types of oils and organic solvents. Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material. Authors’ contributions. G.S., Y.Q., F.T. and Y.C. contributed the conception of the study and design of the experiments, G.S. performed the experiments and wrote the paper, Y.Q. and Y. L. contribute reagents and analysis the data, W.C. and F.T. revised the paper. All the authors read and approved the final manuscript. Competing interests. The authors declare no competing interests. Funding. This study was supported by Liaoning Province Ocean and Fishery office (grant no. 201405) an Science and Technology office (grant no. 2015B11NC078). Acknowledgements. Instrument Analysis centre of Dalian Polytechnic University is gratefully acknowledged for all the equipment employed. Acknowledgements. Instrument Analysis centre of Dalian Polytechnic University is gratefully acknowledged for all the equipment employed. 3.6. Reusability Reusability of the oil absorbent was a critical criterion for its potential pilot-scale application [47]. Here, diesel oil was selected as a model adsorbate to determine the circulating life of three aerogel samples. According to figure 10a–c, the adsorption capacity of HPSA-0 samples maintained at 93.82% after 10 cycles of absorption and regeneration, while the capacity of HPSA-1 and HPSA-2 samples remained 94.66% and 94.92%, respectively. As a result, the modified aerogels were highly worthy of being employed several times without considerable reduction in their efficiency, demonstrating their superior reusability. On the other hand, it was important to note that the recycling life for different oils and organic solvents varied. This was probably due to the various properties of the oil and organic solvents themselves. royalsocietypub 12 cycles of absorption and regeneration, while the capacity of HPSA-1 and HPSA-2 samples remained 94.66% and 94.92%, respectively. As a result, the modified aerogels were highly worthy of being employed several times without considerable reduction in their efficiency, demonstrating their superior reusability. On the other hand, it was important to note that the recycling life for different oils and organic solvents varied. This was probably due to the various properties of the oil and organic solvents themselves. y yp 1 12 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 13 17. Li Y, Liu X, Cai W, Cao Y, Sun Y, Tan F. 2018 Preparation of corn straw based spongy aerogel for spillage oil capture. Korean J. Chem. Eng. 35, 1119–1127. (doi:10.1007/s11814-018-0010-3) 39. Pan Y, Wang W, Peng C, Shi K, Luo Y, Ji X. 2014 Novel hydrophobic polyvinyl alcohol– formaldehyde foams for organic solvents absorption and effective separation. RSC Adv. 4, 660–669. (doi:10.1039/C3RA43907K) 28. Bi H, Huang X, Wu X, Cao X, Tan C, Yin Z, Lu X, Sun L, Zhang H. 2014 Carbon microbelt aerogel prepared by waste paper: an efficient and recyclable sorbent for oils and organic solvents. Small 10, 3544–3550. (doi:10.1002/smll. 201303413) 18. Alaa El-Din G, Amer AA, Malsh G, Hussein M. 2018. Study on the use of banana peels for oil spill removal. Alexandria Eng. J. 57, 2061–2068. (doi:10.1016/j.aej.2017.05.020) 19. Galblaub OA, Shaykhiev IG, Stepanova SV, Timirbaeva GR. 2016 Oil spill cleanup of water surface by plant-based sorbents: Russian practices. Process Saf. Environ. Prot. 101, 88–92. (doi:10.1016/j.psep.2015.11.002) 40. Zhang Z, Tingaut P, Rentsch D, Zimmermann T, Se`be G. 2015 Controlled silylation of nanofibrillated cellulose in water: reinforcement of a model polydimethylsiloxane network. ChemSusChem 8, 2681–2690 (doi:10.1002/cssc. 201500525) 29. 29. Liu J, Li P, Chen L, Feng Y, He W, Yan X, Lu¨ X. 2016 Superhydrophilic and underwater superoleophobic modified chitosan-coated mesh for oil/water separation. Surf. Coat. Technol. 307, 171–176. (doi:10.1016/j.surfcoat.2016.08. 052) 20. Li Y, Wang Z, Yang L, Li X, Zhang Y, Lu C. 2017 Coco peat powder as a source of magnetic sorbent for selective oil-water separation. Ind. Crops Prod. 101, 1–10 (doi:10.1016/j.indcrop. 2017.02.040) 41. 41. Cheng Q, Ye D, Chang C, Zhang L. 2017 Facile fabrication of superhydrophilic membranes consisted of fibrous tunicate cellulose nanocrystals for highly efficient oil/water separation. J. Membr. Sci. 525, 1–8. (doi:10. 1016/j.memsci.2016.11.084) 30. Brown PS, Atkinson ODLA, Badyal JPS. 2014 Ultrafast oleophobic-hydrophilic switching surfaces for antifogging, self-cleaning, and oil- water separation. ACS Appl. Mater. Interfaces 6, 7504–7511. (doi:10.1021/am500882y) 21. Lu Y, Wang Y, Liu L, Yuan W. 2017 Environmental-friendly and magnetic/silanized ethyl cellulose sponges as effective and recyclable oil-absorption materials. Carbohydr. Polym. 173, 422–430. (doi:10.1016/j.carbpol. 2017.06.009) 42. Sehaqui H, Zhou Q, Berglund LA. 2011 High- porosity aerogels of high specific surface area prepared from nanofibrillated cellulose (NFC). Compos. Sci. Technol. 71, 1593–1599 (doi:10. 1016/j.compscitech.2011.07.003) 31. Liu H, Geng B, Chen Y, Wang H. 2017 Review on the aerogel-type oil sorbents derived from nanocellulose. ACS Sustain. Chem. Eng. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 13 5, 49–66. (doi:10.1021/acssuschemeng.6b02301) 22. Sai H, Fu R, Xing L, Xiang J, Li Z, Li F, Zhang T. 2015 Surface modification of bacterial cellulose aerogels’ web-like skeleton for oil/water separation. ACS Appl. Mater. Interfaces 7, 7373–7381. (doi:10.1021/acsami.5b00846) 32. de France KJ, Hoare T, Cranston ED. 2017 Review of hydrogels and aerogels containing nanocellulose. Chem. Mater. 29, 4609–4631. (doi:10.1021/acs.chemmater.7b00531) 43. Nguyen ST, Feng J, Ng SK, Wong JPW, Tan VBC, Duong HM. 2014 Advanced thermal insulation and absorption properties of recycled cellulose aerogels. Colloids Surf. A 445, 128–134. (doi:10.1016/j.colsurfa.2014.01.015) 33. Calcagnile P, Caputo I, Cannoletta D, Bettini S, Valli L, Demitri C. 2017 A bio-based composite material for water remediation from oily contaminants. Mater. Design 134, 374–382. (doi:10.1016/j.matdes.2017.08.063) 23. Wu ZY, Li C, Liang HW, Zhang YN, Wang X, Chen JF, Yu SH. 2014 Carbon nanofiber aerogels for emergent cleanup of oil spillage and chemical leakage under harsh conditions. Sci. Rep. 4, 4079. (doi:10.1038/srep04079) 44. Fumagalli M, Ouhab D, Boisseau SM, Heux L. 2013Versatile gas-phase reactions for surface to bulk esterification of cellulose Microfibrils aerogels. Biomacromolecules 14, 3246–3255 (doi:10.1021/bm400864z) 34. Mulyadi A, Zhang Z, & Deng Y. 2016 Fluorine- free oil absorbents made from cellulose nanofibril aerogels. ACS Appl. Mater. Interfaces 8, 2732–2740. (doi:10.1021/acsami.5b10985) 24. Zhu L, Wang Y, Wang Y, You L, Shen X, Li S. 2017 An environmentally friendly carbon aerogels derived from waste pomelo peels for the removal of organic pollutants/oils. Microporous Mesoporous Mater. 241, 285–292. (doi:10.1016/j.micromeso.2016.12.033) 45. Jiang F, Hsieh Y.-L. 2014 Amphiphilic superabsorbent cellulose nanofibril aerogels. J. Mater. Chem. A 2, 6337–6342. (doi:10.1039/ C4TA00743C) 35. Wu D, Yu Z, Wu W, Fang L, & Zhu H. 2014 Continuous oil–water separation with surface modified sponge for cleanup of oil spills. RSC Adv. 4, 53 514–53 519. (doi:10.1039/ C4RA07583H) 46. Khanjanzadeh H, Behrooz R, Bahramifar N, Gindl-Altmutter W, Bacher M, Edler M, Griesser T. 2018 Surface chemical functionalization of cellulose nanocrystals by 3- aminopropyltriethoxysilane. Int. J. Biol. Macromol. 106, 1288–1296. (doi:10.1016/j. ijbiomac.2017.08.136) 25. Arguna ME, Gu¨clu¨ D, Karatas M. 2014 Adsorption of Reactive Blue 114 dye by using a new adsorbent: pomelo peel. J. Ind. Eng. Chem. 20, 1079–1084. (doi:10.1016/j.jiec.2013.06. 045) 36. 36. Hu H, Zhao Z, Gogotsi Y, Qiu J. 2014 Compressible carbon nanotube–graphene hybrid aerogels with superhydrophobicity and superoleophilicity for oil sorption. Environ. Sci. Technol. Lett. 1, 214–220. (doi:10.1021/ ez500021w) 26. References 451, 223–231. (doi:10.1016/j.apsusc.2018.04.179) 5. Gupta R, Dunderdale G, England M, Hozumi A. 2017 Oil/water separation techniques: a review of recent progresses and future directions. J. Mater. Chem. A 5, 16 025–16 058 (doi:10. 1039/C7TA02070H) 15. Wang J, Zheng Y, Wang A. 2012 Superhydrophobic Kapok fiber oil-absorbent: preparation and high oil absorbency. Chem. Eng. J. 213, 1–7. (doi:10.1016/j.cej.2012.09.116) 10. Feng J, Nguyen ST, Fan Z, Duong HM. 2015 Advanced fabrication and oil absorption properties of super-hydrophobic recycled cellulose aerogels. Chem. Eng. J. 270, 168–175. (doi:10.1016/j.cej.2015.02.034) 16. Cheng H, Gu B, Pennefather MP, Nguyen TX, Phan-Thien N, Duong HM. 2017 Cotton aerogels and cotton-cellulose aerogels from environmental waste for oil spillage cleanup. 6. Ahmad AL, Sumathi S, Hameed BH. 2005 Residual oil and suspended solid removal using natural adsorbents chitosan, bentonite and 6. Ahmad AL, Sumathi S, Hameed BH. 2005 Residual oil and suspended solid removal using natural adsorbents chitosan, bentonite and 11. Maleki H. 2016 Recent advances in aerogels for environmental remediation applications: a Materials & Design 130, 452–458. (doi:10. 1016/j.matdes.2017.05.082) 27. Lin R, Li A, Zheng T, Lu L, Cao Y. 2015 Hydrophobic and flexible cellulose aerogel as an efficient, green and reusable oil sorbent. RSC Adv. 5, 82 027–82 033. (doi:10.1039/ C5RA15194E) 38. Xiao S, Gao R, Lu Y, Li J, Sun Q. 2015 Fabrication and characterization of nanofibrillated cellulose and its aerogels from natural pine needles. Carbohydr. Polym. 119, 202–209. (doi:10.1016/j.carbpol.2014.11.041) royalsoc 13 13 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181823 13 Wang S, Peng X, Zhong L, Tan J, Jing S, Cao X, Chen W, Liu C, Sun R. 2015 An ultralight, elastic, cost-effective, and highly recyclable superabsorbent from microfibrillated cellulose fibers for oil spillage cleanup. J. Mater. Chem. A 3, 8772–8781. (doi:10.1039/C4TA07057G) 47. Korhonen JT, Kettunen M, Ras RHA, Ikkala O. 2011 Hydrophobic nanocellulose aerogels as floating, sustainable, reusable, and recyclable oil absorbents. ACS Appl. Mater. Interfaces 3, 1813–1816. (doi:10.1021/am200475b) 37. Habibi Y. 2014 Key advances in the chemical modification of nanocelluloses. Chem. Soc. Rev. 43, 1519–1542. (doi:10.1039/C3CS60204D)
https://openalex.org/W2979819245	https://discovery.ucl.ac.uk/id/eprint/10089929/1/BJO_Moorfields_AMD_Report_2.pdf	English	null	Moorfields AMD database report 2: fellow eye involvement with neovascular age-related macular degeneration	British journal of ophthalmology	2,019	public-domain	5,403	The Moorfields AMD Database Report 2 - Fellow Eye Involvement with Neovascular Age-related Macular The Moorfields AMD Database Report 2 - Fellow Eye Involvement with Neovascular Age-related Macular Degeneration Katrin Fasler,1,2 Gabriella Moraes,1 Dun Jack Fu,1 Siegfried K. Wagner,1 Eesha Gokhale,1 Karsten U. Kortuem,1,3 Reena Chopra,1 Livia Faes,1,4 Gabriella Preston,1 Nikolas Pontikos,1 Praveen J. Patel,1 Adnan Tufail,1 Aaron Y. Lee,5 Konstantinos Balaskas,1, 6 Pearse A. Keane 1 1 NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK. 2 Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland. 3 University Eye Hospital, Munich, Germany. 4 Eye Clinic, Cantonal Hospital of Lucerne, Lucerne, Switzerland. 5 Department of Ophthalmology, University of Washington, Seattle, USA. 5 Department of Ophthalmology, University of Washington, Seattle, USA. 6 School of Biological Sciences, University of Manchester, Manchester, UK. 6 School of Biological Sciences, University of Manchester, Manchester, UK. Disclosure: Dr. Fasler has received fellowship support from Alfred Vogt Stipendium and Schweizerischer Fonds zur Verhütung und Bekämpfung der Blindheit. She has been an external consultant for DeepMind. Dr. Wagner is an academic clinical fellow funded by the National Institute of Health Research (NIHR). Dr. Keane has received speaker fees from Heidelberg Engineering, Topcon, Carl Zeiss Meditec, Haag-Streit, Allergan, Novartis, and Bayer. He has served on advisory boards for Novartis and Bayer and has been an external consultant for DeepMind and Optos. Dr. Keane is supported by a United Kingdom (UK) NIHR Clinician Scientist Award (NIHR-CS--2014-12-023). Ms Chopra receives studentship support from the College of Optometrists and is a paid intern at DeepMind. Dr. Patel has received speaker fees from Novartis UK, Bayer UK, and Roche UK and has received an advisory board honorarium from Novartis UK, Bayer UK. Dr. Lee has received research funding from Novartis, NVIDIA, and Microsoft Corporation. He is supported by the National Institute of Health (K23EY029246) and Research to Prevent Blindness. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research, Health Education England or the Department of Health. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sector. Correspondence and reprint requests: Pearse A. Keane, MD FRCOphth, NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, United Kingdom. Tel: +44 207 253 3411 Fax: +44 Email: pearse.keane1@nhs.net Competing interests: There are no competing interests for any author. Running Head: The Moorfields AMD Database - Report 2 Keywords: Age-related macular degeneration Choroidal neovascularization Ranibizumab Aflibercept Anti-vascular endothelial growth factor Fellow eye Electronic medical record Visual acuity Word Count: Abstract: 249 Manuscript: 2382 Precis: 35 References: 29 Figures: 4 Tables: 1 Running Head: The Moorfields AMD Database - Report 2 The Moorfields AMD Database - Report 2 The Moorfields AMD Database - Report 2 Keywords: Age-related macular degeneration Choroidal neovascularization Ranibizumab Aflibercept Anti-vascular endothelial growth factor Fellow eye Electronic medical record Visual acuity Word Count: 1 Abbreviations: VEGF - vascular endothelial growth factor AMD - age related macular degeneration nAMD - neovascular age related macular degeneration VA - visual acuity UK - United Kingdom EMR - electronic medical record ETDRS - Early Treatment Diabetic Retinopathy Study ICHOM - International Consortium for Health Outcomes Measurement RCTs - randomized controlled trials ABSTRACT Background/Aims: Neovascular age-related macular degeneration (nAMD) is frequently bilateral, and previous reports on ‘fellow eyes’’ have assumed sequential treatment after a period of treatment of the first eye only. The aim of our study was to analyse baseline characteristics and visual acuity (VA) outcomes of fellow eye involvement with nAMD, specifically differentiating between sequential and non-sequential (due to macular scarring in the first eye) anti-vascular endothelial growth factor treatment and timelines for fellow eye involvement. Methods: Retrospective, electronic medical record database study of the Moorfields AMD database of 8174 eyes/120,756 single entries with data extracted between October 21, 2008 and August 9, 2018. The dataset for analysis consisted of 1180 sequential, 413 nonsequential, and 1110 unilateral eyes. Results: Mean VA of sequentially treated fellow eyes at baseline was significantly higher (62±13), VA gain over two years lower (0.65±14), and proportion of eyes with good VA (≥20/40 or 70 letters) higher (46%) than the respective first eyes (baseline VA 54±16, VA gain at two years 5.6±15, percentage of eyes with good VA 38%). Non-sequential fellow eyes showed baseline characteristics and VA outcomes similar to first eyes. Fellow eye involvement rate was 32% at two years, and median time interval to fellow eye involvement was 71 (IQR 27-147) weeks. Conclusion: This reports shows sequentially treated nAMD fellow eyes have better baseline and final VA than non-sequentially treated eyes after 2 years of treatment. Sequentially treated eyes also had a greater proportion with good VA after 2 years of treatment. PRECIS Depending on age, fellow eye involvement occurs in 32% of patients with neovascular AMD by two years. Fellow eyes generally maintain better vision, except in cases where late-stage disease in the first eye was untreated. INTRODUCTION Anti-vascular endothelial growth factor (VEGF) therapy has revolutionised the treatment of neovascular age-related macular degeneration (nAMD). Two anti-VEGF drugs, ranibizumab and aflibercept, form the mainstay of treatment and received approval from the Food and Drug Administration (FDA) in 2006 and 2011 respectively (bevacizumab is also widely used in an off-licence manner). The pivotal randomised controlled trials (RCTs) that led to the approval of these agents only included one eye per patient as a means of preventing bias due to correlation between eyes.[1,2] This is a necessary step in RCTs, as not accounting for this effect can lead to overestimation of precision and a falsely low p-value.[3] However, this systematically excludes eyes of patients who subsequently develop nAMD in their fellow eye. From a patient’s perspective however, vision-related quality of life does not only depend on the course of visual acuity (VA) in the first treated eye.[4] Legal requirements largely focus on the VA of the better seeing eye. For example, in the United Kingdom, the VA standard for driving is 20/40 and the limit for obtaining a certificate of severe sight impairment is 20/400 (tested binocularly or in the better seeing eye).[5,6] Additionally, patients with bilateral nAMD have functional impairments that lead to a high socioeconomic burden.[7–9] Data on treatment of fellow eyes, specifically sequentially treated fellow eyes, have been reported in small retrospective studies and in one large multicentre electronic medical record (EMR) report.[10–13] These studies concluded that fellow eyes commenced treatment with a higher baseline VA in comparison to the first treated eyes. In addition, they had a smaller gain in VA over time due to the relatively higher baseline VA, i.e., a ceiling effect. However, these studies do not account for non-sequentially treated fellow eyes, i.e., eyes starting treatment for nAMD in fellow eyes with an untreated first eye (e.g. due to development of nAMD in the first eye before anti-VEGF approval or late presentation at first eye involvement). Study Population: Data for this retrospective, comparative, non-randomised cohort study was extracted from the Moorfields AMD Database, consisting of 8174 treatment-naïve eyes / 6664 patients with 120,756 single entries acquired between October 21, 2008 and August 9, 2018. This has been reported in detail elsewhere.[17] The complete dataset for analysis of the current study consisted of the 3880 eyes / 2700 patients (Supplementary sFigure 1). Of these, 1180 patients had sequentially treated fellow eye involvement, while 413 eyes were non-sequentially treated fellow eyes (i.e., untreated macular scarring in their respective first eyes). The 1107 unilateral/singular eyes (only treated in one eye over the observed period without advanced AMD in their fellow eye) were used for the survival analysis of fellow-eye involvement. Definition of sequential involvement was a time interval of ≥28 days between the first injection of first and fellow eye over the course of the observed time period. The presence of a macular scar was manually graded in fundus photographs and optical coherence tomography (OCT) (Topcon 3D OCT, Topcon, Japan) scans. An exemplar case for each group is shown in Figure 1. Approval for data collection and analysis was obtained from the Institutional Review Board at Moorfields (ROAD17/031) and adhered to the tenets set forth in the Declaration of Helsinki. INTRODUCTION Given that involvement of the fellow eye has a substantial impact on vision-related quality of life, accounting for patients that already have poor vision from macular scarring in their first eye clearly is important.[14] The electronic medical record (EMR) database at Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom (UK), represents an ideal source to explore the unanswered questions on fellow eye nAMD outcomes.[Fasler,One and Two Year Visual Outcomes from the Moorfields Age-related Macular Degeneration Database, submitted to BMJ open, 2019 ] This database consists of over 8000 treatment naïve eyes with over 120,000 single entries and has undergone extensive manual data cleaning. Key elements that distinguish its quality compared to others include the completeness of data due to the mandatory input of relevant fields such as VA, the consistency of VA measurements using Early Treatment Diabetic Retinopathy Study (ETDRS) letters, the lack of requirement to merge data from different sites and systems, the standardised treatment scheme following national guidelines, and the ability to directly access the raw imaging data from each patient visit.[15,16] The aim of this study was to analyse baseline characteristics and VA outcomes of fellow eyes (sequentially and non-sequentially treated) undergoing anti-VEGF therapy for nAMD, as well as the timelines for fellow eye involvement. We compare fellow eye outcomes to those of the respective first eyes of sequentially treated fellow eyes. Outcome Measures: The primary outcomes were analogous to the pivotal RCTs, and as recommended by The International Consortium for Health Outcomes Measurement (ICHOM) AMD study group: mean change in VA from baseline as measured using ETDRS letters, proportion of eyes gaining ≥ 5 letters, proportion of eyes with stable vision (change in VA <15 letters to baseline), proportion of eyes with good vision (≥20/40 or 70 letters), and proportion of eyes with poor vision (≤20/200 or 35 letters).[1,2,19,20] Secondary outcomes included the number of injections and time to involvement of fellow eyes. Definitions for one-year and two-year outcome dates were taken from previous real-world studies as visits closest to 52 weeks and 104 weeks post baseline date within ±8 weeks.[21,22] Efforts to Minimise Bias: To minimise survival bias/loss to follow-up (LTFU), all first and fellow eyes that did not complete follow-up were manually validated for the correct date of first injection. All unilateral eyes (1521) underwent manual verification for the presence of a macular scar secondary to end-stage AMD in the fellow eye. We chose not to substitute missing values, but clearly show results for cohorts that complete a certain follow-up period. Visual acuities below measurable ETDRS letters were converted to logMAR 2.0/-15 letters, logMAR 2.3/-30 letters and logMAR 2.7/-50 letters for count fingers, hand movements, and light perception respectively.[18] eyes (1521) underwent manual verification for the presence of a macular scar secondary to end-stage AMD in the fellow eye. We chose not to substitute missing values, but clearly show results for cohorts that complete a certain follow-up period. Visual acuities below measurable ETDRS letters were converted to logMAR 2.0/-15 letters, logMAR 2.3/-30 letters and logMAR 2.7/-50 letters for count fingers, hand movements, and light perception respectively.[18] Statistical Analysis: The data were analysed using the statistics software R (https://www.r-project.org/; provided in the public domain by R Core team 2017 R Foundation for Statistical Computing, Vienna, Austria). The ggplot2 package was used for plots. The eye was defined as unit of analysis. Descriptive statistics included mean +/- 95% confidence interval (CI), and median, where appropriate. Differences between groups were evaluated using Mann Whitney U test and Pearson Chi-Square. A p value of < 0.05 was interpreted as statistically significant. Data Sharing Statement: De-personalised data as well as the code used for analysis for this study will be openly available from the Dryad Digital Repository https://doi.org/.... This should allow both for independent replication of our results as well as additional novel analyses. Depersonalisation was carried out through hash function anonymisation of patient identification numbers, and replacement of appointment dates with follow-up days to baseline. Approval of adequate depersonalisation was obtained by Moorfields Information Governance. Baseline Characteristics: Of the 2710 patients starting treatment in one eye, 1180 (44%) developed fellow eye involvement, 413 (15%) were identified as non-sequentially treated fellow eye involvement, whereas 1117 (41%) were singular/unilateral eyes. Supplementary sFigure 1 shows the flow chart for eyes through the analysis. Mean baseline VA was 54±16 letters for first eyes and 62±13 letters for fellow eyes in sequentially treated patients, and 52±16 letters for non-sequentially treated fellow eyes (Table 1). In sequentially treated patients, fellow eyes had a significantly higher baseline VA than first eyes (p <0.001); more than 40% of fellow eyes had a VA of ≥20/40 compared to 21% of respective first eyes at baseline. Compared to non-sequentially treated fellow eyes, sequentially treated fellow eyes had higher baseline VA (p<0.001). Visual Acuity Outcomes: At one year, mean gain in VA was 5.2±15 letters for first eyes, 2.5±12 letters for sequentially treated fellow eyes, and 4.1±15 letters for non-sequentially treated fellow eyes (Table 1). At two years, mean gain in VA was 5.6±15 letters for first eyes and 0.65±14 letters for fellow eyes in sequentially treated patients, and 3.6±18 letters for non-sequentially treated fellow eyes. Fellow eyes showed a significantly lower gain in VA than first eyes and non-sequentially treated fellow eyes at one and two years (p <0.001). However, percentage of eyes with good vision (VA≥70 letters/>20/40) at presentation was 42%,double that of first or non-sequential fellow eyes (p<0.001) and stayed at 46% at two years, significantly higher than both other groups (p≤0.001). VA and change in VA over time is shown in Figure 2. Percentages of eyes gaining vision (change in VA ≥5 letters), stable vision (change in VA <15 letters), good vision (VA≥70 letters/>20/40), and poor vision (VA ≤35 letters/≤20/200) are shown in Table 1 and Figure 3. Baseline Characteristics: Baseline characteristics n = number of eyes Sequential treatment fellow eye involvement Non-sequential treatment fellow eye involvement, n = 807 First eyes n = 1180 p (paired) first/fellow eyes Fellow eyes n = 1180 p / adjusted p sequential/non-sequen tial fellow eyes Mean VA (letters) ±SD 54±16 p<0.001 63±13 p<0.001 53±16 % of eyes with VA ≥ 20/40 21.5 p<0.001 42.1 p<0.001 21.4 % of eyes with VA ≤20/200 18.1 p<0.001 6.2 p<0.001 20.1 One-year outcomes n = number of eyes First eyes n = 1094 p (paired) first/fellow eyes Fellow eyes n =961 p / adjusted p sequential/non-sequen tial fellow eyes Non-sequential treatment fellow eye involvement, n = 668 Mean VA change (letters)±SD 5.2±15 p<0.001 2.4±12 p<0.001 4.6±15 % of eyes with VA ≥ 20/40 37.9 p<0.001 50.5 p<0.001 36.1 % of eyes with VA ≤20/200 17.0 p<0.001 8.3 p<0.001 16.3 % of eyes with VA gain ≥5 letters 52.6 p<0.001 42.6 p=0.938 47.3 % of eyes with change in VA <15 letters 69.4 p<0.001 81.4 p=0.006 72.9 Mean injection number±SD 7.7±2.1 p=0.503 7.7±1.9 p=0.740 8.0±1.7 Two-year outcomes n = number of eyes First eyes n = 1005 p (paired) first/fellow eyes Fellow eyes n = 781 p / adjusted p sequential/non-sequen tial fellow eyes Non-sequential treatment fellow eye involvement, n = 534 Mean VA change (letters)±SD 5.6±15 p<0.001 0.37± 14 p<0.001 3.4±19 % of eyes with VA≥20/40 38.9 p<0.001 46.1 p=0.001 36.9 % of eyes with VA≤20/200 19.8 p<0.001 10.4 p=0.008 14.4 % of eyes with VA gain ≥5 letters 51.0 p<0.001 39.2 p=0.094 46.3 % of eyes with change in VA <15 letters 69.4 p<0.001 81.4 p=0.005 72.9 Mean injection number±SD 13±4.2 p=0.921 13±3.9 p=0.953 13±3.8 Table 1: Baseline visual acuity and visual acuity outcomes of first and fellow eyes in sequential treatment fellow eye involvement, and non-sequentially treated fellow eyes. Time to Involvement of Second Eye: Median time interval between involvement of first and fellow eye in sequential involvement was 71 weeks (interquartile range: 27-147 weeks). Chance of involvement of fellow eye involvement for eyes starting treatment in one eye 21% (486 eyes) at one year and 32% (742 eyes) at two years, and it was dependent on age at presentation of the first eye: At two years, the risk of fellow eye involvement was 20% for patients younger than 60 years and 40% for patients in their eighties. Survival analysis of fellow-eye involvement is shown in Figure 4. Injection Frequency: Mean number of injections was 8 in all groups at one year and 13 at two years with no significant differences between the groups (Table 1). DISCUSSION Our study shows that fellow eye involvement of nAMD affects 20 to 40% over a two-year period, depending on age at presentation of the first eye, and that there is a significant difference in both baseline VA and VA outcomes depending on characteristics of the first eye. Fellow eye involvement in nAMD is very common, reaching 20-40% depending on age at presentation after two years in our cohort. This rate falls within the range reported in the Comparison of AMD Treatments Trials (20.6% at two years) and other studies.[12,13,23–25] With demographic ageing, sight loss and blindness is predicted to increase by 2.4% from 2013 until 2050, reaching approximately 4 million people in the UK and the share of AMD is estimated to rise from 23% to 30%, representing 1.23 million people.[8] AMD is thus a major and growing contributor to healthcare burden. Considering the annual societal cost per bilaterally treated AMD patient estimated at 5300€ in 2005, the frequency of bilateral involvement makes this patient cohort an important target for vision loss prevention and healthcare cost reduction.[7] In sequential treatment, fellow eyes have a higher baseline VA and maintain good vision over two years of treatment despite the absence of an initial gain in VA comparable to first eyes. This ceiling effect has been well-described and implies a rationale for earliest possible detection and treatment of neovascular changes in AMD.[13,16] Example A (Figure 1) reflects this fellow eye advantage, in which neovascular AMD in the fellow eye was detected pre-symptomatically and treatment was started immediately. Patients might profit from the routinely performed bilateral OCT imaging at every visit, be more vigilant of VA changes in their fellow eye while undergoing treatment for the first eye, and profit from the already in-place pathway to access treatment for the fellow eye quickly. This effect has led to a discussion about strategies for early detection of nAMD and optimal interval of monitoring of AMD patients.[12,13,26] Specifically analysis of imaging biomarkers, possibly aided by artificial intelligence, might prove to be key in risk stratification of fellow eye involvement.[24,27,28] Our study demonstrates that non-sequentially treated fellow eyes do not share the typical fellow eye characteristics. They start treatment with a relatively low baseline VA and their gain in VA is higher, very similar to first eyes of sequentially treated patients. their gain in VA is higher, very similar to first eyes of sequentially treated patients. DISCUSSION Explanations for this could be that patients with non-treatable advanced neovascular disease in the first eye are not regularly monitored or that there is systematic delay in access to treatment. This is supported by the existing lack of awareness of AMD and evidence of substantial delay from symptoms to treatment in the UK AMD care pathways.[29] Interestingly, in this cohort of patients, vision loss secondary to macular scarring in the first eye does not appear to result in increased vigilance that could lead to early detection of fellow eye involvement. One might argue that scarring in the first eye implies more aggressive disease causing worse VA at presentation of fellow eyes, but the similar VA gain over time to first eyes in our cohort does not support this theory. To our knowledge, the findings on non-sequential fellow eye involvement have not been reported before and highlight the arguably most vulnerable cohort of patients in which vision loss in their fellow and better or functionally only seeing eye will lead to significant visual impairment and socioeconomic burden.[7,8] The limitations of this study lie within its retrospective nature based on EMR and the LTFU. However, comparisons of baseline characteristics based on the complete cohorts are unaffected by LTFU and it has been shown that changes in VA are comparable in cohorts of different follow-up periods.[16] Strengths of this study include the large sample size for fellow eye cohorts and the the quality of data coming from one single center and a curated database with additional substantial manual cleaning. Additionally and maybe most importantly, we encourage an open science approach to replicate our results with freely available depersonalized raw data and code. In conclusion, this study highlights the superior visual outcomes of fellow eyes compared to first eyes in the common scenario of sequential fellow eye involvement in nAMD as well as the inferior outcomes of fellow eyes in case of untreated late stage neovascular disease in the first eye. Future research should account for those idiosyncratic subgroups of fellow eyes undergoing treatment for nAMD, as these could prove to represent the span of quality of care in AMD treatment. REFERENCES 1 Rosenfeld PJ, Brown DM, Heier JS, et al. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 2006;355:1419–31. 2 Heier JS, Brown DM, Chong V, et al. Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration. Ophthalmology 2012;119:2537–48. 3 Ray WA, O’Day DM. Statistical analysis of multi-eye data in ophthalmic research. Invest Ophthalmol Vis Sci 1985;26:1186–8. 4 Finger RP, Guymer RH, Gillies MC, et al. The impact of anti--vascular endothelial growth factor treatment on quality of life in neovascular age-related macular degeneration. Ophthalmology 2014;121:1246–51. 5 The Royal College of Opthalmologists. CVI – Certificate of Vision Impairment. https://www.rcophth.ac.uk/professional-resources/certificate-of-vision-impairment/ (accessed 15 Mar 2018). 6 Rees GB. Vision standards for driving: what ophthalmologists need to know. Eye 2015;29:719–20. 7 Cruess AF, Zlateva G, Xu X, et al. Economic burden of bilateral neovascular age-related macular degeneration: multi-country observational study. Pharmacoeconomics 2008;26:57–73. 8 Pezzullo L, Streatfeild J, Simkiss P, et al. The economic impact of sight loss and blindness in the UK adult population. BMC Health Serv Res 2018;18:63. 9 Brown MM, Brown GC, Lieske HB, et al. SOCIETAL COSTS ASSOCIATED WITH NEOVASCULAR AGE-RELATED MACULAR DEGENERATION IN THE UNITED STATES. Retina 2016;36:285–98. 10 Barthelmes D, Walton RJ, Arnold JJ, et al. Intravitreal therapy in bilateral neovascular age-related macular degeneration. Ophthalmology 2014;121:2073–4. 11 Keenan TDL, Kelly SP, Sallam A, et al. Incidence and baseline clinical characteristics of treated neovascular age-related macular degeneration in a well-defined region of the UK. Br J Ophthalmol 2013;97:1168–72. 12 Zarranz-Ventura J, Liew G, Johnston RL, et al. The neovascular age-related macular degeneration database: report 2: incidence, management, and visual outcomes of second treated eyes. Ophthalmology 2014;121:1966–75. 13 Mehta H, Tufail A, Daien V, et al. Real-world outcomes in patients with neovascular age-related macular degeneration treated with intravitreal vascular endothelial growth factor inhibitors. Prog Retin Eye Res Published Online First: 2 January 2018. doi:10.1016/j.preteyeres.2017.12.002 upon conversion to neovascular age-related macular degeneration in second eyes. Qual Life Res 2017;26:2139–51. upon conversion to neovascular age-related macular degeneration in second eyes. Qual Life Res 2017;26:2139–51. 15 Rao P, Lum F, Wood K, et al. Real-World Vision in Age-Related Macular Degeneration Patients Treated with Single Anti-VEGF Drug Type for 1 Year in the IRIS Registry. Ophthalmology Published Online First: 13 November 2017. doi:10.1016/j.ophtha.2017.10.010 16 Writing Committee for the UK Age-Related Macular Degeneration EMR Users Group. The neovascular age-related macular degeneration database: multicenter study of 92 976 ranibizumab injections: report 1: visual acuity. Ophthalmology 2014;121:1092–101. REFERENCES 17 Katrin Fasler, Gabriella Moraes, Siegfried Wagner, Karsten U Kortuem, Reena Chopra, Livia Faes, Gabriella Preston, Nikolas Pontikos, Dun Jack Fu, Praveen Patel, Adnan Tufail, Aaron Lee, Konstantinos Balaskas, Pearse A Keane. One and Two Year Visual Outcomes from the Moorfields AMD Database - an Open Science Resource for the Study of Neovascular Age-related Macular Degeneration. 2018. doi:10.1101/450411 18 Lange C, Feltgen N, Junker B, et al. Resolving the clinical acuity categories ‘hand motion’ and ‘counting fingers’ using the Freiburg Visual Acuity Test (FrACT). Graefes Arch Clin Exp Ophthalmol 2009;247:137–42. 19 Brown DM, Michels M, Kaiser PK, et al. Ranibizumab versus verteporfin photodynamic therapy for neovascular age-related macular degeneration: Two-year results of the ANCHOR study. Ophthalmology 2009;116:57–65.e5. 20 Rodrigues IA, Sprinkhuizen SM, Barthelmes D, et al. Defining a Minimum Set of Standardized Patient-centered Outcome Measures for Macular Degeneration. Am J Ophthalmol 2016;168:1–12. 21 Lotery A, Griner R, Ferreira A, et al. Real-world visual acuity outcomes between ranibizumab and aflibercept in treatment of neovascular AMD in a large US data set. Eye 2017;31:1697–706. 22 Cohen SY, Mimoun G, Oubraham H, et al. Changes in visual acuity in patients with wet age-related macular degeneration treated with intravitreal ranibizumab in daily clinical practice: the LUMIERE study. Retina 2013;33:474–81. 23 Maguire MG, Daniel E, Shah AR, et al. Incidence of choroidal neovascularization in the fellow eye in the comparison of age-related macular degeneration treatments trials. Ophthalmology 2013;120:2035–41. 24 Yanagi Y, Mohla A, Lee SY, et al. Incidence of Fellow Eye Involvement in Patients With Unilateral Exudative Age-Related Macular Degeneration. JAMA Ophthalmol Published Online First: 7 June 2018. doi:10.1001/jamaophthalmol.2018.2154 25 Bek T, Klug SE. Incidence and risk factors for neovascular age-related macular degeneration in the fellow eye. Graefes Arch Clin Exp Ophthalmol 2018;256:2061–8. 26 Chew JK, Zhu M, Broadhead GK, et al. Bilateral Neovascular Age-Related Macular Degeneration: Comparisons between First and Second Eyes. Ophthalmologica 2017;238:23–30. diagnosis of neovascular age-related macular degeneration. Clin Ophthalmol 2015;9:353–66. 28 Schmidt-Erfurth U, Waldstein SM, Klimscha S, et al. Prediction of Individual Disease Conversion in Early AMD Using Artificial Intelligence. Invest Ophthalmol Vis Sci 2018;59:3199–208. 28 Schmidt-Erfurth U, Waldstein SM, Klimscha S, et al. Prediction of Individual Disease Conversion in Early AMD Using Artificial Intelligence. Invest Ophthalmol Vis Sci 2018;59:3199–208. 29 Sim PY, Gajree S, Dhillon B, et al. Investigation of time to first presentation and extrahospital factors in the treatment of neovascular age-related macular degeneration: a retrospective cross-sectional study. 29 Sim PY, Gajree S, Dhillon B, et al. Investigation of time to first presentation and extrahospital factors in the treatment of neovascular age-related macular degeneration: a retrospective cross-sectional study. BMJ Open 2017;7:e017771. 28 Schmidt-Erfurth U, Waldstein SM, Klimscha S, et al. Prediction of Individual Disease Conversion in Early AMD Using Artificial Intelligence. Invest Ophthalmol Vis Sci 2018;59:3199–208. REFERENCES BMJ Open 2017;7:e017771. 29 Sim PY, Gajree S, Dhillon B, et al. Investigation of time to first presentation and extrahospital factors in the treatment of neovascular age-related macular degeneration: a retrospective cross-sectional study. BMJ Open 2017;7:e017771. Figures Figure 1: Representative fundus photographs and OCT-images for each of the groups at involvement of the respective eye(s). Sequential treatment fellow eye involvement (A) of a 83-year-old male. Top row shows findings at first eye involvement: neovascular AMD with intraretinal hemorrhage, intraretinal fluid, and subretinal fluid on OCT in the right eye, and early dry AMD in the left eye. One month later, new neovascular changes with intra- and subretinal fluid on OCT were found incidentally in the left eye, indicating second eye involvement (bottom row). Non-sequential treatment fellow eye involvement (B) of a 86-year-old male. Examination at first presentation revealed neovascular AMD in his right eye with intra- and subretinal fluid on OCT while in the left eye, a disciform scar with a visual acuity of hand movements was present. Unilateral involvement only (C) of a 86-year-old male. Examination at baseline showed neovascular AMD in the right eye with predominantly Figure 1: Representative fundus photographs and OCT-images for each of the groups at involvement of the respective eye(s). Sequential treatment fellow eye involvement (A) of a 83-year-old male. Top row shows findings at first eye involvement: neovascular AMD with intraretinal hemorrhage, intraretinal fluid, and subretinal fluid on OCT in the right eye, and early dry AMD in the left eye. One month later, new neovascular changes with intra- and subretinal fluid on OCT were found incidentally in the left eye, indicating second eye involvement (bottom row). Non-sequential treatment fellow eye involvement (B) of a 86-year-old male. Examination at first presentation revealed neovascular AMD in his right eye with intra- and subretinal fluid on OCT while in the left eye, a disciform scar with a visual acuity of hand movements was present. Unilateral involvement only (C) of a 86-year-old male. Examination at baseline showed neovascular AMD in the right eye with predominantly Figure 1: Representative fundus photographs and OCT-images for each of the groups at involvement of the respective eye(s). Sequential treatment fellow eye involvement (A) of a 83-year-old male. Top row shows findings at first eye involvement: neovascular AMD with intraretinal hemorrhage, intraretinal fluid, and subretinal fluid on OCT in the right eye, and early dry AMD in the left eye. REFERENCES One month later, new neovascular changes with intra- and subretinal fluid on OCT were found incidentally in the left eye, indicating second eye involvement (bottom row). Non-sequential treatment fellow eye involvement (B) of a 86-year-old male. Examination at first presentation revealed neovascular AMD in his right eye with intra- and subretinal fluid on OCT while in the left eye, a disciform scar with a visual acuity of hand movements was present. Unilateral involvement only (C) of a 86-year-old male. Examination at baseline showed neovascular AMD in the right eye with predominantly Figure 1: Representative fundus photographs and OCT-images for each of the groups at Figure 1: Representative fundus photographs and OCT-images for each of the groups at involvement of the respective eye(s). Sequential treatment fellow eye involvement (A) of a 83-year-old male. Top row shows findings at first eye involvement: neovascular AMD with intraretinal hemorrhage, intraretinal fluid, and subretinal fluid on OCT in the right eye, and early dry AMD in the left eye. One month later, new neovascular changes with intra- and subretinal fluid on OCT were found incidentally in the left eye, indicating second eye involvement (bottom row). Non-sequential treatment fellow eye involvement (B) of a Figure 1: Representative fundus photographs and OCT-images for each of the groups at involvement of the respective eye(s). Sequential treatment fellow eye involvement (A) of a Examination at baseline showed neovascular AMD in the right eye with predominantly Examination at baseline showed neovascular AMD in the right eye with predominantly subretinal fluid on OCT and early AMD with drusen in the left eye. Until time of data extraction, subretinal fluid on OCT and early AMD with drusen in the left eye. Until time of data extraction, there has been no progression to neovascular AMD in the left eye. there has been no progression to neovascular AMD in the left eye. OCT, optical coherence tomography; AMD, age related macular degeneration OCT, optical coherence tomography; AMD, age related macular degeneration Figure 2: Mean visual acuity from baseline (a) and change in visual acuity (b) over time and 95% confidence interval stratified by the different groups: first and second eyes in sequentially treated fellow eye involvement and delayed-presentation fellow eyes. VA - visual acuity Figure 3 a: Percentages of eyes with gain in VA (≥ 5 letters) at one and two years for fellow eyes in sequential/non-sequential treatment, and first eyes in sequential treatment. b: Percentages of eyes with stable vision (change in VA ≤±14 letters) for fellow eyes in sequential/non-sequential treatment, and first eyes in sequential treatment. b: Percentages of eyes with stable vision (change in VA ≤±14 letters) for fellow eyes in sequential/non-sequential treatment, and first eyes in sequential treatment. c: Percentages of eyes with poor vision (VA≤ 35 letters or 20/200) for fellow eyes in sequential/non-sequential treatment, and first eyes in sequential treatment. c: Percentages of eyes with poor vision (VA≤ 35 letters or 20/200) for fellow eyes in sequential/non-sequential treatment, and first eyes in sequential treatment. d: Percentages of eyes with good vision (VA≥ 70 letters or 20/40) for fellow eyes in sequential/non-sequential treatment, and first eyes in sequential treatment. d: Percentages of eyes with good vision (VA≥ 70 letters or 20/40) for fellow eyes in sequential/non-sequential treatment, and first eyes in sequential treatment. Figure 4 Survival probability for fellow eye involvement over time (weeks). Figure 4 Survival probability for fellow eye involvement over time (weeks). Survival probability for fellow eye involvement over time (weeks) Supplementary Figure 1: Consolidated Standards of Reporting Trials-style diagram showing patients / eyes included in the analysis VEGF- vascular endothelial growth factor, AMD - age-related macular degeneration, PDT - photodynamic therapy, VA - visual acuity, NICE national institute for health and care excellence
https://openalex.org/W4393037079	https://www.bmj.com/content/bmj/384/bmj-2023-077764.full.pdf	English	null	Community based complex interventions to sustain independence in older people: systematic review and network meta-analysis	BMJ	2,024	cc-by	18,624	DATA SYNTHESIS Interventions were grouped according to a specifically developed typology. Random effects network meta-analysis estimated comparative effects; Cochrane’s revised tool (RoB 2) structured risk of bias assessment. Grading of recommendations WHAT IS ALREADY KNOWN ON THIS TOPIC Older people prioritise maintenance of independence Previous systematic reviews have suggested that community based complex interventions may support independence for older people, but which are most effective is unclear The lack of clear guidance about which services to implement has hampered translation of evidence into policy and practice WHAT THIS STUDY ADDS Individualised care planning with tailored actions, including medicines optimisation and regular follow-ups, probably helps people to stay living at home Although some complex interventions may sustain independence, others may reduce independence Further evidence is needed about who benefits most from which kinds of interventions, which may be provided by individual participant data meta- analysis WHAT IS ALREADY KNOWN ON THIS TOPIC er 2024 by guest. Protected by copyright. Older people prioritise maintenance of independence Previous systematic reviews have suggested that community based complex interventions may support independence for older people, but which are most effective is unclear The lack of clear guidance about which services to implement has hampered translation of evidence into policy and practice RESEARCH RESEARCH RESEARCH RESEARCH MAIN OUTCOMES Living at home, activities of daily living (personal/ instrumental), care home placement, and service/ economic outcomes at 12 months. Community based complex interventions to sustain independence in older people: systematic review and network meta-analysis Thomas F Crocker,1 Joie Ensor,2,3 Natalie Lam,1 Magda Jordão,1 Ram Bajpai,3 Matthew Bond,3 Anne Forster,1 Richard D Riley,2,3 Deirdre Andre,4 Caroline Brundle,1 Alison Ellwood,1 John Green,1 Matthew Hale,1 Lubena Mirza,1 Jessica Morgan,5 Ismail Patel,1 Eleftheria Patetsini,1 Matthew Prescott,1 Ridha Ramiz,1 Oliver Todd,1 Rebecca Walford,5 John Gladman,6,7 Andrew Clegg1 Thomas F Crocker,1 Joie Ensor,2,3 Natalie Lam,1 Magda Jordão,1 Ram Bajpai,3 Matt Anne Forster,1 Richard D Riley,2,3 Deirdre Andre,4 Caroline Brundle,1 Alison Ellwoo John Green,1 Matthew Hale,1 Lubena Mirza,1 Jessica Morgan,5 Ismail Patel,1 Elefth Matthew Prescott,1 Ridha Ramiz,1 Oliver Todd,1 Rebecca Walford,5 John Gladman, Andrew Clegg1 assessment, development and evaluation (GRADE) network meta-analysis structured certainty assessment. assessment, development and evaluation (GRADE) network meta-analysis structured certainty assessment. ABSTRACT OBJECTIVE Correspondence to: T F Crocker medtcro@leeds.ac.uk (or @AUASResearch on Twitter/X; ORCID 0000-0001-7450-3143) To synthesise evidence of the effectiveness of community based complex interventions, grouped according to their intervention components, to sustain independence for older people. WHAT THIS STUDY ADDS Individualised care planning with tailored actions, including medicines optimisation and regular follow-ups, probably helps people to stay living at home For homecare recipients, evidence favoured addition of multifactorial action from individualised care planning and routine review with medication review (0.60, 0.32 to 0.88; low certainty). High risk of bias and imprecise estimates meant that most evidence was low or very low certainty. Few studies contributed to each comparison, impeding evaluation of inconsistency and frailty. Although some complex interventions may sustain independence, others may reduce independence Further evidence is needed about who benefits most from which kinds of interventions, which may be provided by individual participant data meta- analysis the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 RESULTS The review included 129 studies (74 946 participants). Nineteen intervention components, including “multifactorial action from individualised care planning” (a process of multidomain assessment and management leading to tailored actions), were identified in 63 combinations. For living at home, compared with no intervention/placebo, evidence favoured multifactorial action from individualised care planning including medication review and regular follow-ups (routine review) (odds ratio 1.22, 95% confidence interval 0.93 to 1.59; moderate certainty); multifactorial action from individualised care planning including medication review without regular follow-ups (2.55, 0.61 to 10.60; low certainty); combined cognitive training, medication review, nutritional support, and exercise (1.93, 0.79 to 4.77; low certainty); and combined activities of daily living training, nutritional support, and exercise (1.79, 0.67 to 4.76; low certainty). Risk screening or the addition of education and self-management strategies to multifactorial action from individualised care planning and routine review with medication review may reduce odds of living at home. For instrumental activities of daily living, evidence favoured multifactorial action from individualised care planning and routine review with medication review (standardised mean difference 0.11, 95% confidence interval 0.00 to 0.21; moderate certainty). Two interventions may reduce instrumental activities of daily living: combined activities of daily living training, aids, and exercise; and combined activities of daily living training, aids, education, exercise, and multifactorial action from individualised care planning and routine review with medication review and self-management strategies. For personal activities of daily living, evidence favoured combined exercise, multifactorial action from individualised care planning, and routine review with medication review and self-management strategies (0.16, −0.51 to 0.82; low certainty). For homecare recipients, evidence favoured addition of multifactorial action from individualised care planning and routine review with medication review (0.60, 0.32 to 0.88; low certainty). High risk of bias and imprecise estimates meant that most evidence was low or very low certainty. Few studies contributed to each comparison, impeding evaluation of inconsistency and frailty DESIGN Systematic review and network meta-analysis. DATA SOURCES DATA SOURCES Medline, Embase, CINAHL, PsycINFO, CENTRAL, clinicaltrials.gov, and International Clinical Trials Registry Platform from inception to 9 August 2021 and reference lists of included studies. DATA SOURCES Medline, Embase, CINAHL, PsycINFO, CENTRAL, clinicaltrials.gov, and International Clinical Trials Registry Platform from inception to 9 August 2021 and reference lists of included studies. ELIGIBILITY CRITERIA Randomised controlled trials or cluster randomised controlled trials with ≥24 weeks’ follow-up studying community based complex interventions for sustaining independence in older people (mean age ≥65 years) living at home, with usual care, placebo, or another complex intervention as comparators. Community based complex interventions to sustain independence in older people: systematic review and network meta-analysis BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Dow ABSTRACT OBJECTIVE For numbered affiliations see end of the article Correspondence to: T F Crocker medtcro@leeds.ac.uk (or @AUASResearch on Twitter/X; ORCID 0000-0001-7450-3143) Additional material is published online only. To view please visit the journal online. Cite this as: BMJ 2024;384:e077764 http://dx.doi.org/10.1136/ bmj‑2023‑077764 Accepted: 14 February 2024 For numbered affiliations see end of the article DESIGN DESIGN Systematic review and network meta-analysis. Systematic review and network meta-analysis. Cite this as: BMJ 2024;384:e077764 http://dx.doi.org/10.1136/ bmj‑2023‑077764 Accepted: 14 February 2024 Methods BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Do The intervention most likely to sustain independence is individualised care planning including medicines optimisation and regular follow-up reviews resulting in multifactorial action. Homecare recipients may particularly benefit from this intervention. Unexpectedly, some combinations may reduce independence. Further research is needed to investigate which combinations of interventions work best for different participants and contexts. This was a prospectively registered systematic review and network meta-analysis (PROSPERO CRD42019162195) that used Grading of Recommendations Assessment, Development and Evaluation (GRADE) for network meta-analysis to assess certainty14 16-21 and followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 and network meta-analysis guidelines.22 23 We published the protocol before meta-analysis began.24 Only minor changes were made subsequently, detailed in appendix 1. REGISTRATION REGISTRATION PROSPERO CRD42019162195. REGISTRATION PROSPERO CRD42019162195. the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 1 RESEARCH RESEARCH Search strategy and selection criteria Community based complex interventions to support healthy ageing are diverse and may target individuals, their environment, or both. Such interventions typically target factors that contribute to health and wellbeing in older adults, aiming to maximise independence and quality of life. They involve a combination of approaches, which may be tailored to the needs and circumstances of the individual.fi Eligible studies were randomised controlled trials evaluating community based complex interventions to sustain independence in older people that met the criteria in table 1. DA searched the following databases and registers from inception between 9 and 11 August 2021: Cochrane Central Register of Controlled Trials (CENTRAL) (1992-); Medline (1946-); Embase and Embase Classic (1947-); CINAHL (1972-); PsycINFO (1806-); ClinicalTrials.gov (www.clinicaltrials.gov); and WHO International Clinical Trials Registry platform (https://trialsearch.who.int). Search strategies and their development are detailed in appendix 3. We also scanned reference lists of included reports (backward citation searches). The 2023 Chief Medical Officer’s Report on Health in an Ageing Society identifies maximising independence for older people as a policy priority for England.4 8 The report highlights an evidence gap regarding the effectiveness of complex interventions.4 Previous systematic reviews have indicated that, overall, such interventions probably have small but positive effects, despite some limitations in the underlying evidence.9-11 However, they have been unable to indicate which service models or components may be most effective. This field has seen further growth, and systematic review methods have developed strongly.12-14 Network meta-analysis extends traditional pairwise meta-analysis by comparing multiple interventions simultaneously in a single analysis, aiming to generate more precise results and rank them against each other to support policy and commissioning decisions.15 Network meta-analysis has not been used to evaluate community based complex interventions to sustain independence for older people. Therefore, we aimed to provide a rigorous, contemporary synthesis of trial evidence by using network meta-analysis to identify how interventions might best be configured to improve outcomes for older people and inform policy, commissioning, and delivery of evidence based services. Duplicate records were removed with EndNote. Two reviewers independently used Rayyan (https://rayyan. ai/reviews) or Covidence (https://app.covidence.org/ reviews) to evaluate eligibility of records (title and abstract) and, if potentially eligible, their reports (full text). We arranged translation as necessary. Disagreements were resolved by consensus, with guidance from the Project Management Group. est. Protected by copyright. Objectives The global population aged over 65 years is expected to grow from 771 million people (10% of the population) in 2022 to 994 million by 2030 and 1.6 billion by 2050 (16%), necessitating change to healthcare systems.1 As the gap between life expectancy and healthy life expectancy grows, initiatives such as the World Health Organization’s Decade of Healthy Ageing seek to reverse this trend, maximising independence and social participation in later years as a shared global priority.2-4 This is important as an individual right, often diminished by ageism, but also to enable older people’s contribution to society and limit growing healthcare expenditure.2 5-7 Our objectives were to identify randomised controlled trials and cluster randomised controlled trials of community based complex interventions to sustain independence in older people; synthesise evidence of their effectiveness for key outcomes in a meta- analysis of study level data; identify key intervention components and study level frailty to inform groupings for network meta-analysis and meta-regression; compare effectiveness of different intervention configurations by using network meta-analysis; and investigate the impact of frailty and pre-frailty by using meta-regression. Data collection Two reviewers independently extracted data in a custom built Microsoft Access database, with data finalised automatically in case of agreement or resolved by a third reviewer. The main outcomes were living at home, activities of daily living (instrumental/ personal), hospital admission, care home placement, homecare services usage, costs, and cost effectiveness. Additional outcomes were health status, depression, loneliness, falls, and mortality. doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj RESEARCH RESEARCH Table 1 \| Eligibility criteria for studies Characteristic Include Exclude Population Older people (mean age ≥65 years) living at home at study entry Participants living in residential care or nursing homes (care homes)25 Intervention Initiated and mainly provided in the community Interventions delivered in other settings (eg, outpatient, day hospital, intermediate care) Included two or more interacting components (intervention practices, structural elements, and contextual factors) Interventions including only one discrete component (eg, exercise only) Targeted at the individual person, with provision of appropriate specialist care Interventions not targeting the individual, such as general staff education or practice reorganisation Focused on sustaining (maintaining or improving) the person’s independence Interventions not explicitly aimed at sustaining independence in activities of daily living Disease focused interventions (eg, for diabetes, depression) Falls prevention interventions Comparators Usual care, “placebo,” or attention control or a different complex intervention that met criteria Non-complex interventions Outcomes Outcome domains did not form part of eligibility criteria Outcome data were measured only before 24 weeks Study design Randomised controlled trials or cluster randomised controlled trials including all variants such as crossover, waiting list control, and stepped wedge designs Post-crossover data due to likelihood of carryover. Studies for which only one unit (ie, individual or cluster) was randomised to an arm Report characteristics All reports regardless of publication status, date, or language - Table 1 \| Eligibility criteria for studies on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ MJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from revised Cochrane risk of bias tool for randomised trials (RoB 2).12 29 Our effect of interest was assignment to the intervention (intention to treat). We rated risk of bias per domain and overall as low, some concerns, or high (serious or very serious concerns), through consensus between reviewers. Intervention classification We meta-analysed the effect estimates by using Stata modules including metan, mvmeta, and network.30 We synthesised dichotomous outcomes as log transformed odds ratios.31 For continuous outcomes, we used Hedges’ g standardised mean difference. We did random effects meta-analyses.32 We grouped all eligible interventions (including comparators) in preparation for network meta-analysis in a three stage process. Firstly, one reviewer (MJ, NL, RR, LM, IP, or EP) coded and summarised each intervention against the Template for Intervention Description and Replication items,27 28 and TC checked both coding and summaries, with disagreements resolved through discussion and involvement of the Project Management Group as necessary. Secondly, we generated categories of key intervention features through qualitative analysis, iteratively consolidating codes into categories. Thirdly, we grouped interventions on the basis of these categories, using a typology developed through discussion between reviewers, the Project Management Group, and experts including policy makers, commissioners, older people, and carers. The intervention groups became the network meta-analysis nodes. Initially, for each outcome and timeframe, we did a separate meta-analysis for each type of intervention versus control, to provide summary effectiveness results and forest plots based only on direct evidence. We then used network meta-analysis to compare relative effectiveness of all intervention types concurrently, combining direct evidence with indirect evidence, which is based on a network of intermediate comparisons between intervention types.15 We did network meta-analysis (for each outcome and timeframe separately) by using a multivariate random effects meta-analysis framework via the network module in Stata using restricted maximum likelihood estimation.30 We produced summary (pooled) effect estimates for each pair of treatments in the network, with 95% confidence intervals and network plots. guest. Protected by copyright. Assessment of frailty Two reviewers with extensive clinical-academic frailty expertise (AC and JGl) independently categorised study level frailty (robust, pre-frailty, frailty) on the basis of validated measures where available or participants’ characteristics and study inclusion criteria using the phenotype model as a framework.26 Data synthesis We did separate meta-analyses for each timeframe for living at home (dichotomous), personal activities of daily living (continuous), instrumental activities of daily living (continuous), and care home placement (dichotomous) and in the medium term for hospital admission (dichotomous), health status (continuous), and depression (continuous). We narratively synthesised other outcomes owing to a lack of data suitable for meta- analysis. We preferred adjusted effect estimates but calculated effect estimates from extracted data where necessary and possible, applying cluster adjustment where applicable.f Data collection Data were extracted (including treatment effect estimates) and categorised into three timeframes: short term (around six months): 24 weeks to 9 months; medium term (around 12 months): >9 months to 18 months; and long term (around 24 months): >18 months. Medium term was our main timeframe. Other data items collected (including design and participant details) are listed in appendix 4. We did not routinely seek missing data but sought to clarify ambiguities. Intervention characteristicsi Intervention characteristics We identified 19 separate components of included interventions (see box 1 and appendix 9), which were evaluated in 63 combinations including the absence of all these components, which we termed “available care.” “Homecare” was another common control group in populations in which all participants were receiving formal homecare. Homecare was one of 14 action components (further examples include education and exercise). Five other components primarily involved a tailoring process of ascertainment or assessment and planning with potential for subsequent multifactorial action, most often “multifactorial action” from care planning (a process of individualised multidomain assessment and management) with or without routine “review” (scheduled, regular follow-ups). Multifactorial action was further delineated according to the presence or absence of an embedded medication review and specific self-management strategies. Twenty six intervention groups (combinations) were evaluated in more than one study, and these are summarised in relation to Template for Intervention Description and Replication items in appendix 10. Study selection Figure 1 shows the study selection process. Our database and register searches identified 51 180 records. After de-duplication, we screened 40 112 records and then assessed 794 reports for eligibility. Subsequently, we assessed 179 reports identified through searching for additional reports of included studies and backward citation searches. We excluded 477 reports; reasons are listed in appendix 6. We included 129 studies with 266 eligible intervention arms presented in 496 reports39-167; 90 studies contributed to network meta-analyses. We examined the consistency assumption (that direct and indirect evidence are consistent with each other) for each treatment comparison where possible and across the whole network.34 35 We summarised heterogeneity by the estimate of between study variance (τ), where possible.f Risk of bias Two reviewers independently assessed risk of bias in each result of interest from each study, using the 3 the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 RESEARCH RESEARCH on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from summary of our findings, developed with Frailty Oversight Group members, is presented in appendix 2. On the basis of the results, we calculated the ranking of intervention groups by using re-sampling methods. To facilitate interpretation, we transformed each summary odds ratio to a summary risk ratio by using the median risk in the reference comparator arms and corresponding absolute intervention risks and risk differences by using the highest and lowest risk among reference comparator arms (n≥100) as the assumed comparator risks.33 We re-expressed standardised mean differences as the mean difference by using a pooled standard deviation for a common measure of the outcome. Included study characteristics We examined the effect of study level frailty on each intervention group effect where data allowed by using network meta-regression. We did sensitivity analyses excluding results rated as at very serious risk of bias. We examined funnel plots for small study effects if 10 or more studies were available. The 129 studies assigned 74 946 participants (reported by 126 studies; 61% (44 817/72 877) women and 39% (28 060/72 877) men, from 123 studies). Studies were predominantly conducted in developed countries, and most participants were described as white. Nevertheless, the overall population included a broad range of demographic characteristics. Study populations included all frailty levels. Study characteristics are summarised in appendix 7. We judged most results to be at high risk of bias, primarily owing to missing outcome data (appendix 8). or more studies were available. We used the GRADE framework, adapted for network meta-analysis, to rate the certainty of the results of our network meta-analysis and presented summary of findings tables ordered by certainty and ranking.14 16- 19 21 We used plain language terms “probably” and “may” to indicate moderate and low certainty respectively and qualified the size and direction of point estimates (see appendix 5).20 36 We followed the brief economic commentary framework to summarise and compare the principal economic findings reported by included studies.37 We used the GRADE framework, adapted for network meta-analysis, to rate the certainty of the results of our network meta-analysis and presented summary of findings tables ordered by certainty and ranking.14 16- 19 21 19 21 We used plain language terms “probably” and “may” to indicate moderate and low certainty respectively and qualified the size and direction of point estimates (see appendix 5).20 36 We followed the brief economic commentary framework to summarise and compare the principal economic findings reported by included studies.37 Patient and public involvementi This review benefited from the involvement of our established Patient and Public Involvement Frailty Oversight Group in the Bradford Institute for Health Research. The group has a structure that provides connections to the whole spectrum of older people, with a focus on those living with frailty to enable meaningful, public involvement in our research projects.38 We consulted our Frailty Oversight Group throughout the development of the protocol and discussed plans in detail at the group’s quarterly meetings and at our annual consumer research conference. Group members helped to draft and revise the plain language summary for our funding application. Other examples of patient and public involvement include the selection of important outcomes and their prioritisation as main and additional outcomes. Frailty Oversight Group members emphasised that a wide range of outcomes were important to older people, with a particular focus on independence in addition to wellbeing, alongside service orientated outcomes. We also spent time discussing the intervention components that we had identified with group members. Through this work, we developed and refined our descriptions of the components and thus the findings. The plain language Characteristics of network meta-analyses Most networks were small and sparse, with few included studies contributing to most networks (fig 2). We found little evidence of inconsistency or small study effects, but the power to detect this was usually low. All outcomes except mortality needed to be analysed in two separate network meta-analyses, as doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj RESEARCH RESEARCH the networks were disconnected: one with available care as the reference comparator (“available care network”) and one with homecare as the reference comparator (“homecare network”). Estimates are reported here only in comparison with the reference comparator. Patient and public involvementi Comparison with available care is the Reports excluded Ongoing (24 studies) Insuﬃcient information Excluded Study design not RCT/cRCT Particpants Mean age <65 years Not living at home Intervention Not community based Single component only Not targeting individual Not sustaining independence Targets speciﬁc conditions Falls prevention Comparator Not community based Single component intervention Not targeting individual Outcomes No follow-up ≥24 weeks 32 6 427 465 Records identiﬁed Identiﬁcation of studies via databases and registers Databases CENTRAL Medline Embase PsycINFO CINAHL 49 894 Records screened Duplicate records removed before screening 11 068 Records excluded 39 318 Reports not retrieved 40 112 Reports sought for retrieval 51 180 0 Reports not retrieved 0 7003 7005 17 333 7917 10 636 1286 861 425 Registers ClinicalTrials.gov WHO ICTRP Records identiﬁed Identiﬁcation of studies via other methods Backward citation searching from included studies Searching for additional reports of included studies 19 160 76 5 25 113 32 2 102 12 3 7 12 3 35 794 Reports sought for retrieval 179 Reports assessed for eligibility 794 Reports assessed for eligibility 179 Studies included in review 123 Reports of included studies 329 Total studies included in review 129 Total reports of included studies 496 Reports excluded Study design not RCT/cRCT Particpants Mean age <65 years Not living at home Intervention Not community based Single component only Not sustaining independence Comparator Single component intervention 1 1 1 2 1 4 2 Studies included in review from citation searching Reports of included studies from citation searching 179 12 6 7 Additional reports of included studies 160 Fig 1 \| PRISMA flow diagram showing identification, selection, and inclusion of studies from databases, registers, and other sources.23 ICTRP=International Clinical Trials Registry Platform on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from on 23 October 2024 by http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Identiﬁcation of studies via databases and registers on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ ram showing identification, selection, and inclusion of studies from databases, registers, and other sources.23 nical Trials Registry Platform g 1 \| PRISMA flow diagram showing identification, selection, and inclusion of studies from databases, registers, CTRP=International Clinical Trials Registry Platform comparator (“homecare network”). Estimates are reported here only in comparison with the reference comparator. Living at home For living at home in the medium term, the available care network included 21 studies (n=16 937) with 14 intervention groups (fig 4). We found moderate certainty evidence that “multifactorial action and review with medication review” probably results in a slight increase in the odds of living at home (odds ratio 1.22, 95% confidence interval 0.93 to 1.59). We found low certainty evidence that “multifactorial action with medication review” (odds ratio 2.55 (large), 0.61 to 10.60), “cognitive training, medication review, nutrition, and exercise” (1.93 (large), 0.79 to 4.77), and “activities of daily living training, nutrition, and exercise” (1.79 (large), 0.67 to 4.76) may result in an increase in the odds of living at home and that “risk screening” (0.90 (very small), 0.66 to 1.23), “education and multifactorial action and review with medication review” (0.88 (very small), 0.60 to 1.29), and “education, multifactorial action, and review with medication review and self-management strategies” (0.41 (very large), 0.14 to 1.17) may each result in some reduction in the odds of living at home. Other comparisons were very low certainty. For the homecare network, we had one low certainty finding in the short term timeframe of little to no difference for “homecare, activities of daily living training and multifactorial action and review with self- management strategies,” with all other comparisons being of very low certainty. Patient and public involvementi Comparison with available care is the the networks were disconnected: one with available care as the reference comparator (“available care network”) and one with homecare as the reference the networks were disconnected: one with available care as the reference comparator (“available care network”) and one with homecare as the reference the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 5 RESEARCH RESEARCH In the short term and long term timeframes, results were low certainty at best. For “multifactorial action and review with medication review” and “activities of daily living traning, nutrition, and exercise,” estimates were similarly of small increases in the long term but of little to no difference in the short term. For “education, multifactorial action, and review with medication review and self-management strategies” and “risk screening,” we found similar results in other timeframes (may reduce living at home), but for “education and multifactorial action and review with medication review,” we found contrasting evidence of an increase in living at home. Box 1: Nineteen identified components of community based complex interventions* intended to sustain independence in older people Action components • “Activities of daily living training” • Providing “aids” and adaptations • “Alternative medicine” • “Care voucher” provision • “Cognitive training” • Health “education” • Physical “exercise” • Formal “homecare” • Engagement in “meaningful activities” • “Nutrition”(al) support • Psychological (mood) therapy (“psychology”) • “Social skills” training • Technology for communication and engagement (“telecoms”) • “Welfare” rights advice. Tailoring components • “Multifactorial action” from individualised care planning • Routine “review” • “Medication review” • “Monitoring” • Routine “risk screening” Text in quotation marks is short version of name used in results Box 1: Nineteen identified components of community based complex interventions intended to sustain independence in older people i p y p interventions* intended to sustain independence in older people Action components • “Activities of daily living training” • Providing “aids” and adaptations • “Alternative medicine” • “Care voucher” provision • “Cognitive training” • Health “education” • Physical “exercise” • Formal “homecare” • Engagement in “meaningful activities” • “Nutrition”(al) support • Psychological (mood) therapy (“psychology”) • “Social skills” training • Technology for communication and engagement (“telecoms”) • “Welfare” rights advice. Instrumental activities of daily living effect of adding the intervention for a population who are not all receiving any particular care; comparison with homecare is similarly an alternative intervention for a population already in receipt of homecare without associated reablement or multifactorial action from care planning. Most estimates were of low certainty or very low certainty owing to risk of bias, imprecision, or their combination, and we do not detail very low certainty estimates below. Full results are presented in appendix 11 and summarised in figure 3. The medium term instrumental activities of daily living available care network included 16 studies (n=5309) with 14 intervention groups. We found moderate certainty evidence that “multifactorial action and review with medication review” was associated with very slightly increased independence in instrumental activities of daily living versus “available care” (standardised mean difference 0.11, 95% confidence interval 0.00 to 0.21). Two intervention groups may result in some reduction in instrumental activities of daily living: “activities of daily living training, aids, and exercise” and “activities of daily living training, aids, education, exercise, and multifactorial action and review with medication review and self-management strategies.” The findings for “multifactorial action and review with medication review” in the long term were contrasting, with moderate certainty evidence of a very slight reduction in instrumental activities of daily living (standardised mean difference −0.08, −0.21 to 0.05). Action components • Health “education” • Physical “exercise” • Formal “homecare” • Engagement in “meaningful activities” The homecare network for living at home was smaller (five studies; n=1978 in the medium term). In the short and medium term timeframes we found low certainty evidence that “homecare, activities of daily living traing, and multifactorial action and review with self-management strategies” may result in reductions (short term, odds ratio 0.63 (moderate), 0.31 to 1.26; medium term, 0.76 (large), 0.40 to 1.45) in the odds of living at home compared with “homecare” alone. In the short term, “homecare and nutrition” may result in reductions in the odds of living at home compared with “homecare” (odds ratio 0.34 (very large), 0.12 to 0.95). Other comparisons were of very low certainty. • Psychological (mood) therapy (“psychology”) • “Social skills” training Text in quotation marks is short version of name used in results Patient and public involvementi Tailoring components • “Multifactorial action” from individualised care planning • Routine “review” • “Medication review” • “Monitoring” • Routine “risk screening” Text in quotation marks is short version of name used in results Personal activities of daily living For personal activities of daily living, 20 trials (n=8583 participants) with 16 intervention groups contributed to the medium term available care network. One comparison was low certainty: “exercise and multifactorial action and review with medication doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj RESEARCH RESEARCH Living at home in medium term (AC) Personal ADL in medium term (AC) Living at home in medium term (hmcr) Personal ADL in medium term (hmcr) Instrumental ADL in medium term (AC) Hospital admission in medium term (AC) educ and mfar (w/med) (1800) educ and mfar (w/med+slfm) (172) educ and rsk-mfa- (258) educ and rsk-mfa- (308) eng and educ (95) eng and educ (96) educ and psyc (23) exrc and psyc (23) cgn and med and ntr and exrc (170) ADL and ntr and exrc (144) med and ntr and exrc (85) med and ntr and exrc (85) ntr and exrc (61) ADL (141) ADL (141) ADL and aids and educ and exrc and mfar (w/med+slfm) (130) ADL and aids and educ and exrc and mfar (w/med+slfm) (130) educ and exrc and mfar (w/med+slfm) (100) educ and mfar (w/med+slfm) (172) exrc and mfar (w/med+slfm) (120) mfar (w/med+slfm) (130) educ and mfar (w/med) (1523) educ and rsk-mfa- (217) ADL and aids and exrc (149) cgn and ntr and exrc (46) ADL and med and ntr and sst (139) aids (34) rsk-mfa- (1955) rsk-mfa- (1775) mntr-mfa- (77) mfar (w/med) (3016) mfar (w/med) (702) mfar (w/med) (880) mfar (282) mfar (61) mfar (242) mfar (775) mfa- (w/med+slfm) (243) mfa- (w/med+slfm) (288) mfa- (w/med+slfm) (288) educ and mfar (w/med+slfm) (177) hmcr and educ and mfar (374) hmcr and ADL and mfar (w/slfm) (174) hmcr and ADL and mfar (w/slfm) (133) hmcr and mfa- (45) hmcr and mfar (w/med) (99) hmcr and hmnt and exrc (29) mntr-mfa- (102) exrc and mfar (w/med+slfm) (106) mfa- (w/med) (50) mfa- (w/med) (75) mfa- (w/med) (120) mfa- (147) hmcr and mfar (w/sﬂm) (81) mfar (w/med) (1900) hmcr and mfar (w/med) (399) hmcr and mfar (122) hmcr and ntr (96) hmcr and ntr (63) hmcr (890) hmcr (308) AC (8492) AC (3136) AC (4734) AC (4080) ig 2 \| Network plots for analyses of main outcomes in medium term (~12 months) that yielded ≥1 finding of at least low certainty. AC indicates etwork including available care; hmcr indicates network including formal homecare. Each node is labelled with intervention group abbreviation nd number of participants. Personal activities of daily living Node size is proportionate to number of participants; edge thickness is proportionate to number of comparisons. ntervention and control group abbreviations are combination of: ADL=activities of daily living training; aids=provision of aids and adaptations; gn=cognitive training; comm=technology for communication and engagement; educ=health education; eng=engagement in meaningful ctivities; exrc=physical exercise; hmcr=formal homecare; hmnt=alternative medicine; med=medication review; mfa=multifactorial action; mfar=multifactorial action and follow-on routine review; mntr-mfa=monitoring, which may trigger multifactorial action; ntr=nutritional support; syc=psychological therapy; rsk-mfa=risk screening, which may trigger multifactorial action; sst=social skills training; vchr=care voucher provision; wlfr=welfare rights advice; w/med=with medication-review; w/slfm=with self-management strategies on 23 October 2024 by guest. Protected by co http://www.bmj.com/ BMJ: first published as 10.1136/bmj 2023 077764 on 21 March 2024. Downloaded from on 23 October 2024 by guest. Protected by co http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Living at home in medium term (AC) educ and mfar (w/med) (1800) educ and rsk-mfa- (258) cgn and med and ntr and exrc (170) ADL and ntr and exrc (144) rsk-mfa- (1955) mfar (w/med) (3016) mfar (282) mfa- (w/med+slfm) (243) educ and mfar (w/med+slfm) (177) mfa- (w/med) (50) mfa- (147) hmcr and mfar (w/sﬂm) (81) hmcr and mfar (122) AC (8492) Personal ADL in medium term (hmcr) Fig 2 \| Network plots for analyses of main outcomes in medium term (~12 months) that yielded ≥1 finding of at least low certainty. AC indicates network including available care; hmcr indicates network including formal homecare. Each node is labelled with intervention group abbreviation and number of participants. Node size is proportionate to number of participants; edge thickness is proportionate to number of comparisons. Intervention and control group abbreviations are combination of: ADL=activities of daily living training; aids=provision of aids and adaptations; cgn=cognitive training; comm=technology for communication and engagement; educ=health education; eng=engagement in meaningful activities; exrc=physical exercise; hmcr=formal homecare; hmnt=alternative medicine; med=medication review; mfa=multifactorial action; mfar=multifactorial action and follow-on routine review; mntr-mfa=monitoring, which may trigger multifactorial action; ntr=nutritional support; psyc=psychological therapy; rsk-mfa=risk screening, which may trigger multifactorial action; sst=social skills training; vchr=care voucher provision; wlfr=welfare rights advice; w/med=with medication-review; w/slfm=with self-management strategies The homecare network included four trials (n=632 participants) in the medium term. Here too, only one comparison with “homecare” was low certainty: “homecare and multifactorial action and review review and self-management startegies” may result in a very slight increase in personal activities of daily living (standardised mean difference 0.16, −0.51 to 0.82). Personal activities of daily living Downloaded from Moderate certainty Moderate to very large Small Very small Little to no diﬀerence Very small Small Worsening Improvement Moderate to very large Low certainty Fig 3 \| Summary of moderate and low certainty evidence for main outcomes synthesised with network meta-analysis. In comparison with reference comparator. For intervention groups including homecare, reference comparator is homecare; for all other intervention groups, reference comparator is available care. Living at home (+ favoured); instrumental ADL (+ favoured); personal ADL (+ favoured); hospital admission (– favoured); care home placement (– favoured). ADL=activities of daily living; T1=short term timeframe (24 weeks to 9 months); T2=medium term timeframe (>9 months to 18 months; T3=long term timeframe (>18 months); +++++=very large increase; ++++=large increase; +++=moderate increase; ++= slight increase; +=very slight increase; ~=little to no difference; -=very slight reduction;--=slight reduction; ---=moderate reduction; ----=large reduction; -----=very large reduction. Blue shades indicate possible benefit; orange shades indicate possible harm; bold indicates moderate certainty evidence on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Moderate certainty Moderate to very large Small Very small Little to no diﬀerence Very small Small Worsening Improvement Moderate to very large Low certainty Fig 3 \| Summary of moderate and low certainty evidence for main outcomes synthesised with network meta-analysis. In comparison with reference comparator. For intervention groups ncluding homecare, reference comparator is homecare; for all other intervention groups, reference comparator is available care. Living at home (+ favoured); instrumental ADL (+ favoured); personal ADL (+ favoured); hospital admission (– favoured); care home placement (– favoured). ADL=activities of daily living; T1=short term timeframe (24 weeks to 9 months); T2=medium erm timeframe (>9 months to 18 months; T3=long term timeframe (>18 months); +++++=very large increase; ++++=large increase; +++=moderate increase; ++= slight increase; +=very slight ncrease; ~=little to no difference; -=very slight reduction;--=slight reduction; ---=moderate reduction; ----=large reduction; -----=very large reduction. Blue shades indicate possible benefit; orange shades indicate possible harm; bold indicates moderate certainty evidence on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Personal activities of daily living the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 7 7 RESEARCH RESEARCH Low certainty moderate and low certainty evidence for main outcomes synthesised with network meta-analysis. In comparison with reference comparator. For intervention groups , reference comparator is homecare; for all other intervention groups, reference comparator is available care. Living at home (+ favoured); instrumental ADL (+ favoured); oured); hospital admission (– favoured); care home placement (– favoured). ADL=activities of daily living; T1=short term timeframe (24 weeks to 9 months); T2=medium months to 18 months; T3=long term timeframe (>18 months); +++++=very large increase; ++++=large increase; +++=moderate increase; ++= slight increase; +=very slight no difference; -=very slight reduction;--=slight reduction; ---=moderate reduction; ----=large reduction; -----=very large reduction. Blue shades indicate possible benefit; cate possible harm; bold indicates moderate certainty evidence on 23 October 2024 by guest. Protected by c http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from ork meta-analysis. In comparison with reference comparator. For intervention groups e comparator is available care. Living at home (+ favoured); instrumental ADL (+ favoured); L=activities of daily living; T1=short term timeframe (24 weeks to 9 months); T2=medium e increase; ++++=large increase; +++=moderate increase; ++= slight increase; +=very slight tion; ----=large reduction; -----=very large reduction. Blue shades indicate possible benefit; BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded f Moderate certainty Moderate to very large Small Very small Little to no diﬀerence Very small Small Worsening Improvement Moderate to very large Low certainty Fig 3 \| Summary of moderate and low certainty evidence for main outcomes synthesised with network meta-analysis. In comparison with reference comparator. For intervention groups ncluding homecare, reference comparator is homecare; for all other intervention groups, reference comparator is available care. Living at home (+ favoured); instrumental ADL (+ favoured); personal ADL (+ favoured); hospital admission (– favoured); care home placement (– favoured). ADL=activities of daily living; T1=short term timeframe (24 weeks to 9 months); T2=medium erm timeframe (>9 months to 18 months; T3=long term timeframe (>18 months); +++++=very large increase; ++++=large increase; +++=moderate increase; ++= slight increase; +=very slight ncrease; ~=little to no difference; -=very slight reduction;--=slight reduction; ---=moderate reduction; ----=large reduction; -----=very large reduction. Blue shades indicate possible benefit; orange shades indicate possible harm; bold indicates moderate certainty evidence on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Personal activities of daily living Summary of findings table shows relative effects and anticipated absolute effects of each it b d l i t ti t d ith il bl ( ) f li i t h t i di t Li i t h i bi t lt ti t b i ith Intervention group Relative risk (95% CI) Network estimate Anticipated absolute eﬀect (95% CI) Interpretation Ranking (95% CI) Certainty of evidence (GRADE) Multifactorial action and review with medication review Multifactorial action with medication review Cognitive training, medication review, nutrition, and exercise Activities of daily living training, nutrition, and exercise Risk screening Education and multifactorial action and review with medication review Education and multifactorial action and review with medication review and self-management strategies Homecare and multifactorial action and review with self-management strategies Homecare and multifactorial action and review Multifactorial action Multifactorial action and review Education and risk screening Multifactorial action with medication review and self-management strategies OR 1.22 (0.93 to 1.59) Mixed estimate OR 2.55 (0.61 to 10.60) Mixed estimate OR 1.93 (0.79 to 4.77) Mixed estimate OR 1.79 (0.67 to 4.76) Mixed estimate OR 0.90 (0.66 to 1.23) Mixed estimate OR 0.88 (0.60 to 1.29) Mixed estimate OR 0.41 (0.14 to 1.17) Mixed estimate OR 8.89 (1.90 to 41.62) Indirect estimate OR 5.71 (1.65 to 19.83) Mixed estimate OR 2.23 (0.63 to 7.91 Mixed estimate OR 1.15 (0.60 to 2.18) Mixed estimate OR 1.09 (0.60 to 2.01) Mixed estimate OR 1.00 (0.63 to 1.58) Mixed estimate Probably results in slight increase in chance of living at home May result in increase in chance of living at home May result in increase in chance of living at home May result in increase in chance of living at home May result in very slight reduction in chance of living at home May result in very slight reduction in chance of living at home May result in reduction in chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Calculated risk ratio RR 1.01 (1.00 to 1.02) RR 1.04 (0.96 to 1.06) RR 1.03 (0.98 to 1.05) RR 1.03 (0.97 to 1.05) RR 0.99 (0.97 to 1.01) RR 0.99 (0.96 to 1.01) RR 0.91 (0.72 to 1.01) RR 1.06 (1.03 to 1.07) RR 1.06 (1.03 to 1.07) RR 1.04 (0.96 to 1.06) RR 1.01 (0.96 to 1.04) RR 1.01 (0.96 to 1.03) RR 1.00 (0.96 to 1.02) High risk population (833 per 1000 with ac) 26 more per 1000 (10 fewer to 55 more) 94 more per 1000 (79 fewer to 148 more) 73 more per 1000 (36 fewer to 127 more) 66 more per 1000 (62 fewer to 127 more) 15 fewer per 1000 (65 fewer to 27 more) 19 fewer per 1000 (84 fewer to 32 more) 163 fewer per 1000 (420 fewer to 21 more) 145 more per 1000 (71 more to 162 more) 133 more per 1000 (58 more to 157 more) 84 more per 1000 (75 fewer to 142 more) 18 more per 1000 (82 fewer to 83 more) 12 more per 1000 (85 fewer to 76 more) 0 per 1000 (74 fewer to 55 more) Low risk population (981 per 1000 with ac) 3 more per 1000 (1 fewer to 7 more) 11 more per 1000 (12 fewer to 17 more) 9 more per 1000 (5 fewer to 15 more) 8 more per 1000 (9 fewer to 15 more) 2 fewer per 1000 (9 fewer to 3 more) 3 fewer per 1000 (12 fewer to 4 more) 26 fewer per 1000 (102 fewer to 3 more) 17 more per 1000 (9 more to 19 more) 16 more per 1000 (7 more to 18 more) 10 more per 1000 (11 fewer to 17 more) 2 more per 1000 (12 fewer to 10 more) 2 more per 1000 (13 fewer to 9 more) 0 per 1000 (11 fewer to 7 more) 7.5 (5 to 11) 4.6 (1 to 13) 5.3 (2 to 12) 5.9 (2 to 13) 11.1 (7 to 13) 11.2 (6 to 14) 13.5 (8 to 14) 1.5 (1 to 4) 2.4 (1 to 5) 4.9 (1 to 13) 8.4 (4 to 13) 8.9 (4 to 13) 9.8 (5 to 13) Moderate† Low‡ Low‡ Low‡ Low‡ Low‡ Low‡ Very low§ ¶ Very low** †† Very low‡ ‡‡ Very low‡ §§ Very low‡ ‡‡ Very low‡ ¶¶ 4 \| Living at home in medium term: comparisons with available care summary of findings table. Personal activities of daily living Summary of findings table shows relative effects and anticipated absolute effects of each mmunity based complex intervention type compared with available care (ac) for living at home outcome in medium term. Living at home is binary outcome, alternative outcome being either e home placement or death. Relative effects are odds ratio (OR) from network meta-analysis (NMA) and risk ratio (RR) calculated from odds ratio. OR>1 (or RR>1) favours listed community sed complex intervention; OR<1 favours available care. Intervention types are ordered by certainty of evidence (high to very low) and ranking (highest to lowest). NMA included 21 studies, 14 des, and 16 937 participants in total. Follow-up ranged from 12 to 18 months. Heterogeneity was estimated as τ=8.56×10-2. Consistency assumption held. Mean rank of available care was 9.9 5% CI 7 to 12). Calculated from OR and assumed comparator risk of 0.935, median available care risk among these studies. †Serious concerns about imprecision as confidence interval (CI) sses no effect line and includes substantial benefit. CI for absolute effect with high risk population also includes pre-specified definition of very small harm, but given that this was marginal d in light of small lower CI for RR (0.9955), this was not judged as very serious. Downgrade once. ‡Very serious concerns about imprecision as CI includes substantial benefit and substantial rm. Downgrade twice. §Very serious concerns about risk of bias owing to exclusion of participants in per protocol analysis and missing outcome data in indirect evidence via homecare and ltifactorial action and review versus available care comparison. Downgrade twice. ¶Very serious concerns about imprecision as CI is very wide, no closed loop exists, and direct comparison based on indirect evidence from 122 people in homecare and multifactorial action and review and 81 people in homecare and multifactorial action and review with self-management, which es not meet optimal information size. Already downgraded twice for risk of bias; downgrade once. Very serious concerns about risk of bias owing to exclusion of participants in per protocol alysis and missing outcome data. Downgrade twice. ††Very serious concerns about imprecision as CI is very wide, no closed loop exists, and direct comparison is based on indirect evidence m 122 people in homecare and multifactorial action and review, which does not meet optimal information size. Already downgraded twice; downgrade once. ‡‡Serious concerns about risk of s owing to missing outcome data. Downgrade once. Personal activities of daily living Downloaded from Intervention group M d t t S ll V Little to no diﬀerence V S ll Worsening Improvement M d t t Living at home T1 T2 T3 T1 T2 T3 T1 T2 T2 T3 T1 T2 T3 Instrumental ADL Care home placement Hospital admission Personal ADL ADL, aids, education, exercise, and multifactorial action and review with medication review and self-management strategies ADL, aids, and exercise ADL, nutrition, and exercise Cognitive training, medication review, nutrition, and exercise Education, exercise, multifactorial action, and review with medication review and self-management strategies Education and multifactorial action and review with medication review Education and multifactorial action and review with medication review and self-management strategies Exercise and multifactorial action and review (with medication review and self-management) Meaningful activities and multifactorial action with self-management strategies Multifactorial action and review Multifactorial action and review with medication review Multifactorial action with medication review Risk screening Homecare, ADL and multifactorial action and review with self-management strategies Homecare and nutrition Homecare and multifactorial action and review with medication review ~ ~ ~ ~ ++++ ++ ++ ++ + + + + + + ++ + + -- ++++ ----- +++ +++ ++++ +++ --- --- ----- ++++ ++++ --- ---- ----- -- -- - - - -- -- -- -- -- 8 8 doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj RESEARCH RESEARCH Intervention group Relative risk (95% CI) Network estimate Anticipated absolute eﬀect (95% CI) Interpretation Ranking (95% CI) Certainty of evidence (GRADE) Multifactorial action and review with medication review Multifactorial action with medication review Cognitive training, medication review, nutrition, and exercise Activities of daily living training, nutrition, and exercise Risk screening Education and multifactorial action and review with medication review Education and multifactorial action and review with medication review and self-management strategies Homecare and multifactorial action and review with self-management strategies Homecare and multifactorial action and review Multifactorial action Multifactorial action and review Education and risk screening Multifactorial action with medication review and self-management strategies OR 1.22 (0.93 to 1.59) Mixed estimate OR 2.55 (0.61 to 10.60) Mixed estimate OR 1.93 (0.79 to 4.77) Mixed estimate OR 1.79 (0.67 to 4.76) Mixed estimate OR 0.90 (0.66 to 1.23) Mixed estimate OR 0.88 (0.60 to 1.29) Mixed estimate OR 0.41 (0.14 to 1.17) Mixed estimate OR 8.89 (1.90 to 41.62) Indirect estimate OR 5.71 (1.65 to 19.83) Mixed estimate OR 2.23 (0.63 to 7.91 Mixed estimate OR 1.15 (0.60 to 2.18) Mixed estimate OR 1.09 (0.60 to 2.01) Mixed estimate OR 1.00 (0.63 to 1.58) Mixed estimate Probably results in slight increase in chance of living at home May result in increase in chance of living at home May result in increase in chance of living at home May result in increase in chance of living at home May result in very slight reduction in chance of living at home May result in very slight reduction in chance of living at home May result in reduction in chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Evidence is very uncertain about eﬀect on chance of living at home Calculated risk ratio* RR 1.01 (1.00 to 1.02) RR 1.04 (0.96 to 1.06) RR 1.03 (0.98 to 1.05) RR 1.03 (0.97 to 1.05) RR 0.99 (0.97 to 1.01) RR 0.99 (0.96 to 1.01) RR 0.91 (0.72 to 1.01) RR 1.06 (1.03 to 1.07) RR 1.06 (1.03 to 1.07) RR 1.04 (0.96 to 1.06) RR 1.01 (0.96 to 1.04) RR 1.01 (0.96 to 1.03) RR 1.00 (0.96 to 1.02) High risk population (833 per 1000 with ac) 26 more per 1000 (10 fewer to 55 more) 94 more per 1000 (79 fewer to 148 more) 73 more per 1000 (36 fewer to 127 more) 66 more per 1000 (62 fewer to 127 more) 15 fewer per 1000 (65 fewer to 27 more) 19 fewer per 1000 (84 fewer to 32 more) 163 fewer per 1000 (420 fewer to 21 more) 145 more per 1000 (71 more to 162 more) 133 more per 1000 (58 more to 157 more) 84 more per 1000 (75 fewer to 142 more) 18 more per 1000 (82 fewer to 83 more) 12 more per 1000 (85 fewer to 76 more) 0 per 1000 (74 fewer to 55 more) Low risk population (981 per 1000 with ac) 3 more per 1000 (1 fewer to 7 more) 11 more per 1000 (12 fewer to 17 more) 9 more per 1000 (5 fewer to 15 more) 8 more per 1000 (9 fewer to 15 more) 2 fewer per 1000 (9 fewer to 3 more) 3 fewer per 1000 (12 fewer to 4 more) 26 fewer per 1000 (102 fewer to 3 more) 17 more per 1000 (9 more to 19 more) 16 more per 1000 (7 more to 18 more) 10 more per 1000 (11 fewer to 17 more) 2 more per 1000 (12 fewer to 10 more) 2 more per 1000 (13 fewer to 9 more) 0 per 1000 (11 fewer to 7 more) 7.5 (5 to 11) 4.6 (1 to 13) 5.3 (2 to 12) 5.9 (2 to 13) 11.1 (7 to 13) 11.2 (6 to 14) 13.5 (8 to 14) 1.5 (1 to 4) 2.4 (1 to 5) 4.9 (1 to 13) 8.4 (4 to 13) 8.9 (4 to 13) 9.8 (5 to 13) Moderate† Low‡ Low‡ Low‡ Low‡ Low‡ Low‡ Very low§ ¶ Very low** †† Very low‡ ‡‡ Very low‡ §§ Very low‡ ‡‡ Very low‡ ¶¶ Fig 4 \| Living at home in medium term: comparisons with available care summary of findings table. Personal activities of daily living §§Serious concerns about risk of bias owing to recruitment process of participants and missing outcome data in one study. Downgrade ce. ¶¶Very serious concerns about risk of bias owing to randomisation process and missing outcome data. Already downgraded twice; downgrade once on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 9 RESEARCH RESEARCH with medication review” may result in an increase in personal activities of daily living (standardised mean difference 0.60 (moderate), 0.32 to 0.88). medication review, nutrition, and exercise”; and “activities of daily living training, nutrition, and exercise”) may result in an increase in the odds of living at home in this timeframe. For 12 other intervention groups, we found low certainty evidence that they may improve or worsen at least one main outcome, but for other intervention groups evidence was either absent or very uncertain.i Strengths and limitations of review Strengths and limitations of review We used rigorous methods to summarise the contemporary evidence on community based complex interventions for older people as an area of high strategic priority and global policy relevance. The evidence originated from a diverse population of older people, albeit mainly from high income regions. Population subgroups of various socioeconomic status, frailty levels, and ethnicity were included, and the ratio of women to men seemed typical for older populations.1 People with greater resource may be over-represented in many of these trials, given the voluntary nature of participation, which may have limited the effects of interventions and hence contributed to the small effect sizes presented here. Investigations of frailty and risk of bias Across all outcomes, risk of bias sensitivity analyses produced very similar estimates to the main analyses. Funnel plots showed little evidence of asymmetry. Network meta-regressions were unable to estimate the effects of frailty for many comparisons, and those that were estimable had very wide confidence intervals. guest. Protected by copyright. Additional outcomes Additional outcomes are summarised in appendix 13. We found little evidence of any effect on self-reported health status, only low certainty beneficial findings regarding depression, and very little evidence regarding loneliness; more complex interventions were associated with less falling than with more falling (12 v 4 studies). For mortality, we had a large network of 65 studies (n=38 351) and 41 intervention groups. We found low certainty evidence of reductions for two intervention groups and increases for five intervention groups. We developed a data driven grouping of interventions with expert guidance, so the groups were clearly specified to support practice, commissioning, and policy decisions. Our approach has resulted in a very clear typology of interventions, avoiding the historical problem of combining different interventions under unhelpful umbrella terms. We acknowledge that some clinical heterogeneity remains within intervention groups, particularly in the care available to different study populations. However, application of this typology led to the separation of evidence from populations in receipt of homecare and those that were not, reducing heterogeneity and improving the specificity of recommendations. doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj Other main outcomes For the service outcome of hospital admission, we found low certainty estimates of some reductions for “education, exercise, and multifactorial action and review with medication review and self-management strategies” and “education and multifactorial action and review with medication review” and of an increase for “exercise, multifactorial action, and review with medication review and self-management strategies.” For care home placement, all estimates were rated as very low certainty in the medium term. We found some evidence of both increases and decreases in use of homecare services with little pattern (not meta-analysed). We identified moderate certainty evidence in three of our analyses of main outcomes, all related to “multifactorial action and review with medication review.” In comparison with “available care,” we found a probable slight increase in living at home in the medium term and a very slight increase in independence in instrumental activities of daily living in the medium term, but in contrast also a very slight reduction in instrumental activities of daily living in the long term (fig 3). The direct evidence for probable worsening came from studies that also contributed to the beneficial medium term finding (tables 28 and 32 and figures 8 and 14, appendix 11).49 131 For homecare recipients, the addition of “multifactorial action and review with medication review” was associated with low certainty evidence of a moderate increase in personal activities of daily living in the medium term (fig 3). The summary of economic evidence included 39 studies (appendix 12). On the basis of the conclusions of 22 studies that did a full economic evaluation, five intervention groups seemed promising compared with a standard intervention or available care from an economic perspective: “activities of daily living training” (medium term time horizon); “homecare and multifactorial action and review with medication review and self-management strategies” (short term time horizon); “meaningful activities and education” (short and medium term time horizon); “multifactorial action and review with medication review” (short term but not medium or long term time horizon); and “exercise and multifactorial action with medication review” (long term time horizon). Principal findings For consistency, we limited our review to interventions focused explicitly on independence in activities of daily living. Although this differs from preventing institutionalisation or activity limitation, multifactorial action interventions focused on each of these may work similarly. As such interventions can Our review has found evidence that “multifactorial action and review with medication review” probably improves the odds of living at home in the medium term timeframe (around 12 months). We also found that other complex interventions (“multifactorial action with medication review”; “cognitive training, doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj 10 RESEARCH RESEARCH on 23 October 2024 by gu http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from have multiple foci, differences may reflect intervention reporting rather than design. A less rigorous application of the criteria may have enabled us to pool similar evidence from more trials.168 169 older people that incorporate care planning leading to multifactorial action with embedded medication review. The strongest evidence also favoured services incorporating ongoing review of the older person. The combination of exercise and nutritional support was part of two additional favourable intervention groups. Given the overall evidence for their benefits, we recommend access to these interventions in services for older people.172 173 Many of the trials seemed to be well conducted under challenging circumstances, and most declared non- commercial support, limiting the risks of funding bias. However, most results were at some risk of bias owing to missing outcome data. This was often inevitable given the combination of a frail population, long timelines, self-reported outcomes, community based research, and our stringent application of the RoB 2 tool. Many of the identified risks of within study bias were towards no effect or favouring the control group, meaning that true intervention effects may have been underestimated. Unexpectedly, we found evidence that some intervention combinations may reduce independence. This finding was not one that we anticipated but is not one that should be dismissed entirely, despite uncertainty in the evidence. Plausible mechanisms depend on the intervention group details but include invoking disengagement with the person’s health or with services or encouraging an individual to take on more than they can effectively manage. It could also be the case that certain events, such as care home placement or hospital admission, may be part of the best care strategy for an individual. Comparison with other studies Whereas other reviews of similar literature have tended to identify positive effects, albeit small and with some limitations in the underlying evidence,9-11 this review has found evidence of both positive and negative effects, depending on the intervention type (fig 3). This difference is likely to reflect the broad pairwise pooling of comparisons in other reviews, with different combinations of intervention components, different comparators, and heterogeneous effects and time points. These other reviews also did not use GRADE to assess the certainty of the evidence or the RoB 2 tool to assess risk of bias and may therefore have been less likely to conclude that a statistically significant finding was nevertheless very uncertain.f on 23 October 2024 by guest. Protected by copyright. m/ uest. Protected by copyright. Two recently published effectiveness trials have continued the trend of many previous studies in finding no significant difference in outcomes of interest.170 171 Given that many of the effects estimated in this review were relatively small, future definitive trials of similar interventions will likely need to be much larger than most of those included to provide sufficient power to identify plausible effects on independence. Two recently published effectiveness trials have continued the trend of many previous studies in finding no significant difference in outcomes of interest.170 171 AUTHOR AFFILIATIONS 1Academic Unit for Ageing and Stroke Research (University of Leeds), Bradford Institute for Health Research, Bradford Teaching Hospitals NHS Foundation Trust, Bradford, UK if Given that many of the effects estimated in this review were relatively small, future definitive trials of similar interventions will likely need to be much larger than most of those included to provide sufficient power to identify plausible effects on independence. Principal findings Even deterioration in activities of daily living may be in response to provision of assistance for tasks that someone finds difficult or painful and otherwise unrewarding and may therefore be an acceptable trade-off. We recommend that practitioners, commissioners, and policy makers remain mindful of these possibilities when designing systems of care. Owing to the scarcity of both direct and indirect evidence, the power to examine heterogeneity and inconsistency was low in most networks. However, the assessments we were able to do generally indicated little heterogeneity or inconsistency. We were also unable to effectively investigate the effect of population frailty on intervention effects, because of a lack of comparisons that contained different frailty populations.i We carefully assessed certainty in the findings by using GRADE for network meta-analysis guidance, providing clarity for the reader about the strength of the results. For many estimates, certainty was reduced because the confidence intervals included both improvement and worsening. Therefore, some low certainty findings may represent the play of chance around no effect. Despite a very large amount of primary research (129 studies; 74 946 participants) a degree of uncertainty exists about the effects of many types of intervention, although this does not mean that they are ineffective. Although methodological improvements in the primary research could further enhance knowledge, consideration of the scale of effort that would be needed to begin to complete this network of interventions is worthwhile. Therefore, whether embarking on a programme of large scale randomised controlled trials to further examine all interventions would be an effective use of research resources is unclear. Value of information analysis, which aims to quantify the value of new research to examine existing uncertainties, may be a helpful next step to prioritise which interventions to take forward to new trial based evaluation.174 Additionally, conducting individual participant data meta-analysis and realist synthesis may be useful to better explore the factors that may relate to benefit.175 on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ AUTHOR AFFILIATIONS 1Academic Unit for Ageing and Stroke Research (University of Leeds), Bradford Institute for Health Research, Bradford Teaching Hospitals NHS Foundation Trust, Bradford, UK 2Institute of Applied Health Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK 3Centre for Prognosis Research, School of Medicine, Keele University, Keele, UK 4Research Support Team, Leeds University Library, University of Leeds, Leeds, UK 5Geriatric Medicine, Bradford Teaching Hospitals NHS Foundation Trust, Bradford, UK 6Centre for Rehabilitation and Ageing Research, Academic Unit of Injury, Inflammation and Recovery Sciences, University of Nottingham, Nottingham, UK Conclusions, policy implications, and future research AC and JGl assessed frailty. AC, TC, AE, AF, JGl, MJ, and NL did intervention grouping. CB, TC, MJ, NL, and MP assessed risk of bias. MJ liaised with patient and public involvement groups. RB, MB, and JE prepared and analysed effectiveness data. JE and RRi supervised the meta-analyses. TC and NL assessed the certainty of the evidence. TC and NL did economic and narrative synthesis. AC, TC, JE, AF, JGl, NL, RRi, and OT interpreted the findings. TC wrote the first draft of the manuscript with input from AC, JE, AF, and JGl. All authors contributed to critically reviewing or revising the manuscript. CB, MB, RB, AC, TC, JE, JGl, JGr, MH, MJ, NL, JM, LM, EP, IP, MP, RRa, OT, and RW collectively accessed and verified the underlying data reported in the manuscript. JE and NL contributed equally. TC is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Contributors: AC, TC, JGl, and RRi conceived of the study. AC, TC, JE, AF, JGl, MJ, NL, EP, and RRi designed the study. DA, TC, and NL developed the search strategy. DA executed the database and trial register searches. TC, JGr, MJ, NL, JM, EP, RRa, and RW selected the studies. RB, CB, TC, JGr, MH, MJ, NL, JM, LM, EP, IP, RRa, OT, and RW extracted data. AC and JGl assessed frailty. AC, TC, AE, AF, JGl, MJ, and NL did intervention grouping. CB, TC, MJ, NL, and MP assessed risk of bias. MJ liaised with patient and public involvement groups. RB, MB, and JE prepared and analysed effectiveness data. JE and RRi supervised the meta-analyses. TC and NL assessed the certainty of the evidence. TC and NL did economic and narrative synthesis. AC, TC, JE, AF, JGl, NL, RRi, and OT interpreted the findings. TC wrote the first draft of the manuscript with input from AC, JE, AF, and JGl. All authors contributed to critically reviewing or revising the manuscript. CB, MB, RB, AC, TC, JE, JGl, JGr, MH, MJ, NL, JM, LM, EP, IP, MP, RRa, OT, and RW collectively accessed and verified the underlying data reported in the manuscript. JE and NL contributed equally. TC is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Provenance and peer review: Not commissioned; externally peer reviewed. Provenance and peer review: Not commissioned; externally peer reviewed. Provenance and peer review: Not commissioned; externally peer reviewed. Competing interests: All authors have completed the ICMJE uniform disclosure form at https://www.icmje.org/disclosure-of-interest/ and declare: AC, TC, JE, AF, JGl, MJ, NL, and RRi had financial support from the NIHR Health Technology Assessment Programme for the submitted work; MB had financial support from the PhD Graduate Teaching Fund at the University of Liverpool for the submitted work; DA declares payment made to her employer, University of Leeds Library, from the Academic Unit for Ageing and Stroke Research, Bradford Institute for Health Research, for services that included contributions to the submitted work; TC, AC, and AF received research funding from NIHR Programme Grants for Applied Research; AC and AF also received research funding from NIHR HSDR Programme; AC also received research funding from Health Data Research UK, NIHR ARC Yorkshire and Humber, NIHR Leeds BRC, and Dunhill Medical Trust; AF also declares NIHR Senior Investigator award, National Institute for Health (USA) payment for panel membership in 2021 and 2022, and University of Leeds Governor representative on the Governors Board of Bradford Teaching Hospitals NHS Foundation Trust; MB and MP received NIHR pre-doctoral fellowship funding; RB is supported by matched funding awarded to the NIHR Applied Research Collaboration (West Midlands) and is a member of the data monitoring committee for the Predict and Prevent AECOPD Trial and College of Experts, Versus Arthritis; AC is a member of NIHR HTA Commissioned Research Funding Committee and Dunhill Medical Trust Research Grants Committee; RRi received personal payments for training courses provided in-house to universities (Leeds, Aberdeen, Exeter, LSHTM) and other organisations (Roche), has received personal payments from the BMJ and BMJ Medicine as their statistical editor, is a co-convenor of the Cochrane Prognosis Methods Group and on the Editorial Board of Diagnostic and Prognostic Research, and Research Synthesis Methods, but receives no income for these roles, receives personal payment for being the external examiner of the MSc Medical Statistics, London School of Hygiene and Tropical Medicine, was previously an external examiner for the MSc Medical Statistics at University of Leicester, has written two textbooks for which he receives royalties from sales (Prognosis Research in Healthcare, and Individual Participant Data Meta-analysis), is a lead editor on an upcoming book This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for commercial use, provided the original work is properly cited. Conclusions, policy implications, and future research Data sharing: The data associated with this paper will be openly available indefinitely upon publication under a Creative Commons attribution license from the University of Leeds Data Repository. Summary effect estimates and findings from network meta-analyses: https://doi.org/10.5518/1377; risk of bias judgments: https://doi. org/10.5518/1386. Transparency: The lead author (the manuscript’s guarantor) affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as originally planned (and, if relevant, registered) have been explained. RB, MB, and JE prepared and analysed effectiveness data. JE and RRi supervised the meta-analyses. TC and NL assessed the certainty of the evidence. TC and NL did economic and narrative synthesis. AC, TC, JE, AF, JGl, NL, RRi, and OT interpreted the findings. TC wrote the first draft of the manuscript with input from AC, JE, AF, and JGl. All authors contributed to critically reviewing or revising the manuscript. CB, MB, RB, AC, TC, JE, JGl, JGr, MH, MJ, NL, JM, LM, EP, IP, MP, RRa, OT, and RW collectively accessed and verified the underlying data reported in the manuscript. JE and NL contributed equally. TC is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Dissemination to participants and related patient and public communities: To ensure that we clearly communicate our findings with patients and member of the public, we spent time discussing the intervention components we had identified with Frailty Oversight Group members. Through this work, we developed and refined our descriptions of the components. Frailty Oversight Group members helped to draft and revise the plain language summary of our findings (see appendix 2). This will be included in the final report to be published in Health Technology Assessment,176 as well as on our website. We have produced short video presentations of our work that are also available. Our findings will be publicised on social media and at our regular research engagement events in the local community. As this systematic review was secondary research using aggregated, anonymised data, we do not have any contact details for the participants in the primary research to enable us to share the findings directly with them. Conclusions, policy implications, and future research Funding: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme (NIHR128862) and will be published in full in Health Technology Assessment.176 The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. The grant applicants designed the overarching systematic review and network meta-analysis; the funders approved the protocol. The funders have not been involved in any aspect of the collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the article for publication. Conclusions, policy implications, and future research 6Centre for Rehabilitation and Ageing Research, Academic Unit of Injury, Inflammation and Recovery Sciences, University of Nottingham, Nottingham, UK Overall, our best evidence of combinations associated with sustained independence are service models for the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 11 RESEARCH RESEARCH on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 7Health Care of Older People, Nottingham University Hospitals NHS Trust, Nottingham, UK (Cochrane Handbook for Prognosis Reviews, Wiley, 2025), for which he will receive royalties from sales, has received consulting fees for a training course on IPD meta-analysis from Roche in 2018, the NIHR HTA grant paid for travel to Leeds for one meeting, and is a member of the NIHR Doctoral Research Fellowships grant panel, and a member of the MRC Better Methods Better Research grant panel—for the latter, he receives an attendance fee; MH declares NIHR Academic Clinical Fellowship; OT declares NIHR Academic Clinical Lectureship and Dunhill Medical Trust Doctoral Research Fellowship RTF107/0117; no other relationships or activities that could appear to have influenced the submitted work. on 23 October 2024 by guest. Pr http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from We are grateful to Nicola Harrison and Lesley Brown who coordinated and facilitated meetings with our Frailty Oversight Group; members of the Frailty Oversight Group, in particular Anne Grice and Marilyn Foster; members of the NIHR Applied Research Collaboration, Older People with Frailty Theme, Research Implementation Advisory Group; Zubair Arastu and Friederike Ziegler for their contributions to screening reference lists; Andy Mprah for his contribution to data collection; and Jasmin Manik for her support with testing the RoB 2 tool algorithms. We are particularly grateful to all of the authors of studies, both included and excluded, who responded to requests for information. Ethical approval: Ethics approval was not sought as this systematic review was secondary research using aggregated, anonymised data available in the public domain. Contributors: AC, TC, JGl, and RRi conceived of the study. AC, TC, JE, AF, JGl, MJ, NL, EP, and RRi designed the study. DA, TC, and NL developed the search strategy. DA executed the database and trial register searches. TC, JGr, MJ, NL, JM, EP, RRa, and RW selected the studies. RB, CB, TC, JGr, MH, MJ, NL, JM, LM, EP, IP, RRa, OT, and RW extracted data. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 32 Riley RD, Higgins JP, Deeks JJ. Interpretation of random effects meta- analyses. BMJ 2011;342:d549. doi:10.1136/bmj.d549 10 Luker JA, Worley A, Stanley M, Uy J, Watt AM, Hillier SL. The evidence for services to avoid or delay residential aged care admission: a systematic review. BMC Geriatr 2019;19:217. doi:10.1186/s12877- 019-1210-3 33 Schünemann H, Higgins J, Vist G, et al. Completing ‘Summary of findings’ tables and grading the certainty of the evidence. In: Higgins J, Thomas J, Chandler J, et al, eds. Cochrane Handbook for Systematic Reviews of Interventions version 6.3 (updated February 2022). Cochrane, 2022. 33 Schünemann H, Higgins J, Vist G, et al. Completing ‘Summary of findings’ tables and grading the certainty of the evidence. In: Higgins J, Thomas J, Chandler J, et al, eds. Cochrane Handbook for Systematic Reviews of Interventions version 6.3 (updated February 2022). Cochrane, 2022. 11 Whitehead PJ, Worthington EJ, Parry RH, Walker MF, Drummond AER. Interventions to reduce dependency in personal activities of daily living in community dwelling adults who use homecare services: a systematic review. Clin Rehabil 2015;29:1064-76. doi:10.1177/0269215514564894 34 Dias S, Welton NJ, Sutton AJ, Caldwell DM, Lu G, Ades AE. Evidence synthesis for decision making 4: inconsistency in networks of evidence based on randomized controlled trials. Med Decis Making 2013;33:641-56. doi:10.1177/0272989X12455847 12 Sterne JAC, Savović J, Page MJ, et al. RoB 2: a revised tool for assessing risk of bias in randomised trials. BMJ 2019;366:l4898. doi:10.1136/bmj.l4898 35 Veroniki AA, Vasiliadis HS, Higgins JP, Salanti G. Evaluation of inconsistency in networks of interventions. Int J Epidemiol 2013;42:332-45. doi:10.1093/ije/dys222 13 Higgins JPT, Thomas J, Chandler J, et al. Cochrane handbook for systematic reviews of interventions. 2nd ed. John Wiley & Sons, 2019doi:10.1002/9781119536604 . p / j / y 36 Glenton C, Santesso N, Rosenbaum S, et al. Presenting the results of Cochrane Systematic Reviews to a consumer audience: a qualitative study. Med Decis Making 2010;30:566-77. doi:10.1177/0272989X10375853 14 Brignardello-Petersen R, Bonner A, Alexander PE, et al, GRADE Working Group. Advances in the GRADE approach to rate the certainty in estimates from a network meta-analysis. J Clin Epidemiol 2018;93:36-44. doi:10.1016/j.jclinepi.2017.10.005 37 Aluko P, Graybill E, Craig D, et al. Economic evidence. In: Higgins J, Thomas J, Chandler J, et al, eds. Cochrane Handbook for Systematic Reviews of Interventions version 6.3 (updated February 2022). Cochrane, 2022. 15 Riley RD, Jackson D, Salanti G, et al. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Multivariate and network meta- analysis of multiple outcomes and multiple treatments: rationale, concepts, and examples. BMJ 2017;358:j3932. doi:10.1136/bmj. j3932 38 Heaven A, Brown L, Foster M, Clegg A. Keeping it credible in cohort multiple Randomised Controlled Trials: the Community Ageing Research 75+ (CARE 75+) study model of patient and public involvement and engagement. Res Involv Engagem 2016;2:30. doi:10.1186/s40900-016-0044-9f 16 Brignardello-Petersen R, Guyatt GH, Mustafa RA, et al. GRADE guidelines 33: Addressing imprecision in a network meta- analysis. J Clin Epidemiol 2021;139:49-56. doi:10.1016/j. jclinepi.2021.07.011 39 Alegría M, Frontera W, Cruz-Gonzalez M, et al. Effectiveness of a Disability Preventive Intervention for Minority and Immigrant Elders: The Positive Minds-Strong Bodies Randomized Clinical Trial. Am J Geriatr Psychiatry 2019;27:1299-313. doi:10.1016/j. jagp.2019.08.008 17 Brignardello-Petersen R, Murad MH, Walter SD, et al, GRADE Working Group. GRADE approach to rate the certainty from a network meta-analysis: avoiding spurious judgments of imprecision in sparse networks. J Clin Epidemiol 2019;105:60-7. doi:10.1016/j. jclinepi.2018.08.022 j gp 40 Arthanat S. Promoting information communication technology adoption and acceptance for aging-in-place: A randomized controlled trial. J Appl Gerontol 2021;40:471-80. doi:10.1177/0733464819891045 18 Guyatt GH, Oxman AD, Schünemann HJ, Tugwell P, Knottnerus A. GRADE guidelines: a new series of articles in the Journal of Clinical Epidemiology. J Clin Epidemiol 2011;64:380-2. doi:10.1016/j. jclinepi.2010.09.011 19 Puhan MA, Schünemann HJ, Murad MH, et al, GRADE Working Group. A GRADE Working Group approach for rating the quality of treatment effect estimates from network meta-analysis. BMJ 2014;349:g5630. doi:10.1136/bmj.g5630 41 Auvinen K, Voutilainen A, Jyrkkä J, Lönnroos E, Mäntyselkä P. Interprofessional medication assessment among home care patients: any impact on functioning? Results from a randomised controlled trial. BMC Geriatr 2020;20:390. doi:10.1186/s12877-020-01796-1 42 Balaban DJ, Goldfarb NI, Perkel RL, Carlson BL. Follow-up study of an urban family medicine home visit program. J Fam Pract 1988;26:307-12.f 20 Santesso N, Glenton C, Dahm P, et al, GRADE Working Group. GRADE guidelines 26: informative statements to communicate the findings of systematic reviews of interventions. J Clin Epidemiol 2020;119:126-35. doi:10.1016/j.jclinepi.2019.10.014 43 Barenfeld E, Dahlin-Ivanoff S, Wallin L, Gustafsson S. Promoting aging migrants’ capabilities: A randomized controlled trial concerning activities of daily living and self-rated health. AIMS Public Health 2018;5:173-88. doi:10.3934/publichealth.2018.2.173 21 Yepes-Nuñez JJ, Li S-A, Guyatt G, et al. Development of the summary of findings table for network meta-analysis. J Clin Epidemiol 2019;115:1-13. doi:10.1016/j.jclinepi.2019.04.018 22 Hutton B, Salanti G, Caldwell DM, et al. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from The PRISMA extension statement for reporting of systematic reviews incorporating network meta-analyses of health care interventions: checklist and explanations. Ann Intern Med 2015;162:777-84. doi:10.7326/M14-2385 44 Bernabei R, Landi F, Gambassi G, et al. Randomised trial of impact of model of integrated care and case management for older people living in the community. BMJ 1998;316:1348-51. doi:10.1136/ bmj.316.7141.1348f 45 Bleijenberg N, Drubbel I, Schuurmans MJ, et al. Effectiveness of a proactive primary care program on preserving daily functioning of older people: a cluster randomized controlled trial. J Am Geriatr Soc 2016;64:1779-88. doi:10.1111/jgs.14325f 23 Page MJ, McKenzie JE, Bossuyt PM, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ 2021;372:n71. doi:10.1136/bmj.n71 24 Crocker TF, Clegg A, Riley RD, et al. Community-based complex interventions to sustain independence in older people, stratified by frailty: a protocol for a systematic review and network meta-analysis. BMJ Open 2021;11:e045637. doi:10.1136/ bmjopen-2020-045637 46 Blom J, den Elzen W, van Houwelingen AH, et al. Effectiveness and cost-effectiveness of a proactive, goal-oriented, integrated care model in general practice for older people. A cluster randomised controlled trial: Integrated Systematic Care for older People--the ISCOPE study. Age Ageing 2016;45:30-41. doi:10.1093/ageing/afv174 25 Crocker T, Forster A, Young J, et al. Physical rehabilitation for older people in long-term care. Cochrane Database Syst Rev 2013;(2):CD004294. doi:10.1002/14651858.CD004294. pub3 g g g 47 Borrows A, Holland R. Independent living centre occupational therapy (OT) versus routine community OT. Int J Ther Rehabil 2013;20:187- 94doi:10.12968/ijtr.2013.20.4.187 . 48 Botjes E. Methodebeschrijving EigenKrachtWijzer. Databank Effectieve sociale interventies Movisie, 2013. 26 Fried LP, Tangen CM, Walston J, et al, Cardiovascular Health Study Collaborative Research Group. Frailty in older adults: evidence for a phenotype. J Gerontol A Biol Sci Med Sci 2001;56:M146-56. doi:10.1093/gerona/56.3.M146f f 49 Bouman A, van Rossum E, Ambergen T, Kempen G, Knipschild P. Effects of a home visiting program for older people with poor health status: a randomized, clinical trial in The Netherlands. J Am Geriatr Soc 2008;56:397-404. doi:10.1111/j.1532-5415.2007.01565.x 27 Hoffmann TC, Glasziou PP, Boutron I, et al. Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ 2014;348:g1687. doi:10.1136/ bmj.g1687f Soc 2008;56:397-404. doi:10.1111/j.1532-5415.2007.01565.x 50 Brettschneider C, Luck T, Fleischer S, et al. Cost-utility analysis of a preventive home visit program for older adults in Germany. BMC Health Serv Res 2015;15:141. doi:10.1186/s12913-015-0817-0 28 Hoffmann TC, Oxman AD, Ioannidis JPA, et al. Provenance and peer review: Not commissioned; externally peer reviewed. See: http://creativecommons.org/licenses/by/4.0/. 1 United Nations Department of Economic and Social Affairs. Population Division. World Population Prospects 2022: Summary of Results. 2022. https://www.un.org/development/desa/pd/sites/ www.un.org.development.desa.pd/files/wpp2022_summary_of_ results.pdf. 2 World Health Organization. Decade of Healthy Ageing: 2020-2030. https://www.who.int/ageing/decade-of-healthy-ageing. 3 World Health Organization. Global Health Estimates: Life expectancy and leading causes of death and disability. 2023. https://www.who. int/data/gho/data/themes/mortality-and-global-health-estimates/ ghe-life-expectancy-and-healthy-life-expectancy.fi 4 Whitty CJM. Chief medical officer’s annual report 2023: Health in an ageing society. 2023. https://www.gov.uk/government/publications/ chief-medical-officers-annual-report-2023-health-in-an-ageing- society. y 5 Han L, Clegg A, Doran T, Fraser L. The impact of frailty on healthcare resource use: a longitudinal analysis using the Clinical Practice Research Datalink in England. Age Ageing 2019;48:665-71. doi:10.1093/ageing/afz088f 6 de Meijer C, Wouterse B, Polder J, Koopmanschap M. The effect of population aging on health expenditure growth: a critical review. Eur J Ageing 2013;10:353-61. doi:10.1007/s10433-013-0280-x 7 Pot AM, Rabheru K, Chew M. Person-centred long-term care for older persons: a new Lancet Commission. Lancet 2023;401:1754-5. doi:10.1016/S0140-6736(23)00920-0 8 Mahase E. Generalist skills are vital to serve ageing population, says England’s CMO. BMJ 2023;383:2640. doi:10.1136/bmj.p2640 9 Beswick AD, Rees K, Dieppe P, et al. Complex interventions to improve physical function and maintain independent living in elderly people: a systematic review and meta-analysis. Lancet 2008;371:725-35. doi:10.1016/S0140-6736(08)60342-6 12 12 doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj RESEARCH RESEARCH on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 75 Gitlin LN, Winter L, Dennis MP, Corcoran M, Schinfeld S, Hauck WW. A randomized trial of a multicomponent home intervention to reduce functional difficulties in older adults. J Am Geriatr Soc 2006;54:809- 16. doi:10.1111/j.1532-5415.2006.00703.x 54 Challis D, Clarkson P, Williamson J, et al. The value of specialist clinical assessment of older people prior to entry to care homes. Age Ageing 2004;33:25-34. doi:10.1093/ageing/afh007 g g g gh 55 Clark F, Azen SP, Zemke R, et al. Occupational therapy for independent-living older adults. A randomized controlled trial. JAMA 1997;278:1321-6. doi:10.1001/jama.1997.03550160041036f 76 Grimmer K, Luker J, Beaton K, Kumar S, Crockett A, Price K. TRialing individualized interventions to prevent functional decline in at-risk older adults (TRIIFL): study protocol for a randomized controlled trial nested in a longitudinal observational study. Trials 2013;14:266. doi:10.1186/1745-6215-14-266if 56 Clark F, Jackson J, Carlson M, et al. Effectiveness of a lifestyle intervention in promoting the well-being of independently living older people: results of the Well Elderly 2 Randomised Controlled Trial. J Epidemiol Community Health 2012;66:782-90. doi:10.1136/ jech.2009.099754 77 Gustafson DHSr, Kornfield R, Mares M-L, et al. Effect of an eHealth intervention on older adults’ quality of life and health- related outcomes: a randomized clinical trial. J Gen Intern Med 2022;37:521-30. doi:10.1007/s11606-021-06888-1 57 Coleman EA, Grothaus LC, Sandhu N, Wagner EH. Chronic care clinics: a randomized controlled trial of a new model of primary care for frail older adults. J Am Geriatr Soc 1999;47:775-83. doi:10.1111/j.1532-5415.1999.tb03832.x 78 Gustafsson S, Eklund K, Wilhelmson K, et al. Long-term outcome for ADL following the health-promoting RCT--elderly persons in the risk zone. Gerontologist 2013;53:654-63. doi:10.1093/geront/ gns121 j 58 Counsell SR, Callahan CM, Clark DO, et al. Geriatric care management for low-income seniors: a randomized controlled trial. JAMA 2007;298:2623-33. doi:10.1001/jama.298.22.2623 j 58 Counsell SR, Callahan CM, Clark DO, et al. Geriatric care management for low-income seniors: a randomized controlled trial. JAMA 2007;298:2623-33. doi:10.1001/jama.298.22.2623 79 Hall N, De Beck P, Johnson D, Mackinnon K, Gutman G, Glick N. Randomized trial of a health promotion program for frail elders. Can J Aging 1992;11:72-91doi:10.1017/S0714980800014537 .f 59 Cutchin MP, Coppola S, Talley V, Svihula J, Catellier D, Shank KH. Feasibility and effects of preventive home visits for at-risk older people: design of a randomized controlled trial. BMC Geriatr 2009;9:54. doi:10.1186/1471-2318-9-54 80 Harari D, Iliffe S, Kharicha K, et al. Promotion of health in older people: a randomised controlled trial of health risk appraisal in British general practice. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Age Ageing 2008;37:565-71. doi:10.1093/ ageing/afn150f 60 Dalby DM, Sellors JW, Fraser FD, Fraser C, van Ineveld C, Howard M. Effect of preventive home visits by a nurse on the outcomes of frail elderly people in the community: a randomized controlled trial. CMAJ 2000;162:497-500. 81 Hattori S, Yoshida T, Okumura Y, Kondo K. Effects of reablement on the independence of community-dwelling older adults with mild disability: A randomized controlled trial. Int J Environ Res Public Health 2019;16:3954. doi:10.3390/ijerph16203954 61 de Craen AJ, Gussekloo J, Blauw GJ, Willems CG, Westendorp RG. Randomised controlled trial of unsolicited occupational therapy in community-dwelling elderly people: the LOTIS trial. PLoS Clin Trials 2006;1:e2. doi:10.1371/journal.pctr.0010002 82 Hay WI, van Ineveld C, Browne G, et al. Prospective care of elderly patients in family practice. Is screening effective?Can Fam Physician 1998;44:2677-87.fi 62 Dorresteijn TA, Zijlstra GA, Ambergen AW, Delbaere K, Vlaeyen JW, Kempen GI. Effectiveness of a home-based cognitive behavioral program to manage concerns about falls in community-dwelling, frail older people: results of a randomized controlled trial. BMC Geriatr 2016;16:2. doi:10.1186/s12877-015-0177-y 83 Hébert R, Robichaud L, Roy PM, Bravo G, Voyer L. Efficacy of a nurse-led multidimensional preventive programme for older people at risk of functional decline. A randomized controlled trial. Age Ageing 2001;30:147-53. doi:10.1093/ageing/30.2.147 63 Dupuy L, Froger C, Consel C, Sauzéon H. Everyday functioning benefits from an assisted living platform amongst frail older adults and their caregivers. Front Aging Neurosci 2017;9:302. doi:10.3389/fnagi.2017.00302 84 Henderson MJ. In-home preventive health assessment and telephone case management for over 75s living alone in independent living units: A cluster randomized controlled trial [PhD thesis]. Queensland University of Technology, 2005. 64 Fabacher D, Josephson K, Pietruszka F, Linderborn K, Morley JE, Rubenstein LZ. An in-home preventive assessment program for independent older adults: a randomized controlled trial. J Am Geriatr Soc 1994;42:630-8. doi:10.1111/j.1532-5415.1994.tb06862.xf 85 Hendriksen C, Lund E, Strømgård E. Consequences of assessment and intervention among elderly people: a three year randomised controlled trial. Br Med J (Clin Res Ed) 1984;289:1522-4. doi:10.1136/bmj.289.6457.1522 65 Fairhall N, Kurrle SE, Sherrington C, et al. Effectiveness of a multifactorial intervention on preventing development of frailty in pre-frail older people: study protocol for a randomised controlled trial. BMJ Open 2015;5:e007091. doi:10.1136/ bmjopen-2014-007091 86 Hogg W, Lemelin J, Dahrouge S, et al. Randomized controlled trial of anticipatory and preventive multidisciplinary team care: for complex patients in a community-based primary care setting. Can Fam Physician 2009;55:e76-85. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from y 87 Holland SK, Greenberg J, Tidwell L, Malone J, Mullan J, Newcomer R. Community-based health coaching, exercise, and health service utilization. J Aging Health 2005;17:697-716. doi:10.1177/0898264305277959f 66 Faul AC, Yankeelov PA, Rowan NL, et al. Impact of geriatric assessment and self-management support on community- dwelling older adults with chronic illnesses. J Gerontol Soc Work 2009;52:230-49. doi:10.1080/01634370802609288 88 Howel D, Moffatt S, Haighton C, et al. Does domiciliary welfare rights advice improve health-related quality of life in independent- living, socio-economically disadvantaged people aged ≥60 years? Randomised controlled trial, economic and process evaluations in the North East of England. PLoS One 2019;14:e0209560. doi:10.1371/journal.pone.0209560f 67 Fernández-Barrés S, García-Barco M, Basora J, Martínez T, Pedret R, Arija V, Project ATDOM-NUT group. The efficacy of a nutrition education intervention to prevent risk of malnutrition for dependent elderly patients receiving Home Care: A randomized controlled trial. Int J Nurs Stud 2017;70:131-41. doi:10.1016/j.ijnurstu.2017.02.020 89 Imhof L, Naef R, Wallhagen MI, Schwarz J, Mahrer-Imhof R. Effects of an advanced practice nurse in-home health consultation program for community-dwelling persons aged 80 and older. J Am Geriatr Soc 2012;60:2223-31. doi:10.1111/jgs.12026f 68 Fischer G, Sandholzer H, Perschke-Hartmann C. Final report of the scientific support of “Getting Healthy Elderly (GÄW)”. A prevention project of the AOK Lower Saxony.)[German] (Abschlussbericht der wissenschaftlichen Begleitung von “Gesund Älter Werden (GÄW)“). AOK Niedersachsen, 2009. 90 Jing L, Jin Y, Zhang X, Wang F, Song Y, Xing F. The effect of Baduanjin qigong combined with CBT on physical fitness and psychological health of elderly housebound. Medicine (Baltimore) 2018;97:e13654. doi:10.1097/ MD.0000000000013654 69 Ford AB, Katz S, Downs TD, Adams M. Results of long-term home nursing: the influence of disability. J Chronic Dis 1971;24:591-6. doi:10.1016/0021-9681(71)90047-6f 70 Fox PJ, Breuer W, Wright JA. Effects of a health promotion program on sustaining health behaviors in older adults. Am J Prev Med 1997;13:257-64. doi:10.1016/S0749-3797(18)30172-7 91 Jitapunkul S. A randomised controlled trial of regular surveillance in Thai elderly using a simple questionnaire administered by non- professional personnel. J Med Assoc Thai 1998;81:352-6. 71 Fristedt S, Nystedt P, Skogar Ö. Mobile geriatric teams - a cost- effective way of improving patient safety and reducing traditional healthcare utilization among the frail elderly? A randomized controlled trial. Clin Interv Aging 2019;14:1911-24. doi:10.2147/ CIA.S208388 92 Kerse N, McLean C, Moyes SA, et al. The cluster-randomized BRIGHT trial: Proactive case finding for community-dwelling older adults. Ann Fam Med 2014;12:514-24. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Enhancing the usability of systematic reviews by improving the consideration and description of interventions. BMJ 2017;358:j2998. doi:10.1136/bmj.j2998 51 Cameron ID, Fairhall N, Langron C, et al. A multifactorial interdisciplinary intervention reduces frailty in older people: randomized trial. BMC Med 2013;11:65. doi:10.1186/1741-7015- 11-65 29 Crocker TF, Lam N, Jordão M, et al. Risk-of-bias assessment using Cochrane’s revised tool for randomized trials (RoB 2) was useful but challenging and resource-intensive: observations from a systematic review. J Clin Epidemiol 2023;161:39-45. doi:10.1016/j. jclinepi.2023.06.015 52 Carpenter GI, Demopoulos GR. Screening the elderly in the community: controlled trial of dependency surveillance using a questionnaire administered by volunteers. BMJ 1990;300:1253-6. doi:10.1136/bmj.300.6734.1253 30 White IR. Network meta-analysis. Stata J 2015;15:951-85doi:10.117 7/1536867X1501500403 . 53 Cesari M, Demougeot L, Boccalon H, Guyonnet S, Vellas B, Andrieu S. The Multidomain Intervention to preveNt disability in ElDers (MINDED) project: rationale and study design of a pilot study. Contemp Clin Trials 2014;38:145-54. doi:10.1016/j. cct.2014.04.006 31 Doi SA, Furuya-Kanamori L, Xu C, et al. The Odds Ratio is “portable” across baseline risk but not the Relative Risk: Time to do away with the log link in binomial regression. J Clin Epidemiol 2022;142:288- 93. doi:10.1016/j.jclinepi.2021.08.003 13 the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 RESEARCH RESEARCH on 23 October 2024 by guest. Protect http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from A clinimetric evaluation of specialized geriatric care for rural dwelling, frail older people. J Am Geriatr Soc 2000;48:1080-5. doi:10.1111/j.1532-5415.2000.tb04783.xif 107 Mann WC, Ottenbacher KJ, Fraas L, Tomita M, Granger CV. Effectiveness of assistive technology and environmental interventions in maintaining independence and reducing home care costs for the frail elderly. A randomized controlled trial. Arch Fam Med 1999;8:210-7. doi:10.1001/archfami.8.3.210 129 Romera-Liebana L, Orfila F, Segura JM, et al. Effects of a primary care- based multifactorial intervention on physical and cognitive function in frail, elderly individuals: A randomized controlled trial. J Gerontol A Biol Sci Med Sci 2018;73:1688-1674. doi:10.1093/gerona/glx259f 108 Markle-Reid M, Weir R, Browne G, Roberts J, Gafni A, Henderson S. Health promotion for frail older home care clients. J Adv Nurs 2006;54:381-95. doi:10.1111/j.1365-2648.2006.03817.x 130 Rooijackers TH, Kempen GIJM, Zijlstra GAR, et al. Effectiveness of a reablement training program for homecare staff on older adults’ sedentary behavior: A cluster randomized controlled trial. J Am Geriatr Soc 2021;69:2566-78. doi:10.1111/jgs.17286 /j 109 Melis RJ, van Eijken MI, Teerenstra S, et al. A randomized study of a multidisciplinary program to intervene on geriatric syndromes in vulnerable older people who live at home (Dutch EASYcare Study). J Gerontol A Biol Sci Med Sci 2008;63:283-90. doi:10.1093/ gerona/63.3.283f 131 Rubenstein LZ, Alessi CA, Josephson KR, Trinidad Hoyl M, Harker JO, Pietruszka FM. A randomized trial of a screening, case finding, and referral system for older veterans in primary care. J Am Geriatr Soc 2007;55:166-74. doi:10.1111/j.1532-5415.2007.01044.x 110 Meng H, Friedman B, Wamsley BR, Mukamel D, Eggert GM. Effect of a consumer-directed voucher and a disease-management- health-promotion nurse intervention on home care use. Gerontologist 2005;45:167-76. doi:10.1093/geront/45.2.167 132 Ryvicker M, Feldman PH, Rosati RJ, Sobolewski S, Maduro GAJr, Schwartz T. Improving functional outcomes in home care patients: impact and challenges of disseminating a quality improvement initiative. J Healthc Qual 2011;33:28-36. doi:10.1111/j.1945- 1474.2011.00156.xf 111 Messens L, Quinn S, Saez I, Cuidad Mas MJ, Squillace P, Laura AG. Health monitoring and sOcial integration environMent for Supporting WidE ExTension of independent life at HOME (Home Sweet Home): Final Trial Evaluation Report. Zorgbedrijf Antwerpen, 2014.f 133 Serra-Prat M, Sist X, Domenich R, et al. Effectiveness of an intervention to prevent frailty in pre-frail community-dwelling older people consulting in primary care: a randomised controlled trial. Age Ageing 2017;46:401-7. doi:10.1093/ageing/afw242f 112 Metzelthin SF, van Rossum E, de Witte LP, et al. on 23 October 2024 by guest. Protect http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from doi:10.1093/ageing/afr118 f 122 Parsons M, Senior H, Kerse N, Chen MH, Jacobs S, Anderson C. Randomised trial of restorative home care for frail older people in New Zealand. Nurs Older People 2017;29:27-33. doi:10.7748/ nop.2017.e897 102 Liddle J, March L, Carfrae B, et al. Can occupational therapy intervention play a part in maintaining independence and quality of life in older people? A randomised controlled trial. Aust N Z J Public Health 1996;20:574-8. doi:10.1111/j.1467-842X.1996. tb01068.xf p 123 Parsons M, Senior H, Kerse N, et al. Should care managers for older adults be located in primary care? A randomized controlled trial. J Am Geriatr Soc 2012;60:86-92. doi:10.1111/j.1532- 5415.2011.03763.x 103 Liimatta H, Lampela P, Laitinen-Parkkonen P, Pitkala KH. Effects of preventive home visits on health-related quality-of-life and mortality in home-dwelling older adults. Scand J Prim Health Care 2019;37:90-7. doi:10.1080/02813432.2019.1569372 124 Pathy MS, Bayer A, Harding K, Dibble A. Randomised trial of case finding and surveillance of elderly people at home. Lancet 1992;340:890-3. doi:10.1016/0140-6736(92)93294-Wf 104 Loh DA, Hairi NN, Choo WY, et al. MultiComponent Exercise and theRApeutic lifeStyle (CERgAS) intervention to improve physical performance and maintain independent living among urban poor older people--a cluster randomised controlled trial. BMC Geriatr 2015;15:8. doi:10.1186/s12877-015-0002-7f 125 Phelan EA, Balderson B, Levine M, et al. Delivering effective primary care to older adults: a randomized, controlled trial of the senior resource team at group health cooperative. J Am Geriatr Soc 2007;55:1748-56. doi:10.1111/j.1532-5415.2007.01416.xf 126 Ploeg J, Brazil K, Hutchison B, et al. Effect of preventive primary care outreach on health related quality of life among older adults at risk of functional decline: randomised controlled trial. BMJ 2010;340:c1480. doi:10.1136/bmj.c1480 105 Lood Q, Gustafsson S, Dahlin Ivanoff S. Bridging barriers to health promotion: a feasibility pilot study of the ‘Promoting Aging Migrants’ Capabilities study’. J Eval Clin Pract 2015;21:604-13. doi:10.1111/ jep.12345 127 Pröfener F, Anders J, Dapp U, Minder CE, Golgert S, von Renteln- Kruse W. [Acceptance of preventive home visits among frail elderly persons : Participants an non-participants in a Follow-up after 2 and 4 years within the LUCAS longitudinal study][German]. Z Gerontol Geriatr 2016;49:596-605. 106 Mann J, Thompson F, McDermott R, Esterman A, Strivens E. Impact of an integrated community-based model of care for older people with complex conditions on hospital emergency presentations and admissions: a step-wedged cluster randomized trial. BMC Health Serv Res 2021;21:701. doi:10.1186/s12913-021-06668-x 128 Rockwood K, Stadnyk K, Carver D, et al. on 23 October 2024 by guest. Protect http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 116 Morey MC, Peterson MJ, Pieper CF, et al. The Veterans Learning to Improve Fitness and Function in Elders Study: a randomized trial of primary care-based physical activity counseling for older men. J Am Geriatr Soc 2009;57:1166-74. doi:10.1111/j.1532- 5415.2009.02301.x 96 Kono A, Kanaya Y, Fujita T, et al. Effects of a preventive home visit program in ambulatory frail older people: a randomized controlled trial. J Gerontol A Biol Sci Med Sci 2012;67:302-9. doi:10.1093/ gerona/glr176 g g 97 Kukkonen-Harjula K, Karmeniemi P, Suikkanen S, et al. Long-term home-based physiotherapy for older people with signs of frailty-RCT (NCT02305433)[P-229]. Eur Geriatr Med 2017;8:S105. 117 Morgan GS, Haase AM, Campbell RM, Ben-Shlomo Y. A pilot randomised controlled trial of physical activity facilitation for older adults: feasibility study findings. Pilot Feasibility Stud 2019;5:40. doi:10.1186/s40814-019-0414-9 on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 98 Lambotte D, De Donder L, De Roeck EE, et al. Randomized controlled trial to evaluate a prevention program for frail community-dwelling older adults: a D-SCOPE protocol. BMC Geriatr 2018;18:194. doi:10.1186/s12877-018-0875-3i 118 Newbury JW, Marley JE, Beilby JJ. A randomised controlled trial of the outcome of health assessment of people aged 75 years and over. Med J Aust 2001;175:104-7. doi:10.5694/j.1326-5377.2001. tb143541.x 99 Leung AC, Liu C, Chow NW, Chi I. Cost-Benefit Analysis of a Case Management Project for the Community-Dwelling Frail Elderly in Hong Kong. J Appl Gerontol 2004;23:70- 85doi:10.1177/0733464804263088 . 119 Newcomer R, Maravilla V, Faculjak P, Graves MT. Outcomes of preventive case management among high-risk elderly in three medical groups: a randomized clinical trial. Eval Health Prof 2004;27:323-48. doi:10.1177/0163278704270011 100 Leveille SG, Wagner EH, Davis C, et al. Preventing disability and managing chronic illness in frail older adults: a randomized trial of a community-based partnership with primary care. J Am Geriatr Soc 1998;46:1191-8. doi:10.1111/j.1532-5415.1998. tb04533.x f 120 Ng TP, Feng L, Nyunt MS, et al. Nutritional, physical, cognitive, and combination interventions and frailty reversal among older adults: A randomized controlled trial. Am J Med 2015;128:1225-1236.e1. doi:10.1016/j.amjmed.2015.06.017 101 Lewin G, De San Miguel K, Knuiman M, et al. A randomised controlled trial of the Home Independence Program, an Australian restorative home-care programme for older adults. Health Soc Care Community 2013;21:69-78. doi:10.1111/j.1365- 2524.2012.01088.x 121 Parsons J, Rouse P, Robinson EM, Sheridan N, Connolly MJ. Goal setting as a feature of homecare services for older people: does it make a difference?Age Ageing 2012;41:24-9. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from doi:10.1370/afm.1696 93 King AII, Parsons M, Robinson E, Jörgensen D. Assessing the impact of a restorative home care service in New Zealand: a cluster randomised controlled trial. Health Soc Care Community 2012;20:365-74. doi:10.1111/j.1365-2524.2011.01039.xf 72 Gené Huguet L, Navarro González M, Kostov B, et al. Pre Frail 80: Multifactorial intervention to prevent progression of pre-frailty to frailty in the elderly. J Nutr Health Aging 2018;22:1266-74. doi:10.1007/s12603-018-1089-2 94 Kono A, Izumi K, Yoshiyuki N, Kanaya Y, Rubenstein LZ. Effects of an updated preventive home visit program based on a systematic structured assessment of care needs for ambulatory frail older adults in Japan: A randomized controlled trial. J Gerontol A Biol Sci Med Sci 2016;71:1631-7. doi:10.1093/gerona/glw068f 73 Gill TM, Baker DI, Gottschalk M, Peduzzi PN, Allore H, Byers A. A program to prevent functional decline in physically frail, elderly persons who live at home. N Engl J Med 2002;347:1068-74. doi:10.1056/NEJMoa020423 74 Giné-Garriga M, Sansano-Nadal O, Tully MA, et al. Accelerometer- measured sedentary and physical activity time and their correlates in European older adults: The SITLESS study. J Gerontol A Biol Sci Med Sci 2020;75:1754-62. doi:10.1093/gerona/glaa016 95 Kono A, Kai I, Sakato C, Harker JO, Rubenstein LZ. Effect of preventive home visits for ambulatory housebound elders in Japan: a pilot study. Aging Clin Exp Res 2004;16:293-9. doi:10.1007/ BF03324554 95 Kono A, Kai I, Sakato C, Harker JO, Rubenstein LZ. Effect of preventive home visits for ambulatory housebound elders in Japan: a pilot study. Aging Clin Exp Res 2004;16:293-9. doi:10.1007/ BF03324554 14 doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj RESEARCH RESEARCH on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 137 Stewart S, Harvey I, Poland F, Lloyd-Smith W, Mugford M, Flood C. Are occupational therapists more effective than social workers when assessing frail older people? Results of CAMELOT, a randomised controlled trial. Age Ageing 2005;34:41-6. doi:10.1093/ageing/afh230 157 van Rossum E, Frederiks CM, Philipsen H, Portengen K, Wiskerke J, Knipschild P. Effects of preventive home visits to elderly people. BMJ 1993;307:27-32. doi:10.1136/bmj.307.6895.27 on 23 October 2024 by guest. Protected by copyright. http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 158 Vass M, Avlund K, Lauridsen J, Hendriksen C. Feasible model for prevention of functional decline in older people: municipality- randomized, controlled trial. J Am Geriatr Soc 2005;53:563-8. doi:10.1111/j.1532-5415.2005.53201.xf h 138 Stuck AE, Aronow HU, Steiner A, et al. A trial of annual in-home comprehensive geriatric assessments for elderly people living in the community. N Engl J Med 1995;333:1184-9. doi:10.1056/ NEJM199511023331805 159 Vetter NJ, Jones DA, Victor CR. Effect of health visitors working with elderly patients in general practice: a randomised controlled trial. Br Med J (Clin Res Ed) 1984;288:369-72. doi:10.1136/ bmj.288.6414.369ff 139 Stuck AE, Minder CE, Peter-Wüest I, et al. A randomized trial of in-home visits for disability prevention in community-dwelling older people at low and high risk for nursing home admission. Arch Intern Med 2000;160:977-86. doi:10.1001/archinte.160.7.977f 160 von Bonsdorff MB, Leinonen R, Kujala UM, et al. Effect of physical activity counseling on disability in older people: a 2-year randomized controlled trial. J Am Geriatr Soc 2008;56:2188-94. doi:10.1111/ j.1532-5415.2008.02000.x 140 Stuck AE, Moser A, Morf U, et al. Effect of health risk assessment and counselling on health behaviour and survival in older people: a pragmatic randomised trial. PLoS Med 2015;12:e1001889. doi:10.1371/journal.pmed.1001889 161 Wallace JI, Buchner DM, Grothaus L, et al. Implementation and effectiveness of a community-based health promotion program for older adults. J Gerontol A Biol Sci Med Sci 1998;53:M301-6. doi:10.1093/gerona/53A.4.M301 141 Suijker JJ, van Rijn M, Buurman BM, Ter Riet G, Moll van Charante EP, de Rooij SE. Effects of nurse-led multifactorial care to prevent disability in community-living older people: Cluster randomized trial. PLoS One 2016;11:e0158714. doi:10.1371/journal.pone.0158714 162 Walters K, Frost R, Kharicha K, et al. Home-based health promotion for older people with mild frailty: the HomeHealth intervention development and feasibility RCT. Health Technol Assess 2017;21:1- 128. doi:10.3310/hta21730 142 Szanton SL, Thorpe RJ, Boyd C, et al. on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Community aging in place, advancing better living for elders: a bio-behavioral-environmental intervention to improve function and health-related quality of life in disabled older adults. J Am Geriatr Soc 2011;59:2314-20. doi:10.1111/j.1532-5415.2011.03698.xff 163 Whitehead PJ, Walker MF, Parry RH, Latif Z, McGeorge ID, Drummond AE. Occupational Therapy in HomEcare Re-ablement Services (OTHERS): results of a feasibility randomised controlled trial. BMJ Open 2016;6:e011868. doi:10.1136/ bmjopen-2016-011868 143 Szanton SL, Xue QL, Leff B, et al. Effect of a biobehavioral environmental approach on disability among low-income older adults: A randomized clinical trial. JAMA Intern Med 2019;179:204- 11. doi:10.1001/jamainternmed.2018.6026 164 Williams EI, Greenwell J, Groom LM. The care of people over 75 years old after discharge from hospital: an evaluation of timetabled visiting by Health Visitor Assistants. J Public Health Med 1992;14:138-44. 144 Takahashi PY, Pecina JL, Upatising B, et al. A randomized controlled trial of telemonitoring in older adults with multiple health issues to prevent hospitalizations and emergency department visits. Arch Intern Med 2012;172:773-9. doi:10.1001/ archinternmed.2012.256f 165 Wolter A, Stolle C, Roth G, Rothgang H. [Does the resident care assessment instrument improve long-term home care? - results of a nation-wide study in Germany][German]. Gesundheitswesen 2013;75:29-32.f 145 Teut M, Schnabel K, Baur R, et al. Effects and feasibility of an Integrative Medicine program for geriatric patients-a cluster-randomized pilot study. Clin Interv Aging 2013;8:953-61. doi:10.2147/CIA.S45242 166 Wong AKC, Wong FKY, Chang K. Effectiveness of a community- based self-care promoting program for community-dwelling older adults: a randomized controlled trial. Age Ageing 2019;48:852-8. doi:10.1093/ageing/afz095 146 Thiel C, Braun T, Grüneberg C. [Physical training as core component of multimodal treatment of older frail people-study protocol of a randomized controlled pilot study]. Z Gerontol Geriatr 2019;52:45- 60. doi:10.1007/s00391-018-1443-3 167 Yamada Y, Ikegami N. Preventive home visits for community- dwelling frail elderly people based on minimum data set- home care: randomized controlled trial. Geriatr Gerontol Int 2003;3:236-42doi:10.1111/j.1444-1586.2003.00103.x . 147 Thomas R, Worrall G, Elgar F, Knight J. Can they keep going on their own? A four-year randomized trial of functional assessments of community residents. Can J Aging 2007;26:379-90. doi:10.3138/ cja.26.4.379 168 Di Pollina L, Guessous I, Petoud V, et al. Integrated care at home reduces unnecessary hospitalizations of community-dwelling frail older adults: a prospective controlled trial. BMC Geriatr 2017;17:53. doi:10.1186/s12877-017-0449-9 148 Tomita MR, Mann WC, Stanton K, Tomita AD, Sundar V. Use of currently available smart home technology by frail elders: process and outcomes. Top Geriatr Rehabil 2007;23:24- 34doi:10.1097/00013614-200701000-00005 . on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from 169 Byles JE, Tavener M, O’Connell RL, et al. Randomised controlled trial of health assessments for older Australian veterans and war widows. Med J Aust 2004;181:186-90. doi:10.5694/j.1326-5377.2004. tb06233.xi 149 Tulloch AJ, Moore V. A randomized controlled trial of geriatric screening and surveillance in general practice. J R Coll Gen Pract 1979;29:733-40. 150 Tuntland H, Aaslund MK, Espehaug B, Førland O, Kjeken I. Reablement in community-dwelling older adults: a randomised controlled trial. BMC Geriatr 2015;15:145. doi:10.1186/s12877-015-0142-9 170 Bloomfield K, Wu Z, Broad JB, et al. Learning from a multidisciplinary randomized controlled intervention in retirement village residents. J Am Geriatr Soc 2022;70:743-53. doi:10.1111/jgs.17533f 151 van der Pols-Vijlbrief R, Wijnhoven HAH, Bosmans JE, Twisk JWR, Visser M. Targeting the underlying causes of undernutrition. Cost-effectiveness of a multifactorial personalized intervention in community-dwelling older adults: A randomized controlled trial. Clin Nutr 2017;36:1498-508. doi:10.1016/j.clnu.2016.09.030 171 Teh R, Barnett D, Edlin R, et al. Effectiveness of a complex intervention of group-based nutrition and physical activity to prevent frailty in pre-frail older adults (SUPER): a randomised controlled trial. Lancet Healthy Longev 2022;3:e519-30. doi:10.1016/S2666- 7568(22)00124-6 152 van Dongen EJI, Haveman-Nies A, Doets EL, Dorhout BG, de Groot LCPGM. Effectiveness of a diet and resistance exercise intervention on muscle health in older adults: ProMuscle in Practice. J Am Med Dir Assoc 2020;21:1065-1072.e3. doi:10.1016/j. jamda.2019.11.026f 172 Shlisky J, Bloom DE, Beaudreault AR, et al. Nutritional Considerations for Healthy Aging and Reduction in Age-Related Chronic Disease. Adv Nutr 2017;8:17-26. doi:10.3945/an.116.013474fifi 173 UK Chief Medical Officers. UK Chief Medical Officers’ physical activity guidelines. 2019. https://assets.publishing.service.gov. uk/government/uploads/system/uploads/attachment_data/ file/832868/uk-chief-medical-officers-physical-activity- guidelines.pdf.ff 153 van Heuvelen MJ, Hochstenbach JB, Brouwer WH, et al. Differences between participants and non-participants in an RCT on physical activity and psychological interventions for older persons. Aging Clin Exp Res 2005;17:236-45. doi:10.1007/BF03324603 174 Tuffaha HW, Gordon LG, Scuffham PA. Value of information analysis in healthcare: a review of principles and applications. J Med Econ 2014;17:377-83. doi:10.3111/13696998.2014. 907170 p / 154 van Hout HP, Jansen AP, van Marwijk HW, Pronk M, Frijters DF, Nijpels G. Prevention of adverse health trajectories in a vulnerable elderly population through nurse home visits: a randomized controlled trial [ISRCTN05358495]. J Gerontol A Biol Sci Med Sci 2010;65:734-42. doi:10.1093/gerona/glq037 155 van Leeuwen KM, Bosmans JE, Jansen AP, et al. Cost-effectiveness of a chronic care model for frail older adults in primary care: Economic evaluation alongside a stepped-wedge cluster-randomized trial. J Am Geriatr Soc 2015;63:2494-504. on 23 October 2024 by guest. Protect http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from Effectiveness of interdisciplinary primary care approach to reduce disability in community dwelling frail older people: cluster randomised controlled trial. BMJ 2013;347:f5264. doi:10.1136/bmj.f5264f 134 Shapiro A, Taylor M. Effects of a community-based early intervention program on the subjective well-being, institutionalization, and mortality of low-income elders. Gerontologist 2002;42:334-41. doi:10.1093/geront/42.3.334 113 Moll van Charante EP, Richard E, Eurelings LS, et al. Effectiveness of a 6-year multidomain vascular care intervention to prevent dementia (preDIVA): a cluster-randomised controlled trial. Lancet 2016;388:797-805. doi:10.1016/S0140-6736(16) 30950-3 135 Sherman H, Söderhielm-Blid S, Forsberg C, Karp A, Törnkvist L. Effects of preventive home visits by district nurses on self-reported health of 75-year-olds. Prim Health Care Res Dev 2016;17:56-71. doi:10.1017/S1463423614000565f 114 Monteserín Nadal R, Altimir Losada S, Brotons Cuixart C, et al. [Randomized clinical trial on the efficacy of global geriatric assessment in primary care][Spanish]. Rev Esp Geriatr Gerontol 2008;43:5-12. 136 Siemonsma PC, Blom JW, Hofstetter H, et al. The effectiveness of functional task exercise and physical therapy as prevention of functional decline in community dwelling older people with complex health problems. BMC Geriatr 2018;18:164. doi:10.1186/s12877- 018-0859-3 115 Morey MC, Ekelund C, Pearson M, et al. Project LIFE: a partnership to increase physical activity in elders with multiple chronic illnesses. J Aging Phys Act 2006;14:324-43. doi:10.1123/japa.14.3.324 115 Morey MC, Ekelund C, Pearson M, et al. Project LIFE: a partnership to increase physical activity in elders with multiple chronic illnesses. J Aging Phys Act 2006;14:324-43. doi:10.1123/japa.14.3.324 15 the bmj \| BMJ 2024;384:e077764 \| doi: 10.1136/bmj-2023-077764 RESEARCH RESEARCH on 23 October 2024 by guest. Protecte http://www.bmj.com/ BMJ: first published as 10.1136/bmj-2023-077764 on 21 March 2024. Downloaded from doi:10.1111/jgs.13834 156 van Lieshout MRJ, Bleijenberg N, Schuurmans MJ, de Wit NJ. The effectiveness of a PRoactive multicomponent intervention program on disability in independently living older people: A randomized controlled trial. J Nutr Health Aging 2018;22:1051-9. doi:10.1007/ s12603-018-1101-x 154 van Hout HP, Jansen AP, van Marwijk HW, Pronk M, Frijters DF, Nijpels G. Prevention of adverse health trajectories in a vulnerable elderly population through nurse home visits: a randomized controlled trial [ISRCTN05358495]. J Gerontol A Biol Sci Med Sci 2010;65:734-42. doi:10.1093/gerona/glq037f 175 Clegg A, Bandeen-Roche K, Farrin A, et al. New horizons in evidence-based care for older people: individual participant data meta-analysis. Age Ageing 2022;51:afac090. doi:10.1093/ageing/ afac090 155 van Leeuwen KM, Bosmans JE, Jansen AP, et al. Cost-effectiveness of a chronic care model for frail older adults in primary care: Economic evaluation alongside a stepped-wedge cluster-randomized trial. J Am Geriatr Soc 2015;63:2494-504. doi:10.1111/jgs.13834 176 Crocker TF, Lam N, Ensor J, et al. Community-based complex interventions to sustain independence in older people, stratified by frailty: a systematic review and network meta-analysis. Health Technol Assess [forthcoming]. 176 Crocker TF, Lam N, Ensor J, et al. Community-based complex interventions to sustain independence in older people, stratified by frailty: a systematic review and network meta-analysis. Health Technol Assess [forthcoming]. 156 van Lieshout MRJ, Bleijenberg N, Schuurmans MJ, de Wit NJ. The effectiveness of a PRoactive multicomponent intervention program on disability in independently living older people: A randomized controlled trial. J Nutr Health Aging 2018;22:1051-9. doi:10.1007/ s12603-018-1101-x Web appendix: Appendix 1-13 16 16 doi: 10.1136/bmj-2023-077764 \| BMJ 2024;384:e077764 \| the bmj
https://openalex.org/W4316345169	https://www.qeios.com/read/87PNT3/pdf	English	null	Review of: "Theory of a Chemical Kinetic Approach for the Estimation of the Age of Fingerprints"	null	2,023	cc-by	190	Submission date: 15-Jan-2023 06:34PM (UTC-0800) Submission ID: 1884362971 File name: theoretical_kinetics.pdf (82.57K) Word count: 2063 Character count: 10591 Submission date: 15-Jan-2023 06:34PM (UTC-0800) Submission ID: 1884362971 File name: theoretical_kinetics.pdf (82.57K) Word count: 2063 Character count: 10591 2 4 4 5 1 1 1 1 1 1 1 1 10% SIMILARITY INDEX 8% INTERNET SOURCES 2% PUBLICATIONS 1% STUDENT PAPERS 1 6% 2 1% 3 1% 4 1% 5 <1% theoretical kinetics ORIGINALITY REPORT PRIMARY SOURCES forensicscience.jacobspublishers.com Internet Source www.qeios.com Internet Source Submitted to Dubai Medical University Student Paper Samuel Cadd, Meez Islam, Peter Manson, Stephen Bleay. "Fingerprint composition and aging: A literature review", Science & Justice, 2015 Publication link.springer.com Internet Source 10% SIMILARITY INDEX 8% INTERNET SOURCES 2% PUBLICATIONS 1% STUDENT PAPERS 1 6% 2 1% 3 1% 4 1% 5 <1% theoretical kinetics ORIGINALITY REPORT PRIMARY SOURCES forensicscience.jacobspublishers.com Internet Source www.qeios.com Internet Source Submitted to Dubai Medical University Student Paper Samuel Cadd, Meez Islam, Peter Manson, Stephen Bleay. "Fingerprint composition and aging: A literature review", Science & Justice, 2015 Publication link.springer.com Internet Source theoretical kinetics % link.springer.com Internet Source % Exclude quotes On Exclude bibliography On Exclude matches Oﬀ
https://openalex.org/W2765840877	https://www.epj-conferences.org/articles/epjconf/pdf/2017/24/epjconf-jdn22_00007.pdf	English	null	Inelastic neutron scattering	EPJ web of conferences	2,017	cc-by	7,075	1. Introduction Condensed matter physics has beneﬁted tremendously from both elastic and inelastic neutron scattering techniques for more than half a century. Since then, these techniques have been used to study a wide variety of materials. The main reasons arise from the unique physical properties of the neutron itself, zero electric charge, spin half... Also, since the energies of the neutrons with “thermal” wavelengths match the energy scales of many condensed matter systems, it is possible to use them to probe the dynamics. Excitations that can be studied via neutron scattering range in energy from a few meV to a fraction of an electron volt. As a result, inelastic neutron scattering provides crucial information about the energetic terms that appear in a model Hamiltonian. While interesting from a pure fundamental point of view, it should be emphasized that these terms also determine the response of these systems to an external perturbation, hence allowing one to predict the functional capabilities of materials. To give the ﬂavor and introduce the basics of inelastic neutron scattering, it is useful to describe ﬁrst simple examples, either in the context of lattice dynamics or of spin dynamics. DOI: 10.1051/epjconf/201715500007 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) JDN 22 Inelastic neutron scattering Sylvain Petita Laboratoire Léon Brillouin, CEA-CNRS, Université Paris-Saclay, CE-Saclay, 91191 Gif-sur-Yvette, France Abstract. The goal of the JDN22 school was to propose a progressive teaching eager to improve the expertise of students in neutron diffraction. Neutron-based techniques have indeed proved for decades to be essential tools in the investigation of condensed matter. This lecture is however concerned with inelastic neutron scattering and is thus somehow apart. In the context of this school, it should then only be considered as a brief introduction. We give simple examples along with the basics of the spectrometers, and ﬁnally useful formula for the inelastic cross sections in different situations. We strongly encourage interested readers to refer to the bibliography for more detailed information. C ⃝The Authors, published by EDP Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). a e-mail: sylvain.petit@cea.fr 1.1 Lattice dynamics Quantiﬁcation of these modes leads to the concept of “phonons”, a set of harmonic oscillators, characterized by the energies k and polarization ek (that correspond to the direction of vibrations of the atoms). The displacements un are linear combinations of the different phonon modes: This dispersion is a periodic function of k, goes to zero at the Brillouin zone centers and reaches extrema at the zone boundaries. From this simple calculation, we conclude that the excited states of the chain consist in harmonic displacements of the atoms. k This dispersion is a periodic function of k, goes to zero at the Brillouin zone centers and reaches extrema at the zone boundaries. From this simple calculation, we conclude that the excited states of the chain consist in harmonic displacements of the atoms. k This dispersion is a periodic function of k, goes to zero at the Brillouin zone centers and reaches extrema at the zone boundaries. From this simple calculation, we conclude that the excited states of the chain consist in harmonic displacements of the atoms. Energies (frequencies) and wavevectors (wavelength) are related by k = 2 M sin ka 2 . Quantiﬁcation of these modes leads to the concept of “phonons”, a set of harmonic oscillators, characterized by the energies k and polarization ek (that correspond to the direction of vibrations of the atoms). The displacements un are linear combinations of the different phonon modes: Energies (frequencies) and wavevectors (wavelength) are related by k = 2 M sin ka 2 . Quantiﬁcation of these modes leads to the concept of “phonons”, a set of harmonic oscillators, characterized by the energies k and polarization ek (that correspond to the direction of vibrations of the atoms). The displacements un are linear combinations of the different phonon modes: un(t) = k e i(nka−kkt) ¯h √2Mk (ak + a+ −k) ek where the {ak, a+ k } are the annihilation and creation phonon operators. At this point, the question that naturally arises is what would be the outcome of an inelastic neutron scattering experiment on that chain? The response is simple: it would allow one to observe directly the dispersion relation k. 1.1 Lattice dynamics Practically, as we will see later, inelastic neutron scattering instruments allow one to deﬁne trajectories in the (k, ) space; the number of neutrons recorded in the detector is essentially zero along such trajectories except when they cross the k map. As shown in Fig. 1(a–d), a series of constant energy or constant Q cuts will, step by step, reconstruct the dispersion. Figure 1f shows an example of such a mapping obtained in the case of PbTe [1] by gathering cuts together: the dispersion becomes visible, above some background. Note that the intensity along the dispersion is not constant and depends on k. Back to the example of the chain, ﬁtting Eq. (1) to the measured k would allow to measure . In some sense, this reasoning is very similar to what we do when we want to analyze diffraction data. Here also the positions or relative orientations of the spins are “measured” based on a model that best ﬁts the diffraction pattern. 1.1 Lattice dynamics Let’s consider a single mono-atomic chain sketched in Fig. 1(a). Here, the atoms with mass M are at distances a, and bound together by springs (constant ). In other words, the Hamiltonian describing this system is of the form: H = n 1 2Mv2 n + 1 2(xn+1 −xn −a)2 vn and xn are the velocity and position of the individual mass at site labeled n. A diffraction experiment on such a sample (if it would exist!) would give Bragg spots at positions such that vn and xn are the velocity and position of the individual mass at site labeled n. A diffraction experiment on such a sample (if it would exist!) would give Bragg spots at positions such that EPJ Web of Conferences 155, 00007 (2017) JDN 22 DOI: 10.1051/epjconf/201715500007 Qn = 2 a n, characterizing the spatial periodic arrangement of the atoms. Information about the energy scales of the problem, such as inertia M and springs is clearly absent. Qn = 2 a n, characterizing the spatial periodic arrangement of the atoms. Information about the energy scales of the problem, such as inertia M and springs is clearly absent. To get more insight into this question, it is useful to examine the excited states of the chain. Writing the atomic position as xn = un + n a, where the n a are the equilibrium positions, classical mechanics shows that the un displacements of the atoms are governed by the equations of motion: M d2un dt2 = (un+1 −un −un + un−1) whose solutions are plane waves with energy k and wave-vector k. Introducing these solutions into the above equation, we get the dispersion relation: whose solutions are plane waves with energy k and wave-vector k. Introducing these solutions into the above equation, we get the dispersion relation: −M2 u = e ika + e−ika −2 −M2 u = e ika + e−ika −2 hence k = 2 M sin ka 2 (1) (1) This dispersion is a periodic function of k, goes to zero at the Brillouin zone centers and reaches extrema at the zone boundaries. From this simple calculation, we conclude that the excited states of the chain consist in harmonic displacements of the atoms. Energies (frequencies) and wavevectors (wavelength) are related by k = 2 M sin ka 2 . 1.2 Spin dynamics Let’s now consider the example of the two sublattices antiferromagnet depicted in Fig. 2(a). From diffraction alone, the relative orientation of spins is easily determined, but the exchange that couples two neighboring spins, along with the amplitude of a single ion anisotropy term, would be impossible to guess. From inelastic neutron scattering, however, taking advantage of 2 EPJ Web of Conferences 155, 00007 (2017) JDN 22 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) DOI: 10.1051/epjconf/201715500007 JDN 22 Figure 1. Lattice dynamics of a one 1D chain. (a) Chain model. (b) Shows the dispersion relation, while (c–e) show constant-energy scans across this dispersion. Each peak correspond to a different crossing point in (b). (f) Presents a map of longitudinal phonon propagating along c∗in PbTe, obtained by gathering constant-Q scans together. Figure 1. Lattice dynamics of a one 1D chain. (a) Chain model. (b) Shows the dispersion relation, while (c–e) show constant-energy scans across this dispersion. Each peak correspond to a different crossing point in (b). (f) Presents a map of longitudinal phonon propagating along c∗in PbTe, obtained by gathering constant-Q scans together. the magnetic interaction, it is possible to determine the dispersion of elementary excitations of such a magnet. The latter, called spin waves, are collective excitations that physically correspond to precession motions of the spins around the average direction. From theory, based on the Heisenberg model: the magnetic interaction, it is possible to determine the dispersion of elementary excitations of such a magnet. The latter, called spin waves, are collective excitations that physically correspond to precession motions of the spins around the average direction. From theory, based on the Heisenberg model: H = i,j JSi.Sj + D(Sz i )2 we learn that this dispersion is given by: k = S (6J + D)2 −J 2(2 cos kxa + 2 cos kya + 2 cos kza)2 3 EPJ Web of Conferences 155, 00007 (2017) JDN 22 DOI: 10.1051/epjconf/201715500007 Figure 2(b) and (c) illustrate these dispersions respectively without and with a single ion anisotropy D that forces the spins to lie down along . In close analogy with the phonon case, a series of constant energy or constant Q cuts will, step by step, reconstruct this dispersion. Fitting it to the above equation will provide numerical values of the exchange J and of the anisotropy D. Note especially the presence of a spin gap for D ̸= 0. 1.2 Spin dynamics Figure 2(d) shows an example of such a mapping obtained in the case of the multiferroic YMnO3 by gathering such cuts together: as for PbTe, the dispersion becomes visible, above some background. Note also that the intensity along the dispersion is not constant and depends on k. This theory assumes that there is a long range ordering (a phase transition towards a Néel state). In analogy with ﬁeld theory, the “spin waves” are (bosonic) S = 1 particles and the Néel state is considered as the “vacuum” from which these particles emerge. It should be stressed, however, that a number of magnets never order, owing to strong quantum ﬂuctua- tions, low dimensionality and frustration effects. For instance, recent progress in the physics of low dimensional systems have shown that spin waves are actually made of two spin-half quantum entities called spinons, which under usual circumstances remain conﬁned and thus not visible. Actually, this concept of conﬁnement, when particles are bound together by an interaction whose strength increases with increasing particle separation, is a fundamental idea of modern physics. The most widely studied model in which this phenomenon takes place is the spin-half Heisenberg antiferromagnetic chain with ﬁrst-neighbor interactions. In agree- ment with the Wagner and Mermin theorem, quantum ﬂuctuations prevent static long-range order and the ground state remains a spin liquid, with power-law decaying antiferromagnetic correlations. In such a chain, spinons can be seen as domain walls between reversed antiferro- magnetic domains; they can move freely and independently along the chain, as no energy cost is involved: in other words, they do not conﬁne. In this case, the magnetic response shows no well-deﬁned spin wave excitations but an incoherent spectrum called two-spinons continuum, which reﬂects the range of energies corresponding to the different spinon pairs (see Fig. 2(e)). 2. Principles We now turn to the description of spectrometers dedicated to inelastic neutron scattering experiments [2–10]. As for diffraction, most of those experiments demand a monochromatic beam, to be prepared from the white beam produced by the neutron source (reactor or pulsed source). The main idea is then to determine the ﬁnal energy of the neutrons by analyzing the scattered beam. Taking the energy difference between incident and outgoing neutrons gives the energy Ei −Ef = given to the sample (note that this quantity can be negative, which correspond to a situation where the neutrons receive energy from the sample and are thus accelerated). There are essentially two methods for discerning the energy of the neutrons (see Figs. 3 and 4): the ﬁrst one is by measuring the time it takes for neutrons to travel the distance between the sample to the detector, known as time-of-ﬂight neutron spectroscopy (TOF) [9]. This method assumes that the incident beam is chopped to deﬁne the time reference, hence reducing the effective average ﬂux on the sample. The second method is by Bragg scattering from a single crystal, known as triple-axis spectroscopy (TAS) [8, 10, 11]: = 2 \|kf \| = 2d sin = 2 \|kf \| = 2d sin It takes advantage of the continuous ﬂux of the source. It was invented by Brockhouse in the mid 1950s at Chalk River [7]. Although there have been many improvements, the principle behind this technique has not changed from that originally developed. The term triple-axis It takes advantage of the continuous ﬂux of the source. It was invented by Brockhouse in the mid 1950s at Chalk River [7]. Although there have been many improvements, the principle behind this technique has not changed from that originally developed. The term triple-axis 4 EPJ Web of Conferences 155, 00007 (2017) JDN 22 EPJ Web of Conferences 155, 00007 (2017) DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) DOI: 10.1051/epjconf/201715500007 JDN 22 Figure 2. Spin dynamics in a simple two sublattices antiferromagnet. (a) displays the antiferromagnetic chain. (b-c) Show the neutron scattering expected intensity in two different cases, without and with single ion anisotropy respectively. (d) Presents a map of the dispersion of spin waves in the multiferroic material YMnO3 and (e) a map of the two spinons continuum in a 1D chain (M. Arai, M. Fujita, M. Motokawa, J. Akimitsu, and S.M. 2. Principles Bennington, Phys. Rev. Lett. 77, 3649 1996). JDN 22 Figure 2. Spin dynamics in a simple two sublattices antiferromagnet. (a) displays the antiferromagnetic chain. (b-c) Show the neutron scattering expected intensity in two different cases, without and with single ion anisotropy respectively. (d) Presents a map of the dispersion of spin waves in the multiferroic material YMnO3 and (e) a map of the two spinons continuum in a 1D chain (M. Arai, M. Fujita, M. Motokawa, J. Akimitsu, and S.M. Bennington, Phys. Rev. Lett. 77, 3649 1996). derives from the fact that neutrons are scattered from three crystals as they travel from the source to the detector. derives from the fact that neutrons are scattered from three crystals as they travel from the source to the detector. Depending on the possible processes that take place in the sample, the scattered neutrons may have a different energy from the incident one. This is sketched in Fig. 3(a) using rays of several colors. At this point, the conservation of energy and momentum writes: Ei −Ef = ki −kf = Q Ei −Ef = ki −kf = Q ki −kf = Q with neutron incident and ﬁnal energies and momentum related by: with neutron incident and ﬁnal energies and momentum related by: Ei,f = ¯h2k2 i,f 2Mn 5 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) EPJ Web of Conferences 155, 00007 (2017) JDN 22 Figure 3. Sketches of a neutron scattering experiment. (a) Pointing out that under a given scattering angle, the beam contains neutrons of different energies; (b) illustrates the conservation of energy and momentum and (c) the rotation necessary to let coincide Q deﬁned by ki −kf and the reciprocal space of the sample. Importantly, we notice that the scattering angle = (ki, kf ) is determined by the modulus of the wavevector Q: Importantly, we notice that the scattering angle = (ki, kf ) is determined by the modulus of the wavevector Q: cos = k2 i + k2 f −Q2 2kikf The wavevector Q is determined, for the moment, by ki −kf , and has no special meaning in terms of reduced reciprocal coordinates (h, k, ℓ) of the sample itself. This becomes only true if two particular directions of the sample, within the scattering plane, are known. 2. Principles In that case, Q = ki −kf can be projected onto the sample’s reciprocal lattice, so that (h, k, ℓ) are uniquely deﬁned. Rotating the sample around the vertical axis allows to address a new set of (h′, k′, ℓ′) coordinates, corresponding to the same modulus. Each of these methods, TAS or TOF (see Fig. 4(a) and (b) respectively) is most useful in studying a particular type of problem. Traditionally, TAS has been the preferred method for studying single crystals, and TOF for studying powders. Yet, in principle, nothing forbids to measure a single crystal on a TOF instrument. It is just necessary to rotate the sample around the vertical axis, to “reconstruct”, for each scattering angle and each time of ﬂight value, the (h, k, ℓ, ) coordinates. Today, the main advantage of TAS spectrometer consists in the fact 6 EPJ Web of Conferences 155, 00007 (2017) DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) JDN 22 JDN 22 Figure 4. Sketch of TAS and TOF spectrometers. that it is quite easy to vary external parameters like temperature, magnetic ﬁeld, pressure or electric ﬁeld and to measure the response in a small region of (Q, ) space. In contrast, TOF techniques are useful to obtain a survey of excitations in a rather extended (Q, ) region. Figure 4. Sketch of TAS and TOF spectrometers. Figure 4. Sketch of TAS and TOF spectrometers. that it is quite easy to vary external parameters like temperature, magnetic ﬁeld, pressure or electric ﬁeld and to measure the response in a small region of (Q, ) space. In contrast, TOF techniques are useful to obtain a survey of excitations in a rather extended (Q, ) region. that it is quite easy to vary external parameters like temperature, magnetic ﬁeld, pressure or electric ﬁeld and to measure the response in a small region of (Q, ) space. In contrast, TOF techniques are useful to obtain a survey of excitations in a rather extended (Q, ) region. 2.1 TAS in practice As is clear from the Bragg law, monochromators and analysers select ko, 2ko, 3ko ... (ko standing for ki or a kf ). Even if the measured intensities are normalized to the same number of incident neutrons with the help of a low sensibility detector placed in the incident beam, called “monitor”, it remains important to correct these data for those “bad” neutrons with 2ko, 3ko ... This supposes that we know the proportion of harmonics in the incident beam, a quantity that especially depends on the incident energy, the kind of source (thermal, hot, cold, see Fig. 5(a)), and also on the installation of the spectrometer on a neutron guide or not. This correction, called “monitor correction”, is shown in Fig. 5(b). Another issue arising because of harmonics is the possible contamination of the measurement by elastic processes ( = 0) occurring in the sample provided that: nko = mkf (n, m) integers 7 7 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) EPJ Web of Conferences 155, 00007 (2017) JDN 22 JDN 22 Figure 5. Monitor and ﬁlters in a TAS experiment. (a–b) show the incident ﬂux, depending on the kind of source, and the monitor correction obtained for 2T [10]. (c) illustrates the ﬁltering with pyrolithic graphite, high transmission for ko and almost zero for the harmonics. Figure 5. Monitor and ﬁlters in a TAS experiment. (a–b) show the incident ﬂux, depending on the kind of source, and the monitor correction obtained for 2T [10]. (c) illustrates the ﬁltering with pyrolithic graphite, high transmission for ko and almost zero for the harmonics. The intensity of such processes can be extremely large compared to the “true” inelastic expected signal. To partly eliminate this problem, ﬁlters are used, either placed in the incident or in the scattered beam. The idea is to use a peculiarity of the transmission of pyrolithic Graphite, which is almost 1 for a given kf and 0 for the harmonics, at least (in practice) for 2kf . This happens for “magic” values, that are widely used on TAS, 4.1, 3.85, 2.662, 2.57, 1.97, 1.64, and 1.48 Å−1 (see Fig. 5(c)). The Beryllium is another material widely used on cold TAS. If cooled at 70 K, its transmission is about 1 for wavevectors lower than 1.55 Å−1 and 0 above. Finally, it is of fundamental importance to introduce the concept of resolution. with R(, Q) = Vi Vf exp −{Q, }T ¯A {Q, } {Q, } is a 4 dimensional vector with components Q and . ¯A is a 4 × 4 matrix. Explicated in its eigen-directions, becomes a nice 4D-ellipsoid with long and short axes, called resolution ellipsoid. A constant energy scans across the dispersion relation gives en enlarged peak as sketched in Fig. 6(a). There exists softwares able to calculate R [10] but we shall give here only guidelines: • the z-axis can be considered as decoupled from the other quantities. The vertical resolution is generally quite poor. • the z-axis can be considered as decoupled from the other quantities. The vertical resolution is generally quite poor. • because of the inclination of the ellipsoid in (qx, qy, ) space, the analysis of dispersion can become tricky, with especially a focused and an unfocused side: as sketched in Fig. 6(b) and (c), the apparent width of branches can look very different. • because of the inclination of the ellipsoid in (qx, qy, ) space, the analysis of dispersion can become tricky, with especially a focused and an unfocused side: as sketched in Fig. 6(b) and (c), the apparent width of branches can look very different. • the smaller kf or ki, the better the resolution; however, since Vi,f ≈k3 i,f , the less intense is the expected signal. 3.1 Nuclear interaction In the case of nuclear interaction, this cross section can be written as follows: 2 Ef = kf ki i,j bi bj +∞ −∞ dt ⟨e i Q Ri(t=0) e−i Q Rj(t)⟩e−it where bi is the scattering length of ion at site i with time dependent position Ri(t). where bi is the scattering length of ion at site i with time dependent position Ri(t). 3. Cross sections The quantity that we ultimately measure is the neutron cross section. It is proportional to the number of neutrons scattered in a solid angle with a ﬁnal energy Ef . 2.1 TAS in practice Actually, a spectrometer cannot isolate a single point in (Q, ) space; rather, it integrates over a small volume around that point, an effect which can be modeled by the following convolution: I(Q, ) = dQ′ d′ R( −′, Q −Q′) S(′, Q′) 8 EPJ Web of Conferences 155, 00007 (2017) EPJ Web of Conferences 155, 00007 (2017) JDN 22 DOI: 10.1051/epjconf/201715500007 JDN 22 Figure 6. Illustration of the ellipsoid resolution on a TAS [10]. The effect of the inclination of the long axis with respect to the slope of the dispersion is especially illustrated. If these are parallel, then the signal is narrow and intense, while it is broad and weak in the opposite situation. These two possibilities are called focused and unfocused conditions. Figure 6. Illustration of the ellipsoid resolution on a TAS [10]. The effect of the inclination of the long axis with respect to the slope of the dispersion is especially illustrated. If these are parallel, then the signal is narrow and intense, while it is broad and weak in the opposite situation. These two possibilities are called focused and unfocused conditions. with JDN 22 new indexes i = (m, ℓ). new indexes i = (m, ℓ). R (t) Ro + xes i = (m, ℓ). Ri(t) = Ro m + rℓ Note that because of the spatial periodicity, the bi’s only depend on ℓ. The cross section becomes: 2 Ef = kf ki () m,n,ℓ,ℓ′ bℓbℓ′ e i Q (Ro m+rℓ−Ro n−rℓ′) = () m,n e i Q (Ro m−Ro n) ℓ,ℓ′ bℓbℓ′ e i Q (rℓ−rℓ′) = () m,n e i Q (Ro m−Ro n) \|FN(Q)\|2 = () (Q − ) \|FN(Q)\|2 where is a reciprocal lattice vector and FN(Q) is the familiar nuclear structure factor: where is a reciprocal lattice vector and FN(Q) is the familiar nuclear structure factor: FN(Q) = ℓ bℓe i Q rℓ As a result, in this approximation, the cross section is purely elastic ( = 0) and consists of Dirac functions at the nodes of the reciprocal lattice. Their intensities are given by the squared modulus of the structure factor. 3.1.1 The frozen lattice Moving in gradual steps, let’s ﬁrst assume that the lattice is frozen, so that these positions do not depend on time. The vector Ri is decomposed into a vector that labels the unit cell Ro m and a second vector within the unit cell rℓ. Concomitantly, the site i is decomposed into two 9 9 EPJ Web of Conferences 155, 00007 (2017) DOI: 10.1051/epjconf/201715500007 3.1.2 The harmonic approximation We now turn to the ”vibrating” lattice approximation. The Ri(t) become: Ri(t) = Ro m + rℓ+ um,ℓ(t) where um,ℓ(t) describes the harmonic oscillations, in terms of phonon modes, of the atom labeled (m, ℓ). The cross section writes: 2 Ef =kf ki m,n ei Q (Ro m−Ro n) ℓ,ℓ′ bℓbℓ′ei Q (rℓ−rℓ′) +∞ −∞ dt⟨ei Q um,ℓe−i Q un,ℓ′(t)⟩e−it Using quantum properties of the phonon creation and annihilation operators, the expectation value ⟨..⟩can be rearranged: ⟨e i Q um,ℓe−i Q un,ℓ′(t)⟩= e−Wℓ−Wℓ′ e⟨Q um,ℓQ un,ℓ′(t)⟩ Wℓis the so called Debye-Waller factor, that physically describes the average squared deviations of the ions at temperature T with respect to their equilibrium position: Wℓ= 1 2 Q um,ℓ 2 Wℓ= 1 2 Q um,ℓ 2 The cross section becomes: 2 Ef =kf ki m,n ei Q (Ro m−Ro n) ℓ,ℓ′ bℓbℓ′ ei Q (rℓ−rℓ′) e−Wℓ−Wℓ′ +∞ −∞ dt e⟨Q um,ℓQ un,ℓ′(t)⟩e−it Expanding the exponential function to second order, one obtains: e⟨Q um,ℓQ un,ℓ′(t)⟩≈1 + ⟨Q um,ℓQ un,ℓ′(t)⟩+ ... 10 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) JDN 22 The zeroth order leads to a slight modiﬁcation of the structure factor which now incorporates the Debye-Waller factor: The zeroth order leads to a slight modiﬁcation of the structure factor which now incorporates the Debye-Waller factor: 2 Ef = () (Q − ) \|F(Q)\|2 FN(Q) = ℓ bℓe i Q rℓe−Wℓ The second order term is the phonon cross section: The second order term is the phonon cross section: 2 Ef = s As [ 1+n(Q,s) ( −Q,s) + n(Q,s) (+Q,s)] 2 Ef = s As [ 1+n(Q,s) ( −Q,s) + n(Q,s) (+Q,s)] and consists in a sum of Dirac functions on the different phonon branches. This ﬁnally demonstrates that an inelastic neutron scattering experiment allows a direct measurement of the dispersion relations. The intensity along the dispersion is given by the “dynamical” structure factor: As = \|Fs\|2 with with The scalar product between the wavevector Q and the polarization eq,ℓ,s is especially useful as it allows to distinguish transverse and longitudinal vibrations. Longitudinal phonons correspond to vibrations parallel to the propagation vector q, while transverse phonons correspond to vibrations perpendicular to q. To this end, it sufﬁces to measure in different Brillouin zones. With Q = + q ( is the zone center), different conﬁgurations can be chosen so that Q is approximatively parallel or perpendicular to a given polarization. Figure 7(a) shows an example of this trick in the case of CaF2, a sample very often used for practicals at LLB: maps obtained for transverse (Fig. 7(c)) and longitudinal (Fig. 7(d)) phonons propagating along (hh0) are shown as well as a typical individual Q-scan used to produce the maps. Note that positive energy processes correspond to the creation of a phonon by a neutron, while negative energy process correspond to the annihilation of a phonon. n(Q,s) is the Bose number at temperature T: n(Q,s) = 1 e ¯hQ,s kB T −1 Further, the two processes do not have the same intensity: the creation is always possible, even at low temperature, while the annihilation supposes that the initial quantum state of the sample contains phonons. For instance, the annihilation is forbidden at very low temperature since there are no phonons in the ground state conﬁguration. JDN 22 This is called the “detailed 11 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) EPJ Web of Conferences 155, 00007 (2017) JDN 22 JDN 22 Figure 7. Inelastic neutron scattering tutorial in CaF2. (a) Sketch of the reciprocal plane (hk0), along with two Brillouin zone appropriate ﬁr the study of transverse and longitudinal phonon modes. (b) Shows an individual constant-energy scan while (c) and (d) show the maps obtained by gathering such scans in transverse and longitudinal conditions. Figure 7. Inelastic neutron scattering tutorial in CaF2. (a) Sketch of the reciprocal plane (hk0), along with two Brillouin zone appropriate ﬁr the study of transverse and longitudinal phonon modes. (b) Shows an individual constant-energy scan while (c) and (d) show the maps obtained by gathering such scans in transverse and longitudinal conditions. balance”; this is an extremely important concept, also known in other spectroscopy methods like IR or Raman scattering for instance. 3.3 Frozen spins If the spins are frozen, S⊥,ℓdo not depend on time, and we are left with a pure static ( = 0) contribution: 2 Ef = ( Nro)2 () m (Q − m) \|Fm(Q)\|2 m is a reciprocal magnetic lattice vector and Fm is the magnetic structure factor: Fm(Q) = ℓ S⊥,ℓe i Q rℓe−Wℓfℓ(Q) Fm(Q) = ℓ S⊥,ℓe i Q rℓe−Wℓfℓ(Q) 3.2 Magnetic interaction In the case of the magnetic interaction, the cross section is given by: 2 Ef = kf ki ( Nro)2 i,j +∞ −∞ dt ⟨S⊥,i S⊥,j(t) e i Q Ri e−i Q Rj(t)⟩e−it N = 1.913 is the gyromagnetic ratio of the neutron, ro is the classical electron radius. S⊥is the component of the spin perpendicular to the scattering vector Q. It is worth noting that: N = 1.913 is the gyromagnetic ratio of the neutron, ro is the classical electron radius. S⊥is the component of the spin perpendicular to the scattering vector Q. It is worth noting that: S⊥,i S⊥,j(t) = , S,i , −Q Q Q2 S,j(t) The exact derivation of the cross section goes well beyond this lecture; we shall however give the ﬁnal result: The exact derivation of the cross section goes well beyond this lecture; we shall however give the ﬁnal result: 2 Ef =kf ki ( Nro)2 m,n ei Q (Ro m−Ro n) ℓ,ℓ′ fℓ(Q)f ∗ ℓ′(Q)eiQ(rℓ−rℓ′)e−Wℓ−Wℓ′ +∞ −∞ dt⟨S⊥,ℓS⊥,ℓ′(t)⟩e−it 2 Ef =kf ki ( Nro)2 m,n ei Q (Ro m−Ro n) ℓ,ℓ′ fℓ(Q)f ∗ ℓ′(Q)eiQ(rℓ−rℓ′)e−Wℓ−Wℓ′ +∞ −∞ dt⟨S⊥,ℓS⊥,ℓ′(t)⟩e−it JDN 22 In other words, diffraction measures instantaneous correlations. Diffraction at zero energy measures elastic (or inﬁnite time) correlations; inelastic scattering measures the whole (ﬁnite time) spectrum of these correlations. 3.1.3 Connection with diffraction As already pointed out, a diffraction experiment do not analyze the ﬁnal energy of the neutrons but rather integrates over these ﬁnal energies. As a result, this kind of measurement is sensitive to both the pure elastic Bragg scattering, but also to the excitations. As the cross section is very small, it appears as a kind of diffuse scattering, which can be difﬁcult to distinguish from the background. Note that cannot be larger than the incident energy Ei, so that the corresponding cross section reads: = Ei −∞ d kf ki i,j bi bj +∞ −∞ dt ⟨e i Q Ri e−i Q Rj (t)⟩e−it If Ei is large enough compared to the dynamics, one usually integrates out the dependence to write: If Ei is large enough compared to the dynamics, one usually integrates out the dependence to write: = i,j bi bj ⟨e i Q Ri(t=0) e−i Q Rj (t=0)⟩ 12 EPJ Web of Conferences 155, 00007 (2017) JDN 22 EPJ Web of Conferences 155, 00007 (2017) DOI: 10.1051/epjconf/201715500007 3.4 Spin waves In the spin wave approximation, the elementary excitations, called spin waves, correspond to precession motions of the spins around the magnetization direction [12–17]. These spin waves are free particles carrying a spin 1. Tough calculations show that: 2 Ef = s As[ 1 + n(Q,s) ( −Q,s) + n(Q,s)( + Q,s)] In close analogy with the case of phonons, the cross section takes the form of a sum of Dirac functions on the different spin waves branches. Again, this formula shows that an inelastic neutron scattering experiment allows a direct measurement of the dispersion 13 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) EPJ Web of Conferences 155, 00007 (2017) JDN 22 Figure 8. Spin wave dispersion in the cubic ferromagnet La1−xSrxMnO3 compounds. A simple cosine dispersion is expected. Note the ﬂattening close to the zone boundary which has not been explained yet despite a huge theoretical work. Figure 8. Spin wave dispersion in the cubic ferromagnet La1−xSrxMnO3 compounds. A simple cosine dispersion is expected. Note the ﬂattening close to the zone boundary which has not been explained yet despite a huge theoretical work. relations. The intensity of the modes is given by a “dynamical” structure factor As, whose general expression, however, is rather complex; its derivation goes far beyond the scope of this lecture. Figure 8 shows the mapping measured for instance in the La1−xSrxMnO3 compounds, very well know for their magneto-resistance properties. These systems are simple cubic ferromagnets (lattice parameter a), and one expects a dispersion relation reading as: k = 2JS(3 −cos kxa −cos kyb −cos kza) As can be observed from Fig. 8, this simple form ﬁts very well the experiment at small q, but is clearly not adapted close to the zone boundary: the dispersion becomes rather ﬂat, an effect which remains mysterious so far, not reproduced by theoretical calculations. This illustrates the relevance and simplicity of inelastic neutron scattering, which deﬁnitely provides valuable information about the spectrum of elementary excitations even in very complex condensed matter. 3.6 Metal In a metal, or by the way, in any material where the elementary excitations are fermions carrying a spin half (like spinons in 1D chains, for instance), the spin excitations is composed of a continuum that is nothing but a kind of two particles density of states. Indeed, since the particles carry a spin half, two “ﬂips”, hence a pair of particles, is necessary to match the S = 1 transition of the neutron spin. For a given (k, ), different pairs of fermions contribute provided their energies Eq and Eq′ fulﬁll the condition = Eq′ + Eq and provided the momentum is conserved k = q + q′. The corresponding spectral weight depends however on details of the Hamiltonian [18]. 3.5 Crystal ﬁeld levels The response due to the inelastic scattering by crystal ﬁeld excitations is also very useful in practice. The latter correspond to the different transition between states describing the electronic conﬁgurations of the (usually 4f or 5f) electrons. Denoting En and n the energies and wave functions, we get: +∞ −∞ dt ⟨S⊥,ℓS⊥,ℓ(t) ⟩e−it = +∞ −∞ dt n,m e−En/T Z ⟨n\|S⊥,ℓ\|m⟩⟨m\|S⊥,ℓ\|n⟩e i(Em−En−)t = n,m e−En/T Z \|⟨n\|S⊥,ℓ\|m⟩\|2(Em −En −) Z = n e−En/T 14 DOI: 10.1051/epjconf/201715500007 EPJ Web of Conferences 155, 00007 (2017) JDN 22 JDN 22 from which one deduces: 2 Ef = kf ki ( Nro)2 ℓ \|fℓ(Q)\|2 e−2Wℓ n,m e−En/T Z \|⟨n\|S⊥,ℓ\|m⟩\|2(Em −En −) As a result, the scattering due to crystal ﬁeld shows Dirac functions at all possible transitions between two eigenstates = Em −En. Note that there is no particular Q dependence: indeed, no correlations are expected since the moments do not interact with each other, except the Debye Waller and the form factor. At low temperature, most of the possible transitions disappear since e−En/T Z ≈n,1, so that we are left with the transition from the ground state only. The cross section becomes: 2 Ef = kf ki ( Nro)2 ℓ \|fℓ(Q)\|2 e−2Wℓ m \|⟨1\|S⊥,ℓ\|m⟩\|2(Em−E1−) 4. Fluctuation dissipation We would like now to close this lecture by pointing out the link between two equivalent languages: correlations and response function or generalized susceptibility [19]. Actually, the response of a system under a time dependent perturbation B(t) results in the emergence of an observable A. Applying a magnetic ﬁeld will for instance result in a magnetization, or applying a force will result in atomic displacements. In the framework of linear response theory, A and B are “proportional”, related by a generalized susceptibility G: A(t) = dt′G(t −t′)B(t′) or in frequency representation: A() = G()B() A() = G()B() where the Fourier transform G() is deﬁned as: G() = lim ϵ→0+ dte it−ϵt G(t) The theory shows that [19]: G(t) = i(t)⟨[A, B(t)]⟩o where the expectation value is to determined with the unperturbed Hamiltonian of the system (B = 0). If one denotes En and n the eigen-energies and wave functions of the system, one 15 DOI: 10.1051/epjconf/201715500007 G(t) = i(t) n e−En/T Z ⟨n\| [A, B(t)] \|n⟩o Introducing ⟨n\|A\|m⟩= Anm, and calculating the commutator, this becomes: G(t)=i(t) n,m e−En/T Z (AnmBmn e i(Em−En)t−BnmAmn e−i(Em−En)t) or or G(t) = i(t) n,m e−En/T Z AnmBmn e i(Em−En)t −e−Em/T Z BmnAnm e−i(En−Em)t = i(t) n,m e−En/T −e−Em/T Z AnmBmn e i(Em−En)t = i(t) n,m e−En/T Z 1 −e−(Em−En)/T AnmBmn e i(Em−En)t G(t) = i(t) n,m e−En/T Z AnmBmn e i(Em−En)t −e−Em/T Z BmnAnm e−i(En−Em)t = i(t) n,m e−En/T −e−Em/T Z AnmBmn e i(Em−En)t Using: lim ϵ→0+ dte it−ϵt i(t) e ixt = lim ϵ→0+ i[0 −1] i( + x) −ϵ = lim ϵ→0+ −1 ( + x) + iϵ = −Pf 1 + x + i( + x) We have for the imaginary part of G(): We have for the imaginary part of G(): We have for the imaginary part of G(): G′′() = n,m e−En/T Z 1−e−(Em−En)/T AnmBmn (+(Em−En)) = 1−e−/T n,m e−En/T Z AnmBmn ( + (Em −En)) On the other hand, correlations between A and B are deﬁned by: C(t) = ⟨AB(t)⟩o On the other hand, correlations between A and B are deﬁned by: C(t) = ⟨AB(t)⟩o C(t) = ⟨AB(t)⟩o which can be rewritten as: which can be rewritten as: which can be rewritten as: C() = n,m e−En/T Z AnmBmn( + (Em −En)) As a result: G() = 1 −e−/T C() C() = 1 1 1 −e−/T G() As a result: As a result: G() = 1 −e−/T C() C() = 1 1 1 −e−/T G() 16 EPJ Web of Conferences 155, 00007 (2017) EPJ Web of Conferences 155, 00007 (2017) JDN 22 DOI: 10.1051/epjconf/201715500007 These calculations demonstrate the relations between the generalized susceptibility (also called response function) and the correlations. 1 −e−/T −1 is called the detailed balance factor. A() = G()B() The magnetic inelastic neutron cross section is a measure of correlations between spins; it is also nothing but the imaginary part of the frequency dependent magnetic susceptibility. 5. Conclusion In this lecture, the basics of inelastic neutron scattering were presented. We have especially pointed out that diffraction measures instantaneous correlations, while inelastic scattering determine the time dependence of those correlations. This allows a direct measurement of the dispersion of elementary excitations, like phonons or spin waves. References [1] W. Szuszkiewicz, unpublished [2] G.L. Squires, Introduction to the Theory of Neutron Scattering (Dover Publications, 1996) [2] G.L. Squires, Introduction to the Theory of Neutron Scattering (Dover Publications, 1996) [3] P.G. de Gennes, Theory of Neutron Scattering by Magnetic Crystals, in Magnetism, Vol. III, Eds. G.T. Rado and H. Suhl (New York: Academic Press, 1963), p.115–147 [4] Neutron Scattering, ed. by K. Sköld, D.L. Price, Methods in Experimental Physics, Vol. 23C (Academic Press, New York, 1987) [5] S.W. Lovesey, Theory of Neutron Scattering from Condensed Matter, Vol 2 (Oxford Science Publications, 1984) [6] Y.A. Izyumov and N.A. Chernoplekov, Neutron Spectroscopy (Plenum, 1994) [7] B.N. Brockhouse. Inelastic scattering of neutrons in solids and liquids, Vienna. 1961. p. 113; http://nobelprize.org/nobel_prizes/physics/laureates/1994/ brockhouse-lecture.html [8] G. Shirane, S.M. Shapiro and J. Tranquada, Neutron Scattering with a Triple-Axis Spectrometer, Basic Techniques (Cambridge University Press, 2002) [9] Diffusion inélastique de neutrons par temps de vol. J. Ollivier et J.-M. Zanotti. JDN 16, Collection SFN 10, 379–423 (2010) https://doi.org/10.1051/sfn/2010006 [10] La diffusion inélastique des neutrons sur monocristal. Le spectromètre 3-axes. B. Hennion. JDN 16, Collection SFN 10, 357–378 (2010) https://doi.org/10.1051/sfn/2010005 p g [11] Z. Yamani, Z. Tun, and D.H. Ryan, Can. J. Phys. 88, 771–797 (2010) [12] Magnetic Excitations, W.G. Stirling et K.A. McEwen, Methods of Experimental Physics, Vol. 23 Part C (Academic Press, 1987) [13] D.C. Mattis, Theory of Magnetism I (Springer Verlag, 1988) [14] R.M. White, Quantum Theory of Magnetism (Springer Verlag, 1987) ite, Quantum Theory of Magnetism (Springer [15] A. Auerbach, Interacting electrons and Quantum Magnetism (Springer Verlag, 1994) [16] D.C. Wallace, PR 128, 1614 (1962) Spin waves in complex lattice [17] Numerical simulations and magnetism. S. Petit, JDN 18, Collection SFN 12, 105–121 (2011) https://doi.org/10.1051/sfn/201112006 [17] Numerical simulations and magnetism. S. Petit, JDN 18, Collection SFN 12, 105–121 (2011) // / / / [18] Magnétisme de bande et Supraconductivité: l’apport de la diffusion inélastique des neutrons. Yvan Sidis, JDN 20, Collection SFN 13, 04003 (2014) https://doi.org/10.1051/sfn/20141304003 [19] Physique statistique hors d’équilibre: Processus irréversibles linéaires. N. Pottier (EDP Sciences, 2007) 17
https://openalex.org/W4319452444	https://zenodo.org/records/7619277/files/MMMS0208.pdf	English	null	THE EFFECT OF TILLAGE IN VARIOUS WAYS ON THE THICKNESS OF A POROUS SEEDLING	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,951	MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference THE EFFECT OF TILLAGE IN VARIOUS WAYS ON THE THICKNESS OF A POROUS SEEDLING Karabaev Ikramjan Turaevich Research Institute of Cotton Breeding, Seed Production and Cultivation Agrotechnology. Tashkent, Uzbekistan E-mail: ikromjon@gmail.com Atabaeva Mamura Sadridin qizi DSc Doctoral student of Andijan Institute of Agriculture and Agrotechnology. Andijan, Uzbekistan E-mail: mamura@gmail.com https://doi.org/10.5281/zenodo.7619277 Introduction. Today, thanks to the widespread introduction of advanced soil- protecting resource-saving technologies in agriculture into world agricultural practice, a reduction in the cost of production is achieved by improving the agronomic properties of land, growing high and high-quality crops and saving fuel and lubricants. Soil protection technologies are being implemented worldwide on a total area of more than 10 million hectares. THE EFFECT OF TILLAGE IN VARIOUS WAYS ON THE THICKNESS OF A POROUS SEEDLING Decree of the President of the Republic of Uzbekistan dated June 17, 2019 No. 5742 "On the effective use of land and water resources in agriculture", Decree of the President of the Republic of Uzbekistan dated January 28, 2020 No. 4575 "On measures to implement in 2020 the tasks set in the strategy for the development of agriculture of the Republic of Uzbekistan. The degree of study of the problem: It is known that the wheels of high- performance technical means, as a result of the negative impact on the physical properties of the soil, microbiological processes occurring in it, lead to seed germination and plant growth, as well as a sharp decrease in yield. To Uzbekistan P .Nazarov, F.Khasanov, A.Sindirov, A.Khamrakulov [1; B. 2-3], F.Khasanova [2; B. 5], A.Yakubdzhonov [3; B. 391, 137; B. 358], H.Tursunav [4; B. 23], A.Yangiboev [5; B. 11-12] it is noted that when applying the technology of sowing seeds in seedlings, the ground is leveled in autumn, which is due to a reduction in agrotechnical measures in early spring, saving fuel and lubricants, a higher temperature than on flat ground, up to 1-30c, germination 2-4 days ago, reduced water consumption during irrigation. Research methodology. Light gray, mechanical composition of the OK Water Experimental farm Kurgontepa district of Andijan region - medium loamy, unsalted, groundwater is located at a depth of 4-5 meters, the amount of humus (humus) in the Haidovsky (0-30 cm) soil layer is 0.8-0.9%, the nitrogen content of the total forms of nutrients is 0.05-0.09%, is. According to the mechanical composition, the medium and light clod varies from the composition to the 45 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference composition of a heavy clod, the proportion of large dust particles is 44-51%, and small dust particles is 30-40%. MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference composition of a heavy clod, the proportion of large dust particles is 44-51%, and small dust particles is 30-40%. THE EFFECT OF TILLAGE IN VARIOUS WAYS ON THE THICKNESS OF A POROUS SEEDLING Studies have shown that in 2018-2020, with a 2:1 crop rotation system (grain:cereals) varieties "Antonina" from autumn wheat, "salomat" from peanuts were grown as a repeat crop after autumn wheat, "Dormon" from lentils, "Rovot" from beans, "Jahangir" from sunflower., next year, the care of the wheat variety "Andijan 36" as the main crop was carried out on based on the following experimental system (Table 1) 46 EXPERIENCE SYSTEM № Processing methods and depths The main type of culture Types of repeated crops The main type of culture The main type of culture 2018/2019 y 2019 y. 2020 y. 2021 y. 1 Plowing the earth to a constant depth of 35-40 cm +boronization+molting+getting pus + planting (control) Winter wheat Lentil Cotton Cotton 2 peanut 3 Sunflower 4 Beans 5 Planting a bush to a height of 30- 35 cm in one step, cultivating the ground to a constant depth of 35-40 cm. Winter wheat Lentil Cotton Cotton 6 peanut 7 Sunflower 8 Beans 9 Cultivation in a minimal way for winter wheat and repeated sowing (to a depth of 10-12 cm) + plowing to a depth of 35-40 cm when caring for a goose + getting a bush + planting. Winter wheat Lentil Cotton Cotton 10 peanut 11 Sunflower 12 Beans 13 Autumn crops with a desk and doubles are limited to processing by the food method (drinking 10-12 cm) + processing of a gift of 35-40 cm KA'ri hihi, take one pashto 30-35 cm high + planting Winter wheat Lentil Cotton Cotton 14 peanut 15 Sunflower 16 Beans MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference 17 Cultivation of the earth to a constant depth of 35-40 cm, unilateral bringing of the bush to a height of 30-35 cm, nitrogen application under the bush in the form of liquid ammonia in the amount of 100 kg / ha + planting Winter wheat Lentil Cotton Cotton 18 peanut 19 Sunflower 20 Beans 21 For winter wheat and repeated crops, the minimum processing method (to a depth of 10-12 cm) + processing to a depth of 35-40 cm. MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference during processing with a combined unit, the thickness of seedlings was observed up to 254.9-249.9 in lentils, 99.8-102.9 in peanuts, 54.3-55.1 in sunflower and 227.2-214.9 thousand units/ha in legumes. Winter wheat, repeated sowing of peanuts, then next year again the continuous area for caring for the goose is 35-40 cm deep in 2 variants of Haydov, the thickness of goose seedlings is 85.8 thousand pcs./ ha, and other repeated crops (lentils, sunflower, beans), which are cared for during this processing method were determined in the course of observations at the end. For the cultivation of winter wheat and repeated sowing, a set of measures was carried out for plowing to a depth of 35-40 cm to care for a goose planted in one way, tillage to a depth of 10-12 cm (minimum method), as a repeat crop on the 3rd background, when planting seeds, it was noticed that the thickness of seedlings by 0.2-4.8 thousand pcs/ha is higher compared to the background. Cultivation of the earth to a depth of 35-40 cm with constant (when caring for all crops) one-sided planting in pink, taking pink to a height of 30-35 CM.in in the 6th variant, where peanuts were grown as a repeat crop, the thickness of goose seedlings was 86.8 thousand pieces/ha, compared with other repeat crops (lentils, high to/ha. Like autumn wheat and a crop for transplanting, the plum is treated in the soil for care in a minimal way (to a depth of 10-12 cm), treated with a combined unit in Goose care to a depth of 35-40 cm, in the 14th variant, where seeds are planted with a bush 30-35 cm high. It was noticed that it was 3.2-7.8 thousand pieces higher than the background. When working in the soil using combined aggregates, processing to a depth of 35-40 cm, unilateral reception of pushta to a height of 30-35 cm and nitrogen support in autumn under a bush without liquid ammonia in the amount of 100 kg / ha.- in the variant, the density of goose seedlings is 92.3 thousand pcs. / ha, which, compared with other repeated crops that were cared for on these backgrounds, were processed to a depth of 4.3-7.1 thousand pcs., with a constant depth of 35-40 cm, planted in a lump to a height of 30-35 cm. THE EFFECT OF TILLAGE IN VARIOUS WAYS ON THE THICKNESS OF A POROUS SEEDLING When caring for a goose in one step, take a feather at a height of 30-35 cm and put nitrogen under the bush in the form of liquid ammonia in the amount of 100 kg / ha + planting Winter wheat Lentil Cotton Cotton 22 peanut 23 Sunflower 24 Beans 17 In the course of the 2018/2019 research, a field of winter wheat was carried out to a depth of 35-40 cm, Haid and other measures were carried out, and then seeds were sown (2018) against the background of 1 (1-4 var.). It was noted that the thickness of seedlings is 326.9 thousand pcs/ha, and the soil is 323.0 on 3-4- 6 backgrounds sown in special workshops in one way, when processing 323.6 thousand pcs/ha using a combined unit, and 336.8-335.2 thousand pcs/ha on 2- 5 backgrounds obtained in one way. After harvesting autumn wheat, repeated crops were processed in the ground in various ways and to a depth before planting to maintain, seeds of lentils, peanuts, sunflower and beans were sown and the thickness of seedlings was analyzed. In fund 1, where the land is processed in the traditional way, the thickness of seedlings is 220.8 thousand pieces/ha of lentils, 99.6 thousand pieces/ha of peanuts, 50.1 thousand pieces/ha of sunflower and 214.7 thousand pieces/hectares of legumes, with minimal cultivation of the land at a time, are planted in special seeders 3-4-6. 206,7; 202,1; 205,7 in lentil crops, 89,6 in peanut crops, 89,1; 88,9 in sunflower crops, 49,6; 49,1 48,7 in bean crops, 211,7; 207,0; Up to 209,8 thousand. pcs/ha.On 2-5 backgrounds obtained at a time 47 47 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference When caring for a goose in one step, take a feather at a height of 30-35 cm and put nitrogen under the bush in the form of liquid ammonia in the 323,6 205,7 91,6 22 88,9 94,5 23 48,7 86,8 24 209,8 90,1 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference To grow peanuts as autumn wheat and spring crops, the soil is treated in a minimal way (10-12 cm), and then the area for caring for the goose is treated to a depth of 35-40 cm, bringing the bush to a height of 30-35 cm in one step and introducing nitrogen under the bush in the form of- in the variant, the density of goose seedlings was 94.5 thousand pcs / ha, compared with other repeated crops (lentils, sunflower, beans) grown on these backgrounds, up to 2.9-7.7 thousand pcs./ha, compared with the 1st background, where constant processing was carried out, up to 8.7-13.3 thousand. pcs/ha, compared with the 2nd in relation to the background, up to 8.4-13.1 thousand pcs/ha, winter wheat, minimum for growing repeated crops, it was noticed that the combination for pit care is up to 5.5-10.3 thousand units compared to the 4th background treated with filler, and a continuous combination of 2.2-9.3 thousand. Doan/ha is higher than the 5th background with liquid ammonia under the bush. In conclusion, we note that mumkinki, Yorga Minal ishlov berib method, deer bodies and its ecological reporter minimal ishlov berishiak method resurstezhakora agrologiyasin ullaba, daily grown as a combination unit elamida ishlov berib, bir illa pushta olsh, pushta ostiga azottate ammoate 100 kg relative to a mountain fire in the mountainous region of kalinligakar etishtirilganda 6,2, the traditional method of islov berylganda 11,4, permanent Barch ekinstvuyushchy combinator unit assistant ishlow berylganda 9,6, steam kuzg and duplicated ekinnisht, at least a method, he also grew up in haydov 11,8, permanently combined unit bidan ishlov berib, pushta ostiga Siuk ammonium 3.2 grain size less, bu this description of ethylaetgan technology is based on the fact that berysh jaraenid bergenid kuznein-kuznein-dehkandin, as well as on the basis of ethylaetgan technology as in the case of this disease, currently there are a number of reasons why you should avoid using ammonium. MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference Li t f d lit t To grow peanuts as autumn wheat and spring crops, the soil is treated in a minimal way (10-12 cm), and then the area for caring for the goose is treated to a depth of 35-40 cm, bringing the bush to a height of 30-35 cm in one step and introducing nitrogen under the bush in the form of- in the variant, the density of goose seedlings was 94.5 thousand pcs / ha, compared with other repeated crops (lentils, sunflower, beans) grown on these backgrounds, up to 2.9-7.7 thousand pcs./ha, compared with the 1st background, where constant processing was carried out, up to 8.7-13.3 thousand. pcs/ha, compared with the 2nd in relation to the background, up to 8.4-13.1 thousand pcs/ha, winter wheat, minimum for growing repeated crops, it was noticed that the combination for pit care is up to 5.5-10.3 thousand units compared to the 4th background treated with filler, and a continuous combination of 2.2-9.3 thousand. Doan/ha is higher than the 5th background with liquid ammonia under the bush. In conclusion, we note that mumkinki, Yorga Minal ishlov berib method, deer bodies and its ecological reporter minimal ishlov berishiak method resurstezhakora agrologiyasin ullaba, daily grown as a combination unit elamida ishlov berib, bir illa pushta olsh, pushta ostiga azottate ammoate 100 kg relative to a mountain fire in the mountainous region of kalinligakar etishtirilganda 6,2, the traditional method of islov berylganda 11,4, permanent Barch ekinstvuyushchy combinator unit assistant ishlow berylganda 9,6, steam kuzg and duplicated ekinnisht, at least a method, he also grew up in haydov 11,8, permanently combined unit bidan ishlov berib, pushta ostiga Siuk ammonium 3.2 grain size less, bu this description of ethylaetgan technology is based on the fact that berysh jaraenid bergenid kuznein-kuznein-dehkandin, as well as on the basis of ethylaetgan technology as in the case of this disease, currently there are a number of reasons why you should avoid using ammonium. List of used literature: 1.Nazirov R., Gasanov F., Sindarov A., Khamrakulov I. Sozdanie optimal toltshini vskhodov na polyax. // Magazine selskoy ekonomiki Uzbekistana. 2010. № 3. B. 2-3. List of used literature: 1.Nazirov R., Gasanov F., Sindarov A., Khamrakulov I. Sozdanie optimal toltshini vskhodov na polyax. // Magazine selskoy ekonomiki Uzbekistana. 2010. № 3. B. 2-3. 2.Khasanova F.Technology polucheniya M-push. // Magazine selskogo hozyaystva Uzbekistana. Tashkent-2009 № 12. B. 5. 2.Khasanova F.Technology polucheniya M-push. MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference T bl 2 48 Table 2 Introduction into the soil by various methods and to a depth, winter wheat, repeated sowing and thickness of goose seedlings, thousand pcs./ha (2018-2020) № Processing methods and depths Winter wheat (2018.) Repeated crops (2019 y.) The goose (2020 Introduction into the soil by various methods and to a depth, winter wheat, repeated sowing and thickness of goose seedlings, thousand pcs./ha (2018-2020) 48 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference y.) 1 Plowing the earth to a constant depth of 35-40 cm +boronization+molting+getting pus + planting (control) 326,9 220,8 83,9 2 99,6 85,8 3 50,1 81,2 4 214,7 83,5 5 Planting a bush to a height of 30-35 cm in one step, cultivating the ground to a constant depth of 35-40 cm. 336,8 204,9 84,9 6 99,8 86,8 7 54,3 82,1 8 227,2 84,4 9 Cultivation in a minimal way for winter wheat and repeated sowing (to a depth of 10-12 cm) + plowing to a depth of 35-40 cm when caring for a goose + getting a bush + planting. 323,0 206,7 84,1 10 89,6 86,0 11 49,6 81,4 12 211,7 83,7 13 Autumn crops with a desk and doubles are limited to processing by the food method (drinking 10-12 cm) + processing of a gift of 35-40 cm KA'ri hihi, take one pashto 30- 35 cm high + planting 325,1 202,1 87,1 14 89,1 89,0 15 49,1 84,2 16 207,0 86,6 17 Cultivation of the earth to a constant depth of 35-40 cm, unilateral bringing of the bush to a height of 30-35 cm, nitrogen application under the bush in the form of liquid ammonia in the amount of 100 kg / ha + planting 335,2 209,9 88,0 18 102,9 92,3 19 55,1 85,2 20 214,9 87,6 21 For winter wheat and repeated crops, the minimum processing method (to a depth of 10-12 cm) + processing to a depth of 35-40 cm. MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference // Magazine selskogo hozyaystva Uzbekistana. Tashkent-2009 № 12. B. 5. 3.Yakubjanov O.- "Method poseva semyan I podkormki khlopka" yavlyayutsya osnovnimi napravleniyami uglubleniya I razvitiya fermerskikh khozyaystv v agrarnoy SERE. (Sbornik statey Republican nauchno-prakticheskoy konferentii) Andijan. 2007. B. 358 3.Yakubjanov O.- "Method poseva semyan I podkormki khlopka" yavlyayutsya osnovnimi napravleniyami uglubleniya I razvitiya fermerskikh khozyaystv v agrarnoy SERE. (Sbornik statey Republican nauchno-prakticheskoy konferentii) Andijan. 2007. B. 358 50 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference 4.Tursunav X. "Method posadki I Vliyanie toltshini prorostkov na sort gusya Andijan-37" Uzbeksky magazine selskogo hozyaystva. 2012. №8. B. 23 5.Yangiboev A. - "Vodasberegayutshee Arashenie povishaet urojaynost xlopka". Magazine agronauki No. 1. 2009. B. 11-12 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference 4.Tursunav X. "Method posadki I Vliyanie toltshini prorostkov na sort gusya Andijan-37" Uzbeksky magazine selskogo hozyaystva. 2012. №8. B. 23 5.Yangiboev A. - "Vodasberegayutshee Arashenie povishaet urojaynost xlopka". Magazine agronauki No. 1. 2009. B. 11-12 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference 4.Tursunav X. "Method posadki I Vliyanie toltshini prorostkov na sort gusya Andijan-37" Uzbeksky magazine selskogo hozyaystva. 2012. №8. B. 23 5.Yangiboev A. - "Vodasberegayutshee Arashenie povishaet urojaynost xlopka". Magazine agronauki No. 1. 2009. B. 11-12 MODELS AND METHODS IN MODERN SCIENCE International scientific-online conference 4.Tursunav X. "Method posadki I Vliyanie toltshini prorostkov na sort gusya Andijan-37" Uzbeksky magazine selskogo hozyaystva. 2012. №8. B. 23 5.Yangiboev A. - "Vodasberegayutshee Arashenie povishaet urojaynost xlopka". Magazine agronauki No. 1. 2009. B. 11-12 4.Tursunav X. "Method posadki I Vliyanie toltshini prorostkov na sort gusya Andijan-37" Uzbeksky magazine selskogo hozyaystva. 2012. №8. B. 23 5.Yangiboev A. - "Vodasberegayutshee Arashenie povishaet urojaynost xlopka". Magazine agronauki No. 1. 2009. B. 11-12 5.Yangiboev A. - "Vodasberegayutshee Arashenie povishaet urojaynost xlopka". Magazine agronauki No. 1. 2009. B. 11-12 51 51
https://openalex.org/W3186327404	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0249323&type=printable	English	null	Brief communication: Plasma cortisol concentration is affected by lactation, but not intra-nasal oxytocin treatment, in beef cows	PloS one	2,021	cc-by	4,671	RESEARCH ARTICLE Brief communication: Plasma cortisol concentration is affected by lactation, but not intra-nasal oxytocin treatment, in beef cows Brooklyn K. WagnerID1,2, Alejandro E. Relling2, Justin D. Kieffer3, Anthony J. ParkerID2* 1 Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America, 2 Department of Animal Sciences, The Ohio State University, Wooster, Ohio, United States of America, 3 Department of Animal Sciences, The Ohio State University, Columbus, Ohio, United States of America * parker.1203@osu.edu * parker.1203@osu.edu * parker.1203@osu.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract In mammals, including sheep and mice, lactation attenuates the hypothalamo-pituitary- adrenal axis and plasma cortisol concentration. Oxytocin, one neuropeptide present in the blood during lactation, may contribute to such stress attenuation. Providing oxytocin intra- nasally increases plasma oxytocin concentration in cattle and can be used in non-lactating cows to mirror plasma oxytocin concentration of lactating cows. Therefore, our hypothesis was that there would be no difference in plasma cortisol between non-lactating beef cows intra-nasally administered oxytocin and lactating beef cows intra-nasally treated with saline. Twenty Bos taurus cows were randomly allocated by lactational status to one of four treat- ments, in a 2×2 factorial arrangement: 1) Non-lactating, saline (NL-S; n = 5); 2) Non-lactat- ing, oxytocin (NL-OXT; n = 5); 3) Lactating, saline (L-S; n = 5); and 4) Lactating, oxytocin (L- OXT; n = 5). Two hours pre-treatment, cows were catheterized, moved to their appropriate chute and baseline blood samples were collected at -60, -45, -30, and 0 minutes before treatments were administered. Directly following the 0-minute sample, cows were adminis- tered their intra-nasal treatment via a mucosal atomization device. Subsequently, blood was collected at 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 minutes. Non-lac- tating cows had greater (P = 0.02) plasma cortisol concentration compared with lactating cows. There was no lactation by treatment interactions for either plasma cortisol (P = 0.55) or oxytocin (P = 0.89) concentration. Although a treatment by time interaction was identified for oxytocin (P < 0.0001), there was no main effect of lactation on plasma oxytocin concen- tration (P = 0.34). Similar oxytocin and dissimilar cortisol concentration in lactating and non- lactating cows indicate that oxytocin alone cannot be responsible for reduced plasma corti- sol in lactating ruminants. Further investigations are needed to elucidate alternative mecha- nisms that may be involved in the stress hypo-responsive condition of lactating mammals. PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Wagner BK, Relling AE, Kieffer JD, Parker AJ (2021) Brief communication: Plasma cortisol concentration is affected by lactation, but not intra- nasal oxytocin treatment, in beef cows. PLoS ONE 16(7): e0249323. https://doi.org/10.1371/journal. pone.0249323 Editor: Juan J. Loor, University of Illinois, UNITED STATES PLOS ONE PLOS ONE Animal management and experimental design Twenty Bos taurus cows of Angus genetic type (2–10 years of age, 510 ± 72 kg mean ± SEM bodyweight) were used in the present study. Lactating cows were 4 to 6 weeks into lactation. Cows were selected from the Ohio Agricultural Research and Development Center’s beef herd at the Eastern Agricultural Research Station in Caldwell, Ohio. By age and lactational status, cows were randomly allocated to one of four treatment groups, in a 2×2 factorial arrangement: 1) Non-lactating, saline (NL-S; 0.015 mL/kg of bodyweight 0.9% isotonic saline; n = 5); 2) Non-lactating, oxytocin (NL-OXT; 0.60 IU/kg of bodyweight oxytocin; n = 5); 3) Lactating, saline (L-S; n = 5); and 4) Lactating, oxytocin (L-OXT; n = 3). Four cows were tested at a time (i.e., one from each treatment group), either in the morning or afternoon, over a three-day period. All cows were restrained in a cattle chute with a head bail. The calves (1–2 months of age) of lactating cows were kept in front of their dam to allow for visual and nose-to-nose con- tact between the calf and dam for the duration of the study. Materials and methods All experimental procedures were reviewed and approved by The Ohio State University Insti- tutional Animal Care and Use Committee (#2017A00000012). Introduction In mammals, including sheep and mice, lactation attenuates the hypothalamo-pituitary-adrenal (HPA) axis and plasma cortisol concentration [1, 2]. One important neuropeptide present in blood during lactation is oxytocin [3]. In a number of species, oxytocin attenuates the HPA axis activation [1, 4–6]; however, oxytocin can potentiate the HPA axis as well, resulting in greater plasma cortisol, in some species [7, 8]. Additionally, intra-nasal oxytocin treatment results in widespread dispersal of oxytocin within the brain in rodents and non-human primates [9, 10] and has attenuated increases in adrenocorticotropin hormone (another effector within the HPA axis) in non-human primates [11]. Given these results, supplementing oxytocin intra-nasally may benefit production cattle undergoing routine, yet stressful, husbandry procedures (e.g. transpor- tation) via stress reduction and subsequent improvements to both production and welfare out- comes. However, the consequence of oxytocin treatment is highly dependent upon species [8], stressor [1, 4], physiological state [12], and route of administration [4, 13]. Competing interests: The authors have declared that no competing interests exist. In ruminants, sufficient plasma oxytocin concentration is critical to promoting parturition and ensuring milk excretion. In dairy cows, complete milk removal requires plasma oxytocin con- centration to be greater than basal concentration [14]. In addition, Ralph and Tilbrook report greater plasma oxytocin concentration and reduced plasma cortisol concentration in lactating ewes exposed to psycho-social stress for four hours compared with non-lactating ewes [1]. How- ever, non-lactating beef cattle given oxytocin intra-nasally and exposed to restraint and isolation stress did not experience HPA axis attenuation [8]. Given that intra-nasal oxytocin supplementa- tion results in a plasma concentration comparable to or greater than those reported in lactating ruminants [1, 15], the stressor is likely playing a substantial role [8]. Therefore, authors of the present study aimed to examine the effects of oxytocin administration independent of stressor and hypothesized that there would be no difference in plasma cortisol between non-lactating beef cows intra-nasally administered oxytocin and lactating beef cows intra-nasally treated with saline. PLOS ONE PLOS ONE Cortisol and oxytocin in lactating and non-lactating beef cattle Funding: The author(s) received no specific funding for this work. This study was funded with discretionary funds allocated by Dr. Anthony Parker, the corresponding author, of The Ohio State University. Salaries support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Editor: Juan J. Loor, University of Illinois, UNITED STATES Received: October 1, 2020 Accepted: March 15, 2021 Published: July 30, 2021 Received: October 1, 2020 Accepted: March 15, 2021 Published: July 30, 2021 Received: October 1, 2020 Accepted: March 15, 2021 Published: July 30, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0249323 Copyright: © 2021 Wagner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. 1 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 Catheterization, treatment administration and sampling Two hours prior to treatment, cows were catheterized and then moved to their assigned chute area. Briefly, their heads were restrained to the side of the head chute to allow open access to 2 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 PLOS ONE Cortisol and oxytocin in lactating and non-lactating beef cattle the jugular vein. A 5 cm × 5 cm area was clipped over the jugular vein and a baseline blood sample was collected. To begin catheter placement, a local anesthetic (3 mL, 2% Lidocaine Hydrochloride injectable, Vedco Inc., St. Joseph, Missouri, USA) was injected subcutaneously at the site of catheter placement. Betadine1 surgical scrub (Purdue Pharma L.P., Stamford, CT, USA) was used to clean the area (minimum of three passes), followed by the removal of surgical scrub by alcohol (70% ethanol). A 14-gauge × 13.3cm indwelling intravenous catheter (Becton, Dickinson, and Co., Franklin Lakes, NJ, USA) was placed into the jugular vein. Cath- eters were secured to the skin with sutures and extension sets (i.v. extension set 36”, Interna- tional WIN Ltd, PA, USA) were secured to the top of the neck using Elastikon1 (Thermo Fisher Scientific, Waltham, MA, USA). Cows were then moved to their appropriate chute and allowed to rest, during which time cows were able to move their head from side to side. Blood sampling was staggered based on the time of catheterization for each animal. Baseline blood samples were taken prior to intra-nasal administration of oxytocin or saline treatments, at -60, -30, -15, and 0 minutes into a 10 mL syringe via jugular catheter. Directly following the 0-minute sample, the cow’s head was restrained by placing the cow’s head into the head bail, fitting a halter to her head, and tying the halter to the right side of the head bail. The assigned intra-nasal treatment (S, isotonic saline or OXT, Oxytocin, 20 IU/mL Vetone1, Bimeda-MTC Animal Health Inc., Ontario, Canada) was administered with a mucosal atomi- zation device (Nasal™Teleflex1 Inc., Morrisville, NC) by the attending veterinarian. The dose rate of oxytocin (0.60 IU/kg of bodyweight) was chosen based on a previous report in beef cat- tle [8]. Doses ranged from 12 to 19 mL, dependent upon body weight. Half of each dose was administered into each nostril. Catheterization, treatment administration and sampling Subsequently, 10 mL of blood was collected via the jugular vein catheter into a 10 mL syringe at 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 minutes. Collected blood was transferred into two Vacutainer™tubes (lithium heparin and EDTA), inverted a minimum of eight times, and immediately placed on ice (4˚C) until centri- fugation. After sampling was complete, catheters were removed, and the animals were released back into the herd after visual assessment by the attending veterinarian. Sample analyses All blood tubes were centrifuged at 3,000 × g at 4˚C for 15 minutes, after which plasma was removed with a disposable pipette and stored at -20˚C until required for analysis. Recovered plasma from lithium heparin tubes was used, along with a slightly modified commercially available RIA kit (MP Biomedicals, LLC., Solon, OH, USA), to measure plasma cortisol concentration. Following kit instructions for volume of sample (25 μL) yielded results below the normal limit of detection (< 10.0 ng/mL). Therefore, different volumes of plasma sample (25, 50, 100, and 150 μL) were eval- uated. Using parallel displacement and recovery (101.59 ± 10%) it was determined that using 50 μL of sample yielded results between minimum and maximum concentration of 5.0 and 500.0 ng/mL, respectively. The intra-assay and inter-assay variations were 2.8 and 11.1%, respectively. Oxytocin concentration were also determined using a commercially available double-anti- body RIA kit (Oxytocin (Human, Rate, Mouse, Bovine) RIA Kit, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) using plasma recovered from EDTA tubes. The inter-assay varia- tion was 19.3% and the intra-assay variation ranged from 3.1 to 9.6%. The minimum and max- imum concentrations of detection were 2.5 and 160.0 pg/mL, respectively. The analysis for plasma cortisol and oxytocin were completed using duplicate samples. Data analysis Data were analyzed using MIXED procedure in SAS 9.4 Software (SAS Institute, 1999). Cow was treated as a random effect; treatment (S or OXT), lactational status (NL or L), time, and 3 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 PLOS ONE Cortisol and oxytocin in lactating and non-lactating beef cattle their interaction were treated as fixed effects. Day and time of day (AM or PM) were included in the model as fixed effects; however, no effect of either day or time of day was detected and these variables were removed from the model. Repeated measures were assessed using the first-order autoregressive covariance structure. Selection of this covariance structure was based on lowest Bayesian information criterion between first-order autoregressive, compound symmetry, and heterogenous compound symmetry structures. Main effects of treatment, lactational status, and their interaction were assessed by ANOVA. In instances in which an interaction between these contrasts and time were identified (P < 0.05), an ANOVA was performed at each sampling time point. Two lactating cows from the oxytocin treatment (L-OXT) were removed from the study for uncharacteristically uncooperative behavior. Significance was determined at P 0.05 and trends are reported at 0.05 < P 0.10. A log transformation was used on oxytocin data due to non-normality; results are presented as back transformed values. Results The mean plasma cortisol concentration for all four treatments is displayed in Fig 1. Non-lac- tating cows had greater (Fig 2; P = 0.02) plasma cortisol concentration (25.3 ng/mL) compared Fig 1. Mean plasma cortisol concentrations in Bostaurus cows assigned to one of the following treatment. s. 1) Non-lactating, saline (NL-S, – – Δ – –; n = 5), 2) Non-lactating, oxytocin (NL-OXT, – – – –; n = 5); 3) Lactating, saline (L-S, ──▲──; n = 5); and 4) Lactating, oxytocin (L-OXT, ──●──; n = 3). Intra-nasal treatments (S or OXT) were administered at 0 minutes and head restraint was applied for two hours (indicated by the black bar). Oxytocin was administered intra-nasally at a rate of 0.60 IU/kg bodyweight. The pooled SEM was 5.6. Only an effect of lactation (P = 0.02) was detected (see Fig 2). https://doi.org/10.1371/journal.pone.0249323.g001 Fig 1. Mean plasma cortisol concentrations in Bostaurus cows assigned to one of the following treatment. s. 1) Non-lactating, saline (NL-S, – – Δ – –; n = 5), 2) Non-lactating, oxytocin (NL-OXT, – – – –; n = 5); 3) Lactating, saline (L-S, ──▲──; n = 5); and 4) Lactating, oxytocin (L-OXT, ──●──; n = 3). Intra-nasal treatments (S or OXT) were administered at 0 minutes and head restraint was applied for two hours (indicated by the black bar). Oxytocin was administered intra-nasally at a rate of 0.60 IU/kg bodyweight. The pooled SEM was 5.6. Only an effect of lactation (P = 0.02) was detected (see Fig 2). https://doi.org/10.1371/journal.pone.0249323.g001 Fig 1. Mean plasma cortisol concentrations in Bostaurus cows assigned to one of the following treatment. s. 1) Non-lactating, saline (NL-S, – – Δ – –; n = 5), 2) Non-lactating, oxytocin (NL-OXT, – – – –; n = 5); 3) Lactating, saline (L-S, ──▲──; n = 5); and 4) Lactating, oxytocin (L-OXT, ──●──; n = 3). Intra-nasal treatments (S or OXT) were administered at 0 minutes and head restraint was applied for two hours (indicated by the black bar). Oxytocin was administered intra-nasally at a rate of 0.60 IU/kg bodyweight. The pooled SEM was 5.6. Only an effect of lactation (P = 0.02) was detected (see Fig 2). https://doi.org/10.1371/journal.pone.0249323.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 4 / 8 PLOS ONE Cortisol and oxytocin in lactating and non-lactating beef cattle Fig 2. Mean plasma cortisol concentration in Bos taurus cows. Results Cows were lactating (L, ──■──; n = 8) or non-lactating (NL, – – □– –; n = 10). Head restraint start time and duration are indicated by the black bar. The pooled SEM was 4.0. An effect of lactation (P = 0.02) was detected, however there was no interaction between lactational status and intra-nasal treatment (P = 0.55). https://doi org/10 1371/journal pone 0249323 g002 Fig 2. Mean plasma cortisol concentration in Bos taurus cows. Cows were lactating (L, ──■──; n = 8) or non-lactating (NL, – – □– –; n = 10). Head restraint start time and duration are indicated by the black bar. The pooled SEM was 4.0. An effect of lactation (P = 0.02) was detected, however there was no interaction between lactational status and intra-nasal treatment (P = 0.55). htt //d i /10 1371/j l 0249323 002 Fig 2. Mean plasma cortisol concentration in Bos taurus cows. Cows were lactating (L, ──■──; n = 8) or non-lactating (NL, – – □– –; n = 10). Head restraint start time and duration are indicated by the black bar. The pooled SEM was 4.0. An effect of lactation (P = 0.02) was detected, however there was no interaction between lactational status and intra-nasal treatment (P = 0.55). https://doi.org/10.1371/journal.pone.0249323.g002 https://doi.org/10.1371/journal.pone.0249323.g002 with lactating cows (12.9 ng/mL), however no interaction between lactational status and intra- nasal treatment (P = 0.55) was detected. Neither intra-nasal treatment (P = 0.93), nor time (P = 0.53), affected plasma cortisol concentration. There was no effect of lactation (P = 0.34) on plasma oxytocin concentration. However, an intra-nasal treatment by time interaction (Fig 3; P < 0.001) was detected such that oxytocin concentration increased with intra-nasal administration of oxytocin. No interactions between lactational status and intra-nasal treatment (P = 0.89) or time (P = 0.39) were detected. Discussion The present study confirms that lactating beef cows with access to their calf have a reduced plasma cortisol concentration compared with non-lactating cows without a calf; and this dis- crepancy between cows in different physiological states is not solely a result of plasma oxytocin concentration. An attenuated response in plasma cortisol concentration has been observed previously in lactating mammals [1, 12, 16, 17], supporting lactation as a stress hypo-respon- sive condition. Plasma cortisol data resulting from the present study supports previous find- ings in ruminants [1, 17], while contradicting other reports [18]. For example, Ralph and Tilbrook [1] reported attenuated responses in plasma cortisol in sheep exposed to restraint PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 5 / 8 PLOS ONE Cortisol and oxytocin in lactating and non-lactating beef cattle Fig 3. Back transformed mean plasma oxytocin concentration in Bos taurus cows assigned to one of four treatments. Treatments included 1) Non-lactating, saline (NL-S, – – Δ – –; n = 5), 2) Non-lactating, oxytocin (NL-OXT, – – – –; n = 5); 3) Lactating, saline (L-S, ──▲──; n = 5); and 4) Lactating, oxytocin (L-OXT, ──●──; n = 3). Intra-nasal treatments (S or OXT) were administered at 0 minutes followed by two hours of head restraint, as indicated by the black bar. Oxytocin was administered intra-nasally at a rate of 0.60 IU/kg bodyweight. A treatment by time interaction was detected from 60 to 90 minutes (P < 0.01) and from 90 to 110 minutes (P < 0.05). https://doi.org/10.1371/journal.pone.0249323.g003 Fig 3. Back transformed mean plasma oxytocin concentration in Bos taurus cows assigned to one of four treatments. Treatments included 1) Non-lactating, saline (NL-S, – – Δ – –; n = 5), 2) Non-lactating, oxytocin (NL-OXT, – – – –; n = 5); 3) Lactating, saline (L-S, ──▲──; n = 5); and 4) Lactating, oxytocin (L-OXT, ──●──; n = 3). Intra-nasal treatments (S or OXT) were administered at 0 minutes followed by two hours of head restraint, as indicated by the black bar. Oxytocin was administered intra-nasally at a rate of 0.60 IU/kg bodyweight. A treatment by time interaction was detected from 60 to 90 minutes (P < 0.01) and from 90 to 110 minutes (P < 0.05). https://doi org/10 1371/journal pone 0249323 g003 Fig 3. Back transformed mean plasma oxytocin concentration in Bos taurus cows assigned to one of four treatments. PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 Discussion Treatments included 1) Non-lactating, saline (NL-S, – – Δ – –; n = 5), 2) Non-lactating, oxytocin (NL-OXT, – – – –; n = 5); 3) Lactating, saline (L-S, ──▲──; n = 5); and 4) Lactating, oxytocin (L-OXT, ──●──; n = 3). Intra-nasal treatments (S or OXT) were administered at 0 minutes followed by two hours of head restraint, as indicated by the black bar. Oxytocin was administered intra-nasally at a rate of 0.60 IU/kg bodyweight. A treatment by time interaction was detected from 60 to 90 minutes (P < 0.01) and from 90 to 110 minutes (P < 0.05). htt //d i /10 1371/j l 0249323 003 https://doi.org/10.1371/journal.pone.0249323.g003 and isolation stress, while Cook [18] reported no differences in cortisol concentration between lactating and non-lactating ewes exposed to a predator stress. The present study utilized a mild head restraint in a cattle chute, with which the cows were familiar, and therefore no direct comparisons can be made between this and previous studies due to stressor differences and the stressor-dependent nature of the HPA axis response [1, 4]. Furthermore, the results of the present study support earlier published studies that state oxytocin infusion has no detectable effects on plasma cortisol concentration [8, 18]. The hypo-responsive state of the HPA axis observed in lactating mammals is likely facili- tated by several hormones working in concert [2]. Oxytocin, prolactin, and the combination of oxytocin and prolactin suppress plasma cortisol concentration [2, 18, 19] and these hormones likely play a direct role in the stress-hypo-responsive state observed during lactation. However, similar plasma oxytocin concentration in both lactating and non-lactating cows in the present study indicates that lactational status alone does not support greater oxytocin concentration. Tactile stimulation of the teat occurring during suckling may be necessary to stimulate oxyto- cin production and release. To date and in contrast to the present study, previous investiga- tions into the relationship between lactation and oxytocin in ruminants have allowed offspring 6 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 PLOS ONE Cortisol and oxytocin in lactating and non-lactating beef cattle to suckle throughout sampling [1] which may explain discrepancies between studies. Never- theless, similar plasma oxytocin concentration and dissimilar cortisol concentration in lactat- ing and non-lactating cows indicate that oxytocin alone cannot be responsible for reduced plasma cortisol in lactating ruminants. Author Contributions Conceptualization: Brooklyn K. Wagner, Anthony J. Parker. Data curation: Brooklyn K. Wagner. Formal analysis: Brooklyn K. Wagner. Methodology: Brooklyn K. Wagner, Alejandro E. Relling, Justin D. Kieffer, Anthony J. Parker. Methodology: Brooklyn K. Wagner, Alejandro E. Relling, Justin D. Kieffer, Anthony J. Parker. Project administration: Brooklyn K. Wagner, Justin D. Kieffer. Project administration: Brooklyn K. Wagner, Justin D. Kieffer. Resources: Justin D. Kieffer, Anthony J. Parker. Supervision: Alejandro E. Relling, Anthony J. Parker. Supervision: Alejandro E. Relling, Anthony J. Parker. Validation: Alejandro E. Relling. Validation: Alejandro E. Relling. Visualization: Brooklyn K. Wagner, Anthony J. Parker. Visualization: Brooklyn K. Wagner, Anthony J. Parker. Writing – original draft: Brooklyn K. Wagner. Writing – review & editing: Alejandro E. Relling, Anthony J. Parker. Writing – review & editing: Alejandro E. Relling, Anthony J. Parker. Discussion Another plausible explanation for the stress attenuation observed during lactation is that milk within the mammary gland may be acting as a sink for cortisol concentration during times of stress. Verkerk et al. [20] report increases in cortisol concentrations in the composite milk of dairy cows that coincide with a decrease in plasma cortisol concentrations. Equilib- rium between plasma and alveolar milk is re-established within one hour of peak plasma corti- sol concentrations [20] and an association between average cortisol concentrations in blood and milk has been established [21]. It is possible that alveolar milk may be acting as a sink for cortisol in lactating mammals. To the authors’ knowledge, there are no reports of milk cortisol concentrations in beef cows. Overall, non-lactating cows had greater plasma circulating cortisol concentration compared with lactating cows with their calf present. Additional hormones present during lactation, such as prolactin, or teat stimulation may be involved in this comparative decrease in plasma corti- sol. It is also plausible that alveolar milk may be acting as a sink for cortisol in lactating mam- mals. Further investigations are needed to elucidate the specific mechanisms impacting the stress state of lactating mammals. PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 References 1. Ralph CR, Tilbrook AJ. The hypothalamo-pituitary-adrenal (HPA) axis in sheep is attenuated during lac- tation in response to psychosocial and predator stress. Domest Anim Endocrin. 2016; 55:66–73. https:// doi.org/10.1016/j.domaniend.2015.11.003 PMID: 26773370 1. Ralph CR, Tilbrook AJ. The hypothalamo-pituitary-adrenal (HPA) axis in sheep is attenuated during lac- tation in response to psychosocial and predator stress. Domest Anim Endocrin. 2016; 55:66–73. https:// doi.org/10.1016/j.domaniend.2015.11.003 PMID: 26773370 2. Slattery DA, Neumann ID. No stress please! Mechanisms of stress hyporesponsiveness of the maternal brain. J Physiol. 2008; 586:377–85. https://doi.org/10.1113/jphysiol.2007.145896 PMID: 17974588 2. Slattery DA, Neumann ID. No stress please! Mechanisms of stress hyporesponsiveness of the maternal brain. J Physiol. 2008; 586:377–85. https://doi.org/10.1113/jphysiol.2007.145896 PMID: 17974588 7 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 PLOS ONE Cortisol and oxytocin in lactating and non-lactating beef cattle 3. Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function, and regulation. Physiol Rev. 2001; 81:629–83. https://doi.org/10.1152/physrev.2001.81.2.629 PMID: 11274341 4. Windle RJ, Shanks N, Lightman SL, Ingram CD. Central oxytocin administration reduces stress- induced corticosterone release and anxiety behavior in rats. Endocrinology. 1997; 138:2829–34. https://doi.org/10.1210/endo.138.7.5255 PMID: 9202224 5. Neumann ID, Wigger A, Torner L, Holsboer F, Landgraf R. Brain oxytocin inhibits basal and stress- induced activity of the hypothalamo-pituitary-adrenal axis in male and female rates: Partial action within the paraventricular nucleus. J Neuroendocrinol. 2000; 12(3):235–43. https://doi.org/10.1046/j.1365- 2826.2000.00442.x PMID: 10718919 6. Ditzen B, Schaer M, Gabriel B, Bodenmann G, Ehlert U, Heinrichs M. Intranasal oxytocin increases pos- itive communication and reduces cortisol levels during couple conflict. Biol Psychiat. 2009; 65(9):728– 31. https://doi.org/10.1016/j.biopsych.2008.10.011 PMID: 19027101 7. Rault J, Carter CS, Garner JP, Marchant-Forde JN, Richert BT, Lay DC. Repeated intranasal oxytocin administration in early life dysregulates the HPA axis and alters social behavior. Physiol Behav. 2013; 112–113:40–8. https://doi.org/10.1016/j.physbeh.2013.02.007 PMID: 23481917 8. Wagner BK, Relling AE, Kieffer JD, Parker AJ. Intranasal oxytocin treatment does not attenuate the hypothalamo-pituitary-adrenal axis in beef heifers subjected to isolation stress or restraint and isolation stress. Domest Anim Endocrin. 2020; 70:1–6. https://doi.org/10.1016/j.domaniend.2019.07.007 PMID: 31479924 9. Carson DS, Yuan H, Labuschagne I. Improving Research Standards to Restore Trust in Intranasal Oxy- tocin. Biol Psychiat. 2016; 79(8):e53–4. Epub 2015/10/06. https://doi.org/10.1016/j.biopsych.2015.08. 031 PMID: 26435223. 10. Lochhead JJ, Throne RG. Intranasal delivery of biologics to the central nervous system. Adv Drug Deliver Rev. 2012; 64:614–28. https://doi.org/10.1016/j.addr.2011.11.002 PMID: 22119441 11. Parker KJ, Buckmaster CL, Schatzberg AF, Lyons DM. Intranasal oxytocin administration attenuates the ACTH stress response in monkeys. Psychoneuroendocrino. 2005; 30(9):924–9. PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021 References https://doi.org/10. 1016/j.psyneuen.2005.04.002 PMID: 15946803. 12. Neumann ID, Torner L, Wigger A. Brain oxytocin: differential inhibition of neuroendocrine stress responses and anxiety-related behaviour in virgin, pregnant and lactating rats. Neuroscience. 1999; 95:567–75. https://doi.org/10.1016/S0306-4522(99)00433-9 13. Petersson M, Eklund M, Uvna¨s-Moberg K. Oxytocin decreases corticosterone and nociception and increases motor activity in OVX rats. Maturitas. 2005; 51(4):426–33. https://doi.org/10.1016/j.maturitas. 2004.10.005 PMID: 16039417 14. Bruckmaier RM, Schams D, Blum JW. Continuously elevated concentrations of oxytocin during milking are necessary for complete milk removal in dairy cows. J Dairy Res. 1994; 61:323–34. https://doi.org/ 10.1017/s0022029900030740 PMID: 7962834 15. Wagner BK, Relling AE, Kieffer JD, Moraes LE, Parker AJ. Short communication: pharmacokinetics of oxytocin administered intranasally to beef cattle. Domest Anim Endocrin. 2020; 71:106387. Epub 2019/ 12/13. https://doi.org/10.1016/j.domaniend.2019.106387 Epub 2019 Aug 15. PMID: 31830691. 16. Toufexis DJ, Tesolin S, Huang N, Walker C. Altered pituitary sensitivity to corticotropin-releasing factor and arginine vasopressin participates in the stress hyporesponsiveness of lactation in the rat. J Neu- roendocrinol. 1999; 11:757–64. Epub 1999/10/16. https://doi.org/10.1046/j.1365-2826.1999.00381.x PMID: 10520124. 17. Tilbrook AJ, Turner AI, Ibbott MD, Clarke IJ. Activation of the hypothalamo-pituitary-adrenal axis by iso- lation and restraint stress during lactation in ewes: Effect of the presence of the lamb and suckling. Endocrinology. 2006; 147(7):3501–9. https://doi.org/10.1210/en.2005-1632 PMID: 16574791 18. Cook CJ. Oxytocin and prolactin suppress cortisol responses to acute stress in both lactating and non- lactating sheep. J Dairy Res. 1997; 64(3):327–39. Epub 1997/08/01. https://doi.org/10.1017/ s0022029997002240 PMID: 9275253. 19. Torner L, Neumann ID. The brain prolactin system: involvment in stress response adaptations in lacta- tion. Stress. 2002; 5(4):249–57. https://doi.org/10.1080/1025389021000048638 PMID: 12475729 20. Verkerk GA, Phipps AM, Carragher JF, Matthews LR, Stelwagen K. Characterization of milk cortisol concentrations as a measure of short-term stress responses in lactating dairy cows. Animal Welfare. 1998; 7:77–86. 21. Bremel RD, Gangwer MI. Effect of Adrenocorticotropin Injection and Stress on Milk Cortisol Content. J Dairy Sci. 1978; 61:1103–8. https://doi.org/10.3168/jds.S0022-0302(78)83693-5 PMID: 214474 8 / 8 PLOS ONE \| https://doi.org/10.1371/journal.pone.0249323 July 30, 2021
https://openalex.org/W3080841707	https://www.repository.cam.ac.uk/bitstreams/4f266d42-5fcd-419b-b170-75d948d26d89/download	English	null	Engagement and adherence trade-offs for SARS-CoV-2 contact tracing	Philosophical transactions - Royal Society. Biological sciences	2,021	cc-by	8,966	Research 1Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, UK 2MathSys CDT, University of Warwick, Coventry, UK 3Department of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK 4Centre for Mathematical Modelling of Infectious Disease and Department of Global Health and Development, London School of Hygiene and Tropical Medicine, London, UK 5Department for Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge, UK 6School of Psychology, University of Southampton, Southampton, UK 7School of Psychological Science, University of Bristol, Bristol, UK 1Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, UK 2MathSys CDT, University of Warwick, Coventry, UK 1Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford, UK 2MathSys CDT, University of Warwick, Coventry, UK 3Department of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK 4Centre for Mathematical Modelling of Infectious Disease and Department of Global Health and Development, London School of Hygiene and Tropical Medicine, London, UK 5Department for Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge, UK 6School of Psychology, University of Southampton, Southampton, UK 7School of Psychological Science, University of Bristol, Bristol, UK TCDL, 0000-0003-4694-8107; TC, 0000-0003-2934-1063; JG, 0000-0003-1240-7214; Cite this article: Lucas TCD et al. 2021 Engagement and adherence trade-offs for SARS-CoV-2 contact tracing. Phil. Trans. R. Soc. B 376: 20200270. https://doi.org/10.1098/rstb.2020.0270 TCDL, 0000-0003-4694-8107; TC, 0000-0003-2934-1063; JG, 0000-0003-1240-7214; TDH, 0000-0001-5962-4238 TCDL, 0000-0003-4694-8107; TC, 0000-0003-2934-1063; JG, 0000-0003-1240-7214; TDH, 0000-0001-5962-4238 Accepted: 24 October 2020 Accepted: 24 October 2020 Contact tracing is an important tool for allowing countries to ease lockdown policies introduced to combat SARS-CoV-2. For contact tracing to be effective, those with symptoms must self-report themselves while their contacts must self-isolate when asked. However, policies such as legal enforcement of self-isolation can create trade-offs by dissuading individuals from self- reporting. We use an existing branching process model to examine which aspects of contact tracing adherence should be prioritized. We consider an inverse relationship between self-isolation adherence and self-reporting engagement, assuming that increasingly strict self-isolation policies will result in fewer individuals self-reporting to the programme. Tim C. D. Lucas1,†, Emma L. Davis1,†, Diepreye Ayabina1, Anna Borlase1, Thomas Crellen1, Li Pi1, Graham F. Medley2,4, Lucy Yardley6,7, Petra Klepac3,5, Julia Gog5 and T. Déirdre Hollingsworth1 Tim C. D. Lucas1,†, Emma L. Davis1,†, Diepreye Ayabina1, Anna Borlase1, Thomas Crellen1, Li Pi1, Graham F. Medley2,4, Lucy Yardley6,7, Petra Klepac3,5, Julia Gog5 and T. Déirdre Hollingsworth1 Keywords: Keywords: COVID-19, contact tracing, SARS-CoV-2, adherence, case isolation, quarantine This article is part of the theme issue ‘Modelling that shaped the early COVID-19 pandemic response in the UK’. Engagement and adherence trade-offs for SARS-CoV-2 contact tracing royalsocietypublishing.org/journal/rstb Tim C. D. Lucas1,†, Emma L. Davis1,†, Diepreye Ayabina1, Anna Borlase1, Thomas Crellen1, Li Pi1, Graham F. Medley2,4, Lucy Yardley6,7, Petra Klepac3,5, Julia Gog5 and T. Déirdre Hollingsworth1 Research We find that policies which increase the average duration of self-isolation, or that increase the probability that people self-isolate at all, at the expense of reduced self- reporting rate, will not decrease the risk of a large outbreak and may increase the risk, depending on the strength of the trade-off. These results suggest that policies to increase self-isolation adherence should be implemented carefully. Policies that increase self-isolation adherence at the cost of self-reporting rates should be avoided. One contribution of 21 to a theme issue ‘Modelling that shaped the early COVID-19 pandemic response in the UK’. 1. Background The contact tracing team must then manage to contact the close contacts. The close con- tacts must self-isolate when asked and remain in self-isolation for the full isolation period (14 days in the UK). In some systems, the isolated individual is given a self-administered swab test which must be administered correctly. There is imperfect adherence or performance at each of these stages. In this paper, we focus on trade-offs between self-report rate (stage 1) and self- isolation adherence (stages 4 and 5). In our model, stages 2 and 3 are incorporated into a parameter which we call control effectiveness. g The exact form that these trade-offs would take are difficult to know. Adherence to self-isolation requirements might lar- gely be binary with people complying for the full 14 days (as requested in the UK) or not adhering at all. In this case, legal enforcement would be expected to increase the pro- portion of people that self-isolate. Alternatively, it is possible that self-isolation adherence is more continuous with people adhering for a few days instead of the full 14 days. Similarly, legal enforcement might be expected to increase the duration of isolation. Finally, if swab tests are being self-administered, people might be less careful or less willing to endure discom- fort if the consequences of a positive test are more severe (though this might change as saliva tests are produced [27,28]). While it is difficult to know the functional effects of different levels of compliance, it is even more difficult to quan- tify the strengths of the trade-offs. Legal enforcement might have a weak effect on improving self-isolation adherence [23] but a strong deterrent effect on self-reporting. Alternatively, perhaps legal mandation has a strong effect on self-isolation adherence without being a strong deterrent to self-reporting rates. Furthermore, the shapes of these trade-offs are likely to differ in different countries and social groups based on cul- ture, trust in the government and other factors. Careful quantitative and qualitative studies will need to be conducted to quantify these effects. recently been in close proximity with, as well as asking for information on which public areas the infected person has visited. The tracers will then identify as many contacts as possible and ask them to self-isolate for a period. Adherence to the contact tracing system is an important determinant of its efficacy [6,10,12]. 1. Background Since the first cases of SARS-CoV-2 in China in late 2019 [1], the virus has spread globally, resulting in over 600 000 confirmed deaths by August 2020 [2]. Lockdown in the UK began in March 2020 [3] and reduced R0 below 1 while also triggering unprecedented reductions in economic activity [4]. As lockdown restrictions are relaxed, both in the UK and in other countries, other methods for keeping R0 below 1 are needed. Large-scale contact tracing is one of the potential methods for keeping virus spread under control [5–7]. During the current SARS-CoV-2 outbreak, contact tracing has been used to great effect in a number of countries, including Vietnam and South Korea [8,9]. Two broad classes of contact tracing include manual tracing and digital tracing using a smartphone app [10]. Manual contract tracing is the only system currently running in the UK though it is expected that a contact tracing app will be launched soon [11]. In manual contact tracing, trained public health staff ask a case for the names and contact details of people they have †These authors contributed equally to this study. †These authors contributed equally to this study. Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare. c.5429378. © 2021 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. index case close contact case self-reports to test and trace case gives name and contact details of close contacts close contact is contacted and asked to self-isolate close contact self-isolates close contact self-isolates for full duration period correctly perform self-administered swab test 1 2 3 4 5 6 Figure 1. Overview of adherence in test and trace. An untraced individual must self-report and give the name and details of close contacts. The contact tracing team must then manage to contact the close contacts. The close con- tacts must self-isolate when asked and remain in self-isolation for the full isolation period (14 days in the UK). In some systems, the isolated individual is given a self-administered swab test which must be administered correctly. There is imperfect adherence or performance at each of these stages. In this paper, we focus on trade-offs between self-report rate (stage 1) and self- isolation adherence (stages 4 and 5). 1. Background In our model, stages 2 and 3 are incorporated into a parameter which we call control effectiveness. someone or not knowing any details about the close contact. Eighty-two per cent of close contacts were reached and asked to self-isolate. 2 However, these adherence rates are not fixed parameters and can be influenced by policy. For example, economic support for those missing work [15,19], daily phonecalls to monitor adherence [20] or legal ramifications for breaking self-isolation, such as those implemented in Singapore and Taiwan [20], might be expected to increase self-isolation rates [19]. In particular, this work was originally undertaken in response to a question from policy makers asking whether legally mandating self-isolation for close-contacts would reduce transmission rates. Furthermore, there are likely to be trade-offs and dependencies between parameters. In par- ticular, contact tracing relies on self-reporting of symptoms in order to initially identifyachain of transmission but introdu- cing penalties for non-compliance to self-isolation might be expected to decrease the proportion of people that report themselves to the system in the first instance. In general, there are few direct, individual benefits to self-reporting one- self to a contact tracing system; instead the benefits are communal and the drivers for self-reporting are likely to be altruism or social norms [21,22]. However, there are direct costs both to the individual that self-reports and to their close contacts. Self-isolation is mentally difficult [23] and will come with economic costs for many [15,21,24–26]. Legally enforcing self-isolation exacerbates these costs. Figure 1. Overview of adherence in test and trace. An untraced individual must self-report and give the name and details of close contacts. The contact tracing team must then manage to contact the close contacts. The close con- tacts must self-isolate when asked and remain in self-isolation for the full isolation period (14 days in the UK). In some systems, the isolated individual is given a self-administered swab test which must be administered correctly. There is imperfect adherence or performance at each of these stages. In this paper, we focus on trade-offs between self-report rate (stage 1) and self- isolation adherence (stages 4 and 5). In our model, stages 2 and 3 are incorporated into a parameter which we call control effectiveness. Figure 1. Overview of adherence in test and trace. An untraced individual must self-report and give the name and details of close contacts. 1. Background Adherence applies to a number of different aspects of contact tracing [13] as shown in figure 1. Untraced individ- uals with symptoms must get themselves tested (the process of which automatically reports them to the contact tracing system) and they and their household should self-isolate. If they test positive they are contacted by the test and trace team and must give identifying information about the people they have been in close proximity with. Then, both the index case, and the traced contacts, must self-isolate for a period [14,15]. If the contact tracing system uses home swab tests, the swabs must be taken carefully [16,17]. Adherence to each of these steps will be imperfect. Although there are many unobserved variables involved, we can start to examine some of these adherence rates using public statistics from the UK tracing system [18]. For example, of the 6923 people who were referred to the contact tracing system between the 11 and 17 June, 70% were reached. How- ever, these 6923 cases certainly do not represent 100% of the new cases in the country that week. Of the 6923, 74% gave details of at least one contact though it is not possible to tell how many of the remaining 26% actually had no contacts. Of those that gave details of at least one contact, it is unknown what proportion gave details of all their close con- tacts. Someone might not give contact information of a close contact deliberately, for reasons of privacy, embarrassment or to save a contact from being asked to self-isolate, or acciden- tally through not remembering that they were in contact with Here we use a previously published branching process model [6,12] to examine the effects of these trade-offs on the risk of a large outbreak of SARS-CoV-2. We examine trade-offs between self-isolation duration and self-isolation probability with self-reporting rates, contact information reporting probabilities and sensitivity of home swab tests. It is important to note that we do not consider the societal costs [29] of legal enforcement of self-isolation; we aim to quantify the benefits of these policies without considering the costs noting that the costs are not easy to directly compare to the benefits. Table 1. Model parameters values/ranges. (Parameters taken from the literature are ﬁxed and for other parameters a range of values are explored.) (a) Secondary case distribution ( ) y The heterogeneity in the number of potential secondary cases caused by an individual is modelled as a negative binomial distribution. For symptomatic cases, we use a mean value of 1.3 secondary cases while asymptomatic cases are given a 50% lower infection rate. This relates to a scenario where strong social distancing and good hygiene is still being observed. Earlier work [6,7] and preliminary analyses indicated that contact tracing is unable to keep the risk of an outbreak low without being paired with social distancing so this is the scenario we focus on. Estimates for the dispersion parameter, k, for SARS-CoV-2 range from k = 0.1 (0.05–0.2) for pre-lockdown UK [38] to k = 0.25 (0.13–0.88) for Tianjin, China during lockdown measures [39]. Given this range, we have kept the parameter as used in [12,34] setting k = 0.16. This value of k yields a strongly skewed distribution with most individuals causing zero potential secondary cases. processes. For infections transmitted on surfaces, the primary case will rarely know who else touched the same surface. For face-to-face contact in small groups, the primary contact must remember that they were in contact with the secondary case, know their name and chose to divulge this information to the contact tracing team. In the UK the definition of a close contact is being within 2 m for more than 15 min, which probably encompasses most small group infections. For transmission in larger groups, such as at restaurants and bars, contact tracing effectiveness depends on how well the venue recorded who vis- ited. The control effectiveness parameter in the model encodes all of these processes and is varied between 40 and 80%. If contact tracing is successful, the traced individual is asked to self-isolate. We assume it takes one day to contact a contact. If a traced con- tact subsequently shows symptoms or returns a positive test the next round of contact tracing is initiated. That is, the contacts of the traced contact are then traced. processes. For infections transmitted on surfaces, the primary case will rarely know who else touched the same surface. For face-to-face contact in small groups, the primary contact must remember that they were in contact with the secondary case, know their name and chose to divulge this information to the contact tracing team. (b) Infection profile ( ) p Individuals are labelled as symptomatic or asymptomatic with a probability of 50% [35,36]. The onset time of symptoms is mod- elled as a lognormal distribution with mean 1.43 days and s.d. of 0.66 [40]. All individuals, whether symptomatic or asymptomatic are given a symptom onset time as the exposure time of second- ary cases is calculated relative to this time. The exposure time for each new potential case is drawn from a gamma distribution with shape parameter of 17.77 and a rate of 1:39 d1. This distri- bution is centred 12.98 days before the onset of symptoms. If this randomly sampled value yields a negative generation interval (i.e. the secondary case being infected by the primary case before the primary case is infected) the value is resampled. The parameters for this gamma distribution were estimated by fitting to the data in He et al. [37] in a maximum likelihood framework that accounts for this resampling process. The model fitting does not ignore any data as discussed by Ashcroft et al. [41]. The esti- mated distributions are qualitatively similar to the original fitted models (electronic supplementary material, figure S2). If the exposure time of a potential secondary case occurs during the primary case’s self-isolation, the infection event does not occur and the potential secondary case does not become a case. (d) Testing As a baseline we assume that tests have a sensitivity of 65% and that it takes 1 day for results to be returned. This reflects the sensitivity of tests observed in the community [31,32]. Given a positive test result contact tracing for the tested individual is initiated. A negative test allows the tested individual to be immediately released from quarantine. Any contacts of a nega- tive-testing case that were successfully identified prior to receiving the test result are still isolated and tested. This process is different to the UK contact tracing system in which the trace team only ask for contact details if a positive test has been returned. In a branching process model, only infected individuals are modelled. Therefore we do not track the number of uninfected royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 376: 20200270 parameter values refs self-isolation probability 10–70% [30] self-reporting probability 10–70% test sensitivity 35–65% [31–33] minimum isolation duration 1–14 days maximum isolation duration 7, 14 days contact tracing coverage (%) 40–80% number of initial cases 20 symptomatic RS under physical distancing 1.3 asymptomatic RS under physical distancing 0.65 dispersion of RS, k 0.16 [12,34] proportion asymptomatic 50% [35,36] delay: onset to isolation 1 day incubation period (lognormal) mean log: 1.43, s.d. log: 0.66 [37] infection time (gamma) shape: 2.12, rate: 0.69 d−1 [37] infection time shift 12.98 days [37] time to trace contacts (days) 1 day delay: isolate to test result 1 days (a) Secondary case distribution In the UK the definition of a close contact is being within 2 m for more than 15 min, which probably encompasses most small group infections. For transmission in larger groups, such as at restaurants and bars, contact tracing effectiveness depends on how well the venue recorded who vis- ited. The control effectiveness parameter in the model encodes all of these processes and is varied between 40 and 80%. If contact tracing is successful, the traced individual is asked to self-isolate. We assume it takes one day to contact a contact. If a traced con- tact subsequently shows symptoms or returns a positive test the next round of contact tracing is initiated. That is, the contacts of the traced contact are then traced. 2. Methods 3 In this paper, we extend a previous model of SARS-CoV-2 trans- mission [12]. An overview of the model is given in the electronic supplementary material, figure S1 while parameter values and references are given in table 1. At the individual-level, the number of potential secondary contacts are modelled by a nega- tive binomial distribution while the exposure times of these new infections are modelled as a gamma distribution. Self-isolating individuals are assumed to be unable to transmit the disease (assuming isolation within households) and therefore potential secondary cases are avoided if the gamma-distributed exposure time occurs during self-isolation of the primary case. The timing of self-isolation depends on whether the case was traced as a potential contact or not and a number of factors affecting adher- ence as described in detail below. The model proceeds as a branching process with each simulation being seeded with 20 untraced, infected individuals. (c) Contact tracing The first stage in the contact tracing system is an untraced, symp- tomatic individual self-reporting themselves by seeking a test. We define the control effectiveness of the contact tracing system as the proportion of secondarily infected people that are contacted by the contact tracers. In practice, this variable is never observed, but it can be broken down to a number of min isolation = 1 4 7 14 self-rep = 70% 50 % 30 % 10 % 50 70 50 70 50 70 50 70 2 4 6 2 4 6 2 4 6 2 4 6 control effectiveness (%) risk of large outbreak (%) Figure 2. Trade-off between self isolation time (columns) and self-report rate (rows) with error bars denoting 95% confidence intervals. Individuals self isolate for a randomly selected duration between min isolation and 14 days. Untraced, symptomatic individuals self-report with a probability that varies across the rows. The proportion of close contacts that are divulged and asked to self-isolate varies across the x-axis of each subplot. The y-axis shows the risk of a large outbreak (greater than 2000 cases) over 15 000 simulations. The probability that an individual self-isolates at all is fixed at 70%. If we assume we are currently near the top left we expect that introducing legal ramifications for breaking self isolation would move us down and right. This generally increases risk. risk of large outbreak (%) control effectiveness (%) Figure 2. Trade-off between self isolation time (columns) and self-report rate (rows) with error bars denoting 95% confidence intervals. Individuals self isolate for a randomly selected duration between min isolation and 14 days. Untraced, symptomatic individuals self-report with a probability that varies across the rows. The proportion of close contacts that are divulged and asked to self-isolate varies across the x-axis of each subplot. The y-axis shows the risk of a large outbreak (greater than 2000 cases) over 15 000 simulations. The probability that an individual self-isolates at all is fixed at 70%. If we assume we are currently near the top left we expect that introducing legal ramifications for breaking self isolation would move us down and right. This generally increases risk. people that are unnecessarily asked to quarantine. Test specificity affects the number of uninfected people asked to quarantine but does not directly affect the spread of the disease and therefore we do not define a test specificity. (c) Contact tracing In this study, we are concerned with quantifying the benefits of contact tracing and do not attempt to weigh the epidemiological benefits against the sociological costs. below the target rate for other national contact tracing programmes [30]. Furthermore, the very strict restrictions applied to travellers entering countries such as Singapore could also be considered an upper bound on feasible policies. Many of the policies used in these areas, such as enforced isolation in government run hotels, GPS ankle bracelets, and daily video calls, would be considered draconion if applied to the population at large but could be reason- ably expected to produce self-isolation rates of 90%. In contrast to the first trade-off, we assume that everyone who does self-isolate does so for the full maximum value of either 7 or 14 days. (e) Adherence trade-offs y Finally, we assume that policies which increase self-isolation probability will decrease test sensitivity. This scenario applies to the case of home administered tests. With strong incentives to test negative, people will be less likely to perform swabs cor- rectly. We therefore examine a range of test sensitivities from a baseline of 65% down to 35% in increments of 10%. We consider three main trade-offs. As we do not have good data to define the shapes of these trade-offs, we run simulations for all combinations of parameters. First, we assume that without policies to encourage self-iso- lation most people attempt some self-isolation but the lack of adherence is with respect to the duration of self-isolation that decreases. We keep the probability of self-isolation constant at 70%. We assume that each person that does self-isolate isolates for an amount of time taken from a uniform distribution between a minimum and maximum value. For the maximum values, we use either the full 14 days currently recommended in the UK or a shorter 7 day maximum isolation. We vary the minimum duration of self-isolation from 1 day to being equal to the maximum duration. self-report probability In our first comparison we assumed that increasing the dur- ation of self-isolation would reduce the self-reporting probability. We found that increasing the duration of self-iso- lation increases the risk of a large outbreak in the presence of reductions in self-reporting rates. The probability of a large outbreak for all combinations of self-isolation duration and self-report rates are shown in figure 2. If we assume that we are currently in the top left panel (high self-report rates but iso- lation taken uniformly between 1 and 14 days), policies that move us down and right generally increase the risk of a large outbreak. For example, if we consider a control effective- ness of 60%, with a self-isolation duration of between 1 and 14 days and a self-report rate of 70%, the risk of a large outbreak is 1%. If we increase the self-isolation duration to always be 14 days but reduce the self-report rate to 10%, the probability of a large outbreak increases from 1 to 6%. If the trade-off is very weak, such that increasing self-isolation duration to always be 14 days only decreases self-report rates to 50%, we see no change in the probability of an outbreak. self-report probability (%) Figure 3. Trade-off between (a) minimum self isolation time and self-report probability, and (b) self-isolation probability and self-report probability. The con- trol effectiveness is held constant at 60%. The results are a subset of those in figures 2 and 4, with each line being a slice through a column of those plots. The y-axis shows the risk of a large outbreak (greater than 2000 cases) over 15 000 simulations. Error bars show the 95% confidence intervals. In (a) if we optimistically assume we currently have 70% self-report probability but 1 day minimum isolation (red), legally mandating isolation would be expected to move us to the left and to the purple line which gives an increased risk of an outbreak. In (b) if we optimistically assume we currently have 70% self-report probability but 10% self isolation probability (red), legally mandating isolation would be expected to move us to the left and to the purple line which gives a marginal decrease in risk of an outbreak. (f) Simulation process Results presented are the combined output of 15 000 simulations for each parameter combination, or scenario, considered. We define a simulation as leading to a large outbreak if it has more than 2000 cumulative cases or if there are still infected cases after 300 days. The threshold of 2000 cases was chosen by running simulations with a maximum of 5000 cases and noting that of the simulated epidemics that went extinct, 99% of extinction events occurred before reaching 2000 cases. Nearly all simulations either went extinct or reached 2000 cases with very few simu- lations lasting longer than 300 days. These simulations were then used to calculate the probability of a large outbreak given a certain set of conditions. Here, 95% Clopper–Pearson exact confi- dence intervals were also calculated. To test the sensitivity of our Second, we examine the trade-off between self-report prob- ability and self-isolation probability. We expect that policies which increase self-isolation probability will reduce self-report probability. We use values of self-isolation from 10 to 70% in incre- ments of 20% and examine all combinations with self-report probabilities from 10 to 70%, also in increments of 20%. The upper bound for self-isolation here is certainly above the rate of self-isolation currently being achieved in the UK. However, it is 2 4 6 10 30 50 70 risk of large outbreak (%) min isolate 1 4 7 14 (a) 2 4 6 8 10 30 50 70 self-report probability (%) risk of large outbreak (%) self-isolation 10% 30% 50% 70% (b) Figure 3. Trade-off between (a) minimum self isolation time and self-report probability, and (b) self-isolation probability and self-report probability. The con- trol effectiveness is held constant at 60%. The results are a subset of those in figures 2 and 4, with each line being a slice through a column of those plots. The y-axis shows the risk of a large outbreak (greater than 2000 cases) over 15 000 simulations. Error bars show the 95% confidence intervals. In (a) if we optimistically assume we currently have 70% self-report probability but 1 day minimum isolation (red), legally mandating isolation would be expected to move us to the left and to the purple line which gives an increased risk of an outbreak. (f) Simulation process In (b) if we optimistically assume we currently have 70% self-report probability but 10% self isolation probability (red), legally mandating isolation would be expected to move us to the left and to the purple line which gives a marginal decrease in risk of an outbreak. (Online version in colour.) royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 376: 20200270 5 results to the relative asymptomatic transmission rate and the delay between being contacted by the contact tracers and starting self-isolation, we ran simulations at 60% control effectiveness and varied each parameter in turn. The model was written in R and the code and testing suite [42] is publicly available on GitHub (https://github.com/timcdlucas/ringbp/tree/adherence_trade- off_runs). 2 4 6 10 30 50 70 risk of large outbreak (%) min isolate 1 4 7 14 (a) (b) (a) risk of large outbreak (%) ( ) self-report probability (Online version in colour.) If we assume a more pessimistic starting scenario of a self-isolation duration of between 1 and 14 days and self- reporting rates of 10% and given a control effectiveness of 60% we have a 6% risk of a large outbreak (figure 3a, red line). We find that increasing self-report rates gives a larger reduction in risk. Increasing self-report rates from 10 to 70% reduces risk from 6 to 1% (moving right along the x-axis in figure 3a). By contrast, increasing the duration of isolation to always being 14 days does not change the risk of a large outbreak (purple line in figure 3a). We find that reducing the maximum isolation duration from 14 days to 7 days con- sistently increases the risk of a large outbreak (electronic supplementary material, figures S3–S5). Altering the relative asymptomatic transmission rate has a strong effect on the overall risk of a large outbreak, but the effects of minimum isolation length and self-report probability remain similar (electronic supplementary material, figure S6). Furthermore, these results are qualitatively robust to changes in the delay between being asked to self-isolate and doing so (electronic supplementary material, figure S7). currently in the top left panel (high self-report rates but low self-isolation rates), policies that increase self-isolation rates but decrease self-report rates would move us down and right. However, whether this decreases the risk of an out- break depends on the strength of the trade-off. For example, if we consider a control effectiveness of 60%, with a self-isolation rate of 10% and a self-report rate of 70% the risk of a large outbreak is 6%. If we increase the self-isolation rate to 70% and equivalently reduce the self-report rate to 10%, the probability of a large outbreak is still 6%. If the trade-off is weak, such that increasing self-isolation from 10 to 70% only incurs a reduction in self-report rate to 50%, the reduction in risk of a large outbreak is substantial, redu- cing from 6 to 1.5%. However, if the trade-off is strong, such that increasing self-isolation from 10 to 30% causes a reduction in self-reporting rate from 70 to 10%, the risk of an outbreak instead marginally increases from 6 to 7%. (a) Trade-off between self-isolation duration against (b) 2 4 6 8 10 30 50 70 self-report probability (%) risk of large outbreak (%) self-isolation 10% 30% 50% 70% (b) Trade-off between self-isolation probability against self-report probability (b) Trade-off between self-isolation probability against self-report probability We could instead assume a more pessimistic starting scen- ario of self-isolation rates of 10% and self-reporting rates of 10%. Given a control effectiveness of 60% we have a 7% risk of a large outbreak (figure 3b, red line). However, from this scenario we can consider whether it is better to increase self- isolation or to increase self-reporting. Increasing self isolation probability to 70% reduces risk to 6% (move right along the x-axis) and increasing self-report probability to 70% also reduces risk to 6% (purple line). Increasing both to 30% In our second comparison, we assumed that increasing the probability of self-isolation will decrease the self-report prob- ability. We find that increasing self-isolation probability while decreasing self-report probability does not strongly alter the probability of a large outbreak. The probability of a large out- break for all combinations of self-isolation rates and self- report rates are shown in figure 4. If we assume that we are isolate = 10% 30% 50% 70% self-rep = 70% 50% 30% 10% 50 70 50 70 50 70 50 70 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 control effectiveness (%) risk of large outbreak (%) Figure 4. Trade-off between self-isolation probability (columns) and self-report probability (rows) with error bars denoting 95% confidence intervals. The y-axis shows the risk of a large outbreak (greater than 2000 cases) over 15 000 simulations. If we assume we are currently near the top left we expect that introducing legal ramifications for breaking self isolation to move us down and right. Whether this decreases risk depends on the strength of the trade-off. If the trade-off is weak, such that as we move from the top left to isolation probability of 70% and self-report probability of 50%, risk is reduced. By contrast, if increasing isolation probability from 10% to 30% incurs a drop in self-reporting from 70 to 10%, risk does not change. risk of large outbreak (%) control effectiveness (%) Figure 4. Trade-off between self-isolation probability (columns) and self-report probability (rows) with error bars denoting 95% confidence intervals. The y-axis shows the risk of a large outbreak (greater than 2000 cases) over 15 000 simulations. If we assume we are currently near the top left we expect that introducing legal ramifications for breaking self isolation to move us down and right. 4. Discussion reduces risk to 5%. Overall, these two parameters are relatively evenly balanced. The overall risk of a large outbreak changes with relative asymptomatic transmission rate, but the effects of self-isolation probability and self-report probability remain similar (electronic supplementary material, figure S8). The results are similar with different values for the delay between being asked to self-isolate and doing so (elec- tronic supplementary material, figures S9). Overall we have found that policies which increase self-iso- lation rates at the expense of self-report rates are unlikely to improve the effectiveness of contact tracing systems. If the primary trade-off is between the duration of self-isolation and the probability of self-reporting, we find that policies which increase self-isolation and reduce self-report rates will cause either an increase or no change in the probability of a large outbreak depending on the strength of the trade- off. When the primary trade-off was instead between the probability of self-isolation and the rate of self-report, policies which increase self-isolation rates and reduce self-report rates can increase or marginally decrease the probability of a large outbreak depending on the strength of the trade-off. Overall this implies that policies such as fines, and police enforce- ment of self-isolation will have either little benefit or a negative effect. Broadly, policies that improve self-report rates, even at the expense of self-isolation rates should be used. This might include publicity that encourages people to self-report by reminding them that there are no legal consequences to them or their contacts for doing so. (b) Trade-off between self-isolation probability against self-report probability Whether this decreases risk depends on the strength of the trade-off. If the trade-off is weak, such that as we move from the top left to isolation probability of 70% and self-report probability of 50%, risk is reduced. By contrast, if increasing isolation probability from 10% to 30% incurs a drop in self-reporting from 70 to 10%, risk does not change. (c) Trade-off between self-isolation duration against test sensitivity Untraced, sympto- matic individuals self-report with a probability that varies across the rows. The proportion of close contacts that are divulged and asked to self-isolate varies across the x-axis of each subplot. If we assume we are currently near the top left, introducing legal ramifications for breaking self isolation might move us down and right. This generally decreases risk unless the trade off is very strong such that a small increase in isolation probability incurs a large decrease in test sensitivity. One of the core assumptions to this work is that legal conse- quences for breaking self-isolation would improve self-isolation rates. However, the evidence for this is not strong and there is evidence that feelings of shame do not promote adherence [22,26]. By contrast, there is good evidence that other factors such as income and boredom [43] do affect self-isolation rates. How effectively legal consequences for breaking self-isolation can increase self-isolation rates is a complex question that will depend on cultural norms, perceived enforcability, and the strength of economic and psychological consequences for self- isolation. An important consequence of this is that self-isolation rates and the effectiveness of policies aimed to improve these rates will be strongly correlated, such that individuals who are most likely to infect each other are also likely to have similar self-isolation rates. This is not included in our model but has the potential to strongly reduce contact tracing efficacy in certain groups and locations. more effective system for measuring the incidence of SARS- CoV-2 in the community. This gives better early warning for when an outbreak is beginning in an area or group and allows for health care resources to be deployed more efficiently. By contrast, self-isolation comes with many economic and social costs both for the individual and the community. These costs are not the same for all people; the monetary costs to someone who is self-employed or working on very short-term contracts is much higher than for someone who is working at home anyway. Avoiding strict penalties for break- ing self-isolation allows those most affected by these costs to self-isolate less (i.e. for a 7 instead of 14 days) and may increase buy-in to the system as a whole. Furthermore, enforcement of self-isolation policies are an infringement on a basic liberty. (c) Trade-off between self-isolation duration against test sensitivity In our final comparison, we assumed that increasing self- isolation probabilities would decrease the probability of careful administration of home swab tests and therefore decrease the test sensitivity. We found that increasing self-isolation rates decreases the risk of a large outbreak even if this occurs in com- bination with reductions in test sensitivity. The probability of a large outbreak for all combinations of self-isolation rate and test sensitivity are shown in figure 5. If we assume that we are cur- rently in the top left panel (relatively high test sensitivity but low self-isolation rates), policies that increase self-isolation rates but decrease test sensitivity would move us down and right and this in general yields reduced risks of a large outbreak. Forexample, if we consideracontrol effectiveness of 60%, with a self-isolationrateof10%and atest sensitivityof65%,theriskofa large outbreak is 6%. If we increase the self-isolation rate to 70% while reducing the test sensitivity to 35%, the probability of a large outbreak reduces from 6 to 3%. Policies that improve self-report rates or self-isolation rates without an associated trade-off will also improve contact tracing efficacy. For example, economic support and employ- ment protection for individuals that self-isolate would be expected to improve self-isolation rates [15,19,26] without decreasing self-report rates. Similarly, efforts to communicate the reasons why people should self-report and self-isolate may improve both of these rates simultaneously [19,26]. isolate = 10% 30% 50% 70% sensitive = 65% 55% 45% 35% 50 70 50 70 50 70 50 70 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 control effectiveness (%) risk of large outbreak (%) Figure 5. Trade-off between self isolation probability (columns) and test sensitivity (rows) with error bars denoting 95% confidence intervals. Untraced, sympto- matic individuals self-report with a probability that varies across the rows. The proportion of close contacts that are divulged and asked to self-isolate varies across the x-axis of each subplot. If we assume we are currently near the top left, introducing legal ramifications for breaking self isolation might move us down and right. This generally decreases risk unless the trade off is very strong such that a small increase in isolation probability incurs a large decrease in test sensitivity. control effectiveness (%) Figure 5. Trade-off between self isolation probability (columns) and test sensitivity (rows) with error bars denoting 95% confidence intervals. (c) Trade-off between self-isolation duration against test sensitivity While we have not tried to compare these costs to the epide- miological benefits, they must always be taken into account when implementing policy. With regards to test sensitivity, our results are relevant only to self-administered swab-tests. Swab-tests may be replaced with reliable paper-based tests. Given that we found that optimizing self-isolation rates over test-sensitivity minimizes risk, other considerations such as test timing and access are probably more important. Furthermore, currently in the UK, traced contacts are not allowed out of quarantine after a negative test so the system is more robust to low test sensitivity than in our simulations. 5. In context This paper was taken into consideration by the Department for Health and Social Care when deciding whether to impose a legal duty to self-isolate and was referenced in a recent SPI-B report [44]. On the 28th September 2020, the UK government introduced fines for breaching self-isolation rules either after testing positive for SARS-CoV-2 or after being contacted by NHS Test and Trace [45]. The core assumption in our analysis was that introducing penalties for not self-isolating would drive down self-report rates. Our results suggest that increasing self-isolation rates at the Here we have focused solely on the probability of a large outbreak as a consequence of policy change. However, there are other costs and benefits to changing values of self-report rates and self-isolation rates. High self-report rates not only improves contact tracing efficacy directly, it also creates a Data accessibility. All the code used for the simulations are available at https://github.com/timcdlucas/ringbp/tree/adherence_trade- off_runs. Data accessibility. All the code used for the simulations are available at https://github.com/timcdlucas/ringbp/tree/adherence_trade- off_runs. expense of reduced self-report rates would make SARS-CoV- 2 outbreaks harder to control. However, given that many other restrictions are changing simultaneously, it is unlikely that we will be able tell whether the results from our analysis are borne out after this change. The introduction of legal penalties for breaking isolation also changes the important policy question. The original policy question was whether self-isolation should be legally mandated. Now the more rel- evant question is when should these restrictions be lifted. 8 Authors’ contributions. Conceptualization: all authors; formal analysis: E.L.D. and T.C.D.L.; funding acquisition: T.D.H.; investigation: E.L.D., T.C.D.L., A.B., L.P., D.A., T.C. and L.Y.; methodology: E.L.D. and T.C.D.L.; software: E.L.D. and T.C.D.L.; visualization: E.L.D., T.C.D.L. and L.Y.; writing—original draft: E.L.D. and T.C.D.L.; writing—review and editing: all authors. Competing interests. The authors declare that they have no known com- peting financial interests or personal relationships that could have appeared to influence the work reported in this paper. Since the first submission of this paper, results from a large study of adherence in the UK have been released [46]. The study contains self-reported behaviour (rather than intentions) of 42 thousand people between March and August 2020. While the sample was not random, quotas based on age, gender and region were used. References 1. South China Morning Post, Coronavirus: China’s first confirmed COVID-19 case traced back to November 17. See https://www.scmp.com/news/china/society/ article/3074991/coronavirus-chinas-first-confirmed- covid-19-case-traced-back (accessed 2 June 2020). Catalyst Innovations in Care Delivery. (doi:10.1056/ Cat.20.0317) Impact of delays on effectiveness of contact tracing strategies for COVID-19: a modelling study. The Lancet Public Health 5, e452–e459. 14. 14. Smith LE, Amlôt R, Lambert H, Oliver I, Robin C, Yardley L, Rubin GJ. 2020 Factors associated with adherence to self-isolation and lockdown measures in the UK; a cross-sectional survey. Pub. Health 187, 41–52. 8. Le SM. 2020 Containing the coronavirus (COVID-19): lessons from Vietnam. See https://blogs.worldbank. org/health/containing-coronavirus-covid-19-lessons- vietnam (accessed 3 June 2020). 2. Roser M, Ritchie H, Ortiz-Ospina E, Hasell J. 2020 Coronavirus pandemic (COVID-19). See https:// ourworldindata.org/covid-deaths (accessed 10 August 2020). 15. Bodas M, Peleg K. 2020 Self-isolation compliance in the COVID-19 era influenced by compensation: Findings from a recent survey in Israel: public attitudes toward the COVID-19 outbreak and self- isolation: a cross sectional study of the adult population of Israel. Health Affairs 39, 936–941. (doi:10.1377/hlthaff.2020.00382) 9. Park S, Choi GJ, Ko H. 2020 Information technology–based tracing strategy in response to COVID-19 in South Korea–privacy controversies. Jama 323, 2129–2130. 3. Coronavirus: Strict new curbs on life in UK announced by PM. See https://www.bbc.co.uk/ news/uk-52012432 (accessed 27 June 2020). 3. Coronavirus: Strict new curbs on life in UK announced by PM. See https://www.bbc.co.uk/ news/uk-52012432 (accessed 27 June 2020). 10. Ferretti L, Wymant C, Kendall M, Zhao L, Nurtay A, Abeler-Dörner L, Parker M, Bonsall D, Fraser C. 2020 Quantifying SARS-CoV-2 transmission suggests epidemic control with digital contact tracing. Science 368, eabb6936. (doi:10.1126/science. abb6936) 4. GDP monthly estimate, UK: April 2020. See https://www.ons.gov.uk/economy/ grossdomesticproductgdp/bulletins/ gdpmonthlyestimateuk/april2020 (accessed 27 June 2020). 4. GDP monthly estimate, UK: April 2020. See https://www.ons.gov.uk/economy/ grossdomesticproductgdp/bulletins/ gdpmonthlyestimateuk/april2020 (accessed 27 June 2020). 16. Seaman CP, Tran LTT, Cowling BJ, Sullivan SG. 2019 Self-collected compared with professional-collected swabbing in the diagnosis of influenza in symptomatic individuals: a meta-analysis and assessment of validity. J. Clin. Virol. 118, 28–35. (doi:10.1016/j.jcv.2019.07.010) 5. Aleta A et al. 2020 Modelling the impact of testing, contact tracing and household quarantine on second waves of COVID-19. Nat. Hum. Behav. 4, 1–8. (doi:10.1038/s41562-020-0931-9) 11. Coronavirus: NHS virus-tracing app downloaded 55, 000 times. See https://www.bbc.co.uk/news/uk- england-hampshire-52617236 (accessed 3 June 2020). 17. Dhiman N et al. 2012 Effectiveness of patient- collected swabs for influenza testing. Mayo Clinic Proc. 87, 548–554. 6. Davis EL et al. 5. In context Of those with COVID-19 symptoms in the previous seven days, 12% (95% CI 10–14%) requested a test (this measurement corresponds to the self-report parameter used in our analysis) which places the UK in the bottom row of figures 2 and 4. Of those contacted by the track and trace system, 11% (95% CI 8–14%) self-reported as having not left home at all in the following 14 days. This corresponds to the self-isolation prob- ability parameter used in our analysis and places the UK in the left-hand column of figure 4. These adherence rates did not change between March and August. Based on this study, the baseline assumptions made in our analysis were broadly correct. However, further understanding of adher- ence as a multifaceted continuous variable, rather than a binary variable is required. Measurements of aspects of adherence such as minimum isolation time, as used in our analysis, are still needed. Funding. This research was partly funded by the Bill & Melinda Gates Foundation (NTD Modelling Consortium OPP1184344: G.F.M.). This project has received funding from the European Union’s Horizon 2020 research and innovation programme — project EpiPose (101003688: P.K.). Royal Society (RP/EA/180004: P.K.). Wellcome Trust (210758/Z/18/Z: J.H. and S.A.). T.C. is funded by a Sir Henry Wellcome Fellowship from the Wellcome Trust (reference 215919/Z/19/Z). L.Y. is an NIHR Senior Investigator and her research programme is partly supported by NIHR Applied Research Collaboration (ARC)-West, NIHR Health Protection Research Unit (HPRU) for Behavioural Science and Evaluation, and the NIHR Southampton Biomedical Research Centre (BRC) All funders had no role in the study design, collection, analysis, interpretation of data, writing of the report, or decision to submit the manuscript for publication. This work was undertaken as a contribution to the Rapid Assistance in Modelling the Pandemic (RAMP) initiative, coordinated by the Royal Society. Acknowledgements. E.L.D., T.C.D.L., A.B., D.A., L.P., G.F.M. and T.D.H. gratefully acknowledge funding of the NTD Modelling Consortium by the Bill & Melinda Gates Foundation (BMGF) (grant number OPP1184344). The following funding sources are acknowl- edged as providing funding for the named authors. Views, opinions, assumptions or any other information set out in this article should not be attributed to BMGF or any person connected with them. References 2020 An imperfect tool: COVID-19’ test & trace’ success relies on minimising the impact of false negatives and continuation of physical distancing. medRxiv. (doi:10.1101/2020.06.09.20124008) 12. Hellewell J et al. 2020 Feasibility of controlling COVID-19 outbreaks by isolation of cases and contacts. The Lancet Global Health 8, e488–e496. 18. Weekly NHS Test and Trace bulletin, England: 11 to 17 June 2020. https://www.gov.uk/government/ publications/nhs-test-and-trace-statistics-england- 11-june-to-17-june-2020/weekly-nhs-test-and- 13. Beidas RS et al. 2020 Optimizing and implementing contact tracing through behavioral economics. NEJM 7. Kretzschmar ME, Rozhnova G, Bootsma MC, van Boven M, van de Wijgert JH, Bonten MJ. 2020 trace-bulletin-england-11-to-17-june-2020#fn:1 (accessed 27 June 2020). outside China. Wellcome Open Research 5, 67. (doi:10.12688/wellcomeopenres.15842.3) outside China. Wellcome Open Research 5, 67. (doi:10.12688/wellcomeopenres.15842.3) 28. Vogels CB et al. 2020 Salivadirect: simple and sensitive molecular diagnostic test for SARS-CoV-2 surveillance. medRxiv. (doi:10.1101/2020.08.03.20167791) 39. Zhang Y, Li Y, Wang L, Li M, Zhou X. 2020 Evaluating transmission heterogeneity and super-spreading event of COVID-19 in a metropolis of China. Int. J. Environ. Res. Public Health 3705. (doi:10.3390/ijerph17103705) 19. Michie S, West R, Rogers MB, Bonell C, Rubin GJ, Amlôt R. 2020 Reducing SARS-CoV-2 transmission in the UK: a behavioural science approach to identifying options for increasing adherence to social distancing and shielding vulnerable people. Br. J. Health Psychol. 29. Schuklenk U, Gartland KM. 2006 Confronting an influenza pandemic: ethical and scientific issues. Biochem. Soc. Trans. 34, 1151–1154. (doi:10.1042/ BST0341151) 40. Li Q et al. 2020 Early transmission dynamics in Wuhan, China, of novel coronavirus–infected pneumonia. New Engl. J. Med. 382, 1199–1207. 30. Verrall A. 2020 Rapid audit of contact tracing for COVID-19 in New Zealand. Ministry of Health 310. 20. Chung S-C, Marlow S, Tobias N, Alogna I, Alogna A, You S-L. 2020 A rapid systematic review and case study on test, contact tracing, testing, and isolation policies for COVID-19 prevention and control. medRxiv. (doi:10.1101/2020.06.04.20122614) 31. Kucirka LM, Lauer SA, Laeyendecker O, Boon D, Lessler J. 2020 Variation in false-negative rate of reverse transcriptase polymerase chain reaction– based SARS-CoV-2 tests by time since exposure. Ann. Intern. Med 173, 262–267. 41. Ashcroft P, Huisman JS, Lehtinen S, Bouman JA, Althaus CL, Regoes RR, Bonhoeffer S. 2020 COVID-19 infectivity profile correction. (http://arxiv. org/abs/2007.06602). 42. Lucas TCD, Pollington TM, Davis EL, Hollingsworth TD. 2020 Responsible modelling: unit testing for infectious disease epidemiology. Epidemics 33, 100425. 21. Baum NM, Jacobson PD, Goold SD. References 2009 ‘listen to the people’: public deliberation about social distancing measures in a pandemic. Am. J. Bioethics 9, 4–14. (doi:10.1080/15265160903197531) 32. Menni C et al. 2020 Real-time tracking of self- reported symptoms to predict potential COVID-19. Nat. Med. 26, 1037–1040. (doi:10.1038/s41591- 020-0916-2) 43. Wolff W, Martarelli CS, Schüler J, Bieleke M. 2020 High boredom proneness and low trait self-control impair adherence to social distancing guidelines during the covid-19 pandemic. Int. J. Environ. Res. Public Health 17, 5420. (doi:10. 3390/ijerph17155420) 22. Travaglino GA, Moon C. 2020 Explaining compliance with social distancing norms during the COVID-19 pandemic: the roles of cultural orientations, trust and self-conscious emotions in the US, Italy, and South Korea. (doi:10.31234/osf.io/8yn5b) 33. van Kasteren P, van der Veer B, van den Brink S, Wijsman L, de Jonge J, van den Brandt A, Molenkamp R, Reusken C, Meijer A. 2020 Comparison of seven commercial RT-PCR diagnostic kits for COVID-19. J. Clin. Virol. 128, 104412. (doi:10.1016/j.jcv.2020.104412) 23. DiGiovanni C, Conley J, Chiu D, Zaborski J. 2004 Factors influencing compliance with quarantine in Toronto during the 2003 sars outbreak. Biosecur. Bioterror 2, 265–272. (doi:10.1089/bsp.2004.2.265) 44. SPI-B. The impact of financial and other targeted support on rates of self-isolation or quarantine. 2020 https://assets.publishing.service.gov.uk/ government/uploads/system/uploads/attachment_ data/file/925133/S0759_SPI-B__The_impact_of_ financial_and_other_targeted_support_on_rates_ of_self-isolation_or_quarantine_.pdf (accessed 19 October 2020). 34. Lloyd-Smith JO, Schreiber SJ, Kopp PE, Getz WM. 2005 Superspreading and the effect of individual variation on disease emergence. Nature 438, 355–359. (doi:10.1038/nature04153) 24. Blake KD, Blendon RJ, Viswanath K. 2010 Employment and compliance with pandemic influenza mitigation recommendations. Emerg. Infect. Dis. 16, 212. (doi:10.3201/eid1602.090638) 35. Mizumoto K, Kagaya K, Zarebski A, Chowell G. 2020 Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship, Yokohama, Japan, 2020. Eurosurveillance 25, 2000180. (doi:10.2807/1560-7917.ES.2020.25.10. 2000180) 25. Horney JA, Moore Z, Davis M, MacDonald PD. 2010 Intent to receive pandemic influenza a (H1N1) vaccine, compliance with social distancing and sources of information in NC, 2009. PLoS ONE 5, e11226. (doi:10.1371/journal.pone.0011226) 45. Department of Health and Social Care. 2020 New legal duty to self-isolate comes into force today. https://www.gov.uk/government/news/new-legal- duty-to-self-isolate-comes-into-force-today (accessed 14 October 2020). 26. Baggaley RF et al. 2020 Recommendations for augmenting contact tracing in the UK: learning from other diseases. Isaac Newton Institute for Mathematical Sciences (accessed 18 August 2020). 36. Lavezzo E et al. 2020 Suppression of COVID-19 outbreak in the municipality of Vo, Italy. medRxiv. (doi:10.1101/2020.04.17.20053157) 46. References Smith LE, Potts HWW, Amlot R, Fear NT, Michie S, Rubin J. 2020 Adherence to the test, trace and isolate system: results from a time series of 21 nationally representative surveys in the UK (the COVID-19 rapid survey of adherence to interventions and responses [CORSAIR] study). medRxiv. 37. He X et al. 2020 Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat. Med. 1–4. 37. He X et al. 2020 Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat. Med. 1–4. 38. Endo A, Abbott S, Kucharski AJ, Funk S. 2020 Estimating the overdispersion in COVID-19 transmission using outbreak sizes 27. Wyllie AL et al. 2020 Saliva or nasopharyngeal swab specimens for detection of SarS-CoV-2. New Eng. J. Med. 383, 1283–1286. 38. Endo A, Abbott S, Kucharski AJ, Funk S. 2020 Estimating the overdispersion in COVID-19 transmission using outbreak sizes
https://openalex.org/W4381591442	https://zenodo.org/records/8071151/files/784-791.pdf	Quechua	null	NOGIRONLAR UCHUN AVTOMOBILLARNI MODIFIKATSIYALASH VA ERGONOMIKA	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	3,733	ANNOTATSIYA Yillar mobaynida muhandis konstruktorlar imkoniyati cheklangan shaxslar avtomobillarni boshqarishi uchun turli xil qurilma va yordamchi vositalarni ishlab chiqib, avtomobilni erkin boshqarishini ta’minlash ustida ishlab kelishmoqda. Ush bu maqolada Andijon shahridagi 30 ta imkniyati cheklangan shaxslar tomonidan so‘rovnomalar o‘tkazilgan bo‘lib, ularning avtomobilga o‘tirish va boshqarishigacha bo‘lgan holatlar bo‘yicha mavjud muammolar o‘rganilgan. Hozrigi kunda avtomobil saloni oyoq harakatlarini takrorlovchi turli xil uskunalar bilan jixozlangan. So‘rovda baholangan asosiy komponentlar ergonomikaning besh jihatini, ya’ni xavfsizlik, foydalanish qulayligi, qulaylik, mahsuldorlik va ishlash va estetikani o‘z ichiga oladi. Komponentlardagi har bir savolni baholash uchun Likert shkalasi (1 dan 5 gacha) dan foydalanilgan. Tadqiqot shuni ko‘rsatdiki, boshqaruv elementlari, qoplamalar va rul boshqaruvi kabi ichki qismlar ergonomik qayta loyihalashga zarur. Kalit so‘zlar: nogiron oyoq-qo‘llar, qayta loyihalash, avtomobil ichki qismi, ergonomika, inklyuziv dizayn. Kalit so‘zlar: nogiron oyoq-qo‘llar, qayta loyihalash, avtomobil ichki qismi, ergonomika, inklyuziv dizayn. Educational Research in Universal Sciences ISSN: 2181-3515 Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 NOGIRONLAR UCHUN AVTOMOBILLARNI MODIFIKATSIYALASH VA ERGONOMIKA Qayumov Baxrom Abdullajonovich Andijon mashinasozlik instituti, t.f.f.d., dotsent Yoqubov Yoqubjon Odil o‘g‘li Andijon mashinasozlik instituti tayanch doktoranti ABSTRACT Over the years, engineers and designers have developed various devices and aids for people with disabilities to drive and work to ensure that they can freely drive a car. In this article, a survey was conducted of 30 disabled people in the city of Andijan and their problems associated with landing and driving a car were studied. Today, the interior of the car is equipped with various equipment that imitates the movements of 784 Multidisciplinary Scientific Journal May, 2023 https://t.me/Erus_uz Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 the legs. The key components assessed in the survey include five aspects of ergonomics: safety, ease of use, comfort, productivity and performance, and aesthetics. A Likert scale (from 1 to 5) was used to rate each question in the components. The study found that internal components such as controls, upholstery and steering wheel needed an ergonomic redesign. Key words:disabled limbs, redesign, car interior, ergonomics, inclusive design. 1.KIRISH. Ayni damda avtomobil sanoati yangi muammolar va iqtisodiy cheklovlarga duch kelmoqda [1], bunday muammolardan biri inklyuziv dizaynni yaratish uchun inklyuziv guruh ehtiyojlarini aniqlashdir. Inklyuziv dizayn jismoniy imkoniyatlarida cheklovlari bo‘lgan odamlarning ehtiyoj yoki talablarni aniqlashga yordam beradi, bu ularning avtomobil boshqarish tajribasini yaxshilaydi [2], shuningdek, turli jismoniy, kognitiv va hissiy nogironligi bo‘lgan foydalanuvchilarning ehtiyojlarini qondirish uchun muhimdir[3]. Qaysidir ma’noda, yaratilgan dizayn “umum foydalanuv” bo‘lishi kerak. Jismoniy nuqsonlar bilan birga keladigan salomatlik holati va nisbiy individual funktsional ko‘rsatkichlar (bu shaxsiy xolatlarga qarab o‘zgaradi) ko‘pincha nogironlarning avtomobil boshqarish qobiliyatini baholashda qiyinchiliklarga olib keladigan sabablardir [4]. Bundan tashqari, avtomashinani mustaqil boshqarish qobiliyati, ayniqsa, nogironlar uchun kundalik hayotda muhim jihat [5]. Hayot sifatini yaxshilash uchun ko‘pchilik nogironlar moslashtirilgan yoki modifikatsiyalangan avtomobildan shaxsiy transport vositasi [6] sifatida boshqaradilar [7]. Modifikatsiyalangan yoki yordamchi boshqaruv qurilmalari nogiron haydovchining imkoniyatlarini yaxshilashga yordam beradi. Bundan tashqari ko‘plab xorijiy davlatlarda yordamchi qurilmalarni oson toppish mumkin. Olib borilgan so‘rovlar natijasida hozirda O‘zbekistonda nogironligi bo‘lgan shaxslar boshqarishi uchun avtomobillarni modefikatsiyalash yoki yordamchi qurilmalarni ishlab chiqaruvchi korxona va mutaxassislar mavjud emas. Standart avtomobillar an’anaviy boshqaruv organlari bilan jixozlangan bo‘lib; rul, akselarator, tormoz, ilashish muftasi pedali. Pedal o‘z navbatia haydovchi bilan aks aloqani ta’minlashni xisobga olgan holda ishlab chiqilgan. Masalan, rul va pedal aylanish orqali boshqaruvni haydovchining qo‘llari va oyoqlari tomon taqsimlash uchun mo‘ljallangan. Biroq shunday holatlar yoki nogironlikning turlari borki, odatiy boshqaruv tizimlari avtomobilni boshqarishni cheklaydi [6]. Moslashuvchan modifikatsiyalangan qurilmalar nogironlarning avtomobil boshqaruvida erkin va mustaqil harakatlanishini ta’minlaydi [8]. Umuman olganda, moslashuv yoki modifikatsiyaning foydaliligi Sherer [10], Torkildsen [11] va Batavia va boshqalar [12] ga koʻra bir qancha omillarga bogʻliq. 785 May, 2023 Multidisciplinary Scientific Journal https://t.me/Erus_uz https://t.me/Erus_uz Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 O‘rganishlar shuni ko‘rsatdiki O‘zbekistondagi moslashuvchi modefikatsiya qilingan avtomobillar nogironlarning shaxsiy extiyojlaridan kelib chiqib ishlanmagan yoki qo‘lbola usulda ishlab chiqilgan. Ma’lum bo‘lishicha respublikada ishlab chiqarilgan avtomobillarni maxsus uskunalar bilan qayta jixozlash “UzautoMotors” AJ rasmiy dillerlarining ixtisoslashtirilgan servis markazlari orqali amalga oshiriladi. Shunday qilib, ushbu tadqiqot tananing oyoq qismida nogironligi bo‘lgan haydovchilar avtomobillarining interyer dizayni sohasini o‘z ichiga oladi. 2. Metodika. Respondentlarning demografik ma’lumotlari, respondentlarning kelib chiqishi, avtomobil ichki o‘zgarishlari va avtomobil ergonomikasi tajribasidan iborat so‘rovnomalar to‘plami o‘tkazildi. Respondentlar “Andijon avtotex xizmat”, “O‘zbekiston nogironlar jamiyati”, Andijon mashinasozlik instituti talaba va xodimlari. So‘rovnomada Andijon viloyatidan 30 nafar turli xil darajadagi nogironligi bor bo‘lgan avtomobil haydovchilari qatnashishdi. So‘rovnoma anketasi 4 ta katta bo‘limlardan iborat bo‘lib, so‘ngi bo‘lim ergonomikaga bag‘ishlangan. Ushbu so‘rov natijalari uchun normallik va tavsifli tahlilni tekshirish amalga oshirildi. Javoblar ijtimoiy fanlar (SPS) tijorat dasturi uchun statistik paketdan foydalangan holda jadval ko‘rinishida shakillantirildi va natijalar chastota va foizlar shaklida ifodalandi. 3.1. Respondent xususiyatlari Umumiy 55 ta anketa tarqatilgan bo‘lib, 30 ta anketa nogironlar tomonidan talab darajasida to‘ldirilgan. So‘rovnoma 30 ta respondentga asoslangan bo‘lib, ular mustaqil ravishda modifikatsiya qilingan va modifikatsiya qilinmagan avtomobilda harakatlanishgan. 1-jadvalda demografik ma’lumotlarning bir qismi, nogironlik turi, maxsus boshqaruv uskunalariga bo‘lgan ehtiyoj, boshqaruv paytida noxush alomatlar va ularning transport vositalariga o‘zgartirishlar turi kiradi. May, 2023 Multidisciplinary Scientific Journal 786 https://t.me/Erus_uz Educational Research in Universal Sciences SSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 1-jadval Respondentlarning xususiyatlari Respondentlar demografiyasi N (%) Jins Erkak 30 (100%) Ayol - - Respondent tavsilotlari N (%) Nogironlik turi Tananing yuqori harakat tayanch tizimida nogironlik 4 (13%) Tananing quyi harakat tayanch tizimida nogironlik 18 (60%) Umumiy oyoq-qo‘l nogironligi 8 (27%) Maxsus jixozga bo‘lgan extiyoj Ha 16 (53%) Yo‘q 14 (47%) Nojo‘ya ta’sirlar Yuqori tizim 4 (13%) Quyi tizim 11 (37%) Umum tizim 7 (23%) Hech kim 8 (27%) O‘zgartirish kiritildi O‘ng pedaldan chapga 8 (27%) Pedaldan joystikka 1 (3%) O‘rindiq balandligini sozlash 6 (20%) Har bir tugma uchun signallar 3 (10%) Boshqalar 1 (3%) O‘zgartirishlarsiz 1 (3%) 2-jadval Ergonomika bo‘limi uchun Likert reytingi Bo‘lim Ballar darajasi Likert shkalasi A Avtomobil ichki qismi NE-LE-N-E-VE 1-2-3-4-5 B Xavfsizlik VD-D-N-E-VE 1-2-3-4-5 C Qulaylik VU-U-N-C-VC 1-2-3-4-5 D Foydalanishda osonlik VD-D-N-E-VE 1-2-3-4-5 E Samaradorlik va ishlash VD-D-N-E-VE 1-2-3-4-5 F Estetika VNI-NI-N-I-VI 1-2-3-4-5 Juda qulay (VC); Juda oson (VE); Neytral (N); Noqulay (U); Juda qiyin (VD); qiyin (D); juda noqulay (VU); Juda ergonomika (VE); Oson (E); juda muhim (VI); Kamroq ergonomika (LE); Muhim (I); muhim emas (NI); Ergonomika emas (NE); Ergonomika (E); Juda muhim emas (VNI); Qulay (C) Jadvalda ko‘rsatilganidek, respondentlarning ko‘p qismini (87%) erkaklar tashlik etdi, ayol haydovchilarning atigi 13 foizi ishtirok etdi. 3.1. Respondent xususiyatlari Bundan tashqari, respondentlarning 53 foizi nogironlar aravachasi, qo‘ltiq tayoqchalari va sun’iy yuqori va pastki oyoq-qo‘llari kabi harakatchan bo‘lish uchun maxsus jihozlarga muhtoj bo‘lsa, respondentlarning 73 foizi avtomobilni boshqarish uchun yordamchi Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 1-jadval Respondentlarning xususiyatlari Respondentlar demografiyasi N (%) Jins Erkak 30 (100%) Ayol - - Respondent tavsilotlari N (%) Nogironlik turi Tananing yuqori harakat tayanch tizimida nogironlik 4 (13%) Tananing quyi harakat tayanch tizimida nogironlik 18 (60%) Umumiy oyoq-qo‘l nogironligi 8 (27%) Maxsus jixozga bo‘lgan extiyoj Ha 16 (53%) Yo‘q 14 (47%) Nojo‘ya ta’sirlar Yuqori tizim 4 (13%) Quyi tizim 11 (37%) Umum tizim 7 (23%) Hech kim 8 (27%) O‘zgartirish kiritildi O‘ng pedaldan chapga 8 (27%) Pedaldan joystikka 1 (3%) O‘rindiq balandligini sozlash 6 (20%) Har bir tugma uchun signallar 3 (10%) Boshqalar 1 (3%) O‘zgartirishlarsiz 1 (3%) 2-jadval Ergonomika bo‘limi uchun Likert reytingi Bo‘lim Ballar darajasi Likert shkalasi A Avtomobil ichki qismi NE-LE-N-E-VE 1-2-3-4-5 B Xavfsizlik VD-D-N-E-VE 1-2-3-4-5 C Qulaylik VU-U-N-C-VC 1-2-3-4-5 D Foydalanishda osonlik VD-D-N-E-VE 1-2-3-4-5 E Samaradorlik va ishlash VD-D-N-E-VE 1-2-3-4-5 F Estetika VNI-NI-N-I-VI 1-2-3-4-5 2-jadval Ergonomika bo‘limi uchun Likert reytingi Juda qulay (VC); Juda oson (VE); Neytral (N); Noqulay (U); Juda qiyin (VD); qiyin (D); juda noqulay (VU); Juda ergonomika (VE); Oson (E); juda muhim (VI); Kamroq ergonomika (LE); Muhim (I); muhim emas (NI); Ergonomika emas (NE); Ergonomika (E); Juda muhim emas (VNI); Qulay (C) Jadvalda ko‘rsatilganidek, respondentlarning ko‘p qismini (87%) erkaklar tashlik etdi, ayol haydovchilarning atigi 13 foizi ishtirok etdi. Bundan tashqari, respondentlarning 53 foizi nogironlar aravachasi, qo‘ltiq tayoqchalari va sun’iy yuqori va pastki oyoq-qo‘llari kabi harakatchan bo‘lish uchun maxsus jihozlarga muhtoj bo‘lsa, respondentlarning 73 foizi avtomobilni boshqarish uchun yordamchi texnologiyalar va modifikatsiyaga muhtoj. 787 May, 2023 Multidisciplinary Scientific Journal https://t.me/Erus_uz Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 3-jadvalda oyoq-qo‘llari nogironligi bo‘lgan shaxsning asosiy ishiga va uning transport vositalarining bir qismiga nisbatan ergonomik ehtiyojiga javob ko‘rsatilgan. 4. Nogironlar uchun ergonomik avtomobil Shunisi qiziqki, oyoq-qo‘llari nogironligi bo‘lgan haydovchilar xavfsizlik va foydalanish qulayligi (B va D bo‘limlari) nuqtai nazaridan hech qanday muammo tug‘dirmagan, garchi bu avtomobilni boshqarish ko‘rsatkichlariga, ayniqsa noma’lum yo‘l sharoitlarida va noma’lum ob-havo sharoitida ta’sir qiladi. Respondentlardan bir biriga o‘xshash javovlar ko‘plab olingan. 3-jadvalning A bo‘limida eshik tutqichi, atrof-muhit tutqichi, pedallar va transport vositasidan chiqish kabi yuqori rejim va past ergonomik ball olgan umumiy avtomobil ichki muhiti va uskunalari ro‘yxati keltirilgan. 3-jadvalning C, E, F bo‘limlarida 2- jadvalda (C bo‘lim) ajratib ko‘rsatilgan ergonomik aspektlar. Bu shuni ko‘rsatadiki, muayyan vaziyatlarda respondentlar eshiklarni ochishda, o‘tirishda (C bo‘limi), shuningdek, haydash paytida mediatekani boshqarishda qiyinchiliklarga duch kelishadi (E bo‘limi). 3-jadval (F bo‘limi) muhim estetika uchun yuqori rejim reytingini ko‘rsatadi, bu yerda qoplama (pastki), qoplama (orqa), tutqich, rul chambarafi va uzatmalar qutisi tutqichi muhim ahamiyatli deb baholangan. Shunisi qiziqki, oyoq-qo‘llari nogironligi bo‘lgan haydovchilar xavfsizlik va foydalanish qulayligi (B va D bo‘limlari) nuqtai nazaridan hech qanday muammo tug‘dirmagan, garchi bu avtomobilni boshqarish ko‘rsatkichlariga, ayniqsa noma’lum yo‘l sharoitlarida va noma’lum ob-havo sharoitida ta’sir qiladi. Respondentlardan bir biriga o‘xshash javovlar ko‘plab olingan. 4. Nogironlar uchun ergonomik avtomobil A. Vudkuk “Shaxsga yo‘naltirilgan transport loyihalash” nomli maqolasida “Transport jamiyatning asosiy muammosi” ekanligi, bunda u harakatni soddalashtiradi, iqtisodiyotni, shuningdek, aholi salomatligini yaxshilaydi deb ta’kidlagan [13]. Loyihalash orqali xavfsizlik, samaradorlik va qulaylikni oshirish uchun ergonomikani ta’minlash muhimdir [13]. Shu sababli, kundalik hayotida transport vositasiga ega bo‘lgan har bir kishi xavfsizlikni oshirish va xavfni kamaytirish uchun tegishli ergonomik talablarga rioya qilishi juda muhimdir [14]. Bundan tashqari, bir nechta tadqiqotlar ergonomikaning yaxshi sifatga ta’sirini ko‘rsatdi [7, 15]. Loyihadagi ergonomika inson va mashina o‘rtasidagi o‘zaro ta’sirga bog‘liq [18]. Ergonomik omilni hisobga olgan holda, insonning imkoniyatlari va muayyan tuzilmalarning cheklovlari hisobga olinadi [4]. Ergonomik avtomobil dizaynini ko‘rib chiqayotganda, 2B-F jadvallarida keltirilgan ergonomikaning besh jihati mavjud. Ushbu besh omil nogironlarning ehtiyojlarini qondirish uchun eng munosib yaxshilanishni aniqlash uchun joriy ichki konstruksiyadagi zaif tomonlarni aniqlash uchun so‘rovnomada ishlatilgan. Ergonomika bo‘limida 2-jadvalda ko‘rsatilgandek Likert shkalasi (5 ballli shkala) ishlatilgan. A, B, C, D va E bo‘limlari uchun yuqori rejim yoki past tanlovning yuqori chastotasi ergonomikani yanada takomillashtirishga e’tibor berish kerakligini ko‘rsatadi. Ayni paytda, qarama-qarshi tushunchalar ball darajasi boshqa yo‘nalishda bo‘lganligi sababli F bo‘limiga tushadi. Jadval 3. Baholash asosidagi darajaga qarab shikastlangan a’zoning ergonomik talablari A bo‘lim Rejim Ball darajasi Tutqich (eshik) 9 LE Tutqich (atrofda) 9 LE Pedallar 9 LE Chiqish 10 LE C bo‘limi Rejim Ball darajasi Ichkaridan eshikni ochish 9 U Beldan pastki qismni ta’minlash 9 U E bo‘limi Rejim Ball darajasi Haydash paytida navigatsiyadan foydalanish 8 D F bo‘limi Rejim Ball darajasi Mebel (pastki) 12 I Mebel (orqa) 11 I Tutqich 13 VI Rul qoplamasi 11 I Uzatmalar qutisi tutqichi 11 I Jadval 3. Baholash asosidagi darajaga qarab shikastlangan a’zoning ergonomik talablari Jadval 3. Baholash asosidagi darajaga qarab shikastlangan a’zoning ergonomik talablari 3-jadvalda oyoq-qo‘llari nogironligi bo‘lgan shaxsning asosiy ishiga va uning transport vositalarining bir qismiga nisbatan ergonomik ehtiyojiga javob ko‘rsatilgan. 788 Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 3-jadvalning A bo‘limida eshik tutqichi, atrof-muhit tutqichi, pedallar va transport vositasidan chiqish kabi yuqori rejim va past ergonomik ball olgan umumiy avtomobil ichki muhiti va uskunalari ro‘yxati keltirilgan. 3-jadvalning C, E, F bo‘limlarida 2- jadvalda (C bo‘lim) ajratib ko‘rsatilgan ergonomik aspektlar. Bu shuni ko‘rsatadiki, muayyan vaziyatlarda respondentlar eshiklarni ochishda, o‘tirishda (C bo‘limi), shuningdek, haydash paytida mediatekani boshqarishda qiyinchiliklarga duch kelishadi (E bo‘limi). 3-jadval (F bo‘limi) muhim estetika uchun yuqori rejim reytingini ko‘rsatadi, bu yerda qoplama (pastki), qoplama (orqa), tutqich, rul chambarafi va uzatmalar qutisi tutqichi muhim ahamiyatli deb baholangan. 5. Xulosa Xulosada O‘zbekistonda oyoq-qo‘llari nogironligi bo‘lgan haydovchilar o‘rtasida o‘tkazilgan tadqiqot natijalari taqdim etildi. Natijada, oyoq-qo‘llari cheklangan haydovchiga qulay bo‘lgan ergonomik transport vositasini yaratish uchun haydovchi hududining ichki qismini qayta loyihalashda ba’zi muhim jihatlarni hisobga olish zarurligi to‘g‘risida xulosa qilindi. Respondentlarning ba’zi javoblari ta’kidlanmagan bo‘lsa-da, ularning fikrlarini nogironlar ehtiyojlarigni qondirishda yechim sifatida foydalanish kerak. Tadqiqot natijalarini inobatga olgan holda, nogironlar uchun qulay bo‘lgan avtomobil dizaynini yanada ergonomik qilish maqsadida o‘zgartirish bo‘yicha yechim ishlab chiqiladi. Hozirda bu davom etayotgan loyiha bo‘lib, kelajakda imkoniyati cheklangan shaxslar avtomobildan foydalanishda qulaylik va mamnunlikni ta’minlashda xizmat qiladi. FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES) 1. Каюмов Б. А., Вохобов Р. А. Внесение изменений в конструкцию автомобилей по результатам испытаний //Бюллетень науки и практики. – 2019. – Т. 5. – №. 11. – С. 249-254. FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES) 1. Каюмов Б. А., Вохобов Р. А. Внесение изменений в конструкцию автомобилей по результатам испытаний //Бюллетень науки и практики. – 2019. – Т. 5. – №. 11. – С. 249-254. FOYDALANILGAN ADABIYOTLAR RO YXATI: (REFERENCES) 1. Каюмов Б. А., Вохобов Р. А. Внесение изменений в конструкцию автомобилей по результатам испытаний //Бюллетень науки и практики. – 2019. – Т. 5. – №. 11. – С. 249-254. 2. H. S. Bae, J. Ryu, and C. Gerdes, “Road grade and vehicle parameter estimation for longitudinal control using GPS,” presented at the Proceedings of IEEE Conference on Intelligent Transportation Systems, San Francisco, CA, 2001. 3. Захаров Ю. А., Ремзин Е. В., Мусатов Г. А. Основные дефекты корпусных деталей автомобилей и способы их устранения, применяемые в авторемонтном производстве //Инженерный вестник Дона. – 2014. – Т. 31. – №. 4-1. – С. 43. 2. H. S. Bae, J. Ryu, and C. Gerdes, “Road grade and vehicle parameter estimation for longitudinal control using GPS,” presented at the Proceedings of IEEE Conference on Intelligent Transportation Systems, San Francisco, CA, 2001. 3. Захаров Ю. А., Ремзин Е. В., Мусатов Г. А. Основные дефекты корпусных деталей автомобилей и способы их устранения, применяемые в авторемонтном производстве //Инженерный вестник Дона. – 2014. – Т. 31. – №. 4-1. – С. 43. 789 https://t.me/Erus_uz Multidisciplinary Scientific Journal May, 2023 Multidisciplinary Scientific Journal https://t.me/Erus_uz Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 4. Вахобов Р. А., Ёкубов Ё. О., Нумонов М. З. Виртуальное тестирование вспомогательных частей автомобиля //The Scientific Heritage. – 2021. – №. 62-1. – С. 42-46. 4. Вахобов Р. А., Ёкубов Ё. О., Нумонов М. З. Виртуальное тестирование вспомогательных частей автомобиля //The Scientific Heritage. – 2021. – №. 62-1. – С. 42-46. 5. Вохобов Р. ПРОЦЕСС ПРОЕКТИРОВАНИЕ ДЕТАЛЕЙ С ПОМОЩЬЮ РЕИНЖИНИРИНГА //Danish Scientific Journal. – 2020. – №. 36-1. – С. 58-61. 5. Вохобов Р. ПРОЦЕСС ПРОЕКТИРОВАНИЕ ДЕТАЛЕЙ С ПОМОЩЬЮ РЕИНЖИНИРИНГА //Danish Scientific Journal. – 2020. – №. 36-1. – С. 58-61. 5. Вохобов Р. ПРОЦЕСС ПРОЕКТИРОВАНИЕ ДЕТАЛЕЙ С ПОМОЩЬЮ РЕИНЖИНИРИНГА //Danish Scientific Journal. – 2020. – №. 36-1. – С. 58-61. 6. Каюмов Б. А., Шарипов К. А. FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES Моделирование закономерностей распределения отказов элементов инжекционной системы питания двигателей методом сплайн- функций //Архив научных исследований. – 2020. – №. 30. 7. Каюмов Б. А. Разработка методик испытаний электромеханических датчиков автомобилей, используемых для эксплуатации в регионах с жарким климатом //Вестник Мурманского государственного технического университета. – 2022. – Т. 25. – №. 4. – С. 345-353. 5. Вохобов Р. ПРОЦЕСС ПРОЕКТИРОВАНИЕ ДЕТАЛЕЙ С ПОМОЩЬЮ РЕИНЖИНИРИНГА //Danish Scientific Journal. – 2020. – №. 36-1. – С. 58-61. 6. Каюмов Б. А., Шарипов К. А. Моделирование закономерностей распределения отказов элементов инжекционной системы питания двигателей методом сплайн- функций //Архив научных исследований. – 2020. – №. 30. 7. Каюмов Б. А. Разработка методик испытаний электромеханических датчиков автомобилей, используемых для эксплуатации в регионах с жарким климатом //Вестник Мурманского государственного технического университета. – 2022. – Т. 25. – №. 4. – С. 345-353. РЕИНЖИНИРИНГА //Danish Scientific Journal. – 2020. – №. 36-1. – С. 58-61. 6. Каюмов Б. А., Шарипов К. А. Моделирование закономерностей распределения отказов элементов инжекционной системы питания двигателей методом сплайн- функций //Архив научных исследований. – 2020. – №. 30. 7. Каюмов Б. А. Разработка методик испытаний электромеханических датчиков РЕИНЖИНИРИНГА //Danish Scientific Journal. 2020. №. 36-1. С. 58-61. 6. Каюмов Б. А., Шарипов К. А. Моделирование закономерностей распределения отказов элементов инжекционной системы питания двигателей методом сплайн- функций //Архив научных исследований. – 2020. – №. 30. 7. Каюмов Б. А. Разработка методик испытаний электромеханических датчиков автомобилей, используемых для эксплуатации в регионах с жарким климатом //Вестник Мурманского государственного технического университета 2022 6. Каюмов Б. А., Шарипов К. А. Моделирование закономерностей распределения отказов элементов инжекционной системы питания двигателей методом сплайн- функций //Архив научных исследований. – 2020. – №. 30. 7. Каюмов Б. А. Разработка методик испытаний электромеханических датчиков автомобилей, используемых для эксплуатации в регионах с жарким климатом //Вестник Мурманского государственного технического университета. – 2022. – Т. 25. – №. 4. – С. 345-353. 8. Балабин И.В., Куров Б.А., Лаптев С.А. Испытания автомобилей. - М.: Машиностроение, 1988. 9. Бахмутов С.В. Научные основы параметрической оптимизации автомобиля по критериям управляемости и устойчивости: дисс. на соиск. учен. степ. д.т.н. - НАТИ-М., 2001. - 320 с. 10. Каюмов Б. А. ВНЕСЕНИЯ ИЗМЕНЕНИЙ, В КОНСТРУКЦИЮ АВТОМОБИЛЕЙ ИСХОДЯ ИЗ РЕЗУЛЬТАТОВ ИСПЫТАНИЙ //Архив научных исследований. – 2020. – №. 30. 11. Ёкубов Ё. О. У., Эргашев Д. П. Оптимальное проектирование конструкций с 10. Каюмов Б. А. ВНЕСЕНИЯ ИЗМЕНЕНИЙ, В КОНСТРУКЦИЮ АВТОМОБИЛЕЙ ИСХОДЯ ИЗ РЕЗУЛЬТАТОВ ИСПЫТАНИЙ //Архив научных исследований. – 2020. – №. 30. 11. Ёкубов Ё. О. FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES Обзор конструкции ручного управление автомобилей для людей с ограниченными возможностями = Overview of the design of manual control of cars for people with disabilities / Б. А. Каюмов, Е. О. Екубов // Автотракторостроение и автомобильный транспорт : сборник научных трудов : в 2 томах / Белорусский национальный технический университет, Автотракторный факультет ; редкол.: Д. В. Капский (отв. ред.) [и др.]. – Минск: БНТУ, 2022. – Т. 1. – С. 34-38. 18. Каюмов, Б. А. Обзор конструкции ручного управление автомобилей для людей с ограниченными возможностями = Overview of the design of manual control of cars for people with disabilities / Б. А. Каюмов, Е. О. Екубов // Автотракторостроение и автомобильный транспорт : сборник научных трудов : в 2 томах / Белорусский национальный технический университет, Автотракторный факультет ; редкол.: Д. В. Капский (отв. ред.) [и др.]. – Минск: БНТУ, 2022. – Т. 1. – С. 34-38. 18. Каюмов, Б. А. Обзор конструкции ручного управление автомобилей для людей с ограниченными возможностями = Overview of the design of manual control of cars for people with disabilities / Б. А. Каюмов, Е. О. Екубов // Автотракторостроение и автомобильный транспорт : сборник научных трудов : в 2 томах / Белорусский национальный технический университет, Автотракторный факультет ; редкол.: Д. В. Капский (отв. ред.) [и др.]. – Минск: БНТУ, 2022. – Т. 1. – С. 34-38. 19. Книга: "Assistive Technology for People with Disabilities" / A. M. Cook. - 2009. - 1-е изд. - Springer Publishing Company. - 352 с. 20. Документ: "Accessible Vehicle Design Standards" // - 1995. - Первая версия стандартов. - Society of Automotive Engineers. 20. Документ: "Accessible Vehicle Design Standards" // - 1995. - Первая версия стандартов. - Society of Automotive Engineers. 21. Постановление Кабинета Министров Республики Узбекистан: «О мерах по созданию благоприятных условий для пользования лицами с инвалидностью автотранспортными средствами» // - 07.03.2018 г. № 183 2. Программа: "Adaptive Driving Program" // - 1989. - Первое описание. 23. Книга: "Designing Inclusive Futures" / A. Lucas, R. Cooper. - 2008. - 1-е изд. - JohnWiley & Sons. - 320 с. 23. Книга: "Designing Inclusive Futures" / A. Lucas, R. Cooper. - 2008. - 1-е изд. - JohnWiley & Sons. - 320 с. 24. Книга: "Universal Design for Learning in the Classroom" / T. Tobin. - 2014. - 1-е изд. - Routledge. - 168 с. 24. Книга: "Universal Design for Learning in the Classroom" / T. Tobin. - 2014. - 1-е изд. - Routledge. FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES У., Эргашев Д. П. Оптимальное проектирование конструкций с помощью цифровых интегрированных технологии //Universum: технические науки. – 2020. – №. 11-1 (80). – С. 21-24. 11. Ёкубов Ё. О. У., Эргашев Д. П. Оптимальное проектирование конструкций с помощью цифровых интегрированных технологии //Universum: технические науки. – 2020. – №. 11-1 (80). – С. 21-24. 12. Yoqubjon Y. Methodology of optimal design of mechanical structures //Universum: технические науки. – 2021. – №. 12-6 (93). – С. 86-89. 12. Yoqubjon Y. Methodology of optimal design of mechanical structures //Universum: технические науки. – 2021. – №. 12-6 (93). – С. 86-89. 13. Каюмов, Б. А., Эргашев Д. П. Анализ воздушной силы цилиндров и конусов в программе виртуальных испытаний // Автотракторостроение и автомобильный транспорт : сборник научных трудов : в 2 томах. – Минск : БНТУ, 2022. – Т. 2. – С. 337-340. 13. Каюмов, Б. А., Эргашев Д. П. Анализ воздушной силы цилиндров и конусов в программе виртуальных испытаний // Автотракторостроение и автомобильный транспорт : сборник научных трудов : в 2 томах. – Минск : БНТУ, 2022. – Т. 2. – С. 337-340. 14. Кушвид Р.П.К96 Испытания автомобиля: учебник. - М.: МГИУ, 2011. - 351 с. C 341-346 14. Кушвид Р.П.К96 Испытания автомобиля: учебник. - М.: МГИУ, 2011. - 351 с. C 341-346 15. Qayumov B. A., Ergashev D. P. MINIVEN TIPIDAGI AVTOMOBIL KUZOVIGA HAVONING QARSHILIK KUCHINI ANIQLASH //RESEARCH AND EDUCATION. – 2023. – Т. 2. – №. 1. – С. 110-118. 16. Журнал: "Ergonomics and Human Factors: The International Journal of Human Factors and Ergonomics" // - 1958. - Т. 1. - Вып. 1. - Стр. 1-15. 16. Журнал: "Ergonomics and Human Factors: The International Journal of Human Factors and Ergonomics" // - 1958. - Т. 1. - Вып. 1. - Стр. 1-15. 790 Multidisciplinary Scientific Journal May, 2023 790 May, 2023 Multidisciplinary Scientific Journal https://t.me/Erus_uz Educational Research in Universal Sciences ISSN: 2181-3515 VOLUME 2 \| ISSUE 5 \| 2023 17. Руководство: "Driving and Community Mobility for Older Adults: Strategies for Success" / K. Frost. - 2006. - 1-е изд. - American Occupational Therapy Association. - 80 с. 17. Руководство: "Driving and Community Mobility for Older Adults: Strategies for Success" / K. Frost. - 2006. - 1-е изд. - American Occupational Therapy Association. - 80 с. 18. Каюмов, Б. А. FOYDALANILGAN ADABIYOTLAR RO‘YXATI: (REFERENCES - 168 с. 25. Книга: "Human Factors and Ergonomics in Practice: Improving System Performance and Human Well-Being in Transport" / J. Bridges. - 2013. - 1-е изд. - CRC Press. - 416 с. 25. Книга: "Human Factors and Ergonomics in Practice: Improving System Performance and Human Well-Being in Transport" / J. Bridges. - 2013. - 1-е изд. - CRC Press. - 416 с. 26. Документ: "Accessible Transportation and Mobility" // - 2013. - Первая версия. - Всемирная организация здравоохранения. 26. Документ: "Accessible Transportation and Mobility" // - 2013. - Первая версия. - Всемирная организация здравоохранения. - Всемирная организация здравоохранения. 791 Multidisciplinary Scientific Journal https://t.me/Erus_uz
https://openalex.org/W2972954402	https://fr.copernicus.org/articles/22/51/2019/fr-22-51-2019.pdf	English	null	A new bizarre dysoneurid species (Insecta, Trichoptera) in Burmese amber	Fossil record	2,019	cc-by	3,032	2 Materials and methods The amber bearing the embedded caddisﬂy belongs to Burmese amber from northern Myanmar. Most Burmese am- ber comes from the amber mine located near Noije Bum Vil- lage, Tanaing Town (Kania et al., 2015: ﬁg. 1; Jarzembowski et al., 2017: Fig. S1). The age provided by U–Pb dating of zircons from the volcaniclastic matrix of the amber is early Cenomanian (98.8 ± 0.6 Ma) (Shi et al., 2012). A new bizarre dysoneurid species (Insecta, Trichoptera) in Burmese amber Wilfried Wichard1 and Christian Neumann2 1Institute of Biology and its Didactic, University of Cologne, Herbert Lewinstrasse 2, 50931 Cologne, Ge 2Museum für Naturkunde – Leibniz Institute for Evolution and Biodiversity Science, Invalidenstrasse 43, 10115 Berlin, Germany 1Institute of Biology and its Didactic, University of Cologne, Herbert Lewinstrasse 2, 50931 Cologne, Germany 2Museum für Naturkunde – Leibniz Institute for Evolution and Biodiversity Science, Invalidenstrasse 43, 10115 Berlin Germany Correspondence: Christian Neumann (christian.neumann@mfn-berlin.de) Received: 15 April 2019 – Revised: 29 July 2019 – Accepted: 30 July 2019 – Published: 10 September 2019 Received: 15 April 2019 – Revised: 29 July 2019 – Accepted: 30 July 2019 – Published: 10 September 2019 Abstract. We describe an extinct caddisﬂy species, Cre- tapsyche palpinova sp. nov., from mid-Cretaceous Burmese amber. The species belongs to the extinct family Dysoneuri- dae within the superfamily Sericostomatoidea. The family in- cludes some species with unusual maxillary palps. Cretapsy- che palpinova sp. nov. is characterized by ﬁve-segmented maxillary palps, the second segment being the longest, and the third segment bearing an unusual pin-shaped appendage. including antennae, the maxillary and labial palps, the com- pound eyes and the ocelli, the legs with characteristic spurs, and the genitalia (Wichard et al., 2018). 1 Introduction The extinct trichopteran (caddisﬂy) family Dysoneuridae was established by Sukatsheva (1968). The description is merely based on fossil wings, which are preserved as im- pressions in sedimentary rocks from the Upper Jurassic of the Karatau Mountains in southern Kazakhstan. Sukatsheva and Vassilenko (2013) once again described the family us- ing further fossil wing impressions found at various Eurasian sites from the Middle Jurassic to the Lower Cretaceous. The fossil specimen represents a nearly complete adult, visible in ventral and dorsal views. The hind wings are par- tially covered by the forewings. The male genitalia are visible in ventral view. Photos were taken using a Leica stereomicro- scope M 420 Apozoom in combination with a Canon EOS 80D DSLR, EOS utility software, and Zerene Stacker software. All images and ﬁgures were processed with Adobe Photoshop CS4 and Photoshop Elements 15. The preliminary diagnostic forewing characters consist of the absence of apical forks I and IV; the radius R thick- ened, running straight, without bifurcation; the discoidal and medium cells closed or open; the thyridial cell closed and long; Cu2 reaching a desclerotized crossvein running be- tween Cu1b and the wing margin. Abbreviations Wing venation terminology generally follows Holzenthal et al. (2007): The description of the family Dysoneuridae was reviewed when new and complete specimens were found in mid- Cretaceous Burmese amber. An expanded set of important morphological characters were added to the preliminary de- scription of the family. Fore- and hind wings are often excel- lently preserved, as well as the head, thorax, and abdomen I denotes apical fork I, II denotes apical fork II, III denotes apical fork III, V denotes apical fork V, R denotes radius, Rs denotes radius sectori, M denotes media, Cu1 denotes cubi- tus anterior, Cu2 denotes cubitus posterior, A denotes analis, DC denotes discoidal cell, MC denotes median cell, and TC denotes thyridial cell. Foss. Rec., 22, 51–56, 2019 https://doi.org/10.5194/fr-22-51-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Foss. Rec., 22, 51–56, 2019 https://doi.org/10.5194/fr-22-51-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Remarks The family Dysoneuridae as established by Sukat- sheva (1968) is based merely on the venation of an isolated forewing preserved in sedimentary rocks from the Upper Jurassic in southern Kazakhstan. The forewing venation was interpreted as a reduced venation because of the absence of apical forks I and V; in addition, Cu2 reaches a desclerotized line running from the wing margin (arculus) to Cu1b; both characteristics correspond approximately to those of the wings of the family Kokiriidae (Sukatsheva and Vassilenko, 2013). However, for a complete and signiﬁcant description and classiﬁcation of Trichoptera, a complete set of morphological characters is imperatively needed. In the Family Dysoneuridae Sukatsheva, 1968 Cretapsyche palpinova sp. nov. Cretapsyche palpinova sp. nov. Type genus and species: Dysoneura trifurcata Sukat- sheva, 1968. Figs. 1–2. Etymology Cretapsyche palpinova sp. nov. is named for its novel maxil- lary and labial palpi. The family Dysoneuridae belongs to superfamily Seri- costomatoidea indicated by the absence of ocelli, with the terminal maxillary and labial segments being nonannulated and inﬂexible and the tibial spur formula being 2/2/4. The Dysoneuridae are distinct from all other families of Sericos- tomatoidea by their typical wing venation of fore- and hind wings. Diagnosis Scapus about as long as the head and bearing a brush of long setae; the pedicellus short, thickened at end; ﬁrst ﬂag- ellomere almost twice as long as the following ﬂagellomeres and basally thickened. The new species is distinguished from other dysoneurid species by the maxillary and labial palps: maxillary palps ﬁve-segmented; second segment longest, narrow, and slightly bent; third segment hairy, pin-shaped pendant (pilifer); third to ﬁfth segments successively de- creasing in size. Labial palps three-segmented; the terminal segment long, oval, and ﬂattened, with an apicolateral bulge. Family diagnosis Kept in the amber collection of the Museum für Naturkunde, Berlin (MfN: holotype inventory number MB.I. 7340 coll., former Patrick Müller collection, BUB 2587). Fossil from Upper Cretaceous, Cenomanian. Embedded in Burmese am- ber, body well preserved; forewing in dorsal view, hind wing covered dorsally by forewing. Antennae and legs present. Male genitalia visible in ventral view. Dysoneuridae clearly distinguished from all extant and ex- tinct families of Trichoptera by the tibial spur formula 2/2/4 in combination with the following fore- and hind wing ve- national characters. In forewing, forks I and IV absent and forks II, III, and V present; the nygma absent; discoidal and median cells closed or open; the thyridial cell long and closed; Cu2 reaches the desclerotized line (crossvein) run- ning from the wing margin to Cu1b. In the hind wing, forks I, III, and V are present; and forks II and IV and the discoidal cell are absent. Antennae about as long as the forewings or longer; the scapus as long as the head or longer, often bearing a brush of long dark setae. Systematic paleontology Genus diagnosis: the ﬁve-segmented maxillary palps give a clear and striking characteristic – second segment longest, longer than the following three segments to- gether, which are successively smaller and shorter; ﬁfth segment shortest. Order Trichoptera Kirby, 1815 Published by Copernicus Publications on behalf of the Museum für Naturkunde Berlin. Published by Copernicus Publications on behalf of the Museum für Naturkunde Berlin. 52 Suborder Integripalpia Martynov, 1924 Suborder Integripalpia Martynov, 1924 Family Dysoneuridae Sukatsheva, 1968 W. Wichard and C. Neumann: A new bizarre dysoneurid species In male genitalia, inf ap denotes inferior appendages, pre ap denotes preanal appendages, and X ap denotes mesodorsal processes of tergum X. case of the family Dysoneuridae, this became recently avail- able by excellently preserved specimens from Cretaceous Burmese amber, subsequently leading to a redescription of the family Dysoneuridae and their assignment to the superfamily Sericostomatoidea (Wichard et al., 2018). This published work and the nomenclatural acts it contains have been registered in ZooBank on 26 July 2019 and have received the LSID number urn:lsid:zoobank.org:act:1161465B-1937-4580-B02F- 8ED51C484A79. Cretapsyche Wichard et al., 2018 Type species: Cretapsyche circula Wichard et al., 2018 Description Male head without ocelli. Each ﬂagellum about 4 mm in length, consisting of 26 rod-shaped ﬂagellomeres; scapus strong, narrow, distally slightly broader than basally, about 0.5 mm in length and slightly longer than head, hairy, api- cally with tuft of hair; pedicellus short, thickened at ends; ﬁrst ﬂagellomere twice as long as following ﬂagellomeres, basally thickened. Maxillary palpus ﬁve-segmented: ﬁrst www.foss-rec.net/22/51/2019/ Foss. Rec., 22, 51–56, 2019 W. Wichard and C. Neumann: A new bizarre dysoneurid species 53 W. Wichard and C. Neumann: A new bizarre dysoneurid species 53 Figure 1. Cretapsyche palpinova sp. nov. (a) Male in ventral view. (b) Forewing venation: II, III, and V denote apical forks II, III, and V; TC denotes thyridial cell. W. Wichard and C. Neumann: A new bizarre dysoneurid species 53 Figure 1. Cretapsyche palpinova sp. nov. (a) Male in ventral view. (b) Forewing venation: II, III, and V denot denotes thyridial cell. Figure 1. Cretapsyche palpinova sp. nov. (a) Male in ventral view. (b) Forewing venation: II, III, and V denote apical forks II, III, and V; TC denotes thyridial cell. a sp. nov. (a) Male in ventral view. (b) Forewing venation: II, III, and V denote apical forks II, III, and V; TC visible, r-m short and aligned diagonally, m absent. Dis- coidal and median cells open, thyridial cell long and closed. Cu2 running to Cu1b and reaching desclerotized crossvein connecting Cu1b to margin; this crossvein not clearly visible. Hind wings smaller than forewings, each with wing venation and setation as follows (in ventral view, so far as visible): fork III clearly visible, petiolate; crossvein r-m also clearly visible; fork II absent; forks I and V probably present; dorsal surface covered with small scale-shaped hairs. segment long but shorter than second segment; second seg- ment longest, slim and slightly curved, longer than third, fourth, and ﬁfth segments together. Rod-shaped appendage (pilifer) originating from third segment. Labial palpus three- segmented: ﬁrst segment longest, almost as long as second and third segments together; third segment about as long as second segment but wider, ﬂattened, oval, rounded margin, apicolaterally with small rounded tongue. Forewings each 3 mm in length (Fig. 1b). Apical forks I and IV absent; II, III, and V present; fork III petiolate; fork V probably slightly petiolate. Crossveins r and s not clearly Male genitalia visible in ventral view (Fig. 2c, d). 3 Discussion In general fossil insects in Burmese Amber exhibit a surpris- ingly great range of variations and morphological character- istics, which are often unknown to today’s insects. This also applies to the Trichoptera. A comparison of the recent cad- disﬂies with the fossil caddisﬂies embedded in the 40- to 50- million-year-old Baltic Amber points to clear differences but also shows an amazing closeness and relationship to today’s forms (Ulmer 1912, Wichard 2013). Even clearer are the dif- ferences compared to the almost 100-million-year-old cad- disﬂies preserved in Burmese amber. Of course, with their greater age, Burmese Trichoptera are more distantly related to recent caddisﬂies. However, morphological variants were also tested, which obviously did not survive in the course of time and evolution, so that modern species lack them. 2. Labial palps three-segmented, terminal segment oval ﬂattened; maxillary palps with pilifer arising from third segment: C. palpinova sp. nov. – Labial palps three-segmented, simple; maxillary palps without pilifer on third segment: C. circula. 3. Forewing with median cell open; in male genitalia dor- somedian process of tergum X distinct, ventrally pro- truding: C. insueta. – Forewing with median cell closed; in male genitalia dorsomedian process of tergum X simple: C. elegans. As proposed by Wichard et al. (2018), the extinct family Dysoneuridae belongs to the superfamily Sericostomatoidea, characterized by the absence of ocelli, with the terminal max- illary and labial segments both being nonannulated and in- ﬂexible, and with the tibial spur formula being 2/2/4 (Johan- son et al., 2017). The family Dysoneuridae is distinguished from all other families of Sericostomatoidea by its wing ve- nation with the absence of forks I and IV in the forewings and of forks II and IV in the hind wings. C. elegans. Data availability. The material described herein is deposited in the amber collection of the Museum für Naturkunde, Berlin (inventory number MB.I.7340). Author contributions. WW and CN prepared the paper and con- tributed to the editing. The Dysoneuridae found in mid-Cretaceous Burmese am- ber consist of two interesting genera, Burmapsyche and Cre- tapsyche. Burmapsyche has maxillary palps which are three- segmented. In B. palpifurcata each palpus is forked into a one-segmented branch and a two-segmented branch, an un- usual small fork, unknown in modern caddisﬂies. The genus Cretapsyche is characterized by ﬁve-segmented maxillary palps, equipped with a remarkably long, slightly curved sec- ond segment. Description In- ferior appendages two-segmented, each consisting of cox- www.foss-rec.net/22/51/2019/ Foss. Rec., 22, 51–56, 2019 Foss. Rec., 22, 51–56, 2019 54 W. Wichard and C. Neumann: A new bizarre dysoneurid species 54 W. Wichard and C. Neumann: A new bizarre dysoneurid species Figure 2. Cretapsyche palpinova sp. nov. (a) Drawing of the scapi, pedicelli, and ﬁrst antennal segments; the ﬁve-segmented maxillary palps (max palp), the three-segmented labial palps (lab palp); and a pair of pilifers arising from the maxillary palps. (b) Head with palps and antennae in ventral view. (c) Male genitalia in ventral view. (d) Drawing of the male genitalia in ventral view with the inferior appendages (inf ap) consisting of the basal coxopodite and the apical harpago, with the preanal appendages (pre ap) and with tergum X divided in two apical lobes (X ap). Foss Rec 22 51 56 2019 www foss rec net/22/51/2019/ Figure 2. Cretapsyche palpinova sp. nov. (a) Drawing of the scapi, pedicelli, and ﬁrst antennal segments; the ﬁve-segmented maxillary palps (max palp), the three-segmented labial palps (lab palp); and a pair of pilifers arising from the maxillary palps. (b) Head with palps and antennae in ventral view. (c) Male genitalia in ventral view. (d) Drawing of the male genitalia in ventral view with the inferior appendages (inf ap) consisting of the basal coxopodite and the apical harpago, with the preanal appendages (pre ap) and with tergum X divided in two apical lobes (X ap). Figure 2. Cretapsyche palpinova sp. nov. (a) Drawing of the scapi, pedicelli, and ﬁrst antennal segments; the ﬁve-segmented maxillary palps (max palp), the three-segmented labial palps (lab palp); and a pair of pilifers arising from the maxillary palps. (b) Head with palps and antennae in ventral view. (c) Male genitalia in ventral view. (d) Drawing of the male genitalia in ventral view with the inferior appendages (inf ap) consisting of the basal coxopodite and the apical harpago, with the preanal appendages (pre ap) and with tergum X divided in two apical lobes (X ap). Foss. Rec., 22, 51–56, 2019 www.foss-rec.net/22/51/2019/ 55 W. Wichard and C. Neumann: A new bizarre dysoneurid species 4 Key to species of genus Cretapsyche from Cretaceous Burmese amber opodite and harpago; small subtriangular accessory ap- pendage basoventrally; coxopodite strong, about 0.2 mm in length, about as long as harpago; harpago originating from coxopodite, basally narrow, thicker mesally near middle, and tapered apically; curved mesad, enclosing central phallic ap- paratus. Tergum X with mesodorsal lobe divided apparently into two small parallel processes covering phallic appara- tus dorsally. In ventral view dorsolateral preanal appendages also deeply behind central phallic apparatus, each forming small, elongate, triangular spatula. (Family and genus characters: ﬁve-segmented maxillary palps each with second segment longest; in forewing forks II, III, and V and in hind wing forks I, III, and V present; tibial spurs 2/2/4). 1. In forewing, Rs and M branching on about the same level, fork V petiolate; in male genitalia coxopodite and harpago at a ratio of about 1 : 1 in length: 2 – In forewing, M branching before Rs, fork V nearly sessile; in male genitalia coxopodite and harpago at a ratio of about 2 : 1 in length: 3 3 Discussion Additionally, in Cretapsyche palpinova a pin- shaped appendage originates from the third segment of each maxillary palpus that slightly towers above the palpus dis- tally. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. We especially thank Patrick Müller for the ex- cellent collection of Burmese amber inclusions and for making this newly described species available. Further, we wish to express our sincere gratitude to John Morse (Clemson University, USA) and Bo Wang (Academy of Sciences, Nanjing, China), who provided criti- cal reviews that helped in improving the present contribution of the manuscript. Review statement. This paper was edited by Florian Witzmann and reviewed by Bo Wang and John C. Morse. www.foss-rec.net/22/51/2019/ www.foss-rec.net/22/51/2019/ Foss. Rec., 22, 51–56, 2019 W. Wichard and C. Neumann: A new bizarre dysoneurid species 56 References www.foss-rec.net/22/51/2019/ Foss. Rec., 22, 51–56, 2019 References Shi, G., Grimaldi, D. A., Harlow, G. E., Wang, J., Wang, J., Yang, M., Lei, W., Li, Q., and Li, X.: Age constraint on Burmese amber based on U-Pb dating of zircons, Cretaceous Res., 37, 155–163, 2012. Holzenthal, R. W., Blahnik, R. J., Prather, A. L., and Kjer, K. M.: 2007. Order Trichoptera Kirby, 1813 (Insecta), Caddisﬂies, Zootaxa, 1668, 639–698, 2004. Sukatsheva, I. D.: New Jurassic Caddisﬂies from the Kara Tau (Tri- choptera), Yurskie nasekomye Karatau, 175–179, 1968 (in Rus- sian). Jarzembowski, E. A., Wang, B., and Zheng, D.: A new ommatin beetle (Insecta: Coleoptera) with unusual genitalia from mid- Cretaceous Burmese amber, Cretaceous Res., 71, 113–117, 2017 Sukatsheva, I. D. and Vassilenko, D. V.: New Taxa of Caddisﬂies (Insecta, Trichoptera) with reduced forewing venation from the Mesozoic of Asia, Paleontologicheskii Zh., 2013, 57–63, 2013. Johanson, K. A., Malm, T., and Espeland, M.: Molecular phylogeny of Sericostomatoidea (Trichoptera) with the establishment of three new families, Syst. Entomol., 42, 240–266, 2017. Ulmer, G.: Trichopteren des Baltischen Bernsteins, Beiträge zur Naturkunde Preussens, 10, 1–380, 1912. Kania, I., Wang, B., and Szwedo, J.: Dicranoptycha Osten Sacken, 1860 (Diptera, Limoniidae) from the earliest Upper Cretaceous Burmese amber, Cretaceous Res., 52, 522–530, 2015. Wichard, W.: Overview and descriptions of Trichoptera in Baltic Amber: Spicipalpia and Integripalpia, Kessel, Remagen, Ger- many, 230 pp., 2013. Kirby, W.: Strepsiptera, a new order of insects proposed, and the characters of the order, with those of its genera, Transactions of the Linnean Society of London Zoology, 11, 86–122, 1813. Wichard, W., Neumann, C., Müller, P., and Wang, B.: Family Dysoneuridae (Insecta, Trichoptera) in Cretaceous Burmese am- ber, Cretaceous Res., 82, 138–146, 2018. Martynov, A. V.: Rucheiniki (caddisﬂies), Prakticheskaya Ento- mologiya, 5, 1–384, 1924 (in Russian). Foss. Rec., 22, 51–56, 2019 www.foss-rec.net/22/51/2019/ www.foss-rec.net/22/51/2019/
https://openalex.org/W2952777086	http://cds.cern.ch/record/687891/files/0306087v1.pdf	English	null	Reconstruction of electrons with the Gaussian-sum filter in the CMS tracker at the LHC	Journal of physics. G, Nuclear and particle physics	2,005	cc-by	4,906	1. Introduction Modern track detectors based on semiconductor technologies contain larger amounts of material than gaseous detector types, partially due to the detector elements themselves and partially due to additional material required for on-sensor electronics, power, cooling, and mechanical support. A precise mod- elling of material eﬀects in track reconstruction is therefore necessary to obtain the best estimates of the track parameters. Such material eﬀects are par- ticularly relevant for the reconstruction of electrons which, in addition to ionization energy loss and multi- ple Coulomb scattering, suﬀer from large energy losses due to bremsstrahlung. arXiv:physics/0306087v1 [physics.d Figure 1: Probability density function f(z) for diﬀerent thickness values. A well-known model of the bremsstrahlung energy loss is due to Bethe and Heitler [1]. In this model, the probability density function (PDF), f(z), of the energy loss of an electron is the mean value of energy loss and to increase the vari- ance of the momentum by adding the variance of the energy loss distribution. This procedure should en- sure unbiased estimates of the track parameters and of the associated uncertainties [3]. The Kalman ﬁlter is a linear least-squares estimator, and is proved to be optimal only when all probability densities encoun- tered during the track reconstruction procedure are Gaussian. The implicit assumption of approximating the Bethe-Heitler distribution with a single Gaussian is quite crude. It is therefore plausible that a non- linear estimator which takes the actual shape of the distribution into account can do better. the mean value of energy loss and to increase the vari- ance of the momentum by adding the variance of the energy loss distribution. This procedure should en- sure unbiased estimates of the track parameters and of the associated uncertainties [3]. The Kalman ﬁlter is a linear least-squares estimator, and is proved to be optimal only when all probability densities encoun- tered during the track reconstruction procedure are Gaussian. The implicit assumption of approximating the Bethe-Heitler distribution with a single Gaussian is quite crude. It is therefore plausible that a non- linear estimator which takes the actual shape of the distribution into account can do better. f(z) = [−ln z]c−1 Γ(c) , (1) (1) where c = t/ ln 2, t is the thickness of material tra- versed by the electron (in units of radiation length), and z is the fraction of energy remaining after the material layer is traversed. Reconstruction of electrons with the Gaussian-sum ﬁlter in the CMS tracker at LHC 0306087v1 [physics.data-an] 11 Jun 2003 arXiv:physics/0306087v1 [physics.data-an] 11 Jun 2003 The bremsstrahlung energy loss distribution of electrons propagating in matter is highly non Gaussian. Because the Kalman ﬁlter relies solely on Gaussian probability density functions, it might not be an optimal reconstruc- tion algorithm for electron tracks. A Gaussian-sum ﬁlter (GSF) algorithm for electron track reconstruction in the CMS tracker has therefore been developed. The basic idea is to model the bremsstrahlung energy loss dis- tribution by a Gaussian mixture rather than a single Gaussian. It is shown that the GSF is able to improve the momentum resolution of electrons compared to the standard Kalman ﬁlter. The momentum resolution and the quality of the estimated error are studied with various types of mixture models of the energy loss distribution. iv:physics/0306087v1 [physics.data-an] 11 Ju 0 0.2 0.4 0.6 0.8 1 10 −2 10 −1 10 0 10 1 10 2 z f(z) t=0.2 t=0.1 t=0.05 t=0.02 Figure 1: Probability density function f(z) for diﬀerent thickness values. 1. Introduction The probability of a given fractional energy loss is assumed to be independent of the energy of the incoming particle. This PDF is shown in Fig. 1 for diﬀerent thickness values. A non-linear generalization of the Kalman ﬁlter (KF), the Gaussian-sum ﬁlter (GSF) [4, 5], has there- fore been implemented in the reconstruction software of the CMS tracker [6]. In the GSF the distributions of all state vectors are Gaussian mixtures, i.e. weighted sums of Gaussians instead of single Gaussians. The algorithm is therefore appropriate if the probability densities involved in track reconstruction can be ad- equately described by Gaussian mixtures. The basic The baseline for track reconstruction in the CMS tracker is the Kalman ﬁlter [2]. Throughout the ﬁlter tracks are described by a ﬁve-dimensional state vec- tor, containing the information about the momentum, the direction and the position at some reference sur- face. The material eﬀects are currently assumed to be concentrated in the active elements of the detector layers. In this context the optimal treatment of ra- diative energy loss is to correct the momentum with CHEP03, La Jolla, California, March 24-28, 2003 CHEP03, La Jolla, California, March 24-28, 2003 1 Reconstruction of electrons with the Gaussian-sum ﬁl tracker at LHC W. Adam, R. Fr¨uhwirth Institute for High-Energy Physics, Austrian Academy of Sciences, Vienna, Austria A. Strandlie CERN, Geneva, Switzerland T. Todorov IReS, Strasbourg, France TULT009 CHEP03, La Jolla, California, March 24-28, 2003 2 0 0.05 0.1 0.15 0.2 0 1 2 3 4 5 6 7 8 t Dkl nc=2 nc=3 nc=4 nc=5 nc=6 0 0.05 0.1 0.15 0.2 0 0.005 0.01 0.015 0.02 0.025 t Dcdf Figure 2: The distances DKL and DCDF as a function of the thickness t, for CDF-mixtures, with diﬀerent numbers of components. 0 0.05 0.1 0.15 0.2 0 1 2 3 4 5 6 7 8 t Dkl nc=2 nc=3 nc=4 nc=5 nc=6 0 0.05 0.1 0.15 0.2 0 0.005 0.01 0.015 0.02 0.025 t Dcdf idea of the present work is to approximate the Bethe- Heitler distribution as a Gaussian mixture rather than a single Gaussian, in which the diﬀerent components of the mixture model diﬀerent degrees of hardness of the bremsstrahlung in the layer under consideration. The resulting estimator resembles a set of Kalman ﬁlters running in parallel, where each Kalman ﬁlter corresponds to one of the components of the mixture describing the distribution of the state vector. 2. Approximating the fractional energy loss distribution Figure 2: The distances DKL and DCDF as a function of the thickness t, for CDF-mixtures, with diﬀerent numbers of components. An important issue with the GSF reconstruction of electrons is to obtain a good Gaussian-mixture ap- proximation of the Bethe-Heitler distribution. The parameters to be obtained are the weights, the mean values and the variances of each of the components in the approximating mixture. The parameters are de- termined by minimizing the following two distances: 0 0.05 0.1 0.15 0.2 0 2 4 6 8 10 12 t Dkl nc=1 nc=2 nc=3 nc=4 nc=5 nc=6 0 0.05 0.1 0.15 0.2 0 0.02 0.04 0.06 0.08 0.1 0.12 t Dcdf Figure 3: The distances DKL and DCDF as a function of the thickness t, for KL-mixtures, with diﬀerent numbers of components. 0 0.05 0.1 0.15 0.2 0 2 4 6 8 10 12 t Dkl nc=1 nc=2 nc=3 nc=4 nc=5 nc=6 0 0.05 0.1 0.15 0.2 0 0.02 0.04 0.06 0.08 0.1 0.12 t Dcdf DCDF = Z ∞ −∞ \|F(z) −G(z)\| dz, (2) DKL = Z ∞ −∞ ln [f(z)/g(z)]f(z)dz, (3) (2) (3) where f(z) and F(z) are the PDF and cumulative distribution function (CDF) of the model distribution and g(z) and G(z) are the PDF and CDF of the Gaus- sian mixture, respectively. The distance DKL is the so-called Kullback-Leibler distance between the model distribution and the mixture. Hereafter, the mix- tures obtained by minimizing DCDF are called CDF- mixtures, whereas the mixtures obtained by minimiz- ing DKL are called KL-mixtures. The minimizations have been done independently on a set of discrete val- ues of t, ranging from 0.02 to 0.20. Figures 2 and 3 show the resulting distances as a function of thickness for a varying number of components in the approxi- mating mixture. In order to obtain mixtures for arbi- trary values of the thickness, ﬁfth-degree polynomials have been ﬁtted to the parameters as a function of t. Due to the fast access to the parameters from the polynomials, the calculation of the mixture is done on the ﬂy during reconstruction, using the eﬀective thickness of a detector layer from the knowledge of the incident angle of inclination. Figure 3: The distances DKL and DCDF as a function of the thickness t, for KL-mixtures, with diﬀerent numbers of components. 2. Components are merged into clusters, according to a given metric. 2. Components are merged into clusters, according to a given metric. The ﬁrst option has the advantage of being compu- tationally light, but it turns out to be inferior. Even the ﬁrst two moments of the estimated parameters are not described correctly. 2. Approximating the fractional energy loss distribution prohibitively large number of components due to the combinatorics involved each time a layer of material is traversed. In a realistic implementation of the GSF the num- ber of components must repeatedly be reduced to a predeﬁned maximum. As little information as possi- ble should be lost in this procedure. Two strategies have been tested: 1. Only the N components with the largest weights are kept; 1. Only the N components with the largest weights are kept; 3. Reducing the number of components In the second approach, the component with the largest weight is merged with the one closest to it, and this procedure is repeated until the required num- ber of components is reached. The results below have been obtained by using the Kullback-Leibler distance – deﬁned in Equation (3) – as a measure of distance. The approximation of energy loss by a Gaussian mixture amounts to a convolution of this mixture with the current state, which in general is also composed of several Gaussian components. The strict applica- tion of the GSF algorithm therefore quickly leads to a TULT009 CHEP03, La Jolla, California, March 24-28, 2003 3 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 300 350 400 450 KF GSF probability Figure 5: Probability distribution for the estimated q/p for the KF (solid) and the GSF with a maximum of six (dashed-dotted), twelve (dashed), 18 (solid) and 36 (dotted) components kept during reconstruction. In this case the same six-component CDF-mixture has been used both in the simulation of the disturbance of the momentum in a detector unit and in reconstruction. Keeping 36 components yields estimates quite close to the correct distribution of the parameter. 4. Results from simulated tracks in the CMS tracker 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 300 350 400 450 KF GSF probability 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 300 350 400 450 KF GSF probability First, results from the reconstruction of data origi- nating from a simpliﬁed simulation are shown. In this simulation multiple scattering and ionization energy loss are turned oﬀ, all the material is concentrated on the detector units, and the exact amount of material used in the simulation is known by the reconstruction program. Single electron tracks with pT = 10 GeV/c have been simulated for absolute values of η less than 1.0 . Reconstructed hits have been collected using the knowledge of the associated simulated hits, so no pattern recognition has been involved. The following results all refer to the quantity q/p (charge over abso- lute value of the momentum) recorded at the point of closest approach to the vertex in the transverse plane – the transverse impact point (TIP) – after a ﬁt go- ing from the outside towards the inside of the tracker. Figure 4 shows an example of the estimated q/p for one single track, both for the KF and for the GSF. Figure 5: Probability distribution for the estimated q/p for the KF (solid) and the GSF with a maximum of six (dashed-dotted), twelve (dashed), 18 (solid) and 36 (dotted) components kept during reconstruction. In this case the same six-component CDF-mixture has been used both in the simulation of the disturbance of the momentum in a detector unit and in reconstruction. Keeping 36 components yields estimates quite close to the correct distribution of the parameter. -0.14 -0.12 -0.1 -0.08 -0.06 -0.04 0 0.02 0.04 0.06 0.08 0.1 0.12 predicted PDF at TIP in q/p q/p in inverse GeV/c -0.14 -0.12 -0.1 -0.08 -0.06 -0.04 0 0.02 0.04 0.06 0.08 0.1 0.12 predicted PDF at TIP in q/p q/p in inverse GeV/c Figure 4: Estimated q/p of one single track for the GSF (solid), the KF (dashed) and the combined GSF state (dotted). The combined GSF state refers to the ﬁrst and the second moments of the GSF estimate, here visualized as a single Gaussian. The arrow denotes the true value of q/p. It can be seen that the estimated PDF of the GSF is a non-Gaussian function. 4. Results from simulated tracks in the CMS tracker The distributions for the GSF are seen to be less ﬂat than those shown in Fig. 5. ( ) ( ) (dotted). The combined GSF state refers to the ﬁrst and the second moments of the GSF estimate, here visualized as a single Gaussian. The arrow denotes the true value of q/p. It can be seen that the estimated PDF of the GSF is a non-Gaussian function. Figures 5 and 6 show probability distributions for the estimated q/p of the KF and the GSF with a varying maximum number of components kept dur- ing the reconstruction. Given the estimated PDF (a single Gaussian for the KF, a Gaussian mixture for the GSF), each entry in the histogram amounts to the integral from −∞to the true value of q/p. If the estimated PDF is a correct description of the real distribution of the parameter, the corresponding his- togram should be ﬂat. The CDF-mixtures are superior to the KL-mixtures concerning the quality of the estimated q/p. The main trend seems to be related to the maximum number of components kept rather than the number of compo- nents in the mixture describing the energy loss, even though the mixtures with ﬁve and six components are best in the limit of keeping a large number of compo- nents. g The deviation from ﬂatness can be quantiﬁed by the χ2 of the diﬀerence between the probability distribu- tions of q/p and the ﬂat distribution. This χ2 per bin is shown in Fig. 7 for a set of diﬀerent mixtures as a function of the maximum number of components kept. Figure 8 shows the residuals of the estimated q/p of the GSF and the KF with respect to the true value of TULT009 4. Results from simulated tracks in the CMS tracker 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 300 350 400 450 KF GSF probability Figure 6: Probability distribution for the estimated q/p for the KF (solid) and the GSF with a maximum of six (dashed-dotted), twelve (dashed), 18 (solid) and 36 (dotted) components kept during reconstruction. The same six-component mixture as the one described in the caption of Fig. 5 has been used in reconstruction, but the simulation of the disturbance of the momentum in a detector unit has been done by sampling from the Bethe-Heitler distribution. The distributions for the GSF are seen to be less ﬂat than those shown in Fig. 5. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 300 350 400 450 KF GSF probability Figure 4: Estimated q/p of one single track for the GSF (solid), the KF (dashed) and the combined GSF state (dotted). The combined GSF state refers to the ﬁrst and the second moments of the GSF estimate, here visualized as a single Gaussian. The arrow denotes the true value of q/p. It can be seen that the estimated PDF of the GSF is a non-Gaussian function. Figure 4: Estimated q/p of one single track for the GSF (solid), the KF (dashed) and the combined GSF state Figure 6: Probability distribution for the estimated q/p for the KF (solid) and the GSF with a maximum of six (dashed-dotted), twelve (dashed), 18 (solid) and 36 (dotted) components kept during reconstruction. The same six-component mixture as the one described in the caption of Fig. 5 has been used in reconstruction, but the simulation of the disturbance of the momentum in a detector unit has been done by sampling from the Bethe-Heitler distribution. The distributions for the GSF are seen to be less ﬂat than those shown in Fig. 5. Figure 6: Probability distribution for the estimated q/p for the KF (solid) and the GSF with a maximum of six (dashed-dotted), twelve (dashed), 18 (solid) and 36 (dotted) components kept during reconstruction. The same six-component mixture as the one described in the caption of Fig. 5 has been used in reconstruction, but the simulation of the disturbance of the momentum in a detector unit has been done by sampling from the Bethe-Heitler distribution. TULT009 CHEP03, La Jolla, California, March 24-28, 2003 4 Maximum number of components 5 10 15 20 25 30 35 0 20 40 60 80 100 120 χ2/ndof w.r.t. flat distribution Figure 7: Values of χ2 per bin of the probability distribution for CDF-mixtures with four (circles), ﬁve (squares) and six (triangles pointing upwards) components, as well as a KL-mixture with six (triangles pointing downwards) components. The corresponding value for the KF is 146. Maximum number of components 5 10 15 20 25 30 35 0.004 0.0045 0.005 0.0055 0.006 0.0065 0.007 0.0075 0.008 FWHM (∆q/p) (GeV/c)-1 Figure 9: Full-width at half-maximum for the GSF as a function of the maximum number of components kept during reconstruction. The corresponding value of the KF is 0.013. Maximum number of components 5 10 15 20 25 30 35 0.004 0.0045 0.005 0.0055 0.006 0.0065 0.007 0.0075 0.008 FWHM (∆q/p) (GeV/c)-1 Figure 9: Full-width at half-maximum for the GSF as a function of the maximum number of components kept during reconstruction. The corresponding value of the KF is 0.013. Maximum number of components 5 10 15 20 25 30 35 0 20 40 60 80 100 120 χ2/ndof w.r.t. flat distribution Figure 7: Values of χ2 per bin of the probability distribution for CDF-mixtures with four (circles), ﬁve (squares) and six (triangles pointing upwards) components, as well as a KL-mixture with six (triangles pointing downwards) components. The corresponding value for the KF is 146. Maximum number of components 5 10 15 20 25 30 35 0 20 40 60 80 100 120 χ2/ndof w.r.t. flat distribution Maximum number of components 5 10 15 20 25 30 35 0.004 0.0045 0.005 0.0055 0.006 0.0065 0.007 0.0075 0.008 FWHM (∆q/p) (GeV/c)-1 Figure 9: Full-width at half-maximum for the GSF as a function of the maximum number of components kept during reconstruction. The corresponding value of the KF is 0.013. Figure 7: Values of χ2 per bin of the probability distribution for CDF-mixtures with four (circles), ﬁve (squares) and six (triangles pointing upwards) components, as well as a KL-mixture with six (triangles pointing downwards) components. The corresponding value for the KF is 146. TULT009 Maximum number of components 5 10 15 20 25 30 35 0.006 0.0061 0.0062 0.0063 0.0064 0.0065 0.0066 0.0067 0.0068 0.0069 0.007 half width of interval (GeV/c)-1 50% coverage Figure 10: Half-widths of the symmetric intervals covering 50% of the distribution for the GSF as a function of the maximum number of components kept during reconstruction. The corresponding value of the KF is 0.0080. Maximum number of components 5 10 15 20 25 30 35 0.006 0.0061 0.0062 0.0063 0.0064 0.0065 0.0066 0.0067 0.0068 0.0069 0.007 half width of interval (GeV/c)-1 50% coverage the parameter. The estimated q/p for the GSF is the mean value of the state vector mixture, and the mix- ture used for this speciﬁc plot is a CDF-mixture with six components. In order to quantify the diﬀerence between the GSF and the KF residuals, the full-width at half-maximum (FWHM) and the half-width of in- tervals covering 50% and 90% of the distribution have been considered. The covering intervals have been chosen to be symmetric about zero. The FWHM and the half-widths of the covering intervals are shown in Figs. 9, 10 and 11. The diﬀerent ﬂavours of the GSF in these ﬁgures are the same as those described in the caption of Fig. 7. Figure 10: Half-widths of the symmetric intervals covering 50% of the distribution for the GSF as a function of the maximum number of components kept during reconstruction. The corresponding value of the KF is 0.0080. gsfRes Entries 9847 Mean 0.003567 RMS 0.008471 Underflow 764 Overflow 69 Residuals of q/p (in inverse GeV/c) -0.03 -0.02 -0.01 0 0.01 0.02 0.03 0 100 200 300 400 500 600 GSF residuals Entries 9847 Mean 0.003567 RMS 0.008471 Underflow 764 Overflow 69 GSF KF Figure 8: Residuals of estimated q/p with respect to the true value of the parameter for the GSF and the KF at the transverse impact point. A maximum number of twelve components has been kept during reconstruction. Long tails extending outside the limits of the histogram exist both for the KF and for the GSF. These tails are due to hard radiation in the innermost layers of the tracker. TULT009 gsfRes Entries 8988 Mean 0.000924 RMS 0.006759 Underflow 181 Overflow 169 Residuals of q/p with vertex constraint (in inverse GeV/c) -0.03 -0.02 -0.01 0 0.01 0.02 0.03 0 100 200 300 400 500 600 700 GSF residuals Entries 8988 Mean 0.000924 RMS 0.006759 Underflow 181 Overflow 169 GSF KF ability distribution of the GSF exhibits no large de- viation from ﬂatness, indicating that the estimated PDF of q/p describes reasonably well the actual PDF of q/p. This observation is all the more remarkable since, with the full simulation, the energy loss is not generated by the simple Bethe-Heitler model, and nei- ther the exact amount nor the exact location of the material are known to the GSF. Residuals of q/p with vertex constraint (in inverse GeV/c) 0.02 -0.01 0 0.01 0.02 0.03 Figure 14: Residuals of the estimated q/p with respect to the true value at the transverse impact point for the KF and the GSF. A vertex constraint has been included in the ﬁts. The reconstruction algorithms have been run on tracks from a full simulation. The corresponding residuals of the estimated q/p with respect to the true value are shown in Figs. 13 and 14. The residuals shown in Fig. 14 have been obtained by including a vertex constraint in the ﬁt. Such a constraint allows the momentum to be mea- sured in the innermost part of the track and thus gives a handle on possible radiation in the ﬁrst two layers. The result of including this constraint is a less skew distribution with the mode being moved closer towards zero, and the amount of tracks in the tails is also reduced. Even though the results from the full simulation qualitatively seem to conﬁrm those from the simpliﬁed simulation, more studies are needed to understand the diﬀerences in detail. location are known to the reconstruction program. It has been shown that the quality of the momentum estimate depends mainly on the number of mixture components kept during reconstruction, and to some extent also on the number of components in the mix- ture approximation to the energy loss distribution. A comparison with the best linear unbiased estima- tor, the Kalman ﬁlter, shows a clear improvement of the momentum resolution. TULT009 Remarkably, a similar im- provement can be seen with electron tracks from the full simulation, although in this case neither the exact energy loss distribution nor the precise amount and location of material are known to the reconstruction program. More systematic studies with electrons from the full simulation are clearly needed, but it seems safe to conclude that in electron reconstruction the Gaussian-sum ﬁlter yields a substantial gain in preci- sion as compared to the Kalman ﬁlter. TULT009 gsfRes Entries 9847 Mean 0.003567 RMS 0.008471 Underflow 764 Overflow 69 Residuals of q/p (in inverse GeV/c) -0.03 -0.02 -0.01 0 0.01 0.02 0.03 0 100 200 300 400 500 600 GSF residuals Entries 9847 Mean 0.003567 RMS 0.008471 Underflow 764 Overflow 69 GSF KF Maximum number of components 5 10 15 20 25 30 35 0.024 0.0242 0.0244 0.0246 0.0248 0.025 0.0252 0.0254 0.0256 0.0258 0.026 half width of interval (GeV/c)-1 90% coverage Figure 11: Half-widths of the symmetric intervals covering 90% of the distribution for the GSF as a function of the maximum number of components kept during reconstruction. The corresponding value of the KF is 0.0295. Maximum number of components 5 10 15 20 25 30 35 0.024 0.0242 0.0244 0.0246 0.0248 0.025 0.0252 0.0254 0.0256 0.0258 0.026 half width of interval (GeV/c)-1 90% coverage Residuals of q/p (in inverse GeV/c) 0 0.01 0.02 0.03 Figure 8: Residuals of estimated q/p with respect to the true value of the parameter for the GSF and the KF at the transverse impact point. A maximum number of twelve components has been kept during reconstruction. Long tails extending outside the limits of the histogram exist both for the KF and for the GSF. These tails are due to hard radiation in the innermost layers of the tracker. Figure 11: Half-widths of the symmetric intervals covering 90% of the distribution for the GSF as a function of the maximum number of components kept during reconstruction. The corresponding value of the KF is 0.0295. The GSF and the KF have also been run on tracks from a full simulation using the oﬃcial CMS simula- TULT009 CHEP03, La Jolla, California, March 24-28, 2003 5 gsfRes Entries 8988 Mean 0.002396 RMS 0.007577 Underflow 481 Overflow 320 Residuals of q/p (in inverse GeV/c) -0.03 -0.02 -0.01 0 0.01 0.02 0.03 0 100 200 300 400 500 600 GSF residuals Entries 8988 Mean 0.002396 RMS 0.007577 Underflow 481 Overflow 320 GSF KF Figure 13: Residuals of the estimated q/p with respect to the true value at the transverse impact point for the KF and the GSF. The reconstruction algorithms have been run on tracks from a full simulation. TULT009 gsfRes Entries 8988 Mean 0.002396 RMS 0.007577 Underflow 481 Overflow 320 Residuals of q/p (in inverse GeV/c) -0.03 -0.02 -0.01 0 0.01 0.02 0.03 0 100 200 300 400 500 600 GSF residuals Entries 8988 Mean 0.002396 RMS 0.007577 Underflow 481 Overflow 320 GSF KF tion program [7]. The pT and the η range are the same as in the simpliﬁed simulation, but the amount and spatial distribution of the material are diﬀerent. Probability distributions of the estimated q/p for the GSF and the KF are shown in Fig. 12. The prob- 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 300 350 400 450 probability Figure 12: Probability distribution for the estimated q/p for the KF (dashed) and the GSF (solid). The speciﬁc mixture used in the GSF is a CDF-mixture with six components. A maximum number of twelve components has been kept during reconstruction. The reconstruction algorithms have been run on tracks from a full simulation. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 50 100 150 200 250 300 350 400 450 probability Figure 13: Residuals of the estimated q/p with respect to the true value at the transverse impact point for the KF and the GSF. The reconstruction algorithms have been run on tracks from a full simulation. Figure 12: Probability distribution for the estimated q/p for the KF (dashed) and the GSF (solid). The speciﬁc mixture used in the GSF is a CDF-mixture with six components. A maximum number of twelve components has been kept during reconstruction. The reconstruction algorithms have been run on tracks from a full simulation. gsfRes Entries 8988 Mean 0.000924 RMS 0.006759 Underflow 181 Overflow 169 Residuals of q/p with vertex constraint (in inverse GeV/c) -0.03 -0.02 -0.01 0 0.01 0.02 0.03 0 100 200 300 400 500 600 700 GSF residuals Entries 8988 Mean 0.000924 RMS 0.006759 Underflow 181 Overflow 169 GSF KF Figure 14: Residuals of the estimated q/p with respect to the true value at the transverse impact point for the KF and the GSF. A vertex constraint has been included in the ﬁts. The reconstruction algorithms have been run on tracks from a full simulation. 5. Conclusion The Gaussian-sum ﬁlter has been implemented in the CMS reconstruction program. It has been vali- dated with electron tracks with a simpliﬁed simulation in which the energy loss distribution (Bethe-Heitler model), the exact amount of material and its exact TULT009 TULT009 CHEP03, La Jolla, California, March 24-28, 2003 6 CHEP03, La Jolla, California, March 24-28, 2003 References [1] H. Bethe and W. Heitler, Proc. R. Soc. London A 146 (1934) 83. [2] R. Fr¨uhwirth, Nucl. Instrum. and Methods A 262 (1987) 444. [3] D. Stampfer, M. Regler and R. Fr¨uhwirth, Comp. Phys. Comm. 79 (1994) 157. [4] R. Fr¨uhwirth, Comp. Phys. Comm. 100 (1997) 1. [5] R. Fr¨uhwirth and S. Fr¨uhwirth-Schnatter, Comp. Phys. Comm. 110 (1998) 80. [6] http://cmsdoc.cern.ch/orca/ [7] http://cmsdoc.cern.ch/cmsim/cmsim.html References Phys. Comm. 79 (1994) 157. [4] R. Fr¨uhwirth, Comp. Phys. Comm. 100 (1997) 1. [1] H. Bethe and W. Heitler, Proc. R. Soc. London A 146 (1934) 83. ( ) [2] R. Fr¨uhwirth, Nucl. Instrum. and Methods A 262 (1987) 444. ( ) [2] R. Fr¨uhwirth, Nucl. Instrum. and Methods A 262 (1987) 444. ( ) [3] D. Stampfer, M. Regler and R. Fr¨uhwirth, Comp. [3] D. Stampfer, M. Regler and R. Fr¨uhwirth, Comp. TULT009
https://openalex.org/W2044830738	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0110740&type=printable	English	null	Whole Exome Sequencing Identifies Novel Genes for Fetal Hemoglobin Response to Hydroxyurea in Children with Sickle Cell Anemia	PloS one	2,014	cc-by	5,581	Abstract Funding: This work was supported by the National Human Genome Research Institute (U54-HGOO3273)(EB, RAG), National Heart, Lung and Blood Institute (U01- HL078787, R01-HL090941)(REW), Doris Duke Charitable Foundation (2010036)(REW), and the Russell and Diana Hawkins Family Foundation Discovery Fellowship (JRC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: vsheehan@bcm.edu approved pharmacologic treatment for induction of HbF in adult patients with SCA, and is approved by the European Medicines Agency for both children and adults with SCA. Hydroxyurea significantly reduces pain and acute chest episodes, the need for blood transfusions and hospitalizations, and most importantly, reduces mortality [4–6]. While hydroxyurea has suspected disease modulating effects outside of HbF induction, the majority of its benefit is directly related to the amount of HbF produced in response to the drug [7,8]. There is an inverse relationship between levels of drug-induced HbF and number of pain episodes, hospitalizations, and overall mortality [9,10]. Abstract Hydroxyurea has proven efficacy in children and adults with sickle cell anemia (SCA), but with considerable inter-individual variability in the amount of fetal hemoglobin (HbF) produced. Sibling and twin studies indicate that some of that drug response variation is heritable. To test the hypothesis that genetic modifiers influence pharmacological induction of HbF, we investigated phenotype-genotype associations using whole exome sequencing of children with SCA treated prospectively with hydroxyurea to maximum tolerated dose (MTD). We analyzed 171 unrelated patients enrolled in two prospective clinical trials, all treated with dose escalation to MTD. We examined two MTD drug response phenotypes: HbF (final %HbF minus baseline %HbF), and final %HbF. Analyzing individual genetic variants, we identified multiple low frequency and common variants associated with HbF induction by hydroxyurea. A validation cohort of 130 pediatric sickle cell patients treated to MTD with hydroxyurea was genotyped for 13 non-synonymous variants with the strongest association with HbF response to hydroxyurea in the discovery cohort. A coding variant in Spalt-like transcription factor, or SALL2, was associated with higher final HbF in this second independent replication sample and SALL2 represents an outstanding novel candidate gene for further investigation. These findings may help focus future functional studies and provide new insights into the pharmacological HbF upregulation by hydroxyurea in patients with SCA. Editor: Wilbur Lam, Emory University/Georgia Insititute of Technology, United States of America Received July 17, 2014; Accepted September 15, 2014; Published October 31, 2014 Copyright: 2014 Sheehan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Sheehan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All 171 exome sequence files are available from dbGaP(phs000691.v1.p1). Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All 171 exome s from dbGaP(phs000691.v1.p1). Whole Exome Sequencing Identifies Novel Genes for Fetal Hemoglobin Response to Hydroxyurea in Children with Sickle Cell Anemia Vivien A. Sheehan1*, Jacy R. Crosby2,3, Aniko Sabo4, Nicole A. Mortier5, Thad A. Howard5, Donna M. Muzny4, Shannon Dugan-Perez4, Banu Aygun6, Kerri A. Nottage7, Eric Boerwinkle3,4, Richard A. Gibbs4, Russell E. Ware5, Jonathan M. Flanagan1 1 Hematology Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America, 2 The University of Texas Graduate School of Biomedical Sciences at Houston, Department of Biostatistics, Bioinformatics, and Systems Biology, University of Texas, Houston, Texas, United States of America, 3 Human Genetics Center, University of Texas, Houston, Texas, United States of America, 4 Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, United States of America, 5 Division of Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 6 Steven and Alexandra Cohen Children’s Medical Center of New York, New Hyde Park, New York, United States of America, 7 Department of Hematology, St. Jude Children’s Research Hospital, Memphis, Tennessee, United States of America October 2014 \| Volume 9 \| Issue 10 \| e110740 Introduction Sickle cell anemia (SCA) is an inherited blood disorder, affecting 1 in 400 African Americans, causing significant morbidity and mortality. Although a monogenic disease, individuals with SCA (usually homozygous HbSS) exhibit wide variability in their laboratory and clinical phenotypes. One of the most powerful and reproducible modifiers of disease severity is an individual’s endogenous level of fetal hemoglobin (HbF) [1]. If produced in sufficient amounts, HbF is able to prevent the intracellular polymerization of deoxygenated sickle hemoglobin (HbS), which is the nidus of the clinical disease process [2,3]. Pharmacologic induction of HbF is clinically beneficial, and the most widely used and safest method for increasing HbF levels in patients with SCA is treatment with hydroxyurea. Currently, it is the only FDA- Several clinical studies have shown that individual hematolog- ical responses to hydroxyurea treatment are highly variable, with induced HbF levels ranging from 10% to greater than 30% HbF even for compliant patients on similar dosing regimens [11–14]. October 2014 \| Volume 9 \| Issue 10 \| e110740 October 2014 \| Volume 9 \| Issue 10 \| e110740 1 PLOS ONE \| www.plosone.org Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Previous efforts to identify predictors associated with final HbF produced in response to hydroxyurea have identified higher baseline HbF values, higher white blood cell count (WBC), and absolute reticulocyte count (ARC) as important factors [13,15,16]. However, none of these parameters can accurately predict the degree of HbF induction by hydroxyurea in an individual patient. From analysis of sibling pairs, it is known that the degree of HbF induction by hydroxyurea has a strong heritable component [17], indicating that genetic modifiers may have a large effect on drug response. Identification of specific genetic variants associated with HbF induction may elucidate reasons for this phenotypic variability and provide new insights into the drug’s mechanisms of action related to HbF induction. Introduction Discovery Cohort Validation Cohort Discovery Cohort Validation Cohort Age (years) 10.4 64.5 8.1 64.0 - - WBC (x109/L) 13.5 64.2 13.1 65.7 6.5 62.0 6.9 62.0 ANC (x109/L) 7.2 63.2 6.2 64.3 3.0 61.2 2.7 60.9 Hb (g/dL) 9.1 60.9 8.0 61.7 9.4 61.0 9.5 61.3 ARC (x109/L) 0.28 60.13 0.29 60.12 0.17 60.09 0.12 60.05 MCV (fL) 86.1 64.8 82.1 68.3 116.4 613.0 97.7 611.1 HbF (%) 8.0 64.9 11.9 65.9 27.6 67.3 25.8 68.2 DHbF (%) - - 19.5 66.6 13.9 +7.0 HU dose (mg/kg/day) - - 25.1 64.5 27.1 64.3 WBC: white blood cell count; ANC: absolute neutrophil count; ARC: absolute reticulocyte count; MCV: mean corpuscular volume; HU: hydroxyurea. The discovery cohort was composed of 120 patients from HUSTLE and 51 from SWiTCH. The validation cohort was collected from patients treated at TCCH. doi:10.1371/journal.pone.0110740.t001 October 2014 \| Volume 9 \| Issue 10 The aim of this study was to use a whole exome sequencing (WES) pharmacogenomics approach to identify genetic predictors of HbF response to hydroxyurea. Using two prospective pediatric cohorts with robust HbF phenotype data and standardized dose escalation regimen to MTD as a discovery cohort, we undertook a novel unbiased screen to test the entire exome for variants that are associated with hydroxyurea-induced HbF response levels (as measured by maximum %HbF at MTD [final HbF] or the change in %HbF from baseline to final [DHbF]). We focused on genetic variants with predicted functional effects on protein coding regions and identified several non-synonymous mutations that may influence the HbF response to hydroxyurea in children with SCA. We then validated a coding variant in SALL2 in an unrelated, ‘‘real-world’’ cohort of children treated with hydroxy- urea. Patient characteristics Overall, both cohorts showed robust response to hydroxyurea with evidence of substantial individual variability in drug response (Table 1). All discovery cohort samples were genotyped for variants in BCL11A (rs1427407, rs4671393, rs11886868, rs7599488) and HBSIL-MYB (rs9399137, rs9402686); we found an association with baseline HbF for all BCL11A variants tested other than rs7599488. There was no significant association between the BCL11A variants tested and final HbF. No association with baseline, final or HbF was seen for either HBSIL-MYB variants tested. Linear association was performed with BCL11A variants as a covariate, without a significant change in associations. At the time of hydroxyurea initiation, the average age of the 171 patients in the discovery cohort was 10.464.5 years of age. The average age of the 130 patients in the validation cohort was 8.164.0 years. All patients were treated under a similar dose escalation to MTD regimen according to protocol, or similar institutional guidelines 18, 19. After a minimum of 6 months on hydroxyurea therapy, all patients reached a stable MTD (average 25.164.5 mg/kg/day in the discovery cohort, 27.164.3 mg/kg/ day in the validation cohort) with predictable laboratory benefits (Table 1). The mean increase in HbF was 19.566.6% in the discovery cohort and 13.967.0% in the validation cohort, reflecting slight differences between the two groups of patients. There was evidence of consistent myelosuppression across both cohorts, however. The baseline HbF, distribution of DHbF at MTD and final HbF at MTD were all similar to prior reports (Figure 1, A–C). [15,20]. Whole exome sequencing All 171 samples in the discovery cohort passed stringent WES quality control parameters with an average of 92% of the targeted PLOS ONE \| www.plosone.org 2 Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Figure 1. Comparison of discovery and validation cohorts. A, Baseline, or endogenous HbF for the discovery cohort is shown in binned histogram, and distribution of baseline HbF in validation cohort by a line plot. B, Delta HbF for the discovery cohort is shown in binned histogram, and distribution of delta HbF in validation cohort by a line plot. C, Final, or MID HbF for the discovery cohort is shown in binned histogram, and distribution of final, or MID in validation cohort by a line plot. doi:10.1371/journal.pone.0110740.g001 Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Figure 1. Comparison of discovery and validation cohorts. A, Baseline, or endogenous HbF for the discovery cohort is shown in binned histogram, and distribution of baseline HbF in validation cohort by a line plot. B, Delta HbF for the discovery cohort is shown in binned histogram, and distribution of delta HbF in validation cohort by a line plot. C, Final, or MID HbF for the discovery cohort is shown in binned histogram, and distribution of final, or MID in validation cohort by a line plot. doi:10.1371/journal.pone.0110740.g001 drug response by performing burden analysis with SKAT and T2 tests using the DHbF and final HbF phenotypes, but none of the gene level p-values were significant. exonic regions sequenced at greater than 206 coverage per individual. We identified a total of 278,639 autosomal variants, and 127,238 of these variants were non-synonymous or splice site variants expected to introduce an amino acid change in their encoded proteins (Table S1). For single variant association testing, we further filtered the non-synonymous variants (n = 127,238) for those with a minor allele frequency (MAF) greater than or equal to 2% (n = 38,012). We corrected for any population stratification using principal component analysis (PCA) performed by the EIGENSTRAT method. We identified 12 variants associated with DHbF with a p-value less than 561024 (Table 2). In addition, we identified 13 variants associated with final HbF, also with a p-value less than 561024 (Table 3). Although none of the p-values achieved genome wide significance level (p,1.3X1026), these results offered suggestive signals of potential associations. October 2014 \| Volume 9 \| Issue 10 \| e110740 Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Gene SNP ID Amino Acid Change Protein Function Prediction MAF (%) Function Beta Value SE P-value EML1 rs141631682 Gly109Asp Damaging 2 Microtubule assembly 10.9 2.7 7.4161025 SESN1 rs2273668 Leu103Ile Damaging 2 Peroxiredoxin reduction 9.3 2.3 8.8861025 PAPLN rs17126352 Val416Ile Benign 3 Metalloprotease 7.1 1.8 1.3561024 DCHS2 rs61746132 Pro1676Lys Damaging 12 Calcium dependent cell adhesion 3.9 1.0 1.7361024 KRT80 rs61749462 Arg364Ser Damaging 3 Keratin 80 8.0 2.1 1.8061024 SALL2 rs61743453 Pro840Arg Damaging 4 Transcription factor 6.7 1.8 2.3761024 NOM1 rs61742645 Arg779Cys Benign 2 PP1 interacting protein 8.7 2.3 2.4361024 N4BP2L2 rs35108810 Asp246Val Damaging 15 ELA2 transcription inhibitor 23.3 0.9 3.4761024 RNF113B rs16955011 Val92Met Damaging 18 Ring finger protein 3.1 0.8 3.7661024 FTSJ2 rs55904231 Ser11Phe Benign 7 RNA methyltransferase 5.2 1.4 3.7661024 RHPN2 rs28626308 Arg70Gln Damaging 7 Rho GTPase binding protein 5.2 1.4 3.9161024 ADAR rs17843865 Tyr587Cys Benign 4 Adenosine deaminase 26.4 1.8 4.6561024 Variants selected were predicted damaging with a p-value ,0 001 in the discovery cohort n = 171 composed of patients from HUSTLE and SWiTCH trials Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Gene SNP ID Amino Acid Change Protein Function Prediction MAF (%) Function Beta Value SE P-value EML1 rs141631682 Gly109Asp Damaging 2 Microtubule assembly 10.9 2.7 7.4161025 SESN1 rs2273668 Leu103Ile Damaging 2 Peroxiredoxin reduction 9.3 2.3 8.8861025 PAPLN rs17126352 Val416Ile Benign 3 Metalloprotease 7.1 1.8 1.3561024 DCHS2 rs61746132 Pro1676Lys Damaging 12 Calcium dependent cell adhesion 3.9 1.0 1.7361024 KRT80 rs61749462 Arg364Ser Damaging 3 Keratin 80 8.0 2.1 1.8061024 SALL2 rs61743453 Pro840Arg Damaging 4 Transcription factor 6.7 1.8 2.3761024 NOM1 rs61742645 Arg779Cys Benign 2 PP1 interacting protein 8.7 2.3 2.4361024 N4BP2L2 rs35108810 Asp246Val Damaging 15 ELA2 transcription inhibitor 23.3 0.9 3.4761024 RNF113B rs16955011 Val92Met Damaging 18 Ring finger protein 3.1 0.8 3.7661024 FTSJ2 rs55904231 Ser11Phe Benign 7 RNA methyltransferase 5.2 1.4 3.7661024 RHPN2 rs28626308 Arg70Gln Damaging 7 Rho GTPase binding protein 5.2 1.4 3.9161024 ADAR rs17843865 Tyr587Cys Benign 4 Adenosine deaminase 26.4 1.8 4.6561024 V i t l t d di t d d i ith l ,0 001 i th di h t 171 d f ti t f HUSTLE d SWiTCH t i l introduce an amino acid change that has a damaging effect on protein structure or function. We genotyped these 13 variants by TaqMan PCR in our independent validation cohort of 130 patients with SCA. We found that one of the 13 variants, located in the SALL2 gene, maintained association with hydroxyurea response. In the discovery cohort, the P840R variant in the SALL2 gene (rs61743453) was associated with a higher change in HbF in response to hydroxyurea (p = 2.3761024, beta value 6.7). In the validation cohort, this same P840R variant was associated with a higher final HbF, with a p-value of 0.05, and a beta value of 4.2. Using Fisher’s combined probability test method, a meta analysis of the association of the SALL2 variant with DHbF in the discovery and validation sample groups (n = 301) leads to a combined p-value of 8.3061024. The meta analysis of the association of SALL2 with final HbF in the discovery and validation sample groups resulted in a combined p-value of 1.4861024. Table 2. Variants associated with DHbF on hydroxyurea. Whole exome sequencing We used the existing methods of SIFT and PolyPhen2 [21] for predicting the functional impact of each non-synonymous variant to estimate which of the 25 variants had a predicted damaging or benign effect on encoded protein function (Tables 2 and 3). As our phenotypes of interest are continuous variables, we used linear regression analysis to test the association of the 38,012 common (MAF$2%) non-synonymous variants using final HbF, and DHbF as independent, continuous variables. In addition, we attempted to find rare variants with MAF,2% associated with From these 25 variants, we identified 13 variants with strongest association with response to hydroxyurea and predicted to October 2014 \| Volume 9 \| Issue 10 \| e110740 October 2014 \| Volume 9 \| Issue 10 \| e110740 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 Discussion Many individuals with SCA are prescribed hydroxyurea, and there is evidence that genetic modifiers affect individual response [17,22]. In order to identify novel candidate genes and variants associated with hydroxyurea response, we sequenced the exomes of 171 individuals enrolled in two prospective clinical trials and related their sequence variant data to HbF response. This discovery cohort was obtained from patients treated on protocol, with the highest level of drug compliance supervision, including monthly pill counts. Our validation cohort (n = 130) was treated under guidelines similar to that of the discovery cohort. As expected, individual MTD was achieved at different hydroxyurea doses, within a range of 10–35 mg/kg/d, reflecting the typical range in bioavailability among patients. There was no correlation between hydroxyurea dose and HbF response (p = 0.56), support- ing the conclusion that differences in pharmacokinetics and pharmacodynamics affect HbF levels achieved on hydroxyurea [22]. Adenosine deaminase Whole exome sequencing permitted analysis of genes beyond a usual set of a priori biologic candidate genes for this phenotype and variants across a broad allele frequency spectrum. We identified multiple non-synonymous variants associated with DHbF or final HbF (Tables 2 and 3) in the discovery cohort. We then performed genotyping on a validation cohort for 13 candidate variants with the lowest p-values and were also predicted to be damaging. Of the 13 variants genotyped, the variant in SALL2 was associated with a higher HbF in the discovery cohort, and a higher final HbF at MTD in the discovery cohort. The validated variant in SALL2 represents a novel variant not previously implicated in -globin expression or HbF response to hydroxyurea. Further studies of other sickle cell cohorts treated with hydroxyurea are needed to confirm this promising associa- tion. SALL2 is a multi-zinc finger transcription factor implicated in hematopoietic cell maturation and cell cycle arrest [23]. It contains the same conserved 12 amino acid N-terminal motif as BCL11A [24,25]. This motif has been shown to be essential for binding of the nucleosome remodeling and deacetylase co- repressor complex, or NuRD, which includes the histone deacelylases HDAC1 and HDAC2. Both HDAC1 and HDAC2 have been shown to act as co-repressors of gamma globin [26]. The variant identified here (rs61743453), causes a proline to arginine change at residue 840 and is predicted to bedamaging to protein function. This P840R SALL2 variant was associated with a higher HbF response to hydroxyurea (Figure 2). Further function- October 2014 \| Volume 9 \| Issue 10 \| e110740 4 Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Table 3. Variants associated with final HbF on hydroxyurea. October 2014 \| Volume 9 \| Issue 10 \| e110740 October 2014 \| Volume 9 \| Issue 10 \| e110740 Adenosine deaminase Gene SNP ID Amino Acid Change Protein Function Prediction MAF (%) Function Beta Value SE P-value RSPH3 rs61750777 Ala154Val Damaging 6 Radial spoke protein 26.5 1.6 6.5561025 OLR1 rs11053646 Lys167Asn Benign 2 Opioid receptor 3.4 0.9 1.2261024 SEC31B rs11819496 Arg478Thr Damaging 2 ER transport 212.0 3.1 1.5561024 COPE rs34510432 Arg85 His Damaging 2 ER transport 9.9 2.6 1.8861024 RNF113B rs16955011 Val92Met Damaging 18 Ring finger protein 3.4 0.9 1.9361024 CDHR3 rs6967330 Cys529Tyr Benign 29 Calcium dependent cell adhesion 3.1 0.8 2.5861024 ETAA1 rs3770655 Pro771Ser Benign 89 Ewing’s tumor associated antigen 4.3 1.2 2.8461024 TTLL10 rs113596156 Arg35Gln Benign 2 Polyglycylase 9.8 2.7 2.9161024 DOCK1 rs869801 Ala1793Thr Benign 21 Cytokinesis 23.5 1.0 3.6061024 MYBBP1A rs899441 Lys637Glu Benign 6 MYB associated 25.4 1.5 4.2661024 MARCH10 rs116835087 Gly587Ser Damaging 6 Membrane-associated ring finger 26.0 1.7 4.3361024 PKD1L1 rs11972142 Thr879Ala Benign 15 Polycystic kidney disease like 3.6 1.0 4.7861024 ADCY10 rs16859886 Thr234Met Benign 12 Adenyl cyclase 3.9 1.1 4.9661024 Variants selected were predicted damaging, with a p-value ,0.001 in the discovery cohort, n = 171, composed of patients from HUSTLE and SWiTCH trials. doi:10 1371/journal pone 0110740 t003 LOS ONE \| www.plosone.org October 2014 \| Volume 9 \| Issue 10 \| e110740 5 Genetic Modifiers of Hemoglobin F Response to Hydroxyurea Figure 2. Effect of SALL2 variant rs61743453 on HbF response to hydroxyurea. A, Effect of rs61743453 on delta HbF in discovery cohort. B, Effect of rs61743453 on MTD HbF in validation cohort. Variant refers to the Pro840Arg variant; no individuals were homozygous for this change. doi:10.1371/journal.pone.0110740.g002 Figure 2. Effect of SALL2 variant rs61743453 on HbF response to hydroxyurea. A, Effect of rs61743453 on delta HbF in discovery cohort. B, Effect of rs61743453 on MTD HbF in validation cohort. Variant refers to the Pro840Arg variant; no individuals were homozygous for this change. doi:10.1371/journal.pone.0110740.g002 al studies will help establish the role of SALL2 in HbF induction in the context of hydroxyurea. dose-escalated to mild myelosuppression using a standardized regimen [18,19]. Despite the relatively small sample size, we used the best genotype-phenotype pairs available from prospectively treated pediatric patients from two clinical trials for the discovery cohort. We assembled a validation cohort from patients treated with hydroxyurea according to standard of care and expert guidelines in a pediatric hematology center with an established sickle cell program. The validation cohort contained 130 unrelated children with SCA followed at the Texas Children’s Hospital Hematology Center (TCHHC). Adenosine deaminase All patients receiving hydroxyurea at TCHHC were approached for enrollment in an Internal Review Board- approved protocol for genetic analysis. They were treated with hydroxyurea using institutional guidelines rather than a specific protocol, but all were escalated to MTD following a standardized regimen as previously described [19]. All patients were treated with hydroxyurea for at least 6 months prior to the designated MTD timepoint. Total HbF levels for both discovery and validation cohorts were measured by HPLC. This study may have failed to detect loci with modest effects because of low statistical power and some associations identified in the discovery cohort may have failed validation given the small size of the validation cohort. Accordingly, all of the mutations identified in the discovery cohort may represent coding variants worth pursuing in future studies. The data presented here bodes well for the success of larger collaborative efforts aimed at identifying genetic modifiers of hydroxyurea response, and serves as a call for coordinated collaboration among pediatric sickle cell centers to increase sample size and increase the odds for novel discovery and translational potential. Ethics Statement All patients and their families gave informed consent for genomic DNA sample collection, storage, and sequencing. The WES genomic analyses were approved by the Baylor College of Medicine Institutional Review Board. All DNA samples and data in this study were denominalized for analysis. Whole exome sequencing DNA concentrations were quantified using picogreen fluores- cent detection method (Quant-iT, Invitrogen). For each DNA sample, the entire exome was captured using the NimbleGen VCRome 2.1 capture reagent followed by sequencing on an Illumina platform using standard chemistries. The sequencing reads were mapped to Hg19 reference genome using the BWA [27]. Sample level genome variants were identified and annotated using the Human Genome Sequencing Center’s integrated Mercury pipeline which includes quality score recalibration and insertion/deletion (Indel) realignment, genome variant identifica- tion by AtlasSNP [28], and annotation using Cassandra software. A project level variant call format (VCF) was generated for all the samples, and included variants that were present in at least one sample. Variants with more than 5% missed genotyping calls were excluded from analysis. Error threshold for alignment was two base errors per read with penalties for indels (maximum of 1) are much more costly than the penalties for SNVs (maximum of 2). We allowed for read-trimming to 35 bp. References (2013) The effect of hydroxcarbamide therapy on survival of children with sickle cell disease. Br J Haematol 161: 852–860. 19. Ware RE (2009) How I use hydroxyurea to treat young patients with sickle cell anemia. Blood 115: 5300–5311. 20. Steinberg MH (2001) Modulation of fetal hemoglobin in sickle cell anemia. Hemoglobin 25: 195–211. 7. Lebensburger J, Johnson SM, Askenazi DJ, Rozario NL, Howard TH, et al. (2011) Protective role of hemoglobin and fetal hemoglobin in early kidney disease for children with sickle cell anemia. Am J Hematol 86: 430–432. g 21. Flanagan SE, Patch AM, Ellard S (2010) Using SIFT and PolyPhen to predict loss-of-function and gain-of-function mutations. Genet Test Mol Biomarkers 14: 533–537. 8. Lebensburger JD, Pestina TI, Ware RE, Boyd KL, Persons DA (2010) Hydroxyurea therapy requires HbF induction for clinical benefit in a sickle cell mouse model. Haematologica 95: 1599–1603. 22. Ware RE, Despotovic JM, Mortier NA, Flanagan JM, He J, et al. (2011) Pharmacokinetics, pharmacodynamics, and pharmacogenetics of hydroxyurea treatment for children with sickle cell anemia. Blood 118: 4985–4991. 9. Steinberg MH, Barton F, Castro O, Pegelow CH, Ballas SK, et al. (2003) Effect of hydroxyurea on mortality and morbidity in adult sickle cell anemia: risks and benefits up to 9 years of treatment. JAMA 289: 1645–1651. 23. Chai L (2011) The role of HSAL (SALL) genes in proliferation and differentiation in normal hematopoiesis and leukemogenesis. Transfusion 51 Suppl 4: 87S–93S. 10. Smith WR, Ballas SK, McCarthy WF, Bauserman RL, Swerdlow PS, et al. (2011) The association between hydroxyurea treatment and pain intensity, analgesic use, and utilization in ambulatory sickle cell anemia patients. Pain Med 12: 697–705. pp 24. Sankaran VG, Menne TF, Xu J, Akie TE, Lettre G, et al. (2008) Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 322: 1839–1842. 11. Maier-Redelsperger M, de Montalembert M, Flahault A, Neonato MG, Ducrocq R, et al. (1998) Fetal hemoglobin and F-cell responses to long-term hydroxyurea treatment in young sickle cell patients. The French Study Group on Sickle Cell Disease. Blood 91: 4472–4479. 25. Lauberth SM, Rauchman M (2006) A conserved 12-amino acid motif in Sall1 recruits the nucleosome remodeling and deacetylase corepressor complex. J Biol Chem 281: 23922–23931. 26. Bradner JE, Mak R, Tanguturi SK, Mazitschek R, Haggarty SJ, et al. (2010) Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease. References 1. Platt OS, Brambilla DJ, Rosse WF, Milner PF, Castro O, et al. (1994) Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med 330: 1639–1644. 1. Platt OS, Brambilla DJ, Rosse WF, Milner PF, Castro O, et al. (1994) Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med 330: 1639–1644. 13. Zimmerman SA, Schultz WH, Davis JS, Pickens CV, Mortier NA, et al. (2004) Sustained long-term hematologic efficacy of hydroxyurea at maximum tolerated dose in children with sickle cell disease. Blood 103: 2039–2045. dose in children with sickle cell disease. Blood 103: 2039–2045 14. Ware RE, Aygun B (2009) Advances in the use of hydroxyurea. Hematology Am Soc Hematol Educ Program: 62–69. 2. Cheetham RC, Huehns ER, Rosemeyer MA (1979) Participation of haemoglo- bins A, F, A2 and C in polymerisation of haemoglobin S. J Mol Biol 129: 45–61. 15. Ware RE, Eggleston B, Redding-Lallinger R, Wang WC, Smith-Whitley K, et al. (2002) Predictors of fetal hemoglobin response in children with sickle cell anemia receiving hydroxyurea therapy. Blood 99: 10–14. 3. Powars DR, Weiss JN, Chan LS, Schroeder WA (1984) Is there a threshold level of fetal hemoglobin that ameliorates morbidity in sickle cell anemia? Blood 63: 921–926. 16. Green NS, Ender KL, Pashankar F, Driscoll C, Giardina PJ, et al. (2013) Candidate sequence variants and fetal hemoglobin in children with sickle cell disease treated with hydroxyurea. PLoS One 8: e55709. 4. Steinberg MH, McCarthy WF, Castro O, Ballas SK, Armstrong FD, et al. (2010) The risks and benefits of long-term use of hydroxyurea in sickle cell anemia: A 17.5 year follow-up. Am J Hematol 85: 403–408. disease treated with hydroxyurea. PLoS One 8: e55709. 17. Steinberg MH, Voskaridou E, Kutlar A, Loukopoulos D, Koshy M, et al. (2003) Concordant fetal hemoglobin response to hydroxyurea in siblings with sickle cell disease. Am J Hematol 72: 121–126. 5. Voskaridou E, Christoulas D, Bilalis A, Plata E, Varvagiannis K, et al. (2010) The effect of prolonged administration of hydroxyurea on morbidity and mortality in adult patients with sickle cell syndromes: results of a 17-year, single- center trial (LaSHS). Blood 115: 2354–2363. 18. Ware RE, Helms RW (2011) Stroke With Transfusions Changing to Hydroxyurea (SWiTCH). Blood 119: 3925–3932. 6. Lobo CL, Pinto JF, Nascimento EM, Moura PG, Cardoso GP, et al. Subjects The discovery cohort was composed of 171 unrelated children with SCA; 120 were enrolled in the Hydroxyurea Study of Long- Term Effects (HUSTLE, NCT00305175); and 51 were enrolled in the NHLBI-sponsored Stroke with Transfusions Changing to Hydroxyurea (SWiTCH, NCT00122980). HUSTLE was a single center trial investigating long term effects of hydroxyurea in SCA, while SWiTCH was a multi-center trial investigating the use of hydroxyurea on stroke prevention. HUSTLE and SWiTCH study patient samples were used with approval from the Baylor College of Medicine Internal Review Board, protocol H-29047. Patients and their families in both clinical trials provided written consent for DNA sample collection, storage and sequencing. Texas Children’s Hospital Hematology Center patients and their families provided written consent to whole exome sequencing, posting of sequences to dbGAP, and data collection under BCM Internal Review Board protocol H31356. All DNA samples and data in this study were denominalized for analysis. All 171 individuals had a known baseline HbF level measured at greater than 3 years of age, were initially treated with hydroxyurea at 20 mg/kg, and then WES genotyping of variants of interest with heterozygosity scores less than 0.45 were verified by TaqMan genotyping. TaqMan genotyping assays were performed on an Applied Biosystem’s StepOne instrument (AB, Foster City, CA). After 40 October 2014 \| Volume 9 \| Issue 10 \| e110740 PLOS ONE \| www.plosone.org 6 Genetic Modifiers of Hemoglobin F Response to Hydroxyurea amplification cycles, threshold cycle values were automatically calculated, and the individual SNP genotypes were called by the StepOne v2.0 software (AB, Foster City, CA). sequencing for 13 SNPs with the lowest p-values that were identified as associated with HbF response to hydroxyurea, non- synonymous, and damaging. The relationship between genotype and HbF response to hydroxyurea was analyzed with a one directional t-test. Acknowledgments We thank all the patients and families, as well as the clinical investigators and research staff, for their participation in the SWiTCH and HUSTLE trials. We would like to thank the patients, their families, and the research staff at Texas Children’s Hematology Center for their participation in FWES, which provided the validation cohort. To analyze the effect of rare variants (MAF,2%) on the phenotypes DHbF and final HbF, we used two gene based tests, a simple burden test (T2) and SKAT [29,30]. In SKAT and T2 testing, a collection of rare variants within a single gene are tested for association with the phenotype. The T2 considers those rare variants with MAF less than 2% and assumes that the effects of all variants are in the same direction. SKAT is a kernel-based test that considers rare variants having effects in either direction [29,31]. Both tests considered only non-synonymous variants. Supporting Information Table S1 Quality control filters used in WES analysis. Sex chromosomes were removed, as gender did not impact HbF response to hydroxyurea. Sites with heterzygousto homozygous ration.0.4 were removed.Variants with MAF,2% were analyzed by burden testing. (PDF) Statistical Analysis Linear regression analysis was used to test the association of the filtered variants using final HbF and DHbF as independent, continuous variables. The DHbF and final HbF phenotypes both had normal distributions, indicating they were suitable for linear regression analysis (Figure 1); values were adjusted for age and gender. Principal component analysis (PCA) was performed using the EIGENSTRAT method, and applied to all models. Quality control filtering steps, including SNP missingness check, removal of sex chromosomes, monomorphic site, synonymous and intronic variant removal, excess heterozygosity filter and minor allele frequency (MAF) cut-off of 2% and the impact of these filtering steps on the total number of SNPs, are described in Table S1. Validation Genomic DNA from 130 patients from TCHHC collected as a validation cohort were genotyped by TaqMan or Sanger Genomic DNA from 130 patients from TCHHC collected as a validation cohort were genotyped by TaqMan or Sanger Author Contributions Conceived and designed the experiments: VAS JMF EB RAG REW. Performed the experiments: TAH AS DMM SDP. Analyzed the data: JRC EB. Contributed reagents/materials/analysis tools: RAG EB. Wrote the paper: VAS JMF REW EB. Clinical trial data analysis and sample collection: BA KAN NAM REW. 31. Morgenthaler S, Thilly WG (2007) A strategy to discover genes that carry multi- allelic or mono-allelic risk for common diseases: a cohort allelic sums test (CAST). Mutat Res 615: 28–56. 30. Li B, Leal SM (2008) Methods for detecting associations with rare variants for common diseases: application to analysis of sequence data. Am J Hum Genet 83: 311–321. 28. Challis D, Yu J, Evani US, Jackson AR, Paithankar S, et al. (2012) An integrative variant analysis suite for whole exome next-generation sequencing data. BMC Bioinformatics 13: 8. 29. Lee S, Wu MC, Lin X (2012) Optimal tests for rare variant effects in sequencing association studies. Biostatistics 13: 762–775. Genetic Modifiers of Hemoglobin F Response to Hydroxyurea References Proc Natl Acad Sci U S A 107: 12617–12622. 12. Kinney TR, Helms RW, O’Branski EE, Ohene-Frempong K, Wang W, et al. (1999) Safety of hydroxyurea in children with sickle cell anemia: results of the HUG-KIDS study, a phase I/II trial. Pediatric Hydroxyurea Group. Blood 94: 1550–1554. 27. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows- Wheeler transform. Bioinformatics 25: 1754–1760. PLOS ONE \| www.plosone.org 7 October 2014 \| Volume 9 \| Issue 10 \| e110740 October 2014 \| Volume 9 \| Issue 10 \| e110740 Genetic Modifiers of Hemoglobin F Response to Hydroxyurea October 2014 \| Volume 9 \| Issue 10 \| e110740 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 8
https://openalex.org/W4327529147	https://link.springer.com/content/pdf/10.1007/s11257-023-09359-w.pdf	English	null	Recommending on graphs: a comprehensive review from a data perspective	User modeling and user-adapted interaction	2,023	cc-by	40,427	User Modeling and User-Adapted Interaction (2023) 33:803–888 https://doi.org/10.1007/s11257-023-09359-w User Modeling and User-Adapted Interaction (2023) 33:803–888 https://doi.org/10.1007/s11257-023-09359-w Abstract Recent advances in graph-based learning approaches have demonstrated their effec- tiveness in modelling users’ preferences and items’ characteristics for Recommender Systems (RSs). Most of the data in RSs can be organized into graphs where various objects (e.g. users, items, and attributes) are explicitly or implicitly connected and inﬂuence each other via various relations. Such a graph-based organization brings beneﬁts to exploiting potential properties in graph learning (e.g. random walk and network embedding) techniques to enrich the representations of the user and item nodes, which is an essential factor for successful recommendations. In this paper, we provide a comprehensive survey of Graph Learning-based Recommender Systems (GLRSs). Speciﬁcally, we start from a data-driven perspective to systematically cate- gorize various graphs in GLRSs and analyse their characteristics. Then, we discuss the state-of-the-art frameworks with a focus on the graph learning module and how they address practical recommendation challenges such as scalability, fairness, diversity, explainability, and so on. Finally, we share some potential research directions in this rapidly growing area. Keywords Recommender system · Graph learning · Graph neural network Recommending on graphs: a comprehensive review from a data perspective Lemei Zhang1 · Peng Liu1 · Jon Atle Gulla1 Received: 29 March 2021 / Accepted in revised form: 15 January 2023 / Published online: 13 March 2023 © The Author(s) 2023 1 Department of Computer Science, NTNU, 7491 Trondheim, Norway B Lemei Zhang lemei.zhang@ntnu.no Peng Liu peng.liu@ntnu.no Jon Atle Gulla jon.atle.gulla@ntnu.no 1 Introduction In the last few decades, the rapid development of Web 2.0 and smart mobile devices has resulted in the dramatic proliferation of online unstructured data, such as news articles. 1 Department of Computer Science, NTNU, 7491 Trondheim, Norway 123 804 L. Zhang et al. They are explicitly or implicitly connected with each other and can naturally be formed into graphs representing objects and their relationships in varied domains, including e-commerce, social networks, and so on. On the one hand, the interconnection of objects shows a direct (e.g. social relations in a social network) or indirect interactive relationship (e.g. item co-occurrence in an item homogeneous network), which pro- vides a more intuitive and effective way for recommendation systems to explore the hidden relationships between the target user and the recommended items. On the other hand, the data structure of graphs breaks the independent interaction assumption1 by linking users or items with their associated attributes such that the recommender sys- tems are able to capture not only the user–item interactions but also the rich underlying connections by mining item–item/user–user relations to make more accurate recom- mendations. Moreover, most recommendation models work as black boxes that only provide predictive results rather than exhibiting the reasons behind a recommendation, such as collaborative signals in collaborative ﬁltering or knowledge-aware reasoning in knowledge graph-based recommendation. Some recommender systems give users such as “users who bought A also bought B” as an explanation of the recommended results. However, no in-depth explanations of the intrinsic recommendation mecha- nism for the selected items may result in the users over-relying on the recommendation system and ignoring that the purpose of the recommendation is to make the recom- mendation platform proﬁtable (Jannach et al. 2019). Recent advances in graph-based recommender systems have demonstrated their effectiveness in improving the explain- ability of the recommender systems by using explicit connections between objects in graphs to reveal the recommendation results (He et al. 2015; Ma et al. 2019; Hu et al. 2018). Therefore, it is of crucial signiﬁcance to fully explore the semantic connec- tions and potential relations of the graphs to improve the performance on both the explainability and accuracy of recommendations. However, there exist some problems and challenges in how recommender systems (RSs) can make full use of these data: (1) Heterogeneous Objects: Unstructured data can be organized into a graph includ- ing different-typed objects and links. 1 The techniques, e.g. factorization machine (Koren et al. 2009), Neural FM (He and Chua 2017), based upon the independent interaction assumption model the user or item as a single object, but ignore the connection relationship among them. 1 Introduction Modelling and abstracting such a space of information have been a challenging task encountered in RSs. (2) Large-scale Volume: Real graphs, such as social networks, can easily have millions even billions of nodes and edges, which renders most traditional recommendation algorithms computationally infeasible. (3) Dynamic Contents: Most real-world graphs are intrinsically dynamic with addi- tion/deletion of edges and nodes. Meanwhile, similar to a graph structure, node attributes also change naturally such that new content patterns may emerge and outdated content patterns will fade. Recently, graph learning (GL) has exhibited the potential to obtain knowledge embedded in different kinds of graphs. Many GL techniques, such as random walk, graph embedding, and graph neural networks, have been developed to learn the com- plexrelationsmodelledongraphsandachieveagreatimprovementinrecommendation Recently, graph learning (GL) has exhibited the potential to obtain knowledge embedded in different kinds of graphs. Many GL techniques, such as random walk, graph embedding, and graph neural networks, have been developed to learn the com- plexrelationsmodelledongraphsandachieveagreatimprovementinrecommendation 123 123 12 Recommending on graphs: a comprehensive review from a data… 805 performance. An emerging RS paradigm built on GL, namely Graph Learning-based Recommender Systems (GLRSs), has attracted growing attention in both research and industry communities. For example, researchers leverage random walk to propagate users’ preference scores from historical item nodes and output a preference distribu- tion over unobserved items, such as ItemRank (Gori et al. 2007) over the item–item correlation graph, RecWalk (Nikolakopoulos and Karypis 2019) over the user–item bipartite graph, and TriRank (He et al. 2015) over the user–item–aspect tripartite graph. Moreover, various graph embedding techniques and graph neural networks have been proposed and incorporated into the representation learning of RSs, using direct or multi-hop connections within graphs to enrich the representations of the user and item nodes. These approaches further improve the recommendation performance. pp p p To date, there are only a handful of literature reviews related to our paper. Wang et al. (2021a) survey KG-embedding models for link prediction. In Guo et al. (2020), Liu and Duan (2021), the authors summarize recent works utilizing KGs as side information for accurate and explainable recommendations. The authors of (Wu et al. 2020a) summarize the most recent works on GNN-based recommender systems and propose a classiﬁcationschemafororganizingexistingworks.Ourworkdiffersfromtheprevious works in that we give a systematic and comprehensive review of recommendation techniques starting with different types of data-driven graphs rather than focusing on one speciﬁc branch. 2 https://en.wikipedia.org/wiki/Linked_data. 1 Introduction Another closely related topic is linked data-based recommendation. Figueroa et al. (2015) present a systematic literature review to summarize the state of the art in RS that uses structured data published as Linked Data for providing recommendations of items from diverse domains. Tarus et al. (2018) present a comprehensive review of ontology-based recommendations for e-learning. The main distinction is that these works discuss making recommendations with the subject of linked data, which is structured interlinked data that manifest as a Web of Data from multiple sources. 2 In fact, both works focus on knowledge-based recommendations without referring to recent deep learning-based technologies. In this paper, we focus on how raw data can be extracted into a wider range of graphs (e.g. tree-based graphs, homogeneous graphs, and hypergraphs) and how traditional as well as state-of-the-art graph learning techniques can be applied to these graphs for recommendation purposes. Although there is a variety of literature on this subject, only one study analysed the role of GLRSs (Wang et al. 2020e). The limitations lie in that they only cover a limited number of references and do not go into the technical details on graph learning modules in GLRS and how they address those challenges. Furthermore, they do not give a systematic summarization of existing datasets adopted by graph learning-based recommendation research. To overcome such an information gap, in this paper, we contribute the most comprehensive overview of state-of-the-art GLRSs. We system- atically analyse the benchmark datasets in GLRSs, provide detailed descriptions of representative models, make the necessary comparisons, and discuss their solutions to practical recommendation issues such as scalability, fairness, diversity, and explain- ability. 123 123 806 L. Zhang et al. Contributions: This survey provides a thorough literature review on the approaches of graph learning-based recommender systems and the involved various types of graphs from a data perspective. It provides a panorama starting from various data charac- teristics to applied technologies, with the hope that both academic researchers and industrial practitioners can have a rough guideline and step into the ﬁeld of graph learning-based recommendation from the data resources available at hand. This sur- vey serves to promote the innovation and development in the ﬁeld of GLRSs, while exploring the possibility of enhancing the richness by discussing and summarizing existing open issues in the ﬁeld. 1 Introduction To this end, the main contributions of this work are threefold: • We explore different data input categories based on their acquisition and intrinsic characteristics and further proposed a novel taxonomy to categorize various graphs in GLRSs from data perspectives. Meanwhile, we summarize resources regarding GLRSs, including benchmark datasets and open-source knowledge graphs. • We conduct a systematic literature review on traditional and recent developments and progress of graph learning-based techniques for recommender systems, which correspond to associated different graph taxonomies. • We analyse the limitations of existing works and suggest future research directions of GLRSs such as dynamicity, interpretability, and fairness, for giving references for this community. Organization of Our Survey: The rest of the survey is organized as follows. In Sect.2, we review our research methodology on how we collected the related papers and provide an initial analysis of datasets adopted by reference papers. In Sect.3, we introduce the deﬁnitions of the basic concepts required to understand the graph learning-based recommendation problem, followed by a formal problem deﬁnition of graph learning-based recommendation. Section4 provides a new taxonomy of graphs that are related to speciﬁc datasets. Section5 provides an overview of state-of-the- art GLRSs techniques. Section6 discusses the current challenges and suggests future directions, followed by the conclusions in Sect.7. 4 https://ieeexplore.ieee.org. 3 https://dl.acm.org. 3 https://dl.acm.org. 4 https://ieeexplore.ieee.org. 3 https://dl.acm.org. 4 https://ieeexplore.ieee.org. 1 3 Recommending on graphs: a comprehensive review from a data… 807 Table 1 Search string Key words Search string graph, hypergraph, recommender, recommendation, recommender system, recommendation system, recommendation service, recommendation approach, recommendation model, recommendation method, recommendation algorithm, recommendation application, recommendation engine, recommendation agent, recommendation framework, collaborative ﬁltering, social recommendation, representation learning, knowledge graph, graph neural network (TITLE-ABS-KEY⋆(recommender) OR TITLE-ABS-KEY(recommendation system) OR TITLE-ABS-KEY(recommendation service) OR TITLE-ABS-KEY(recommendation approach) OR TITLE-ABS-KEY(recommendation model) OR TITLE-ABS-KEY(recommendation method) OR TITLE-ABS-KEY(recommendation algorithm) OR TITLE-ABS-KEY(recommendation application) OR TITLE-ABS-KEY(recommendation engine) OR TITLE-ABS-KEY(recommendation agent) OR TITLE-ABS-KEY(recommendation framework) OR TITLE-ABS-KEY(collaborative ﬁltering) OR TITLE-ABS-KEY(social recommendations)) AND (TITLE-ABS-KEY(graph) OR TITLE-ABS-KEY(hypergraph) OR TITLE-ABS-KEY(knowledge graph) OR TITLE-ABS-KEY(graph neural network) OR TITLE-ABS-KEY(representation learning)) ⋆TITLE-ABS-KEY is a combined ﬁeld that searches abstracts, keywords, and document titles Table 1 Search string Key words Search string graph, hypergraph, recommender, recommendation, recommender system, recommendation system, recommendation service, recommendation approach, recommendation model, recommendation method, recommendation algorithm, recommendation application, recommendation engine, recommendation agent, recommendation framework, collaborative ﬁltering, social recommendation, representation learning, knowledge graph, graph neural network (TITLE-ABS-KEY⋆(recommender) OR TITLE-ABS-KEY(recommendation system) OR TITLE-ABS-KEY(recommendation service) OR TITLE-ABS-KEY(recommendation approach) OR TITLE-ABS-KEY(recommendation model) OR TITLE-ABS-KEY(recommendation method) OR TITLE-ABS-KEY(recommendation algorithm) OR TITLE-ABS-KEY(recommendation application) OR TITLE-ABS-KEY(recommendation engine) OR TITLE-ABS-KEY(recommendation agent) OR TITLE-ABS-KEY(recommendation framework) OR TITLE-ABS-KEY(collaborative ﬁltering) OR TITLE-ABS-KEY(social recommendations)) AND (TITLE-ABS-KEY(graph) OR TITLE-ABS-KEY(hypergraph) OR TITLE-ABS-KEY(knowledge graph) OR TITLE-ABS-KEY(graph neural network) OR TITLE-ABS-KEY(representation learning)) ⋆TITLE-ABS-KEY is a combined ﬁeld that searches abstracts, keywords, and document titles Search string (TITLE-ABS-KEY⋆(recommender) OR TITLE-ABS-KEY(recommendation system) OR TITLE-ABS-KEY(recommendation service) OR TITLE-ABS-KEY(recommendation approach) OR TITLE-ABS-KEY(recommendation model) OR TITLE-ABS-KEY(recommendation method) OR TITLE-ABS-KEY(recommendation algorithm) OR TITLE-ABS-KEY(recommendation application) OR TITLE-ABS-KEY(recommendation engine) OR TITLE-ABS-KEY(recommendation agent) OR TITLE-ABS-KEY(recommendation framework) OR TITLE-ABS-KEY(collaborative ﬁltering) OR TITLE-ABS-KEY(social recommendations)) AND (TITLE-ABS-KEY(graph) OR TITLE-ABS-KEY(hypergraph) OR TITLE-ABS-KEY(knowledge graph) OR TITLE-ABS-KEY(graph neural network) OR TITLE-ABS-KEY(representation learning)) Springer, 5 ResearchGate, 6 and Web of Science. 7 We conducted the same keyword- based search in these search engines. Springer, 5 ResearchGate, 6 and Web of Science. 7 We conducted the same keyword- based search in these search engines. We ﬁrst checked the paper titles and then reviewed the abstracts, keywords, results, and conclusions to obtain the ﬁrst list of studies. We then double-checked the reference list in those papers to identify additional studies that were relevant to our review topic. After that, the publications retrieved needed to be further ﬁltered in order to eliminate false positives, which are irrelevant to the current survey. Therefore, a pre-deﬁned set of inclusion and exclusion criteria displayed in Table 2 were applied to the retrieved papers. 5 https://www.springer.com. 6 https://www.researchgate.net/. 7 https://www.webofknowledge.com. 2.1 Paper collection Toachieveasystematicstructureofexistingresearchongraph-basedrecommendation, this study was performed based on a bibliographic review proposed by Webster and Watson (2002), Kitchenham (2004), Wolfswinkel et al. (2013). Speciﬁcally, we ﬁrst conducted a comprehensive review of previously published papers concerning GLRSs and used Scopus as the main source of information. The search strings are listed in Table 1. Other bibliographic databases and archives also constitute the auxiliary sources used for literature search, such as ACM Digital Library,3 IEEE Xplore, 4 123 Recommending on graphs: a comprehensive review from a data… ⋆TITLE-ABS-KEY is a combined ﬁeld that searches abstracts, keywords, and document titles 7 https://www.webofknowledge.com. Recommending on graphs: a comprehensive review from a data… Finally, we obtained a collection of 182 papers that meet the mentioned criteria and then are summarized in Sects.3 and 4. Figure1 gives the statistics of the collected papers with the publication time and venue. Note that these papers were mainly selected according to the two criteria: (1) pub- lication time; (2) impact. Therefore, the references cited in this paper on the ﬁeld of GLRS are representative, but still limited. The uncited literature is only limited by the length of this paper and the pre-set ﬁltering criteria. 123 L. Zhang et al. 808 808 L. Zhang et al. Table 2 Inclusion criteria and exclusion Criteria Criteria Inclusion Criteria Exclusion Criteria Recommender System The study focused on recommender system in multiple domains The study presents a system or technique other than a recommender system The use of graph as input The study presents a system that uses a type of graph as the input, and other data structures such as texts, image or acoustic information can also be the auxiliary means of system input The study presents a system using a data structure except graph as input Publication Date The paper is published between 2007 and September 2021 The paper is published before 2007 or after September 2021 Language The paper is written in English The paper is written in a language different than English Publication type The paper has been peer reviewed and published in prestigious and top-tier international conferences and journals e.g. SIGIR, NIPS, ICML, RecSys, CIKM, ICLR, AAAI, IJCAI, WWW, WSDM, KDD, UMAP, TOIS, TKDE, and UMUAI etc. The paper is a primary study The paper has not been peer reviewed (e.g. theses, books, technical reports, (extended) abstracts, talks, presentations, tutorials, guidelines) or not published in top-tier conferences or journals. The paper is a secondary study (e.g. systematic literature review, survey) Accessibility The paper’s content can be accessed from a technical university (e.g. Norwegian University of Science and Technology) without additional payment The paper’s content cannot be accessed from a technical university (e.g. Norwegian University of Science and Technology) without additional payment Fig. Recommending on graphs: a comprehensive review from a data… 1 Statistics of publications related to GLRSs grouped by the publication year and venue Table 2 Inclusion criteria and exclusion Criteria Criteria Inclusion Criteria Exclusion Criteria Recommender System The study focused on recommender system in multiple domains The study presents a system or technique other than a recommender system The use of graph as input The study presents a system that uses a type of graph as the input, and other data structures such as texts, image or acoustic information can also be the auxiliary means of system input The study presents a system using a data structure except graph as input Publication Date The paper is published between 2007 and September 2021 The paper is published before 2007 or after September 2021 Language The paper is written in English The paper is written in a languag different than English Publication type The paper has been peer reviewed and published in prestigious and top-tier international conferences and journals e.g. SIGIR, NIPS, ICML, RecSys, CIKM, ICLR, AAAI, IJCAI, WWW, WSDM, KDD, UMAP, TOIS, TKDE, and UMUAI etc. The paper is a primary study The paper has not been peer reviewed (e.g. theses, books, technical reports, (extended) abstracts, talks, presentations, tutorials, guidelines) or not published in top-tier conferences or journals. The paper is a secondary study (e.g systematic literature review, survey) Accessibility The paper’s content can be accessed from a technical university (e.g. Norwegian University of Science and Technology) without additional payment The paper’s content cannot be accessed from a technical university (e.g. Norwegian University of Science and Technology) without additiona payment Table 2 Inclusion criteria and exclusion Criteria Criteria Inclusion Criteria Recommender System The study focused on recommender system in multiple domains The use of graph as input The study presents a system that uses a type of graph as the input, and other data structures such as texts, image or acoustic information can also be the auxiliary means of system input Publication Date The paper is published between 2007 and September 2021 Language The paper is written in English Publication type The paper has been peer reviewed and published in prestigious and top-tier international conferences and journals e.g. SIGIR, NIPS, ICML, RecSys, CIKM, ICLR, AAAI, IJCAI, WWW, WSDM, KDD, UMAP, TOIS, TKDE, and UMUAI etc. The paper is a primary study Accessibility The paper’s content can be accessed from a technical university (e.g. The paper is published before 2007 or after September 2021 The paper is written in a language different than English The paper has not been peer reviewed (e.g. theses, books, technical reports, (extended) abstracts, talks, presentations, tutorials, guidelines) or not published in top-tier conferences or journals. The paper is a secondary study (e.g. systematic literature review, survey) 2.2 Data analysis Analyzing the collected papers, we made two observations on the utilized datasets: (1) They were across different domains, such as e-commerce and entertainment domains; (2) Some datasets could be used to construct multiple types of graphs for different recommendation purposes, while some were only used to construct one type of graph. For instance, we found nearly all classiﬁed graph types were utilized for the Amazon dataset, while only a multi-source graph could be found for the Epinions dataset. To clearly make comparisons and show the difference of these datasets in terms of both domains and graph types, we made a detailed comparison of all datasets in Table 5 (“Appendix A”). Recent advances in GLRS either focus on incorporating explicit or implicit user/item information into the process of mapping, and design learning algorithms for speciﬁc graph structures (Christoforidis et al. 2021; Pang et al. 2022), or focus on how to incorporate time information into graph forming and learning processes to bet- ter capture the dynamic needs of users to improve recommendation performance (Fan et al. 2021; Zhang et al. 2022). In Lv et al. (2021), the authors make reproductions of 12 heterogeneous graph neural network based modes and test them on 11 public available datasets regarding node classiﬁcation, link prediction and knowledge-aware recom- mendation tasks. Their reported experimental results reveal that the superiority of the performance of the most advanced HGNN models rely on the lack of fair comparison with the homogeneous GNNs and other baselines in their original paper. Beside, some works also reveal issues such as data leakage, tuning on test set, cost of large amount of memory, and time for training. Meanwhile, the authors of Lv et al. (2021) release a heterogeneous graph benchmark (HGB) for open, reproducible heterogeneous graph research, and present a GAT-based heterogeneous GNN model resulting in promising results on three aforementioned tasks. We count the corresponding datasets for GLRS technologies adopted in this survey and present the results in Table 7 (“Appendix C”). From the table, we can observe that both datasets and their leveraged GLRS technologies are distributed extremely unbalanced. The reason for the former one may be attributed to whether the dataset is public, the date of publication, whether the dataset contains various attributes of users/items and so forth. 2.2 Data analysis The reason for the uneven distribution of technology can be roughly attributed to the development of deep learning in the ﬁeld of graph learning to bring more possibilities. Recommending on graphs: a comprehensive review from a data… Norwegian University of Science and Technology) without additional payment The paper is published before 2007 or after September 2021 The paper is written in a language different than English The paper has not been peer reviewed (e.g. theses, books, technical reports, (extended) abstracts, talks, presentations, tutorials, guidelines) or not published in top-tier conferences or journals. The paper is a secondary study (e.g. systematic literature review, survey) The paper is published before 2007 or after September 2021 The paper’s content cannot be accessed from a technical university (e.g. Norwegian University of Science and Technology) without additional payment Fig. 1 Statistics of publications related to GLRSs grouped by the publication year and venue h bli i d Fig. 1 Statistics of publications related to GLRSs grouped by the publication year and venue 123 12 Recommending on graphs: a comprehensive review from a data… 809 The deﬁnitions related to GLRSs are as follows. The deﬁnitions related to GLRSs are as follows. Deﬁnition 1 (Graph) A graph is G = (V , E), where v ∈V is a node and e ∈E is an edge. Each edge ei j is a pair between vertex vi and v j. Each edge of G can be mapped to a real number (if any), denoted as W : E →R+ from edge e ∈E to a real number w ∈R+. Such weights can represent e.g. costs, lengths or capabilities, depending on the speciﬁc problem. G is associated with a node type mapping function fv : V →A and an edge type mapping function fe : E →R. A and R denote the set of node types and edge types, respectively. Each node vi ∈V belongs to one particular type, i.e. fv(vi) ∈A. Similarly, for ei j ∈E, fe(ei j) ∈R. When a graph has ei j ̸≡e ji and fe(ei j) ̸≡fe(e ji), it is a directed graph. Otherwise, the graph is undirected. Deﬁnition 2 (Network Schema) The network schema (Sun and Han 2013), denoted as TG = (A, R), is a meta template for a heterogeneous network G = (V , E) with the node type mapping fv and the edge mapping fe, which is a directed graph deﬁned over node type set A with edges as relations from R. Deﬁnition 3 (HomogeneousGraph)AhomogeneousgraphGhomo = (V , E)isagraph in which \|A\| = \|R\| = 1. This is to say that all nodes in G belong to a single type and all edges to one single type. Deﬁnition 4 (Tree Graph) A tree graph Gtree = (V , E) is a graph in which all nodes are connected with each other and there is no cycles in G. The leaf node in a tree graph has degree 1, where degree of a vertex v, denoted as d(v), is deﬁned as the number of vertices that are adjacent to v. Deﬁnition 5 (Heterogeneous Graph) A heterogeneous graph Gheter = (V , E) can be deﬁned as a graph in which \|A\| > 1 and/or \|R\| > 1. Deﬁnition 6 (k-partite Graph) A k-partite graph Gkpar = (V , E) is a graph in which nodes are partitioned into k different disjoint sets {A1, A2 . . . Ak} where A = A1 ∪ A2∪. . 3 Problem formalization In this section, we ﬁrst introduce the deﬁnition of the basic concepts in graph-based recommendations and then provide a formal deﬁnition of the graph-based recommen- dation problem. 123 123 L. Zhang et al. 810 The deﬁnitions related to GLRSs are as follows. .∪Ak and Ai ∩A j = ∅, where i ̸= j; i, j ∈{1, . . . , k}. In Gkpar, no two nodes within the same set are adjacent. If ∃emn between nodes vm and vn where fv(vm) ∈Ai and fv(vn) ∈A j, then i ̸= j; i, j ∈{1, . . . , k}. Deﬁnition 7 (Knowledge Graph) A knowledge graph Gknow = (V , E) is a directed graph whose nodes are entities and edges are subject–property–object triple facts. Each edge of the form (head entity, relation, tail entity), denoted as < h,r, t >, indicates a relationship of r from entity h to entity t. h, t ∈V are entities and r ∈E is the relation. Entities and relations in a knowledge graph are usually of different types, such that A ∩R = ∅. Knowledge graphs can be viewed as another type of heterogeneous graphs. The network schema of a heterogeneous graph speciﬁes type constraints on graph objects/nodes and relationships of links/edges between the objects/nodes. The con- straints make the heterogeneous graph semi-structured data, guiding the exploration of the semantics of the graph. 811 Recommending on graphs: a comprehensive review from a data… 3.2 Problem definition Given a data source X, normally a user set U and an item set I, for each user u ∈ U, the recommendation problem can generally be seen as a mapping function Y = argmaxu f (U, I) generating the corresponding recommendation results from I that are of interests to the user u. However, there is no formal deﬁnition of GLRSs to date due to different implementations of various models on different datasets with speciﬁc characteristics. Graphs in GLRSs can be built upon input data sources, e.g. user–item interactions as well as other auxiliary information, for instance, considering the graph G = (V , E), where nodes in V can represent e.g. users, items and other named entities, while edges in E can represent e.g. purchases, clicks, social relations as well as other relationships among entities. In this survey, we formulate the GLRS problem from a general perspective. Speciﬁcally, for the input data source(s) X, we would like to ﬁnd a mapping M(X) →G, which is used as the input to generate the corresponding recommendation results Y by modelling graph properties as the main way complemented with other auxiliary features of graphs: Y = argmaxu f (M(X) →G\|) (1) (1) where G can be of different types, e.g. homogeneous, k-partite, tree-based, complex heterogeneous graph, etc., based on speciﬁc recommendation scenarios, while Y can be of different forms, e.g. rating scores, possible links, classiﬁcations, or ranked lists. is the model parameter set to be optimized during model training. In this survey, we will focus on different types of input data sources X, different types of graphs G formed from X, the main technologies used for recommendation purposes f , and the connections between these aspects to elaborate on GLRSs-related studies. 4 From data to graphs Most of the existing reviews on GLRSs only focus on the input graph types and related recommendation technologies, but none of them associated different graph types with original datasets. In fact, the construction of the graph is largely determined by the dataset at hand. A large amount of semantic and structural information is hidden in the graphs constructed by analyzing the dataset, and more and more research results show that the performance of recommendation can be improved in many aspects, such as accuracy (Wang et al. 2020d; Sun et al. 2021), fairness (Farnadi et al. 2018), diversity (Mansoury et al. 2020; Isuﬁet al. 2021), and explainability (Liu et al. 2021a), by appropriate learning and modelling the graphs. Based on this, before diving into the speciﬁc types of graphs, it is necessary to understand the input data structures, their attributes, and how these attributes are related to the formation of graphs. Broadly speaking, there are two types of input data: user–item interaction and side information (Shi et al. 2014), from which side information can also be further classiﬁed into user/item attributes and external resources. Hence, we classify the input data into three major categories: interactive data, context data, and external data. The rationale of the classiﬁcation is twofold: (1) If it is interactive data for GLRS, e.g. user–item 123 L. Zhang et al. 812 Table 3 A summary of the data, corresponding graph types, recommendation tasks and representative techniques in GLRS Data Class Graph Type Recommendation Task Representative Technique Category Interactive Data Homogeneous graph Sequential Recommendation, Top-N Recommendation PRM (Gori et al. 2007), TEM (Wang et al. 2020b), DNN (Xu et al. 2019a; Isuﬁet al. 2021), GNN (Qiu et al. 2020b), DHM (Wu et al. 2019c; Abugabah et al. 2020) K-partite graph Link Prediction, Top-N Recommendation KML (Li and Chen 2013), RWM (Eksombatchai et al. 2018), DNN (Zheng et al. 2018), AM (Wang et al. 2019d), GNN (Wang et al. 2019f; Chen et al. 2020a) Hypergraph Top-N Recommendation DHM (Wang et al. 2020c) Multiple graph Link Prediction GDRM (Verma et al. 2019) Contextual Data Tree-based graph Sequential Recommendation, Rating Prediction LFM (Koenigstein et al. 2011), GDRM (He et al. 2016), DNN (Huang et al. 2019), AM (Gao et al. 2019b) Homogeneous graph Sequential Recommendation, Top-N Recommendation RWM (Jamali and Ester 2009), TEM (Gao et al. 2018), DHM (Song et al. 2019b; Chen et al. 4 From data to graphs 2019a) K-partite graph Next-item Recommendation, Link Prediction, Rating Prediction MPM (Yu et al. 2014), GDRM (Cen et al. 2019), TEM (He et al. 2015), AE (Zhang et al. 2019a), AM (Xin et al. 2019), GNN (Kim et al. 2019; Ying et al. 2018), Others (KNN) (Phuong et al. 2019) 813 Recommending on graphs: a comprehensive review from a data… g g p p Data Class Graph Type Recommendation Task Representative Technique Category Complex heterogeneous graph Top-N Recommendation, Rating Prediction LFM (Zhao et al. 2017), RWM (Bagci and Karagoz 2016; Ma et al. 2019), RM (Shi et al. 2020), MPM (Shi et al. 2015), GDRM (Shi et al. 2018; Fu et al. 2020), TEM (Chen et al. 2019d), AE (Zhang et al. 2016), AM (Han et al. 2018), GNN (Wang et al. 2019e), Others (Spectral Clustering) (Farseev et al. 2017) Hypergraph Top-N Recommendation RTGR (Bu et al. 2010), GNN (Yu et al. 2021), DHM (Gharahighehi et al. 2020) Multiple graph Top-N Recommendation, Rating Prediction LFM (Ma et al. 2011a), GDRM (Ali et al. 2020), AM (Vijaikumar et al. 2019), GNN (Fan et al. 2019b; Wu et al. 2019b), DHM (Monti et al. 2017) External Data K-partite graph Rating Prediction RWM (Jiang et al. 2018a), MPM (Lu et al. 2020; Yu et al. 2013) Complex heterogeneous graph Top-N Recommendation, Sequential Recommendation, Rating Prediction PRM (Catherine et al. 2017; Catherine and Cohen 2016), MPM (Ostuni et al. 2013), GDRM (Palumbo et al. 2017; Jiang et al. 2018b), TEM (Wang et al. 2018b, 2019a; Ai et al. 2018), DNN (Sun et al. 2018; Wang et al. 2019g), GNN (Wang et al. 2019c), DRL (Song et al. 2019a; Lei et al. 2020a), DHM (Sheu and Li 2020; Wang et al. 2020g; Zhou et al. 2020a; Wang et al. 2018c) Multiple graph Top-N Recommendation DNN (Wang et al. 2017) 12 814 L. Zhang et al. interactions, or context data on user/item side. (2) Whether the information should be from additional data sources, e.g. external knowledge bases. Table 3 illustrates the taxonomy of datasets, and their relations to graph types as well as techniques in GLRSs. Interactive Data in GLRSs. Recommendation is inheritly a tool and technique that provides users with potentially interesting items based on past user–item inter- actions. The user–item interaction data as a prerequisite for GLRS naturally form a relational connection between the user and item. 4 From data to graphs The connections can appear in explicit or implicit form depending on whether obvious numerical numbers or posi- tive/negative responses are directly observed. User implicit feedback data are inferred by indirect user behaviour such as clicking, page viewing, purchasing, watching and listening (example datasets are Last.fm (Cantador et al. 2011), Bing-News (Wang et al. 2018b,c), and YahooMusic (Dror et al. 2012)), whereas explicit interactive data are collected directly by prompting users to provide numerical feedback or clear attitudes on items such as ratings, likes, and dislikes (example datasets are MovieLens (Canta- dor et al. 2011), Amazon (McAuley et al. 2015; He and McAuley 2016), and Douban (Ma et al. 2011a; Zheng et al. 2017)). Such connections most commonly appear in the form of a user–item matrix, where each row encodes the preferences of a user with his/her interacted items. Each element in the user–item matrix represents the user interaction with the item, which can be binary numbers if implicit interactive data were found or non-negative numbers if explicit interactive data were found. Contextual Data in GLRSs. Context refers to the information collected during the interaction between the user and the item, such as timestamps, locations, or textual reviews, serving as an additional information source appended to the user–item inter- actions. Contextual data8 are rich information attached to an individual user or item that depicts user characteristics such as job and gender (in e.g. dataset) or item prop- erties such as description and product categories (in e.g. Foursquare (Gao et al. 2012), Yahoo! trafﬁc stream (Menon et al. 2011) datasets). Such information associated with the user/item can form natural connections in certain relationships and therefore result in a network structure that can be used as GLRSs. In addition to the directly col- lected data attached to users/items, indirect information can also be obtained by a preliminary analysis of the dataset, such as user similarities, and entities derived from texts (Phuong et al. 2019). Undoubtedly, there exist obvious relationships between the implicitly derived information and the analysed user/item. For instance, the user similarity exhibits a hidden connection between users, and the entities extracted from an item text are originally attached to the item. It is indisputable that by rationally taking the graphs formed from contextual data, it can enrich information sources, pro- vide more possibilities for graph learning-based recommendations, and improve the performance other than the accuracy of GLRSs (e.g. diversity). 8 Other terms may be used to indicate contextual data interchangeably such as side information, features, demographic data, categories, contexture information, etc. (Chen et al. 2020b). We do not distinguish them in this paper due to the same mathematical representation they share. 4 From data to graphs External Data in GLRSs. To obtain more valuable resources apart from user– item interactions and their attached data from datasets, one can also seek external sources for GLRSs. A typical example can be found in knowledge graphs (KGs) which are graph structured data that describe entities or concepts and connect them 123 Recommending on graphs: a comprehensive review from a data… 815 Fig. 2 An example tree structure from the Amazon and Last.fm websites. From the root node to the leaf node, the hierarchical structure gradually reﬁnes and embodies the types of goods and music Fig. 2 An example tree structure from the Amazon and Last.fm websites. From the root node to the leaf node, the hierarchical structure gradually reﬁnes and embodies the types of goods and music with different types of semantic relationships (Liu et al. 2019a). External data can also provide complementary information to overcome data sparsity issues though some of them e.g. KGs may require domain knowledge for recommendation in speciﬁc domains such as e-commerce. Cross-domain knowledge is another type of external data, which refers to user/item side information from multiple sources. For instance, user proﬁles across different networks connected through anchor links (e.g. the link which connects the same entity from different platforms is called an anchor link) (Wang et al. 2017), item proﬁles from different communities (Farseev et al. 2017), user social relationships through information sharing platforms (Shi et al. 2021) are leveraged to improve both recommendation accuracy and diversify recommendation output. Accordingly, multiple subgraphs are built upon various sources which are then jointly learned for recommendation tasks (Wang et al. 2017). Depending on different data shown above, one or more speciﬁc graph structures can be applied to form the input network to GLRSs. Based on this, we further innovatively propose to classify different graph types as tree-based graphs, homogeneous graphs, K-partite graphs, complex heterogeneous graphs, hypergraphs, and multiple graphs. The relations between different data and graph types are shown in Table 5 (“Appendix A”). 4.1 Tree-based graphs A tree-based graph where the items are organized in a hierarchy by a certain attribute of them (e.g. the category) is a natural yet powerful structure for human knowledge. It provides a machine- and human-readable description of a set of items and their par- allel or hierarchical relationships like afﬁliatedTo, subClass and isAPartOf relations. Such hierarchical relations between items have been widely studied and proven to be effective in generating high-quality recommendations (Menon et al. 2011; Koenigstein et al. 2011; Mnih 2012; Kanagal et al. 2012; He et al. 2016; Yang et al. 2016; Sun et al. 2017). Tree-structured data are mostly obtained in a user/item context in explicit con- textual data. The most common example is the categories of items. Typical domains of tree-based graphs in GLRSs consist of online products [e.g. the Amazon web store (McAuley et al. 2015)], foods (e.g. Gowalla (Liu et al. 2013)), movies (e.g. IMDB), and music (e.g. Last.fm). 123 123 L. Zhang et al. 816 Fig. 3 Homogeneous graph illustration. a An example attributed homogeneous graph with nodes repre- senting users and edges representing social relations between users in Song et al. (2019b). Node attributes are from implicit interactive data. b An example of a session graph with nodes representing interacted items and edges connecting consecutive interacted items in one session (Xu et al. 2019a) Fig. 3 Homogeneous graph illustration. a An example attributed homogeneous graph with nodes repre- senting users and edges representing social relations between users in Song et al. (2019b). Node attributes are from implicit interactive data. b An example of a session graph with nodes representing interacted items and edges connecting consecutive interacted items in one session (Xu et al. 2019a) Figure2 illustrates an example of tree-based graphs in Amazon and Last.fm to organize electronics or music by categories/genres. If a user buys a Monitor, she may possibly prefer Power Strips to match her Monitor instead of Aviation Electronics. This is due to both Monitor and Power Strips belonging to a higher layer category— Computers according to their intrinsic electrical characteristics. If a user prefers one song under a certain genre, she is more likely to favor other songs under this genre. Representative algorithms coping with such graphs include Latent Factor Models (LFM) (Kanagal et al. 2012), Graph Distributed Representation-based Techniques (GDRM) (He et al. 2016), Deep Neural Networks (DNN) (Huang et al. 4.1 Tree-based graphs 2019), and Attention Mechanism (AM) (Gao et al. 2019b). 9 Some articles also categorize graphs into directed and undirected graphs. In our point of view, the undi- rected graph can be readily converted into a directed graph by replacing each edge with two oppositely directed edges. Thus, in this survey, without loss of generality, we assume that all graphs are directed graphs. Recommending on graphs: a comprehensive review from a data… (2020), by considering the dynamic tran- sitions in the interactive sequence within one session, the authors construct directed homogeneous session graphs in which a node represents an interacted item and an edge connects adjacent interacted items while retaining the order of interaction. The nodes of these homogeneous graphs are without attribute information, which is referred to as non-attributed homogeneous graphs. In practice, many real-world networks usually have attributes with their nodes that are also important for making sense of modelling network topological as well as contextual information for recommendation purposes. Such networks with node attributes and a single type of nodes as well as edges are named attributed homogeneous graphs9 (Gao et al. 2018; Jamali and Ester 2009). An example can be found in a friend network where edges represent friendship (e.g. follow, like) between two users, and nodes represent users with attributes e.g. demographic information, or a sequence of items the user interacted with (Song et al. 2019b). In such a case, both social inﬂuence and user attributes can help to learn user preferences and thus affect the recommendation performance (Song et al. 2019b). Figure3 illustrates examples of non-/attributed homogeneous graphs to help resolve the cold start issue of recommender systems. Typical approaches in coping with such graphs include PageRank-based Models (PRM) (Gori et al. 2007), Translation-based Embedding Models (TEM) (Wang et al. 2020b; Gao et al. 2018), Random Walk-based Models (RWM) (Jamali and Ester 2009), Deep Neural Networks (DNN) (Xu et al. 2019a; Isuﬁet al. 2021), Graph Neural Networks (GNN) (Qiu et al. 2020b), and Deep Hybrid Models (DHM) (Wu et al. 2019c; Abugabah et al. 2020; Song et al. 2019b; Chen et al. 2019a). Recommending on graphs: a comprehensive review from a data… 817 Fig. 4 K-partite graph illustration. a A user–item bipartite graph with nodes representing user and items and edges representing interactions between users and items in Wang et al. (2019f). b A k-partite graph with nodes separated into four parts representing user, genre, movie, actor and director, and edges only existing among different node sets (Jiang et al. 2018a) Fig. 4 K-partite graph illustration. a A user–item bipartite graph with nodes representing user and items and edges representing interactions between users and items in Wang et al. (2019f). b A k-partite graph with nodes separated into four parts representing user, genre, movie, actor and director, and edges only existing among different node sets (Jiang et al. 2018a) it becomes possible to capture consecutive transitions among nodes while generat- ing accurate node embeddings for recommendation use. Based on this, in Xu et al. (2019a), Wu et al. (2019c), Abugabah et al. (2020), by considering the dynamic tran- sitions in the interactive sequence within one session, the authors construct directed homogeneous session graphs in which a node represents an interacted item and an edge connects adjacent interacted items while retaining the order of interaction. The nodes of these homogeneous graphs are without attribute information, which is referred to as non-attributed homogeneous graphs. In practice, many real-world networks usually have attributes with their nodes that are also important for making sense of modelling network topological as well as contextual information for recommendation purposes. Such networks with node attributes and a single type of nodes as well as edges are named attributed homogeneous graphs9 (Gao et al. 2018; Jamali and Ester 2009). An example can be found in a friend network where edges represent friendship (e.g. follow, like) between two users, and nodes represent users with attributes e.g. demographic information, or a sequence of items the user interacted with (Song et al. 2019b). In such a case, both social inﬂuence and user attributes can help to learn user preferences and thus affect the recommendation performance (Song et al. 2019b). Figure3 illustrates examples of non-/attributed homogeneous graphs to help resolve the cold start issue of recommender systems. it becomes possible to capture consecutive transitions among nodes while generat- ing accurate node embeddings for recommendation use. Based on this, in Xu et al. (2019a), Wu et al. (2019c), Abugabah et al. 4.2 Homogeneous graphs A homogeneous graph is a graph with a single type of objects and links in GLRSs. Typical examples are user graphs in social networks, which include different types of social relations between users (Verma et al. 2019; Fan et al. 2019b). User–user relations can be derived from interactive data if recommending other users as the ultimate goal of the recommender system such as friend recommendation (Fan et al. 2019b), or contextual data if social relations are potential critical factors affecting users’ next choices (Fan et al. 2019b; Farseev et al. 2017). The hypothesis behind tak- ing a user social effect as essential contextual information is that two connected users in a user graph usually share similar preferences and inﬂuence each other by recom- mending items. Besides, various relationships among items, e.g. item co-occurrence and substitution items (Wang et al. 2020b; Xin et al. 2019; Gori et al. 2007) in a user behaviour sequence, connect all the items together and thus result in a homoge- neous item graph. The co-occurrence relations in item graphs implicitly derived from interactive data not only reﬂect certain latent relations between items, but also reveal some behaviour patterns of users. It has been proved that the fusion of co-occurrence relations between items can yield signiﬁcant performance enhancements (Wang et al. 2020b). With the development of Graph Neural Network (GNN) and its varieties, 123 Recommending on graphs: a comprehensive review from a data… 4.3 K-partite graphs A k-partite graph, also called multipartite graph, is a kind of graph whose nodes can be partitioned into k different independent sets so that no two nodes within the same set are adjacent. In the GLRSs scenario, the k-partite graphs are unipartite graphs when 9 Some articles also categorize graphs into directed and undirected graphs. In our point of view, the undi- rected graph can be readily converted into a directed graph by replacing each edge with two oppositely directed edges. Thus, in this survey, without loss of generality, we assume that all graphs are directed graphs. 12 3 818 L. Zhang et al. k = 1, bipartite graphs when k = 2 and tripartite graphs when k = 3. In Xin et al. (2019), the authors constructed unipartite graphs where nodes are items, and different relations exist between two nodes. Relations can be extracted from contextual data or external sources like knowledge graphs. Especially, bipartite graphs have attracted signiﬁcant attention in areas like social network analysis (Tay and Lin 2014). They divide network nodes into two types and edges exist only between different types of nodes. User–item interactions can be naturally considered a bipartite graph, where the nodes represent users and items, and user nodes are linked with those interacted item nodes. Figure4a gives an example of a bipartite graph formed from user–item interactive data (Wang et al. 2019f). The edges of the bipartite graph can either be a single type or multiple types of interactions, e.g. click, like, purchase or view (Li and Chen 2013; Zheng et al. 2018; Wang et al. 2019f; Zhang et al. 2019a; Phuong et al. 2019; Nikolakopoulos and Karypis 2019; Gori et al. 2007; Sun et al. 2020). In addition to user–item interactions, auxiliary information of user/item can also be constructed as a bipartite graph. For instance, items and their attributes (e.g. pin-boards for Pinterest dataset (Ying et al. 2018)) can also be seen as two types of nodes in forming an item–entity bipartite graph. Apart from user–item interactions, bipartite graphs can be extended to multi-partite graphs by mining the contextual information of user/item and leveraging beneﬁcial attributes or related information such as user query, actors and actresses of movies as other groups of nodes (Kim et al. 2019; Fan et al. 2019a; Berg et al. 2017; Cheng et al. 2007; Lei et al. 2020a; Jiang et al. 2018a). 4.3 K-partite graphs For instance, the authors of Cheng et al. (2007) built a user–movie–genre tripartite graph for personalized recommendation. In Fig.4b, a multi-partite graph is formed by utilizing contextual information of items (movies), such as genre, actor, director (Jiang et al. 2018a). K-partite graphs can be weighted by assigning numerical points like a rating or similarity score on corresponding edges (Chen et al. 2020a; Zhang et al. 2019a; Yao et al. 2015). In Yao et al. (2015), the authors constructed a multi-partite graph incor- porating user, item, and their related contextual features. Each number on the edge represents the co-occurrence of two end-nodes. Figure4 shows examples of k-partite graphs in the GLRS scenario. Various GLRS-based approaches have been proposed to learn such kind of graphs, such as Kernel-based Machine learning methods (KML) (Li and Chen 2013), RWM (Eksombatchai et al. 2018; Jiang et al. 2018a), Meta-path based Methods (MPM) (Yu et al. 2014; Lu et al. 2020; Yu et al. 2013), GDRM (Cen et al. 2019), TEM (He et al. 2015), DNN (Zheng et al. 2018), Auto-Encoder (AE) (Zhang et al. 2019a), AM (Wang et al. 2019d), GNN (Wang et al. 2019f; Chen et al. 2020a) (see Table 3 for more details). 4.4 Complex heterogeneous graphs Complex heterogeneous graphs are related to graphs with multiple types of nodes and edges. The connections among nodes do not follow speciﬁc rules (Xu et al. 2019b; Jiang et al. 2018b; Shi et al. 2018; Lu et al. 2020; Wang et al. 2020f; Kyriakidi et al. 2020; Feng and Wang 2012; Zheng et al. 2017). For instance, in Wang et al. (2020f) 123 Recommending on graphs: a comprehensive review from a data… 819 Fig. 5 Complex heterogeneous graph illustration. a An example of complex heterogeneous graphs in which there are three kinds of nodes representing group, user, and item, respectively, and group–user, user–item two kinds of edges, for social recommendations (Shi et al. 2018). b An illustration of KGs with multiple kinds of entity nodes relation edges for exploring high-order user preferences for recommendations (Wang et al. 2019a) Fig. 5 Complex heterogeneous graph illustration. a An example of complex heterogeneous graphs in which there are three kinds of nodes representing group, user, and item, respectively, and group–user, user–item two kinds of edges, for social recommendations (Shi et al. 2018). b An illustration of KGs with multiple kinds of entity nodes relation edges for exploring high-order user preferences for recommendations (Wang et al. 2019a) the authors constructed long- and short-term graphs in which nodes are divided into user and item nodes, and edges can exist either between user and item, or among items. Complex heterogeneous graphs are usually non-weighted by default, but they can also be weighted. In Zheng et al. (2017), Shi et al. (2016), numbers on user–item edges depict the user’s rating on the item. Figure5a, b gives examples of weighted and unweighted complex heterogeneous graphs, respectively. The goal of complex heterogeneous graph learning is to obtain the latent vertex representations by mapping vertexes into a low-dimensional space, which then can be leveraged for recommendations (Xu et al. 2019b; Jiang et al. 2018b; Shi et al. 2018; Lu et al. 2020). The authors of Jiang et al. (2018b) constructed a complex heterogeneous graph with four types of nodes and ten types of edges. The node representations are learned for citation recommendations. In Shi et al. (2018), the authors constructed a heterogeneous graph with user, item, and various types of attributes as nodes. Finally, the learned user/item representation is adopted to predict the user’s rating score of the item to make recommendations. Differently, in Kyriakidi et al. 4.4 Complex heterogeneous graphs The resources of all KGs used for GLRSs have been collected and are displayed in Table 4. Typical technologies related to learn complex heterogeneous graphs include LFM approaches(Zhaoetal.2017),PRM(Catherineetal.2017;CatherineandCohen2016), RWM approaches (Bagci and Karagoz 2016; Ma et al. 2019), Rule-based Model (RM) (Shi et al. 2020), MPM (Shi et al. 2015; Ostuni et al. 2013), GDRM (Shi et al. 2018; Fu et al. 2020; Palumbo et al. 2017; Jiang et al. 2018b), TEM (Chen et al. 2019d; Wang et al. 2018b, 2019a; Ai et al. 2018), AE (Zhang et al. 2016), AM (Han et al. 2018), Graph Neural Network (GNN) (Wang et al. 2019e,c), Deep Reinforcement Learning (DRL) (Song et al. 2019a; Lei et al. 2020a), and DHM (Sheu and Li 2020; Wang et al. 2020g; Zhou et al. 2020a; Wang et al. 2018c). Other techniques such as Spectral Clustering (Farseev et al. 2017) are also adopted to learn such kind of graphs. (Refer to Table 3 for more details) 4.4 Complex heterogeneous graphs (2020), the connection mode can be revealed via path traversing, so that the similarities between two nodes can be calculated for a recommendation. Another typical example falling into this category is a Knowledge Graph (KG). KG is a multi-relational graph composed of entities as nodes and relations as different types of edges as illustrated in Fig.5b. Each edge of KG represents a triple of the form (head entity, relation, tail entity), also called a fact, indicating that two entities are connected by a speciﬁc relation. Recent years have witnessed a rapid growth in KG application in recommendation, resulting in promising improvements in both recommendation accuracy and explainability due to the rich structured information that KG provides about the items. Existing KGs, e.g. Yago (Suchanek et al. 2007), DBPedia (Lehmann et al. 2015), provide auxiliary information apart from user–item interactions. The relational properties in KGs break down the independent interaction assumption by linking items with their attributes. Meanwhile, the introduction of KGs alleviates the data sparsity and cold-start issues raised in recommender systems (Wang et al. 2019b; Ma et al. 2019; Wang et al. 2020g; Zhou et al. 2020b; Song et al. 2019a; Yang and Dong 2020; Wang et al. 2020a; Ai et al. 2018; Zhang et al. 2016; Ma et al. 2019; Catherine and Cohen 2016; Shi et al. 2020; Chen et al. 2019d; Wang et al. 2020g; 123 12 820 L. Zhang et al. Zhou et al. 2020a,b; Cao et al. 2019; Yu et al. 2013; Ostuni et al. 2013). However, various types of entities and relations in KGs also pose the challenge of capturing semantically interconnected information for effective recommendations. In addition, how to reasonably and vividly provide recommendation results through KG internal reasoningandthelinkageamonguser–iteminteractionsdeservesmoreattention(Wang et al. 2019g), for instance, how to impartially and convincingly explain the reasoning process of the recommendation list to the target user. The resources of all KGs used for GLRSs have been collected and are displayed in Table 4. Zhou et al. 2020a,b; Cao et al. 2019; Yu et al. 2013; Ostuni et al. 2013). However, various types of entities and relations in KGs also pose the challenge of capturing semantically interconnected information for effective recommendations. In addition, how to reasonably and vividly provide recommendation results through KG internal reasoningandthelinkageamonguser–iteminteractionsdeservesmoreattention(Wang et al. 2019g), for instance, how to impartially and convincingly explain the reasoning process of the recommendation list to the target user. 123 4.5 Hypergraphs Hypergraphs are deﬁned as a generalization of graphs in which the edges are arbitrary non-empty subsets of the vertex set (Agarwal et al. 2006). Instead of having edges between pairs of vertices, hypergraphs have edges that connect sets of two or more vertices. Correspondingly, such edges of hypergraphs are called hyperedge. If the hyperedge always has the degree of 2, the hypergraph reduces to an ordinary graph. For clarity, we only refer to hypergraphs with multiple hyperedge degrees. The moti- vation for introducing hypergraphs for recommendations is twofold (Feng et al. 2019): ﬁrst, the data correlations can be more complex than the pair-wise relationship, which is difﬁcult to model with traditional graph structures; second, the data representations can be multi-modal, which means that data can be connected through e.g. text infor- mation, visual information, or social connections, which is difﬁcult to capture with the traditional graphs. Thus, a hypergraph is a way to model a more general data structure, and the recommendation performance can be improved through the modelling of the high-order proximity in the constructed hypergraphs (Yu et al. 2021). Recent years witness the learning of hypergraphs in promoting the development of recommender systems (Wang et al. 2020c; Bu et al. 2010; Li and Li 2013; Tan et al. 2011; Zhu et al. 2016; Mao et al. 2019; Yu et al. 2021; Gharahighehi et al. 2021, 2020). Hypergraphs can be generalized explicitly from data sources or derived implicitly indi- rectly through, for instance, a clustering technique. In Bu et al. (2010), the authors constructed a hypergraph with six types of vertices and nine types of hyperedges rep- resenting complex relationships of nodes rather than a pair-wise form. Speciﬁcally, user–item interactive data, contextual data such as social relations, tagging and album information, are adopted. Meanwhile, the group information obtained through 123 Recommending on graphs: a comprehensive review from a data… 821 Table 4 A collection of commonly used knowledge graphs Name Domain Type Main Knowledge Source General KG YAGO (Suchanek et al. 2007) Cross-Domain Wikipedia Freebase (Bollacker et al. 2008) Cross-Domain Wikipedia, NNDB, FMD, MusicBrainz DBpedia (Lehmann et al. 2015) Cross-Domain Wikipedia Satori20 Cross-Domain Web Data CN-DBPedia (Xu et al. 2017) Cross-Domain Baidu Baike, Hudong Baike, Wikipedia (Chi- nese) NELL (Carlson et al. 2010) Cross-Domain Web Data Wikidata21 Cross-Domain Wikipedia, Freebase Google Knowledge Graph22 Cross-Domain Web data Facebooks Entities Graph23 Cross-Domain Wikipedia, Facebook data ConceptNet (Speer et al. 4.5 Hypergraphs 2017) Cross-Domain Web data MultiWordNet (Pianta et al. 2002) Cross-Domain Princeton WordNet Babelfy (Moro et al. 2014) Cross-Domain BabelNet Open Multilingual Wordnet (Bond and Paik 2012) Cross-Domain Wiktionary, Unicode Common Locale Data Repository Domain Speciﬁc KG Bio2RDF (Belleau et al. 2008) Biological Domain Public bioinformatics databases, NCBIs databases KnowLife (Ernst et al. 2014) Biomedical Domain Scientiﬁc literature, Web portals IMDB24 Movie Domain Web data KnowIME (Yan et al. 2020) Intelligent Manufacturing Domain Internet, Baidu Encyclopedia, and related intelligent manufacturing websites Table 4 A collection of commonly used knowledge graphs Web Data Baidu Baike, Hudong Baike, Wikipedia (Chi- nese) Web Data Wikipedia, Freebase Web data K nearest neighbour and K-means also forms part of the hyperedges. Pliakos and Kotropoulos (2014) used a uniﬁed hypergraph model boosted by group sparsity opti- mization and encapsulated the high order connections among users, images, tags, and geo-information for tag recommendation. An hyperedge can be weighted by binary values indicating whether to participate or whether the link exists between two nodes, or integer values indicating the frequency of participation (Yu et al. 2021). Besides, 123 L. Zhang et al. 822 Fig. 6 a An example hypergraph in which nodes are represented in different colours and edges exist between colours of nodes (Wang et al. 2020c). b An example of multiple graphs in which there is a user–item–attribute tripartite graph and a homogeneous social graph (Wang et al. 2017) Fig. 6 a An example hypergraph in which nodes are represented in different colours and edges exist between colours of nodes (Wang et al. 2020c). b An example of multiple graphs in which there is a user–item–attribute tripartite graph and a homogeneous social graph (Wang et al. 2017) decimal values such as similarity scores can also be assigned as edge weight depend- ing on recommendation context (Bu et al. 2010; Tan et al. 2011). Different weights assigned to a hypergraph differentiate the impact of each hyperedge. An example of a hypergraph is illustrated in Fig.6a, in which the hypergraph consists of three nodes with one hyperedge in September 2017 and four nodes with two hyperedges in September 2019 (Wang et al. 2020c). Representative approaches in dealing with hypergraphs in GLRS include Regular- ization Theory-based Graph Ranking (RTGR) (Bu et al. 2010), GNN (Yu et al. 2021), and DHM (Wang et al. 2020c; Gharahighehi et al. 2020). More speciﬁc details are found in Sect.5 and Table 3. 4.6 Multiple graphs Incorporating various types of information involved into a graph will often make the composed graph too complicated and not conducive to subsequent algorithms. There- fore, many researchers choose to split complex heterogeneous graphs into multiple subgraphs for learning separately. While such a “divide and conquer" strategy disas- sembles the complexity of the graph, it can also be adapted to certain graph learning algorithms (Verma et al. 2019; Fan et al. 2019b; Wu et al. 2019b; Vijaikumar et al. 2019; Wang et al. 2017). For instance, in Fan et al. (2019b), the authors disassem- bled the complex multigraph with more than one type of edge between two nodes, into three homogeneous subgraphs, which are then learned separately through the DeepWalk technique for friend recommendation. More researches choose to separate interactive data from contextual data/external data as a powerful supplementary information for learning user/item representations and meanwhile to some extent alle- viating the cold-start issue (Liu et al. 2020; Ali et al. 2020). The authors of (Fan et al. 2019b; Wu et al. 2019b) extract the user social relations from original data to build a homogeneous graph of the social network. Similarly, in Fig.6b, the authors of Wang et al. (2017) use the users as anchor links (e.g. the link which connects the same entity from different platforms is called an anchor link) to obtain the user social informa- tion from cross-domain knowledge resulting in homogeneous subgraphs to improve recommendation performance. In Monti et al. (2017), Isuﬁet al. (2021), the authors Recommending on graphs: a comprehensive review from a data… 823 construct two homogeneous graphs, namely an item graph and a user graph in which nodes represent items and users, respectively, and edges represent similarities between adjacent nodes. construct two homogeneous graphs, namely an item graph and a user graph in which nodes represent items and users, respectively, and edges represent similarities between adjacent nodes. Another advantage of leveraging a multi-graph for recommendation is scalability. When a new data source is added, it can be constructed as a separate graph independent of the original data source, and the learned embedding representations can be merged in the upper layer. In such cases, different data sources can be separated into a more concise and clear form for subsequent maintenance and learning (Vijaikumar et al. 2019). ) Various ways can be adopted to learn multiple graphs for recommendations. For graphs with similar structures (e.g. 4.6 Multiple graphs either homogeneous or heterogeneous), similar learning strategies are usually adopted to obtain the low-dimensional representation of nodes (Ali et al. 2020; Monti et al. 2017), while it is usually necessary to adopt different learning strategies for graphs with different structures. This is reasonable because usually a certain learning strategy should be performed under certain assumptions or rules (Ma et al. 2011a; Chen et al. 2019a). To deal with multiple graphs, the most common way is to learn each independent graph separately and then aggregate the results for recommendation purposes. Typical examples are GDRM (Verma et al. 2019; Ali et al. 2020), LFM (Ma et al. 2011a), AM (Vijaikumar et al. 2019), GNN (Fan et al. 2019b; Wu et al. 2019b), DHM (Monti et al. 2017), and DNN (Wang et al. 2017). 4.7 Graph discussion Graphs, ranging from a ﬂat tree-based structure to a complex network structure, from a homogeneous network to a heterogeneous one, evolve from both structural and con- textual sides. As summarized in “Appendix Table 5”, though many different datasets overlap across various graphs and recommendation tasks, it is undeniable that there is no perfect graph type that can embrace all types of data or solve all problems of the recommender system. However, we can still make several observations: First, tree-based graphs are mainly derived from e-commerce datasets (e.g. Amazon and JD) in which types of commodities can be broken down by the category’s level of granularity.Suchkindoftreestructureprovidesrecommendersystemswithaparadigm which can be further reﬁned in a “top-down” manner. Second, homogeneous graphs can be formed by leveraging user social networks (user graph) or interlinking of items (item graph). A session graph is a kind of directed item graph with nodes of items that are clicked by the user and links of relations that are according to the clicked order. Homogeneous graphs can be used to mine the relationships between a single type of node (e.g. users/items/sessions) to make targeted modelling for recommendations. Third, most k-partite graphs, especially for k = 2, are constructed based on user– item interactions, which are the preliminary requirements for RS and can be naturally formed into a graph with users and items as two groups of nodes. For generalized k-partite graphs, they normally formalize by extending the user–item bipartite graph 123 824 L. Zhang et al. with contextual knowledge w.r.t. users/items that are deemed to potentially beneﬁt recommendation performance. Besides, complex heterogeneous graphs incorporating multi-types of nodes and relations reveal the intricate relationship between user, item, and other related infor- mation from contextual/external data sources in real scenarios. Especially, KGs are generally leveraged in the domains of movies, music, books, or news, where named entities are highly recognizable and have been witnessed, and most of which can be found in the corresponding entries in e.g. Wikipedia, DBpedia, Yago, etc. Furthermore, hypergraphs have recently been introduced to represent the connec- tions between sample groups such as user groups according to social relationships, or item groups according to a user’s co-purchase history, which breaks the traditional node-to-node pattern, pursuing a higher-level representation of data structure. 4.7 Graph discussion With their diversity, heterogeneous graphs with various aspects of information can be used to model more complex situations than other types of graphs so as to better solve the cold-start and data sparsity issues of recommender systems. However, such complex- ity of both graph structure and content leads to a more complicated modelling process and brings challenges that cannot be ignored. One possible solution is to break down such complexity into multiple subgraphs. In this way, the complex relationship of the graph is degraded, and the relationship between nodes is more concise and clear. To reveal the group knowledge among nodes and meanwhile incorporate multi-modal information for better recommendations, a hypergraph is proposed to express multiple relationships beyond the pair-wise relations. With the development of technology, the types of graphs that computers can process tend to be increasingly complex and ﬁne- grained, which suggests that RS can handle more sophisticated and multi-dimensional problems and scenarios. 5 Graph-based models for GLRS In this section, we will go through various models adopted in GLRS and analyse them from the technical perspective, as shown in Fig.7. Many GLRS recommendation algorithms are not only based on a speciﬁc technology but often include multiple different types of technologies. Therefore, our classiﬁcation is mainly based on the technology used by the key components of the model pointed out in the paper. A comprehensive technology summary in GLRS is found in Table 6 (“Appendix B”). 5.1 Traditional techniques Traditional machine learning techniques used in GLRS can generally be classi- ﬁed into PageRank-based models (PRM), regularization theory-based graph ranking techniques (RTGR), kernel-based machine learning techniques (KML), latent factor models (LFM), and others (e.g. association mining and clustering). Graph learning- based traditional techniques can be used for multiple types of graphs from various domains of data sources. The classic PageRank algorithm computes an importance score for every node according to the graph connectivity, which is usually exploited to 123 12 Recommending on graphs: a comprehensive review from a data… Recommending on graphs: a comprehensive review from a data… 825 Fig. 7 A categorization of GLRS techniques from the technical perspective Fig. 7 A categorization of GLRS techniques from the technical perspective tackle the recommendation as a ranking problem. Many adapted PageRank algorithms have been proposed and widely applied to a variety of graphs like homogeneous item graphs (Gori et al. 2007), K-partite graphs (Shams and Haratizadeh 2017; Jäschke et al. 2007; Musto et al. 2017, 2021), and complex heterogeneous graphs (Catherine and Cohen 2016; Lee et al. 2013) for recommendation. A separate line of work in this area—regularization theory-based graph ranking techniques—is widely exploited to learn the ranking results on hypergraphs for recommendations (Bu et al. 2010; Li and Li 2013; Tan et al. 2011; Mao et al. 2019). They usually use hypergraphs to model high-order relations among various types of objects in social networks and use the regularization framework (Zhou and Schölkopf 2004) for ranking graph data. Some research works use kernel-based machine learning techniques to compute similarities between structured objects, such as nodes of a graph, that cannot be naturally repre- sented by a simple set of numbers, and demonstrate their effectiveness in a variety of graphs in RS (Li and Chen 2013; Yajima 2006; Li et al. 2014; Fouss et al. 2012; Ostuni et al. 2014) (homogenous graph, K-partite graph and knowledge graph). For instance, Yajima (2006) used a Laplacian kernel to capture the positional relations among nodes on a homogenous item graph and built one-class SVM models for each user to recommend items that are positionally closer to their previously bought items. Li and Chen (2013) propose a generic kernel-based machine learning approach of link prediction in bipartite graphs and apply it in recommender systems. In a later work, Li et al. (2014) inspect a spectrum of social network theories to systematically model the multiple facets of a homogeneous user graph in social networks and infer user preferences. They design and select kernels corresponding to major social network theories and then adapt a non-linear multiple kernel learning technique to combine tackle the recommendation as a ranking problem. Many adapted PageRank algorithms have been proposed and widely applied to a variety of graphs like homogeneous item graphs (Gori et al. 2007), K-partite graphs (Shams and Haratizadeh 2017; Jäschke et al. 2007; Musto et al. Recommending on graphs: a comprehensive review from a data… 2017, 2021), and complex heterogeneous graphs (Catherine and Cohen 2016; Lee et al. 2013) for recommendation. A separate line of work in this area—regularization theory-based graph ranking techniques—is widely exploited to learn the ranking results on hypergraphs for recommendations (Bu et al. 2010; Li and Li 2013; Tan et al. 2011; Mao et al. 2019). They usually use hypergraphs to model high-order relations among various types of objects in social networks and use the regularization framework (Zhou and Schölkopf 2004) for ranking graph data. Some research works use kernel-based machine learning techniques to compute similarities between structured objects, such as nodes of a graph, that cannot be naturally repre- sented by a simple set of numbers, and demonstrate their effectiveness in a variety of graphs in RS (Li and Chen 2013; Yajima 2006; Li et al. 2014; Fouss et al. 2012; Ostuni et al. 2014) (homogenous graph, K-partite graph and knowledge graph). For instance, Yajima (2006) used a Laplacian kernel to capture the positional relations among nodes on a homogenous item graph and built one-class SVM models for each user to recommend items that are positionally closer to their previously bought items. Li and Chen (2013) propose a generic kernel-based machine learning approach of link prediction in bipartite graphs and apply it in recommender systems. In a later work, Li et al. (2014) inspect a spectrum of social network theories to systematically model the multiple facets of a homogeneous user graph in social networks and infer user preferences. They design and select kernels corresponding to major social network theories and then adapt a non-linear multiple kernel learning technique to combine 123 826 L. Zhang et al. the multiple kernels for recommendation. Latent factor models are usually adopted by many researchers to learn the latent feature of users and items for recommendation purposes. In GLRS, LFM can be accompanied by regularization terms constraining the trust/distrust relations between users to integrate contextual information extracted from users’ social networks (Du et al. 2011; Ma et al. 2009; Sun et al. 2015). Ma et al. (2008), Ma et al. (2011b) proposed a factor analysis based on probabilistic matrix factorization to alleviate the data sparsity and poor prediction accuracy problems in recommender systems by incorporating social contextual information, such as users’ social trust networks and social tags. Recommending on graphs: a comprehensive review from a data… In addition, there are many other traditional machine learning techniques used to model the relations between the nodes in GLRS, such as clustering (Farseev et al. 2017; Song et al. 2011), association mining (Huang et al. 2004) and graphing complex numbers (Xie et al. 2015), and applied to K-partite graphs (Song et al. 2011), complex heterogeneous graphs (Huang et al. 2004; Xie et al. 2015), and multiple graphs (Farseev et al. 2017). 123 5.2 Path-based techniques 8 Bibliographic network schema and meta paths deﬁned in Sun et al. (2011) favored for a long period in the GLRS domain for generating paths in homogeneous as well as heterogeneous graphs (Jamali and Ester 2009; Feng and Wang 2012; Jiang et al. 2018a; Eksombatchai et al. 2018; Nikolakopoulos and Karypis 2019; Gori et al. 2007; Li and Chen 2013; Yin et al. 2010; Vijaikumar et al. 2019; Sharma et al. 2016; Mao et al. 2019; Bagci and Karagoz 2016; Ma et al. 2019; Cheng et al. 2007; Yao et al. 2015). For instance, Jiang et al. (2018a) propose a generalized random walk with restart model on a k-partite graph to extract the paths. Then, a BPR-based machine learning technique is leveraged to learn the weights of links in the graph. favored for a long period in the GLRS domain for generating paths in homogeneous as well as heterogeneous graphs (Jamali and Ester 2009; Feng and Wang 2012; Jiang et al. 2018a; Eksombatchai et al. 2018; Nikolakopoulos and Karypis 2019; Gori et al. 2007; Li and Chen 2013; Yin et al. 2010; Vijaikumar et al. 2019; Sharma et al. 2016; Mao et al. 2019; Bagci and Karagoz 2016; Ma et al. 2019; Cheng et al. 2007; Yao et al. 2015). For instance, Jiang et al. (2018a) propose a generalized random walk with restart model on a k-partite graph to extract the paths. Then, a BPR-based machine learning technique is leveraged to learn the weights of links in the graph. To integrate different types of objects and links in heterogeneous networks, the work of Sun et al. (2011) proposed the concept of a meta-path, which is adopted by many later researches (Yu et al. 2013; Cen et al. 2019; Lu et al. 2020; Shi et al. 2018, 2015; Zheng et al. 2017; Shi et al. 2016; Ostuni et al. 2013; Yu et al. 2014). Speciﬁcally, a meta-path is a path deﬁned on the graph of network schema TG = (A, R),25 and normally denoted in the form of A1 R1 −→A2 R2 −→A3 . . . Rl −→Al+1, which deﬁnes a composite relation R = R1 ◦R2 ◦. . . ◦Rl between types A1 and Al+1, where →explicitly shows the direction of a relation from graph G, ◦denotes the composition operator on relations. Figure8 illustrates two examples of meta-paths Fig. 8b, c derived from network schema Fig. 8a. 5.2 Path-based techniques For heterogeneous graphs with multiple types of nodes and relations, the basic idea of earlier recommendation strategies is to leverage path-based semantic relatedness between users and items over the constructed graphs. Different from similarity- based techniques based upon the item/user attributes, path-based techniques especially emphasize the essential role of links in graphs, and links between start node and end node can form a path serving a recommendation purpose. In this case, the underlying relationships via network propagation show particularly important for indirectly con- nected objects. They can be used mainly for complex heterogeneous graphs (Bagci and Karagoz 2016; Ma et al. 2019; Feng and Wang 2012; Shi et al. 2015, 2018, 2020; Zheng et al. 2017; Shi et al. 2016; Ostuni et al. 2013) and k-partite graphs (Li and Chen 2013; Cheng et al. 2007; Yao et al. 2015; Jiang et al. 2018a; Nikolakopoulos and Karypis 2019; Sharma et al. 2016; Eksombatchai et al. 2018; He et al. 2015; Cen et al. 2019; Lu et al. 2020; Yu et al. 2013, 2014), but can also be adopted for multiple and homogeneous graphs (Yin et al. 2010; Vijaikumar et al. 2019; Jamali and Ester 2009; Gori et al. 2007), covering various domains such as POI recommendation, academic and book, e-commerce, and entertainment domains. Earlier studies leverage a series of predeﬁned rules to generate a path on the constructed graphs followed by different similarity measurements for ranking the candidate items for recommendation (He et al. 2015; Catherine and Cohen 2016; Catherine et al. 2017; Kyriakidi et al. 2020). Another graph tracing algorithm initially designed for homogeneous networks is a random walk-based algorithm (Andersen et al. 2008). It starts at a node and follows outgoing edges, uniformly at random or according to predeﬁned transition probability, until the stop condition is reached. The output paths indicate the context of connected vertices. The randomness of walks gives the ability to explore the graph while considering both the global and local structural information by walking through neighbouring vertices. The random walk mechanism enables capturing complex, high-order, and indirect relations between nodes for rec- ommendations. Due to these advantages, random walk and its various variants are 123 12 Recommending on graphs: a comprehensive review from a data… Recommending on graphs: a comprehensive review from a data… 827 Fig. 8 Bibliographic network schema and meta paths deﬁned in Sun et al. (2011) Fig. 25 Please refer to Deﬁnition 1 and 2 in Section 3.1 for the meaning of the symbols. 26 Meta-path-based approaches usually require handcrafted features to represent path semantics and thus further require domain knowledge (Sun et al. 2011). 26 Meta-path-based approaches usually require handcrafted features to represent path semantics and thu further require domain knowledge (Sun et al. 2011). 25 Please refer to Deﬁnition 1 and 2 in Section 3.1 for the meaning of the symbols. 5.2 Path-based techniques When a user-speciﬁc meta-path e.g. P = (A1A2 . . . Al) has been given, several similarity measures can be deﬁned for a pair-wise nodes comparison, namely to compare vi ∈A1 and v j ∈Al according to a series of paths derived based on P, referred to path instances. Random walk is one representative to generate paths instances p ∈P following the predeﬁned meta-path schema (Shi et al. 2015). To further learn the attributed heterogeneous information network (HIN) for better recommendations, later studies attempt to combine meta- paths with a traditional latent model, e.g. FM (Zhao et al. 2017), MF (Shi et al. 2018; Yu et al. 2014). Though random-walk-based similarity measures require less domain knowledge compared to meta-path-based measures,26 the latter turn out to be more meaningful and interpretable in most GLRSs (Sun et al. 2011). 25 Please refer to Deﬁnition 1 and 2 in Section 3.1 for the meaning of the symbols. 26 Meta-path-based approaches usually require handcrafted features to represent path semantics and thus further require domain knowledge (Sun et al. 2011). 12 3 828 L. Zhang et al. Fig. 9 A toy example of embedding a graph into 2D space with different granularities (Cai et al. 2018). G1,2,3 denotes the substructure containing node v1, v2, v3 Fig. 9 A toy example of embedding a graph into 2D space with different granularities (Cai et al. 2018). G1,2,3 denotes the substructure containing node v1, v2, v3 Despite that path-based similarity strategies have achieved initial success in improving RS accuracy to some extent, challenges still exist. First, meta-path-based similarities rely on explicit path reachability and the quality would be affected by the sparse and noisy input data, especially for links that are accidentally formed but do not convey meaningful information for recommendations. Second, the explicit path relatedness derived from the path-based similarity does not necessarily have a positive impact on recommendation performance. For instance, the work of Yu et al. (2013) learns a linear weighting mechanism to integrate the extracted meta-paths for the subsequent recommendations, ignoring the complicated mapping mechanism of the constructed k-partite graphs. Third, path-based similarity strategies need to generate similarity scores for all candidate items at each step for every user which reduces the effectiveness of the system and thus makes it difﬁcult to be applied in a large-scale scenario. 5.3 Graph embedding-based techniques p p p The recent advances on GE-based GLRSs have been largely inﬂuenced by the skip- gram model (Mikolov et al. 2013a) designed originally to learn word representations w.r.t. the words context in a sequence e.g. a sentence. For a speciﬁc type of graph, skip-gram can be used on path sequences extracted from the graph in which nodes can be analogous to words, and paths can be analogous to sentences. Inspired by this, a series of graph distributed representation-based GLRSs using skip-gram-related algo- rithms, e.g. DeepWalk (Perozzi et al. 2014), LINE (Tang et al. 2015), and Node2vec (Grover and Leskovec 2016), gradually emerged and achieved encouraging success (Verma et al. 2019; Palumbo et al. 2017; Jiang et al. 2018b; Vijaikumar et al. 2019; Gao et al. 2018; Ali et al. 2020; He et al. 2016; Wang et al. 2020a; Gharahighehi et al. 2021; Fu et al. 2020). For instance, the authors of Gao et al. (2018) apply DeepWalk which aims to maximize the average logarithmic probability of all vertex context pairs in a random walk sequence, to learn user and item representations on a multi-source homogeneous item graph to consider item structure, textual content and tag informa- tion simultaneously which are then used for collaborative ﬁltering. In Palumbo et al. (2017) the authors generate user and item representations with Node2vec, an exten- sion of DeepWalk by leveraging a biased random walk to navigate the neighbourhood nodes, on a complex heterogeneous knowledge graph, which are then used to com- pute property-speciﬁc relatedness scores between users and items as the input for the learning to rank approach, resulting in optimizing top-N item recommendations. Another research line of GE-based technique adopts translation-based embedding models inspired by Mikolov et al. (2013b), e.g. TransE (Bordes et al. 2013). Different from DeepWalk-related approaches, TransE explicitly models entities and relation- ships among entities into the same space or different spaces while preserving certain information of the graph, which is later generalized into a hyperplane translation (TransH (Wang et al. 2014)) and a translation in separate entity space and relation spaces (TransR (Lin et al. 2015)). The basic idea behind TransE is that the relation- ship between two entities corresponds to a translation between the embeddings of entities, that is, h + r = t where h, t, and r represent head entity, tail entity and relation between h and t in triplet (h,r, t) in a graph. 5.3 Graph embedding-based techniques The motivation for applying graph embedding (GE) strategies lies in that they can provide an effective yet efﬁcient way to solve the graph analytics problem (Cai et al. 2018). GE-based techniques are mainly applied to complex heterogeneous graphs (Fu et al. 2020; Wang et al. 2020a; Jiang et al. 2018b; Palumbo et al. 2017; Wang et al. 2018b; Cao et al. 2019; Wang et al. 2021b; Cao et al. 2019; Ai et al. 2018; Chen et al. 2019d) from multiple data sources but can also be applied to homogeneous graphs (Gao et al. 2018; Wang et al. 2020b), tree graphs (He et al. 2016), k-partite graphs (Li et al. 2021b), and multiple graphs (Verma et al. 2019; Ali et al. 2020) in different recommendation domains. Speciﬁcally, graph embedding converts a graph into a low dimensional space in which the graph information can be retained as much as possible. By representing a graph as a (or a set of) low-dimensional vector(s), graph algorithms can be applied efﬁciently. Figure9 illustrates how graph embedding projects a graph into the vector space with different granularities, e.g. w.r.t. node/edge/substructure/whole graph (Cai et al. 2018). Some researches differentiate graph representation learning and graph embedding by comparing the dimension of the output embedding vectors with the dimension of the inputs (Cai et al. 2018). Graph embedding focuses on learning the low-dimensional representations, while graph representation learning does not require the learned representations to be low dimensional. Though they have slight differences, Recommending on graphs: a comprehensive review from a data… Recommending on graphs: a comprehensive review from a data… 829 we do not make a special distinction in this survey. Essentially, the two approaches aim to project a graph into the vector space while preserving the graph structure and capturing the connectivity information within the graph to serve the recommendation task. The mapping can be deﬁned as: we do not make a special distinction in this survey. Essentially, the two approaches aim to project a graph into the vector space while preserving the graph structure and capturing the connectivity information within the graph to serve the recommendation task. The mapping can be deﬁned as: f : vi →xi ∈Rd (2) (2) where d ≪\|V\|, and xi = {x1, x2, . . . , xd} is the embedded or learned vector that captures the structural properties of node vi. 5.3 Graph embedding-based techniques Researchers attempted to adopt such translation-based models for e.g. a knowledge graph embedding for recommen- dation (Ai et al. 2018; Wang et al. 2020b; Chen et al. 2019d; Cao et al. 2019; Wang et al. 2018b, 2019a). For example, Wang et al. (2020b) assign a basic representa- tion and various relational ones for each item from a directed homogeneous graph via TransE, which are then combined dynamically by temporal kernel functions, providing both recommendations and explanations. Chen et al. (2019d) adopt TransH to embed the objects’ social relationships from the homogeneous graph into a shared lower- 123 830 L. Zhang et al. dimensional space and learn a user’s dynamic preference via a probabilistic model from the user–item bipartite graph. Finally, the recommendation list is generated with item-based collaborative ﬁltering. 5.4 Deep learning-based techniques Deep learning (DL) has driven a remarkable revolution in recommender applications as can be seen by the number of research publications on deep learning-based rec- ommendation techniques having increased exponentially recently. It has been applied to multiple types of graphs from single graph to multiple graphs, from homogeneous to heterogeneous graphs from different recommendation domains. To draw an overall concept of this ﬁeld, we further classify the existing DL-based approaches into DNN, auto-encoder, attention mechanism, reinforcement learning, graph neural network, transformer-based approaches and deep hybrid models as shown in Fig.7. 5.4.1 Deep neural network (DNN) A deep neural network (DNN) is adopted to model complex non-linear relationships with generated compositional models where the object is expressed as a layered com- position of primitives. By piling up layers, composition of features from lower layers can be extracted and learned (Bengio 2009). DNNs are typically feedforward networks in which data ﬂow from input layer, are transformed into vector representations, and projected into a different space to an output layer without looping back. DNN-based techniques can be used for tree graphs (Huang et al. 2019) and complex heteroge- neous graphs (Sun et al. 2018; Mezni et al. 2021; Wang et al. 2019g) in e-commerce and entertainment domains, or homogeneous graphs (Wu et al. 2019a) in social net- work domain. Taking graphs as input, DNN can learn high-order interactions among nodes by stacking layers with non-linear transformations (Wang et al. 2017; Wu et al. 2019a). For instance, Wang et. al (Wang et al. 2017) apply several multiple layers with a pooling operation to explore interaction patterns between user, item and their attributes from multiple cross-domain graphs taking nodes’ one hot encoding as input. To learn the propagation effect of social inﬂuence of users, they further employ the smoothness regularization term to closely embed users connected by social networks from different data sources. Another variation in DNN is Recurrent neural networks (RNNs) (Cho et al. 2014; Hochreiter and Schmidhuber 1997). The original superiority of RNNs can well capture the dependencies among items from graphs in time-sensitive user–item interaction sequences or in session-based recommendation settings. However, the limitations lie in that it is difﬁcult to model dependencies in a longer sequence, and training is burdened with high cost, especially with the increase in sequence length. Thus, some works combine RNN with other mechanisms to balance this disadvantage of RNN. For instance, Huang et al. (2019) design a memory-module to extract a user’s ﬁne-grained preferenceonataxonomyfrommultiplehopsreasoningonatree-basedgraph,together with a GRU layer to learn the sequential pattern. Wang et al. (2019g) make it different by adopting an LSTM layer to model the sequential dependencies of entities and Recommending on graphs: a comprehensive review from a data… 831 Fig. 10 Illustration of the graph auto-encoder framework in GLRS (Berg et al. 2017).The system input bipartite user–item graph which is represented an input data source X and adjacency matrix M. 5.4.1 Deep neural network (DNN) Then, the graph encoder learns node representations of users and items U, V , which go through the decoder to derive the predicted rating matrix ˆM Fig. 10 Illustration of the graph auto-encoder framework in GLRS (Berg et al. 2017).The system input bipartite user–item graph which is represented an input data source X and adjacency matrix M. Then, the graph encoder learns node representations of users and items U, V , which go through the decoder to derive the predicted rating matrix ˆM relations on a complex heterogeneous KG, generating path representations followed by a pooling operation to obtain a prediction signal for user–item pairs. Besides, using RNN for long sequence modelling also suffers from the vanishing and exploding gradient problem because of the choice of the number of layers and the activation functions, which is a common problem in many types of neural networks, e.g. feed- forward neural network, and CNN. Despite its limitations, the RNN-based approach still dominates in sequential recommendations due to its recurrent nature that matches the natural way of our brain to read one after another in a sequence mode. Convolutional neural networks (CNNs) (Krizhevsky et al. 2012) are capable of extracting local and global representations from heterogeneous data sources such as textual and visual information. To leverage CNN for extracting graph structured data, Wang et al. (2018c) extend traditional CNN which allows ﬂexibility in incorporating symbolic knowledge from a complex heterogeneous knowledge graph for learning sentence representations. 5.4.2 Auto-encoder A basic auto-encoder (AE) contains an encoder which encodes (projects) high- dimensional inputs X to low dimensional hidden representations Z, and a decoder which decodes (re-projects) hidden representations Z to the output ˆX that looks like the original input X. The objective is to minimize the reconstruction error, and ﬁnd the most efﬁcient and informative compact representations for the inputs. In most GLRSs studies, AE is applied to learn a complex heterogeneous graph (Zhang et al. 2016) in the entertainment and book recommendation domains. To apply AE to graph-structured data for a recommendation purpose, Zhang et al. (2016) ﬁrst use TransE to learn graph topological information from the complex het- erogeneous knowledge graph. Then, stacked denoising auto-encoders and stacked convolutional auto-encoders are adopted to learn textual and visual representations of items, which are the input for the collaborative ﬁltering framework. Later, Berg et al. (2017) consider a recommender system as a matrix completion task, and pro- pose to apply a graph auto-encoder to produce latent features of user and item nodes through a form of message passing on the bipartite user–item interaction graph. The learned latent user and item representations are used to reconstruct the rating links through a bilinear decoder. Generally speaking, a graph auto-encoder takes node fea- ture embedding X and adjacency matrix A as inputs, generating latent variable Z as 123 123 832 L. Zhang et al. output through the encoder (inference model). To reconstruct the graph structure data, the decoder (generative model) takes Z as input and outputs a reconstructed adja- cency matrix ˆA. Based on Berg et al. (2017), Zhang et al. (2019a) go a step further by proposing a new stacked and reconstructed graph convolutional network for a user– item bipartite graph, which takes low-dimensional user and item embeddings as the input to the model and solves the cold start problem by reconstructing the masked node embeddings with a block of graph encoder-decoder in the training phase. Figure10 illustrates how an auto-encoder operates on graph-structured data for a recommenda- tion purpose. The problem of the auto-encoder framework is that it usually leads to a local optimum due to the back-propagation algorithm it employs (Tian et al. 2014), which is also the common problem of most deep learning-based techniques that adopt back-propagation as a training procedure. 5.4.2 Auto-encoder Besides, the encoder-decoder architecture requires that the complete sequence of information must be captured by a single vector, which poses problems in holding on to information at the beginning of the sequence and encoding long-range dependencies. 123 5.4.3 Attention mechanism The attention mechanism (Bahdanau et al. 2014) is motivated by human visual atten- tion. For example, people only need to focus on speciﬁc parts of visual inputs to understand or recognize them. The attention mechanism is proposed to determine the signiﬁcance of the inputs e.g. sequences. The effectiveness of the attention-based techniques in RSs has been veriﬁed and aroused considerable attention over recent years. In attention-based GLRSs, inputs are weighted with attention scores and out- puts are normally vectors that combine different importance of the inputs. Attention mechanism can be used to allow the learning process to focus on parts of a graph that are more relevant to a speciﬁc task. Generally, it can be used in conjunction with MLP, CNN, RNN and other deep learning-based architectures. Thus, the heart of the attention-based techniques is how to obtain and calculate the attention weight of each input part. In this paper, attention mechanism can be used for multiple different types of graphs in various recommendation domains. For instance, in the e-commerce domain, the Amazon dataset can be formed into a tree graph (Gao et al. 2019b), k- partite graph (Wang et al. 2019d) and complex graph (Han et al. 2018) learned with an attention network. The Ciao, Epinions, Taobao and Kuaishou datasets can be formed into homogeneous graphs (Chen et al. 2019a; Chang et al. 2021b) learned with atten- tion mechanisms. Data sources in the entertainment domain can be constructed into k-partite graphs (Wang et al. 2019d; Xin et al. 2019), complex heterogeneous graphs (Han et al. 2018; Wang et al. 2019a), homogeneous graphs (Hao et al. 2021b; Chen et al. 2019a) and multiple graphs (Xia et al. 2021a) learned with an attention network. Attention mechanisms can also be applied in POI (Wang et al. 2019d; Hao et al. 2021b) and book (Wang et al. 2019a) domains. There are three attention mechanisms commonly used in recent studies: (1) the vanilla attention mechanism learns the attention scores for the input data by trans- forming the representations of input data via fully connected layers, and then adopting a softmax layer to normalize the scores (Han et al. 2018; Wang et al. 2019d; Gao et al. 2019b). Han et al. (2018) propose to use multi-layer MLP to learn a user/item aspect- Recommending on graphs: a comprehensive review from a data… 833 Fig. 11 Illustration of different attention mechanisms in GLRS. 5.4.3 Attention mechanism (2019b), the authors design a parallel co-attention mechanism to dynamically infer the primary reasons of the user purchase decision, assigning higher attention weights to more relevant meta-paths extracted on the k-partite graph. Other studies adopt attention variations based on these three categories. For instance, in Verma et al. (2019) the authors adapt a skip-gram to a merged heterogeneous user–item interaction and use social networks followed by a multi-layer and multi-head atten- tion (Vaswani et al. 2017) mechanism to learn the different importance of entities. Multi-head attention improves self-attention mechanisms to draw global dependen- cies between inputs and outputs by eschewing the use of recurrence in neural network and running through an attention mechanism several times in parallel. In Liu et al. (2020) the authors adopt Sentence-BERT (Reimers and Gurevych 2019), a language model that is based on multi-head attention and a bidirectional training procedure, to explore the potential links between item based on reviews. The learned item represen- tations from BERT are then used to generate an item subgraph according to the cosine similarities between all items. level representation based on extracted meta-paths from the complex heterogeneous graph. Then an attention mechanism is adopted to weigh the contribution of differ- ent aspect-level latent factors to ﬁnal user/item representations. (2) The self-attention mechanism (Vaswani et al. 2017) gained exposure recently as it can replace RNN and CNN in sequence learning, achieving better accuracy with lower computational com- plexity. It focuses on the self-matching of a sequence whereby the attention weights are calculated by the multiplication between key and query vectors transformed from the input sequence (Cen et al. 2019; Wu et al. 2019b). For instance, Cen et al. 2019 adopt self-attention to capture the inﬂuential factors between different edge types of the neighbours of a speciﬁc node on the attributed bipartite graph. (3) The co-attention mechanism focuses on co-learning and co-matching of two sequences whereby the attention weights of one sequence are conditioned on the other sequence, and vice versa. Some studies prefer to classify co-attention and self-attention as one category (Zhang et al. 2019b; Sun et al. 2019), but for clarity, in this survey we describe them separately. In Xu et al. (2019b), the authors design a parallel co-attention mechanism to dynamically infer the primary reasons of the user purchase decision, assigning higher attention weights to more relevant meta-paths extracted on the k-partite graph. 5.4.3 Attention mechanism a In Xu et al. (2019b), the co-attention component takes embeddings derived from different meta-paths as inputs. Then the query vectors qu, qi transformed from user and selling agent embeddings, as well as the item embeddings hρ vi go through the parallel co-attention network to learn the item embedding ˜yvi . b To update the representation of the ring node 2 in the context of the annular-graph, a self-attention mechanism takes two context vector and the node 2 vectors as input to learn the importance of context nodes to the central node (Hao et al. 2021b) Fig. 11 Illustration of different attention mechanisms in GLRS. a In Xu et al. (2019b), the co-attention component takes embeddings derived from different meta-paths as inputs. Then the query vectors qu, qi transformed from user and selling agent embeddings, as well as the item embeddings hρ vi go through the parallel co-attention network to learn the item embedding ˜yvi . b To update the representation of the ring node 2 in the context of the annular-graph, a self-attention mechanism takes two context vector and the node 2 vectors as input to learn the importance of context nodes to the central node (Hao et al. 2021b) level representation based on extracted meta-paths from the complex heterogeneous graph. Then an attention mechanism is adopted to weigh the contribution of differ- ent aspect-level latent factors to ﬁnal user/item representations. (2) The self-attention mechanism (Vaswani et al. 2017) gained exposure recently as it can replace RNN and CNN in sequence learning, achieving better accuracy with lower computational com- plexity. It focuses on the self-matching of a sequence whereby the attention weights are calculated by the multiplication between key and query vectors transformed from the input sequence (Cen et al. 2019; Wu et al. 2019b). For instance, Cen et al. 2019 adopt self-attention to capture the inﬂuential factors between different edge types of the neighbours of a speciﬁc node on the attributed bipartite graph. (3) The co-attention mechanism focuses on co-learning and co-matching of two sequences whereby the attention weights of one sequence are conditioned on the other sequence, and vice versa. Some studies prefer to classify co-attention and self-attention as one category (Zhang et al. 2019b; Sun et al. 2019), but for clarity, in this survey we describe them separately. In Xu et al. 5.4.3 Attention mechanism Other studies adopt attention variations based on these three categories. For instance, in Verma et al. (2019) the authors adapt a skip-gram to a merged heterogeneous user–item interaction and use social networks followed by a multi-layer and multi-head atten- tion (Vaswani et al. 2017) mechanism to learn the different importance of entities. Multi-head attention improves self-attention mechanisms to draw global dependen- cies between inputs and outputs by eschewing the use of recurrence in neural network and running through an attention mechanism several times in parallel. In Liu et al. (2020) the authors adopt Sentence-BERT (Reimers and Gurevych 2019), a language model that is based on multi-head attention and a bidirectional training procedure, to explore the potential links between item based on reviews. The learned item represen- tations from BERT are then used to generate an item subgraph according to the cosine similarities between all items. Figure11 illustrates several above mentioned attention mechanism architectures for GLRSs. The core of the attention mechanism of focusing on the most relevant parts of 123 123 834 L. Zhang et al. Fig. 12 Deep reinforcement learning-based GLRS with a knowledge graph (Lei et al. 2020a). The system ﬁrst performs the walking starting with the target user u0 over the adjacent attribute vertices on the complex heterogeneous graph, resulting in a path to the desired item. Two reasoning function f and g score attributes and items. Then, the policy network takes the state vector s as input and outputs the values Q(s, a), indicating the estimated rewards for two actions aask and arec nforcement learning-based GLRS with a knowledge graph (Lei et al. 2020a). The system e walking starting with the target user u0 over the adjacent attribute vertices on the complex raph, resulting in a path to the desired item. Two reasoning function f and g score attributes the policy network takes the state vector s as input and outputs the values Q(s, a), indicating wards for two actions aask and arec Fig. 12 Deep reinforcement learning-based GLRS with a knowledge graph (Lei et al. 2020a). The system ﬁrst performs the walking starting with the target user u0 over the adjacent attribute vertices on the complex heterogeneous graph, resulting in a path to the desired item. Two reasoning function f and g score attributes and items. 5.4.3 Attention mechanism Then, the policy network takes the state vector s as input and outputs the values Q(s, a), indicating the estimated rewards for two actions aask and arec the input by providing a direct path to the input helps to alleviate the bottleneck prob- lem of the vanishing gradient and to resolve the disadvantage of the encoder-decoder architecture that has the problem of remembering long sequence dependencies. How- ever, one believes that the attention mechanism adds more weight parameters to the model, which increases training time, especially for long input sequences. 5.4.4 Deep reinforcement learning (DRL) Reinforcement learning (RL) uses a trial-and-error experience with an agent that learns a good behaviour by modifying or acquiring new behaviours and skills incrementally. During such a learning process, the agent interacts with the environment and must make value judgements so as to select good actions over bad. Actions that get them to the target outcome are rewarded (reinforced). Deep reinforcement learning (Mnih et al. 2015) goes a step further by incorporating deep neural networks to represent the knowledge acquisition progress. It has been mainly adopted to learn k-partite (Song et al. 2019a) and complex heterogeneous graphs (Lei et al. 2020a; Liu et al. 2021a; Xian et al. 2019) in entertainment, book and e-commerce domain for recommendation purposes. In GLRSs, one can take a path generation procedure as a decision-making process for training with RL, so that the optimal recommendation results as well as the inter- pretation of the results can be generated at the same time (Song et al. 2019a; Xian et al. 2019; Lei et al. 2020a; Wang et al. 2020g). For instance, Xian et al. (2019) propose to use an RL approach where an agent starts from a given user, and learns to navigate to the potential items of interest on the complex heterogeneous KG. After that, the rea- 123 Recommending on graphs: a comprehensive review from a data… 835 soning path history can serve as a genuine explanation for the recommendation results. Similarly, Song et al. (2019a) formulate the generation of user-to-item paths extracted from a k-partite graph as a sequential decision process. Speciﬁcally, it deﬁnes the target user as the initial state and then walks on the constructed heterogeneous user– item–entity graph as actions. In the work of Zhou et al. (2020b), the authors adopt a complex heterogeneous KG to improve the sample efﬁciency as well as interactive recommendation performance by applying a deep Q-network to ﬁt on samples from the local graph of the KG rather than the whole graph. Interestingly, we can ﬁnd that most DRL-based recommendation approaches utilize a KG as an important medium to learn user-to-item inference. Figure12 illustrates a typical example of adopting a KG with DRL for recommendation purpose. 5.4.4 Deep reinforcement learning (DRL) It is probably due to the explicit asso- ciation between the target user and items which reveals the user’s potential interests, compared with traditional recommendation systems, a KG-based recommender sys- tem can mine more potential relationships between nodes for learning user and item representations. DRL-based approaches have great potential in decision-making and long-term plan- ning in a dynamic environment (Silver et al. 2016). However, the ideal way to train a DRL model to learn the optimal recommendation policy is to train the agent online, which cannot always be satisﬁed. One commonly used training strategy is to make use of ofﬂine logged data directly, but it will suffer from the estimation bias problem under the real-time interaction setting (Chen et al. 2019c). Besides, similar to other deep learning-based techniques, DRL-based approaches also lack interpretability. More importantly, few appropriate platforms or resources for developing and testing DRL- based techniques in academia exist (Fang et al. 2020). 5.4.5 Graph neural networks Graph neural network (GNN) enjoys a massive hype as recent works have witnessed a boostofperformanceinRSs.TheyaremotivatedfromCNNandgraphembeddingsand designed speciﬁcally on graph-structured data in the non-Euclidean domain (Zhang et al. 2020). GNN can be applied from homogeneous graphs (Zhu et al. 2021b; Isuﬁ et al. 2021), k-partite graphs (Wang et al. 2019f; Chen et al. 2020a; Wang et al. 2019e; Sun et al. 2020; Wu et al. 2021b; Fan et al. 2019a; Chen et al. 2021a; Ying et al. 2018; Wei et al. 2021; Li et al. 2021a) to complex heterogeneous (Zhao et al. 2019; Zheng et al. 2021; Wang et al. 2019c,b; Wu et al. 2021a; Zhang et al. 2021d) and multiple ones (Zhu et al. 2021a; Zhang et al. 2021c; Liu et al. 2020; Huang et al. 2021b; Tang et al. 2021; Wu et al. 2019b; Tian et al. 2021; Chang et al. 2021a) in different recommendation domains. GNN achieve improvements in recommendation results by capturing the higher- order interaction in user–item relationships through iterative propagation resulting in better user/item representations. Speciﬁcally, GNN aim to iteratively aggregate feature information from neighbours and integrate the aggregated information with the current node representation (Wu et al. 2020b). Further, they can simultaneously model the diffusion process on the graph with the RNN kernel. Following the existing work of Wu et al. (2020a), we categorize GNN as spectral (Zheng et al. 2018; Wang et al. 2020a; Farseev et al. 2017) and non-spectral approaches (Sun et al. 2020; Isuﬁ 123 L. Zhang et al. 836 Fig. 13 Illustration on GNN-based GLRSs. (I) The representation of the central node updates by incor- porating the inﬂuence of its neighbourhood representation in GNN algorithm (Wang et al. 2019c). (II) GGAT takes into account the different effects of neighbour nodes on the central node, and combines the attention mechanism with the GNN node propagation process to update the representation of the central node (Veliˇckovi´c et al. 2017) Fig. 13 Illustration on GNN-based GLRSs. (I) The representation of the central node updates by incor- porating the inﬂuence of its neighbourhood representation in GNN algorithm (Wang et al. 2019c). (II) GGAT takes into account the different effects of neighbour nodes on the central node, and combines the attention mechanism with the GNN node propagation process to update the representation of the central node (Veliˇckovi´c et al. 2017) et al. 2021; Kim et al. 2019; Chen et al. 2020a; Wang et al. αi j = exp(LeakyReLU(−→ a T [W−→ hi \|\|W−→ h j ])) k∈Ni exp(LeakyReLU(−→ a T [W−→ hi \|\|W−→ hk ])) (3) (3) where −→ a and W is the weight matrix. hk is the neighbour node embedding of node ni whose node embedding is hi. Figure13(II) illustrates the schematic diagram of the attention operation of GAT. where −→ a and W is the weight matrix. hk is the neighbour node embedding of node ni whose node embedding is hi. Figure13(II) illustrates the schematic diagram of the attention operation of GAT. Despite their veriﬁed effectiveness in the community of graph-based recommen- dations, they suffer from the expensive computation overhead with the exponential growth of the neighbourhood size as the layers stacked up (Ying et al. 2018). Besides, researchers empirically show that the performance of GNN quickly degenerate when the number of layers is deep owing to that the effectiveness of informative neighbours will be diminished in large amount irrelevant neighbours (Liu et al. 2019b). To solve this, Xu et al. (2019b) design a relation-aware GNN with an attention mechanism to prioritize neighbours based on their importance. Then a meta-path deﬁned receptive ﬁeld sampler is integrated to derive the node embeddings as well as address the rapid growth of the multiple-hop neighbourhood of each node from a k-partite graph, which is followed by a co-attention mechanism for differentiating purchase motivations. In Zhang et al. (2019a), the authors also point out that training GCN-based models for rating prediction faces the label leakage issue, which results in the overﬁtting problem and signiﬁcantly degrades the ﬁnal performance, which can be improved by removing the sampled edges. Figure13a, b illustrates examples of how GNN can be used for recommender systems in Wang et al. (2019c), and the main component of GAT in Veliˇckovi´c et al. (2017), respectively. Although some problems have been proposed by researchers for improvement or solutions, other challenges still exist and deserve more attention from both academia and industry. First, though GNN-based graph recommendation strategies can incor- porate a high-order proximity of vertices, it suffers from the problem of performance degradation and complexity increase with the increase of the number of layers. As a result, it is more prone to encounter the over-smoothing problem with the increase in the network depth (Liu et al. 2019b; Li et al. 2018a; Yu et al. 2021). This phenom- ena can become a pervasive problem when learning large-scale graph/networks when aggregating high-order information from distant neighbours is necessary. 5.4.5 Graph neural networks 2019c,f,b; Fan et al. 2019a; Liu et al. 2020; Ying et al. 2018; Zhao et al. 2019). Spectral GNN are based on spectral graph theory (Shuman et al. 2013) which studies connections between combinatorial properties of a graph and the eigenvalues of matrices associated with the graph, e.g. laplacian matrix. They focus on the connectivity of the graph rather than geometrical proximity. For instance, Farseev et al. (2017) performs spectral clustering to form user community w.r.t. user side information e.g. geographical regions, user’s active timestamp from a complex heterogeneous graph, which are then considered to sort all candidate items to a generate ranked list for recommendation. Zheng et al. (2018) propose to use a spectral convolution operation in the spectral domain of the bipartite user–item graph to alleviate the cold-start problem of RS. The non-spectral approaches mainly include aggregator and updater to learn a multi-layer graph. The aggregator is responsible for collecting information from neigh- bourhood nodes and related edges, while the updater aims to merge the propagation information around the central node and collected through the aggregator. Normally, GNN are utilized to learn the representations of nodes and links of the graphs, which are then used for the following recommendation strategies, e.g. rating prediction and link prediction etc. For instance, Monti et al. (2017) propose a GCN-based technique for recommender systems for the ﬁrst time, in which GCN is a variant of GNN and used to aggregate information from two auxiliary user–user and item–item homoge- neous graphs with the convolutional operation. The latent factors of users and items were updated after each aggregation step, and a combined objective function of GCN and MF was used to train the model. Ying et al. (2018) propose to use GCN to generate item embeddings from both a bi-partite graph structure as well as item feature infor- mation with random walks for recommendations. It can be applied to very large-scale web recommenders and has been deployed in Pinterest to address a variety of real- world recommendation tasks. In Chen et al. (2019a) the authors adopt a GNN layer for modelling both the local and global inﬂuence of user social relations on constructed homogeneous user social graph. Graph attention networks (GATs) (Veliˇckovi´c et al. 5.4.5 Graph neural networks Recommending on graphs: a comprehensive review from a data… 837 2017) are an enhanced version of GNN which utilize masked self-attention layers to limit the shortcomings of prior graph convolutional-based approaches. An attention weight αi ∈[0, 1] is assigned to the neighbourhood nodes of a target node nt, where i∈N(t) αi = 1 and N(t) denotes the set of neighbouring nodes of nt. One advantage of applying attention to graphs is to avoid the noisy part of a graph so as to increase the signal-to-noise ratio in information processing. Speciﬁcally, GAT aims to compute the attention coefﬁcients 2017) are an enhanced version of GNN which utilize masked self-attention layers to limit the shortcomings of prior graph convolutional-based approaches. An attention weight αi ∈[0, 1] is assigned to the neighbourhood nodes of a target node nt, where i∈N(t) αi = 1 and N(t) denotes the set of neighbouring nodes of nt. One advantage of applying attention to graphs is to avoid the noisy part of a graph so as to increase the signal-to-noise ratio in information processing. Speciﬁcally, GAT aims to compute the attention coefﬁcients αi j = exp(LeakyReLU(−→ a T [W−→ hi \|\|W−→ h j ])) k∈Ni exp(LeakyReLU(−→ a T [W−→ hi \|\|W−→ hk ])) (3) 5.4.6 Deep hybrid models In order to deal with more complicated and diverse problems, as well as process more complex graphs, many graph-based recommendation models utilize more than one deep learning technique. The ﬂexibility of neural blocks in deep neural networks makes it possible to combine several neural components to complement one another and form a more powerful hybrid model. The use of a variety of different DL-based components can also maximize the strengths and improve the defects of a single technology to a certain extent. Such hybrid models can be leveraged to learn homogeneous graphs (Song et al. 2019b; Abugabah et al. 2020; Xu et al. 2019a; Wu et al. 2019c; Huang et al. 2021a), k-partite graphs (Kim et al. 2019; Zhang et al. 2019a; Wang et al. 2020f; Liu et al. 2021c; Xie et al. 2021; Xu et al. 2019b; Xia et al. 2021b), complex heterogeneous graphs (Wang et al. 2020g; Xie et al. 2021; Yang and Dong 2020; Zhou et al. 2020b; Sang et al. 2021; Sheu and Li 2020; Shi et al. 2021; Wang et al. 2018c; Zhou et al. 2020a; Yang et al. 2021a), hypergraphs (Wang et al. 2020c; Gharahighehi et al. 2020), andmultiplegraphs (Monti et al. 2017; Zhanget al. 2021a; Xiaet al. 2021c; Fan et al. 2019b; Liu et al. 2021b; Monti et al. 2017) in social network, e-commerce, entertainment, academic, book and many other recommendation domains. In Sun et al. (2018), the authors employ a batch of bi-directional recurrent net- works (Schuster and Paliwal 1997) to learn the semantic representations of each path extracted from complex heterogeneous KG. Then an attention gated hidden layer is applied to learn the different importance of the derived paths between two entities followed by a pooling operation and a fully connected layer for rating prediction. Zhang et al. (2019a) propose to leverage multi-link GCN as an encoder and two-layer feedforward neural network as a decoder to learn the user and item (users and items are denoted as nodes) representations, considering both node’s content information as well as structural information of the undirected user–item bipartite graph. Some studies leverage different techniques to learn various graph features, such as node attributes and graph structure. Zhang et al. (2016) construct a complex heterogeneous knowledge graph to learn a user’s potential preferences, where the item nodes asso- ciate with textual and visual features as their attributes. 5.4.6 Deep hybrid models To model such multi-modal information, the authors ﬁrst apply a network embedding (TransR) approach to extract items’ structural representations by considering the heterogeneity of both nodes and relationships, followed by a stacked denoising auto-encoder and stacked convolutional auto-encoder to extract items’ textual and visual representations, respectively. Finally, the pair-wise ranking between items is considered to learn the CF architecture. Deep hybrid approaches have become a trend in solving complex recommendation prob- lems, facing the complicated and changeable network structure for modelling dynamic user preferences. αi j = exp(LeakyReLU(−→ a T [W−→ hi \|\|W−→ h j ])) k∈Ni exp(LeakyReLU(−→ a T [W−→ hi \|\|W−→ hk ])) (3) Second, cur- rent GNN are mainly applied for a static graph, but how to apply GNN for dynamic graphs with changing structures is still an open challenge. 123 123 L. Zhang et al. 838 5.5 Discussion of graph-based recommendation models In this section, we present the main ideas and the basic technical details of each class of graph learning-based recommendation approaches. From the descriptions above, we make several observations: (1) traditional graph learning-based approaches may 123 12 Recommending on graphs: a comprehensive review from a data… 839 suffer from information loss, e.g. nearest neighbour-based approaches. Some of them ignore long-term or high-order dependencies such as the latent factor model (Tao et al. 2021). However, they laid a theoretical foundation for the later development of graph learning-based technologies, so that many recent advanced deep learning-based techniques for graphs still use traditional algorithms as the basic framework. (2) For path-basedapproaches,theyeitherrelyondomainknowledgewhichmaynotalwaysbe applicable e.g. meta-path-based similarities, and/or require explicit path reachability which may incorporate noisy, meaningless paths and thus do not always have a positive impact on recommendation results (Noia et al. 2016). Besides, recommendations that rely on similarity measures cannot be easily applied to large-scale networks. (3) Graph embedding-based approaches pave the way for more complex and high-order features among nodes and links modelling. More and more state-of-the-art GLRSs leverage GE combined with deep learning approaches such as an attention network for more efﬁcient recommendation tasks. (4) With the many achievements of the deep learning- based GLRSs, the number of research works in the ﬁeld has become exponentially increased. Deep learning-based approaches can be applied to more complicated graphs with multi-type nodes and links, as well as additional attributes associated with graphs (Song et al. 2019b). Besides, deep learning-based approaches are more robust to sparse data and can adapt to the varied magnitude of the input (with the help of e.g. attention mechanism) (Fang et al. 2019). However, interpretability and efﬁciency are still the main concerns for most GE-based and deep learning-based GLRSs which need to be furtherstudiedinthefuture.(5)FromTable5,byrelatinggraphtypestotheirassociated modelling technologies, we observe that tree graphs are modelled mainly by traditional techniques, while other types of graphs are modelled and learned mainly through deep learning and graph embedding-based approaches. Besides, attention mechanisms become especially prevalent and are adopted for nearly all types of graphs for selecting branches, ﬁltering noisy nodes, and learning better nodes and edges representations for recommendation purposes. 6 Challenges and open issues in GLRS For some kinds of graphs, e.g. hypergraphs and multiple graphs, it is more difﬁcult to provide explanations with such graphs due to the dispersion of edges or nodes. Besides, how to show user dynamic preferences on graphs is also worth studying. Besides, we need to consider the target population who we provide explanations for. This can be the end-users or researchers. For both groups, whether they are satisﬁed with this explanation and whether this explanation is sufﬁcient still requires further empirical veriﬁcation. Such veriﬁcation cannot just stay on a small-scale user study or case study. It requires more target audience participation and different situations should also be taken into account. Fairness. Fairness in recommendation has gradually attracted increasing attention in recentyearsandhasbeenstudiedmainlyasanequityandparityproblemforindividuals or groups of users. Current RSs discriminate unfairly among the users in terms of recommendation performance, and further, the systems may discriminate between users in terms of explanation diversity (Farnadi et al. 2018). One reason is probably related to the issue of data imbalance. E.g. economically disadvantaged customers tend to make fewer purchases, leading to imbalanced data. However, such imbalances may lead to biased models that exhibit unfairness w.r.t. the recommendation quality and explanation (Fu et al. 2020). Though active users tend to interact with more items, an empirical study (Fu et al. 2020) shows that the majority of users are inactive users who are easily disregarded by recommendation engines. The imbalanced data can easily lead to biased observation on graphs where path inference from user-to-item usually participates in the process of graph learning and meanwhile provide recommendation explanations. Many researches have been done to alleviate data sparsity issue. Farnadi et al. (2018) propose to solve the fairness on the user side for both the individual- and group-level for KG enhanced explainable RSs. Speciﬁcally, they reveal that the unfairness issue is due to data imbalance through an empirical study on e-commerce In addition, most existing graph leaning-based recommendation models generally consider paths along with nodes in the KG as pertinent signals for recommendation (Hu et al. 2018). User–item paths in the KG can directly serve as explanations that provide the reason why an item is recommended (Xian et al. 2019). In (Fu et al. 2020), Fu et al. considered a relational structure serving as explanation across different paths with similar semantics w.r.t. relations in each path. 6 Challenges and open issues in GLRS Graph-based recommendation is an exciting and rapidly growing research area that attracts attention from both industrial and academic domains. While existing works have established a solid foundation for GLRSs research, this section reveals several challenges and promising prospective research directions. Speciﬁcally, there are two types of challenges: (1) Challenges still unsolved by graphs, which include explain- ability, fairness and generality issues; (2) Challenges caused by graphs and their limitations, including scalability, dynamic graph, and complex heterogeneity learning issues. We will explain these issues separately. Explainability on graphs. A good explanation for recommendation results can help to improve the transparency, persuasiveness, effectiveness, trustworthiness and satisfac- tion of recommender systems, facilitate system designers for better system debugging, and allow a user to control how the system utilizes her proﬁles making the RS scrutable. Earlier studies provide explanations of GLRSs highlighting top keywords or aspects 123 840 L. Zhang et al. as an explanation for recommendation results (He et al. 2015), but none of them shows the constructed graphs. With the surge of deep learning-based approaches in GLRSs, it is even harder to provide convincing explanations and calibrate why the recommendation models are effective and thus yield a robust model for varied scenarios. Ma et al. (2019) provide recommendation explanations according to the learned reasoning rules on heterogeneous graphs with ground-truth item associations in the knowledge graph. The emergence of attention mechanisms has more or less eased the non-interpretable concerns of deep learning-based recommendations on graphs. The learned attention weights can tell which parts of the input graph contribute more than others with higher attention scores to the recommendation results. However, the faithfulness of the higher attention weights in contributing to the performance of the recommendation is still doubtful (Liu et al. 2022). doubtful (Liu et al. 2022). In addition, most existing graph leaning-based recommendation models generally consider paths along with nodes in the KG as pertinent signals for recommendation (Hu et al. 2018). User–item paths in the KG can directly serve as explanations that provide the reason why an item is recommended (Xian et al. 2019). In (Fu et al. 2020), Fu et al. considered a relational structure serving as explanation across different paths with similar semantics w.r.t. relations in each path. We argue that this lacks reliable evaluation metrics for the explainability and persuasiveness for target users, such as GLUE (Wang et al. 2018a) for language understanding. 6 Challenges and open issues in GLRS We argue that this lacks reliable evaluation metrics for the explainability and persuasiveness for target users, such as GLUE (Wang et al. 2018a) for language understanding. For some kinds of graphs, e.g. hypergraphs and multiple graphs, it is more difﬁcult to provide explanations with such graphs due to the dispersion of edges or nodes. Besides, how to show user dynamic preferences on graphs is also worth studying. Besides, we need to consider the target population who we provide explanations for. This can be the end-users or researchers. For both groups, whether they are satisﬁed with this explanation and whether this explanation is sufﬁcient still requires further empirical veriﬁcation. Such veriﬁcation cannot just stay on a small-scale user study or case study. It requires more target audience participation and different situations should also be taken into account. Fairness. Fairness in recommendation has gradually attracted increasing attention in recentyearsandhasbeenstudiedmainlyasanequityandparityproblemforindividuals or groups of users. Current RSs discriminate unfairly among the users in terms of recommendation performance, and further, the systems may discriminate between users in terms of explanation diversity (Farnadi et al. 2018). One reason is probably related to the issue of data imbalance. E.g. economically disadvantaged customers tend to make fewer purchases, leading to imbalanced data. However, such imbalances may lead to biased models that exhibit unfairness w.r.t. the recommendation quality and explanation (Fu et al. 2020). Though active users tend to interact with more items, an empirical study (Fu et al. 2020) shows that the majority of users are inactive users who are easily disregarded by recommendation engines. The imbalanced data can easily lead to biased observation on graphs where path inference from user-to-item usually participates in the process of graph learning and meanwhile provide recommendation explanations. Many researches have been done to alleviate data sparsity issue. Farnadi et al. (2018) propose to solve the fairness on the user side for both the individual- and group-level for KG enhanced explainable RSs. Speciﬁcally, they reveal that the unfairness issue is due to data imbalance through an empirical study on e-commerce Recommending on graphs: a comprehensive review from a data… 841 dataset, namely Amazon. Then they propose fairness metrics in terms of path diversity as well as recommendation performance disparity based on KG. In Gharahighehi et al. (2021), the authors constructed a hypergraph taking into account multiple stakeholders in the news domain to mitigate the imbalance problem caused by stakeholders with few articles. 6 Challenges and open issues in GLRS Exposure bias can be caused by the users being only aware of a very small fraction of items in a large dataset. Chen et al. (2019a) pointed out the exposure bias that users are only aware of a very small fraction of items in a large dataset so that they infer data conﬁdence with the help of users’ social network and draw different weights on training instances via personalized random walk to alleviate it. Another possible reason lies in the incomplete evaluation metrics, which renders the inclination of the learning objectives on accuracy driven for recommendation purposes. Despite some studies considering the inﬂuence of fairness in GLRSs, related research is still quite limited with many issues remaining to be focused on. For instance, whether the construction and learning process of the graphs affects the fairness and discrimination of the ranking of recommendation results, whether there exists algo- rithmic bias among various graph learning technologies, and whether fairness, bias, and discrimination conﬂict with the accuracy of graph-based recommendations. Some biases can be dynamic. For instance, in the real world, users’ preferences, exposure, and relations may evolve over time (Chen et al. 2019a). How to solve the dynamic bias problem in GLRS is also one of the research directions worth thinking about in the future. Scalability on large-scale graphs. Scalability is an essential factor that affects the applicability of recommendation models in real-world scenarios. To deal with large- scale graphs, most existing models choose to adopt a sampling scheme to construct subgraphs following the sampling strategy proposed in GraphSage (Hamilton et al. 2017). Some use the random walk strategy to get the neighbourhood nodes and links, while others consider using the shortest path algorithm for subgraph construction. Another algorithm to increase model scalability is to use a clustering scheme. Whether using sampling or clustering, a model will lose part of the graph information. The scalability is gained at the price of corrupting graph completeness. A node may miss its inﬂuential neighbours with a bad sampling strategy, and a graph may be deprived of a distinct structural pattern by clustering. Though the subgraph strategy makes GNN-based algorithms applicable no matter how large-scale the whole graph is, the shortcomingis that thenoderepresentationshouldberecalculatedfor eachpropagation layer.Thus,howtotradeoffthealgorithmscalabilityandgraphintegritycouldbeoneof thefurther researchdirections. Moreresearches canbestudiedonthesamplingstrategy inintegratingmoreinformativeinformationfromneighbourhoodnodesandlinkswhile minimizing the harm to the graph integrity. Recently, Kyriakidi et al. 6 Challenges and open issues in GLRS (2020) propose to adopt graph databases as a base to improve the data scalability and meanwhile build recommendation models on top. Different from other graph-based recommender systems which focus on model complexity when considering the efﬁciency problem, the work of Kyriakidi et al. (2020) transforms the recommendation problem into a path optimization problem from start nodes to end nodes on heterogeneous graphs, which sheds light on a new perspective for improving the scalability of GL-based recommendations. 123 123 842 L. Zhang et al. Data Sparsity on Graphs. Data sparsity issues will cause the graph’s adjacency matrix to be sparse and affect recommendation performance. In order to alleviate this problem, one way is to construct auxiliary graphs by mining contextual information of users or items, such as friendship connections amongst users, co-purchase networks associated with products and services to the end-users, or trust relationships associ- ated with users, entities associated with items. On the other hand, data augmentation techniques can be used. The former has been widely used and studied (Wu et al. 2019b), while the latter has not been extensively explored. Recently, some studies try to alleviate the data sparsity problem with item/segment dropout to augment data (Wu et al. 2021b), with which edges are randomly dropped out in the constructed graph, resulting in the robustness of recommendation models facing noisy input data such as unsatisﬁed clicking or viewing behaviour. However, it may also lead to sparser data. In Xia et al. (2021c), the authors adopted a self-supervised graph co-training strategy for learning session representations with two different encoders in the session-based rec- ommendation. As the initial attempt in many graph learning-based recommendation domains, more in-depth attempts are still needed, and the advantages and disadvan- tages of different data augmentation technologies with different types of graphs in different recommendation domains also require a comprehensive analysis. Recommendation on Dynamic Graphs. Most research focuses on the modelling and learning of static graphs, but neglects the dynamic properties of graphs. In many real-world scenarios, the graphs changes over time, such as people may be followed or unfollowed day to day in a social network, or newly published news articles appear everyday resulting in changes of news-related graphs for news recommendation. As a result, some works crop the dynamic graph as a sequence of graph snapshots (Abugabah et al. 2020; Xia et al. 2021d; Huang et al. 6 Challenges and open issues in GLRS For existing GLRS approaches, given a data source, the common way is to take graph(s) formed by data objects and their explicit or implicit relationships as input, and the model usually needs to be reformulated and retrained under certain conditions, or a general model is applied for a speciﬁc task in a speciﬁc scenario, rather than extending the existing models for new tasks. This led to thousands of new models corresponding to thousands of different kinds of input graphs, which is far from the true generality of the graph model for recommendations. Therefore, one possible future research direction is whether there is a model suitable for learning all kinds of graphs. The dynamic property of online graphs shows the inevitability of changes in modelling input graphs. When additional attributes are added to the input data, for instance, when contextual or external information is attached to interactive data, resulting in the expansion of the graph, a model with generality should be expanded in a small range on the basis of the previously learned content to adapt to the change of the input graph rather than retraining new models. This should be one of the future research directions in the long run. item–item relations may be from the co-interaction pattern from a speciﬁc user or user group (Xia et al. 2021a). To integrate multi-sourced side information for recommen- dation purposes, some works learn representations from different graphs separately and combine the vectors from different sources (Zhang et al. 2021c). Some works combine different graphs into a large-scale heterogeneous graph which is then learned in a uniﬁed way (Wu et al. 2021a). These two kinds of integration strategies can both contribute to the improvement in recommendations, but there is no evidence showing which one is better. This is thus another research question for further study. Generality of Graph Learning. Currently, no model can be applied for all types of input graphs. For existing GLRS approaches, given a data source, the common way is to take graph(s) formed by data objects and their explicit or implicit relationships as input, and the model usually needs to be reformulated and retrained under certain conditions, or a general model is applied for a speciﬁc task in a speciﬁc scenario, rather than extending the existing models for new tasks. 6 Challenges and open issues in GLRS This led to thousands of new models corresponding to thousands of different kinds of input graphs, which is far from the true generality of the graph model for recommendations. Therefore, one possible future research direction is whether there is a model suitable for learning all kinds of graphs. The dynamic property of online graphs shows the inevitability of changes in modelling input graphs. When additional attributes are added to the input data, for instance, when contextual or external information is attached to interactive data, resulting in the expansion of the graph, a model with generality should be expanded in a small range on the basis of the previously learned content to adapt to the change of the input graph rather than retraining new models. This should be one of the future research directions in the long run. Privacy Issue Of Graph Learning. Though existing GLRSs reveal promising improvements on recommendation tasks, the graph topological and node binding fea- tures may cause privacy issues. The users’ private information may be inferred from such a recommendation, which falls into the attribute inference attack problem. To deal with such an issue, in Zhang et al. (2021b) a privacy-preserving graph learning-based recommender is designed with a two-stage perturbation on input feature encoding and an optimization process to defend against attribute inference attacks. However, the authors also point out the challenge of balancing the personalized recommenda- tion performance and the extent of the privacy protection mechanism, which means the privacy-preserving recommender systems are far from mature and deserve more attention in the future. 6 Challenges and open issues in GLRS 2021a), most of which are in session settings. Though a surge of works consider modelling dynamic graphs from the changes of the adjacency matrix (Li et al. 2017), leveraging dynamic random walk sampling (Nguyen et al. 2018), Hawkess process (Huang et al. 2020) or combining with other algorithms capturing the dynamic properties of the graph (Xu et al. 2020), they do not adapt to recommendation scenarios. Thus, we believe it is an important research direction with signiﬁcance and practical value. Complex Heterogeneity Learning. Apart from the user–item bipartite graph, most heterogeneity of graphs is reﬂected in the integration of different side information. Side information has demonstrated a high degree of effectiveness in improving rec- ommendation performance, especially for data sparsity and cold-start issues (Zhang et al. 2016). It can appear in different forms: textual, visual, or audio information; structure or non-structure. In current studies of graph-based recommendations, side information is extensively involved either as extra attributes of nodes or edges (Song et al. 2019b), or as sources being learned to construct heterogeneous graphs (Sheu and Li 2020), or as external resources outside the graph, which are learned in parallel with the graph and then integrated at a high level. Despite the variety of utilizing side information, no study can indicate which fusion strategy is better in general cases, or suitable for which recommendation scenarios (Farseev et al. 2017). These we believe are extremely signiﬁcant references and guidance for future researchers and technol- ogy users. Besides, some side information is from multi-sources such as user social relationships pointing out user–user interlinks directly (Farseev et al. 2017), while Recommending on graphs: a comprehensive review from a data… 843 item–item relations may be from the co-interaction pattern from a speciﬁc user or user group (Xia et al. 2021a). To integrate multi-sourced side information for recommen- dation purposes, some works learn representations from different graphs separately and combine the vectors from different sources (Zhang et al. 2021c). Some works combine different graphs into a large-scale heterogeneous graph which is then learned in a uniﬁed way (Wu et al. 2021a). These two kinds of integration strategies can both contribute to the improvement in recommendations, but there is no evidence showing which one is better. This is thus another research question for further study. Generality of Graph Learning. Currently, no model can be applied for all types of input graphs. 7 Conclusion The study systematically investigated graph learning-based recommendation. The rec- ommendation algorithms based on graph-structured data can be well applied to solve the sparsity and cold-start problems with improved accuracy by mining and leverag- ing the explicit as well as implicit relations revealed in graphs. In GLRSs, the core is how to process graph-structured data, how to learn and obtain adequate informa- tion from the graph to fulﬁl the ﬁnal recommendation purpose, and how to adapt the graph operation process to more complex and diverse graph structures as well 123 L. Zhang et al. 844 as large-scale node and edges in the real world. Looking at the changes of GLRSs in recent years, the graph structure has gone from homogeneous to heterogeneous, the graph attribute from zero to multiplex, the technology used from the traditional recommendation algorithm to deep learning-based models, and the evaluation of rec- ommendation performance from focusing only on accuracy and click-through rate to increasingly multidimensional development. Such developments shed light on a new perspective for the community of recommendation researchers and practitioners. We argue that the current graph-based recommendation algorithms are far from being fully developed, and further research investment and empirical studies are still needed. Acknowledgements This work is supported by the Research Council of Norway under Grant Nos. 245469 and 309834. Funding Open access funding provided by NTNU Norwegian University of Science and Technology (incl St. Olavs Hospital - Trondheim University Hospital) OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Appendix A Statistics of datasets commonly used in GLRS See Table 5. See Table 5. See Table 5. 123 Recommending on graphs: a comprehensive review from a data… Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) 123 Recommending on graphs: a comprehensive review from a data… 845 Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) Yelp1 12 L. Zhang et al. 846 5 continued ain Dataset Graph Type Related Papers Complex Heterogeneous Graph Zhao et al. (2017), Sun et al. (2018), Shi et al. (2018), Shi et al. (2015), Kyriakidi et al. (2020), Catherine and Cohen (2016), Lei et al. (2020a), Wang et al. (2020g), Zheng et al. (2017), Shi et al. (2016), Mezni et al. (2021), Xie et al. (2021), Liu et al. (2021c) Hypergraph Mao et al. (2019), Yu et al. (2021) Multiple Graphs Liu et al. (2020), Xia et al. (2021a), Huang et al. (2021b), Tang et al. (2021), Zhu et al. (2021a), Guo et al. (2021a) Epinions (Ma et al. 2011a; Tang et al. 2012; Massa and Avesani 2007; Zhao et al. 2014; Richardson and Domingos 2002) Homogeneous Graph Chen et al. (2019a), Jamali and Ester (2009), Ma et al. (2009), Ma et al. (2008), Ma et al. (2011b) K-partite Graph Mansoury et al. Recommending on graphs: a comprehensive review from a data… Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) 123 (2020) Complex Heterogeneous Graph Salamat et al. (2021) Multiple Graphs Fan et al. (2019b), Wu et al. (2019b), Liu et al. (2020), Ma et al. (2011a), Zhang et al. (2021a), Huang et al. (2021b) Diginetica2 Homogeneous Graph Xu et al. (2019a), Wu et al. (2019c), Qiu et al. (2020a), Pan et al. (2020), Huang et al. (2021a) Multiple Graphs Wang et al. (2020h), Xia et al. (2021c) Ciao (Tang et al. 2012) Homogeneous Graph Chen et al. (2019a) Complex Heterogeneous Graph Salamat et al. (2021) Multiple Graphs Fan et al. (2019b), Zhang et al. (2021a) JD (Wang et al. 2015) Tree-based Graph Huang et al. (2019) Recommending on graphs: a comprehensive review from a data… 847 le 5 continued main Dataset Graph Type Related Papers ommerce Retailrocket3 Homogeneous Graph Xu et al. (2019a), Huang et al. (2021a) Multiple Graphs Xia et al. (2021c) Bbookstore (Huang et al. 2007b) K-partite Graph Li and Chen (2013) Clothing retail (Huang et al. 2007a) K-partite Graph Li and Chen (2013) Alibaba (Cen et al. 2019) K-partite Graph Cen et al. (2019), Tan et al. (2020), Wu et al. (2021b) Complex Heterogeneous Graph Wang et al. (2021b) Multiple Graphs Guo et al. (2021b) Taobao (Fan et al. 2019a; Zhao et al. 2019) Homogeneous Graph Chang et al. (2021b), Ouyang et al. (2021) K-partite Graph Fan et al. (2019a); Xia et al. (2021b); Chen et al. (2021a) Complex Heterogeneous Graph Zhao et al. (2019) Multiple Graphs Zhao et al. (2019), Wang et al. (2020d) Beidian (Xu et al. 2019b) K-partite Graph Xu et al. (2019b) YOOCHOOSE4 Homogeneous Graph Wu et al. (2019c), Qiu et al. (2020a), Pan et al. (2020), Huang et al. (2021a) Multiple Graphs Xia et al. (2021c) Etsy (Wang et al. 2020c) Hypergraph Wang et al. (2020c) Beibei (Xia et al. 2021b; Gao et al. 2019a) K-partite Graph Xia et al. (2021b), Chen et al. (2021a) HOOPS (Fu et al. 2021) Complex Heterogeneous Graph Fu et al. (2021) Tmall5 Multiple Graphs Wang et al. (2020h), Xia et al. (2021c), Guo et al. (2021b) Domain 12 L. Zhang et al. 848 main Dataset Graph Type Related Papers MALib dataset (He et al. 2020) K-partite Graph Li et al. (2021a) Cosmetics6 Multiple Graphs Liu et al. (2021b) UserBehavior7 Multiple Graphs Liu et al. (2021b) Criteo8 Complex Heterogeneous Graph Zheng et al. (2021) Avazu9 Complex Heterogeneous Graph Zheng et al. Recommending on graphs: a comprehensive review from a data… Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) 123 (2021) USCFC10 Complex Heterogeneous Graph Zheng et al. (2021) Adult11 Complex Heterogeneous Graph Zheng et al. (2021) Google Play (Liang et al. 2017) Complex Heterogeneous Graph Xie et al. (2021) AppChina (Xie et al. 2015) Complex Heterogeneous Graph Xie et al. (2015) Advertising (PVC, PCC, Click) (Agarwal et al. 2010) Tree-based Graph Menon et al. (2011) Yahoo! shopping dataset (Kanagal et al. 2012) Tree-based Graph Kanagal et al. (2012) Homogeneous Graph Gori et al. (2007); Isuﬁet al. (2021); Hao et al. (2021b); Du et al. (2011); Ma et al. (2020); Ouyang et al. (2021) Recommending on graphs: a comprehensive review from a data… 849 able 5 continued omain Dataset Graph Type Related Papers ntertainment MovieLens (Cantador et al. 2011; Harper and Konstan 2015) K-partite Graph Zheng et al. (2018), Wang et al. (2019d), Wang et al. (2020f), Zhang et al. (2019a), Lu et al. (2020), Phuong et al. (2019), Song et al. (2019a), Yu et al. (2014), Xin et al. (2019), Cheng et al. (2007), Yu et al. (2013), Jiang et al. (2018a), Nikolakopoulos and Karypis (2019), Wei et al. (2021), Musto et al. (2017), Fouss et al. (2012), Ostuni et al. (2014), Lei et al. (2020b), Tan et al. (2020), Mansoury et al. (2020), Zhang et al. (2021b), Wu et al. (2021c), Zhang et al. (2022), Fan et al. (2021), Hao et al. (2021a), Yang et al. (2021b), Hsu and Li (2021) Complex Heterogeneous Graph Palumbo et al. (2017), Han et al. (2018), Wang et al. (2018b), Sun et al. (2018), Wang et al. (2019a), Wang et al. (2019c), Wang et al. (2019b), Wang et al. (2019g), Yang and Dong (2020), Wang et al. (2020a), Zhang et al. (2016), Catherine and Cohen (2016), Zhou et al. (2020b), Cao et al. (2019), Shi et al. (2016), Ostuni et al. (2013), Sang et al. (2021), Lee et al. (2013), Xie et al. (2015), Palumbo et al. (2020) Multiple Graphs Monti et al. (2017), Wang et al. (2020d), Xia et al. (2021a), Tang et al. (2021) Tree-based Graph Huang et al. (2019) Last.fm (Schedl 2016; Cantador et al. 2011) Homogeneous Graph Chen et al. (2019a); Qiu et al. (2020b) Entertainment Domain 12 L. Zhang et al. 850 able 5 continued omain Dataset Graph Type Related Papers K-partite Graph Wang et al. (2019e), Song et al. (2019a), Yao et al. (2015), Lei et al. (2020b), Wu et al. (2021c), Hao et al. Recommending on graphs: a comprehensive review from a data… Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) 123 (2021a) Complex Heterogeneous Graph Wang et al. (2019c), Wang et al. (2019b), Lei et al. (2020a), Wang et al. (2020g), Feng and Wang (2012), Ostuni et al. (2013), Sang et al. (2021), Wang et al. (2021b), Palumbo et al. (2020), Pang et al. (2022) Hypergraph Bu et al. (2010), Tan et al. (2011), Yu et al. (2021) Multiple Graphs Tian et al. (2021) YahooMusic(Monti et al. 2017; Nikolakopoulos et al. 2019; Dror et al. 2012) Tree-based Graph Koenigstein et al. (2011), Mnih (2012) K-partite Graph Nikolakopoulos and Karypis (2019) Multiple Graphs Monti et al. (2017), Tang et al. (2021) Flixster (Monti et al. 2017; Jamali and Ester 2010) Homogeneous Graph Isuﬁet al. (2021) K-partite Graph Zhang et al. (2019a) Multiple Graphs Monti et al. (2017), Tang et al. (2021) ntertainment Bing-News (Wang et al. 2018b,c) Complex Heterogeneous Graph Wang et al. (2018c), Wang et al. (2018b), Wang et al. (2019a), Liu et al. (2021a) KKBox’s music12 K-partite Graph Xin et al. (2019) Complex Heterogeneous Graph Wang et al. (2019g) Recommending on graphs: a comprehensive review from a data… 851 inued Dataset Graph Type Related Papers HetRec Delicious (Cantador et al. 2011) Homogeneous Graph Song et al. (2019b) Complex Heterogeneous Graph Feng and Wang (2012) Hypergraph Zhu et al. (2016) Xing13 14 Homogeneous Graph Abugabah et al. (2020) Complex Heterogeneous Graph Wang et al. (2020a), Pang et al. (2022) DepaulMovie (Zheng et al. 2015) K-partite Graph Phuong et al. (2019), Musto et al. (2021) InCarMusic (Zheng et al. 2015) K-partite Graph Phuong et al. (2019) Dianping-Food (Wang et al. 2019b) Complex Heterogeneous Graph Wang et al. (2019b) InMind Movie Agent (Catherine et al. 2017) Complex Heterogeneous Graph Catherine et al. (2017) IntentBooks (Zhang et al. 2016) Complex Heterogeneous Graph Zhang et al. (2016) IMDB (Yin et al. 2010) Multiple Graphs Yin et al. (2010) YouTube (Tang et al. 2009) K-partite Graph Cen et al. (2019) Adressa (Gulla et al. 2017) Complex Heterogeneous Graph Sheu and Li (2020), Shi et al. (2021) Hypergraph Gharahighehi et al. (2021) Roularta (Gharahighehi et al. 2021) Hypergraph Gharahighehi et al. (2021), Gharahighehi et al. (2020) MIND15 Complex Heterogeneous Graph Wu et al. (2021a), Liu et al. (2021a), Zhang et al. (2021d) Multiple News Portal (Li et al. 2011) Hypergraph Li and Li (2013) nued Dataset Graph Type Related Papers HetRec Delicious (Cantador et al. 2011) Homogeneous Graph Song et al. Recommending on graphs: a comprehensive review from a data… Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) 123 (2019b) Complex Heterogeneous Graph Feng and Wang (2012) Hypergraph Zhu et al. (2016) Xing13 14 Homogeneous Graph Abugabah et al. (2020) Complex Heterogeneous Graph Wang et al. (2020a), Pang et al. (2022) DepaulMovie (Zheng et al. 2015) K-partite Graph Phuong et al. (2019), Musto et al. (2021) InCarMusic (Zheng et al. 2015) K-partite Graph Phuong et al. (2019) Dianping-Food (Wang et al. 2019b) Complex Heterogeneous Graph Wang et al. (2019b) InMind Movie Agent (Catherine et al. 2017) Complex Heterogeneous Graph Catherine et al. (2017) IntentBooks (Zhang et al. 2016) Complex Heterogeneous Graph Zhang et al. (2016) IMDB (Yin et al. 2010) Multiple Graphs Yin et al. (2010) YouTube (Tang et al. 2009) K-partite Graph Cen et al. (2019) Adressa (Gulla et al. 2017) Complex Heterogeneous Graph Sheu and Li (2020), Shi et al. (2021) Hypergraph Gharahighehi et al. (2021) Roularta (Gharahighehi et al. 2021) Hypergraph Gharahighehi et al. (2021), Gharahighehi et al. (2020) MIND15 Complex Heterogeneous Graph Wu et al. (2021a), Liu et al. (2021a), Zhang et al. (2021d) Multiple News Portal (Li et al. 2011) Hypergraph Li and Li (2013) Domain 12 L. Zhang et al. 852 Table 5 continued Domain Dataset Graph Type Related Papers Sougou News (Shi et al. 2021) Complex Heterogeneous Graph Shi et al. (2021) Filmtrust (Guo et al. 2013) Multiple Graphs Zhang et al. (2021a) Mtime (Li et al. 2014) Homogeneous Graph Li et al. (2014) Netease (Cao et al. 2017) Multiple Graphs Chang et al. (2021a) Kuaishou (Chang et al. 2021b) Homogeneous Graph Chang et al. (2021b) Tiktok (Wei et al. 2021) K-partite Graph Wei et al. (2021) Kwai (Wei et al. 2021) K-partite Graph Wei et al. (2021) Restaurant&consumer (Vargas-Govea et al. 2011) K-partite Graph Jiang et al. (2018a) Social Network Douban (Song et al. 2019b; Ma et al. 2011a; Zheng et al. 2017; Shi et al. 2018; Monti et al. 2017) Homogeneous Graph Song et al. (2019b), Isuﬁet al. (2021) K-partite Graph Zhang et al. (2019a) Complex Heterogeneous Graph Shi et al. (2018), Shi et al. (2015), Salamat et al. (2021) Hypergraph Yu et al. (2021) Multiple Graphs Ma et al. (2011a), Zheng et al. (2017), Shi et al. (2016), Monti et al. (2017), Zhang et al. (2021a), Tian et al. (2021), Guo et al. (2021a) WeChat (Wu et al. 2019b; Wang et al. 2020f) K-partite Graph Wang et al. (2020f) Multiple Graphs Wu et al. Recommending on graphs: a comprehensive review from a data… Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) 123 (2019b) Social Network Recommending on graphs: a comprehensive review from a data… 853 Table 5 continued Domain Dataset Graph Type Related Papers Twitter (Sharma et al. 2016; De Domenico et al. 2013) K-partite Graph Cen et al. (2019); Sharma et al. (2016) Reddit16 Homogeneous Graph Abugabah et al. (2020) Complex Heterogeneous Graph Pang et al. (2022) Pinterest(Ying et al. 2018; Eksombatchai et al. 2018) K-partite Graph Ying et al. (2018); Eksombatchai et al. (2018); Lei et al. (2020b); Tan et al. (2020); Yang et al. (2021b) Flickr (Wu et al. 2019a) Homogeneous Graph Wu et al. (2019a) Hike network (Verma et al. 2019) Multiple Graphs Verma et al. (2019) Academic or Book BookCrossing (Ziegler et al. 2005) K-partite Graph Fouss et al. (2012); Li and Chen (2013); Hsu and Li (2021) Complex Heterogeneous Graph Wang et al. (2018b), Wang et al. (2019a), Wang et al. (2019c), Wang et al. (2019b), Yang and Dong (2020), Zhou et al. (2020b), Sang et al. (2021) DBbook17 (Lu et al. 2020) K-partite Graph Song et al. (2019a); Musto et al. (2017); Lu et al. (2020) Complex Heterogeneous Graph Cao et al. (2019) DBLP (Tang et al. 2008) Complex Heterogeneous Graph Zhu et al. (2021c) Multiple Graphs Ali et al. (2020); Yin et al. (2010) CiteULike (Wang et al. 2013) Homogeneous Graph Gao et al. (2018) K-partite Graph Yao et al. (2015); Song et al. (2011) Hypergraph Zhu et al. (2016) 123 continued Dataset Graph Type Related Papers Twitter (Sharma et al. 2016; De Domenico et al. 2013) K-partite Graph Cen et al. (2019); Sharma et al. (2016) Reddit16 Homogeneous Graph Abugabah et al. (2020) Complex Heterogeneous Graph Pang et al. (2022) Pinterest(Ying et al. 2018; Eksombatchai et al. 2018) K-partite Graph Ying et al. (2018); Eksombatchai et al. (2018); Lei et al. (2020b); Tan et al. (2020); Yang et al. (2021b) Flickr (Wu et al. 2019a) Homogeneous Graph Wu et al. (2019a) Hike network (Verma et al. 2019) Multiple Graphs Verma et al. (2019) mic or Book BookCrossing (Ziegler et al. 2005) K-partite Graph Fouss et al. (2012); Li and Chen (2013); Hsu and Li (2021) Complex Heterogeneous Graph Wang et al. (2018b), Wang et al. (2019a), Wang et al. (2019c), Wang et al. (2019b), Yang and Dong (2020), Zhou et al. (2020b), Sang et al. (2021) DBbook17 (Lu et al. 2020) K-partite Graph Song et al. (2019a); Musto et al. Recommending on graphs: a comprehensive review from a data… Table 5 Statistics of datasets commonly used in GLRS Domain Dataset Graph Type Related Papers E-commerce Amazon (He and McAuley 2016; McAuley et al. 2015) Tree-based Graph He et al. (2016), Sun et al. (2017), Gao et al. (2019b), Huang et al. (2019) Homogeneous Graph Wang et al. (2020b), Ma et al. (2020), Zhu et al. (2021b) K-partite Graph Cen et al. (2019), Zheng et al. (2018), Wang et al. (2019f), Chen et al. (2020a), Wang et al. (2019d), Vijaikumar et al. (2019), Wang et al. (2019e), He et al. (2015), Sun et al. (2020), Wu et al. (2021b), Sun et al. (2021), Yang et al. (2021b), Fan et al. (2021), Zhang et al. (2022) Complex Heterogeneous Graph Han et al. (2018), Zhao et al. (2017), Zhao et al. (2019), Xian et al. (2019), Wang et al. (2020a), Ai et al. (2018), Ma et al. (2019), Wang et al. (2020g), Wang et al. (2021b), Xie et al. (2021), Liu et al. (2021c), Chen et al. (2021b) Hypergraph Wang et al. (2020c) Multiple Graphs Zhang et al. (2021c), Zhu et al. (2021a) Tree-based Graph Gao et al. (2019b) Homogeneous Graph Wu et al. (2019a); Song et al. (2019b); Zhu et al. (2021b) Yelp1 K-partite Graph Wang et al. (2019f), Vijaikumar et al. (2019), Wang et al. (2019e), Lu et al. (2020), He et al. (2015), Yu et al. (2014), Wu et al. (2021b), Sun et al. (2021) 123 (2017); Lu et al. (2020) Complex Heterogeneous Graph Cao et al. (2019) DBLP (Tang et al. 2008) Complex Heterogeneous Graph Zhu et al. (2021c) Multiple Graphs Ali et al. (2020); Yin et al. (2010) CiteULike (Wang et al. 2013) Homogeneous Graph Gao et al. (2018) K-partite Graph Yao et al. (2015); Song et al. (2011) Hypergraph Zhu et al. (2016) Academic or Book Domain 12 L. Zhang et al. 854 e 5 continued ain Dataset Graph Type Related Papers Goodreads (Wang and Caverlee 2019) Homogeneous Graph Ma et al. (2020) Hypergraph Wang et al. (2020c) User-Tag (Pan et al. 2008; Sun et al. 2015; Song et al. 2011) Homogeneous Graph Du et al. (2011); Sun et al. (2015) K-partite Graph Song et al. (2011) Librarything (Zhao et al. 2014) Complex Heterogeneous Graph Palumbo et al. (2020) Multiple Graphs Liu et al. (2020) Youshu (Chen et al. 2019b) Multiple Graphs Chang et al. (2021a) ACL Anthology Network (Radev et al. 2013) Multiple Graphs Ali et al. (2020) OHSUMED (Hersh et al. 1994) K-partite Graph Cheng et al. (2007) K-partite Graph Wang et al. (2019f); Chen et al. (2020a); Tan et al. (2020) Table 5 continued Domain Dataset Graph Type Related Papers Goodreads (Wang and Caverlee 2019) Homogeneous Graph Ma et al. (2020) Hypergraph Wang et al. (2020c) User-Tag (Pan et al. 2008; Sun et al. 2015; Song et al. 2011) Homogeneous Graph Du et al. (2011); Sun et al. (2015) K-partite Graph Song et al. (2011) Librarything (Zhao et al. 2014) Complex Heterogeneous Graph Palumbo et al. (2020) Multiple Graphs Liu et al. (2020) Youshu (Chen et al. 2019b) Multiple Graphs Chang et al. (2021a) ACL Anthology Network (Radev et al. 2013) Multiple Graphs Ali et al. (2020) OHSUMED (Hersh et al. 1994) K-partite Graph Cheng et al. (2007) K-partite Graph Wang et al. (2019f); Chen et al. (2020a); Tan et al. (2020) Table 5 continued Domain Dataset Graph Type Related Papers Goodreads (Wang and Caverlee 2019) Homogeneous Graph Ma et al. (2020) Hypergraph Wang et al. (2020c) User-Tag (Pan et al. 2008; Sun et al. 2015; Song et al. 2011) Homogeneous Graph Du et al. (2011); Sun et al. (2015) K-partite Graph Song et al. (2011) Librarything (Zhao et al. 2014) Complex Heterogeneous Graph Palumbo et al. (2020) Multiple Graphs Liu et al. (2020) Youshu (Chen et al. 2019b) Multiple Graphs Chang et al. (2021a) ACL Anthology Network (Radev et al. 2013) Multiple Graphs Ali et al. (2020) OHSUMED (Hersh et al. 1994) K-partite Graph Cheng et al. (2007) K-partite Graph Wang et al. (2019f); Chen et al. (2020a); Tan et al. (2020) Recommending on graphs: a comprehensive review from a data… 855 omain Dataset Graph Type Related Papers Gowalla (Liang et al. 2016; Cho et al. 2011; Liu et al. Academic or Book 2013) Homogeneous Graph Qiu et al. (2020b); Hao et al. (2021b) Complex Heterogeneous Graph Bagci and Karagoz (2016) Multiple Graphs Christoforidis et al. (2021) OI Trip.com+Facebook+Twitter (Wang et al. 2017) Multiple Graphs Wang et al. (2017) Foursquare (Gao et al. 2012) Tree-based Graph Yang et al. (2016) K-partite Graph Wang et al. (2019d) Complex Heterogeneous Graph Bagci and Karagoz (2016) Multiple Graphs Christoforidis et al. (2021) Brightkite (Cho et al. 2011) Complex Heterogeneous Graph Bagci and Karagoz (2016) Google Local (He et al. 2017) Homogeneous Graph Zhu et al. (2021b) Multiple Graphs Zhu et al. (2021a) Tripadvisor18 K-partite Graph Musto et al. (2021) NUS-MSS (Farseev et al. 2015) Homogeneous Graph Farseev et al. (2017) thers Educational website and textbooks (Shi et al. 2020) Complex Heterogeneous Graph Shi et al. (2020) MIT AI + CASAS (Chen et al. 2019d) Complex Heterogeneous Graph Chen et al. (2019d) REDIAL (Li et al. 2018b) Complex Heterogeneous Graph Zhou et al. (2020a) VizML corpus (Hu et al. 2019) K-partite Graph Li et al. (2021b) Others POI 12 L. Zhang et al. 856 Table 5 continued Domain Dataset Graph Type Related Papers Legal recommendation dataset (Yang et al. 2021a) Complex Heterogeneous Graph Yang et al. (2021a) Yahoo! trafﬁc stream (Menon et al. 2011) Tree-based Graph Menon et al. (2011) 1https://www.yelp.com/dataset/documentation/main 2https://competitions.codalab.org/competitions/11161 3https://www.kaggle.com/retailrocket/ecommerce-dataset 4https://2015.recsyschallenge.com/challenge.html 5https://tianchi.aliyun.com/dataset/dataDetail?dataId=42 6https://www.kaggle.com/mkechinov/ecommerce-events-history-in-cosmetics-shop 7https://tianchi.aliyun.com/dataset/dataDetail?dataId=649&userId=1 8https://www.kaggle.com/c/criteo-display-ad-challenge 9https://www.kaggle.com/c/avazu-ctr-prediction 10https://www.kaggle.com/kaggle/us-consumer-ﬁnance-complaints 11http://archive.ics.uci.edu/ml/datasets/Adult 12https://wsdm-cup-2018.kkbox.events/ 13https://2016.recsyschallenge.com/ 14http://www.recsyschallenge.com/2017/ 15https://msnews.github.io/ 16https://www.kaggle.com/colemaclean/subreddit-interactions 17http://2014.eswc-conferences.org/important-dates/call-RecSys.html 18https://github.com/irecsys/CARSKit/tree/master/context-aware_data_sets 123 Recommending on graphs: a comprehensive review from a data… 857 Appendix B Technology summary in GLRS See Table 6. 6 Technology summary in GLRS ology Class Tech Subclass Domain Dataset Graph Type onal Machine ning niques PRM E-commerce Yelp Complex Heterogeneous Graph: Catherine and Cohen (2016) Entertainment InMind Movie Agent Complex Heterogeneous Graph: Catherine et al. (2017) MovieLens Complex Heterogeneous Graph: Catherine and Cohen (2016), Lee et al. (2013) K-partite Graph: Shams and Haratizadeh (2017), Musto et al. (2017) Last.fm K-partite Graph: Jäschke et al. (2007) DePaulMovie K-partite Graph: Musto et al. (2021) Academic or Book BibSonomy K-partite Graph: Jäschke et al. (2007) DBbook K-partite Graph: Musto et al. (2017) POI Tripadvisor K-partite Graph: Musto et al. (2021) RTGR E-commerce Yelp Hypergraph: Mao et al. (2019), Yu et al. (2021) Entertainment Last.fm Hypergraph: Bu et al. (2010), Tan et al. (2011), Yu et al. (2021) Multiple News Portal Hypergraph: Li and Li (2013) HetRec Delicious Hypergraph: Zhu et al. (2016) Adressa Hypergraph: Gharahighehi et al. (2021) Roularta Hypergraph: Gharahighehi et al. (2021) Social Network Douban Hypergraph: Yu et al. (2021) Academic or Book CiteULike Hypergraph: Zhu et al. (2016) 3 12 L. Zhang et al. 858 ontinued y Class Tech Subclass Domain Dataset Graph Type POI Trip.com+Face book+Twitter Multiple Graphs: Gao et al. (2019b) KML Entertainment MovieLens K-partite Graph: Fouss et al. (2012) Complex Heterogeneous Graph: Ostuni et al. (2014) mtime Homogeneous Graph: Li et al. (2014) Academic or Book Book-Crossing K-partite Graph: Fouss et al. (2012) LFM E-commerce Amazon Complex Heterogeneous Graph: Zhao et al. (2017) Tree-based Graph: Sun et al. (2017) Yelp Complex Heterogeneous Graph: Zhao et al. (2017), Kyriakidi et al. (2020) Epinions Multiple Graphs: Ma et al. (2011a) Homogeneous Graph: Ma et al. (2009), Ma et al. (2008), Ma et al. (2011b) Yahoo! Shopping Tree-based Graph: Kanagal et al. (2012) LFM Entertainment Yahoo!Music Tree-based Graph: Koenigstein et al. (2011), Mnih (2012) MovieLens Complex Heterogeneous Graph: Du et al. (2011) Social Network Douban Multiple Graphs: Ma et al. (2011a) Academic or Book User-Tag Homogeneous Graph: Du et al. (2011), Sun et al. (2015) POI Foursquare Tree-based Graph: Yang et al. (2016) Other Yahoo! trafﬁc stream Tree-based Graph: Menon et al. (2011) l Machine ues Others (Spectral graph) E-commerce Amazon K-partite Graph: Zheng et al. (2018) Entertainment MovieLens K-partite Graph: Zheng et al. (2018) HetRec Delicious K-partite Graph: Zheng et al. (2018) Koenigstein et al. (2011), Mnih (2012) MovieLens Complex Heterogeneous Graph: Du et al. (2011) Social Network Douban Multiple Graphs: Ma et al. Appendix B Technology summary in GLRS (2011a) Academic or Book User-Tag Homogeneous Graph: Du et al. (2011), Sun et al. (2015) POI Foursquare Tree-based Graph: Yang et al. (2016) Other Yahoo! trafﬁc stream Tree-based Graph: Menon et al. (2011) Traditional Machine Learning Techniques Others (Spectral graph) E-commerce Amazon K-partite Graph: Zheng et al. (2018) Entertainment MovieLens K-partite Graph: Zheng et al. (2018) HetRec Delicious K-partite Graph: Zheng et al. (2018) 3 12 Recommending on graphs: a comprehensive review from a data… 859 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type POI NUS-MSS Homogeneous Graph: Farseev et al. (2017) Others (Similarity) Entertainment DepaulMovie K-partite Graph: Phuong et al. (2019) MovieLens K-partite Graph: Phuong et al. (2019) InCarMusic K-partite Graph: Phuong et al. (2019) Others (Clustering) Social Network User-Tag K-partite Graph: Song et al. (2011) BbSonomy K-partite Graph: Song et al. (2011) Academic or Book CiteULike K-partite Graph: Song et al. (2011) Others (Complex Number) E-commerce AppChina Complex Heterogeneous Graph: Xie et al. (2015) Entertainment MovieLens Complex Heterogeneous Graph: Xie et al. (2015) E-commerce Clothing-Retail K-partite Graph: Li and Chen (2013) Amazon Multiple Graphs: Vijaikumar et al. (2019) Complex Heterogeneous Graph: Ma et al. (2019) Path-based Techniques RWM Yelp Multiple Graphs: Vijaikumar et al. (2019) Epinions Homogeneous Graph: Jamali and Ester (2009) Entertainment IMDB Multiple Graphs: Yin et al. (2010) MovieLens K-partite Graph: Cheng et al. (2007), Jiang et al. (2018a), Nikolakopoulos and Karypis (2019) Homogeneous Graph: Gori et al. (2007) Last.fm K-partite Graph: Yao et al. (2015) Complex Heterogeneous Graph: Feng and Wang (2012) Yahoo2RMusic K-partite Graph: Nikolakopoulos and Karypis (2019) Path-based Techniques 123 3 12 L. Zhang et al. 860 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type HetRec Delicious Complex Heterogeneous Graph: Feng and Wang (2012) Restaurant & Consumer K-partite Graph: Jiang et al. (2018a) Social Network Twitter K-partite Graph: Sharma et al. (2016) Pinterest K-partite Graph: Eksombatchai et al. (2018) Path-based Techniques RWM Academic or Book Book-Crossing K-partite Graph: Li and Chen (2013) DBLP Multiple Graphs: Yin et al. (2010) OHSUMED K-partite Graph: Cheng et al. (2007) CiteULike K-partite Graph: Yao et al. (2015) POI Brightkite Complex Heterogeneous Graph: Bagci and Karagoz (2016) Gowalla Complex Heterogeneous Graph: Bagci and Karagoz (2016) Foursquare Complex Heterogeneous Graph: Bagci and Karagoz (2016) RM E-commerce Amazon K-partite Graph: He et al. (2015) Yelp K-partite Graph: He et al. (2015) Academic or Book Educational Website and Textbooks Complex Heterogeneous Graph: Shi et al. Appendix B Technology summary in GLRS (2020) MPM E-commerce Amazon K-partite Graph: Cen et al. (2019) Alibaba K-partite Graph: Cen et al. (2019) Yelp K-partite Graph: Lu et al. (2020); Yu et al. (2014) Complex Heterogeneous Graph: Shi et al. (2018), Shi et al. (2015), Zheng et al. (2017), Shi et al. (2016) Path-based Techniques 123 12 Recommending on graphs: a comprehensive review from a data… Deep Learning-based Techniques AM Recommending on graphs: a comprehensive review from a data… 861 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type Entertainment YouTube K-partite Graph: Cen et al. (2019) MovieLens K-partite Graph: Lu et al. (2020), Yu et al. (2014), Yu et al. (2013) Complex Heterogeneous Graph: Shi et al. (2016), Ostuni et al. (2013) Last.fm Complex Heterogeneous Graph: Ostuni et al. (2013) Social Network Twitter K-partite Graph: Cen et al. (2019) Douban Complex Heterogeneous Graph: Shi et al. (2018), Shi et al. (2015), Zheng et al. (2017), Shi et al. (2016) Academic or Book DBook K-partite Graph: Lu et al. (2020) Graph Embedding-based Techniques GDRM E-commerce Amazon Complex Heterogeneous Graph: Fu et al. (2020), Wang et al. (2020a) Tree-based Graph: He et al. (2016) Taobao Multiple Graphs: Wang et al. (2020d) Entertainment MovieLens Complex Heterogeneous Graph: Wang et al. (2020a); Palumbo et al. (2020) Multiple Graphs: Wang et al. (2020d) Last.fm Complex Heterogeneous Graph: Palumbo et al. (2020) Xing Complex Heterogeneous Graph: Wang et al. (2020a) GDRM Social Network Hike Network Multiple Graphs: Verma et al. (2019) Academic or Book Citation Complex Heterogeneous Graph: Jiang et al. (2018b) LibraryThing Complex Heterogeneous Graph: Palumbo et al. (2020) 12 3 L. Zhang et al. 862 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type DBLP Multiple Graphs: Ali et al. (2020) ACL Anthology Network Multiple Graphs: Ali et al. (2020) POI Foursquare Multiple Graphs: Christoforidis et al. (2021) Graph Embedding-based Techniques Gowalla Multiple Graphs: Christoforidis et al. (2021) E-commerce Amazon Complex Heterogeneous Graph: Ai et al. (2018) Homogeneous Graph: Wang et al. (2020b) TEM Alibaba Complex Heterogeneous Graph: Wang et al. (2021b) Entertainment MovieLens Complex Heterogeneous Graph: Palumbo et al. (2017), Wang et al. (2018b), Cao et al. (2019) Bing-News Complex Heterogeneous Graph: Wang et al. (2018b) Last.fm Complex Heterogeneous Graph: Wang et al. (2021b) Book-Crossing Complex Heterogeneous Graph: Wang et al. (2018b) Academic or Book CiteULike Homogeneous Graph: Gao et al. (2018) DBBook Complex Heterogeneous Graph: Cao et al. (2019) Other MIT AI+CASAS Complex Heterogeneous Graph: Chen et al. (2019d) VizML Corpus K-partite Graph: Li et al. (2021b) E-commerce Amazon Tree-based Graph: Huang et al. (2019) JD Tree-based Graph: Huang et al. (2019) DNN Yelp Homogeneous Graph: Wu et al. (2019a) Graph Embedding-based Techniques 123 12 Recommending on graphs: a comprehensive review from a data… 863 Recommending on graphs: a comprehensive review from a data… Technology Class Tech Subclass Domain Dataset Graph Type Complex Heterogeneous Graph: Sun et al. (2018); Mezni et al. (2021) Entertainment MovieLens Complex Heterogeneous Graph: Sun et al. (2018), Wang et al. (2019g) Last.fm Tree-based Graph: Huang et al. (2019) KKBox Complex Heterogeneous Graph: Wang et al. (2019g) Social Network Flickr Homogeneous Graph: Wu et al. (2019a) AE Entertainment MovieLens Complex Heterogeneous Graph: Zhang et al. (2016) Academic or Book IntentBooks Complex Heterogeneous Graph: Zhang et al. (2016) Amazon Tree-based Graph: Gao et al. (2019b) K-partite Graph: Wang et al. (2019d); Zhang et al. (2022) Complex Heterogeneous Graph: Han et al. (2018) Yelp Tree-based Graph: Gao et al. (2019b) Multiple Graphs: Xia et al. (2021a) Deep Learning-based Techniques AM E-commerce Epinions Homogeneous Graph: Chen et al. (2019a) Ciao Homogeneous Graph: Chen et al. (2019a) HOOPS Complex Heterogeneous Graph: Fu et al. (2021) Taobao Homogeneous Graph: Chang et al. (2021b) Kuaishou Homogeneous Graph: Chang et al. (2021b) Entertainment MovieLens Complex Heterogeneous Graph: Han et al. (2018), Wang et al. (2019a) Homogeneous Graph: Hao et al. (2021b) Deep Learning-based Techniques AM Deep Learning-based Techniques AM 123 3 12 L. Zhang et al. 864 Class Tech Subclass Domain Dataset Graph Type K-partite Graph: Wang et al. (2019d); Xin et al. (2019); Zhang et al. (2022) Multiple Graphs: Xia et al. (2021a) Bing-News Complex Heterogeneous Graph: Wang et al. (2019a) KKBox K-partite Graph: Xin et al. (2019) Last.fm Homogeneous Graph: Chen et al. (2019a) Academic or Book Book-Crossing Complex Heterogeneous Graph: Wang et al. (2019a) POI Foursquare K-partite Graph: Wang et al. (2019d) Gowalla Homogeneous Graph: Hao et al. (2021b) DRL E-commerce Amazon Complex Heterogeneous Graph: Xian et al. (2019) Yelp Complex Heterogeneous Graph: Lei et al. (2020a) Entertainment Last.fm K-partite Graph: Song et al. (2019a) Complex Heterogeneous Graph: Lei et al. (2020a) DRL MovieLens K-partite Graph: Song et al. (2019a) MIND Complex Heterogeneous Graph: Liu et al. (2021a) Bing-News Complex Heterogeneous Graph: Liu et al. (2021a) Academic or Book DBbook K-partite Graph: Song et al. (2019a) Amazon K-partite Graph: Wang et al. (2019f); Chen et al. (2020a); Wang et al. (2019e); Sun et al. (2020); Wu et al. (2021b) Homogeneous Graph: Zhu et al. (2021b), Ma et al. (2020) Homogeneous Graph: Zhu et al. (2021b), Ma et al. (2020) 123 123 12 Recommending on graphs: a comprehensive review from a data… 865 Multiple Graphs: Liu et al. (2020), Huang et al. Recommending on graphs: a comprehensive review from a data… (2021b), Tang et al. (2021), Zhu et al. (2021a), Guo et al. (2021a) Multiple Graphs: Wu et al. (2019b); Liu et al. (2020); Huang et al. (2021b) Homogeneous Graph: Ouyang et al. (2021) K-partite Graph: Fan et al. (2019a), Chen et al. (2021a) Complex Heterogeneous Graph: Zhao et al. (2019) Alibaba K-partite Graph: Tan et al. (2020), Wu et al. (2021b) K-partite Graph: Tan et al. (2020), Wu et al. (2021b) Multiple Graphs: Guo et al. (2021b) Beibei K-partite Graph: Chen et al. (2021a) 3 12 L. Zhang et al. 866 Multiple Graph: Tang et al. (2021) Deep Learning-based Techniques GNN Last.fm Homogeneous Graph: Qiu et al. (2020b) K-partite Graph: Wang et al. (2019e), Lei et al. (2020b) 123 12 Recommending on graphs: a comprehensive review from a data… Recommending on graphs: a comprehensive review from a data… 867 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type MIND Complex Heterogeneous Graph: Wu et al. (2021a), Zhang et al. (2021d) Netease Multiple Graphs: Chang et al. (2021a) YahooMusic Multiple Graphs: Tang et al. (2021) Tiktok K-partite Graph: Wei et al. (2021) Kwai K-partite Graph: Wei et al. (2021) Social Network WeChat Multiple Graphs: Wu et al. (2019b) Pinterest K-partite Graph: Ying et al. (2018), Lei et al. (2020b), Tan et al. (2020), Yang et al. (2021b) Douban Homogeneous Graph: Isuﬁ et al. (2021) Multiple Graphs: Tian et al. (2021), Guo et al. (2021a) Academic or Book Book-Crossing Complex Heterogeneous Graph: Wang et al. (2019c), Wang et al. (2019b) Librarything Multiple Graphs: Liu et al. (2020) Youshu Multiple Graphs: Chang et al. (2021a) Goodreads Homogeneous Graph: Ma et al. (2020) POI Gowalla Homogeneous Graph: Qiu et al. (2020b) K-partite Graph: Wang et al. (2019f), Chen et al. (2020a), Tan et al. (2020) Google Local Homogeneous Graph: Zhu et al. (2021b) Multiple Graphs: Zhu et al. (2021a) 123 L. Zhang et al. 868 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type Other MALib-Dataset K-partite Graph: Li et al. (2021a) Amazon K-partite Graph: Fan et al. (2021) Complex Heterogeneous Graph: Wang et al. (2020g), Liu et al. (2021c), Xie et al. (2021), Chen et al. (2021b) Hypergraph: Wang et al. (2020c) Yelp Homogeneous Graph: Song et al. (2019b) Complex Heterogeneous Graph: Wang et al. (2020g), Liu et al. (2021c), Xie et al. (2021) Deep Learning-based Techniques DHM E-commerce Diginetica Homogeneous Graph: Xu et al. (2019a); Wu et al. (2019c); Huang et al. (2021a) Multiple Graphs: Xia et al. (2021c) Retailrocket Homogeneous Graph: Xu et al. (2019a), Huang et al. (2021a) Yoochoose Homogeneous Graph: Wu et al. (2019c), Huang et al. (2021a) Ciao Complex Heterogeneous Graph: Salamat et al. (2021) Multiple Graphs: Fan et al. (2019b), Zhang et al. (2021a) Epinions Complex Heterogeneous Graph: Salamat et al. (2021) Multiple Graphs: Fan et al. (2019b); Zhang et al. (2021a) Etsy Hypergraph: Wang et al. (2020c) Deep Learning-based Techniques DHM 123 12 Recommending on graphs: a comprehensive review from a data… Recommending on graphs: a comprehensive review from a data… 869 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type Beidia K-partite Graph: Xu et al. (2019b) Taobao K-partite Graph: Xia et al. (2021b) Beibei K-partite Graph: Xia et al. (2021b) Tmall Multiple Graphs: Xia et al. Recommending on graphs: a comprehensive review from a data… (2021c) RetailRocket Multiple Graphs: Xia et al. (2021c) Cosmetics Multiple Graphs: Liu et al. (2021b) UserBehavior Multiple Graphs: Liu et al. (2021b) Google Play Complex Heterogeneous Graph: Xie et al. (2021) Entertainment MovieLens K-partite Graph: Wang et al. (2020f), Zhang et al. (2019a), Wu et al. (2021c), Fan et al. (2021), Hao et al. (2021a), Hsu and Li (2021) Complex Hetero- geneous Graph: Yang and Dong (2020), Zhou et al. (2020b), Sang et al. (2021) Multiple Graphs: Monti et al. (2017) Deep Learning-based Techniques DHM Entertainment Last.fm K-partite Graph: Wu et al. (2021c), Hao et al. (2021a) Complex Hetero- geneous Graph: Yang and Dong (2020), Wang et al. (2020g), Sang et al. (2021), Pang et al. (2022) Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type Beidia K-partite Graph: Xu et al. (2019b) Taobao K-partite Graph: Xia et al. (2021b) Beibei K-partite Graph: Xia et al. (2021b) Tmall Multiple Graphs: Xia et al. (2021c) RetailRocket Multiple Graphs: Xia et al. (2021c) Cosmetics Multiple Graphs: Liu et al. (2021b) UserBehavior Multiple Graphs: Liu et al. (2021b) Google Play Complex Heterogeneous Graph: Xie et al. (2021) Entertainment MovieLens K-partite Graph: Wang et al. (2020f), Zhang et al. (2019a), Wu et al. (2021c), Fan et al. (2021), Hao et al. (2021a), Hsu and Li (2021) Complex Hetero- geneous Graph: Yang and Dong (2020), Zhou et al. (2020b), Sang et al. (2021) Multiple Graphs: Monti et al. (2017) Deep Learning-based Techniques DHM Entertainment Last.fm K-partite Graph: Wu et al. (2021c), Hao et al. (2021a) Complex Hetero- geneous Graph: Yang and Dong (2020), Wang et al. (2020g), Sang et al. (2021), Pang et al. (2022) Domain Dataset Graph Type Beidia K-partite Graph: Xu et al. (2019b) Taobao K-partite Graph: Xia et al. (2021b) Beibei K-partite Graph: Xia et al. (2021b) Tmall Multiple Graphs: Xia et al. (2021c) RetailRocket Multiple Graphs: Xia et al. (2021c) Cosmetics Multiple Graphs: Liu et al. (2021b) UserBehavior Multiple Graphs: Liu et al. (2021b) Google Play Complex Heterogeneous Graph: Xie et al. (2021) Entertainment MovieLens K-partite Graph: Wang et al. (2020f), Zhang et al. (2019a), Wu et al. (2021c), Fan et al. (2021), Hao et al. (2021a), Hsu and Li (2021) Complex Hetero- geneous Graph: Yang and Dong (2020), Zhou et al. (2020b), Sang et al. (2021) Multiple Graphs: Monti et al. (2017) Entertainment Last.fm K-partite Graph: Wu et al. (2021c), Hao et al. Deep Learning-based Techniques DHM Recommending on graphs: a comprehensive review from a data… (2021a) Complex Hetero- geneous Graph: Yang and Dong (2020), Wang et al. (2020g), Sang et al. (2021), Pang et al. (2022) Deep Learning-based Techniques DHM Deep Learning-based Techniques DHM 123 L. Zhang et al. 870 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type Entertainment HetRec Delicious Homogeneous Graph: Song et al. (2019b) Adressa Complex Heterogeneous Graph: Sheu and Li (2020), Shi et al. (2021) Roularta Hypergraph: Gharahighehi et al. (2020) Bing-News Complex Heterogeneous Graph: Wang et al. (2018c) Flixster K-partite Graph: Zhang et al. (2019a) Multiple Graphs: Monti et al. (2017) Xing Homogeneous Graph: Abugabah et al. (2020) Complex Heterogeneous Graph: Pang et al. (2022) Deep Learning-based Techniques DHM YahooMusic Multiple Graphs: Monti et al. (2017) Filmtrust Multiple Graphs: Zhang et al. (2021a) Sougou Complex Heterogeneous Graph: Shi et al. (2021) Douban Homogeneous Graph: Song et al. (2019b) Social Network K-partite Graph: Zhang et al. (2019a) Complex Heterogeneous Graph: Salamat et al. (2021) Multiple Graphs: Monti et al. (2017), Zhang et al. (2021a) Deep Learning-based Techniques DHM 123 Recommending on graphs: a comprehensive review from a data… Recommending on graphs: a comprehensive review from a data… 871 Table 6 continued Technology Class Tech Subclass Domain Dataset Graph Type WeChat K-partite Graph: Wang et al. (2020f) Reddit Homogeneous Graph.: Abugabah et al. (2020) Complex Heterogeneous Graph: Pang et al. (2022) Others K-partite Graph: Kim et al. (2019) Academic or Book Goodreads Hypergraph: Wang et al. (2020c) Book-Crossing K-partite Graph: Hsu and Li (2021) Complex Heterogeneous Graph: Yang and Dong (2020), Zhou et al. (2020b), Sang et al. (2021) DBLP Complex Heterogeneous Graph: Zhu et al. (2021c) Others REDIAL Complex Heterogeneous Graph: Zhou et al. (2020a) Legal recommendation dataset Complex Heterogeneous Graph: Yang et al. (2021a) 123 872 L. Zhang et al. Appendix C Statistics of datasets and related technologies in GLRS See Table 7. Table 7 Quantitative summarization of different datasets on adopted GLRS technologies Domain Dataset Traditional ML Path-based Graph embedding Deep Learning DNN AE AM DRL GNN DHM E-commerce Amazon 3 4 5 1 4 1 12 6 Yelp 5 8 3 2 1 9 4 Epinions 4 1 1 3 3 Diginetica 4 4 Ciao 1 3 JD 1 Retailrocket 3 Clothing retail 1 Alibaba 1 1 3 Taobao 1 1 4 1 Beidian YOOCHOOSE 3 2 Etsy 1 Beibei 1 1 HOOPS 1 Tmall 2 1 Cosmetics 1 UserBehavior 1 Criteo 1 Avazu 1 USCFC 1 Adult 1 Google Play 1 AppChina 1 Yahoo! shopping dataset 1 MovieLens 10 9 6 2 1 7 1 9 10 Last.fm 4 3 2 1 1 2 6 6 YahooMusic 2 1 1 1 Flixster 2 2 Bing-News 1 1 1 1 KKBox’s music 1 1 Appendix C Statistics of datasets and related technologies in GLRS Appendix C Statistics of datasets and related technologies in GLRS Appendix C Statistics of datasets and related technologies in GLRS See Table 7. 12 3 Recommending on graphs: a comprehensive review from a data… References Abugabah, A., Cheng, X., Wang, J.: Dynamic graph attention-aware networks for session-based recom- mendation. In: 2020 International Joint Conference on Neural Networks (IJCNN), IEEE, pp. 1–7 (2020) Agarwal, S., Branson, K., Belongie, S.: Higher order learning with graphs. In: Proceedings of the 23rd International Conference on Machine Learning, pp. 17–24 (2006) Agarwal,D.,Agrawal,R.,Khanna,R.,etal.:Estimatingratesofrareeventswithmultiplehierarchiesthrough scalable log-linear models. In: Proceedings of the 16th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 213–222 (2010) g y g pp Ai, Q., Azizi, V., Chen, X., et al.: Learning heterogeneous knowledge base embeddings for explainable recommendation. Algorithms 11(9), 137 (2018) Ali, Z., Qi, G., Muhammad, K., et al.: Paper recommendation based on heterogeneous network embedding. Knowl.-Based Syst. 210(106), 438 (2020) Andersen, R., Borgs, C., Chayes, J., et al.: Trust-based recommendation systems: an axiomatic approach In: Proceedings of the 17th international conference on World Wide Web, pp. 199–208 (2008) Bagci, H., Karagoz, P.: Context-aware friend recommendation for location based social networks using random walk. In: Proceedings of the 25th International Conference Companion on World Wide Web, pp. 531–536 (2016) Bahdanau, D., Cho, K., Bengio, Y.: Neural machine translation by jointly learning to align and translate. arXiv preprint arXiv:1409.0473 (2014) Belleau, F., Nolin, M.A., Tourigny, N., et al.: Bio2rdf: towards a mashup to build bioinformatics knowledg systems. J. Biomed. Inform. 41(5), 706–716 (2008) y Bengio, Y.: Learning Deep Architectures for AI. Now Publishers Inc, Norwell (2009) Berg, R.v.d., Kipf, T.N., Welling, M.: Graph convolutional matrix completion. arXiv prepri arXiv:1706.02263 (2017) Bollacker, K., Evans, C., Paritosh, P., et al.: Freebase: a collaboratively created graph database for struc- turing human knowledge. In: Proceedings of the 2008 ACM SIGMOD International Conference on Management of Data, pp. 1247–1250 (2008) Bond, F., Paik, K.: A survey of wordnets and their licenses. Small 8(4), 5 (2012) Bordes, A., Usunier, N., Garcia-Duran, A., et al.: Translating embeddings for modeling multi-relation data. Adv. Neural Inform. Process. Syst. 26 (2013) Bu, J., Tan, S., Chen, C., et al.: (2010) Music recommendation by uniﬁed hypergraph: combining social media information and music content. In: Proceedings of the 18th ACM International Conference on Multimedia, pp. 391–400 Cai, H., Zheng, V.W., Chang, K.C.C.: A comprehensive survey of graph embedding: problems, technique and applications. IEEE Trans. Knowl. Data Eng. 30(9), 1616–1637 (2018) Cantador, I., Brusilovsky, P., Kuﬂik, T.: Second workshop on information heterogeneity and fusion in recommender systems (hetrec2011). Recommending on graphs: a comprehensive review from a data… 873 Table 7 continued Domain Dataset Traditional ML Path-based Graph embedding Deep Learning DNN AE AM DRL GNN DHM Entertainment HetRec Delicious 2 1 1 Xing 1 2 DepaulMovie 2 InCarMusic 1 Dianping- Food 1 InMind Movie Agent 1 IntentBooks 1 IMDB 1 YouTube 1 Adressa 2 2 Roularta 2 1 MIND 1 2 Multiple News Portal 1 Sougou News 1 Filmtrust 1 Mtime 1 Netease 1 Kuaishou 1 Tiktok 1 Kwai 1 Restaurant &consumer 1 Social Network Douban 2 4 3 5 WeChat 1 1 Twitter 2 Reddit 2 Pinterest 1 4 Flickr 1 Hike network 1 BookCrossing 1 1 1 1 2 4 Academic or Book DBbook 1 1 1 1 DBLP 1 1 1 CiteULike 2 1 1 Goodreads 1 1 L. Zhang et al. 874 Table 7 continued Domain Dataset Traditional ML Path-based Graph embedding Deep Learning DNN AE AM DRL GNN DHM User-Tag 3 Librarything 1 1 Youshu 1 ACL Anthology Network 1 OHSUMED 1 POI Gowalla 1 1 1 4 Trip.com+ Facebook+ Twitter 1 Foursquare 1 1 1 1 Brightkite 1 Google Local 2 Tripadvisor 1 NUS-MSS 1 Other Educational website and textbooks 1 MIT AI + CASAS 1 REDIAL 1 VizML corpus 1 Legal recom- mendation dataset 1 Yahootrafﬁc stream 1 123 Recommending on graphs: a comprehensive review from a data… 875 References Frontiers in big Data, p. 49 (2020b) Chen, C., Ma, W., Zhang, M., et al.: Graph heterogeneous multi-relational recommendation. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp 3958–3966 (2021a) Chen, H., Li, Y., Sun, X., et al.: Temporal meta-path guided explainable recommendation. In: Proceeding of the 14th ACM International Conference on Web Search and Data Mining, pp. 1056–1064 (2021 Cheng, H., Tan, P.N., Sticklen, J., et al.: Recommendation via query centered random walk on k-partite graph. In: Seventh IEEE International Conference on Data Mining (ICDM 2007), IEEE, pp. 457–462 (2007) Cho, E., Myers, S.A., Leskovec, J.: Friendship and mobility: user movement in location-based social net- works. In: Proceedings of the 17th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 1082–1090 (2011) Cho, K., Van Merriënboer, B., Bahdanau, D., et al.: On the properties of neural machine translation: Encode decoder approaches. arXiv preprint arXiv:1409.1259 (2014) Christoforidis, G., Kefalas, P., Papadopoulos, A.N., et al.: Reline: point-of-interest recommendations using multiple network embeddings. Knowl. Inf. Syst. 63(4), 791–817 (2021) De Domenico, M., Lima, A., Mougel, P., et al.: The anatomy of a scientiﬁc rumor. Sci. Rep. 3(1), 1–9 (201 De Domenico, M., Lima, A., Mougel, P., et al.: The anatomy of a scientiﬁc rumor. Sci. Rep. 3(1), 1–9 (2013) Dror, G., Koenigstein, N., Koren, Y., et al.: The yahoo! music dataset and kdd-cup’11. In: Proceedings of KDD Cup 2011, PMLR, pp. 3–18 (2012) Dror, G., Koenigstein, N., Koren, Y., et al.: The yahoo! music dataset and kdd-cup’11. In: Proceedings of KDD Cup 2011, PMLR, pp. 3–18 (2012) Du, L., Li, X., Shen, Y.D.: User graph regularized pairwise matrix factorization for item recommendation. In: International Conference on Advanced Data Mining and Applications, Springer, pp. 372–385 (2011) Eksombatchai, C., Jindal, P., Liu, J.Z., et al.: Pixie: A system for recommending 3+ billion items to 200+ million users in real-time. In: Proceedings of the 2018 World Wide Web Conference, pp. 1775–1784 (2018) ( ) Ernst, P., Meng, C., Siu, A., et al.: Knowlife: a knowledge graph for health and life sciences. In: 2014 IEEE 30th International Conference on Data Engineering, IEEE, pp. 1254–1257 (2014) Fan, S., Zhu, J., Han, X., et al.: Metapath-guided heterogeneous graph neural network for intent recommen- dation. In: Proceedings of the 25th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. References In: Proceedings of the ﬁfth ACM Conference on Recommender Systems, pp. 387–388 (2011) Cao, D., Nie, L., He, X., et al.: Embedding factorization models for jointly recommending items and user generated lists. In: Proceedings of the 40th international ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 585–594 (2017) p pp ( ) Cao, Y., Wang, X., He, X., et al.: Unifying knowledge graph learning and recommendation: Towards a better understanding of user preferences. In: The World Wide Web Conference, pp. 151–161 (2019) Carlson, A., Betteridge, J., Wang, R.C., et al.: Coupled semi-supervised learning for information extraction. In: Proceedings of the third ACM International Conference on Web Search and Data Mining, pp. 101–110 (2010) Catherine, R., Cohen, W.: Personalized recommendations using knowledge graphs: a probabilistic logic programming approach. In: Proceedings of the 10th ACM Conference on Recommender Systems, pp. 325–332 (2016) Catherine, R., Mazaitis, K., Eskenazi, M., et al.: Explainable entity-based recommendations with knowledge graphs. arXiv preprint arXiv:1707.05254 (2017) Cen, Y., Zou, X., Zhang, J., et al.: Representation learning for attributed multiplex heterogeneous network. In: Proceedings of the 25th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 1358–1368 (2019) 123 876 L. Zhang et al. Chang, J., Gao, C., He, X., et al.: Bundle recommendation and generation with graph neural networks. IEEE Trans. Knowl. Data Eng. (2021a) Chang,J.,Gao,C.,Zheng,Y.,etal.:Sequentialrecommendationwithgraphneuralnetworks.In:Proceedings of the 44th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 378–387 (2021b) pp Chen, J., Wang, C., Zhou, S., et al.: Samwalker: Social recommendation with informative sampling strategy. In: The World Wide Web Conference, pp. 228–239 (2019a) Chen, L., Liu, Y., He, X., et al.: Matching user with item set: Collaborative bundle recommendation wi deep attention network. In: IJCAI, pp. 2095–2101 (2019b) Chen, M., Beutel, A., Covington, P., et al.: Top-k off-policy correction for a reinforce recommender system. In: Proceedings of the Twelfth ACM International Conference on Web Search and Data Mining, pp. 456–464 (2019c) Chen, Y., Zhou, M., Zheng, Z., et al.: Time-aware smart object recommendation in social internet of things. IEEE Internet Things J. 7(3), 2014–2027 (2019) Chen, L., Wu, L., Hong, R., et al.: Revisiting graph based collaborative ﬁltering: A linear residual graph convolutional network approach. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 27–34 (2020a) Chen, W.H., Hsu, C.C., Lai, Y.A., et al.: Attribute-aware recommender system based on collaborative ﬁltering: Survey and classiﬁcation. References 2478–2486 (2019a) Fan, W., Ma, Y., Li, Q., et al.: Graph neural networks for social recommendation. In: The World Wide Web Conference, pp. 417–426 (2019b) Fan, Z., Liu, Z., Zhang, J., et al.: Continuous-time sequential recommendation with temporal graph col- laborative transformer. In: Proceedings of the 30th ACM International Conference on Information & Knowledge Management, pp. 433–442 (2021) Fang, H., Guo, G., Zhang, D., et al.: Deep learning-based sequential recommender systems: Concepts, algorithms, and evaluations. In: International Conference on Web Engineering. Springer, pp. 574–577 (2019) Fang, H., Zhang, D., Shu, Y., et al.: Deep learning for sequential recommendation: algorithms, inﬂuential factors, and evaluations. ACM Trans. Inf. Syst. (TOIS) 39(1), 1–42 (2020) 123 12 Recommending on graphs: a comprehensive review from a data… 877 Farnadi, G., Kouki, P., Thompson, S.K., et al.: A fairness-aware hybrid recommender system. arXiv prepri arXiv:1809.09030 (2018) Farseev, A., Nie, L., Akbari, M., et al.: Harvesting multiple sources for user proﬁle learning: a big data study. In: Proceedings of the 5th ACM on International Conference on Multimedia Retrieval, pp. 235–242 (2015) Farseev, A., Samborskii, I., Filchenkov, A., et al.: Cross-domain recommendation via clustering on multi- layer graphs. In: Proceedings of the 40th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 195–204 (2017) Feng, W., Wang, J.: Incorporating heterogeneous information for personalized tag recommendation in social tagging systems. In: Proceedings of the 18th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 1276–1284 (2012) Feng, Y., You, H., Zhang, Z., et al.: Hypergraph neural networks. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 3558–3565 (2019) Figueroa, C., Vagliano, I., Rocha, O.R., et al.: A systematic literature review of linked data-based recom- mender systems. Concurr. Comput. Pract. Exp. 27(17), 4659–4684 (2015) Fouss, F., Francoisse, K., Yen, L., et al.: An experimental investigation of kernels on graphs for collaborative recommendation and semisupervised classiﬁcation. Neural Netw. 31, 53–72 (2012) Fu, Z., Xian, Y., Gao, R., et al.: Fairness-aware explainable recommendation over knowledge graphs. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 69–78 (2020) Fu, Z., Xian, Y., Zhu, Y., et al.: Hoops: Human-in-the-loop graph reasoning for conversational recommenda- tion. In: Proceedings of the 44th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. References 2415–2421 (2021) Gao, H., Tang, J., Liu, H.: gscorr: Modeling geo-social correlations for new check-ins on location-based social networks. In: Proceedings of the 21st ACM International Conference on Information and Knowl- edge Management, pp. 1582–1586 (2012) Gao, L., Yang, H., Wu, J., et al.: Recommendation with multi-source heterogeneous information. In: IJCAI International Joint Conference on Artiﬁcial Intelligence (2018) Gao, C., He, X., Gan, D., et al.: Neural multi-task recommendation from multi-behavior data. In: 201 IEEE 35th International Conference on Data Engineering (ICDE), IEEE, pp. 1554–1557 (2019a) Gao, J., Wang, X., Wang, Y., et al.: Explainable recommendation through attentive multi-view learning. I Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 3622–3629 (2019b) Gharahighehi, A., Vens, C., Pliakos, K.: Multi-stakeholder news recommendation using hypergraph learn- ing. In: Joint European Conference on Machine Learning and Knowledge Discovery in Databases. Springer, pp. 531–535 (2020) p g pp Gharahighehi, A., Vens, C., Pliakos, K.: Fair multi-stakeholder news recommender system with hypergrap ranking. Inf. Process. Manag. 58(5), 102,663 (2021) Gori, M., Pucci, A., Roma, V., et al.: Itemrank: A random-walk based scoring algorithm for recommender engines. In: IJCAI, pp. 2766–2771 (2007) Grover, A., Leskovec, J.: node2vec: Scalable feature learning for networks. In: Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 855–864 (2016) Gulla, J.A., Zhang, L., Liu, P., et al.: The adressa dataset for news recommendation. In: Proceedings of th International Conference on Web Intelligence, pp. 1042–1048 (2017) Guo, G., Zhang, J., Yorke-Smith, N.: A novel bayesian similarity measure for recommender systems. In: IJCAI, pp. 2619–2625 (2013) Guo, Q., Zhuang, F., Qin, C., et al.: A survey on knowledge graph-based recommender systems. IEEE Trans. Knowl. Data Eng. (2020) Guo, L., Yin, H., Chen, T., et al.: Hierarchical hyperedge embedding-based representation learning for group recommendation. ACM Trans. Inf. Syst. (TOIS) 40(1), 1–27 (2021) Guo, W., Su, R., Tan, R., et al.: Dual graph enhanced embedding neural network for CTR prediction. In: Proceedings of the 27th ACM SIGKDD Conference on Knowledge Discovery & Data Mining, pp. 496–504 (2021b) Hamilton, W., Ying, Z., Leskovec, J.: Inductive representation learning on large graphs. In: Advances in Neural Information Processing Systems, vol. 30 (2017) Han, X., Shi, C., Wang, S., et al.: Aspect-level deep collaborative ﬁltering via heterogeneous information networks. In: IJCAI, pp. 3393–3399 (2018) 12 3 878 L. Zhang et al. References Hao, B., Zhang, J., Yin, H., et al.: Pre-training graph neural networks for cold-start users and items represen- tation. In: Proceedings of the 14th ACM International Conference on Web Search and Data Mining, pp. 265–273 (2021a) Hao, J., Dun, Y., Zhao, G., et al.: Annular-graph attention model for personalized sequential recommenda- tion. IEEE Trans. Multimedia (2021b) Harper, F.M., Konstan, J.A.: The movielens datasets: History and context. ACM Trans. Interact. Intell. Syst. (TIIS) 5(4), 1–19 (2015) He, X., Chua, T.S.: Neural factorization machines for sparse predictive analytics. In: Proceedings of the 40th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 355–364 (2017) He, R., McAuley, J.: Ups and downs: Modeling the visual evolution of fashion trends with one-class collaborative ﬁltering. In: Proceedings of the 25th International Conference on World Wide Web, pp. 507–517 (2016) He, X., Chen, T., Kan, M.Y., et al.: Trirank: Review-aware explainable recommendation by modeling aspects. In: Proceedings of the 24th ACM International on Conference on Information and Knowledge Management, pp. 1661–1670 (2015) He, R., Lin, C., Wang, J., et al.: Sherlock: sparse hierarchical embeddings for visually-aware one-cla collaborative ﬁltering. arXiv preprint arXiv:1604.05813 (2016) He, R., Kang, W.C., McAuley, J.: Translation-based recommendation. In: Proceedings of the Eleventh ACM Conference on Recommender Systems, pp. 161–169 (2017) He, R., Kang, W.C., McAuley, J.: Translation-based recommendation. In: Pr Conference on Recommender Systems, pp. 161–169 (2017) He, Q., Li, B., Chen, F., et al.: Diversiﬁed third-party library prediction for mobile app development. IEEE Trans. Softw. Eng. (2020) Hersh, W., Buckley, C., Leone, T., et al.: Ohsumed: An interactive retrieval evaluation and new large te collection for research. In: SIGIR’94, Springer, pp. 192–201 (1994) Hochreiter, S., Schmidhuber, J.: Long short-term memory. Neural Comput. 9(8), 1735–1780 (1997) Hsu, C., Li, C.T.: Retagnn: Relational temporal attentive graph neural networks for holistic sequential recommendation. Proc. Web Conf. 2021, 2968–2979 (2021) Hu, B., Shi, C., Zhao, W.X., et al.: Leveraging meta-path based context for top-n recommendation with a neural co-attention model. In: Proceedings of the 24th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 1531–1540 (2018) Hu, K., Bakker, M.A., Li, S., et al.: Vizml: A machine learning approach to visualization recommendation. In: Proceedings of the 2019 CHI Conference on Human Factors in Computing Systems, pp. 1–12 (2019) Huang, Z., Chung, W., Chen, H.: A graph model for e-commerce recommender systems. J. Am. Soc. Inform Sci. Technol. References 55(3), 259–274 (2004) Huang, Z., Zeng, D., Chen, H.: A comparative study of recommendation algorithms in e-commerce appl cations. IEEE Intell. Syst. 22(5), 68–78 (2007) Huang, Z., Zeng, D.D., Chen, H.: Analyzing consumer-product graphs: Empirical ﬁndings and applications in recommender systems. Manage. Sci. 53(7), 1146–1164 (2007) Huang, J., Ren, Z., Zhao, W.X., et al.: Taxonomy-aware multi-hop reasoning networks for sequential recommendation. In: Proceedings of the Twelfth ACM International Conference on Web Search and Data Mining, pp. 573–581 (2019) Huang, H., Fang, Z., Wang, X., et al.: Motif-preserving temporal network embedding. In: IJCAI, pp. 1237 1243 (2020) Huang, C., Chen, J., Xia, L., et al.: Graph-enhanced multi-task learning of multi-level transition dynamics for session-based recommendation. In: AAAI Conference on Artiﬁcial Intelligence (AAAI) (2021a) for session-based recommendation. In: AAAI Conference on Artiﬁcial Intelligence (AAAI) (2021a) Huang, C., Xu, H., Xu, Y., et al.: Knowledge-aware coupled graph neural network for social recommenda- Huang, C., Xu, H., Xu, Y., et al.: Knowledge-aware coupled graph neural network for social recomm tion. In: 35th AAAI Conference on Artiﬁcial Intelligence (AAAI) (2021b) Huang, C., Xu, H., Xu, Y., et al.: Knowledge-aware coupled graph neural network tion. In: 35th AAAI Conference on Artiﬁcial Intelligence (AAAI) (2021b) g Isuﬁ, E., Pocchiari, M., Hanjalic, A.: Accuracy-diversity trade-off in recommender systems via graph convolutions. Inf. Process. Manag. 58(2), 102,459 (2021) Jamali, M., Ester, M.: Trustwalker: a random walk model for combining trust-based and item-based rec- ommendation. In: Proceedings of the 15th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 397–406 (2009) Jamali, M., Ester, M.: A matrix factorization technique with trust propagation for recommendation in social networks. In: Proceedings of the Fourth ACM Conference on Recommender Systems, pp. 135–142 (2010) 12 123 Recommending on graphs: a comprehensive review from a data… 879 Jannach, D., Jugovac, M., Nunes, I.: Explanations and user control in recommender systems. In: Proceedings of the 23rd International Workshop on Personalization and Recommendation on the Web and Beyond, pp. 31–31 (2019) Jäschke, R., Marinho, L., Hotho, A., et al.: Tag recommendations in folksonomies. In: European Conference on Principles of Data Mining and Knowledge Discovery. Springer, pp. 506–514 (2007) Jiang, Z., Liu, H., Fu, B., et al.: Recommendation in heterogeneous information networks based on gener- alized random walk model and Bayesian personalized ranking. In: Proceedings of the Eleventh ACM International Conference on Web Search and Data Mining, pp. References 288–296 (2018a) Jiang, Z., Yin, Y., Gao, L., et al.: Cross-language citation recommendation via hierarchical representation learning on heterogeneous graph. In: The 41st International ACM SIGIR Conference on Research & Development in Information Retrieval, pp. 635–644 (2018b) p pp Kanagal, B., Ahmed, A., Pandey, S., et al.: Supercharging recommender systems using taxonomies for learning user purchase behavior. arXiv preprint arXiv:1207.0136 (2012) Kim, K.M., Kwak, D., Kwak, H., et al.: Tripartite heterogeneous recommendation. arXiv preprint arXiv:1908.02569 (2019) Kim, K.M., Kwak, D., Kwak, H., et al.: Tripartite heterogeneous graph propagation for large-scale social recommendation. arXiv preprint arXiv:1908.02569 (2019) Kitchenham, B.: Procedures for performing systematic reviews. Keele, UK, Keele University 33(2004), 1–26 (2004) Koenigstein, N., Dror, G., Koren, Y.: Yahoo! music recommendations: modeling music ratings with temporal dynamicsanditemtaxonomy.In:ProceedingsoftheFifthACMconferenceonRecommenderSystems, pp. 165–172 (2011) Koren, Y., Bell, R., Volinsky, C.: Matrix factorization techniques for recommender systems. Comput 42(8), 30–37 (2009) Krizhevsky, A., Sutskever, I., Hinton, G.E.: Imagenet classiﬁcation with deep convolutional neural network In: Advances in Neural Information Processing Systems 25 (2012) Krizhevsky, A., Sutskever, I., Hinton, G.E.: Imagenet classiﬁcation with deep In: Advances in Neural Information Processing Systems 25 (2012) Kyriakidi, M., Koutrika, G., Ioannidis, Y.: Recommendations as graph explorations. In: Fourteenth ACM Conference on Recommender Systems, pp. 289–298 (2020) Kyriakidi, M., Koutrika, G., Ioannidis, Y.: Recommendations as grap Conference on Recommender Systems, pp. 289–298 (2020) Lee, S., Park, S., Kahng, M., et al.: Pathrank: Ranking nodes on a heterogeneous graph for ﬂexible hybr recommender systems. Expert Syst. Appl. 40(2), 684–697 (2013) Lehmann, J., Isele, R., Jakob, M., et al.: Dbpedia-a large-scale, multilingual knowledge base extracted fro wikipedia. Semantic Web 6(2), 167–195 (2015) Lei, W., Zhang, G., He, X., et al.: Interactive path reasoning on graph for conversational recommendation. In: Proceedings of the 26th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 2073–2083 (2020a) Lei, Y., Pei, H., Yan, H., et al.: Reinforcement learning based recommendation with graph convolutional q-network. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Devel- opment in Information Retrieval, pp. 1757–1760 (2020b) Li, X., Chen, H.: Recommendation as link prediction in bipartite graphs: a graph kernel-based machine learning approach. Decis. Support Syst. 54(2), 880–890 (2013) Li, L., Li, T.: News recommendation via hypergraph learning: encapsulation of user behavior and news content. In: Proceedings of the sixth ACM International Conference on Web Search and Data Mining, pp. References 4424–4431 (2019b) Liu, S., Ounis, I., Macdonald, C., et al.: A heterogeneous graph neural model for cold-start recommendation. In: Proceedings of the 43rd international ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 2029–2032 (2020) Liu, D., Lian, J., Liu, Z., et al.: Reinforced anchor knowledge graph generation for news recommendation reasoning. In: Proceedings of the 27th ACM SIGKDD Conference on Knowledge Discovery & Data Mining, pp. 1055–1065 (2021a) Liu, L., Wang, L., Lian, T.: Case4sr: Using category sequence graph to augment session-based recommen- dation. Knowl.-Based Syst. 212(106), 558 (2021b) Liu, Y., Yang, S., Zhang, Y., et al.: Learning hierarchical review graph representations for recommendatio IEEE Trans. Knowl. Data Eng. (2021c) Liu, Y., Li, H., Guo, Y., et al.: Rethinking attention-model explainability through faithfulness violation tes arXiv preprint arXiv:2201.12114 (2022) Lu, Y., Fang, Y., Shi, C.: Meta-learning on heterogeneous information networks for cold-start recommenda- tion. In: Proceedings of the 26th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 1563–1573 (2020) Lv, Q., Ding, M., Liu, Q., et al.: Are we really making much progress? revisiting, benchmarking and reﬁning heterogeneous graph neural networks. In: Proceedings of the 27th ACM SIGKDD Conference on Knowledge Discovery & Data Mining, pp. 1150–1160 (2021) Ma, H., Yang, H., Lyu, M.R., et al.: Sorec: social recommendation using probabilistic matrix factorization. In: Proceedings of the 17th ACM Conference on Information and Knowledge Management, pp. 931– 940 (2008) Ma, H., Lyu, M.R., King, I.: Learning to recommend with trust and distrust relationships. In: Proceeding of the third ACM Conference on Recommender Systems, pp. 189–196 (2009) Ma, H., Zhou, D., Liu, C., et al.: Recommender systems with social regularization. In: Proceedings of the Fourth ACM International Conference on Web Search and Data Mining, pp. 287–296 (2011a) Ma, H., Zhou, T.C., Lyu, M.R., et al.: Improving recommender systems by incorporating social contextual information. ACM Trans. Inf. Syst. (TOIS) 29(2), 1–23 (2011) Ma, W., Zhang, M., Cao, Y., et al.: Jointly learning explainable rules for recommendation with knowledg graph. In: The World Wide Web Conference, pp. 1210–1221 (2019) Ma, C., Ma, L., Zhang, Y., et al.: Memory augmented graph neural networks for sequential recommendatio In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 5045–5052 (2020) Mansoury, M., Abdollahpouri, H., Pechenizkiy, M., et al.: Fairmatch: A graph-based approach for improving aggregate diversity in recommender systems. References 305–314 (2013) Li, L., Wang, D., Li, T., et al.: Scene: a scalable two-stage personalized news recommendation system. In: Proceedings of the 34th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 125–134 (2011) pp Li, X., Wang, M., Liang, T.P.: A multi-theoretical kernel-based approach to social network-based recom- mendation. Decis. Support Syst. 65, 95–104 (2014) pp y Li, J., Dani, H., Hu, X., et al.: Attributed network embedding for learning in a dynamic environment. In: Proceedings of the 2017 ACM on Conference on Information and Knowledge Management, pp. 387–396 (2017) Li, Q., Han, Z., Wu, X.M.: Deeper insights into graph convolutional networks for semi-supervised learnin In: Thirty-Second AAAI Conference on Artiﬁcial Intelligence (2018a) Li, R., Ebrahimi Kahou, S., Schulz, H., et al.: Towards deep conversational recommendations. In: Advances in Neural Information Processing Systems, vol. 31 (2018b) Li, B., He, Q., Chen, F., et al.: Embedding app-library graph for neural third party library recommendation. In: Proceedings of the 29th ACM Joint Meeting on European Software Engineering Conference and Symposium on the Foundations of Software Engineering, pp 466–477 (2021a) 12 3 123 880 L. Zhang et al. Li, H., Wang, Y., Zhang, S., et al.: Kg4vis: A knowledge graph-based approach for visualization recom mendation. IEEE Trans. Visual Comput. Graph. 28(1), 195–205 (2021b) Liang, D., Charlin, L., McInerney, J., et al.: Modeling user exposure in recommendation. In: Proceeding of the 25th International Conference on World Wide Web, pp. 951–961 (2016) Liang, T., He, L., Lu, C.T., et al.: A broad learning approach for context-aware mobile application rec- ommendation. In: 2017 IEEE International Conference on Data Mining (ICDM). IEEE, pp. 955–960 (2017) Lin, Y., Liu, Z., Sun, M., et al.: Learning entity and relation embeddings for knowledge graph completio In: Twenty-ninth AAAI Conference on Artiﬁcial Intelligence (2015) Liu, J., Duan, L.: A survey on knowledge graph-based recommender systems. In: 2021 IEEE 5th Advanced Information Technology, Electronic and Automation Control Conference (IAEAC), IEEE, pp. 2450– 2453 (2021) Liu, X., Liu, Y., Aberer, K., et al.: Personalized point-of-interest recommendation by mining users’ pref- erence transition. In: Proceedings of the 22nd ACM International Conference on Information & Knowledge Management, pp. 733–738 (2013) Liu,C.,Li,L.,Yao,X.,etal.:Asurveyofrecommendationalgorithmsbasedonknowledgegraphembedding. In:2019IEEEInternationalConferenceonComputerScienceandEducationalInformatization(CSEI). IEEE, pp. 168–171 (2019a) pp ( ) Liu, Z., Chen, C., Li, L., et al.: Geniepath: Graph neural networks with adaptive receptive paths. In: Pro- ceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. References In: Proceedings of the 28th ACM Conference on User Modeling, Adaptation and Personalization, pp. 154–162 (2020) Mao, M., Lu, J., Han, J., et al.: Multiobjective e-commerce recommendations based on hypergraph ranking. Inf. Sci. 471, 269–287 (2019) 123 12 Recommending on graphs: a comprehensive review from a data… 881 assa, P., Avesani, P.: Trust-aware recommender systems. In: Proceedings of the 2007 ACM Conference on Recommender Systems, pp. 17–24 (2007) McAuley, J., Targett, C., Shi, Q., et al.: Image-based recommendations on styles and substitutes. In: Proceed- ings of the 38th international ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 43–52 (2015) pp Menon, A.K., Chitrapura, K.P., Garg, S., et al.: Response prediction using collaborative ﬁltering with hierarchies and side-information. In: Proceedings of the 17th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 141–149 (2011) g y g pp ( ) Mezni, H., Benslimane, D., Bellatreche, L.: Context-aware service recommendation based on knowledge graph embedding. IEEE Trans. Knowl. Data Eng. (2021) Mikolov, T., Chen, K., Corrado, G., et al.: Efﬁcient estimation of word representations in vector space. arXiv preprint arXiv:1301.3781 (2013a) Mikolov, T., Sutskever, I., Chen, K., et al.: Distributed representations of words and phrases and their compositionality. In: Advances in Neural Information Processing Systems, vol. 26 (2013b) Mnih, A.: Taxonomy-informed latent factor models for implicit feedback. In: Proceedings of KDD Cu 2011, PMLR, pp. 169–181 (2012) Mnih, V., Kavukcuoglu, K., Silver, D., et al.: Human-level control through deep reinforcement learnin Nature 518(7540), 529–533 (2015) ( ) ( ) Monti, F., Bronstein, M., Bresson, X.: Geometric matrix completion with recurrent multi-graph neural networks. In: Advances in Neural Information Processing Systems, vol. 30 (2017) Moro, A., Raganato, A., Navigli, R.: Entity linking meets word sense disambiguation: a uniﬁed approach. Trans. Assoc. Comput. Linguist. 2, 231–244 (2014) Moro, A., Raganato, A., Navigli, R.: Entity linking meets word se Trans. Assoc. Comput. Linguist. 2, 231–244 (2014) Musto, C., Semeraro, G., Gemmis, M.d., et al.: Tuning personalized pagerank for semantics-aware recom- mendations based on linked open data. In: European Semantic Web Conference. Springer, pp. 169–183 (2017) Musto, C., Lops, P., de Gemmis, M., et al.: Context-aware graph-based recommendations exploiting per- sonalized pagerank. Knowl.-Based Syst. 216(106), 806 (2021) Nguyen, G.H., Lee, J.B., Rossi, R.A., et al.: Continuous-time dynamic network embeddings. Companion Proc. Web Conf. 2018, 969–976 (2018) Nikolakopoulos, A.N., Karypis, G.: Recwalk: Nearly uncoupled random walks for top-n recommendation. References In: Proceedings of the twelfth ACM International Conference on Web Search and Data Mining, pp. 150–158 (2019) Nikolakopoulos, A.N., Kalantzis, V., Gallopoulos, E., et al.: Eigenrec: generalizing puresvd for effective and efﬁcient top-n recommendations. Knowl. Inf. Syst. 58(1), 59–81 (2019) y Noia, T.D., Ostuni, V.C., Tomeo, P., et al.: Sprank: Semantic path-based ranking for top-n recommendation using linked open data. ACM Trans. Intell. Syst. Technol. (TIST) 8(1), 1–34 (2016) Ostuni, V.C., Di Noia, T., Di Sciascio, E., et al.: Top-n recommendations from implicit feedback leveraging linked open data. In: Proceedings of the 7th ACM Conference on Recommender Systems, pp. 85–92 (2013) Ostuni, V.C., Noia, T.D., Mirizzi, R., et al.: A linked data recommender system using a neighborhood-based graph kernel. In: International Conference on Electronic Commerce and Web Technologies. Springer, pp. 89–100 (2014) Ouyang, W., Zhang, X., Ren, S., et al.: Learning graph meta embeddings for cold-start ads in click-through rate prediction. In: Proceedings of the 44th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 1157–1166 (2021) Palumbo, E., Rizzo, G., Troncy, R.: Entity2rec: Learning user-item relatedness from knowledge graphs for top-n item recommendation. In: Proceedings of the Eleventh ACM Conference on Recommender Systems, pp. 32–36 (2017) y , pp ( ) Palumbo, E., Monti, D., Rizzo, G., et al.: entity2rec: Property-speciﬁc knowledge graph embeddings for item recommendation. Expert Syst. Appl. 151(113), 235 (2020) Pan, R., Zhou, Y., Cao, B., et al.: One-class collaborative ﬁltering. In: 2008 Eighth IEEE International Conference on Data Mining. IEEE, pp. 502–511 (2008) Pan, Z., Cai, F., Chen, W., et al.: Star graph neural networks for session-based recommendation. In: Pro- ceedings of the 29th ACM International Conference on Information & Knowledge Management, pp. 1195–1204 (2020) 123 882 L. Zhang et al. Pang, Y., Wu, L., Shen, Q., et al.: Heterogeneous global graph neural networks for personalized session- based recommendation. In: Proceedings of the Fifteenth ACM International Conference on Web Search and Data Mining, pp. 775–783 (2022) Perozzi, B., Al-Rfou,R., Skiena, S.: Deepwalk: Online learning of social representations. In: Proceedings of the 20th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 701–710 (2014) Phuong, T.M., Phuong, N.D., et al.: Graph-based context-aware collaborative ﬁltering. Expert Syst. Appl. 126, 9–19 (2019) Pianta, E., Bentivogli, L., Girardi, C.: Multiwordnet: developing an aligned multilingual database. In: Fir International Conference on Global WordNet, pp. References 293–302 (2002) Pliakos, K., Kotropoulos, C.: Simultaneous image tagging and geo-location prediction within hypergraph ranking framework. In: 2014 IEEE International Conference on Acoustics, Speech and Signal Pro- cessing (ICASSP), pp. 6894–6898. IEEE (2014) Qiu, R., Huang, Z., Li, J., et al.: Exploiting cross-session information for session-based recommendation with graph neural networks. ACM Trans. Inf. Syst. (TOIS) 38(3), 1–23 (2020) Qiu, R., Yin, H., Huang, Z., et al.: Gag: Global attributed graph neural network for streaming session-based recommendation. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 669–678 (2020b) p pp Radev, D.R., Muthukrishnan, P., Qazvinian, V., et al.: The ACL anthology network corpus. Lang. Resour. Eval. 47(4), 919–944 (2013) Reimers, N., Gurevych, I.: Sentence-bert: Sentence embeddings using siamese bert-networks. arXiv preprint arXiv:1908.10084 (2019) Richardson, M., Domingos, P.: Mining knowledge-sharing sites for viral marketing. In: Proceedings of the eighth ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 61–70 (2002) Salamat, A., Luo, X., Jafari, A.: Heterographrec: a heterogeneous graph-based neural networks for social recommendations. Knowl.-Based Syst. 217(106), 817 (2021) Sang, L., Xu, M., Qian, S., et al.: Knowledge graph enhanced neural collaborative recommendation. Expe Syst. Appl. 164(113), 992 (2021) Schedl, M.: The lfm-1b dataset for music retrieval and recommendation. In: Proceedings of the 2016 ACM on International Conference on Multimedia Retrieval, pp. 103–110 (2016) Schuster, M., Paliwal, K.K.: Bidirectional recurrent neural networks. IEEE Trans. Signal Process. 45(11), 2673–2681 (1997) ( ) Shams, B., Haratizadeh, S.: Graph-based collaborative ranking. Expert Syst. Appl. 67, 59–70 (2017) Sharma, A., Jiang, J., Bommannavar, P., et al.: Graphjet: Real-time content recommendations at twitte Proc. VLDB Endow. 9(13), 1281–1292 (2016) Sheu, H.S., Li, S.: Context-aware graph embedding for session-based news recommendation. In: Fourteenth ACM Conference on Recommender Systems, pp. 657–662 (2020) Shi, C., Zhang, Z., Luo, P., et al.: Semantic path based personalized recommendation on weighted het- erogeneous information networks. In: Proceedings of the 24th ACM International on Conference on Information and Knowledge Management, pp. 453–462 (2015) Shi, C., Liu, J., Zhuang, F., et al.: Integrating heterogeneous information via ﬂexible regularization frame- work for recommendation. Knowl. Inf. Syst. 49(3), 835–859 (2016) Shi, C., Hu, B., Zhao, W.X., et al.: Heterogeneous information network embedding for recommendation. IEEE Trans. Knowl. Data Eng. 31(2), 357–370 (2018) Shi, D., Wang, T., Xing, H., et al.: A learning path recommendation model based on a multidimensional knowledge graph framework for e-learning. References In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence (2017) Sun, Z., Yang, J., Zhang, J., et al.: Recurrent knowledge graph embedding for effective recommendation. In: Proceedings of the 12th ACM Conference on Recommender Systems, pp. 297–305 (2018) Sun, Z., Guo, Q., Yang, J., et al.: Research commentary on recommendations with side i and research directions. Electron. Commer. Res. Appl. 37(100), 879 (2019) Sun, J., Guo, W., Zhang, D., et al.: A framework for recommending accurate and diverse items using bayesian graph convolutional neural networks. In: Proceedings of the 26th ACM SIGKDD Interna- tional Conference on Knowledge Discovery & Data Mining, pp. 2030–2039 (2020) Sun, J., Cheng, Z., Zuberi, S., et al.: Hgcf: Hyperbolic graph convolutio In: Proceedings of the Web Conference 2021, 593–601 (2021) Sun, J., Cheng, Z., Zuberi, S., et al.: Hgcf: Hyperbolic graph convolution networks for collaborative ﬁltering. In: Proceedings of the Web Conference 2021, 593–601 (2021) Tan, S., Bu, J., Chen, C., et al.: Using rich social media information for music recommendation via hype graph model. ACM Trans. Multimedia Comput. Commun. Appl. (TOMM) 7(1), 1–22 (2011) Tan, Q., Liu, N., Zhao, X., et al.: Learning to hash with graph neural networks for recommender system In: Proceedings of the Web Conference 2020, 1988–1998 (2020) Tang, J., Zhang, J., Yao, L., et al.: Arnetminer: extraction and mining of academic social networks. In: Proceedings of the 14th ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 990–998 (2008) Tang, L., Wang, X., Liu, H.: Uncoverning groups via heterogeneous interaction analysis. In: 2009 Nin IEEE International Conference on Data Mining, pp. 503–512. IEEE (2009) Tang, J., Gao, H., Liu, H.: mtrust: Discerning multi-faceted trust in a connected world. In: Proceedings of the Fifth ACM International Conference on Web Search and Data Mining, pp. 93–102 (2012) Tang, J., Qu, M., Wang, M., et al.: Line: Large-scale information network embedding. In: Proceed the 24th International Conference on World Wide Web, pp. 1067–1077 (2015) ng, M., et al.: Line: Large-scale information network Tang, H., Zhao, G., Bu, X., et al.: Dynamic evolution of multi-graph based collaborative ﬁltering for recommendation systems. Knowl.-Based Syst. 228(107), 251 (2021) Tang, H., Zhao, G., Bu, X., et al.: Dynamic evolution of multi-graph b recommendation systems. Knowl.-Based Syst. 228(107), 251 (2021) Tao, Z., Zhao, X., Tanaka, T., et al.: Bayesian latent factor model for higher-order data. In: Asian Conference on Machine Learning. PMLR, pp. References Knowl.-Based Syst. 195(105), 618 (2020) Shi, S., Ma, W., Wang, Z., et al.: Wg4rec: Modeling textual content with word graph for news recom- mendation. In: Proceedings of the 30th ACM International Conference on Information & Knowledge Management, pp. 1651–1660 (2021) g pp Shi, Y., Larson, M., Hanjalic, A.: Collaborative ﬁltering beyond the user-item matrix: a survey of the state of the art and future challenges. ACM Comput. Surv. (CSUR) 47(1), 1–45 (2014) Shuman, D.I., Narang, S.K., Frossard, P., et al.: The emerging ﬁeld of signal processing on graphs: Extending high-dimensional data analysis to networks and other irregular domains. IEEE Signal Process. Mag. 30(3), 83–98 (2013) Silver, D., Huang, A., Maddison, C.J., et al.: Mastering the game of go with deep neural networks and tree search. Nature 529(7587), 484–489 (2016) 123 12 123 Recommending on graphs: a comprehensive review from a data… 883 Song, W., Duan, Z., Yang, Z., et al.: Explainable knowledge graph-based recommendation via deep rein- forcement learning. arXiv preprint arXiv:1906.09506 (2019a) ng, W., Duan, Z., Yang, Z., et al.: Explainable knowledge graph-based recommendation via deep rein- forcement learning. arXiv preprint arXiv:1906.09506 (2019a) Song, W., Xiao, Z., Wang, Y., et al.: Session-based social recommendation via dynamic graph attention networks. In: Proceedings of the Twelfth ACM International Conference on Web Search and Data Mining, pp. 555–563 (2019b) Song, Y., Zhang, L., Giles, C.L.: Automatic tag recommendation algorithms for social recommender sys- tems. ACM Trans. Web (TWEB) 5(1), 1–31 (2011) Speer, R., Chin, J., Havasi, C.: Conceptnet 5.5: An open multilingual graph of general knowledge. I Thirty-First AAAI Conference on Artiﬁcial Intelligence (2017) Suchanek, F.M., Kasneci, G., Weikum, G.: Yago: a core of semantic knowledge. In: Proceedings of the 16 International Conference on World Wide Web, pp. 697–706 (2007) Sun,Y.,Han,J.:Miningheterogeneousinformationnetworks:astructuralanalysisapproach.ACMSIGKDD Explor. Newsl. 14(2), 20–28 (2013) Sun, Y., Han, J., Yan, X., et al.: Pathsim: Meta path-based top-k similarity search in heterogeneous infor- mation networks. Proc. VLDB Endow. 4(11), 992–1003 (2011) Sun, Z., Han, L., Huang, W., et al.: Recommender systems based on social networks. J. Syst. Softw. 9 109–119 (2015) Sun, Z., Yang, J., Zhang, J., et al.: Exploiting both vertical and horizontal dimensions of feature hierarchy for effective recommendation. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence (2017) Sun, Z., Yang, J., Zhang, J., et al.: Exploiting both vertical and horizontal dimensions of feature hierarchy for effective recommendation. References 1285–1300 (2021) Tarus, J.K., Niu, Z., Mustafa, G.: Knowledge-based recommendation: a review of ontology-based recom- mender systems for e-learning. Artif. Intell. Rev. 50(1), 21–48 (2018) Tay, D.B., Lin, Z.: Design of near orthogonal graph ﬁlter banks. IEEE Signal Process. Lett. 22(6), 701–70 (2014) Tian, F., Gao, B., Cui, Q., et al.: Learning deep representations for graph clustering. In: Proceedings of th AAAI Conference on Artiﬁcial Intelligence (2014) Tian, Z., Liu, Y., Sun, J., et al.: Exploiting group information for personalized recommendation with graph neural networks. ACM Trans. Inf. Syst. (TOIS) 40(2), 1–23 (2021) Tian, Z., Liu, Y., Sun, J., et al.: Exploiting group information for personali neural networks. ACM Trans. Inf. Syst. (TOIS) 40(2), 1–23 (2021) Vargas-Govea, B., González-Serna, G., Ponce-Medellın, R.: Effects of relevant contextual features in the performance of a restaurant recommender system. ACM RecSys 11(592), 56 (2011) Vaswani, A., Shazeer, N., Parmar, N., et al.: Attention is all you need. In: Advances in Neural Information Processing Systems, vol. 30 (2017) 12 3 3 884 L. Zhang et al. Veliˇckovi´c, P., Cucurull, G., Casanova, A., et al.: Graph attention networks. arXiv preprint arXiv:1710.1090 (2017) (2017) Verma, J., Gupta, S., Mukherjee, D., et al.: Heterogeneous edge embedding for friend recommendation. In: Verma, J., Gupta, S., Mukherjee, D., et al.: Heterogeneous edge embedding for friend recommendation. I European Conference on Information Retrieval. Springer, pp. 172–179 (2019) Vijaikumar, M., Shevade, S., Murty, M.N.: Sorecgat: Leveraging graph attention mechanism for top-n social recommendation. In: Joint European Conference on Machine Learning and Knowledge Discovery in Databases. Springer, pp. 430–446 (2019) Databases. Springer, pp. 430–446 (2019) Wang, J., Caverlee, J.: Recurrent recommendation with local coherence. In: Proceedings of the Twelf ACM International Conference on Web Search and Data Mining, pp. 564–572 (2019) Wang, H., Chen, B., Li, W.J.: Collaborative topic regression with social regularization for tag recomme dation. In: Twenty-Third International Joint Conference on Artiﬁcial Intelligence (2013) Wang, Z., Zhang, J., Feng, J., et al.: Knowledge graph embedding by translating on hyperplanes. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence (2014) Wang, J., Zhao, W., He, Y., et al.: Leveraging product adopter information from online reviews for product recommendation. In: Proceedings of the International AAAI Conference on Web and Social Media, pp. 464–472 (2015) Wang, X., He, X., Nie, L., et al.: Item silk road: Recommending items from information domains to social users. References In: Proceedings of the 40th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 185–194 (2017) Wang, A., Singh, A., Michael, J., et al.: Glue: A multi-task benchmark and analysis platform for natur language understanding. arXiv preprint arXiv:1804.07461 (2018a) Wang, H., Zhang, F., Wang, J., et al.: Ripplenet: Propagating user preferences on the knowledge graph for recommender systems. In: Proceedings of the 27th ACM International Conference on Information and Knowledge Management, pp. 417–426 (2018b) Wang, H., Zhang, F., Xie, X., et al.: Dkn: Deep knowledge-aware network for news recommendation. In: Proceedings of the 2018 World Wide Web Conference, pp. 1835–1844 (2018c) Wang, H., Zhang, F., Wang, J., et al.: Exploring high-order user preference on the knowledge graph f recommender systems. ACM Trans. Inf. Syst. (TOIS) 37(3), 1–26 (2019a) Wang, H., Zhang, F., Zhang, M., et al.: Knowledge-aware graph neural networks with label smoothness regularization for recommender systems. In: Proceedings of the 25th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 968–977 (2019b) Wang, H., Zhao, M., Xie, X., et al.: Knowledge graph convolutional networks for recommender systems. In: The World Wide Web Conference, pp. 3307–3313 (2019c) Wang, P., Chen, H., Zhu, Y., et al.: Uniﬁed collaborative ﬁltering over graph embeddings. In: Proceedings of the 42nd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 155–164 (2019d) Wang, X., He, X., Cao, Y., et al.: Kgat: Knowledge graph attention network for recommendation. In: Proceedings of the 25th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 950–958 (2019e) Wang, X., He, X., Wang, M., et al.: Neural graph collaborative ﬁltering. In: Proceedings of the 42nd international ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 165–174 (2019f) Wang, X., Wang, D., Xu, C., et al.: Explainable reasoning over knowledge graphs for recommendation. I Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 5329–5336 (2019g) Wang, C., Ma, W., Zhang, M., et al.: Toward dynamic user intention: temporal evolutionary effects of item relations in sequential recommendation. ACM Trans. Inf. Syst. (TOIS) 39(2), 1–33 (2020a) Wang, C., Zhang, M., Ma, W., et al.: Make it a chorus: knowledge-and time-aware item modeling for sequential recommendation. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 109–118 (2020b) Wang, J., Ding, K., Hong, L., et al.: Next-item recommendation with sequential hypergraphs. References In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 1101–1110 (2020c) pp Wang, M., Lin, Y., Lin, G., et al.: M2grl: A multi-task multi-view graph representation learning frame- work for web-scale recommender systems. In: Proceedings of the 26th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 2349–2358 (2020d) g y g pp ( ) Wang, S., Hu, L., Wang, Y., et al.: Graph learning approaches to recommender systems: a review. arXiv preprint arXiv:2004.11718 (2020e) 123 12 Recommending on graphs: a comprehensive review from a data… 885 Wang, W., Zhang, W., Rao, J., et al.: Group-aware long-and short-term graph representation learning for sequential group recommendation. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 1449–1458 (2020f) Wang, X., Xu, Y., He, X., et al.: Reinforced negative sampling over knowledge graph for recommendation. Proceedings of the Web Conference 2020, 99–109 (2020) Wang, Z., Wei, W., Cong, G., et al.: Global context enhanced graph neural networks for session-based recommendation. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 169–178 (2020h) recommendation. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 169–178 (2020h) p pp Wang, M., Qiu, L., Wang, X.: A survey on knowledge graph embeddings for link prediction. Symmetry 13(3), 485 (2021a) Wang, X., Huang, T., Wang, D., et al.: Learning intents behind interactions with knowledge graph for recommendation. Proceedings of the Web Conference 2021, 878–887 (2021) Webster, J., Watson, R.T.: Analyzing the past to prepare for the future: Writing a literature review. MIS Quarterly xiii–xxiii (2002) Wei, Y., Wang, X., He, X., et al.: Hierarchical user intent graph network for multimedia recommendatio IEEE Trans. Multimedia (2021) Wolfswinkel, J.F., Furtmueller, E., Wilderom, C.P.: Using grounded theory as a method for rigorous reviewing literature. Eur. J. Inf. Syst. 22(1), 45–55 (2013) Wu, L., Sun, P., Fu, Y., et al.: A neural inﬂuence diffusion model for social recommendation. In: Proceedings f h 42 d i l AC S G C f h d l i f i Wu, L., Sun, P., Fu, Y., et al.: A neural inﬂuence diffusion model for social recommendation. In: Proceedings of the 42nd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. References Zhang et al. Xin, X., He, X., Zhang, Y., et al.: Relational collaborative ﬁltering: Modeling multiple item relations for recommendation. In: Proceedings of the 42nd international ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 125–134 (2019) Xu, B., Xu, Y., Liang, J., et al.: Cn-dbpedia: A never-ending chinese knowledge extraction system. In: International Conference on Industrial, Engineering and Other Applications of Applied Intelligent Systems, pp. 428–438. Springer (2017) Xu, C., Zhao, P., Liu, Y., et al.: Graph contextualized self-attention network for session-based recomme dation. In: IJCAI, pp. 3940–3946 (2019a) Xu, D., Ruan, C., Korpeoglu, E., et al.: Inductive representation learning on temporal graphs. arXiv preprint arXiv:2002.07962 (2020) Xu, F., Lian, J., Han, Z., et al.: Relation-aware graph convolutional networks for agent-initiated social e-commerce recommendation. In: Proceedings of the 28th ACM International Conference on Infor- mation and Knowledge Management, pp. 529–538 (2019b) Yajima, Y.: One-class support vector machines for recommendation tasks. In: Paciﬁc-Asia Conference on Knowledge Discovery and Data Mining, pp. 230–239. Springer (2006) Yan, H., Yang, J., Wan, J.: Knowime: a system to construct a knowledge graph for intelligent manufacturing equipment. IEEE Access 8, 41,805-41,813 (2020) Yang, J., Sun, Z., Bozzon, A., et al.: Learning hierarchical feature inﬂuence for recommendation by recursive regularization. In: Proceedings of the 10th ACM Conference on Recommender Systems, pp. 51–58 (2016) Yang, J., Ma, W., Zhang, M., et al.: Legalgnn: Legal information enhanced graph neural network for recommendation. ACM Trans. Inf. Syst. (TOIS) 40(2), 1–29 (2021) Yang, Y., Wu, L., Hong, R., et al.: Enhanced graph learning for collaborative ﬁltering via mutual information maximization. In: Proceedings of the 44th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 71–80 (2021b) p pp ( ) Yang, Z., Dong, S.: Hagerec: hierarchical attention graph convolutional network incorporating knowledge graph for explainable recommendation. Knowl.-Based Syst. 204(106), 194 (2020) Yao, W., He, J., Huang, G., et al.: A graph-based model for context-aware recommendation using implicit feedback data. World Wide Web 18(5), 1351–1371 (2015) Yin, Z., Gupta, M., Weninger, T., et al.: Linkrec: a uniﬁed framework for link recommendation with user attributes and graph structure. In: Proceedings of the 19th International Conference on World Wide Web, pp. 1211–1212 (2010) Ying, R., He, R., Chen, K., et al.: Graph convolutional neural networks for web-scale recommender systems. In: Proceedings of the 24th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. References 235–244 (2019a) Wu, Q., Zhang, H., Gao, X., et al.: Dual graph attention networks for deep latent representation of multi- faceted social effects in recommender systems. In: The World Wide Web Conference, pp. 2091–2102 (2019b) Wu, S., Tang, Y., Zhu, Y., et al.: Session-based recommendation with graph neural networks. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 346–353 (2019c) Wu, S., Sun, F., Zhang, W., et al.: Graph neural networks in recommender systems: a survey. arXiv preprint arXiv:2011.02260 (2020a) Wu, Z., Pan, S., Chen, F., et al.: A comprehensive survey on graph neural networks. IEEE Trans. Neur Netw. Learn. Syst. 32(1), 4–24 (2020) Wu, C., Wu, F., Huang, Y., et al.: User-as-graph: User modeling with heterogeneous graph pooling for news recommendation. In: Proceedings of the Thirtieth International Joint Conference on Artiﬁcial Intelligence, pp. 1624–1630 (2021a) Wu, J., Wang, X., Feng, F., et al.: Self-supervised graph learning for recommendation. In: Proceedings of the 44th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 726–735 (2021b) pp Wu, L., Chen, L., Shao, P., et al.: Learning fair representations for recommendation: A graph-based per- spective. Proceedings of the Web Conference 2021, 2198–2208 (2021) Xia, L., Huang, C., Xu, Y., et al.: Knowledge-enhanced hierarchical graph transformer network for multi- behavior recommendation. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp 4486–4493 (2021a) Xia, L., Xu, Y., Huang, C., et al.: Graph meta network for multi-behavior recommendation. In: Proceedings of the 44th International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 757–766 (2021b) Xia, X., Yin, H., Yu, J., et al.: Self-supervised graph co-training for session-based recommendation. In: Proceedings of the 30th ACM International Conference on Information & Knowledge Management, pp. 2180–2190 (2021c) Xia, X., Yin, H., Yu, J., et al.: Self-supervised hypergraph convolutional networks for session-based rec- ommendation. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 4503–4511 (2021d) Xian, Y., Fu, Z., Muthukrishnan, S., et al.: Reinforcement knowledge graph reasoning for explainable recommendation. In: Proceedings of the 42nd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 285–294 (2019) Xie, F., Chen, Z., Shang, J., et al.: A link prediction approach for item recommendation with comple number. Knowl.-Based Syst. 81, 148–158 (2015) Xie, F., Zheng, A., Chen, L., et al.: Attentive meta-graph embedding for item recommendation in hetero- geneous information networks. Knowl.-Based Syst. 211(106), 524 (2021) 12 3 886 L. References 974–983 (2018) Yu, J., Yin, H., Li, J., et al.: Self-supervised multi-channel hypergraph convolutional network for soci recommendation. Proceedings of the Web Conference 2021, 413–424 (2021) Yu, X., Ren, X., Gu, Q., et al.: Collaborative ﬁltering with entity similarity regularization in heterogeneous information networks. In: IJCAI HINA, 27 (2013) Yu, X., Ren, X., Sun, Y., et al.: Personalized entity recommendation: A heterogeneous information network approach. In: Proceedings of the 7th ACM International Conference on Web Search and Data Mining, pp. 283–292 (2014) Zhang, C., Wang, Y., Zhu, L., et al.: Multi-graph heterogeneous interaction fusion for social recommend tion. ACM Trans. Inf. Syst. (TOIS) 40(2), 1–26 (2021) Zhang, F., Yuan, N.J., Lian, D., et al.: Collaborative knowledge base embedding for recommender systems. In: Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 353–362 (2016) Zhang, J., Shi, X., Zhao, S., et al.: Star-gcn: Stacked and reconstructed graph convolutional networks f recommender systems. arXiv preprint arXiv:1905.13129 (2019a) Zhang, M., Wu, S., Yu, X., et al.: Dynamic graph neural networks for sequential recommendation. IEE Trans. Knowl. Data Eng. (2022) Zhang, S., Yao, L., Sun, A., et al.: Deep learning based recommender system: a survey and new perspectives. ACM Comput. Surv. (CSUR) 52(1), 1–38 (2019) Zhang, S., Yin, H., Chen, T., et al.: Graph embedding for recommendation against attribute inference attacks. Proceedings of the Web Conference 2021, 3002–3014 (2021) 123 12 Recommending on graphs: a comprehensive review from a data… 887 Zhang, W., Liu, H., Liu, Y., et al.: Semi-supervised hierarchical recurrent graph neural network for city-wide parking availability prediction. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 1186–1193 (2020) Zhang, W., Liu, H., Liu, Y., et al.: Semi-supervised hierarchical recurrent graph neural network for city-wide parking availability prediction. In: Proceedings of the AAAI Conference on Artiﬁcial Intelligence, pp. 1186–1193 (2020) Zhang, W., Chen, Z., Zha, H., et al.: Learning from substitutable and complementary relations for graph- based sequential product recommendation. ACM Trans. Inf. Syst. (TOIS) 40(2), 1–28 (2021) Zhang, X., Yang, Q., Xu, D.: Combining explicit entity graph with implicit text information for news recommendation. Companion Proceedings of the Web Conference 2021, 412–416 (2021) Zhao, T., McAuley, J., King, I.: Leveraging social connections to improve personalized ranking for col- laborative ﬁltering. In: Proceedings of the 23rd ACM International Conference on Information and Knowledge Management, pp. References 261–270 (2014) Zhao, H., Yao, Q., Li, J., et al.: Meta-graph based recommendation fusion over heterogeneous informa- tion networks. In: Proceedings of the 23rd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining, pp. 635–644 (2017) Zhao, J., Zhou, Z., Guan, Z., et al.: Intentgc: a scalable graph convolution framework fusing heterogeneous information for recommendation. In: Proceedings of the 25th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 2347–2357 (2019) Zheng, Y., Mobasher, B., Burke, R.: Carskit: A java-based context-aware recommendation engine. In: 2015 IEEE International Conference on Data Mining Workshop (ICDMW), pp. 1668–1671. IEEE (2015) Zheng, Y., Mobasher, B., Burke, R.: Carskit: A java-based context-aware recommendation engine. In: 2015 IEEE International Conference on Data Mining Workshop (ICDMW), pp. 1668–1671. IEEE (2015) Zheng, J., Liu, J., Shi, C., et al.: Recommendation in heterogeneous information network via dual similarity regularization. Int. J. Data Sci. Anal. 3(1), 35–48 (2017) g p pp Zheng, J., Liu, J., Shi, C., et al.: Recommendation in heterogeneous information network via dual similarity regularization. Int. J. Data Sci. Anal. 3(1), 35–48 (2017) g ( ) ( ) Zheng, L., Lu, C.T., Jiang, F., et al.: Spectral collaborative ﬁltering. In: Proceedings of the 12th ACM Conference on Recommender Systems, pp. 311–319 (2018) Zheng, Y., Wei, P., Chen, Z., et al.: Graph-convolved factorization machines for personalized recommen- dation. IEEE Trans. Knowl. Data Eng. (2021) Zhou, D., Schölkopf, B.: A regularization framework for learning from graph data. In: ICML 2004 Workshop on Statistical Relational Learning and Its Connections to Other Fields (SRL 2004), pp. 132–137 (2021) Zhou, K., Zhao, W.X., Bian, S., et al.: Improving conversational recommender systems via knowledge graph based semantic fusion. In: Proceedings of the 26th ACM SIGKDD International Conference on Knowledge Discovery & Data Mining, pp. 1006–1014 (2020a) Zhou, S., Dai, X., Chen, H., et al.: Interactive recommender system via knowledge graph-enhanced rein- forcement learning. In: Proceedings of the 43rd International ACM SIGIR Conference on Research and Development in Information Retrieval, pp. 179–188 (2020b) Zhu, Y., Guan, Z., Tan, S., et al.: Heterogeneous hypergraph embedding for document recommendation. Neurocomputing 216, 150–162 (2016) p g Zhu, T., Sun, L., Chen, G.: Embedding disentanglement in graph convolutional networks for recommenda- tion. IEEE Trans. Knowl. Data Eng. (2021a) Zhu, T., Sun, L., Chen, G.: Embedding disentanglement in graph convolutional networks for recommenda- tion. IEEE Trans. Knowl. Data Eng. References (2021a) Zhu, T., Sun, L., Chen, G.: Graph-based embedding smoothing for sequential recommendation. IEEE Trans. Knowl. Data Eng. (2021b) Zhu, T., Sun, L., Chen, G.: Graph-based embedding smoothing for sequential recommendation. IEEE Trans. Knowl. Data Eng. (2021b) Zhu, Y., Lin, Q., Lu, H., et al.: Recommending scientiﬁc paper via heterogeneous knowledge embedding based attentive recurrent neural networks. Knowl.-Based Syst. 215(106), 744 (2021c) Zhu, Y., Lin, Q., Lu, H., et al.: Recommending scientiﬁc paper via heterogeneous knowledge embedding based attentive recurrent neural networks. Knowl.-Based Syst. 215(106), 744 (2021c) Ziegler, C.N., McNee, S.M., Konstan, J.A., et al.: Improving recommendation lists through topic diversiﬁ- cation. In: Proceedings of the 14th International Conference on World Wide Web, pp. 22–32 (2005) Ziegler, C.N., McNee, S.M., Konstan, J.A., et al.: Improving recommendation lists through topic diversiﬁ- cation. In: Proceedings of the 14th International Conference on World Wide Web, pp. 22–32 (2005) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published map and institutional afﬁliations. Lemei Zhang is a postdoctoral fellow at the at the Norwegian Research Center for AI Innovation (NorwAI) at NTNU, Norway. Her research topics include recommender system and user modelling. Speciﬁcally, she is focusing on applying data mining and machine learning techniques to design effective algorithms to enhance performance of recommender systems in various domains, such as e-commerce, social networks, and so forth. Peng Liu works as a postdoctoral fellow at the Norwegian Research Center for AI Innovation (NorwAI) at NTNU. His research focuses on natural language processing and recommender systems. His primary interests lie in the areas of language modelling, sentiment analysis, topic modeling, lexical semantics, and 3 L. Zhang et al. 888 recommendation algorithms based on data streams and multimodal contexts such as text, image, and so forth. Jon Atle Gulla is a professor of information systems at the Norwegian University of Science and Technol- ogy (NTNU) since 2002 and the director of the Norwegian Research Center for AI Innovation. He has a Ph.D. in computer science from 1993 and holds three M.Sc. degrees in computer science, linguistics, and management. His research is on natural language processing and semantics in the context of recommender systems, search engines, and conversational systems. 123
https://openalex.org/W2000534628	https://europepmc.org/articles/pmc4119291?pdf=render	English	null	‘We do not do any activity until there is an outbreak’: barriers to disease prevention and health promotion at the community level in Kongwa District, Tanzania	Global health action/Global health action. Supplement	2,014	cc-by	10,583	Tumaini Nyamhanga1, Gasto Frumence1, Mughwira Mwangu1 and Anna-Karin Hurtig2 Tumaini Nyamhanga1, Gasto Frumence1, Mughwira Mwangu1 and Anna-Karin Hurtig2 1Department of Development Studies, School of Public Health and Social Sciences, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania; 2Epidemiology and Global Health, Umea˚ International School of Public Health, Umea˚ University, Umea˚ , Sweden Background: Little is known about the barriers to disease prevention and health promotion at the community level within a decentralized health system. Objective: This paper, therefore, presents and discusses findings on barriers (and opportunities) for instituting disease prevention and health promotion activities. Design: The study was conducted in Kongwa District, Tanzania, using an explorative case study approach. Data were collected through document reviews and in-depth interviews with key informants at district, ward, and village levels. A thematic approach was used in the analysis of the data. Results: This study has identified several barriers, namely decision-makers at the national and district levels lack the necessary political will in prioritizing prevention and health promotion; the gravity of prevention and health promotion stated in the national health policy is not reflected in the district health plans; gross underfunding of community-level disease prevention and health promotion activities; and limited community participation. Conclusion: In this era, when Tanzania is burdened with both communicable and non-communicable diseases, prevention and health promotion should be at the top of the health care agenda. Despite operating in a neoliberal climate, a stronger role of the state is called for. Accordingly, the government should prioritize higher health-protecting physical, social, and economic environments. This will require a national health promotion policy that will clearly chart out how multisectoral collaboration can be put into practice. Keywords: barriers; disease prevention; health promotion; Tanzania Keywords: barriers; disease prevention; health promotion; Tanzania Correspondence to: Tumaini Nyamhanga, Department of Development Studies, Muhimbili University of Health and Allied Sciences, P.O. Box 65454, Dar es Salaam, Tanzania, Email: tumaininyamhanga@yahoo. com T he World Health Organization (WHO) and minis- tries of health in low- and middle-income coun- tries have recently albeit rhetorically sounded a need to revitalize the primary health care (PHC) approach for health improvement (1). This can partly be attributed to the challenge of the double burden of disease experienced by low-income countries (2). Global Health Action 2014. # 2014 Tumaini Nyamhanga et al. This is an Open Access article distributed under the terms of the Creative Commons CC-BY 4.0 License (http://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. 1 Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 (page number not for citation purpose) Global Health Action ORIGINAL ARTICLE ‘We do not do any activity until there is an outbreak’: barriers to disease prevention and health promotion at the community level in Kongwa District, Tanzania Tumaini Nyamhanga1, Gasto Frumence1, Mughwira Mwangu1 and Anna-Karin Hurtig2 From the above-cited definition, two core values universal health care (social justice) and community participation guide PHC (9). As such, success of PHC is determined by collaboration between health sector and related sectors such as water, agriculture, education, finance, and com- munication. The collaboration should be people-centered that is, community members should be involved in agenda setting, analyzing problems, planning, and im- plementation of interventions (10). The health policy also states that health sector reforms seek to operationalize the same basic principles of PHC in the provision of health care services. The response in most low-income countries like Tanzania was the development of essential health care package which is an integrated collection of cost-effective interventions. The package comprises the following: reproductive and child health services, control of communicable and NCDs, treat- ment of common conditions of local prevalence within the district, community health promotion and disease prevention through environmental sanitation, and man- agement and occupational health services. Although Tanzania’s Health policy (11) theoretically embraces the above-described principles of PHC which are key to the health for all strategy there has not been sufficient conformity with these principles to bring about the desired improvement in people’s lives. This may partly be a result of limited intersectoral collaboration for health development and limited community involvement. This deficiency is evidenced by poor development in- dicators such as low life expectancy and high child and maternal morbidity and mortality (2). Tanzania’s health policy (11) categorically states that PHC remains the cornerstone in the delivery of essen- tial health care in the country. PHC as a value-based systems approach for all levels (Alma Ata 1978) thus still remains a point of reference that is emphasized by the government in the National Health Policy, as indicated in Box 1. PHC in Tanzania: before the Alma-Ata declaration Tanzania started implementing the principles of PHC long before the 1978 Alma-Ata Declaration (12). Right after independence, the first president of Tanzania, Mwalimu Julius Kambarage Nyerere, issued a philoso- phical statement saying that Tanzania (then Tanganyika) had three enemies that she had to fight: poverty, illiteracy, and ill health (13). This philosophical commitment was operationalized by being integrated into the national policy popularly known as the Arusha Declaration of 1967 (14). One of the aims of the Arusha Declaration was that government should exercise effective control over the principal means of production and pursue policies that facilitate collective beneficence from national resources. The policy thus ordered free provision of social services, notably health, education, and water (14). It is worth noting that the implementation of the Arusha Declara- tion marked a shift in the focus of service delivery from a predominantly curative to a more comprehensive PHC approach. That is, more resources were directed toward training middle- and lower-level cadres of health workers, and the construction of health facilities in rural settings (dispensaries and health centers). In addition, community health programs on nutrition, literacy, and sanitation received both financial and political support. Tumaini Nyamhanga1*, Gasto Frumence1, Mughwira Mwangu1 and Anna-Karin Hurtig2 That is, non-communicable diseases (NCDs) for example, heart disease, cancer, and diabetes are increasingly becoming a cause of morbidity and mortality in poor countries, while communicable diseases remain highly prevalent and are also predisposing some NCDs. The impact will be particularly felt in sub-Saharan Africa, where NCDs will account for 46% of all deaths by 2030, up from 28% in 2008, and in South Asia, which will see the share of deaths from NCDs increase from 51 to 72% during the same period (3). These poor regions of the world will, at the same time, continue to grapple with the widespread prevalence of communicable diseases such as HIV, malaria, tuberculosis, and mother and child conditions, and so face a ‘double burden’ of disease not experienced by their wealthier counterparts. T The magnitude of the challenge notwithstanding, there is hope for turning the tide if prevention and health Tumaini Nyamhanga et al. promotion at the community level are made a priority (47), as advocated in the PHC approach. According to WHO, PHC refers to ‘essential health care based on practical, scientifically sound, and socially acceptable method and technology; universally accessible to all in the community through their full participation; at an affordable cost; and geared toward self-reliance and self- determination’ (8). It is a comprehensive approach to service provision that involves activities targeting the individual (curative services) and the general population (health promotion and disease prevention). From the above-cited definition, two core values universal health care (social justice) and community participation guide PHC (9). As such, success of PHC is determined by collaboration between health sector and related sectors such as water, agriculture, education, finance, and com- munication. The collaboration should be people-centered that is, community members should be involved in agenda setting, analyzing problems, planning, and im- plementation of interventions (10). promotion at the community level are made a priority (47), as advocated in the PHC approach. According to WHO, PHC refers to ‘essential health care based on practical, scientifically sound, and socially acceptable method and technology; universally accessible to all in the community through their full participation; at an affordable cost; and geared toward self-reliance and self- determination’ (8). It is a comprehensive approach to service provision that involves activities targeting the individual (curative services) and the general population (health promotion and disease prevention). 2 (page number not for citation purpose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 2 (page number not for citation purpose) Conceptual framework This paper is guided by the WHO’s system framework (28) as interpreted from the perspective of PHC by Saachy et al. (29) (Fig. 1). Although the original adaptation by Saachy et al. focuses on the national health system, the current study applies the framework at both national and district levels. This is because the two levels of governance work interdependently. Whereas the central (national level) government develops a health policy and allocates resources, the local (district level) government is entrusted with a duty of implementing the policy. Ideological reforms involved reducing the role of the state from being the sole provider of health services to that of being a partner and a creator of a conducive environment for other actors private, civil society, and non-governmental organizations (NGOs) (21). More importantly, in the Tanzanian context, the ideological reform involved disposing of the egalitarian policy of free provision of health services put in place by the Arusha Declaration (22). Thus, in the context of Tanzania, the objectives of the district health system are to deliver accessible, equitable, and good-quality health services that are both responsive to community demands and based on the principle of inter-sectoral collaboration. Organizational reforms entailed decentralization, which involved transfer of power, authority, and functions from the central to local government authorities (23). Decen- tralization took place in three areas: fiscal, political, and managerial/administrative. Fiscal decentralization involved allowing local authorities to collect taxes from their own sources for covering recurrent expenditures (4). On the contrary, the central government retained the authority to mobilize and disburse health block grants and basket funds. Health block grants come from the central government’s internal revenues and are spent on personnel emoluments, other charges, and a development grant, whereas basket funds are finances that are con- tributed to the country by several bilateral and multi- lateral development partners and spent on other charges (24, 25). Moreover, political decentralization involved the transfer of political authority and function from central government ministries to the local government authorities cities, municipalities, and district councils (26). The Prime Minister’s office Regional Administration and Local Government and Health ministries and the Ministry of Health and Social Welfare retained the The framework illustrates that for the district health system to attain the set objectives there are specific functions that must be fulfilled. Health sector reforms and PHC Little is known about local realities regarding the impact that health sector reforms have had on broader prevention and health promotion programs. The aim of this paper, therefore, is to shed light on barriers to implementation of comprehensive primary health specifically disease prevention and health promotion activities at the community level in the context of health sector reforms. Health sector reforms are defined as intensive long-term efforts to strengthen and improve health systems and, ultimately, improve the nation’s health (17, 18). Health sector reforms are aimed at increasing the effectiveness, efficiency, quality, equity, and financial soundness of health systems (19). In Tanzania, as in other developing countries, the reforms were prompted by a number of factors, including the movement from a state-controlled economy to market-oriented economies; insufficient fund- ing for health; and the poor quality, low accountability, and inefficiency of existing health services (20). The reforms measures can be grouped into three broad cate- gories: ideological and policy changes, organizational changes, and financing changes. Barriers to disease prevention and health promotion Barriers to disease prevention and health promotion socioeconomic reforms (15). The reforms involved the adoption and implementation of structural adjustment programs (SAPs) prescribed by the International Mone- tary Fund and the World Bank (16). The SAPs specifi- cally involved implementation of the following measures: removal of subsidies; budget; liberalization; privatiza- tion; currency devaluation; and cuts in social services (16). authority for formulating policies, guidelines, standards, and regulation of health practice. However, the local government authorities were entrusted with the respon- sibility of planning and utilizing available resources to meet health needs in their areas of jurisdiction (27). Reports on evaluation of PHC show that many countries that have been implementing health sector reforms adop- ted selective PHC with curative care and few aspects of disease prevention such as vaccination and encourage- ment of long-term breastfeeding (9). 3 (page number not for citation purpose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Box 1. Primary health care In its endeavor to ensure success in delivery of essen- tial health care in the country, the Government [of Tanzania] through Primary Health Care emphasizes on: . Community involvement and ownership through active participation in identification of problem areas, planning, implementation, monitoring, and evaluation of health care services; . Multisectoral collaboration by establishment of committees involving other sectors such as water, agriculture, and education, and ministries such as community development, gender, and children; . Equity and accessibility to health care by ensuring that every individual has the right to health care and equitable distribution of health resources in the country; . Empowerment through decentralization of health services to regions and districts and communities to ensure effective coordination, implementation, supervision, and provision of quality health care to the community; However, a gradual shift away from investing in pre- vention and health promotion started in the 1990s when Tanzania embarked on neoliberal-oriented health sector reforms. These reforms were preceded by the economic crisis of the late 1970s and early 1980s that necessita- ted Tanzania, like many African countries, to undertake y . Providing promotive, preventive, curative, and reha- bilitative interventions to all individuals and fam- ilies with their active participation. 2 (pag Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Conceptual framework These functions include the development of human resources, a health informa- tion system, service delivery, financing, and gover- nance. The current study will focus on the latter three functions. In Tanzania, there are well-established structures at the national, district, and community levels for managing the above-mentioned functions of the health system (Fig. 2). The core structure at the district level is the council health management team (CHMT), which carries out the following key functions: planning, coordination, implementation, monitoring, and evaluation of all health activities. In essence, the CHMT oversees service delivery at the hospital, health center, dispensary, and community levels (23). The communities are linked to the health system through health facility governing committees, which have been formed by a local government act (30). It was envisaged that the committees would assist health 3 pose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 3 (page number not for citation purpose) Tumaini Nyamhanga et al. Fig. 1. Conceptual Framework. Source: Adapted from Saachy et al. (29). Fig. 1. Conceptual Framework. Source: Adapted from Saachy et al. (29). 4 (page number not for citation purpose) Methods center and dispensary management teams in planning and supervising community health programs (31) and that they would facilitate the empowerment of house- holds and communities with the knowledge and skills needed to reduce the burden of disease (21). In addition, there are government structures at ward and village levels center and dispensary management teams in planning and supervising community health programs (31) and that they would facilitate the empowerment of house- holds and communities with the knowledge and skills needed to reduce the burden of disease (21). In addition, there are government structures at ward and village levels ward development and village health committees that play a role in providing political leadership and enhan- cing inter-sectoral linkages for health development in the communities they serve. center and dispensary management teams in planning and supervising community health programs (31) and that they would facilitate the empowerment of house- holds and communities with the knowledge and skills needed to reduce the burden of disease (21). In addition, there are government structures at ward and village levels ward development and village health committees that play a role in providing political leadership and enhan- cing inter-sectoral linkages for health development in the communities they serve. 4 (page number not for citation purpose) Study setting This study was conducted from January to February, 2013, in Kongwa District. Kongwa is a site for the implementation of a collaborative health system research project between Muhimbili University of Health and Allied Sciences in Tanzania and Umea University in Sweden. The choice of Kongwa was purposively made to represent a typical rural district that has a moderate level Fig. 2. Organization structure of a decentralized health system. Source: Modiﬁed from the ﬁgure on interlinks between central and local government structures (Molel, 2010). Fig. 2. Organization structure of a decentralized health system. Source: Modiﬁed from the ﬁgure on interlinks between central and local government structures (Molel, 2010). Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 4 ( Barriers to disease prevention and health promotion Health Committee; and three members of the Health Facility Governing Committee. of socioeconomic development and is fairly accessible in terms of transportation and communication networks. Kongwa is one of the districts in Dodoma region. It is bordered by the Manyara region to the northern part, to the east by the Morogoro region, to the south by the Mpwapwa district, and to the west by the Dodoma rural district. Administratively, the district has 14 wards and, according to the 2002 Tanzania National Census, the population of Kongwa District was 249,760, with an annual growth rate of 2.4% (12). The health care system in Kongwa District is largely based on public health facilities, including one district hospital, three health centers, and 27 dispensaries. Study design This paper is based on an explorative case study. Quali- tative case study methodology enables researchers to explore and explain a phenomenon within its context using multiple sources of data (32). The approach was relevant to this study because it involved in-depth investigation of barriers and opportunities for instituting disease prevention and health promotion activities at the community level in the context of health sector reforms in the study district. Additionally, several supporting documents that were cited by the respondents, and others that were deemed rele- vant by the investigators, were reviewed. These included: the National Health Policy of 2007 and the council’s comprehensive health plan (CCHP) for Kongwa. These documents were read with a view of understanding articulation of and efforts put on health promotion and disease prevention activities at the community level. Study population and their sampling criteria Tape-recorded in-depth interviews were transcribed verbatim, translated from Kiswahili into English. Sup- plementary field notes were typed immediately after data collection, using a word processor. Additional infor- mation that was obtained later on the phone was also included. A thematic analysis approach was used in analyzing the study’s findings through the following steps (33). First, transcripts and field notes were read several times in order to identify patterns of experience (cate- gories of common ideas). Second, all data that relate to the already classified patterns were identified and placed within the corresponding pattern. Third, patterns of experience were examined to determine similarities and differences among them with respect to the mean- ings they conveyed in relation to the research question. Fourth, the patterns of experience were combined into study themes that represented a higher level of patterned response or meaning within the data set (34). These study themes are highlighted in the introductory part of the results section. The recruitment of the study participants in this sub- study was purposive, as it took account of their positions and experience in dealing with prevention and health promotion at the community level. Table 1 summarizes the details of the study respondents. As Table 1 shows, 23 respondents were involved in the study. Out of these, there were seven CHMT members, four key district officials (District Planning Officer, District Treasurer, Chair of Council Health Services); four heads of programs (RCH Coordinator, District AIDS Coordinator, Malaria Coordinator, and TB/ Leprosy Coordinator); two Ward Executive Officers; one Ward Health Officer, five members of the Village Table 1. Study respondents Number S/N Category Male Female 1 CHMT members 5 2 2 Other key district officials (DPLO, DT, CCHS) 3 1 3 Heads of programs 2 2 4 Ward executive officers 2 0 5 Members of Village health committee 1 1 6 Members of health facility governing committee 2 1 6 Ward health officer 1 0 Total 16 7 Cit ti Gl b H lth A ti 2014 7 23878 // / / Data collection methods The data were collected through conversation by face-to- face in-depth key informant interviews with the officers concerned, when they could find a convenient time. In addition, telephone interviews were also undertaken when further clarification was sought on data collected face-to-face. An interview guide with a list of open-ended questions was used. These questions covered major relevant areas such as: priority in allocation of resources prevention versus curative care; planning for prevention and health promotion activities at community level; community participation; and local leadership structures for prevention and health promotion. prevention versus curative care; planning for prevention and health promotion activities at community level; community participation; and local leadership structures for prevention and health promotion. 5 (page number not for citation purpose) Results In this section, we present results on barriers to imple- mentation of disease prevention and health promotion activities at the community level in the context of health sector reforms. The results are organized around the study themes that reflect the three elements of the district health system in the conceptual framework, namely: service delivery, financing, and governance. It is worth noting that there is an inevitable overlap that shows a close interdependence among the constituent elements of the health system. The study themes are lack of political will toward delivery of comprehensive PHC; underfund- ing of prevention and health promotion; Ill-preparedness of the ward and village authorities; and limited commu- nity participation. Another member of the CHMT added that: Another member of the CHMT added that: Another member of the CHMT added that: Ethical considerations Sometimes during budgeting someone at the Minis- try commented that we as the Ministry [government] have decided to put our efforts into medicine so remove this item [health promotion]. So you find that we planned for preventive cure from district level or lower level but at the end of the day the planning process is not participatory; the Ministry dictates which items should be funded and which ones shouldn’t. It is the directive from the Ministry that matters. (KI-4, Kongwa) The study obtained ethical clearance from Muhimbili University of Health and Allied Sciences. The respon- dents were requested to participate in the study, and provided their oral consent. Prior to consenting, they were assured of confidentiality by being told that no names were required and that no unique identifiers would feature in the study report or on publication. Further- more, each potential respondent was informed of his/her right to decline outright to participate, or to withdraw consent at any stage of the interview, without undesirable consequences. In addition, a review of documents shows that although the National Health Policy nicely explains the impor- tance of prevention and health promotion, translation of the same into a district health plan is a major challenge. Prevention and health promotion activities are not well articulated in the comprehensive district health plan. As Table 2 shows, prevention and health promotion activities are scarcely indicated. For instance, for reducing mortality due to malaria the plan contained only one preventive activity: to provide a hati punguzo (a voucher for accessing a subsidized mosquito net) for all pregnant women attending ANC by 100%. The table also shows that preventive activities for diarrhea are missing sug- gesting a neglect of hygiene, water supply, and sanitation. Likewise, activities for addressing NCDs are sparingly indicated. For instance, in the 2011 comprehensive health plan, control of NCDs is missing from the community cost center. It is only indicated under the health center cost center, where it is shown that screening for cervical cancer will be conducted. Lack of political will toward the delivery of comprehensive PHC The readiness for implementing comprehensive PHC was assessed through key informant interviews and a review of important documents the National Health Policy of 2007 and the Council Comprehensive Health Plan. The respondents indicated that both national- and district- level decision-makers lack political will in prioritizing prevention and health promotion activities. Their main focus is on the curative aspect of health care. When asked why prevention and health promotion activities were not a top priority, one of the key informants, a member of the CHMT said: Nevertheless, the CCHP shows a window of opportu- nity for addressing environmental sanitation. There is a statement that the council would facilitate the private sector to invest in environmental health activities. It reads: ‘The council will facilitate private sector and civil society organizations to invest in the collection and disposal of refuse’ (2010/2011 CCHP, Kongwa). 6 (page number not for citation purpose) Trustworthiness The trustworthiness of the findings can be secured through different approaches such as: triangulation, pro- longed engagement, peer-debriefing, and member checks (35). In this study, key informant interviews were com- plemented with document reviews to triangulate sources of information. The interview guide was piloted to im- prove the questions and the moderating skills. The data collection period was prolonged to allow the researchers Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 5 ose) 5 ose) 5 (page number not for citation purpose) Tumaini Nyamhanga et al. Here curative care takes precedence over preventive, while in other countries it is vice versa, so we have to focus now on preventive rather than curative care. (KI-1, Kongwa) enough time for reflection between field visits and to allow them to perform a preliminary analysis that guided their subsequent data collection. The final data analysis was facilitated by peer-debriefing sessions involving a multidisciplinary research team. Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Target Target 2) Training of 20 COs on syndomic case management. Source: Kongwa Comprehensive Council Health Plan for the Year 2010/2011. Several respondents indicated that because of the lack of financial motivation, village health committees are virtually dormant. One member of the committee aptly conveyed a strong message on the inactivity of the village health committee in handling issues of sanitation: and subsistence allowance for the district health staff who carry out outreach visits. Two, waste disposal and water availability in the health facilities. The table makes no mention of support for waste disposal, sanitation, and/or water availability at the household level. As a conse- quence, there is limited community participation in utilizing these funds. It was reported that there are no government funded community activities initiated and implemented by local people themselves. and subsistence allowance for the district health staff who carry out outreach visits. Two, waste disposal and water availability in the health facilities. The table makes no mention of support for waste disposal, sanitation, and/or water availability at the household level. As a conse- quence, there is limited community participation in utilizing these funds. It was reported that there are no government funded community activities initiated and implemented by local people themselves. Yes, the village health committee exists, but we do not do any activity unless there is an outbreak. We work under difficult conditions; we need allowances. (KI-3, Kongwa) Table 3. Allocation ceiling for the health basket fund and block grant (other charges) Cost center Ceiling range for allocation by council (%) Office of District Medical Officer (DMO) 1520 Council hospital/CDH 2530 Voluntary agency hospitals (VAH) 1015 (health basket funds only) Health centers 1520 Dispensaries 2025 Community initiatives 25 Source: URT (24). Citation: Glob Health Action 2014 7: 23878 http://dx doi org/10 3402/gha Table 3. Allocation ceiling for the health basket fund and block grant (other charges) So the little money is spent by the district health depart- ment itself to support community action like the distri- bution of mosquito nets and prophylactic drugs for neglected tropical diseases (e.g. elephantiasis). One of the key CHMT members said: Community health activities take at most about 2% to 5% of the CCHP [council comprehensive health plan] budget and still you can find that the activities do not aim straight at empowering com- munities to address local needs. Underfunding of prevention and health promotion Document reviews show that prevention and health promotion activities are grossly underfunded. From the resource allocation point of view, such activities are marginalized as only 25% of district health funds are allocated for them, as indicated in Table 3. I think maybe it is the system from the ministry down to the district level, as I do remember some- times I went to the ministry for budgeting, and someone at the ministry commented that why don’t we remove the activity of health promotion and instead put procurement of medicines? You know, in Tanzania, we are unlike the developed countries. Additional evidence of under-funding is indicated in Table 4, which shows that money budgeted under community health initiatives and promotion to a large extent targets two main areas. One, meeting travel costs 6 (pag 6 (pag Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Barriers to disease prevention and health promotion Table 2. Objectives, targets, and planned interventions for selected diseases 2010/11 Problem Objective Target Activity High morbidity and mortality rate due to malaria To reduce morbidity and mortality due to malaria by 50% by year, 2015 To reduce morbidity and mortality due to malaria by 7% by June 2011 1) Training to 20 health workers on malaria case mgt. 2) Commemoration of African Malaria day. 3) Computer training to malaria/ IMCI data manager. To provide HATI PUNGUZO for all pregnant woman attending ANC by 100%. 1) Training to 20 health workers on malaria case management. 2) Commemoration of African Malaria day. 3) Computer training to malaria/ IMCI data manager. To provide HATI PUNGUZO for all pregnant woman attending ANC by 100%. High morbidity and mortality rate due to communicable diseases such as, Diarrhea Tuberculosis and HIV/AID/ STI To increase proportion of TB cases completed treatment from 97 to 100% by June 2009 To reduce STI/HIV/AID cases To increase case mgt ability and case detection in all 41 HF. Reduce HIV infection from 3.1 to 2.0%. To train 20 clinician/nurses on proper case management for 4 days. Tracing of defaulters Conduct supportive supervision to 41 health facilities. 1) Training 20 health providers on proper management of STI Table 2. Objectives, targets, and planned interventions for selected diseases 2010/11 Table 2. High morbidity and mortality rate due to communicable diseases such as, Diarrhea Tuberculosis and HIV/AID/ STI 7 (page number not for citation purpose) Underfunding of prevention and health promotion Objectives, targets, and planned interventions for selected diseases 2010/11 Problem Objective Target Activity High morbidity and mortality rate due to malaria To reduce morbidity and mortality due to malaria by 50% by year, 2015 To reduce morbidity and mortality due to malaria by 7% by June 2011 1) Training to 20 health workers on malaria case mgt. 2) Commemoration of African Malaria day. 3) Computer training to malaria/ IMCI data manager. To provide HATI PUNGUZO for all pregnant woman attending ANC by 100%. 1) Training to 20 health workers on malaria case management. 2) Commemoration of African Malaria day. 3) Computer training to malaria/ IMCI data manager. To provide HATI PUNGUZO for all pregnant woman attending ANC by 100%. High morbidity and mortality rate due to communicable diseases such as, Diarrhea Tuberculosis and HIV/AID/ STI To increase proportion of TB cases completed treatment from 97 to 100% by June 2009 To reduce STI/HIV/AID cases To increase case mgt ability and case detection in all 41 HF. Reduce HIV infection from 3.1 to 2.0%. To train 20 clinician/nurses on proper case management for 4 days. Tracing of defaulters Conduct supportive supervision to 41 health facilities. 1) Training 20 health providers on proper management of STI. 2) Training of 20 COs on syndomic case management. To conduct supportive supervision on syndomic case management to 41 health facility. Source: Kongwa Comprehensive Council Health Plan for the Year 2010/2011. 8 (page number not for citation purpose) 8 (page number not for citation purpose) Target It [the community] is just invited to take part in the implementa- tion. The implementer of the activities is the health department. (KI-2, Kongwa) The observation from the above quote that the little funds do not support community-led disease prevention Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 7 pose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 7 (page number not for citation purpose) 7 (page number not for citation purpose) Tumaini Nyamhanga et al. mance budget framework for 2012/13 Cost Centre E07 Community Health Initiatives/Promotion g y Details Intervention Activity Description Unit Qty Unit cost Output indicator Case management (diagnosis and treatment) of locally important To conduct NTDs Outreach at community and conduct BTRP Surgeries BY June 2013 Petrol Liters 1 600,000.00 Number of outreach conducted diseases and neglected tropical diseases Diesel Liters 776.0 2,500.00 Per diem domestic Person 1.0 1,950,000.00 Promotion and prevention predisposing factors for NTDs at community level To sensitize community on NTDs through commemoration of vision world sight meetings by June 2013 Per diem domestic Person 1.0 195,000.00 Activity report presented Ground travel (Bus, Railway, Taxi) Liters 1.0 20,000.00 Mobilized adequate funding for health and social welfare services (CHF, NHIF, P4P, User Fees etc.) Community sensitization on CHF at 22 wards by June 2013 Per diem domestic Can 1.0 990,000.00 Number of household enrolled Fuel Person 1.0 440,000.00 Proper disposal of hazardous waste, solid and liquid waste To procure waste handling material for Mlali Health Centre by June 2013 Cleaning supplies Person 1.0 1,000,000.00 Number of waste handling materials procured TB DOTS Plus (TB HIV, MDR TB) To sensitize community on TB through commemoration of world TB day by June 2013 Extra-duty Person 1.0 250,000.00 Number of meetings conducted Food and refreshment Person 1.0 125,000.00 entertainment Lump sum 1.0 200,000.00 Fuel Liters 1.0 100,000.00 Integrated management of childhood illnesses (IMCI) To conduct mentoring of IMCI to 80 health staff in 30 HFs by June 2013 Special needs material and supplies Person days 1.0 1,260,000.00 Number of health staff mentored Provision of safe clean water hygiene and sanitation To procure waste handling materials at 54 HFs by June 2013 Water and waste disposal Quarter 1.0 5,928,300.00 Number of waste handling materials procured Source: Kongwa District Council Performance Budget Framework (MTEF) 2012/13. Source: Kongwa District Council Performance Budget Framework (MTEF) 2012/13. Local governance in disease prevention and health promotion Local governance in disease prevention and health promotion The governance function with respect to prevention and health promotion was examined from the perspectives of government authorities at the ward and village levels and that of community participation. The results in the following sections are presented in that order. Ill-preparedness of the ward and village authorities The respondents revealed that governance structures for prevention and health promotion at the village level are weak. We are particularly referring to the village health committee, dispensary governing committee, and ward development committee (WDC). One of the district officials was asked to comment on the role of the WDC. His response was: We receive a health plan from the doctor, and we add certain views about this and that; we discuss together until we agree that this item can be done this period and the other(s) one can be done later. (KI-6, Kongwa) Primarily the WDC support all development activ- ities in the ward, but I can say that the WDC members do not know well their responsibilities despite the fact that we trained them; still they do not perform as expected. (KI-11, Kongwa) It was further reported that platforms for tapping people’s felt health needs and for organizing community-based initiatives are virtually nonexistent. A few attempts made by NGOs led to the formation of some groups, but they could not be sustained beyond the lifespan of the NGOs’ projects. One respondent had this to say regarding the existence of social groups that may organize the commu- nities toward designing and implementing community- based health programs: It was also reported that the lack of transparency in the way the committees are constituted results in having incompetent structures: It is true that some committees are not competent and sometimes others are elected on political bias. You know, we can go there to carry out elections, we tell the village executive officer to follow the laid- down directives, to advertise to the villagers, but for the sake of their interest the village chairman can select a person he wants. You see, then account- ability will not be there. (KI-8, Kongwa) Aaah, there were some groups which were involved in HIV/AIDS for example at Kibaigwa; they were groups organized by NGOs but after the NGOs had elapsed [project ended] the groups also dispersed so we do NOT have any group now. Limited community participation Respondents expressed concern that there is limited involvement of stakeholders at the community level in planning for prevention and health promotion activities. There is insufficient participation of villagers in defining local problems requiring prevention and health promo- tion interventions. The existing ‘participatory planning’, in which the communities are presumed to take part through health facility governing committees, is limited in scope. In practice, as its name suggests, a health facility governing committee serves the purpose of appraising and approving ‘curative’ plans drafted by the health facility medical staff. This was well summarized by one respondent when answering a question on the role of the committee in health planning: Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Target Source: Kongwa District Council Performance Budget Framework (MTEF) 2012/13. empowering the community to carry out disease preven- tion and health promotion activities. and health promotion activities is further corroborated by the information in the performance budget framework for Kongwa district for the year 201213 as indicated in Table 4. The key message in this table with respect to resource allocation is that although the interventions appear to be broad (for instance, provision of safe clean water hygiene and sanitation) the corresponding activi- ties are narrowly focused on strengthening capacity of health facilities (procuring waste handling materials for 54 health facilities). Little or no efforts are directed at and health promotion activities is further corroborated by the information in the performance budget framework for Kongwa district for the year 201213 as indicated in Table 4. The key message in this table with respect to resource allocation is that although the interventions appear to be broad (for instance, provision of safe clean water hygiene and sanitation) the corresponding activi- ties are narrowly focused on strengthening capacity of health facilities (procuring waste handling materials for 54 health facilities). Little or no efforts are directed at The finding that the little funds do not tangibly reach down to the community was corroborated by one of the ward health officers, who complained about the lack of financial support for preventive activities at the ward and village levels. He said: The Ward Health Committee doesn’t have any funding for implementing its activities. I do not 8 (pag 8 (pag Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Barriers to disease prevention and health promotion Barriers to disease prevention and health promotion Barriers to disease prevention and health promotion When a cholera outbreak occurs our committee works in close collaboration with the village govern- ment in making sure that the rules regulating issues like the use of toilets are followed. (KI-7, Kongwa) have a means of transport. So when I have to go and meet the village health attendants I use my own money. Another challenge is that you find a villager telling you he has dug a pit for a latrine but does not have a slab; that’s money. So these are the challenges. (KI-5, Kongwa) 9 (page number not for citation purpose Local governance in disease prevention and health promotion (KI-10, Kongwa) Members of the WDC and village health committee who were interviewed admitted that they lack knowledge and skills in planning and supervising community health programs. Those limitations notwithstanding, it was re- ported that WDCs and village health committees have been instrumental in mobilizing people toward becoming members of the community health fund a voluntary pre-payment scheme that offers a client (household) the opportunity to acquire a ‘health card’ after paying a contribution. Notwithstanding the above-presented shortfalls in parti- cipation, it is worth noting what existing structures do in the area of prevention and health promotion. It is prudent to acknowledge the role of village health workers (VHWs). The VHWs, normally two per village or hamlet, are people who are selected by fellow villagers through their government. Their role is to work in collaboration with a primary-level health facility (dispensary/health center) to facilitate the implementation of prevention and health promotion interventions. They do so through sensitization of community members and through taking part themselves in administering such interventions which include, but are not limited to, the distribution of Furthermore, the committees were reported to exercise government powers in the control of cholera epidemics that used to occur in Kongwa in the recent past. One of the members of the village health committee aptly said: 9 pose) 9 pose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 9 (page number not for citation purpose) Tumaini Nyamhanga et al. parallel to what was going on in the political sphere and reflected the influence of political ideology. mosquito nets, weighing of children under age 5, and vitamin A supplementation. One of the VHWs summari- zed what they do: p gy Similar experiences have been documented in Venezuela and Brazil (5). The difference between health sector reforms in Tanzania and those in Venezuela is that in the former (Tanzania) the reforms were, and still are, pushing the country to the right paying less attention to the plight of the poor because of the introduction of user fees (37) and increasingly paying less attention to disease prevention and health promotion at the community level. On the other hand, in Venezuela, the reforms were a reaction against the poor health conditions of the poor under neoliberal policies. Armada et al. (7) report that by the late 1990s, class-based health disparities were vast. Discussion In this section, we discuss the key findings of the study, placing them in a wider context of existing knowledge. The study has highlighted several barriers and some opportunities regarding the implementation of disease prevention and health promotion activities at the com- munity level in a decentralized health system. The barriers identified are: decision-makers at the national and district levels lack the necessary political will in prioritizing prevention and health promotion; the gravity of preven- tion and health promotion stated in the national health policy is not reflected in the district health plans; gross underfunding of community-level disease prevention and health promotion activities; and limited community participation. These shortfalls notwithstanding, impor- tant opportunities for improving the situation have been highlighted the existence of the National Health Policy that states the importance of prevention and health pro- motion toward having a healthy population; the decen- tralized health system structure; and the existence of structures that serve as a bridge between the health system and communities. g g ( ) Moreover, the study has found that prevention and promotion activities are not adequately articulated in the comprehensive district health plan. This implies that the emphasis on prevention is merely rhetorical. Most activities targeting major diseases such as malaria, TB, and diarrhea are curative presented as ‘case manage- ment’. Consequently, people, particularly the poor, will continue being trapped in the vicious circle of ill health. A similar observation was made by Baum et al. (6) as they argued that the health care sector in many poor countries is dominated by a Western biomedical imagina- tion that results in curative medicine being privileged over strategies that emphasize disease prevention and health promotion. In addition, the study findings also show that the local government has delegated its responsibility to provide environmental sanitation services to the private sector and civil society organizations. Although this is an opportunity for a publicprivate partnership (PPP) in service provision, in a poor country a PPP strategy presents a risk of having a larger part of the population unattended to. This is because a substantial number of people may not be able to afford to pay private providers, thereby failing to dispose of waste properly. Hickel (38) Local governance in disease prevention and health promotion Hence, when Hugo Chavez became president, he spent his first term, between 1998 and 2002, implementing a variety of strategies to eliminate barriers to health care. These included measures such as the implementation of inte- grated health care, and a focus on PHC centers and on prevention activities, thus shifting the emphasis away from curative care. Similarly, in Brazil the changes in the health system occurred toward universality (5). The reforms were as a result of mobilization in health, struggling for better and inclusive health services. These struggles from several groups trade unions, health workers, academics, and other civic groups constituted a health movement that stirred reforms and culminated in a health system that is more democratic and put preven- tion at the top of the agenda. Inspired by Brazil, South Africa’s Health Ministry is reforming post-apartheid a curative-oriented health system engaging more in what has been referred to as re-engineering of PHC (29). We follow up the mother’s and the child’s health. We visit households in the hamlets on a weekly basis to follow up to ensure that all children are sent to the clinic, and if we find a sick child we advise parents to take him/her to the health facility. We use special visit forms to fill in when we visit the hamlet. We weigh children, administer vitamin A supplements for children and birth control tablets for women and educate them how to use them. (KI-9, Kongwa) 10 (page number not for citation purpose) 10 (page number not for citation purpose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Underfunding: a major challenge The study has indicated that prevention and health promotion activities are grossly underfunded. From the resource allocation point of view such activities at the community level are marginalized as only 25% of dis- trict health funds are allocated for them. As a conse- quence, this small budget is spent on narrowly focused activities. For example, although the intervention is stated as ‘provision of safe clean water, hygiene, and sanitation’, the corresponding implementation activity is: procure- ment of waste handling materials for 54 health facilities. This pattern of expenditure leaves broader health promo- tion and disease prevention needs of community mem- bers unattended to. That is, the budget in Table 4 makes no mention of support for waste disposal, sanitation, and/or water safety at the household level. As discussed in the preceding sections, underfunding reflects an inclination toward prioritizing curative over preventive intervention. This pattern of funding has been reported elsewhere (39, 40). It is surprising that this is happening in a poor country that is experiencing a double burden of disease (2). Prominent scientists in the devel- oped world, like the Englishman Thomas McKeown, noted more than three decades ago that major contribu- tions to improved health in those countries over the pre- vious two centuries came more from changes in sanitary conditions than from medical interventions (41). The need to invest in improving living conditions for people for better health has been well consolidated in the report of the Commission on Social Determinants of Health (42). In addition, ill-preparedness probably reflects the lack of health promotion policy that would have clearly pre- scribed an institutional framework for the organization and coordination of prevention and health promotion programs at the national, district, and community levels. The policy would clearly indicate roles of stakeholders at various levels of governance. Tanzania may learn from Nigeria, which has a health promotion policy, apart from the National Health Policy (47). The study has found that there is limited involvement from stakeholders at the community level in planning for prevention and health promotion activities. Although health facility governing committees and VHWs serve as a bridge between the health system and the commu- nities, their involvement is largely in approving facility- based plans. That is, the system of involving communities through committees does not provide enough room to reflect community felt health needs in the CCHP. Local governance and participation: opportunities for the future? The study has discovered that local government struc- tures are not well prepared in handling community prevention and health promotion activities. This defi- ciency may compromise their effectiveness and efficiency in providing political leadership for community-based health programs. The role of governance or political com- mitment in determining the success or failure of health programs has been documented elsewhere in sub-Saharan Africa (45, 46). Also the objectives stated by Saachy et al. (29) are defined for the national level and are not in themselves operational for implementation. Accessible, equitable, and good quality health services are not all possible to achieve to the same extent in any health system rich or even worse if poor. Such fallacy creates disagreements throughout the system and leaves the districts, facilities, and communities without procedural guidance to make ends meet. Are the district plans in essence top down frameworks to get budgets approved rather than flexible tools for the necessary local compro- mises? The focus should therefore rightly shift for plans to be both responsive to community demands and based on the principle of inter-sectoral collaboration (29). Ideology framing approaches to disease prevention and health promotion The study found that respondents pointed out both national- and district-level decision-makers’ lack of political will in prioritizing prevention and health pro- motion activities. This might be attributed to the impact that ideological change has had on the mindsets of health system managers. In the Ujamaa ideology, the health for all strategy under the framework of PHC was at the top of the agenda of the Ministry of Health (36). During this period universal access to health care was the goal, and to that end the government considered health to be a social right rather than a matter of individual effort to seek and achieve health. Thus, health services were provided free of charge and prevention and health promotion activities were given considerable attention (36). However, in the early 1990s changes started in the health sector and they were almost 10 (page Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Barriers to disease prevention and health promotion attributes this pattern of environmental health manage- ment to the current political discourse of market indivi- dualism, which treats sanitation as a matter of individual achievement rather than a social right to be fulfilled by the state. Local governance and participation: opportunities for the future? 11 (page number not for citation purpose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Conclusion In this era, when Tanzania is burdened with both com- municable and NCDs, prevention and health promotion should be at the top of the agenda. This paper has discussed barriers to the realization of this wish ranging from the adoption of neoliberal ideology that promotes a biomedical conception of health to limited social par- ticipation. Those gaps notwithstanding, the paper has highlighted that change is possible. A decentralized health system and existing community structures can be capi- talized on. Despite operating in a neoliberal climate, a stronger role of the state is called for. That is, the state should demonstrate a clearer responsibility for ensuring community-directed health promotion supported by the health facility and district health services and other rele- vant sectors in implementation level collaboration. This may need both a further democratization at health facility and community levels and facilitation through stronger leadership from the state. Similar recommendations are emerging from other recent studies in Tanzania (10, 50). 7. Armada F, Muntaner C, Chung H, Williams-Brennan L, Benach J. Barrio Adentro and the reduction of health inequal- ities in Venezuela: an appraisal of the ﬁrst years. Int J Health Serv 2009; 39: 16187. 8. World Health Organization. Declaration of Alma-Ata. Adopted at the International Conference on Primary Health Care, Alma- Ata, USSR, 612 September 1978. Available from: http://www. who.int/publications/almaata_declaration_en.pdf [cited 16 Sep- tember 2013]. 9. WHO. World Health Report (2008). Primary health care: now more than ever before. Available from: http://www.who.int/whr/ 2008/whr08_en.pdf [cited 5 May 2014]. 10. Kamuzora P, Maluka S, Ndawi B, Byskov J, Hurtig AK. Promoting community participation in priority setting in district health systems: experiences from Mbarali district, Tanzania. Glob Health Action 2013; 6: 22669. Available from: http://dx.doi.org/10.3402/gha.v6i0.22669 [cited 5 May 2014]. 11. MoHSW (2007). Tanzania national health policy (draft). Dar es Salaam: Ministry of Health and Social Welfare. Salaam: Ministry of Health and Social Welfare. 12. WHO. Declaration of Alma Ata. Available from: http://www. euro.who.int/en/publications/policy-documents/declaration-of- alma-ata,-1978 [cited 10 November 2013]. Furthermore, there is a need for a national health promotion policy that will clearly chart out how multi- sectoral collaboration can be put into practice. The Prime Minister’s office may play a stewardship role toward that direction. More importantly, the time is ripe for the evolution of social movements to demand the fulfillment of people’s right to comprehensive health care provision at the community level. 13. Nyerere JK. Man and development. Conclusion Dar es Salaam: Tanzania Publishing House; 1968. 14. Nyerere JK. Arusha declaration: ten years after. Dar es Salaam: Government Printer; 1977. 15. Onimode BA. Political economy of the African crisis. Harare: Zed Books; 1988. 16. Shao I, Kiwara A, Makusi G. Structural adjustment in a socialist country: the case of Tanzania. In: Mwanza AM, ed. Structural adjustment programmes in SADC: experience and lessons from Malawi, Tanzania, Zambia, and Zimbabwe. Harare: SAPES Books; 1992, pp. 99145. Study limitation 2. WHO (2011). World health statistics 2011. Geneva: WHO. Because the study used case study design it lacked representativeness and thus findings cannot be general- ized. However, being a qualitative study, its goal was not to generalize but rather to provide rich information using multiple sources of data on barriers and opportu- nities for instituting disease prevention and health pro- motion activities at the community level in the context of health sector reforms. In addition, it would have been beneficial to have more than one case. However, this case gives indication of the barriers faced at a district level. 3. World Bank. The growing danger of non-communicable dis- eases acting now to reverse course. Washington, DC: World Bank; 2011. 4. Nyamhanga T, Frumence G, Mwangu M, Hurtig KA. Achieve- ments and challenges of resource allocation for health in a decentralized system in Tanzania: perspectives of national and district level ofﬁcers. East Afr J Public Health 2013; 9: 41627. 5. Mahmood Q, Muntaner C. Politics, class actors, and health sector reform in Brazil and Venezuela. Glob Health Promot 2013; 20: 59. DOI: 10.1177/1757975913476902. 6. Baum FE, Begin M, Houweling TAJ, Taylor S. Changes not for the fainthearted: reorienting health care systems toward health equity through action on the social determinants of health. Am J Public Health 2009; 99: 196774. DOI: 10.2105/AJPH.2008. 154856. Acknowledgement 17. WHO (2000). World Health Report 2000. Geneva: WHO. The authors gratefully acknowledge the Swedish International Development Agency (Sida) for funding this study through Grant No. 75000516. We also thank the Muhimbili University of Health and Allied Sciences and the management of Kongwa district councils for their administrative support and the key informants who provided information for this study. 18. Lubben M, Mayhew SH, Collins C, Green A. Reproductive health and health sector reform in developing countries: establishing a framework for Dialogue. Bull World Health Organ 2002; 80: 66774. 19. WHO (2004). Health sector reform and reproductive health report of a technical consultation. Geneva, Switzerland: WHO. 20. Dmytraczenko T, Rao V, Ashford L. Health sector reform: how it affects reproductive health. Policy brief: Population Reference Bureau; 2003. Available from: http://pdf.dec.org/pdf_docs/ Pnact445.pdf [cited 27 September 2013]. References Kerala state of India (48) and in Brazil (5). A stronger community participation is called for now as low-income countries contemplate revitalizing the PHC approach (49). 1. WHO (2008). Closing the gap in a generation: health equity through action on the social determinants of health. Geneva: WHO. 12 (page number not for citation purpose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Underfunding: a major challenge It is therefore not surprising that community-based environ- mental sanitation activities are scantly budgeted for or missed out sometimes. Probably, the situation would have been different if ‘participation’ were expanded to include taking on board the views of social groups (NGOs, community-based organizations, and civil society organi- zations) that are interested in prevention and health promotion. This means communities need to be encour- aged to form such groups where they don’t exist. Such social movements could also mobilize efforts from com- munity members themselves toward making their resi- dences healthy. The role of social movements in bringing about better health conditions has been documented in the In spite of this evidence, proponents of the new public health paradigm (43, 44) express their disappointment that poor countries’ financial resources for health are increasingly being spent on the provision of curative health services, especially hospital services, rather than being invested in prevention and health promotion. They add that even when health promotion activities are funded, the focus is generally on changing the behavior of individuals rather than creating wider physical, social, and economic environments supportive of healthy beha- vior. This is shown by Tanzania’s primary health services development program (PHSDP) 2007 2017. It is worth highlighting that the PHSDP total budget is Tshs 11.8 trillion, but out of this only 7% has been allocated for health promotion activities, and issues of environmental sanitation are completely left out (27). 11 urpose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 11 (page number not for citation purpose) Tumaini Nyamhanga et al. References The authors declare that they have no conﬂict of interests. Conflict of interest and funding The authors declare that they have no conﬂict of interests. Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 12 ( Barriers to disease prevention and health promotion 21. Semali IAJ. Understanding stakeholders’ roles in health sector reform process in Tanzania: the case of decentralizing the immunization program. PhD thesis. University of Basel, Faculty of Science; 2003. 36. Ministry of Health (MoH) (1990). National health policy. Dar es Salaam: MoH. 37. REPOA (2004). Equity implications of health sector user fees in Tanzania: do we retain the user fee or do we set the user f(r)ee? Analysis of Literature and Stakeholder views commissioned by research for poverty alleviation (REPOA). Dar es Salaam: REPOA. 22. Ministry of Health (1994). Proposals for health sector reform. Dar es Salaam: MOH, 179. 23. Crook R. Decentralization and poverty reduction in Africa: the politics of local-central relations. Publ Admin Dev 2003; 17: 36277. 38. Hickel J. Neoliberal plague: the political economy of HIV transmission in Swaziland. J South Afr Stud 2012; 38: 51329. DOI: 10.1080/03057070.2012.699700. 24. United Republic of Tanzania (URT) (2011). Comprehensive health planning guidelines. Dar es Salaam: Ministry of Health and Social Welfare and Prime Minister’s Ofﬁce Regional Administration and Local Government. 39. McGinnis MJ, Williams-Russo P, Knickman JR. The case for more active policy attention to health promotion. Health Aff. (Millwood) 2002; 21: 7893. 25. United Republic of Tanzania (URT) (2012). Guidelines for preparation of annual plan and budget 2012/13 in the imple- mentation of ﬁve year development plan 2011/12 2015/16. Dar es Salaam: Ministry of Finance and The President’s Ofﬁce, Planning Commission. 40. Ostlin P, Eckermann E, Mishra UD, Nkowane M, Wallstam E. Gender and health promotion: a multisectoral policy approach. Health Promot Int 2006; 21: 2535. 41. McKeown T. The role of medicine: dream, mirage, or nemesis? London: Nufﬁeld Provincial Hospitals Trust; 1976. 26. Frumence G, Nyamhanga T, Mwangu MA, Hurtig AK. Challenges to the implementation of health sector decentraliza- tion in Tanzania: experiences from Kongwa district council. Glob Health Action 2013; 6: 20983. DOI: 10.3402/gha.v6i0. 20983. 42. CSDH (2008). Closing the gap in a generation: health equity through action on Social Determinants of Health. Final Report of the Commission on Social Determinants of Health. Geneva: World Health Organization. 43. Baum F. The new public health. 3rd ed. Melbourne: Oxford University Press; 2008. 27. Maluka SO, Hurtig AK, Sebastian MS, Shayo E, Byskov J, Kamuzora P. Conflict of interest and funding Decentralization and health care prioritization process in Tanzania: from a national rhetoric to a local reality. Int J Health Plann Manage 2011; 26: e10220. DOI: 10.1002/ hpm.1048. 44. Raphael D. Social Determinants of Health: Canadian perspec- tives. Toronto: Canadian Scholars Press; 2004. 45. Parkhurst JO, Lush L. The political environment of HIV: lessons from a comparison of Uganda and South Africa. Soc Sci Med 2004; 59: 191324. 28. WHO. Strengthening health systems to improve health out- comes: WHO’s framework for action. Available from: www. who.int/healthsystems/strategy/en [cited 29 September 2013]. 46. Nyanator FK, Awoonor-Williams JK, Philips JF, Jones TC, Miller RA. The Ghana Community-based Health Planning and Services Initiative for scaling up service delivery innovation. Health Policy Plan 2005; 20: 2534. DOI: 10.1093/heapol/ czi003. 29. Saachy N, Sanders D, Kruger N. Overview of health sector reforms in South Africa. Final Report. London: DFID Human Resources Development Centre; 2011. 30. United Republic of Tanzania (URT) (2001). The District Council (Council Health Service Establishment) Instrument Act No, 7 1982. Dar es Salaam: Government Printer; 2001. 47. Federal Ministry of Health Nigeria. National Health Pro- motion Policy 2006. Available from: www.afro.who.int [cited 27 September 2013]. 31. United Republic of Tanzania (URT) (2007). Primary health services development programme (PHSDP) 2007 2017. Dar es Salaam: Ministry of Health and Social Welfare; 2007. 48. Ve´ron R. The ‘‘new’’ Kerala model: lessons for sustainable development. World Dev 2001; 29: 60117. 32. Baxter P, Jack S. Qualitative case study methodology: study design and implementation for novice researchers. Qual Rep 2008; 13: 54459. 49. Ruano AL. The role of social participation in municipal-level health systems: the case of Palencia, Guatemala. Glob Health Action 2013; 6: 20786. http://dx.doi.org/10.3402/gha.v6i0. 20786. 33. Aronson J. A pragmatic view of thematic analysis. Qual Rep 1994; 2 (1). Available from: http://www.nova.edu/ssss/QR/ BackIssues/QR2-1/aronson.html. [cited 2 October 2013]. 50. Mubyazi GM, Bloch P. Psychosocial, behavioural and health system barriers to delivery and uptake of intermittent preventive treatment of malaria in pregnancy in Tanzania viewpoints of service providers in Mkuranga and Muﬁndi districts. BMC Health Serv Res 2014; 14: 15. DOI: 10.1186/1472-6963-14-15. 34. Braun V, Clarke V. Using thematic analysis in psychology. Qual Res Psychol 2006; 3: 77101. 35. Dahlgren L, Emelin M, Winkvist A. Qualitative methodology in international public health. Umea: Umea University; 2004. Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 Conflict of interest and funding 13 (page number not for citation purpose) 13 urpose) Citation: Glob Health Action 2014, 7: 23878 - http://dx.doi.org/10.3402/gha.v7.23878 13 (page number not for citation purpose)
https://openalex.org/W4292079852	https://www.frontiersin.org/articles/10.3389/fenrg.2022.935648/pdf	English	null	Study on multi-objective optimization for nuclear reactor radiation shield design coupling genetic algorithm with paralleling discrete ordinate code	Frontiers in energy research	2,022	cc-by	5,402	Study on multi-objective optimization for nuclear reactor radiation shield design coupling genetic algorithm with paralleling discrete ordinate code OPEN ACCESS EDITED BY Shripad T. Revankar, Purdue University, United States REVIEWED BY Gerardo Maria Mauro, University of Sannio, Italy Jiankai Yu, Massachusetts Institute of Technology, United States CORRESPONDENCE Bin Liu, liubin871204@126.com Yi Tan, sharpty@sina.com Guang Hu, guanghu@mail.xjtu.cn SPECIALTY SECTION This article was submitted to Nuclear Energy, a section of the journal Frontiers in Energy Research RECEIVED 04 May 2022 ACCEPTED 21 July 2022 PUBLISHED 17 August 2022 CITATION Liu B, Tan Y, Jing F, Hu G and Yan M (2022), Study on multi-objective optimization for nuclear reactor radiation shield design coupling genetic algorithm with paralleling discrete ordinate code. Front Energy Res 10:935648 OPEN ACCESS EDITED BY Shripad T. Revankar, Purdue University, United States REVIEWED BY Gerardo Maria Mauro, University of Sannio, Italy Jiankai Yu, Massachusetts Institute of Technology, United States Bin Liu1, Yi Tan1, Futing Jing1, Guang Hu2 and Mingfei Yan3 1Science and Technology on Reactor System Design Technology Laboratory, Nuclear Power Institute of China, Chengdu, China, 2School of Nuclear Science and Technology, Xi’an Jiaotong University, Xi’an, Shannxi, China, 3RIKEN Center for Advanced Photonics, RIKEN, Wako, Saitama, Japan An optimization method combining a genetic algorithm and the paralleling discrete ordinate code is developed and applied to the shielding design of the Savannah reactor. Several approaches are studied to cope with the multi- objective optimization problem, such as transforming into single-objective optimization approach, non-dominated sorting approach, and the approach of adding constraints on sub-objectives. Comparing with the current design methods, the optimization method developed in this study shows better performance since the discrete ordinate calculations are free of statistical ﬂuctuations. The multi-objective optimization approach with constraints on sub-objectives seems to have the best performance. More optimized individuals satisfying the constraints can be obtained, and the optimized objectives show the best improvements comparing with the initial design. CITATION Liu B, Tan Y, Jing F, Hu G and Yan M (2022), Study on multi-objective optimization for nuclear reactor radiation shield design coupling genetic algorithm with paralleling discrete ordinate code. Front. Energy Res. 10:935648. doi: 10.3389/fenrg.2022.935648 COPYRIGHT © 2022 Liu, Tan, Jing, Hu and Yan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Study on multi-objective optimization for nuclear reactor radiation shield design coupling genetic algorithm with paralleling discrete ordinate code No use, distribution or reproduction is permitted which does not comply with these terms. multi-objective optimization, genetic algorithm, Pareto optimality, paralleling SN code, nuclear reactor shielding TYPE Original Research PUBLISHED 17 August 2022 DOI 10.3389/fenrg.2022.935648 TYPE Original Research PUBLISHED 17 August 2022 DOI 10.3389/fenrg.2022.935648 TYPE Original Research PUBLISHED 17 August 2022 DOI 10.3389/fenrg.2022.935648 1 Introduction The radiation shielding design is of great signiﬁcance for high-performance advanced nuclear reactors, such as nuclear marine propulsion systems (Yamaji and Sako, 1994), space reactors, and some other advanced nuclear systems (El-Genket, 2009). The primary purpose of the radiation shielding design is to design a shield enclosing the nuclear reactor to attenuate radiations and make the radiation dose out of the shield as low as reasonably achievable (ALARA) (Chen et al., 2019). Meanwhile, for those nuclear reactors in which space is limited, the shield also needs to be compact, light-weighted, and maybe specialized. As a result, the radiation shielding design problem can be modeled as a multi-objective optimization problem, given by Frontiers in Energy Research 01 frontiersin.org 10.3389/fenrg.2022.935648 Liu et al. 10.3389/fenrg.2022.935648 minimize f ⃗x f1 ⃗x, f2 ⃗x, /, fm ⃗x T subject to ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ fi ⃗x ≤fi,0, i 1, 2, /, m subject to ⎧ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎩ ⃗x (x1, x2, /, xn), x ∈Rn Lj ≤xj ≤Uj, j 1, 2, /, n hkxj ≤0, k 1, 2, /, q vlxj 0, l 1, 2, /, p , (1) of the sub-objectives are not added, so normally there are solutions of which the sub-objectives are beyond tolerable limits. In addition, the commonly used radiation transportation codes are the Monte Carlo code and one- dimensional SN code. Simulations of Monte Carlo codes are always ﬂuctuated as a result of its statistical nature (Zhuang, et al., 2019), causing difﬁculty for the optimization algorithms in searching for global optimal solutions. Also, the one- dimensional SN code cannot simulate the real radiation transportation problems. (1) where ⃗x is a n-dimensional variable vector of the design parameters; fi( ⃗x) represents the sub-objective of the optimization; fi,0 represents the tolerable limit of the sub- objectives; the values Lj and Uj represent the lower and upper bounds of the variables, respectively; and the inequality and equality equations hk and vl represent some domain constraints for the parameters considering the economical or engineering conditions. In this study, an optimization design method for the nuclear reactor radiation shielding structure is developed. The non-dominated sorting algorithm is used for multi- objective optimization. 2 Methodology Based on the optimization method, combining genetic algorithms with the paralleling SN code Hydra, several approaches are studied for the multi-objective optimization problem stated by Eq. 1, which are illustrated as follows. 1 Introduction Constraints of the sub-objectives are added to ensure sub-objectives of the optimized solutions within tolerable limits. In addition, the paralleling three-dimensional SN code Hydra is used to perform the radiation transportation calculations. Hydra was developed by the NECP (Nuclear Engineering Computational Physics) laboratory which uses the Koch–Baker–Alcouffe (KBA) algorithm to perform the parallelization calculation (Wang et al., 2020). The aim of this study is to develop an accurate and efﬁcient optimization method solving the multi-objective optimization problem of the nuclear reactor shielding design, with the optimized solutions of which sub-objectives are within the tolerable limits. Conventionally, the radiation shielding structures are designed through trial-and-error procedure (Cacuci, 2010; Cai et al., 2018; Ahmad et al., 2021), which performs as follows: ﬁrst, an initial shielding design is proposed according to the designer’s expertise; then the initial design is modeled and simulated with radiation transportation codes, such as Monte Carlo codes and SN (discrete ordinate) codes. Second, design objectives are analyzed. If the required objectives are not satisﬁed, the parameters of the shielding structure are modiﬁed according to the designer’s experience, and another solution is obtained, simulated, and analyzed. The conventional method is time- consuming, has low efﬁciency, and the optimal solution is always hard to obtain. In recent years, multi-objective optimization algorithms are introduced into the shielding design ﬁeld. Tunes et al. (2017) combine the Matlab (Matrix Laboratory) optimization toolbox with MCNP (A General Monte Carlo N-Particle Transport Code) to optimize the biological shielding structure of a compact pressurized water nuclear reactor. Chen et al. developed the optimization method combining a non-dominated sorting algorithm with the Monte Carlo code MCNP, which is applied to the shielding design of the Savannah reactor (Chen et al., 2019; Chen et al., 2020). Wu et al. (2021) combined the PSO (Particle Swarm Optimization) multi-objective optimization algorithm with the one-dimensional SN code ANISN (A One-Dimensional Discrete Ordinates Transport Code with Anisotropic Scattering) to optimize the nuclear reactor shielding structure. In addition, the optimization methods combining multi-objective optimization algorithms with radiation transportation codes are also used to the shielding design of the accelerator-driven neutron source (Ma et al., 2021). Frontiers in Energy Research 2.3 Approach 3: Non-dominated sorting with sub-objective constraints gi ⃗x ⎧ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎩ 1 1 + fi ⃗x , if fi ⃗x ≤fi,0 ⃗x 0 else (3) (3) Optimization results beyond the tolerable limits may appear with direct application of the multi-objective optimization algorithm. Hence, the multi-objective optimization method with constraints of sub-objectives is studied. First, the minimization optimization problem is converted into the maximization optimization problem, given as Thus, the minimization problem stated by Eq. 2 can be transformed into the maximization problem given by maximize g ⃗x gi ⃗x T . (4) (4) maximize g ⃗x gi ⃗x T , i 1, 2, /, m, (7) (7) As a result, the multi-objective minimization problem of the shielding design is converted into a single-objective maximization problem by treating the individuals not satisfying the constraints as “lethal genes”, of which the ﬁtness values are set to be 0. A single-objective genetic algorithm based on elitism is applied to solve the single-objective maximization problem stated by Eq. 4 (Zhou and Sun, 1999). where the sub-objectives are deﬁned as where the sub-objectives are deﬁned as gi ⃗x ⎧ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎩ 1 1 + fi ⃗x , if fi ⃗x ≤fi,0 0 else . (8) (8) Then, Eqs 7, 8 are implemented into the non-dominated sorting algorithm NSGA-II for the multi-objective optimization. 2.1 Approach 1: The single-objective method This approach converts the multi-objective optimization problem into a single-objective optimization problem by setting the other sub-objectives as constraints, given as minimize f ⃗x fi ⃗x T , i 1, 2, /, m subject to ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ fi ⃗x ≤fi,0, j 1, 2, /, m; j ≠i subject to ⎧ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎩ ⃗x (x1, x2, /, xn), x ∈Rn Lj ≤xj ≤Uj, j 1, 2, /, n hkxj ≤0, k 1, 2, /, q vlxj 0, l 1, 2, /, p . (2) (2) As is stated previously, the current multi-objective optimization methods of shielding design mainly combine multi-objective algorithms with radiation transportation codes. The non-dominated sorting algorithm is the most widely used algorithm for multi-objective optimization. Two main drawbacks exist among the current optimization methods. First, constraints In order to eliminate the individuals not satisfying the constraints, the subobjectives are transformed by, In order to eliminate the individuals not satisfying the constraints, the subobjectives are transformed by, frontiersin.org Frontiers in Energy Research 02 frontiersin.org Liu et al. 10.3389/fenrg.2022.935648 ∀i ∈{1, 2, /, m}: fi ⃗x1 ≤fi ⃗x2 (5) ∃j ∈{1, 2, /, m}: fj ⃗x1 < fj ⃗x2 (6) FIGURE 1 Pareto front of the two-objective-minimization problem. FIGURE 1 Pareto front of the two-objective-minimization problem. (5) (6) Additionally, a solution ⃗xp is called non-dominated solution if there does not exist another solution that dominates it. The set of the non-dominated solutions is called the Pareto front, e. g. for a minimization problem with two objectives, and the dominated and non-dominated solutions are shown by Figure 1. The NSGA-II (Non-dominated Sorting Genetic Algorithm) is applied to solve the multi-objective optimization problem in this study. NSGA-II is one of the most popular multi-objective optimization algorithms with three special characteristics, fast non-dominated sorting approach, fast crowded distance estimation procedure, and simple crowded comparison operator. Currently, the NSGA-II algorithm is widely used in the multi-objective optimization ﬁeld (Deb et al., 2002). 3 Results and discussion In most cases, sub-objectives of the multi-objective optimization might be conﬂicting with each other, so there is no single optimal solution to a given problem. Therefore, the solutions are non-dominated “Pareto optimal”, which are the trade-offs between the sub-objectives. For arbitrary given two decision vectors ⃗x1 and ⃗x2, it can be said that ⃗x1 dominates ⃗x2 (denoted as ⃗x1 ≺⃗x2) if the following conditions are satisﬁed: 1) the solution in decision vector ⃗x1 is no worse than that of decision vector ⃗x2 in all objectives; 2) the solution in decision vector ⃗x1 is strictly better than that of ⃗x2 in at least one objective. Th d b d b d h ll Frontiers in Energy Research 3.2 The optimization objectives For the Savannah reactor, the purpose of optimizing is to obtain a lightweight and compact shielding structure with the radiation dose in the tally volume lower than the tolerable limit. Hence, the three optimization objectives are the shielding weight, radiation dose in the tally volume, and volume of the shielding structure, denoted as fW, fD, and fV, respectively. In addition, the fast neutron ﬂux (> 1.0 MeV) at the pressure vessel should be lower than the tolerable limit value to ensure the operation life of the reactor, so the fast neutron ﬂux at the pressure vessel, denoted as f4, acts as a constraint during the optimization. shield is a simpliﬁed shield model which consists of an annulus of light water and stainless steel contained in the shield tank surrounding the reactor. The water annulus is supplemented by lead at the shield tank outer wall. The simpliﬁed shield model of the Savannah reactor is shown in Figure 2. The visualization toolkit Vised is used to visualize the geometrical structures of the reactor shielding (X-5 Monte Carlo Team, 2003). The shielding weight, volume, radiation dose in the tally volume, and fast neutron ﬂux at the pressure vessel of the initial design of the Savannah reactor perform as the tolerable limits of the sub-objectives during the optimization. The paralleling SN code Hydra is applied to perform the radiation transport simulation. Fixed-source calculations with one source iteration are performed in the S16-P3 approximation, where S16 indicates the ﬂux angular discretization is 16 and P3 indicates the order of the expansion in Legendre polynomials of the scattering cross- section is 3. The BUGLE-96 (Broad User Group Library ENDF/ B) cross-section library, speciﬁcally produced for LWR (Light- Water Reactor) shielding and dosimetry applications, is used in the Hydra calculations. BUGLE-96 is based on the ENDF/B-VI There are 18 shielding layers surrounding the reactor core. Thicknesses of the pressure vessel, air insulator, and containment vessel are considered to be ﬁxed; thus, the number of the optimization parameters is 13. The initial layers of geometrical structures with different materials and the initial design parameters and ranges of the optimization parameters are listed in Table 1. Ranges of the optimization parameters are TABLE 1 Structure of the Savannah reactor shield and the optimization parameters. TABLE 1 Structure of the Savannah reactor shield and the optimization parameters. 3.1 The savannah reactor shielding model The simpliﬁed Savannah reactor model is introduced to perform radiation shielding design optimization. The Savannah is a single-screw, combination passenger-cargo ship, which is powered by a nuclear reactor (Blizard et al., 1962). The Savannah reactor is of the pressurized water type, fueled with slightly enriched UO2. The critical core is 229.2 cm long overall, with an active fuel length of 167.6 cm and an equivalent core diameter of 157.6 cm. The initial design for the Savannah reactor The conditions can be described mathematically as Frontiers in Energy Research 03 frontiersin.org Liu et al. 10.3389/fenrg.2022.935648 considered based on the engineering needs of the Savannah reactor (Blizard et al., 1962). FIGURE 2 Schematic of the radial and axial cross-section of the simpliﬁed Savannah reactor shield model. FIGURE 2 Schematic of the radial and axial cross-section of the simpliﬁed Savannah reactor shield model. 3.2 The optimization objectives Material Initial values (cm) Optimization parameter Range (cm) — Reactor core 78.7 — — 1 Water (0.8 g/cm3) 9.5 x0 0–20 2 Iron 2.5 x1 0–20 3 Water (0.8 g/cm3) 5.7 x2 0–20 4 Iron 5.1 x3 0–20 5 Water (0.8 g/cm3) 11.9 x4 0–20 6 Iron 2.5 x5 0–20 7 Water (0.8 g/cm3) 7.6 x6 0–20 8 Iron (Pressure vessel) 15.2 — — 9 Air (Insulation) 10.2 — — 10 Fe 1.0 — — 11 Water (1.0 g/cm3) 83.8 x7 50–150 12 Iron 1.5 x8 0–20 13 Lead 7.6 x9 0–20 14 Air (space with equipment) 274.3 x10 274.32–518.16 15 Iron (containment vessel) 6.4 — — 16 Lead 15.2 x11 0–50 17 Polyethylene 15.2 x12 0–50 18 Air (tally volume) 1.0 — — Frontiers in Energy Research 04 frontiersin.org Liu et al. 10.3389/fenrg.2022.935648 TABLE 2 Tolerable limits of the sub-objectives. Sub-objective fW,0 (ton) fD,0 (Sv·h−1) fV,0 (m3) f4,0 (n·cm−2·s−1) Tolerable limit (by Hydra) 202.9 3.8 × 10−5 162.1 7.6 × 109 Tolerable limit (by MCNP) — 4.1 × 10−5 — 8.3 × 109 TABLE 2 Tolerable limits of the sub-objectives. (Evaluated Nuclear Data File) Release 3 evaluated data and is available in the FIDO (Floating Index Data Operation) ANISN format (White et al., 1996). The MCNP code is also carried out to calculate the tolerable limits of the sub-objectives, listed in Table 2. The relative deviation between the Hydra code and the MCNP code for fV,0 and f4,0 are 8 and 8.7%, respectively, indicating that the accuracy of Hydra code is sufﬁcient for optimization. In order to keep the sub-objectives to the same scale, the sub-objectives are normalized using the following method, given by FIGURE 3 Optimized objectives and Pareto fronts of the optimization cases. f1 fW fW, max −fW, min f2 fD fD, max −fD, min , f3 fV fV, max −fV, min (9) (9) where fW, max, fW, min, fD, max, fD, min, fV, max, and fV, min are the maximum and minimum values of the shielding weight, radiation dose, and the shielding volume, respectively, among the optimization range listed in Table 1. FIGURE 3 Population size used in the optimization is 70, and the number of generations is 100. The SBX (Simulated Binary Crossover) operator is adopted with the crossover rate of 0.9. The polynomial mutation operator is used with the mutation rate of 0.2. As is shown in Figure 3, the optimized objectives of case 1 and case 2 are close to the Pareto front of case 3. The Pareto front curve of case 4 overlaps with the Pareto front curve of case 3. For the optimization results of case 3, there are 35 individuals of which the sub-objectives are within the tolerable limits. However, for the optimization results of case 4, there are 70 individuals of which the sub-objectives are within the tolerable limits, indicating that more optimized individuals can be obtained by applying constraints on the sub-objectives for optimization. 3.4.1 Optimization with two objectives 3.4.1 Optimization with two objectives First, situations with two competing sub-objectives are studied, i. e., the shielding weight and radiation dose in the tally volume. Four cases are studied in this section. The optimization approach and objectives of each case are illustrated in Table 3. The optimized objectives (for case 1 and case 2) and the Pareto fronts (for case 3 and case 4) are illustrated in Figure 3. The geometrical structures of the initial design and eight typical individuals picked from the optimization results of the four cases are studied and shown in Figure 4. The eight typical optimized individuals are the optimized individuals of case 1, Frontiers in Energy Research 05 frontiersin.org frontiersin.org Liu et al. 10.3389/fenrg.2022.935648 FIGURE 4 Cross-sections of the geometrical structures of the initial design and typical individuals after optimization; (A) initial design; (B) case 1; (C) case 2; (D) case 3 with minimal weight; (E) case 3 with minimal dose; (F) case 3 with minimal weight within tolerable limits; (G) case 3 with minimal dose within tolerable limits; (H) case 4 with minimal weight; and (I) case 4 with minimal dose. FIGURE 4 Cross-sections of the geometrical structures of the initial design and typical individuals after optimization; (A) initial design; (B) case 1; (C) case 2; (D) case 3 with minimal weight; (E) case 3 with minimal dose; (F) case 3 with minimal weight within tolerable limits; (G) case 3 with minimal dose within tolerable limits; (H) case 4 with minimal weight; and (I) case 4 with minimal dose. TABLE 4 Sub-objectives of the initial design and typical optimized individuals. FIGURE 5 Pareto fronts of the optimization cases. Weight (ton) Dose (Sv/h) (a) 202.9 3.8 × 10−5 (b) 141.2 3.8 × 10−5 (c) 202.9 2.3 × 10−7 (d) 109.9 1.5 × 10−3 (e) 533.2 3.2 × 10−15 (f) 148.6 1.0 × 10−5 (g) 196.2 6.9 × 10−8 (h) 138.3 3.4 × 10−5 (i) 202.8 4.6 × 10−8 Comparing with the initial design, the optimized structures are oriented by the optimization objectives. As for the two competing sub-objectives, the optimized structure with minimal weight appears to have the maximal dose and vice versa, as shown in Figures 4D,E. The other individuals show the same pattern. The sub-objectives of the optimized individuals in Figure 4 are listed in Table 3. Frontiers in Energy Research 3.4.1 Optimization with two objectives It can be seen that the optimization case 2, and case 3 with minimal weight, case 3 with minimal dose, case 3 with minimal weight under tolerable limits, case 3 with minimal dose under tolerable limits, case 4 with minimal weight, and case 4 with minimal dose. Frontiers in Energy Research 06 frontiersin.org Liu et al. 10.3389/fenrg.2022.935648 FIGURE 6 Cross-sections of the geometrical structures of the initial design and typical individuals after optimization; (A) initial design; (B) case 1 with the minimal weight; (C) case 1 with the minimal dose; (D) case 1 with the minimal volume; (E) case 1 with the minimal weight within the tolerable limits; (F) case 1 with the minimal dose within the tolerable limits; (G) case 1 with the minimal volume within the tolerable limits; (H) case 2 with minimal weight; (I) case 2 with minimal dose; (J) case 2 with minimal volume; (K) case 3 with minimal weight; (L) case 3 with minimal dose; and (M) case 3 with minimal volume. FIGURE 6 Cross-sections of the geometrical structures of the initial design and typical individuals after optimization; (A) initial design; (B) case 1 with the minimal weight; (C) case 1 with the minimal dose; (D) case 1 with the minimal volume; (E) case 1 with the minimal weight within the tolerable limits; (F) case 1 with the minimal dose within the tolerable limits; (G) case 1 with the minimal volume within the tolerable limits; (H) case 2 with minimal weight; (I) case 2 with minimal dose; (J) case 2 with minimal volume; (K) case 3 with minimal weight; (L) case 3 with minimal dose; and (M) case 3 with minimal volume. result with approach 3 contains the best individuals with sub- objectives under the tolerable limits, shown (h) and (i) in Table 4. Comparing with the initial design, the optimized weight can be reduced by 31.8% (h) and the radiation dose can be reduced by ~ three orders of magnitude (i). however not equivalent. Three cases are studied in this section. The optimization approach and limit value of the weight of the cases are illustrated in Table 5. Pareto fronts of the three cases are shown in Figure 5. The number of the optimized individuals of which the sub-objectives are under the tolerable limits for the three cases is 9, 13, and 27. 3.4.1 Optimization with two objectives The “relaxation” is effective for the tight limitation that generates too many lethal genes which may cause difﬁculty for the algorithm searching for the optimal solutions. Frontiers in Energy Research Author contributions BL designed the research and wrote the paper; YT raised valuable questions that help improving the paper; FJ performed the HYDRA calculations; GH conducted the research and performed the genetic algorithm; MY performed the MCNP calculations. 3.4.2 Optimization with three objectives Optimizations with two competing sub-objectives are studied in Section 3.4.1, and the results agree well with the theoretical anticipation. In this part, optimizations with three sub-objectives are also studied. The sub-objective dose is competing with the other two sub-objectives, i e., weight and volume. The sub- objectives weight and volume are positively correlated, The geometric structures of the initial design and the typical optimized individuals are shown in Figure 6. The typical optimized individuals are as follows: case 1 with Frontiers in Energy Research 07 frontiersin.org frontiersin.org 10.3389/fenrg.2022.935648 Liu et al. minimal weight, case 1 with minimal dose, case 1 with minimal volume, case 1 with minimal weight within the tolerable limits, case 1 with minimal dose within the tolerable limits, case 1 with minimal volume within the tolerable limits, case 2 with minimal weight, case 2 with minimal dose, case 2 with minimal volume, case 3 with minimal weight, case 3 with minimal dose, and case 3 with minimal volume. Similar to the optimization results in Section 3.4.1, the optimized structures are oriented by the optimization objectives. As for the competing sub-objectives, i. e., weight and dose the optimized structure with minimal TABLE 6 Sub-objectives of the initial design and the typical optimized individuals. Weight (ton) Dose (Sv·h−1) Volume (m3) (a) 202.9 3.8 × 10−5 162.1 (b) 70.9 8.6 × 10−1 130.2 (c) 760.7 2.7 × 10−15 334.9 (d) 74.4 3.8 × 10−1 124.6 (e) 146.5 2.9 × 10−5 155.9 (f) 201.1 4.6 × 10−6 160 (g) 148.3 3.4 × 10−5 153.1 (h) 140.7 3.1 × 10−5 158.1 (i) 156.7 1.1 × 10−5 162 (j) 147.5 3.5 × 10−5 155.7 (k) 140 3.8 × 10−5 158.1 (l) 171.8 4.0 × 10−6 159.2 (m) 166.6 3.8 × 10−5 147.6 TABLE 5 Optimization cases with three objectives. Case Optimization method Limit value of weight (ton) Case 1 Approach 2 None Case 2 Approach 3 162.1 Case 3 Approach 3 300.0 weight (k), the optimized radiation dose (l), and the optimized volume (m) are reduced by 31.0, 89.4, and 9.0%, respectively. Comparing with the optimized results of Section 3.4.1, the optimized individuals with minimum weight and dose are a little higher. Increase of the sub-objectives may cause difﬁculty for the optimization algorithm searching for optimal solutions. TABLE 6 Sub-objectives of the initial design and the typical optimized individuals. 4 Conclusion individuals. Weight (ton) Dose (Sv·h−1) Volume (m3) (a) 202.9 3.8 × 10−5 162.1 (b) 70.9 8.6 × 10−1 130.2 (c) 760.7 2.7 × 10−15 334.9 (d) 74.4 3.8 × 10−1 124.6 (e) 146.5 2.9 × 10−5 155.9 (f) 201.1 4.6 × 10−6 160 (g) 148.3 3.4 × 10−5 153.1 (h) 140.7 3.1 × 10−5 158.1 (i) 156.7 1.1 × 10−5 162 (j) 147.5 3.5 × 10−5 155.7 (k) 140 3.8 × 10−5 158.1 (l) 171.8 4.0 × 10−6 159.2 (m) 166.6 3.8 × 10−5 147.6 An optimization method combining the non-dominated sorting genetic algorithm with the paralleling 3-dimensional SN code is developed and applied to radiation shielding design of the Savannah reactor. Better performances of the radiation shielding structure can be obtained through optimization. For example, as for the optimization with two objectives, the optimized shielding weight and radiation dose can be reduced by 31.8% and ~3 orders of magnitude, respectively. The optimized shielding weight, radiation dose, and shielding volume of the optimization with the three objectives are reduced by 31.0, 8.4, and 9.0%, respectively. The developed method is meaningful for lightweight and compact design of nuclear reactors. However, sub-objectives that the developed method can process are too few to shielding design of some types of reactors with special purposes, so optimization design method processing many sub-objectives (~10) is to be developed in the future. minimal weight, case 1 with minimal dose, case 1 with minimal volume, case 1 with minimal weight within the tolerable limits, case 1 with minimal dose within the tolerable limits, case 1 with minimal volume within the tolerable limits, case 2 with minimal weight, case 2 with minimal dose, case 2 with minimal volume, case 3 with minimal weight, case 3 with minimal dose, and case 3 with minimal volume. Data availability statement The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. Similar to the optimization results in Section 3.4.1, the optimized structures are oriented by the optimization objectives. As for the competing sub-objectives, i. e., weight and dose, the optimized structure with minimal weight appears to have the maximal dose and vice versa, as shown in Figures 6B,C. Also, as for the two positively correlated sub-objectives, i. e., weight and volume, the optimized structures are also positively correlated, as shown in Figures 6B,D.The other individuals show the same pattern. The sub-objectives of the initial design and the typical optimized individual are listed in Table 6. It can be seen that the optimization result with method 3 with “relaxation” contains best individuals with sub-objectives under the tolerable limits, shown as (k), (l), and (m) in Table 6. Comparing with the initial design, the optimized Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Frontiers in Energy Research 08 frontiersin.org 10.3389/fenrg.2022.935648 Liu et al. References sources by application of NSGA-II and MCNP. IEEE Trans. Nucl. Sci. 68 (2), 110–117. doi:10.1109/tns.2020.3040500 Ahmad, S., Chang, B., Li, B., Qi, Y., and Liu, C. (2021). Mass optimization of the radiation shadow shield for space nuclear power system. Prog. Nucl. Energy 131 (1), 103607. doi:10.1016/j.pnucene.2020.103607 Ahmad, S., Chang, B., Li, B., Qi, Y., and Liu, C. (2021). Mass optimization of the radiation shadow shield for space nuclear power system. Prog. Nucl. Energy 131 (1), 103607. doi:10.1016/j.pnucene.2020.103607 Tunes, M. A., de Oliveira, C. R. E., and Schon, C. G. (2017). Multi-objective optimization of a compact pressurized water nuclear reactor computational model for biological shielding design using innovative materials. Nucl. Eng. Des. 313 (1), 20–28. doi:10.1016/j.nucengdes.2016.11.009 Blizard, E. P., Blosser, T. V., and Freestone, R. M. (1962). The radiation leakage survey of the shield of the nuclear ship Savannah. ORNL-3336. United States: Oak Ridge National Laboratory. Blizard, E. P., Blosser, T. V., and Freestone, R. M. (1962). The radiation leakage survey of the shield of the nuclear ship Savannah. ORNL-3336. United States: Oak Ridge National Laboratory. Wang, Y. P., Zheng, Y. Q., Xu, L. F., and Cao, L. (2020). NECP-Hydra: A high- performance parallel SN code for core-analysis and shielding calculation. Nucl. Eng. Des. 366 (1), 1107111–1107118. doi:10.1016/j.nucengdes.2020.110711 Cacuci, D. G. (2010). Handbook of nuclear engineering. Boston, MA, USA: Springer. Cacuci, D. G. (2010). Handbook of nuclear engineering. Boston, MA, USA: Springer. Cai, Y., Hu, H. S., Lu, S. Y., and Jia, Q. (2018). Optimization of radiation shielding material aiming at compactness, lightweight, and low activation for a vehicle- mounted accelerator-driven D-T neutron source. Appl. Radiat. Isotopes 135, 147–154. doi:10.1016/j.apradiso.2018.01.021 White, J. E., Ingersoll, D. T., and Wright, R. Q., (1996). Production and testing of the revised VITAMIN-B6 ﬁne-group and the BUGLE-96 broad-group neutron/ photon cross section libraries derived from ENDF/B-BI.3 nuclear data, NUREG/ CR-6214, Revision 1, ORNL-6795/R1. Chen, Z. P., Zhang, Z. Y., Xie, J. S., Guo, Q., and Yu, T. (2019). Metaheuristic optimization method for compact reactor radiation shielding design based on genetic algorithm. Ann. Nucl. Energy 134 (1), 318–329. doi:10.1016/j.anucene.2019. 06.031 Wu, X., Yang, Y. W., Han, S., Zhao, Z., Fang, P., and Gao, Q. (2021). Multi- objective optimization method for nuclear reactor radiation shielding design based on PSO algorithm. Ann. Nucl. Energy 160 (1), 108404. doi:10.1016/j.anucene.2021. 108404 Chen, Z. P., Zhang, Z. Y., Xie, J. S., Guo, Q., Yu, T., Zhao, P., et al. (2020). Publisher’s note organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated References Multi- objective optimization strategies for radiation shielding design with genetic algorithm. Comput. Phys. Commun. 260 (1), 107267. doi:10.1016/j.cpc.2020.107267 X-5 Monte Carlo Team (2003). MCNP-A general N-particle transport code. New Mexico: Los Alamos National Laboratory. Yamaji, A., and Sako, K. (1994). Shielding design to obtain compact marine reactor. J. Nucl. Sci. Technol. 31 (6), 510–520. doi:10.1080/18811248.1994.9735185 Deb, K., Pratap, A., Agarwal, S., and Meyarivan, T. (2002). A fast and elitist multi- objective genetic algorithm: NSGA-II. IEEE Trans. Evol. Comput. 6 (2), 182–197. doi:10.1109/4235.996017 Zhou, M., and Sun, S. D. (1999). Genetic algorithms: Theory and applications. Beijing, China: National Industrial Press. El-Genk, M. S. (2009). Deployment history and design considerations for space reactor power systems. Acta Astronaut. 64 (1), 833–849. doi:10.1016/j.actaastro.2008.12.016 Zhuang, S. X., He, P., Sun, G. Y., Hao, L., and Wu, Y. (2019). Hybrid Monte Carlo- deterministic method for 3D neutron transport simulation based on energy region division. Ann. Nucl. Energy 130 (1), 271–276. doi:10.1016/j.anucene.2019.02.043 Ma, B. L., Song, L., Yan, M. F., Ikeda, Y., Otake, Y., and Wang, S. (2021). Multi- objective optimization shielding design for compact accelerator-driven neutron Frontiers in Energy Research Frontiers in Energy Research 09 frontiersin.org
https://openalex.org/W4307307146	https://zenodo.org/records/1041493/files/EvaluatingInvestmentRisks.pdf	English	null	Evaluating Investment Risks of Metallic Mines Using an Extended TOPSIS Method with Linguistic Neutrosophic Numbers	Zenodo (CERN European Organization for Nuclear Research)	2,017	cc-by	14,727	Article Received: 26 July 2017; Accepted: 3 August 2017; Published: 8 August 2017 Abstract: The investment in and development of mineral resources play an important role in the national economy. A good mining project investment can improve economic efﬁciency and increase social wealth. Faced with the complexity and uncertainty of a mine’s circumstances, there is great signiﬁcance in evaluating investment risk scientiﬁcally. In order to solve practical engineering problems, this paper presents an extended TOPSIS method combined with linguistic neutrosophic numbers (LNNs). Firstly, considering that there are several qualitative risk factors of mining investment projects, the paper describes evaluation information by means of LNNs. The advantage of LNNs is that major original information is reserved with linguistic truth, indeterminacy, and false membership degrees. After that, a number of distance measures are deﬁned. Furthermore, a common status is that the decision makers can’t determine the importance degrees of every risk factor directly for a few reasons. With respect to this situation, the paper offers a weight model based on maximizing deviation to obtain the criteria weight vector objectively. Subsequently, a decision-making approach through improving classical TOPSIS with LNNs comes into being. Next, a case study of the proposed method applied in metallic mining projects investment is given. Some comparison analysis is also submitted. At last, the discussions and conclusions are ﬁnished. Keywords: metallic mine project; investment risks evaluation; linguistic neutrosophic numbers; maximum deviation; extended TOPSIS Symmetry 2017, 9, 149; doi:10.3390/sym9080149 1. Introduction Furthermore, qualitative information is also not expressed. Smarandache [17] ﬁrstly put forward the concept of neutrosophic sets (NSs) to deal with consistent, hesitant, and inconsistent information simultaneously. After that, many extensions based on NSs have been presented [18–20]. Related decision-making methods include TOPSIS [21], VIKOR [22], TODIM [23], COPRAS [24,25], WASPAS [26], MULTIMOORA [27], ELECTRE [28,29], QUALIFLEX [30], and other approaches [31,32]. Among them, TOPSIS is widely used. The basic idea of this method is that the distance of the optimal alternative with the positive ideal solution is nearest, and the negative-position ideal solution is farthest [21]. It is easy to understand and operate for decision makers. In order to qualitatively evaluate risk, like social environment risk and management risk in a mining project, linguistic variables may be a good description [33,34]. Much literature has focused on risk assessment with linguistic information. A venture analysis method on the basis of Dempster–Shafer theory under linguistic environment was presented in the literature [35]. Liu et al. [36] established a risk linguistic decision matrix and discussed the situation when weight informationis unknown. An unbalanced linguistic weighted geometric average operator was proposed to deal with fuzzy risk evaluation problems in [37]. Peiris et al. [38] built three linguistic models to assess alien plants’ invasion risks. For the sake of keeping as much linguistic evaluation information as possible, multiple extensions about language were suggested. For example, the notion of 2-dimensional uncertain linguistic variables occurred some researchers [39–41]. The idea of single-valued neutrosophic linguistic numbers occurred to Ye [42]. Other extensive forms are intuitionistic linguistic sets [43], hesitant fuzzy linguistic term sets [44,45], probabilistic linguistic term sets [46,47], and so on [48,49]. It is worth noting that Chen et al. [50] proposed a group decision-making method in the light of linguistic intuitionistic fuzzy numbers (LIFNs). They connected linguistic values with intuitionistic fuzzy numbers [51]. Then, the linguistic intuitionistic Frank Heronian average operator [52] and some improved linguistic intuitionistic fuzzy operators [53] were proposed. However, there are only linguistic membership degrees and linguistic non-membership degrees reﬂected in LIFNs. To overcome this shortcoming, Fang and Ye [54] came up with the concept of linguistic neutrosophic numbers (LNNs). They are based on linguistic terms and simpliﬁed neutrosophic numbers [55]. The truth-membership, indeterminacy-membership, and false-membership in a linguistic neutrosophic number (LNN) are found using linguistic information. 1. Introduction The assessment of investment risk has always attracted the attention of many researchers in different ﬁelds [1]. For example, Wu et al. [2] proposed an improved Analytical Hierarchy Process (AHP) approach to select an optimal ﬁnancial investment strategy. An extended TOPSIS method was provided by Hatami-Marbini and Kangi [3], and applied in the Tehran stock exchange. Yazdani-Chamzini et al. [4] constructed a model on the basis of AHP, decision-making trial and evaluation, and TOPSIS to evaluate investment risk in the private sector of Iran. A VIKOR-DANP method was presented by Shen et al. [5] and a case study of Taiwan’s semiconductor industry was also given to demonstrate the effectiveness of the approach. Dincer and Hacioglu [6] discussed the relationships of ﬁnancial stress and conﬂict risk in emerging capital markets with a fuzzy AHP-TOPSIS and VIKOR method. In high-tech ﬁelds, such as nanotechnology, Hashemkhani, Zolfani, and Bahrami [7] provided a SWARA-COPRAS decision-making method. Unlike other general industries, investment in the mining industry usually has a long cycle and large uncertainty [8]. There are a lot of risk factors in the process of mining investment. Consequently, identifying and assessing the investment venture of a mine accurately and properly is vital for any project. Symmetry 2017, 9, 149; doi:10.3390/sym9080149 www.mdpi.com/journal/symmetry www.mdpi.com/journal/symmetry 2 of 18 Symmetry 2017, 9, 149 The widely-used risk evaluation methods of mining investment can be divided into two main categories [9]. Traditional methods include fault tree analysis, Monte Carlo Simulation, breakeven analysis, the decision tree method, and so on. Another kind contains Analytic Hierarchy process (AHP), fuzzy comprehensive evaluation, and so on. Many researchers have paid particular attention to the latter method, which is based on fuzzy mathematics. Chen et al. [10] sorted and summarized the risk elements of metallic mines, and then presented a method based on a fuzzy set and a neural network. Wang et al. [11] constructed the fuzzy comprehensive appraisal model through creating a risk estimation indicator system. San et al. [12] focused on Tongxin mine, and established an investment risk assessment model with a fuzzy analytic hierarchy process. These methods take the ambiguity of the assessment process into consideration. However, the fuzzy numbers, such as interval numbers [13], triangular fuzzy numbers [14,15], and trapezoidal fuzzy numbers [16], used in most approaches have some limitations. On the one hand, they only described limited consistent information, while the hesitant and inconsistent values are not indicated. 1. Introduction The difference of LNNs with neutrosophic linguistic numbers (NLNs) [56] is that there is only a linguistic value in NLNs, and the truth-membership, indeterminacy-membership, and false-membership are crisp numbers. For instance, (s1, s2, s3) is a LNN, while (s1, < 0.1, 0.2, 0.3 >) is a neutrosophic linguistic number (NLN). Of course, they are independent of each other as well. In addition, Fang and Ye [54] deﬁned the operations and comparison rules of LNNs, and then decision-making methods based on of several weighted mean operators were raised. 3 of 18 Symmetry 2017, 9, 149 From this we can see, considering the complicacy of mine environment and the ambiguity of the human mind, assessing the ventures of mining projects on the basis of LNNs may be feasible and advisable. As a result, this paper considers metallic mine investment risk under a linguistic neutrosophic situation with incomplete weight information. A new and reasonable way to evaluate risk degrees by means of LNNs is proposed. In summary, the fundamental innovations of this article are conveyed as follows: (1) Present a number of distance measures between two LNNs, such as the Hamming distance, the Euclidean distance, and the Hausdorff distance. Equally important, prove relevant properties of these formulas; (2) Use the thought of maximum deviation for our reference, build a model with respect to linguistic neutrosophic environment to obtain the values of mine risk evaluation criteria weight; (3) Come up with the extended TOPSIS model with LNNs. Importantly, utilize this method to cope with investment decision-making matter of metallic mine projects; 4) Compare with other methods, in order to demonstrate the signiﬁcance and superiority. We methodize the rest of this article as follows. In Section 2, basic background and knowledge related to risk factors, linguistic information, and LNNs are presented. The extended TOPSIS method with LNNs is depicted after deﬁning the distance measures of LNNs and constructing the weight model in Section 3. Section 4 studies a case of metallic mining investment, and the proposed approach is applied in it. In Section 5, we make comparison with several current literatures. And then, conclusions are made in the last section. 2. Background In this section, some preliminaries about mining investment risk factors, linguistic term sets, linguistic scale functions, and LNNs are presented. 2.1. Risk Factors of Mining Project Investment 2.2. Linguistic Term Sets and Linguistic Scale Function 2.2. Linguistic Term Sets and Linguistic Scale Function Xu [58] ﬁrst put forward the concept of linguistic term sets. For a certain linguistic term set, there are a group of linguistic values si (i = 0, 1, . . . , 2g). Consequently, the linguistic term set can be denoted as S = {si\|i = 0, 1, . . . , 2g}. While the linguistic values in the above-mentioned linguistic term set are discrete, they may not work on aggregated linguistic information. Accordingly, Xu [58] redeﬁned the linguistic term set with S = {si\|i ∈[0, 2u]} (u > g), where the elements are continuous. Moreover, we can compare arbitrary linguistic terms in accordance with their subscripts. Namely, when i > j, si > sj is established. The operational rules of any two linguistic values si, sj ∈S are indicated: (1) the addition operator si ⊕sj = si+j; (2) the scalar multiplication τsi = sτi, 0 ≤τ ≤1; (3) the negation operator ne(si) = s−i. Deﬁnition 1. [59] The linguistic scale function is regarded as a mapping from linguistic values si (i = 0, 1, · · · , 2g) to a corresponding crisp number cni ∈[0, 1]. Furthermore, it should meet the requirement of monotonically increasing, that is to say, 0 ≤cn0 < cn1 < · · · < cn2g ≤1. g As the continuous linguistic term sets are deﬁned, we use f (si) = cni = i 2u (i ∈[0, 2u]) as the linguistic scale function in this essay. The inverse function can be described as f −1(cni) = 2u · cni (i ∈[0, 2u]). 2.1. Risk Factors of Mining Project Investment The economic factors of mines and the risk inﬂuence factors of metallic mines are introduced in this subsection. According to the situation of mining investment in China and the research results of the World Bank’s investment preference, Pan [57] divided the economic venture of mining investment into ﬁve types. They are ﬁnancial risk, production risk, market risk, personnel risk, and environmental risk, respectively. More details can be seen in Table 1. Table 1. The economic risk factors of mines. Table 1. The economic risk factors of mines. Risk Factors Explanations Financial risk Caused by the unexpected changes in the mine’s balance of payments. It largely consists of ﬁnancial balance, exchange rate, interest rate, and other factors. Production risk Caused by accident, which makes it impossible to produce the production plan according to the predetermined cost. Mainly including production cost, technical conditions, selection scheme, and so on. Market risk Caused by the unexpected changes in the market, which makes the mine unable to sell its products according to the original plan. It chieﬂy contains demand forecasting, substitution products, peer competition, and other factors. Personnel risk Caused by accident or change of the important personnel in the mine, which causes a signiﬁcant impact on the production and operation of the mine. The main factors include accidental casualties, conﬁdential leaks, and personnel changes. Environmental risk Caused by the changes of the external environment of the mining industry, which primarily comprises the national policies, geological conditions, and pollution control. 4 of 18 Symmetry 2017, 9, 149 In 2011, Chen et al. [10] summarized and classiﬁed the inﬂuence factors of the metallic mining investment process based on strategic angle of investment implementation. The presented risk system (see Table 2.) includes two levels of indicators, which are ﬁve primary indicators and sixty secondary indicators. The secondary index corresponds to the attributes of the primary one. Table 2. Investment risk evaluation system of metallic mining. Assessment Indicators Primary indicators Secondary indicators Production risk Mining type, production equipment level, and mining technology Geological risk Geological grade, mine reserves, hydrogeology, and surrounding rock conditions Social environment Marco economy, national industrial policy, and international environment Market risk Marketing ability, product market price, and potential competition Management risk Rationality of enterprise organization, scientiﬁc decision, and management personnel Table 2. Investment risk evaluation system of metallic mining. 2.3. Linguistic Neutrosophic Numbers where γi is the related weight value of ηi, 0 ≤γi ≤1 and n ∑ i=1 γi = 1. 3. Extended TOPSIS Method with Incomplete Weight Information In this section, we present the idea of an extended TOPSIS method with LNNs, and discuss the situation in which weight information is completely unknown. 2.3. Linguistic Neutrosophic Numbers Deﬁnition 5. [54] If η1 = (sT1, sI1, sF1) and η2 = (sT2, sI2, sF2) are two LNNs, then the comparison rule is: (1) η1 > η2 if SC(η1) > SC(η2); (2) η1 > η2 if SC(η1) = SC(η2) and AC(η1) > AC(η2); (3) η1 = η2 if SC(η1) = SC(η2) and AC(η1) = AC(η2). (1) η1 > η2 if SC(η1) > SC(η2); ( ) ( ) 2) η1 > η2 if SC(η1) = SC(η2) and AC(η1) > AC(η2); 3) η1 = η2 if SC(η1) = SC(η2) and AC(η1) = AC(η2). Deﬁnition 6. [54] Assume there are a group of LNNs ηi = (sTi, sIi, sFi) (i = 1, 2, . . . , n), the linguistic neutrosophic weight arithmetic mean (LNWAM) operator is: Deﬁnition 6. [54] Assume there are a group of LNNs ηi = (sTi, sIi, sFi) (i = 1, 2, . . . , n), the linguistic neutrosophic weight arithmetic mean (LNWAM) operator is: LNWAM(η1, η2, . . . , ηn) = n ∑ i=1 γiηi = (s 2u−2u n ∏ i=1 (1−Ti 2u ) γi , s 2u n ∏ i=1 ( Ii 2u ) γi , s 2u n ∏ i=1 ( Fi 2u ) γi ), (3) (3) i 1 i 1 i 1 where γi is the corresponding weight value of ηi, 0 ≤γi ≤1 and n ∑ i=1 γi = 1. where γi is the corresponding weight value of ηi, 0 ≤γi ≤1 and n ∑ i=1 γi = 1. where γi is the corresponding weight value of ηi, 0 ≤γi ≤1 and n ∑ i=1 γi = 1. where γi is the corresponding weight value of ηi, 0 ≤γi ≤1 and n ∑ i=1 γi = 1. Deﬁnition 7. [54] Assume ηi = (sTi, sIi, sFi) (i = 1, 2, . . . , n) are a set of LNNs, the linguistic neutrosophic weight geometric mean (LNWGM) operator is: LNWGM(η1, η2, . . . , ηn) = n ∑ i=1 ηi γi = (s 2u n ∏ i=1 ( Ti 2u ) γi , s 2u−2u n ∏ i=1 (1−Ii 2u ) γi , s 2u−2u n ∏ i=1 (1−Fi 2u ) γi ), (4) (4) where γi is the related weight value of ηi, 0 ≤γi ≤1 and n ∑ i=1 γi = 1. 2.3. Linguistic Neutrosophic Numbers Deﬁnition 2. [54] Given the linguistic term set S = {si\|i ∈[0, 2u]}, if sT, sI, sF ∈S, then η = (sT, sI, sF) can be regarded as a LNN, where sT, sI, and sF are independent, and describe the linguistic truth-membership degree, the linguistic indeterminacy-membership degree, and the linguistic falsity-membership degree in turn. Deﬁnition 3. [54] Assume η1 = (sT1, sI1, sF1) and η2 = (sT2, sI2, sF2) are two LNNs, then the operations of them are represented as follows: (1) η1 ⊕η2 = (sT1, sI1, sF1) ⊕(sT2, sI2, sF2) = (sT1+T2−T1T2 2u , s I1I2 2u , s F1F2 2u ); (2) η1 ⊗η2 = (sT1, sI1, sF1) ⊕(sT2, sI2, sF2) = (s T1T2 2u , sI1+I2−I1I2 2u , sF1+F2−F1F2 2u ); (3) qη1 = q(sT1, sI1, sF1) = (s2u−2u(1−T1 2u ) q, s2u( I1 2u ) q, s2u( F1 2u ) q), q > 0; (4) η1q = (sT1, sI1, sF1)q = (s2u( T1 2u ) q, s2u−2u(1−I1 2u ) q, s2u−2u(1−F1 2u ) q), q > 0. (1) η1 ⊕η2 = (sT1, sI1, sF1) ⊕(sT2, sI2, sF2) = (sT1+T2−T1T2 2u , s I1I2 2u , s F1F2 2u ); (2) η1 ⊗η2 = (sT1, sI1, sF1) ⊕(sT2, sI2, sF2) = (s T1T2 2u , sI1+I2−I1I2 2u , sF1+F2−F1F2 2u ); (3) qη1 = q(sT1, sI1, sF1) = (s2u−2u(1−T1 2u ) q, s2u( I1 2u ) q, s2u( F1 2u ) q), q > 0; (4) η1q = (sT1, sI1, sF1)q = (s2u( T1 2u ) q, s2u−2u(1−I1 2u ) q, s2u−2u(1−F1 2u ) q), q > 0. 5 of 18 Symmetry 2017, 9, 149 Deﬁnition 4. [54] Suppose η = (sT, sI, sF) is an optional LLN, the following are the score function and the accuracy function, respectively: SC(η) = (4u + T −I −F)/(6u), (1) AC(η) = (T −F)/(2u). (2) SC(η) = (4u + T −I −F)/(6u), (1) (1) y f p y SC(η) = (4u + T −I −F)/(6u), (1) AC(η) = (T −F)/(2u). (2) SC(η) = (4u + T −I −F)/(6u), (1) AC(η) = (T −F)/(2u). (2) (2) Deﬁnition 5. [54] If η1 = (sT1, sI1, sF1) and η2 = (sT2, sI2, sF2) are two LNNs, then the comparison rule is: (1) η1 > η2 if SC(η1) > SC(η2); (2) η1 > η2 if SC(η1) = SC(η2) and AC(η1) > AC(η2); (3) η1 = η2 if SC(η1) = SC(η2) and AC(η1) = AC(η2). 3.2. Distance Measures of LNNs In this subsection, we intend to introduce several distance formulas of LNNs, so that the discussion behind these can be smoothly advanced. Deﬁnition 8. Let η1 = (sT1, sI1, sF1) and η2 = (sT2, sI2, sF2) be two haphazard LNNs. S = {si\|i ∈[0, 2u]} is the linguistic term set, and f (si) = i 2u is the linguistic scale function. Then, the distance between η1 and η2 are denoted as follows: d(η1, η2) = ( 1 3(\| f (sT1) −f (sT2)\|λ + \| f (s2u−I1) −f (s2u−I2)\|λ + \| f (s2u−F1) −f (s2u−F2)\|λ)) 1 λ , λ > 0. (5) (5) Remarkably: 3.1. Descriptions With respect to the multi-criteria decision-making problems under linguistic neutrosophic situations, k decision makers evaluate a set of options X = {x1, x2, . . . , xn} under some attributes A = {a1, a2, . . . , am}. ωj is the corresponding weight of aj, which is completely unknown, but satisﬁes ωj ∈[0, 1] and m ∑ i=1 ωj = 1. There are k decision makers {b1, b2, . . . , bk} with the related weight k {γ1, γ2, . . . , γk}, 0 ≤γl ≤1 (l = 1, 2, . . . , k) and k ∑ l=1 γl = 1. S = {si\|i ∈[0, 2u]} is the predeﬁ linguistic term set. In order to rank the objects or pick out the optimal one(s), each decision-maker (bl(l = 1, 2, . . . , k)) makes evaluations and then constructs the corresponding decision-making matrix, that is: N(l) = (η(l) ij )n×m =   η(l) 11 · · · η(l) 1m η(l) 21 · · · η(l) 2m ... · · · ... η(l) n1 · · · η(l) nm   =   (s(l) T11, s(l) T11, s(l) T11) · · · (s(l) T1m, s(l) T1m, s(l) T1m) (s(l) T21, s(l) T21, s(l) T21) · · · (s(l) T2m, s(l) T2m, s(l) T2m) ... · · · ... (s(l) Tn1, s(l) Tn1, s(l) Tn1) · · · (s(l) Tnm, s(l) Tnm, s(l) Tnm)   , (l = 1, 2, . . . , k). 6 of 18 Symmetry 2017, 9, 149 The basic elements of the matrix N(l) are by means of LNNs, where η(l) ij = (s(l) Tij, s(l) Tij, s(l) Tij)(s(l) Tij, s(l) Tij, s(l) Tij ∈S) means the assessment information of bl about xi related to criteria aj. Remarkably: (1) when λ = 1, the Hamming distance (1) when λ = 1, the Hamming distance dHm(η1, η2) = 1 3(\| f (sT1) −f (sT2)\| + \| f (s2u−I1) −f (s2u−I2)\| + \| f (s2u−F1) −f (s2u−F2)\|); (6) (6) (2) when λ = 2, the Euclidean distance (2) when λ = 2, the Euclidean distance (2) when λ = 2, the Euclidean distance dEd(η1, η2) = r 1 3(\| f (sT1) −f (sT2)\|2 + \| f (s2t−I1) −f (s2t−I2)\|2 + \| f (s2t−F1) −f (s2t−F2)\|2); (7) (3) the Hausdorff distance (3) the Hausdorff distance dHd(η1, η2) = max \| f (sT1) −f (sT2)\|, \| f (s2t−I1) −f (s2t−I2)\|, \| f (s2t−F1) −f (s2t−F2)\| . (8) Property 1. Given three arbitrary LNNs η1 = (sT1, sI1, sF1), η2 = (sT2, sI2, sF2) and η3 = (sT3, sI3, sF3). The linguistic term set is S = {si\|i ∈[0, 2u]}, and the universal set of LNNs is Ω. For any η1, η2, η3 ∈Ω, the following properties are met: (1) 0 ≤d(η1, η2) ≤1; (2) d(η1, η2) = d(η2, η1); (3) d(η1, η2) = 0 if η1 = η2; (4) d(η1, η3) ≤d(η1, η2) + d(η2, η3). (1) 0 ≤d(η1, η2) ≤1; (2) d(η1, η2) = d(η2, η1); (3) d(η1, η2) = 0 if η1 = η2; (2) d(η1, η2) = d(η2, η1); (3) d(η1, η2) = 0 if η1 = η2; (3) d(η1, η2) = 0 if η1 = η2; (4) d(η1, η3) ≤d(η1, η2) + d(η2, η3). (2) This proof is obvious. Proof. If u = 4, two LNNs η1 = (s1, s2, s4) and η2 = (s5, s3, s6), the Hamming distance is dHm(η1, η2) ≈0.292, the Euclidean distance is dEd(η1, η2) ≈0.331, and the Hausdorff distance is dHd(η1, η2) = 0.500. 3.3. Weight Model Based on Maximum Deviation 3.3. Weight Model Based on Maximum Deviation Because the weight information is completely unknown, we use the maximum deviation approach to determine the weight vector of criteria in this subsection. Th b i id f h i d i i h d i h [60] Because the weight information is completely unknown, we use the maximum deviation approach to determine the weight vector of criteria in this subsection. The basic idea of the maximum deviation method is that [60]: The basic idea of the maximum deviation method is that [60]: The basic idea of the maximum deviation method is that [60]: The basic idea of the maximum deviation method is that [60]: (1) If there is a tiny difference of evaluation values ij η among all objects under criteria ja ( 1,2,..., ) j m = , it indicates that the criteria ja has little effect on the sorting results. Accordingly, it is appropriate to allocate a small value of the related weight j ω . (1) If there is a tiny difference of evaluation values ηij among all objects under criteria aj(j = 1, 2, . . . , m), it indicates that the criteria aj has little effect on the sorting results. Accordingly, it is appropriate to allocate a small value of the related weight ωj. (2) Conversely, if there is a signiﬁcant variance of assessment information ηij among all alternatives under criteria aj(j = 1, 2, . . . , m), then the criteria aj may be very important to the ranking orders. In this case, giving a large weight value ωj is reasonable. (2) Conversely, if there is a signiﬁcant variance of assessment information ηij among all alternatives under criteria aj(j = 1, 2, . . . , m), then the criteria aj may be very important to the ranking orders. In this case, giving a large weight value ωj is reasonable. (3) Notably, if ηij are the same values among all options under criteria aj ((j = 1, 2, . . . , m)), it means that the criteria aj doesn’t affect the ranking results. Therefore, we can make the corresponding weight ωj = 0. (3) Notably, if ηij are the same values among all options under criteria aj ((j = 1, 2, . . . , m)), it means that the criteria aj doesn’t affect the ranking results. Therefore, we can make the corresponding weight ωj = 0. Proof. and 1 2 2 2 \| ( ) ( )\| u F u F f s f s − − − 1 2 2 3 2 2 2 2 \| ( ) ( ) ( ) ( ) \| u F u F u F u F f s f s f s f s − − − − = − + − 1 2 2 3 2 2 2 2 \| ( ) ( )\| \| ( ) ( )\| u F u F u F u F f s f s f s f s − − − − ≤ − + − , 1 2 2 3 2 2 2 2 \| ( ) ( )\| \| ( ) ( )\| u F u F u F u F f s f s f s f s − − − − ≤ − + − , hence, 1 3 1 2 2 3 ( , ) ( , ) ( , ) d d d η η η η η η ≤ + . Example 1. If 4 u = , two LNNs 1 1 2 4 ( , , ) s s s η = and 2 5 3 6 ( , , ) s s s η = , the Hamming distance is 1 2 ( , ) 0.292 Hm d η η ≈ , the Euclidean distance is 1 2 ( , ) 0.331 Ed d η η ≈ , and the Hausdorff distance is 1 2 ( , ) 0.500 Hd d η η = . Example 1. If u = 4, two LNNs η1 = (s1, s2, s4) and η2 = (s5, s3, s6), the Hamming distance is dHm(η1, η2) ≈0.292, the Euclidean distance is dEd(η1, η2) ≈0.331, and the Hausdorff distance is dHd(η1, η2) = 0.500. Example 1. If 4 u = , two LNNs 1 1 2 4 ( , , ) s s s η = and 2 5 3 6 ( , , ) s s s η = , the Hamming distance is 1 2 ( , ) 0.292 Hm d η η ≈ , the Euclidean distance is 1 2 ( , ) 0.331 Ed d η η ≈ , and the Hausdorff distance is 1 2 ( , ) 0.500 Hd d η η = . Example 1. Proof. Thus, d(η1, η2) = ( 1 3(\| f (sT1) −f (sT2)\|λ + \| f (s2u−I1) −f (s2u−I2)\|λ + \| f (s2u−F1) −f (s2u−F2)\|λ)) 1 λ = ( 1 3(\| T1−T2 2u \|λ + \| I2−I1 2u \|λ + \| F2−F1 2u \|λ)) 1 λ = ( 1 3(\| T1−F1+F1−T2 2u \|λ + \| I2−I1 2u \|λ + \| F2−T2+T2−F1 2u \|λ)) 1 λ = ( 1 3(\| T2−F2+F1−T2 2u \|λ + \| I2−I1 2u \|λ + \| F1−T1+T2−F1 2u \|λ)) 1 λ = ( 1 3(\| F1−F2 2u \|λ + \| I1−I2 2u \|λ + \| T2−T1 2u \|λ)) 1 λ = ( 1 3(\| f (sT2) −f (sT1)\|λ + \| f (s2u−I2) −f (s2u−I1)\|λ + \| f (s2u−F2) −f (s2u−F1)\|λ)) 1 λ = d(η2, η1) 1 1 2 2 1 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 T T I I F F u u u λ λ λ λ − − − = + + 1 1 1 1 2 2 1 2 2 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 T F F T I I F T T F u u u λ λ λ λ − + − − − + − = + + 1 2 2 1 2 2 1 1 1 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 T F F T I I F T T F u u u λ λ λ λ − + − − − + − = + + 1 1 2 1 2 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 F F I I T T u u u λ λ λ λ − − − = + + 2 1 2 1 2 1 1 2 2 2 2 1 ( (\| ( ) ( ) \| \| ( ) ( ) \| \| ( ) ( ) \| )) 3 T T u I u I u F u F f s f s f s f s f s f s λ λ λ λ − − − − = − + − + − 2 1 ( , ) d η η = (3) Since η1 = η2, then SC(η1) = SC(η2) and AC(η1) = AC(η2) ⇒(4u + T1 −I1 −F1)/(6u) = (4u + T2 −I2 −F2)/(6u) and (T1 −F1)/(2u) = (T2 −F2)/(2u) ⇒T1 −I1 −F1 = T2 −I2 −F2 and T1 −F1 = T2 −F2 ⇒I1 = I2 and T1 −F1 = T2 −F2. Proof. (1) Because f (si) = i 2u ∈[0, 1] ⇒\| f (sT1) −f (sT2)\| ∈[0, 1], \| f (s2u−I1) −f (s2u−I2)\| and \| f (s2u−F1) − f (s2u−F2)\|, as λ > 0, then 0 ≤d(η1, η2) ≤1. (2) This proof is obvious. 7 of 18 7 of 18 Symmetry 2017, 9, 149  (4u T − (3) Since η1 = η2, then SC(η1) = SC(η2) and AC(η1) = AC(η2) ⇒(4u + T1 −I1 −F1)/(6u) = (4u + T2 −I2 −F2)/(6u) and (T1 −F1)/(2u) = (T2 −F2)/(2u) ⇒T1 −I1 −F1 = T2 −I2 −F2 and T1 −F1 = T2 −F2 ⇒I1 = I2 and T1 −F1 = T2 −F2. Proof. Thus, d(η1, η2) = ( 1 3(\| f (sT1) −f (sT2)\|λ + \| f (s2u−I1) −f (s2u−I2)\|λ + \| f (s2u−F1) −f (s2u−F2)\|λ)) 1 λ = ( 1 3(\| T1−T2 2u \|λ + \| I2−I1 2u \|λ + \| F2−F1 2u \|λ)) 1 λ = ( 1 3(\| T1−F1+F1−T2 2u \|λ + \| I2−I1 2u \|λ + \| F2−T2+T2−F1 2u \|λ)) 1 λ = ( 1 3(\| T2−F2+F1−T2 2u \|λ + \| I2−I1 2u \|λ + \| F1−T1+T2−F1 2u \|λ)) 1 λ = ( 1 3(\| F1−F2 2u \|λ + \| I1−I2 2u \|λ + \| T2−T1 2u \|λ)) 1 λ = ( 1 3(\| f (sT2) −f (sT1)\|λ + \| f (s2u−I2) −f (s2u−I1)\|λ + \| f (s2u−F2) −f (s2u−F1)\|λ)) 1 λ = d(η2, η1) 1 1 2 2 1 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 T T I I F F u u u λ λ λ λ − − − = + + 1 1 1 1 2 2 1 2 2 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 T F F T I I F T T F u u u λ λ λ λ − + − − − + − = + + 1 2 2 1 2 2 1 1 1 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 T F F T I I F T T F u u u λ λ λ λ − + − − − + − = + + 1 1 2 1 2 2 1 1 ( (\| \| \| \| \| \| )) 3 2 2 2 F F I I T T u u u λ λ λ λ − − − = + + 2 1 2 1 2 1 1 2 2 2 2 1 ( (\| ( ) ( ) \| \| ( ) ( ) \| \| ( ) ( ) \| )) 3 T T u I u I u F u F f s f s f s f s f s f s λ λ λ λ − − − − = − + − + − 2 1 ( , ) d η η = (4)(4) As 1 2 \| ( ) ( ) \| T T f s f s − = 1 2 2 3 \| ( ) ( ) ( ) ( )\| T T T T f s f s f s f s − + − 1 2 2 3 \| ( ) ( ) \| \| ( ) ( ) \| T T T T f s f s f s f s ≤ − + − , 1 2 2 2 \| ( ) ( )\| u I u I f s f s − − − 1 2 2 3 2 2 2 2 \| ( ) ( ) ( ) ( ) \| u I u I u I u I f s f s f s f s − − − − = − + − 1 2 2 3 2 2 2 2 \| ( ) ( )\| \| ( ) ( )\| u I u I u I u I f s f s f s f s − − − − ≤ − + − , and 1 2 2 2 \| ( ) ( )\| u F u F f s f s − − − 1 2 2 3 2 2 2 2 \| ( ) ( ) ( ) ( ) \| u F u F u F u F f s f s f s f s − − − − = − + − 1 2 2 3 2 2 2 2 \| ( ) ( )\| \| ( ) ( )\| u F u F u F u F f s f s f s f s − − − − ≤ − + − , hence, 1 3 1 2 2 3 ( , ) ( , ) ( , ) d d d η η η η η η ≤ + . Proof. (4)(4) As 1 2 \| ( ) ( ) \| T T f s f s − = 1 2 2 3 \| ( ) ( ) ( ) ( )\| T T T T f s f s f s f s − + − 1 2 2 3 \| ( ) ( ) \| \| ( ) ( ) \| T T T T f s f s f s f s ≤ − + − , 1 2 2 2 \| ( ) ( )\| u I u I f s f s − − − 1 2 2 3 2 2 2 2 \| ( ) ( ) ( ) ( ) \| u I u I u I u I f s f s f s f s − − − − = − + − 1 2 2 3 2 2 2 2 \| ( ) ( )\| \| ( ) ( )\| u I u I u I u I f s f s f s f s − − − − ≤ − + − , and 1 2 2 2 \| ( ) ( )\| u F u F f s f s − − − 1 2 2 3 2 2 2 2 \| ( ) ( ) ( ) ( ) \| u F u F u F u F f s f s f s f s − − − − = − + − 1 2 2 3 2 2 2 2 \| ( ) ( )\| \| ( ) ( )\| u F u F u F u F f s f s f s f s − − − − ≤ − + − , hence, 1 3 1 2 2 3 ( , ) ( , ) ( , ) d d d η η η η η η ≤ + . 3.3. Weight Model Based on Maximum Deviation 3.3. Weight Model Based on Maximum Deviation For the sake of obtaining the difference values, we deﬁne the deviation degree of a certain object xi(i = 1, 2, . . . , n) to all objects for a certain criteria aj(j = 1, 2, . . . , m) as follows: Dij(ωj) = n ∑ e=1 d(ηij, ηej)ωj, (9) Dij(ωj) = n ∑ e=1 d(ηij, ηej)ωj, (9) where d(ηij, ηej) is the distance measure between ηij and ηej. where d(ηij, ηej) is the distance measure between ηij and ηej. Symmetry 2017, 9, 149 8 of 18 Subsequently, the deviation degrees of all options under the criteria aj(j = 1, 2, . . . , m) can be denoted as: Subsequently, the deviation degrees of all options under the criteria aj(j = 1, 2, . . . , m) can be denoted as: n n n Dj(ωj) = n ∑ i=1 Dij(ωj) = n ∑ i=1 n ∑ e=1 d(ηij, ηej)ωj. (10) (10) Thus, the total deviation of all alternatives with all criteria is proposed in the following: Thus, the total deviation of all alternatives with all criteria is proposed in the following: D(ω) = m ∑ j=1 Dj(ωj) = m ∑ j=1 n ∑ i=1 Dij(ωj) = m ∑ j=1 n ∑ i=1 n ∑ e=1 d(ηij, ηej)ωj. (11) (11) As a result, we can build the weight model based on maximum deviation as follows: max D(ω) = m ∑ j=1 n ∑ i=1 n ∑ e=1 d(ηij, ηej)ωj = m ∑ j=1 n ∑ i=1 n ∑ e=1 ( 1 3(\| f (sTij) −f (sTej)\|λ + \| f (s2u−Iij) −f (s2u−Iej)\|λ + \| f (s2u−Fij) −f (s2u−Fej)\|λ)) 1 λ ωj s.t    m ∑ j=1 ω2 j = 1 0 ≤ωj ≤1, j = 1, 2, . . . 3.3. Weight Model Based on Maximum Deviation 3.3. Weight Model Based on Maximum Deviation , m (12) (12) s.t In order to get the solution, we can construct the Lagrange function as that: L(ω, p) = m ∑ j=1 n ∑ i=1 n ∑ e=1 ( 1 3(\| f (sTij) −f (sTej)\|λ + \| f (s2u−Iij)− f (s2u−Iej)\|λ+ \| f (s2u−Fij) −f (s2u−Fej)\|λ)) 1 λ ωj + p 2 ( m ∑ j=1 ω2 j −1) (13) (13) Taking the partial deviation of this function, we have: Taking the partial deviation of this function, we have:        ∂L(ω,p) ∂ω = m ∑ j=1 n ∑ i=1 n ∑ e=1 ( 1 3(\| f (sTij) −f (sTej)\|λ + \| f (s2u−Iij) −f (s2u−Iej)\|λ+\| f (s2u−Fij) −f (s2u−Fej)\|λ)) 1 λ + pωj = 0 ∂L(ω,p) ∂p = m ∑ j=1 ω2 j −1 = 0 ⇒p = s m ∑ j=1 ( n ∑ i=1 n ∑ e=1 ( 1 3(\| f (sTij) −f (sTej)\|λ + \| f (s2u−Iij) −f (s2u−Iej)\|λ + \|(s2u−Fij) −f (s2u−Fej)\|λ)) 1 λ ) 2 and ωj = n ∑ i=1 n ∑ e=1 ( 1 3 (\| f (sTij)−f (sTej)\|λ+\| f (s2u−Iij)−f (s2u−Iej)\|λ+\|(s2u−Fij)−f (s2u−Fej)\|λ)) 1 λ v u u t m ∑ j=1 ( n ∑ i=1 n ∑ e=1 ( 1 3 (\| f (sTij)−f (sTej)\|λ+\| f (s2u−Iij)−f (s2u−Iej)\|λ+\|(s2u−Fij)−f (s2u−Fej)\|λ)) 1 λ ) 2 . (14) (ω,p) ∂ω = m ∑ j=1 n ∑ i=1 n ∑ e=1 ( 1 3(\| f (sTij) −f (sTej)\|λ + \| f (s2u−Iij) −f (s2u−Iej)\|λ+\| f (s2u−Fij) −f (s2u−Fej)\|λ)) 1 λ + pωj = 0 ( ) m ∂L(ω,p) ∂p = m ∑ j=1 ω2 j −1 = 0 (14) In the end, we can use the following formula to normalize the criteria weights: In the end, we can use the following formula to normalize the criteria weights: ω∗ j = ωj m ∑ j=1 ωj , j = 1, 2, . . . , m. (15) (15) The Extended TOPSIS Method with LNNs 3.4. The Extended TOPSIS Method with LNNs 3.4. The Extended TOPSIS Method with LNNs The bigger the value of Di, the better the alternative xi is. 3.4. The Extended TOPSIS Method with LNNs , m) based on Formula (9), and then normalize the weight information in line with Formula (10), denoted as ω• j (j = 1, 2, . . . , m). (j = 1, 2, . . . , m). (j ) Step 4: Establish the weight standardized decision-making matrix N∗ = (η∗ ij)n×m = (s∗ Tij, s∗ Iij, s∗ Fij) n×m through multiplying the normalized matrix with weight vector, where s∗ Tij = ωjs• Tij, s∗ Iij = ωjs• Iij and s∗ Fij = ωjs• Fij. j ij ij j ij ep 5: Distinguish the positive ideal solution η+ and the negative ideal solution η−, respectively, ij j ij ij j ij Step 5: Distinguish the positive ideal solution η+ and the negative ideal solution η−, respectively, then: η+ = (η+ 1 , η+ 1 , . . . , η+ m), η+ j = max i (η∗ ij), (j = 1, 2, . . . , m) (16) (16) And η−= (η− 1 , η− 1 , . . . , η− m), η− j = min i (η∗ ij), (j = 1, 2, . . . , m) (17) (17) Step 6: Based on Formula (5), calculate the distance measures of the positive ideal solution to all options, and the distance measures of the negative ideal solution to all options in proper sequence. The computation formulas are: d+ = (d+ 1 , d+ 1 , . . . , d+ n ), d+ i = m ∑ j=1 d(η+ j , η∗ ij), (i = 1, 2, . . . , n) (18) (18) And d−= (d− 1 , d− 1 , . . . , d− n ), d− i = m ∑ j=1 d(η− j , η∗ ij), (i = 1, 2, . . . , n). (19) (19) Step 7: For each option xi (i = 1, 2, . . . , n), compute the values of correlation coefﬁcient Di with the following equation: d Step 7: For each option xi (i = 1, 2, . . . , n), compute the values of correlation coefﬁcient Di with the following equation: Di = d− i d+ i + d− i . (20) (20) Step 8: Achieve the ranking orders according to the values of Di (i = 1, 2, . . . , n). 3.4. The Extended TOPSIS Method with LNNs In this subsection, an extended TOPSIS approach under a linguistic neutrosophic environment is proposed. p p The detailed steps are described as follows: (l) (l) (l) The detailed steps are described as follows: p Step 1: Obtain the normalized decision-making matrix N•(l) = (η•(l) ij )n×m = (s•(l) Tij , s•(l) Iij , s•(l) Fij ) n×m. If the criteria belong to cost type, let s•(l) Tij = s(l) 2u−Tij, s•(l) Iij = s(l) 2u−Iij and s•(l) Fij = s(l) 2u−Fij. If the criteria belong to beneﬁt type, then the matrix remains, that is to say s•(l) Tij = s(l) Tij, s•(l) Iij = s(l) Iij and s•(l) Fij = s(l) Fij . Step 1: Obtain the normalized decision-making matrix N•(l) = (η•(l) ij )n×m = (s•(l) Tij , s•(l) Iij , s•(l) Fij ) n×m. If the criteria belong to cost type, let s•(l) Tij = s(l) 2u−Tij, s•(l) Iij = s(l) 2u−Iij and s•(l) Fij = s(l) 2u−Fij. If the criteria belong to beneﬁt type, then the matrix remains, that is to say s•(l) Tij = s(l) Tij, s•(l) Iij = s(l) Iij and s•(l) Fij = s(l) Fij . j j j j j j Step 2: Get the comprehensive decision-making matrix N• = (η• ij)n×m = (s• Tij, s• Iij, s• Fij) n×m using the LNWAM operator or LNWGM operator on the basis of Formula (3) or Formula (4). j j j j j j Step 2: Get the comprehensive decision-making matrix N• = (η• ij)n×m = (s• Tij, s• Iij, s• Fij) n×m using the LNWAM operator or LNWGM operator on the basis of Formula (3) or Formula (4). Step 2: Get the comprehensive decision making matrix N = (ηij)n×m = (sTij, sIij, sFij) n×m using the LNWAM operator or LNWGM operator on the basis of Formula (3) or Formula (4). Symmetry 2017, 9, 149 9 of 18 Step 3: Use the weight model to calculate the weight values ωj (j = 1, 2, . . . , m) based on Formula (9), and then normalize the weight information in line with Formula (10), denoted as ω• j (j = 1, 2, . . . , m). Step 3: Use the weight model to calculate the weight values ωj (j = 1, 2, . . . 4. Case Study In this section, we study a case of evaluating investment risks of a gold mine using the proposed approach. Recently, a construction investment company in Hunan province, called JK MINING Co., Ltd., had a plan for investing in a domestic metal mine. After an initial investigation and screening, four famous metal mines, described as {x1, x2, x3, x4}, have been under consideration. The enterprise establishes a team of three experts to conduct ﬁeld explorations and surveys in depth, so that the optimal mine can be selected. The specialists need to evaluate the investment risk in line with their ﬁndings, professional knowledge, and experience. Assume the importance of each professional is equal, that is to say γ1 = γ2 = γ3 = 1 3. After heated discussions, ﬁve attributions are recognized as the evaluation criteria. They are geological risk (a1), production risk (a2), market risk (a3), management risk (a4), and social environment risk (a5), separately. Then, the experts deﬁned the linguistic term set, S = {si\|i ∈[0, 8]}, where s = {s0 = exceedingly low, s1 = pretty low, s2 = low, s3 = slightly low, s4 = medium, s5 = slightly high, s2 = high, s3 = pretty high, s4 = exceedingly high}. Afterwards, they can give scores (or score ranges) or linguistic information directly of options under each attribute. The corresponding relationships between grade and linguistic term can been seen in Table 3. 10 of 18 10 of 18 Symmetry 2017, 9, 149 Table 3. Reference of investment risk evaluation. Table 3. Reference of investment risk evaluation. Grade 0~19 20~29 30~39 40~49 50~59 60~69 70~79 80~89 90~100 Evaluation exceedingly low pretty low low slightly low medium slightly high high pretty high exceedingly high Linguistic term s0 s1 s2 s3 s4 s5 s6 s7 s8 In order to describe the ambiguity and uncertainty of risks, their evaluation information is represented by LNNs. Subsequently, these assessment matrices are formed as Tables 4–6: In order to describe the ambiguity and uncertainty of risks, their evaluation information is represented by LNNs. Subsequently, these assessment matrices are formed as Tables 4–6: Table 4. Decision-making matrix N(1). 4. Case Study N(1) a1 a2 a3 a4 a5 x1 (s1, s2, s1) (s2, s3, s2) (s4, s4, s3) (s1, s5, s1) (s3, s3, s2) x2 (s2, s6, s2) (s3, s8, s2) (s2, s4, s1) (s3, s1, s2) (s1, s2, s1) x3 (s2, s3, s1) (s3, s2, s3) (s1, s4, s1) (s3, s5, s1) (s5, s2, s4) x4 (s3, s1, s2) (s1, s7, s1) (s4, s6, s3) (s2, s5, s1) (s4, s6, s4) Table 5. Decision-making matrix N(2). N(2) a1 a2 a3 a4 a5 x1 (s1, s6, s1) (s4, s3, s4) (s2, s6, s2) (s3, s5, s2) (s5, s2, s4) x2 (s1, s4, s1) (s3, s2, s1) (s2, s3, s4) (s4, s0, s5) (s2, s6, s4) x3 (s3, s5, s2) (s2, s4, s3) (s1, s6, s5) (s3, s5, s3) (s2, s6, s1) x4 (s2, s7, s2) (s4, s6, s1) (s3, s7, s2) (s4, s4, s2) (s3, s8, s4) Table 6. Decision-making matrix N(3). N(3) a1 a2 a3 a4 a5 x1 (s2, s4, s1) (s3, s5, s2) (s5, s1, s4) (s2, s6, s1) (s3, s3, s2) x2 (s1, s2, s1) (s2, s4, s2) (s1, s5, s3) (s4, s2, s0) (s0, s5, s6) x3 (s2, s3, s3) (s1, s5, s2) (s2, s4, s5) (s0, s4, s6) (s3, s2, s4) x4 (s2, s3, s2) (s4, s2, s1) (s1, s4, s3) (s3, s4, s5) (s0, s4, s5) Table 4. Decision-making matrix N(1). Table 4. Decision-making matrix N(1). Table 4. Decision-making matrix N(1). Table 5. Decision-making matrix N(2). Next, the extended TOPSIS approach presented in Section 3.4 is employed to identify the optimal metal mine. A concrete calculation process is delivered as follows: Step 1: Obtain the normalized decision matrix. As all the criteria are risk element, regarded as a part of cost, then normalizing evaluation values with function s•(l) Tij = s(l) 2u−Tij, s•(l) Iij = s(l) 2u−Iij and s•(l) Fij = s(l) 2u−Fij. The followings (Tables 7–9) are the normalized decision-making matrix of each expert. = s(l) 2u−Fij. The followings (Tables 7–9) are the normalized decision-making matrix of each expert Table 7. Normalized decision-making matrix N•(1). Table 7. Normalized decision-making matrix N•(1). 4. Case Study N•(1) a1 a2 a3 a4 a5 x1 (s7, s6, s7) (s6, s5, s6) (s4, s4, s5) (s7, s3, s7) (s5, s5, s6) x2 (s6, s2, s6) (s5, s0, s6) (s6, s4, s7) (s6, s7, s6) (s7, s6, s7) x3 (s6, s5, s7) (s5, s6, s5) (s7, s4, s7) (s5, s3, s7) (s3, s6, s4) x4 (s5, s7, s6) (s7, s1, s7) (s4, s2, s5) (s6, s3, s7) (s4, s2, s4) 11 of 18 Symmetry 2017, 9, 149 Table 8. Normalized decision-making matrix N•(2). Table 8. Normalized decision-making matrix N•(2). N•(2) a1 a2 a3 a4 a5 x1 (s7, s2, s7) (s4, s5, s4) (s6, s2, s6) (s5, s3, s6) (s3, s6, s4) x2 (s7, s4, s7) (s5, s6, s7) (s6, s5, s4) (s4, s8, s3) (s6, s2, s4) x3 (s5, s3, s6) (s6, s4, s5) (s7, s2, s3) (s5, s3, s5) (s6, s2, s7) x4 (s6, s1, s6) (s4, s3, s7) (s5, s1, s6) (s4, s4, s6) (s5, s0, s4) Table 9. Normalized decision-making matrix N•(3). N•(3) a1 a2 a3 a4 a5 x1 (s6, s4, s7) (s5, s3, s6) (s3, s7, s4) (s6, s2, s7) (s5, s5, s6) x2 (s7, s6, s7) (s6, s4, s6) (s7, s3, s5) (s4, s6, s8) (s8, s3, s2) x3 (s6, s5, s5) (s7, s3, s6) (s6, s4, s3) (s8, s4, s2) (s5, s6, s4) x4 (s6, s5, s6) (s4, s6, s7) (s7, s4, s5) (s5, s4, s3) (s8, s4, s3) Step 2: Using the LNWAM operator in line with Formula (3) to get the comprehensive decision matrix as Table 10: Table 8. Normalized decision-making matrix N•(2). Table 9. Normalized decision-making matrix N•(3). Step 2: Using the LNWAM operator in line with Formula (3) to get the comprehensive decision matrix as Table 10: Step 2: Using the LNWAM operator in line with Formula (3) to get the comprehensive decision matrix as Table 10: Table 10. Comprehensive decision-making matrix N•. Table 10. Comprehensive decision-making matrix N•. N• a1 a2 a3 a4 a5 x1 (s6.74, s3.63, s7) (s5.12, s4.22, s5.24) (s4.58, s3.83, s4.93) (s6.18, s2.62, s6.65) (s4.44, s5.31, s5.24) x2 (s6.74, s3.63, s6.65) (s5.38, s0, s6.32) (s6.18, s3.91, s5.19) (s4.83, s6.95, s5.24) (s8, s3.3, s3.83) x3 (s5.71, s4.22, s5.94) (s6.18, s4.16, s5.31) (s6.74, s3.17, s3.98) (s8, s3.3, s4.12) (s4.89, s4.16, s4.82) x4 (s5.71, s3.27, s6) (s5.48, s2.62, s7) (s5.71, s2, s5.31) (s5.11, s3.63, s5.01) (s8, s0, s3.63) Table 10. Comprehensive decision-making matrix N•. 4. Case Study N• a1 a2 a3 a4 a5 x1 (s6.74, s3.63, s7) (s5.12, s4.22, s5.24) (s4.58, s3.83, s4.93) (s6.18, s2.62, s6.65) (s4.44, s5.31, s5.24) x2 (s6.74, s3.63, s6.65) (s5.38, s0, s6.32) (s6.18, s3.91, s5.19) (s4.83, s6.95, s5.24) (s8, s3.3, s3.83) x3 (s5.71, s4.22, s5.94) (s6.18, s4.16, s5.31) (s6.74, s3.17, s3.98) (s8, s3.3, s4.12) (s4.89, s4.16, s4.82) x4 (s5.71, s3.27, s6) (s5.48, s2.62, s7) (s5.71, s2, s5.31) (s5.11, s3.63, s5.01) (s8, s0, s3.63) Step 3: Calculate the values of the criteria weight ωj (suppose λ = 1) on the basis of Formula (9) as follows: ω1 ≈0.17, ω2 ≈0.42, ω3 ≈0.31, ω4 ≈0.55 and ω1 ≈0.63. Normalize them based on Formula (10): ω• 1 = ω1 ω1+ω2+ω3+ω4+ω5 ≈0.08, ω• 2 ≈0.20, ω• 3 ≈0.15, ω• 4 ≈0.27 and ω• 5 ≈0.30. Step 4: Establish the weight standardized decision-making matrix as Table 11. Step 3: Calculate the values of the criteria weight ωj (suppose λ = 1) on the basis of Formula (9) as follows: ω1 ≈0.17, ω2 ≈0.42, ω3 ≈0.31, ω4 ≈0.55 and ω1 ≈0.63. Normalize them based on Formula (10): ω• 1 = ω1 ω1+ω2+ω3+ω4+ω5 ≈0.08, ω• 2 ≈0.20, ω• 3 ≈0.15, ω• 4 ≈0.27 and ω• 5 ≈0.30. Step 4: Establish the weight standardized decision-making matrix as Table 11. Step 3: Calculate the values of the criteria weight ωj (suppose λ = 1) on the basis of Formula (9) as follows: ω1 ≈0.17, ω2 ≈0.42, ω3 ≈0.31, ω4 ≈0.55 and ω1 ≈0.63. Normalize them based on Formula (10): ω• 1 = ω1 ω1+ω2+ω3+ω4+ω5 ≈0.08, ω• 2 ≈0.20, ω• 3 ≈0.15, ω• 4 ≈0.27 and ω• 5 ≈0.30. Step 4: Establish the weight standardized decision-making matrix as Table 11 1 ω1+ω2+ω3+ω4+ω5 2 3 4 Step 4: Establish the weight standardized decision-making matrix as Table 11. Table 11. Weight standardized decision-making matrix Nw. Nw a1 a2 a3 a4 a5 x1 (s1.1, s7.51, s7.91) (s1.48, s7.04, s7.35) (s0.96, s7.16, s7.44) (s2.64, s5.92, s7.61) (s1.73, s7.07, s7.05) x2 (s1.1, s7.51, s7.88) (s1.6, s0, s7.63) (s1.59, s7.19, s7.5) (s1.77, s7.7, s7.14) (s8, s6.13, s6.41) x3 (s0.76, s7.6, s7.81) (s2.05, s7.02, s7.37) (s1.94, s6.96, s7.2) (s8, s6.3, s6.69) (s1.97, s6.57, s6.87) x4 (s0.76, s7.45, s7.82) (s1.65, s6.4, s7.79) (s1.37, s6.5, s7.53) (s1.92, s6.46, s7.05) (s8, s0, s6.31) Step 5: Identify the positive ideal solution and the negative ideal solution, respectively. See Table 12. Table 11. Weight standardized decision-making matrix Nw. 5. Comparison Analysis In this section, several related studies are compared through solving the same problem of gold mine venture assessment. In this section, several related studies are compared through solving the same problem of gold mine venture assessment. The comparison results can be seen in Table 13, and the particularized discussions and analysis are depicted in the following: The comparison results can be seen in Table 13, and the particularized discussions and analysis are depicted in the following: Table 13. Ranking orders using different approaches. Table 13. Ranking orders using different approaches. Approaches Ranking Orders Optimal Alternatives Worst Alternatives Approach with the LNWAM operator [54] x4 > x2 > x3 > x1 x4 x1 Approach with the LNWGM operator [54] x4 > x3 > x2 > x1 x4 x1 Approach with uij = 1 3sTij [50] x4 > x3 > x2 > x1 x4 x1 Approach with uij = 1 3sTij + 1 6sIij [50] x1 > x3 > x2 > x4 x1 x4 Approach with uij = 1 3sTij + 1 3sFij [50] x2 > x1 > x3 > x4 x2 x4 Approach with SVNLN-TOPSIS [42] x4 > x2 > x3 > x1 x4 x1 The presented approach x4 > x2 > x3 > x1 x4 x1 (1) The information in Reference [54] is LNNs. The multi-criteria group decision-making methods based on the LNWM operator or LNGM operator are presented. If we use the LNWAM operator to deal with the same problem in this paper, we have the comprehensive evaluations of each alternative as follows: c1 = (s5.4, s3.87, s5.67), c2 = (s8, s0, s5.04) c3 = (s8, s3.76, s4.65) c4 = (s8, s0, s4.98). Since the score function SC(c1) ≈0.494, SC(c2) = 0.790, SC(c3) ≈0.650, SC(c4) ≈0.793, then SC(c4) > SC(c2) > SC(c3) > SC(c1)⇒x4 > x2 > x3 > x1. If the LNWGM operator is used, then c# 1 = (s5.19, s4.15, s5.87), c# 2 = (s6.12, s4.6, s5.32), c# 3 = (s6.22, s3.81, s4.75), c# 4 = (s6.08, s2.24, s5.43). As SC(c# 1) ≈0.465, SC(c# 2) ≈0.508, SC(c# 3) ≈0.569, SC(c# 4) ≈0.600, we have SC(c# 4) > SC(c# 3) > SC(c# 2) > SC(c# 1)⇒x4 > x3 > x2 > x1. ( ) h f f [ ] l f b ( ) h (2) The information in Reference [50] is linguistic intuitionistic fuzzy numbers (LIFNs). 4. Case Study Step 8: Since D4 > D2 > D3 > D1, then the ranking order is x4 > x2 > x3 > x1, and the best metal mine is x4. Step 7: Compute the values of correlation coefﬁcient: D1 ≈0.11, D2 ≈0.52, D3 ≈0.34 and D4 ≈0.55. Step 8: Since D4 > D2 > D3 > D1, then the ranking order is x4 > x2 > x3 > x1, and the best metal mine is x4. 4. Case Study Nw a1 a2 a3 a4 a5 x1 (s1.1, s7.51, s7.91) (s1.48, s7.04, s7.35) (s0.96, s7.16, s7.44) (s2.64, s5.92, s7.61) (s1.73, s7.07, s7.05) x2 (s1.1, s7.51, s7.88) (s1.6, s0, s7.63) (s1.59, s7.19, s7.5) (s1.77, s7.7, s7.14) (s8, s6.13, s6.41) x3 (s0.76, s7.6, s7.81) (s2.05, s7.02, s7.37) (s1.94, s6.96, s7.2) (s8, s6.3, s6.69) (s1.97, s6.57, s6.87) x4 (s0.76, s7.45, s7.82) (s1.65, s6.4, s7.79) (s1.37, s6.5, s7.53) (s1.92, s6.46, s7.05) (s8, s0, s6.31) Step 5: Identify the positive ideal solution and the negative ideal solution, respectively. See Table 12. Step 5: Identify the positive ideal solution and the negative ideal solution, respectively. See Table 12. Step 5: Identify the positive ideal solution and the negative ideal solution, respectively. See Table 12. Table 12. Positive ideal solution and negative ideal solution. η+ 1 η+ 2 η+ 3 η+ 4 η+ 5 (s1.1, s7.51, s7.88) (s1.6, s0, s7.63) (s1.94, s6.96, s7.2) (s8, s6.3, s6.69) (s8, s0, s6.31) η− 1 η− 2 η− 3 η− 4 η− 5 (s0.76, s7.6, s7.81) (s1.48, s7.04, s7.35) (s0.96, s7.16, s7.44) (s1.77, s7.7, s7.14) (s1.73, s7.07, s7.05) Step 6: In line with Formula (5), the distances are measured as follows (assume λ = 1): d+ 1 ≈9.88, d+ 2 ≈5.06, d+ 3 = 7.13, d+ 4 = 5.01, d− 1 ≈1.22, d− 2 ≈5.50, d− 3 ≈3.68 and d− 4 ≈6.04. Table 12. Positive ideal solution and negative ideal solution. Step 6: In line with Formula (5), the distances are measured as follows (assume λ = 1): d+ 1 ≈9.88, d+ 2 ≈5.06, d+ 3 = 7.13, d+ 4 = 5.01, d− 1 ≈1.22, d− 2 ≈5.50, d− 3 ≈3.68 and d− 4 ≈6.04. Step 6: In line with Formula (5), the distances are measured as follows (assume λ = 1): d+ 1 ≈9.88, d+ 2 ≈5.06, d+ 3 = 7.13, d+ 4 = 5.01, d− 1 ≈1.22, d− 2 ≈5.50, d− 3 ≈3.68 and d− 4 ≈6.04. Step 6: In line with Formula (5), the distances are measured as follows (assume λ = 1): d+ 1 ≈9.88, d+ 2 ≈5.06, d+ 3 = 7.13, d+ 4 = 5.01, d− 1 ≈1.22, d− 2 ≈5.50, d− 3 ≈3.68 and d− 4 ≈6.04. 12 of 18 Symmetry 2017, 9, 149 Step 7: Compute the values of correlation coefﬁcient: D1 ≈0.11, D2 ≈0.52, D3 ≈0.34 and D4 ≈0.55. Owing to the limited space, we take the ﬁrst idea as an example in the following. The converted decision-making matrices of each expert are shown as Tables 14–16: 5. Comparison Analysis Nco(3) a1 a2 a3 a4 a5 x1 (s2, s11/3) (s5/3, s3) (s1, s11/3) (s2, s3) (s5/3, s11/3) x2 (s7/3, s13/3) (s2, s10/3) (s7/3, s8/3) (s4/3, s14/3) (s8/3, s5/3) x3 (s2, s10/3) (s7/3, s3) (s2, s7/3) (s8/3, s2) (s5/3, s10/3) x4 (s2, s11/3) (s4/3, s13/3) (s7/3, s3) (s5/3, s7/3) (s8/3, s7/3) Then, using the method in Reference [50], the collective evaluations of each option are x1 = (s83/48, s173/52), x2 = (s103/52, s68/21), x3 = (s133/73, s257/84) and x4 = (s173/96, s29/11) (let the position weight w = (0.2, 0.3, 0.5)T). Then the score functions are L(x1) ≈−1.60, L(x2) ≈−1.26, L(x3) ≈−1.24 and L(x4) ≈−0.83. Because L(x4) > L(x3) > L(x2) > L(x1), the ranking result is x4 > x3 > x2 > x1. Likewise, we use the approach in Reference [50] with the second and third thought to deal with the same problem, successively. Afterwards, we get the corresponding ranking orders are x1 > x3 > x2 > x4 and x2 > x1 > x3 > x4, respectively (suppose the position weight is constant and that w = (0.2, 0.3, 0.5)T). (3) The information in Reference [42] consists of single valued neutrosophic linguistic numbers Table 14. Converted decision-making matrix Nco(1). (s4/3, s3) (s2, s11/3) (s7/3, s11/3) (s4/3, s7/3) Table 15. Converted decision-making matrix Nco(2). Table 16. Converted decision-making matrix Nco(3). Table 16. Converted decision-making matrix Nco(3). (s2, s3) (s4/3, s14/3) (s8/3, s2) (s5/3, s7/3) Then, using the method in Reference [50], the collective evaluations of each option are x1 = (s83/48, s173/52), x2 = (s103/52, s68/21), x3 = (s133/73, s257/84) and x4 = (s173/96, s29/11) (let the position weight w = (0.2, 0.3, 0.5)T). Then the score functions are L(x1) ≈−1.60, L(x2) ≈−1.26, L(x3) ≈−1.24 and L(x4) ≈−0.83. Because L(x4) > L(x3) > L(x2) > L(x1), the ranking result is x4 > x3 > x2 > x1. Likewise, we use the approach in Reference [50] with the second and third thought to deal with the same problem, successively. Afterwards, we get the corresponding ranking orders are x1 > x3 > x2 > x4 and x2 > x1 > x3 > x4, respectively (suppose the position weight is constant and that w = (0.2, 0.3, 0.5)T). (3) The information in Reference [42] consists of single valued neutrosophic linguistic numbers (SVNLNs). The ﬁrst step is to change the LNNs into SVNLNs. 5. Comparison Analysis In the ﬁrst place, it is necessary to translate LNNs into LIFNs. However, there is no existing universal conversion method. In this case, we have three ideas. The ﬁrst idea is that all the linguistic indeterminacy-membership degrees in LNNs are allocated to linguistic non-membership degrees in LIFNs. In other words, uij = 1 3sTij and vij = 1 3sIij + 1 3sFij. For example, a LNN (s3, s6, s6) can be changed into a linguistic intuitionistic fuzzy number (LIFN) (s1, s4). The second opinion is that linguistic indeterminacy-membership degrees in LNNs are assigned to linguistic membership degrees and linguistic non-membership degrees in LIFNs on average. That is to say, uij = 1 3sTij + 1 6sIij and vij = 1 6sIij + 1 3sFij. For instance, the LIFN (s2, s3) may take the place of a LNN (s3, s6, s6). On the contrary, the last attitude is that all the linguistic indeterminacy-membership degrees in LNNs are allotted to linguistic membership degrees in LIFNs. So to speak, uij = 1 3sTij + 1 3sFij and vij = 1 3sFij. As an example, a LNN (s3, s6, s6) may be replaced by a LIFN (s3, s2). Owing to the limited space, we take the ﬁrst idea as an example in the following. The converted decision-making matrices of each expert are shown as Tables 14–16: Owing to the limited space, we take the ﬁrst idea as an example i decision-making matrices of each expert are shown as Tables 14–16: Owing to the limited space, we take the ﬁrst idea as an example in the following. The converted decision-making matrices of each expert are shown as Tables 14–16: 13 of 18 Symmetry 2017, 9, 149 Table 14. Converted decision-making matrix Nco(1). Nco(1) a1 a2 a3 a4 a5 x1 (s7/3, s13/3) (s2, s11/3) (s4/3, s3) (s7/3, s10/3) (s5/3, s11/3) x2 (s2, s8/3) (s5/3, s2) (s2, s11/3) (s2, s13/3) (s7/3, s13/3) x3 (s2, s4) (s5/3, s11/3) (s7/3, s11/3) (s5/3, s10/3) (s1, s10/3) x4 (s5/3, s13/3) (s7/3, s8/3) (s4/3, s7/3) (s2, s10/3) (s4/3, s2) Table 15. Converted decision-making matrix Nco(2). Nco(2) a1 a2 a3 a4 a5 x1 (s7/3, s3) (s4/3, s3) (s2, s8/3) (s5/3, s3) (s1, s10/3) x2 (s7/3, s11/3) (s5/3, s13/3) (s2, s3) (s4/3, s11/3) (s2, s2) x3 (s5/3, s3) (s2, s3) (s7/3, s5/3) (s5/3, s8/3) (s2, s3) x4 (s2, s7/3) (s4/3, s10/3) (s5/3, s7/3) (s4/3, s10/3) (s5/3, s4/3) Table 16. Converted decision-making matrix Nco(3). 5. Comparison Analysis In order to attain the ideal ranking order, we can assign grades for alternatives in these seven rankings successively. The better the option is, the higher the score is. That is to say, the optimal alternative in a ranking may be distributed with 4, the second is 3, the third is 2, and the worst is 1. As an illustration, according to the ranking x4 > x2 > x3 > x1 in literature [54] with the LNWAM operator, we have G1(x4) = 4, G1(x2) = 3, G1(x3) = 2 and G1(x1) = 1. Similarly, grades in other ranking methods can be determined. In the end, the overall grades of all alternatives can be earned through summation as follows: G(x1) = 12, G(x2) = 19, G(x3) = 17 and G(x4) = 22. Because G(x4) > G(x2) > G(x3) > G(x1), the ideal ranking result may be regarded as x4 > x2 > x3 > x1. It is obvious that the result is the same with the proposed method in this paper. The feasibility and availability of the presented approach are indicated. Besides, the best and worst objects are identical in the literature [42,54] and our approach. The reasons for the differences between literature [54] with our method may be the decision-making thought. Our measure is based on distance, while the literature [54] is based on aggregation operators. Some initial information may be missing in the process of aggregating. Moreover, diverse conclusions may occur with different aggregation operators, which has been demonstrated in the second and third line in Table 13. Both the method in Reference [42] and ours are in line with TOPSIS, and the same orders are received. However, there may be some limitations in [42]. Because the attribute weight vector is given directly, the positive and negative ideal solutions are absolute. In addition, the rankings in literature [50] are all different from the presented method. The reason for the distinction may be that the indeterminacy-membership information in LNNs is unavoidably distorted in LIFNs to some extent. From the analysis above, the advantages of the proposed method can be summarized as follo (1) Evaluating the risk degree of mining projects under qualitative criteria by means of LNNs is a good choice. As all the consistent, hesitant, and inconsistent linguistic information are taken into account. 5. Comparison Analysis Ntr(2) a1 a2 a3 a4 a5 x1 (s7, < 1, 2/7, 1 >) (s5, < 4/5, 1, 4/5 >) (s6, < 1, 1/3, 1 >) (s6, < 5/6, 1/2, 1 >) (s6, < 1/2, 1, 2/3 >) x2 (s7, < 1, 4/7, 1 >) (s7, < 5/7, 6/7, 1 >) (s6, < 1, 5/6, 2/3 >) (s8, < 1/2, 1, 3/8 >) (s6, < 1, 1/3, 2/3 >) x3 (s6, < 5/6, 1/2, 1 >) (s6, < 1, 2/3, 5/6 >) (s7, < 1, 2/7, 3/7 >) (s5, < 1, 3/5, 1 >) (s7, < 6/7, 2/7, 1 >) x4 (s6, < 1, 1/6, 1 >) (s7, < 4/7, 3/7, 1 >) (s6, < 5/6, 1/6, 1 >) (s6, < 2/3, 2/3, 1 >) (s5, < 1, 0, 4/5 >) Table 19. Transformed decision-making matrix Ntr(3). Ntr(3) a1 a2 a3 a4 a5 x1 (s7, < 6/7, 4/7, 1 >) (s6, < 5/6, 1/2, 1 >) (s7, < 3/7, 1, 4/7 >) (s7, < 6/7, 2/7, 1 >) (s6, < 5/6, 5/6, 1 >) x2 (s7, < 1, 6/7, 1 >) (s6, < 1, 2/3, 1 >) (s7, < 1, 3/7, 5/7 >) (s8, < 1/2, 3/4, 1 >) (s8, < 1, 3/8, 1/4 >) x3 (s6, < 1, 5/6, 5/6 > (s7, < 1, 3/7, 6/7 >) (s6, < 1, 2/3, 1/2 >) (s8, < 1, 1/2, 1/4 >) (s6, < 5/6, 1, 2/3 >) x4 (s6, < 1, 5/6, 1 >) (s7, < 4/7, 6/7, 1 >) (s7, < 1, 4/7, 5/7 >) (s5, < 1, 4/5, 3/5 >) (s8, < 1, 1/2, 3/8 >) After that, the extended SVNLN-TOPSIS approach in literature [42] is employed to assess the metal mine’s investment venture. The relative closeness coefﬁcients of each mine are calculated as follows: rc1 = 21/25, rc2 = 54/67, rc3 = 5/6 and rc4 = 29/36. Because rc4 < rc2 < rc3 < rc1, we have x4 > x2 > x3 > x1. After that, the extended SVNLN-TOPSIS approach in literature [42] is employed to assess the metal mine’s investment venture. The relative closeness coefﬁcients of each mine are calculated as follows: rc1 = 21/25, rc2 = 54/67, rc3 = 5/6 and rc4 = 29/36. Because rc4 < rc2 < rc3 < rc1, we have x4 > x2 > x3 > x1. From Table 13, we can see that there are diverse ranking results with distinct methods. 5. Comparison Analysis For a certain LNN η = (sT, sI, sF), if g = max(T, I, F), we can make the linguistic value in a single valued neutrosophic linguistic number (SVNLN) equal to sg, then the truth-membership, indeterminacy-membership, and false-membership degrees in a SVNLN are described as T/g, I/g and F/g in proper order. So to say, a LNN η = (sT, sI, sF) may be converted into a SVNLN (sg, < T/g, I/g, F/g >). For example, a LNN (s3, s3, s6) and a SVNLN (s6, < 0.5, 0.5, 1 >) are equivalent in manner. The transformed decision-making matrices of each specialist are listed as Tables 17–19: Table 17. Transformed decision-making matrix Ntr(1). Ntr(1) a1 a2 a3 a4 a5 x1 (s7, < 1, 6/7, 1 >) (s6, < 1, 5/6, 1 >) (s5, < 4/5, 4/5, 1 >) (s7, < 1, 3/7, 1 >) (s6, < 5/6, 5/6, 1 >) x2 (s6, < 1, 1/3, 1 >) (s6, < 5/6, 0, 1 >) (s7, < 6/7, 4/7, 1 >) (s7, < 6/7, 1, 6/7 >) (s7, < 1, 6/7, 1 >) x3 (s7, < 6/7, 5/7, 1 >) (s6, < 5/6, 1, 5/6 >) (s7, < 1, 4/7, 1 >) (s7, < 5/7, 3/7, 1 >) (s6, < 1/2, 1, 2/3 >) x4 (s7, < 5/7, 1, 6/7 >) (s7, < 1, 1/7, 1 >) (s5, < 4/5, 2/5, 1 >) (s7, < 6/7, 3/7, 1 >) (s4, < 1, 1/2, 1 >) Table 17. Transformed decision-making matrix Ntr(1). 14 of 18 Symmetry 2017, 9, 149 Table 18. Transformed decision-making matrix Ntr(2). Ntr(2) a1 a2 a3 a4 a5 x1 (s7, < 1, 2/7, 1 >) (s5, < 4/5, 1, 4/5 >) (s6, < 1, 1/3, 1 >) (s6, < 5/6, 1/2, 1 >) (s6, < 1/2, 1, 2/3 >) x2 (s7, < 1, 4/7, 1 >) (s7, < 5/7, 6/7, 1 >) (s6, < 1, 5/6, 2/3 >) (s8, < 1/2, 1, 3/8 >) (s6, < 1, 1/3, 2/3 >) x3 (s6, < 5/6, 1/2, 1 >) (s6, < 1, 2/3, 5/6 >) (s7, < 1, 2/7, 3/7 >) (s5, < 1, 3/5, 1 >) (s7, < 6/7, 2/7, 1 >) x4 (s6, < 1, 1/6, 1 >) (s7, < 4/7, 3/7, 1 >) (s6, < 5/6, 1/6, 1 >) (s6, < 2/3, 2/3, 1 >) (s5, < 1, 0, 4/5 >) Table 19. Transformed decision-making matrix Ntr(3). 5. Comparison Analysis Ntr(3) a1 a2 a3 a4 a5 x1 (s7, < 6/7, 4/7, 1 >) (s6, < 5/6, 1/2, 1 >) (s7, < 3/7, 1, 4/7 >) (s7, < 6/7, 2/7, 1 >) (s6, < 5/6, 5/6, 1 >) x2 (s7, < 1, 6/7, 1 >) (s6, < 1, 2/3, 1 >) (s7, < 1, 3/7, 5/7 >) (s8, < 1/2, 3/4, 1 >) (s8, < 1, 3/8, 1/4 >) x3 (s6, < 1, 5/6, 5/6 > (s7, < 1, 3/7, 6/7 >) (s6, < 1, 2/3, 1/2 >) (s8, < 1, 1/2, 1/4 >) (s6, < 5/6, 1, 2/3 >) x4 (s6, < 1, 5/6, 1 >) (s7, < 4/7, 6/7, 1 >) (s7, < 1, 4/7, 5/7 >) (s5, < 1, 4/5, 3/5 >) (s8, < 1, 1/2, 3/8 >) After that, the extended SVNLN-TOPSIS approach in literature [42] is employed to assess the metal mine’s investment venture. The relative closeness coefﬁcients of each mine are calculated as follows: rc1 = 21/25, rc2 = 54/67, rc3 = 5/6 and rc4 = 29/36. Because rc4 < rc2 < rc3 < rc1, we have x4 > x2 > x3 > x1. From Table 13, we can see that there are diverse ranking results with distinct methods. In order to attain the ideal ranking order, we can assign grades for alternatives in these seven rankings successively. The better the option is, the higher the score is. That is to say, the optimal alternative in a ranking may be distributed with 4, the second is 3, the third is 2, and the worst is 1. As an illustration, according to the ranking x4 > x2 > x3 > x1 in literature [54] with the LNWAM operator, we have G1(x4) = 4, G1(x2) = 3, G1(x3) = 2 and G1(x1) = 1. Similarly, grades in other ranking methods can be determined. In the end, the overall grades of all alternatives can be earned through summation as follows: G(x1) = 12, G(x2) = 19, G(x3) = 17 and G(x4) = 22. Because G(x4) > G(x2) > G(x3) > G(x1), the ideal ranking result may be regarded as x4 > x2 > x3 > x1. It is obvious that the result is Table 18. Transformed decision-making matrix Ntr(2). Table 18. Transformed decision making matrix N . 6. Discussion and Conclusions To evaluate risk is the beginning of a metallic mining project investment. Proper risk assessments have great signiﬁcance on the success of investments. Owing to the uncertainty and complexity in mine surroundings, this paper advised an extended TOPSIS method with LNNs to rise to this challenge. LNNs were suggested to manifest the indeterminate and inconsistent linguistic values, so that the evaluation information can be retained as much as possible. Then, generalized distance formulas were presented to calculate the difference degrees of two LNNs. As it is not easy for the mining investment decision makers to directly determine criteria weight values, a weight model based on maximum deviation was recommended. Afterwards, the method of ranking mines was shown by a case study. Furthermore, the effectiveness and highlights of the presented approach can be reﬂected in the comparison analysis. Even though the extended TOPSIS with LNNs method is a good solution, there are still some limitations. For example, the determination of the criteria weight values does not take the subjective elements into consideration. Hence, a more reasonable weight determination method should be further proposed. Besides, the sub-attribute risk factors may be considered in the future. The presented method with LNNs for evaluating the investment risks may be extended to interval linguistic neutrosophic numbers. Acknowledgments: This work was supported by National Natural Science Foundation of China (51374244) Author Contributions: Weizhang Liang, Guoyan Zhao and Hao Wu conceived and worked together to achieve this work, Weizhang Liang wrote the paper, Guoyan Zhao made contribution to the case study. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Comparison Analysis (1) Evaluating the risk degree of mining projects under qualitative criteria by means of LNNs is a good choice. As all the consistent, hesitant, and inconsistent linguistic information are taken into account. (2) The ﬂexibility has increased because various distance measures, aggregation operators, and linguistic scale functions can be chosen according to the savants’ experience or reality. (3) A common situation, in which the criteria weight information is unknown, is under consideration. There are many complex risk factors in the process of metallic mining investment. Thus, it is difﬁcult or unrealistic for decision makers to give the weight vector directly. The weight (3) A common situation, in which the criteria weight information is unknown, is under consideration. There are many complex risk factors in the process of metallic mining investment. Thus, it is difﬁcult or unrealistic for decision makers to give the weight vector directly. The weight 15 of 18 Symmetry 2017, 9, 149 model based on the thought of maximum deviation may be a simple and suitable way to resolve this problem. l based on the thought of maximum deviation may be a simple and suitable way to resolve roblem. (4) Instead of using absolute ideal points, the extended TOPSIS method deﬁned the relative ideal solutions. The strength of it is that different ideal solutions are calculated corresponding with the different original information of different mining projects. This may be more in line with reality. (4) Instead of using absolute ideal points, the extended TOPSIS method deﬁned the relative ideal solutions. The strength of it is that different ideal solutions are calculated corresponding with the different original information of different mining projects. This may be more in line with reality. References Evaluation of investment risks of mine based on fuzzy neural network. J. Guangxi Univ. 2011, 36, 1030–1035. 11. Wang, Z.G.; Zheng, J.; Li, H.Y. The risk evaluation model of mining project investment based on fuzzy comprehensive method. Appl. Mech. Mater. 2013, 298, 2928–2934. [CrossRef] 12. Shan, R.L.; Zhang, C.J.; Zhao, X.; Wu, S.J.; Huang, F.; Liu, Y. Study on evaluation model for investment risk of coal mine infrastructure based on fuzzy analytic hierarchy process. Coal Eng. 2015, 47, 139–142. 13. Hu, B.Q.; Wang, S. A novel approach in uncertain programing. I: New arithmetic and order relation for interval numbers. J. Ind. Manag. Optim. 2017, 2, 351–371. 14. Buckley, J.J. Possibilistic linear programming with triangular fuzzy numbers. Fuzzy Sets Syst. 1988, 26, 135–138. [CrossRef] 15. Li, F.; Xiao, M.; Zhang, J.; Yang, J.; Zhu, L. Health risk assessment on tunnel workers’ exposure to PM10 based on triangular fuzzy numbers. Adv. Mater. Mach. Electron. 2017, 1820, 040011. 16. Li, J.; Zeng, W. Fuzzy risk analysis based on the similarity measure of generalized trapezoidal fuzzy numbers. J. Intell. Fuzzy Syst. 2017, 32, 1673–1683. [CrossRef] 17. Smarandache, F. Neutrosophy. In Neutrosophic Probability, Set, and Logic; Proquest Information & Le Ann Arbor, MI, USA, 1998; p. 105. 18. Zhang, C.; Li, D.Y.; Sangaiah, A.K.; Broumi, S. Merger and acquisition target selection based on neutrosophic multigranulation rough sets over two universes. Symmetry 2017, 9, 126. [CrossRef] 19. Chen, J.Q.; Ye, J.; Du, S.G.; Yong, R. Expressions of rock joint roughness coefﬁcient using neutrosophic interval statistical numbers. Symmetry 2017, 9, 123. [CrossRef] 20. Lu, Z.K.; Ye, J. Cosine measures of neutrosophic cubic sets for multiple attribute decision-making. Symmetry 2017, 9, 121. [CrossRef] 21. Pouresmaeil, H.; Shivanian, E.; Khorram, E.; Fathabadi, H.S. An extended method using TOPSIS and VIKOR for multiple attribute decision making with multiple decision makers and single valued neutrosophic numbers. Adv. Appl. Stat. 2017, 50, 261–292. [CrossRef] 22. Bausys, R.; Zavadskas, E.K. Multi-criteria decision making approach by VIKOR under interval neutrosophic set environment. Econ. Comput. Econ. Cybern. Stud. Res. 2015, 49, 33–48. 3. Zhang, M.C.; Liu, P.D.; Shi, L.L. An extended multiple attribute group decision-making TODIM met based on the neutrosophic numbers. J. Intell. Fuzzy Syst. 2016, 30, 1773–1781. [CrossRef] 4. Bausys, R.; Zavadskas, E.K.; Kaklauskas, A. Application of neutrosophic set to multi-criteria decision mak by COPRAS. Econ. Comput. Econ. Cybern. Stud. Res. 2015, 49, 91–105. 25. References 1. Li, H.; Dong, K.; Jiang, H.; Sun, R.; Guo, X.; Fan, Y. Risk assessment of China’s overseas oil reﬁning investment using a fuzzy-grey comprehensive evaluation method. Sustainability 2017, 9, 696. [CrossRef] 1. Li, H.; Dong, K.; Jiang, H.; Sun, R.; Guo, X.; Fan, Y. Risk assessment of China’s overseas oil reﬁning investment using a fuzzy-grey comprehensive evaluation method. Sustainability 2017, 9, 696. [CrossRef] 2. Wu, W.S.; Kou, G.; Peng, Y.; Ergu, D. Improved AHP-group decision making for investment strategy selection. Technol. Econ. Dev. Econ. 2012, 18, 299–316. [CrossRef] 2. Wu, W.S.; Kou, G.; Peng, Y.; Ergu, D. Improved AHP-group decision making for investment strategy selection. Technol. Econ. Dev. Econ. 2012, 18, 299–316. [CrossRef] 3. Hatami-Marbini, A.; Kangi, F. An extension of fuzzy TOPSIS for a group decision making with an application to tehran stock exchange. Appl. Soft Comput. 2017, 52, 1084–1097. [CrossRef] 4. Yazdani-Chamzini, A.; Shariati, S.; Yakhchali, S.H.; Kazimieras Zavadskas, E. Proposing a new methodology for prioritising the investment strategies in the private sector of Iran. Econ. Res. Ekonomska Istraživanja 2014, 27, 320–345. [CrossRef] 4. Yazdani-Chamzini, A.; Shariati, S.; Yakhchali, S.H.; Kazimieras Zavadskas, E. Proposing a new methodology for prioritising the investment strategies in the private sector of Iran. Econ. Res. Ekonomska Istraživanja 2014, 27, 320–345. [CrossRef] 5. Shen, K.Y.; Yan, M.R.; Tzeng, G.H. Combining VIKOR-DANP model for glamor stock selection and stock performance improvement. Knowl. Based Syst. 2014, 58, 86–97. [CrossRef] 5. Shen, K.Y.; Yan, M.R.; Tzeng, G.H. Combining VIKOR-DANP model for glamor stock selection and stock performance improvement. Knowl. Based Syst. 2014, 58, 86–97. [CrossRef] 6. Dincer, H.; Hacioglu, U. A comparative performance evaluation on bipolar risks in emerging capital markets using fuzzy AHP-TOPSIS and VIKOR approaches. Eng. Econ. 2015, 26, 118–129. [CrossRef] 6. Dincer, H.; Hacioglu, U. A comparative performance evaluation on bipolar risks in emerging capital markets using fuzzy AHP-TOPSIS and VIKOR approaches. Eng. Econ. 2015, 26, 118–129. [CrossRef] 7. Hashemkhani Zolfani, S.; Bahrami, M. Investment prioritizing in high tech industries based on SWARA-COPRAS approach. Technol. Econ. Dev. Econ. 2014, 20, 534–553. [CrossRef] 8. Davis, G.A. Bias and error in mine project capital cost estimation. Eng. Econ. 2008, 53, 118–139. 9. Bai, L.W.; Chen, G.P. Research on investment risk management of mine projects. Available online: http://kns.cnki.net/KCMS/detail/detail.aspx?dbcode=CJFQ&dbname=CJFD2006&ﬁlename= KYKB200606002&v=MDU4NjdmYitab0Z5SGtWNzdCTGpUQWJMRzRIdGZNcVk5RlpvUjhlWDFMdXhZUzd EaDFUM3FUcldNMUZyQ1VSTDI= (accessed on 19 June 2017). Symmetry 2017, 9, 149 16 of 18 16 of 18 10. Chen, J.H.; Min, H.U.; Xiao, C.; Lu, Z.H. References ˇCereška, A.; Zavadskas, E.K.; Cavallaro, F.; Podvezko, V.; Tetsman, I.; Grinbergien˙e, I. Sustainable assessment of aerosol pollution decrease applying multiple attribute decision-making methods. Sustainability 2016, 8, 586. [CrossRef] 26. Baušys, R.; Juodagalvien˙e, B. Garage location selection for residential house by WASPAS-SVNS method. J. Civ. Eng. Manag. 2017, 23, 421–429. [CrossRef] 27. Stanujkic, D.; Zavadskas, E.K.; Smarandache, F.; Brauers, W.K.M.; Karabasevic, D. A neutrosophic extension of the MULTIMOORA method. Informatica 2017, 28, 181–192. 28. Peng, J.J.; Wang, J.Q.; Zhang, H.Y.; Chen, X.H. An outranking approach for multi-criteria decision-making problems with simpliﬁed neutrosophic sets. Appl. Soft Comput. 2014, 25, 336–346. [CrossRef] 29. Zhang, H.Y.; Wang, J.Q.; Chen, X.H. An outranking approach for multi-criteria decision-making pr with interval-valued neutrosophic sets. Neural Comput. Appl. 2016, 27, 615–627. [CrossRef] 30. Peng, J.J.; Wang, J.Q.; Yang, W.E. A multi-valued neutrosophic qualitative ﬂexible approach based on likelihood for multi-criteria decision-making problems. Int. J. Syst. Sci. 2017, 48, 425–435. [CrossRef] 31. Li, Y.H.; Liu, P.D.; Chen, Y.B. Some single valued neutrosophic number heronian mean operators and their application in multiple attribute group decision making. Informatica 2016, 27, 85–110. [CrossRef] 32. Ye, J. Correlation coefﬁcients of interval neutrosophic hesitant fuzzy sets and its application in a multiple attribute decision making method. Informatica 2016, 27, 179–202. [CrossRef] 33. Zadeh, L.A. The concept of a linguistic variable and its applications to approximate reasoning-1. Inf. Sci. 1975, 8, 199–249. [CrossRef] 33. Zadeh, L.A. The concept of a linguistic variable and its applications to approximate reasoning-1. Inf. Sci. 1975, 8, 199–249. [CrossRef] 34. Yang, C.; Zhang, Q.; Ding, S. An evaluation method for innovation capability based on uncertain linguistic variables. Appl. Math. Comput. 2015, 256, 160–174. [CrossRef] 34. Yang, C.; Zhang, Q.; Ding, S. An evaluation method for innovation capability based on uncertain linguistic variables. Appl. Math. Comput. 2015, 256, 160–174. [CrossRef] Symmetry 2017, 9, 149 17 of 18 17 of 18 35. Deng, Y.; Sadiq, R.; Jiang, W.; Tesfamariam, S. Risk analysis in a linguistic environment: A fuzzy evidential reasoning-based approach. Expert Syst. Appl. Int. J. 2011, 38, 15438–15446. [CrossRef] 36. Liu, P.D.; Zhang, X. The study on multi-attribute decision-making with risk based on linguistic variable. Int. J. Comput. Intell. Syst. 2010, 3, 601–609. [CrossRef] 37. Pei, Z.; Shi, P. Fuzzy risk analysis based on linguistic aggregation operators. Int. J. Innov. Comput. Inf. Control Ijicic 2011, 7, 7105–7117. 38. Peiris, H.O.W.; Chakraverty, S.; Perera, S.S.N.; Ranwala, S. References Novel fuzzy linguistic based mathematical model to assess risk of invasive alien plant species. Appl. Soft Comput. 2017, 59, 326–339. [CrossRef] 39. Liu, P.D.; He, L.; Yu, X. Generalized hybrid aggregation operators based on the 2-dimension uncertain linguistic information for multiple attribute group decision making. Group Decis. Negot. 2016, 25, 103–126. [CrossRef] 40. Liu, W.H.; Liu, H.B.; Li, L.L. A multiple attribute group decision making method based on 2-dimension uncertain linguistic weighted Heronian mean aggregation operator. Int. J. Comput. Commun. Control 2017, 12, 254–264. [CrossRef] 41. Liu, P.D.; Teng, F. An extended TODIM method for multiple attribute group decision-making based on 2-dimension uncertain linguistic variable. Complexity 2016, 21, 20–30. [CrossRef] 2. Ye, J. An extended TOPSIS method for multiple attribute group decision making based on single val neutrosophic linguistic numbers. J. Intell. Fuzzy Syst. 2015, 28, 247–255. 43. Meng, F.Y.; Tang, J.; An, Q.X.; Chen, X.H. Decision making with intuitionistic linguistic preference relations. Int. Trans. Oper. Res. 2017. [CrossRef] 44. Tüysüz, F.; ¸Sim¸sek, B. A hesitant fuzzy linguistic term sets-based AHP approach for analyzing the performance evaluation factors: An application to cargo sector. Complex Intell. Syst. 2017, 1–9. [CrossRef] 45. Gou, X.; Liao, H.C.; Xu, Z.S.; Herrera, F. Double hierarchy hesitant fuzzy linguistic term set and MULTIMOORA method: A case of study to evaluate the implementation status of haze controlling measures. Inf. Fusion 2017, 38, 22–34. [CrossRef] 46. Pang, Q.; Wang, H.; Xu, Z.S. Probabilistic linguistic term sets in multi-attribute group decision making. Inf. Sci. 2016, 369, 128–143. [CrossRef] 46. Pang, Q.; Wang, H.; Xu, Z.S. Probabilistic linguistic term sets in multi-attribute group decision making. Inf. Sci. 2016, 369, 128–143. [CrossRef] 7. Zhang, P.H.; She, S.X. Assessment of service quality in wireless sensor networks with probabilistic lingu term sets. Int. J. Online Eng. 2017, 13, 1861–2121. [CrossRef] 48. Faizi, S.; Rashid, T.; Zafar, S. An outranking method for multi-criteria group decision making using intuitionistic fuzzy linguistic term sets. J. Intell. Fuzzy Syst. 2017, 32, 2153–2164. [CrossRef] 49. Yang, W.; Pang, Y.; Shi, J.; Yue, H. Linguistic hesitant intuitionistic fuzzy linear assignment method based on choquet integral. J. Intell. Fuzzy Syst. 2017, 32, 767–780. [CrossRef] 49. Yang, W.; Pang, Y.; Shi, J.; Yue, H. Linguistic hesitant intuitionistic fuzzy linear assignment method based on choquet integral. J. Intell. Fuzzy Syst. 2017, 32, 767–780. [CrossRef] 50. Chen, Z.C.; Liu, P.D.; Pei, Z. 59. Wang, J.Q.; Wu, J.T.; Wang, J.; Zhang, H.Y.; Chen, X.H. Interval-valued hesitant fuzzy linguistic sets and their applications in multi-criteria decision-making problems. Inf. Sci. 2014, 288, 55–72. [CrossRef] 60. Wang, Y.M. Using the method of maximizing deviations to make decision for multi-indices. Syst. Eng. Electron. 1997, 8, 21–26. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 59. Wang, J.Q.; Wu, J.T.; Wang, J.; Zhang, H.Y.; Chen, X.H. Interval-valued hesitant fuzzy linguistic sets and their applications in multi-criteria decision-making problems. Inf. Sci. 2014, 288, 55–72. [CrossRef] 60 Wang YM Using the method of maximizing deviations to make decision for multi indices Syst Eng References An approach to multiple attribute group decision making based on linguistic intuitionistic fuzzy numbers. Int. J. Comput. Intell. Syst. 2015, 8, 747–760. [CrossRef] 51. Wei, C.P.; Tang, X. A new method for ranking intuitionistic fuzzy numbers. Int. J. Knowl. Syst. Sci. 2017, 2, 43–49. [CrossRef] 52. Peng, H.G.; Wang, J.Q.; Cheng, P.F. A linguistic intuitionistic multi-criteria decision-making method based on the frank heronian mean operator and its application in evaluating coal mine safety. Int. J. Mach. Learn. Cybern. 2017, 1–16. [CrossRef] 3. Liu, P.D.; Wang, P. Some improved linguistic intuitionistic fuzzy aggregation operators and their applicat to multiple-attribute decision making. Int. J. Inf. Technol. Decis. Making 2017, 16, 817–850. [CrossRef] 4. Fang, Z.B.; Ye, J. Multiple attribute group decision-making method based on linguistic neutrosophic num Symmetry 2017, 9, 111. [CrossRef] 55. Peng, J.J.; Wang, J.Q.; Yang, L.J.; Qian, J. A novel multi-criteria group decision-making approach using simpliﬁed neutrosophic information. Int. J. Uncertain. Quantif. 2017. [CrossRef] 56. Tian, Z.P.; Wang, J.; Zhang, H.Y.; Wang, J.Q. Multi-criteria decision-making based on generalized prioritized aggregation operators under simpliﬁed neutrosophic uncertain linguistic environment. Int. J. Mach. Learn. Cybern. 2016, 1–17. [CrossRef] 57. Pan, Y. Fuzzy analysis of the economic risk factor and degree of mining investment. Met. Mine 2001, 1, 000. 58. Xu, Z.S. Deviation measures of linguistic preference relations in group decision making. OMEGA 2005, 33, 249–254. [CrossRef] 58. Xu, Z.S. Deviation measures of linguistic preference relations in group decision making. OMEGA 2005, 33, 249–254. [CrossRef] 18 of 18 Symmetry 2017, 9, 149
https://openalex.org/W2116209745	https://hal-insu.archives-ouvertes.fr/insu-03623459/document	English	null	Organic materials in planetary and protoplanetary systems: nature or nurture?	Astronomy & astrophysics	2,011	cc-by	17,400	To cite this version: C. M. Dalle Ore, M. Fulchignoni, D. P. Cruikshank, M. A. Barucci, R. Brunetto, et al.. Organic materials in planetary and protoplanetary systems: nature or nurture?. Astronomy and Astrophysics - A&A, 2011, 533, 10.1051/0004-6361/201116599. insu-03623459 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: insu-03623459 https://insu.hal.science/insu-03623459v1 Submitted on 30 Mar 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Astronomy & Astrophysics A&A 533, A98 (2011) DOI: 10.1051/0004-6361/201116599 c⃝ESO 2011 A&A 533, A98 (2011) DOI: 10.1051/0004-6361/201116599 c⃝ESO 2011 ABSTRACT Aims. The objective of this work is to summarize the discussion of a workshop aimed at investigating the properties, origins, and evolution of the materials that are responsible for the red coloration of the small objects in the outer parts of the solar system. Because of limitations or inconsistencies in the observations and, until recently, the limited availability of laboratory data, there are still many questions on the subject. Our goal is to approach two of the main questions in a systematic way: – Is coloring an original signature of materials that are presolar in origin (“nature”) or stems from post-formational c or weathering (“nurture”)? g ( ) chemical signature of the material that causes spectra to be sloped towards the red in the visible? – What is the chemical signature of the material that causes spectra to be sloped towards the red in the visible? We examine evidence available both from the laboratory and from observations sampling diﬀerent parts of the solar system and cir- cumstellar regions (disks). What is the chemical signature of the material that causes spectra to be sloped towards the red in the visible? We examine evidence available both from the laboratory and from observations sampling diﬀerent parts of the solar system and cir- cumstellar regions (disks). dial vs. evolutionary origin for the material that reddens the small objects in the outer parts of our, as well as in other, planetary systems. We proceed by ﬁrst summarizing laboratory results followed by observational data collected at various distances from the Sun. Results. While laboratory experiments show clear evidence of irradiation eﬀects, particularly from ion bombardment, the ﬁrst obsta- cle often resides in the ability to unequivocally identify the organic material in the observations. The lack of extended spectral data of good quality and resolution is at the base of this problem. Furthermore, that both mechanisms, weathering and presolar, act on the icy materials in a spectroscopically indistinguishable way makes our goal of deﬁning the impact of each mechanism challenging. Conclusions. Through a review of some of the workshop presentations and discussions, encompassing laboratory experiments as well as observational data, we infer that both “nature” and “nurture” are instrumental in the coloration of small objects in the outer parts y g j p p y y We proceed by ﬁrst summarizing laboratory results followed by observational data collected at various distances from the Sun. 13 University of Hawaii, Institute for Astronomy, USA e-mail: owen@ifa.hawaii.edu 13 University of Hawaii, Institute for Astronomy, USA e-mail: owen@ifa.hawaii.edu 14 Space Telescope Science Institute, Baltimore, MD 21218, USA 14 Space Telescope Science Institute, Baltimore, MD 21218, USA 14 Space Telescope Science Institute, Baltimore, MD 21218, USA 15 p p 15 Department of Physics and Astronomy, Johns Hopkins University, Baltimore, MD 21218, USA e-mail: Pascucci@stsci.edu 15 Department of Physics and Astronomy, Johns Hopkins University, Baltimore, MD 21218, USA e-mail: Pascucci@stsci.edu 16 NASA Lunar Science Institute, Mail Stop 17-1, Moﬀett Field, CA 94035, USA nar Science Institute, Mail Stop 17-1, Moﬀett Field, CA 940 NASA Lunar Science Institute, Mail Stop 17-1, Moﬀett Fi 17 Nasa Postdoctoral Program, NASA Ames Research Center, Moﬀett Field, CA 94035, USA e-mail: noemi.pinilla-alonso@nasa.gov 17 Nasa Postdoctoral Program, NASA Ames Research Center, Moﬀett Field, CA 94035, USA e-mail: noemi.pinilla-alonso@nasa.gov 18 INAF – Osservatorio Astroﬁsico di Catania, via S. Soﬁa 78, 95123 Catania, Italy e-mail: Gianni@oact.inaf.it 18 INAF – Osservatorio Astroﬁsico di Catania, via S. Soﬁa 78, 95123 Catania, Italy e-mail: Gianni@oact.inaf.it Received 27 January 2011 / Accepted 6 June 2011 Article published by EDP Sciences Organic materials in planetary and protoplanetary systems: nature or nurture? C. M. Dalle Ore1,2,3, M. Fulchignoni4, D. P. Cruikshank3, M. A. Barucci4, R. Brunetto5, H. Campins6 C. de Bergh3, J. H. Debes7, E. Dotto8, J. P. Emery9, W. M. Grundy10, A. P. Jones5, V. Mennella11, F. R. Orthous-Daunay12, T. Owen13, I. Pascucci14,15, Y. J. Pendleton16, N. Pinilla-Alonso17, E. Quirico12, and G. Strazzulla18 1 Centre International d’Ateliers Scientiﬁques de l’Observatoire de Paris, Paris, France 2 SETI Institute, 189 Bernardo Ave., Mountain View, CA 94043, USA q 2 SETI Institute, 189 Bernardo Ave., Mountain View, CA 94043, USA 2 SETI Institute, 189 Bernardo Ave., Mountain View, CA 94043, USA e-mail: Cristina.M.DalleOre@nasa.gov 3 NASA Ames Research Center, Mail Stop 245-6, Moﬀett Field, CA 94035, USA e-mail: Dale.P.Cruikshank@nasa.gov 3 NASA Ames Research Center, Mail Stop 245-6, Moﬀett Field, CA 94035, US e-mail: Dale.P.Cruikshank@nasa.gov 4 LESIA, Observatoire de Paris, 92195 Meudon Principal Cedex, France e-mail: [marcello.fulchignoni,Antonella.Barucci, Catherine 4 LESIA, Observatoire de Paris, 92195 Meudon Principal Cedex, France e-mail: [marcello.fulchignoni,Antonella.Barucci, Catherine.deBergh]@obspm.fr 5 5 Institute d’Astrophysique Spatiale (IAS), Université Paris 11 and CNRS, 91405 Orsay, France 5 Institute d’Astrophysique Spatiale (IAS), Université Paris 11 and CNRS, 91405 Orsay, France 6 Physics Department, University of Central Florida, Orlando, FL 32816, USA 7 6 Physics Department, University of Central Florida, Orlando, FL 32816, USA 7 7 NASA Postdoctoral Program Fellow, Goddard Space Flight Center, USA 7 NASA Postdoctoral Program Fellow, Goddard Space Flight Center, USA 8 INAF – Osservatorio Astronomico di Roma, via Frascati 33, 00040 Monteporzio Catone (Roma), Italy e-mail: Dotto@mporzio.astro.it 9 Earth and Planetary Sciences Dept, University of Tennessee, Knoxville, TN 37919, USA e-mail: JEmery2@utk.edu 9 Earth and Planetary Sciences Dept, University of Tennessee, Knoxville, TN 37919, USA il JE 2@ k d y 10 Lowell Observatory, 1400 W.Mars Hill Rd., FlagstaﬀAZ 86001, USA y 10 Lowell Observatory, 1400 W.Mars e-mail: W.Grundy@lowell.edu 10 Lowell Observatory, 1400 W.Mars Hill Rd., FlagstaﬀAZ 86001, USA e-mail: W.Grundy@lowell.edu 11 owell Observatory, 1400 W.Mars Hill Rd., FlagstaﬀAZ 86 e-mail: W.Grundy@lowell.edu 11 11 INAF Osservatorio Astronomico di Capodimonte, via Moiariello, 16, 80131 Napoli, Italy y 12 UJF-Grenoble 1/CNRS-INSU, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG) UMR 5274, France 13 University of Hawaii, Institute for Astronomy, USA e-mail: owen@ifa.hawaii.edu 13 University of Hawaii, Institute for Astronomy, USA e-mail: owen@ifa.hawaii.edu 1. Introduction Similarly, the regions of Saturn’s rings that exhibit red color may acquire their reﬂectance and coloration properties from the presence of iron or iron oxides (Cuzzi et al. 2009). With the exception of the new interpretation by Clark et al. (2008), so far, the coloration of solid bodies in the outer solar system and in debris disks of other planetary systems has been attributed to “organic” materials that can be either native to icy bodies or the products of the irradiation of volatile ices, or a combination of the two. We call the scenario of a surface be- ing reddened by irradiation “nurture”. We note, however, that both Cruikshank et al. (2001) and Emery & Brown (2004) found that scattering models with maﬁc silicates can match the spectral reﬂectance of the red colored jovian Trojan asteroids for wave- lengths shorter than 2.5 μm, as discussed further below. An alter- native view to the “nurture” scenario is oﬀered by Grundy (2009) where the “nurture” idea is matched with a “nature” counter- part, amply supported in recent literature (Grundy & Stansberry 2003; Grundy 2009, and references therein). The “nature” coun- terpart described in detail below consists of the case in which the reddening is caused by sub-micron particles of organic mate- rial of presolar origin trapped in ice from which comes the label “nature”. According to Grundy (2009) the degree of reddening varies with the concentration of the organics with respect to ice. We summarize the idea and oﬀer laboratory and observational evidence to support the two views in an eﬀort to unravel the puzzle that has impacted our understanding of outer solar sys- tem and planetary system chemistry. We also assess whether the evidence weighs in favor of either scenario to provide a guideline for future work and investigations. Solid organic complexes (tholins) produced synthetically by a corona or a spark discharge in a gaseous mixture of N2 and CH4 (and other molecules in some experiments) are strongly colored, and are eﬀective when used in scattering models to reproduce the color of Titan’s atmospheric aerosols (e.g., Imanaka et al. 2004; Bernard et al. 2006). Similarly, tholins are an eﬀective color- ing agent in models of solid surfaces (e.g., Hoﬀman et al. 1993; Dalle Ore et al. 2009; Owen et al. 2001; and Cruikshank et al. 2005). New views of the red coloration of solid bodies in the solar system (Fig. 1. Introduction The colors of solar system bodies are often the main (or sole) source of information available for determining the composi- tion of the surface materials, especially in the absence of spectra showing diagnostic absorption or emission features. These com- bined with information on the absolute reﬂectance (e.g., geomet- ric albedo), the color of reﬂected sunlight measured over a range of wavelengths in the visible and near-infrared (0.3−2.5 μm) spectral region, are usually enough to discriminate between mineral- and ice-covered surfaces. In general terms, surfaces with a high albedo and neutral color are usually ice-covered (with H2O being most common), while medium high albedo and neutral color may indicate pulverized minerals. Low albedo and a moderately red color are suggestive of maﬁc minerals (e.g., olivine and pyroxene), while low albedo and very red color are usually taken to indicate a surface rich in complex, macro- molecular carbonaceous material similar to the kind found in primitive carbonaceous meteorites. This latter material is often referred to by the shorthand terms, kerogen-like or “organics”, but it is necessary to keep in mind the rather longer but more descriptive name given in the previous sentence when using this simpliﬁcation. Furthermore, we use the term “reddening” when referring to either the amount of organics or the eﬀects of “weathering”, as explained in the text. The term “weathering” is used with reference to the gradual changes to the surface of the bodies caused by irradiation. Fig. 1. Measured spectral reﬂectance of several solar system small bod- ies. Key: Pholus = Centaur object, Iapetus = Saturn Satellite, Hektor, Odysseus & Euphrosyne = Trojan asteroids, Himalia = Jupiter Satellite, Borrelly = Jupiter-family comet. Figure from J. P. Emery. The idea of complex organic materials constituting the col- oring agent for low-albedo asteroids appears to have originated with Gradie and Veverka (1980), who suggested that “... very opaque, very red, polymer-type organic compounds, which are structurally similar to aromatic-type kerogen”, mixed with mont- morillonite (clay), magnetite (iron oxide), and carbon black, could match the colors of Trojan asteroids in the spectral range 0.3−1.1 μm. The organic material they used in their laboratory simulations was derived from coal tar by removing the soluble components and leaving the insoluble complex solid material called kerogen. may be material that is intrinsically red, such as metallic iron or iron oxides. ABSTRACT planets and satellites: composition – comets: general – circumstellar matter – Kuiper belt: general – dust, extinction Key words. planets and satellites: composition – comets: general – circumstellar matter – Kuiper belt: general – dust Fig. 1. Measured spectral reﬂectance of several solar system small bod- ies. Key: Pholus = Centaur object, Iapetus = Saturn Satellite, Hektor, Odysseus & Euphrosyne = Trojan asteroids, Himalia = Jupiter Satellite, Borrelly = Jupiter-family comet. Figure from J. P. Emery. ABSTRACT p y g y y Results. While laboratory experiments show clear evidence of irradiation eﬀects, particularly from ion bombardment, the ﬁrst obsta- cle often resides in the ability to unequivocally identify the organic material in the observations. The lack of extended spectral data of good quality and resolution is at the base of this problem. Furthermore, that both mechanisms, weathering and presolar, act on the icy materials in a spectroscopically indistinguishable way makes our goal of deﬁning the impact of each mechanism challenging. y q y y g p good quality and resolution is at the base of this problem. Furthermore, that both mechanisms, weathering and presolar, act on the icy materials in a spectroscopically indistinguishable way makes our goal of deﬁning the impact of each mechanism challenging. Conclusions. Through a review of some of the workshop presentations and discussions, encompassing laboratory experiments as well as observational data, we infer that both “nature” and “nurture” are instrumental in the coloration of small objects in the outer parts p p y g y g g p g g Conclusions. Through a review of some of the workshop presentations and discussions, encompassing laboratory experiments as well as observational data, we infer that both “nature” and “nurture” are instrumental in the coloration of small objects in the outer parts Article published by EDP Sciences A98, page 1 of 14 A&A 533, A98 (2011) of the solar system. While in the case of some observations it is clear that the organic reddening material originated before the solar nebula (i.e. presolar grains found in meteorites), for many other cases pointers are not as clear and indicate a concurrence of both processes. of the solar system. While in the case of some observations it is clear that the organic reddening material origina (i.e. presolar grains found in meteorites), for many other cases pointers are not as clear and indicate a concurrence of b hile in the case of some observations it is clear that the organic reddening material originated before the solar ne nd in meteorites), for many other cases pointers are not as clear and indicate a concurrence of both processes. (i.e. presolar grains found in meteorites), for many other cases pointers are not as clear and indicate a concurrence of both processes. Key words. planets and satellites: composition – comets: general – circumstellar matter – Kuiper belt: general – dust, extinction Key words. 3.1. Laboratory studies of refractory carbonaceous materials Carbonaceous and organic materials in the solar system and in the interstellar medium (ISM) of our own, as well as external galaxies, display a wide range of composition and structure, il- lustrating the rich chemistry that occurs in space. Experimental studies play a fundamental role in understanding this diver- sity: in the laboratory it is possible to analyze collected extrater- restrial carbon-rich materials [meteorites (see the spectrum of Orgueil in Fig. 4), micro-meteorites, IDPs, and cometary grains collected by the Stardust mission], and to synthesize, charac- terize, and process laboratory analogues to gain insight into the formation, origin, and evolution of the natural samples. Among these processes UV irradiation and cosmic ion irra- diation play an important role in the life-cycle of interstellar dust (Mennella et al. 2003), in the production of complex organics in the presolar nebula (Muñoz-Caro et al. 2006) as well as on the surface of TNOs and other minor bodies (Brunetto et al. 2006), and in the alteration of pre-existing carbon materials (Moroz et al. 2004; Baratta et al. 2008). Many studies point out that the early Sun was much more active than today, passing through an active T Tauri phase before reaching the main sequence (e.g., Feigelson & Montmerle 1999); such strong particle and photon radiation could have had major inﬂuences on processing mate- rial during the planetary accretion phase, and more speciﬁcally on the carbon chemistry. Although there is abundant evidence that icy surfaces that include carbon-bearing species are rapidly reddened and dark- ened as a result of radiation damage, there are also several lines of evidence to suggest that the surface colors of TNOs are not due to thin, modern radiolytic veneers, but in fact reﬂect their bulk compositions. Speciﬁcally, the color clusters reported by Barucci et al. (2005) are hard to explain if the surfaces of TNOs are progressively altered by radiation damage, but occasionally refreshed by impact excavation of pristine material. The colors of primary and secondary bodies among small binary TNOs are also very closely correlated (Benecchi et al. 2009). This pattern is inconsistent with time-dependent surface coloration from radiation damage, since impact erosion rates depend on object size. 1. Introduction 1) are emerging with improvements in computa- tional methods for scattering models and new laboratory data, and with a large and homogeneous observational data set for the spectral reﬂectances of Saturn’s satellites and rings. Clark et al. (2008) have shown that a brightness peak in the blue spec- tral region seen in most of Saturn’s satellites can be explained as a Rayleigh scattering phenomenon caused by submicrometer particles that form a thin coating on the ice or other surface ma- terials. For neutral-colored surfaces, these small particles may be composed of carbon, while for red-colored surfaces, the particles A98, page 2 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems 2. Sub-micron organic particles and their possible inﬂuence on the colors of primitive bodies: the “nature” hypothesis The hollow rims of red organic globules such as seen in the Tagish Lake meteorite are also worth mentioning in this context. These shells are thought to have been produced by photolysis of the surfaces of small icy grains prior to their accretion into larger bodies (e.g., Nakamura-Messenger et al. 2006). Subsequently, the remaining ice sublimated away, leaving the less volatile or- ganic shell. These particles tell us that at least some icy grains were already well-irradiated before they accreted into planetes- imals, so we may not require the modern-day radiation envi- ronment to explain the existence of reddish organic materials in primitive bodies (although ongoing radiolysis and photolysis of icy surfaces surely does also occur). Micron and submicron sized organic and carbonaceous parti- cles occur in primitive meteorites, interplanetary dust particles (IDPs), and comet dust. Some could have formed in the proto- planetary nebula, via the irradiation of icy particles. Others could have survived from the cloud of gas and dust from which the Sun formed. In regions where the volatiles condensed to form ices in the outer parts of the proto-planetary nebula, these par- ticles could have served as condensation nuclei, acquiring icy mantles, perhaps with distinct regional compositions very dif- ferent from the mix of ices prevalent in the presolar cloud. Small carbonaceous particles and ices were likely the main solid ingredients from which planetesimals accreted in the outer proto-planetary nebula although comet samples retrieved by the Stardust mission also contain silicates and sulﬁdes as signiﬁcant components (Sandford et al. 2006; Sandford 2008; Clemett et al. 2010). 3. Evidence from laboratory work Both the “nature” and the “nurture” scenarios are likely to have played an important role in the primordial nebula before plan- etesimals formed. We describe some of the processes that aﬀect carbon-rich materials and are thought to be predominant: they are mostly “nurture” mechanisms. Of the large number of diﬀer- ent forms of carbon we consider those that are most relevant and abundant in space. At visible wavelengths, the addition of colorless ice to oth- erwise dark organic material can dramatically increase the red- ness as well as the reﬂectance, if they are mixed at spatial scales of the order of the wavelength or less (Grundy & Stansberry 2003; Grundy 2009). The behavior of such mixtures suggests a resolution to the dilemma posed by the inconsistent behav- ior of the colors and albedos of trans-neptunian objects (TNOs), Centaurs, and ecliptic comet (EC) nuclei (e.g., Jewitt 2002; Lamy & Toth 2009). In fact, although these objects are thought to be genetically linked, much redder colors and higher albedos are found among TNOs and Centaurs than among the EC nu- clei (Doressoundiram et al. 2008), culminating in consistently red colors and high albedos among dynamically cold classical belt TNOs. Cold classical TNOs, with their near circular orbits, presumably formed close to where they currently are. They may or may not be direct progenitors of Centaurs and thence ECs (Volk & Malhotra 2008; Horner & Lykawka 2010) but, because of their distance from the Sun, their surface compositions could oﬀer relatively little-altered samples of the solids accreted in the outermost parts of the nebula. Radiative transfer mixing models can produce a wide variety of albedos and colors. By includ- ing sublimation loss of ice, they can also reproduce the observed color and albedo evolution as TNOs progress from the far fringes of the solar system through the giant planet-crossing Centaur zone to become ECs. 3.1. Laboratory studies of refractory carbonaceous materials TNOs could be more resilient to radiation damage than previously thought, if their surface ices do not include low molecular weight organic molecules, as could happen if they mostly consist of already-highly-processed carbonaceous parti- cles plus relatively clean H2O ice distilled by sublimation and re-condensation in the nebula. Several laboratories have concentrated their eﬀorts on the production, characterization, and alteration of refractory car- bonaceous materials of astrophysical interest, and in particular on the extensively studied amorphous carbons, with diﬀerent degrees of hydrogenation. Diﬀerent types of amorphous car- bon can be produced by several techniques, such as laser py- rolysis, laser ablation or arc discharge (e.g., Scott & Duley 1996; Mennella et al. 1999), and ion and photon irradiation of hydrocarbon-rich ices (e.g., Strazzulla & Baratta 1991; Dartois et al. 2005). Recent studies show that the IR spectra of these materials can be used to interpret observations of the dust in the diﬀuse ISM of galaxies, where an important fraction of the available carbon is locked in the dust (Pendleton & Allamandola 2002; Dartois et al. 2007; Dartois & Muñoz-Caro 2007). Carbon soot is another interesting material (Jäger et al. 2006), due to A98, page 3 of 14 A&A 533, A98 (2011) Fig. 2. Results of irradiation of CH4 ice (Brunetto et al. 2006): eV/16 amu units refer to the energy (eV) adsorbed per 16 atomic mass unit. The dotted line shows the spectrum of the residue at a higher tem- perature (250 K). its possible connection with polycyclic aromatic hydrocarbons (PAHs), and to the fact that its IR spectral features may help to interpret the nature of some of the ubiquitously observed aro- matic IR bands (Pino et al. 2008). Aromatic compounds are stable molecules made of six-fold rings of carbon and hydro- gen atoms (hydrocarbon). The most basic aromatic compound is the benzene ring (C6H6): PAHs are complex molecules made of fused aromatic (benzene) rings and in a terrestrial setting are a product of combustion. Visible-IR spectra of soot and amorphous carbons dif- fer in their chemical (e.g., hydrogen content, sp2/sp3 ratio, i.e. graphite-like to diamond-like bonding character), optical (e.g., variation of the optical gap), and structural (e.g., bond dis- order, crystallites size) properties. These properties can vary ac- cording to diﬀerent formation processes (“nature” scenario) but are also altered by photon and ion irradiation processes (“nur- ture” scenario) similar to that which is observed for irradiated organic residues. 3.3. Laboratory evidence conclusion The observed reddening material could be carbonaceous in na- ture or could be the result of sublimation and irradiation of carbon-rich compounds (nurture). The characteristics of car- bonaceous materials themselves are related to diﬀerent forma- tion processes, but can also be transformed by irradiation: their colors do not seem to correlate directly with their mode of formation, highlighting the diﬃculty in deﬁning the “nature” and “nurture” scenarios. Important constraints should be sought 3.2. Ion irradiation and the colors of C-rich materials Several experiments have been performed to investigate the ef- fects of ion irradiation on carbon-rich materials (e.g. Thompson et al. 1987). Three diﬀerent kinds of materials chosen to best rep- resent those thought to be present on the surfaces of atmosphere- less outer solar system objects (i.e. ices, initially red solids, and polymer-like initially transparent solids), have been irradiated. They are: frozen (16 K) methane (CH4) (Brunetto et al. 2006), asphaltite (a natural bitumen) (Moroz et al. 2004), and a ﬁlm of polystyrene deposited on a silicate (olivine) (Kanuchova et al. 2010). 4.1. Interstellar medium (ISM) Fundamental to the evolution of the biogenic molecules, to the process of planetary system formation, and perhaps to the origin of life, is the connection between the organic material found in the interstellar medium and that which was incorporated in the most primitive solar system bodies. Understanding the connec- tion between interstellar ices and organics can elucidate the in- ventory of materials available as the most primitive solar system bodies formed. Star formation and the subsequent evolution of planetary systems occur in dense molecular clouds, which are comprised, in part, of interstellar dust grains gathered from the diﬀuse inter- stellar medium (DISM). Carbonaceous materials are a primary component of interstellar dust, forming in the outﬂow of car- bon stars and the DISM. Over time, the low density DISM is swept into dense molecular clouds, the principal formation sites and repositories of most interstellar molecules. Organic com- pounds created in these clouds represent the ﬁrst step towards the complex materials that might be responsible of the reddening in the outer solar system. In this section we examine their rela- tionship to ices, an essential ingredient in the “nature” scenario. 3.1. Laboratory studies of refractory carbonaceous materials The colors of amorphous carbon vary non- uniquely as a function of the above properties, suggesting that colors might be a poor parameter to constrain the “nature” and “nurture” scenarios in carbonaceous materials. Fig. 2. Results of irradiation of CH4 ice (Brunetto et al. 2006): eV/16 amu units refer to the energy (eV) adsorbed per 16 atomic mass unit. The dotted line shows the spectrum of the residue at a higher tem- perature (250 K). in the laboratory analysis of collected extraterrestrial primitive carbons (e.g., cometary grains) to further understand the weight of one scenario over the other. On the other hand, the study of carbon-rich irradiated materials shows that weathering plays an important role in modifying the residue of H2O ice sublimation. It becomes apparent that a clear distinction between the “nature” and “nurture” scenario is very diﬃcult when analyzing the ma- terials that we think are responsible for most of the reddening in the solar system. Therefore, when examining the solar sys- tem evidence, we will make the simplifying assumption that the weathering process began with low-order, volatile-rich organics that are presumed to be presolar materials. We then try to in- fer whether “nature” or “nurture” prevails at the later stages of evolution of the materials that constitute the building blocks of planetary systems. 4. Observational evidence outside the solar system 4.1. Interstellar medium (ISM) The irradiation of frozen CH4 shows a strong reddening and darkening of the spectra, due to the formation of an or- ganic (C-rich) refractory residue. The color is preserved after the sublimation of the volatile species (methane and other com- pounds formed by irradiation) as shown by the spectrum of the residue at higher temperatures (250 K, Fig. 2). Further irradia- tion of such a solid polymer-like residue, as well as of natural red solids (e.g., asphaltite), produces a darkening and a ﬂattening of the spectra. Recently, samples of silicates (olivine) covered with polystyrene layers have been considered. Polystyrene is consid- ered a model for an originally transparent polymer-like material. Before irradiation, it is a transparent medium that, as ion ﬂu- ence increases, exhibits spectral reddening and darkening. The reddening increases and reaches a maximum at a given ﬂuence. Further irradiation produces further darkening, but is accompa- nied by a ﬂattening of the spectrum. This process is what we refer through this paper as weathering and is considered one of the main “nurture” mechanisms. 4.1.1. The nature of interstellar organics and their relationship to ices Infrared observational studies provide details of the solid-state features in dust grains. A series of absorption bands have been A98, page 4 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems observed in the 3.4 μm wavelength region towards bright in- frared objects that are seen through high column densities of dust along multiple sightlines throughout the diﬀuse interstellar medium (DISM) (Pendleton et al. 1994). the physical and chemical properties of a-C:H solids are sur- prisingly complex. Their properties can vary in response to the external conditions (e.g., temperature; photon, ion or elec- tron irradiation). For example, a-C:H can undergo an equivalent process known as “photo-darkening”, “graphitisation” or “aro- matisation” when heated or exposed to UV- or ion-irradiation. This transformation leads to a closing of the optical gap and a concomitant change in the optical properties at near-IR to visible-UV wavelengths. Comparisons of organic residues, produced under a vari- ety of laboratory conditions, to the diﬀuse interstellar medium observations have shown that aliphatic chain-like hydrocarbon grains are responsible for the spectral absorption features ob- served near 3.4 μm (∼2940 cm−1). The hydrocarbons in the DISM appear to carry the -CH2- and -CH3 functional groups in the abundance ratio CH2/CH3 ∼2.5, and the amount of car- bon tied up in this component is greater than 4% of the cosmic carbon available (Sandford et al. 1991), highlighting the impor- tance of these materials. The Murchison carbonaceous meteorite has been shown a good analog of the interstellar observations, re- vealing a striking similarity between some of the hydrocarbons in the meteorite and the DISM (Pendleton & Allamandola 2002). Dartois et al. (2004) indeed show that the thermal anneal- ing of hydrogenated amorphous carbon materials is accompa- nied by an increase in the aromatic component, i.e. an “arom- atization”. This type of evolution for interstellar hydrocarbons was proposed previously (Duley et al. 1989; Jones 1990; Jones et al. 1990) and it was suggested to be the result of the photon- driven processing of hydrogen-rich a-C:H mantles accreted onto silicate grains in the ISM. In this model the progressive loss of hydrogen, and the associated “aromatisation” of the struc- ture was assumed to lead to materials with a lower hydrogen content, a smaller band gap, and an increased aromatic content. 4.1.1. The nature of interstellar organics and their relationship to ices The end point of this evolutionary process is expected to be a low-density, aromatic-rich material that can disaggregate into its constituent aromatic molecular components (PAH-like species) with associated sp3 and sp2 carbon and hydrogen atom bridging structures (e.g., Duley 2000; Petrie et al. 2003; Pety et al. 2005; Jones 2005) as a result of UV photo-processing or fragmentation in grain-grain collisions (Jones 2005; Jones et al. 1996). Ground- and space-based telescopic observations trace in- terstellar organics from the diﬀuse to dense interstellar clouds, revealing that organic material in the DISM is predominantly hydrocarbon in nature, possessing little N or O, and with the C distributed between aromatic ring-clusters and aliphatic forms. These data provide the ﬁrst step towards identifying the nature of the reddening material in the DISM and giving us a possible link with solar system materials. The transition between the diﬀuse and dense cloud regions provides an interesting place to study the onset of interstellar ice chemistry. The hardy, refractory com- ponent of the DISM is expected to survive incorporation into the dense cloud environment, but to date there is no observational evidence that the DISM 3.4-μm band is present in dense molec- ular clouds. The deep ice band at 3.0 μm detected toward many deeply embedded protostellar objects and through the quiescent dust through which background ﬁeld stars are observed (Smith et al. 1993) often has a broad wing on the long-wavelength side of the band that is assumed to arise from some type of simple hy- drocarbon (Sellgren et al. 1994). However, the wavelength peak of this feature and its overall structure diﬀer appreciably from the symmetric and asymmetric aliphatic hydrocarbon bands of the DISM (Chiar & Pendleton 2008). Mennella (2008a, 2010) proposed a scenario in which the diﬀerent conditions in dense clouds with respect to diﬀuse regions (e.g. ice coating of the dust grains, H atom temperature) may inhibit the formation of CH2 and CH3 groups, while the formation of the 3.47 μm band can proceed (see also Sect. 4.3). Thus, it seems that a viable source of interstellar, circum- stellar or solar system “PAHs” could be provided by the natural evolution of interstellar hydrogenated amorphous carbon grains. 4.1.1. The nature of interstellar organics and their relationship to ices However, in this case any “released” aromatic rich, “molecular” carriers are not the pristine and classic PAHs generally discussed in the PAH literature, but are probably more complex and con- tain carbon in other than purely aromatic form. 4.2. Dense clouds In order to study the ice chemistry of dense clouds from the ear- liest stages, one needs to look at the transition zone between the diﬀuse and dense clouds where extinction is high enough that the grains are shielded from the harsh interstellar radiation ﬁeld of the DISM allowing for ice formation. The spectroscopy of back- ground stars seen through quiescent clouds with no star forma- tion activity reveals that chemistry occurs before star formation begins. In fact, star formation might well begin with dust already coated with a fairly complex mixture of ices (Chiar et al. 2011). NH3-ice is an important mantle constituent in all dense cloud en- vironments, and therefore an important reservoir of elemental N in these regions. 4.3. Evolution of organics from diffuse interstellar clouds to the solar system The results of experiments aimed at simulating the evolution of the aliphatic carbon component in space have recently shown that an evolutionary link between the organics observed in the two environments is possible (Mennella 2008b, 2010). In fact, the same carbon grain population may absorb at 3.4 μm in dif- fuse regions of the ISM, and at 3.47 μm in dense regions. The carbon materials absorbing at 3.47 μm in dense cloud/protostars, included in a comet during the formation process in the cold outer edge of the solar nebula, may thermally evolve to de- velop the CH2 and CH3 groups, as testiﬁed by laboratory simu- lations. Therefore, a unifying interpretation of how the aliphatic component appears in the main phases of its evolution in space is possible. As the inner part of the disk clears out, the observed infrared spectra dramatically change. No water molecules and no organ- ics are detected, just atomic ionic species and the very high J transition of OH, pointing to dissociation of water by the now stronger UV ﬁeld that is no longer shielded by the dust (Najita et al. 2010). The outer disk does not seem to be aﬀected by these changes: the molecular complexity observed at this stage is the same as that of optically thick gas-rich disks. Studies of the interaction of hydrogen atoms at 80 K with carbon grains covered with a water ice layer at 12 K conﬁrm that exposure of the samples to H atoms induces the activation of the band at 3.47 μm with no evidence for the formation of aro- matic and aliphatic C-H bonds in the CH2 and CH3 functional groups. A penetration depth of 100 nm has been estimated for H atoms in the porous water ice covering carbon grains. Sample warm up to room temperature causes the activation of the IR fea- tures representing the vibrations of the CH2 and CH3 aliphatic functional groups. The presence of the 3.47 μm band carrier is compatible with the evolutionary time scale limit imposed by fast cycling of materials between dense and diﬀuse regions of the interstellar medium. In diﬀuse regions the formation of the CH2 and CH3 aliphatic bands, inhibited in dense regions, takes place, masking the 3.47 μm band. 4.1.2. The evolution of hydrocarbonaceous dust in the ISM Through laboratory simulations, Dartois et al. (2004) have shown that hydrogenated amorphous carbon (denoted a-C:H) can explain not only the 3.4-μm, but also the 6.85- and 7.25-μm interstellar absorption bands if the a-C:H is hydrogen-rich (>50 atomic percent H). Given that recent studies of carbon dust and PAH evolution in the ISM indicate that these materials are rather susceptible to destructive processing in SN-generated shock waves, carbonaceous solids must be rather rapidly re- formed there (Serra Díaz-Cano & Jones 2008; Micelotta et al. 2010a,b, 2011; Jones & Nuth III 2011). Therefore it appears that a large fraction of the carbon dust in the ISM is likely to be in the form of an amorphous, re-accreted, a-C:H-like carbon, rather than in the form of crystalline graphite (pure carbon) that would be very diﬃcult to condense in the low-density ISM. Thus, graphite is probably not an abundant component of inter- stellar carbon dust (e.g. Serra Díaz-Cano & Jones 2008). For a material containing the atoms of only two elements (C and H) g Regarding the dust component of interstellar grains, the pos- sibility of the presence of PAH-like chemicals (aromatic hydro- carbons) in the building blocks of the solar system has been es- tablished. The next step is to understand if and how such basic primitive components could have survived the primordial neb- ula environment to become part of the current outer solar system bodies. Aliphatic hydrocarbonsare observed in the diﬀuse ISM, they are present in IDPs, cometary grains and meteorites, while they are absent in dense cloud environments. There are two possible interpretations: 1. There is no evolutionary link between the aliphatic com- pounds observed in the diﬀuse regions of the ISM and those present in solar system materials. The observation of 1. There is no evolutionary link between the aliphatic com- pounds observed in the diﬀuse regions of the ISM and those present in solar system materials. The observation of A98, page 5 of 14 A&A 533, A98 (2011) organics in the two environments is a mere coincidence. In diﬀuse interstellar clouds, the C-H bonds of the CH2 and CH3 groups form as a result of processing of carbon particles by H atoms (at thermal energies), UV photons and cosmic ions and they are destroyed when carbon grains enter into dense clouds (e.g., Mennella et al. 2002, 2003). 4.1.2. The evolution of hydrocarbonaceous dust in the ISM Other pro- cesses, e.g., photoprocessing of C bearing ices, during solar system formation and evolution, form the aliphatic materials observed in solar system materials. solar system. The chemical transformation of complex organic molecules during accretion and disk evolution and the level of complexity that they achieve as the protoplanetary disk en- ters the planet-formation stage are current research topics being pushed forward by recent dramatic advances in infrared and mil- limeter astronomy. Recent observations point to chemical evolution during the ﬁrst 30 Myr of the disk lifetime. Due to the very high tem- peratures necessary for star formation, all materials, unless far enough from the protostar, are in gaseous form. During the con- densation, planet accretion and formation phases they return to be seen in solid form. Optically thick gas-rich disks around young solar analogs are found to have rich infrared spectra indi- cating the presence of water, OH, and organic molecules such as C2H2 and HCN in the warm (∼700 K) disk atmosphere within a few AU from the star (Carr & Najita 2008; Pontoppidan et al. 2010). The large abundances of these molecules suggest that they form not only from evaporation of inward migrating ices but also from additional chemistry in the gas phase. Atomic and ionic species such as ionized Ne are also frequently detected (Pascucci et al. 2007; Güdel et al. 2010) and trace the very hot disk surface (several thousands K gas) likely photoevaporating from the star-disk system (Pascucci & Sterzik 2009). CN, HCN, H2CO and some deuterated species are also detected at large disk radii (beyond 100 AU) via interferometric millimeter ob- servations (Öberg et al. 2010). 2. An evolutionary link between the aliphatic observed in dif- fuse ISM and solar system materials exists. In this case, a fundamental constraint to be considered is the absence of the aliphatic component in dense clouds: going from diﬀuse ISM to the formation site of comets/parent bodies one has to pass through the dense cloud phase where the solar system formed. 4.3. Evolution of organics from diffuse interstellar clouds to the solar system The activation of the CH2 and CH3 aliphatic vibrational modes at the end of H processing after sample warm up represents the ﬁrst experimental evidence sup- porting an evolutionary connection between the interstellar car- bon grain population, which is responsible for the 3.4 μm band (diﬀuse regions) and contributes to the absorption at 3.47 μm (dense regions), and the organics observed in interplanetary dust particles and cometary Stardust grains. Finally, as the disk clears out most of its primordial dust (and likely gas), for systems older than 10 Myr, infrared and millimeter spectra are strikingly lacking a number of molecules, atomic and ionic species (Pascucci et al. 2006) such as H2 IR gas lines, forbidden atomic lines or CO mm lines, that are instead observed in younger disks. This fact is attributed to a decrease of gas in the disk. According to Pascucci et al. (2006) there is less than 0.1 Jupiter mass of gas in disks that are ∼10 Myr old or older, implying that giant planet formation should occur in less than 10 Myr, in agreement with current giant planet formation theories. Along with time evolution, we also have evidence for the eﬀect of diﬀerent UV ﬁelds from the diﬀerent types of molecu- lar species detected toward stars of diﬀerent luminosities. While stars that are a few times more massive than the Sun with stronger UV lack almost any molecular detections in their spec- tra (Fedele et al. 2011; Pontoppidan et al. 2010), disks around very low-mass stars present a rich chemistry (Pascucci et al. 2009). What remains to be seen is how much of the chemistry in the disk mid-plane is impacted by the diﬀerent radiation ﬁelds and what are the eﬀects on the composition of forming planets. A98, page 6 of 14 4.4. The chemistry of protoplanetary disks and the effect of stellar UV irradiation Porous aggregates of carbon, water ice, and silicates can also ﬁt the scattered light and IR spectral energy distribution (SED) of HR 4796A, providing an alternate explanation (Köhler et al. 2008). However, there are problems with this interpretation, in- cluding whether water ice should survive in the UV environment of an A star (Grigorieva et al. 2007). 0 50 100 150 200 Scattering Angle 100 1000 Normalized Surface Brightness Porous Ice mixtures Tholins Furthermore, neither pure tholins nor porous aggregates ad- equately ﬁt the observed phase function of HR 4796A’s ring in the visible (Debes et al., in prep.). Analysis of the HR 4796A ring surface brightness in the visible as a function of scattering angle for the north-east and south-west lobes of the disk show a remarkably ﬂat inferred phase function for the constituent dust. Figure 3 shows that the data are poorly ﬁt by either a Henyey- Greenstein phase function (as assumed by Köhler et al. 2008) or Mie scattering phase functions of pure micron-sized tholin grains (as assumed by Debes et al. 2008). Further study is re- quired to determine the composition of the dust in the HR 4796A disk and how representative it is compared to other debris disk systems. More information on the “nature” versus “nurture” problem at the late stages of planet formation may come as more debris disks are observed in scattered light in the visible and near-IR. Fig. 3. (Top left) Scattering eﬃciency Fdisk/F⋆as a function of wave- length for the HR 4796A debris disk. Horizontal error bars represent the width of the ﬁlter for each image of the disk, and the solid line is the best ﬁtting mixture of Titan tholins and astronomical silicates (amin = 1 μm (grain size), 20% Silicates, 80% Tholins). (Top right) Contours of the 68.5% and 98% conﬁdence levels for the models of HR 4796A’s scat- tered light as a function of minimum grainsize and Tholin fraction. The X represents the best ﬁtting model with a χ2 ν = 0.98. (Bottom) Relative scattered light surface brightness at visible wavelengths for the HR 4796A debris disk as a function of inferred scattering angle for dust grains in the disk. Grey squares represent the south-west lobe of the ring corrected for the brightness asymmetry present in the ansae of the disk (Schneider et al. 2009) while black squares represent the north-east lobe. 5. Observational evidence in the solar system Having laid the foundations of the investigation by studying the building blocks of the solar system, we proceed to examine the observational evidence at increasing distance from the Sun. We ﬁrst search for evidence of organic signatures and try to quan- tify the organic matter content in the interplanetary material as well as meteoritic samples analyzed in the laboratory. We follow up on this with a systematic search for signs of organics in the spectra and photometric colors of small bodies (i.e. asteroids, satellites, TNOs, comets) from the asteroids in the Main Belt to the region beyond Neptune. Lastly, we analyze the behavior of colors in the outer solar system looking for correlations with the quantity of ice. This scheme is based on the fact that, ac- cording to our current knowledge of the solar nebula formation and evolution, the quantity of H2O ice increases with decreasing temperature and increasing distance from the Sun. chemical routes to its formation have so far not been clearly elucidated. The D and 15N enrichments found in these mate- rials are consistent with synthesis in a cold medium through ion-molecule reactions, but the context where these enrichments took place (molecular cloud, proto-planetary disk) is unknown (e.g., Alexander et al. 2007). The origin of the soluble organic matter (SOM) – i.e. molecules released with organic solvents or water – has been attributed to the eﬀects of hydrothermal pro- cesses in the parent body (Alexander et al. 1998; Yabuta et al. 2005), in which SOM could be the hydrolysis product of IOM. An alternative view considers SOM as a component of the ices that were accreted along with refractory phases, and therefore with no connection with IOM (Remusat et al. 2005). Among and within chondrites, the IOM presents a wide range of isotopic and elemental compositions, as well as large chemical and structural variations (Alexander et al. 2007; Quirico et al. 2003). These variations are controlled by hetero- geneities in the precursors accreted by the parent bodies and by the eﬀects of post-accretion processes (Cody & Alexander 2005; Quirico et al. 2009). Post-accretion processes include low-temperature hydrothermalism (Cody & Alexander 2005; Yabuta et al. 2005; Orthous-Daunay et al. 2010a), long-duration radiogenic thermal metamorphism (Bonal et al. 2006a, 2007; 4.4. The chemistry of protoplanetary disks and the effect of stellar UV irradiation Once the gas disperses in a protoplanetary disk, gas phase chem- istry ceases and rocky bodies and dust are all that remain in a young planetary system. Often the collision or evaporation of planetesimals form second generation dusty debris disks. In these systems, the dust provides a unique remote laboratory to study the composition and thermal processing in the disk Interstellar clouds evolve into proto-planetary disks and then protostars. Observations of proto-planetary disks can be used to monitor the chemical variations during the early phases of planetary evolution and to infer the chemistry in the primordial A98, page 6 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems 0.0 0.5 1.0 1.5 2.0 2.5 Wavelength (μm) 3 4 5 6 7 8 9 Fdisk/F* (x10−4) 30 40 50 60 70 80 90 100 Fraction of Tholin Dust (%) 0 1 2 3 4 5 Grain Size (μm) 0 50 100 150 200 Scattering Angle 100 1000 Normalized Surface Brightness Porous Ice mixtures Tholins Fig. 3. (Top left) Scattering eﬃciency Fdisk/F⋆as a function of wave- length for the HR 4796A debris disk. Horizontal error bars represent the width of the ﬁlter for each image of the disk, and the solid line is the best ﬁtting mixture of Titan tholins and astronomical silicates (amin = 1 μm (grain size), 20% Silicates, 80% Tholins). (Top right) Contours of the 68.5% and 98% conﬁdence levels for the models of HR 4796A’s scat- tered light as a function of minimum grainsize and Tholin fraction. The X represents the best ﬁtting model with a χ2 ν = 0.98. (Bottom) Relative scattered light surface brightness at visible wavelengths for the HR 4796A debris disk as a function of inferred scattering angle for dust grains in the disk. Grey squares represent the south-west lobe of the ring corrected for the brightness asymmetry present in the ansae of the disk (Schneider et al. 2009) while black squares represent the north-east lobe. Overplotted are phase functions of a) a porous mixture of ices, silicates, and carbonaceous material assuming a Henyey-Greenstein ap- proximation to the phase function and b) Tholins assuming a Mie scat- tering model phase function. 4.4. The chemistry of protoplanetary disks and the effect of stellar UV irradiation Overplotted are phase functions of a) a porous mixture of ices, silicates, and carbonaceous material assuming a Henyey-Greenstein ap- proximation to the phase function and b) Tholins assuming a Mie scat- tering model phase function. 4.4. The chemistry of protoplanetary disks and the effect of stellar UV irradiation 0.0 0.5 1.0 1.5 2.0 2.5 Wavelength (μm) 3 4 5 6 7 8 9 Fdisk/F* (x10−4) 30 40 50 60 70 80 90 100 Fraction of Tholin Dust (%) 0 1 2 3 4 5 Grain Size (μm) (see Apai et al. 2010, for a recent review). Both scattered light and thermal emission from these disks can provide composi- tional information, on the processing of organic compounds. Unfortunately, many debris disks are cold and have few spec- tral features in the mid-IR. If they are spatially resolved in scat- tered light, they are often imaged at one or two wavelengths. Therefore, because of the infancy of such observations, more questions than answers remain. The most detailed scattered light analysis of a disk comes from observations of the HR 4796A debris disk observed in seven wavelength bands that span the visible to the near-IR. From the analysis of Debes et al. (2008), a mixture of silicates and tholins were found to be the best- ﬁtting explanation for the red character of the disk, assuming a purely Mie scattering model of solid spheres (See Fig. 3). Porous aggregates of carbon, water ice, and silicates can also ﬁt the scattered light and IR spectral energy distribution (SED) of HR 4796A, providing an alternate explanation (Köhler et al. 2008). However, there are problems with this interpretation, in- cluding whether water ice should survive in the UV environment of an A star (Grigorieva et al. 2007). (see Apai et al. 2010, for a recent review). Both scattered light and thermal emission from these disks can provide composi- tional information, on the processing of organic compounds. Unfortunately, many debris disks are cold and have few spec- tral features in the mid-IR. If they are spatially resolved in scat- tered light, they are often imaged at one or two wavelengths. Therefore, because of the infancy of such observations, more questions than answers remain. The most detailed scattered light analysis of a disk comes from observations of the HR 4796A debris disk observed in seven wavelength bands that span the visible to the near-IR. From the analysis of Debes et al. (2008), a mixture of silicates and tholins were found to be the best- ﬁtting explanation for the red character of the disk, assuming a purely Mie scattering model of solid spheres (See Fig. 3). 5.1. Structural and chemical characterization of organic matter in meteoritic chondrites and interplanetary dust In order to identify the source of reddening in meteoritic chon- drites and interplanetary dust particles (IDPs) we separate the organic matter into its main components. In order to identify the source of reddening in meteoritic chon- drites and interplanetary dust particles (IDPs) we separate the organic matter into its main components. Insoluble organic matter (IOM) is the predominant carbon material in chondrite meteorites, stratospheric IDPs and antarc- tic micrometeorites (AMMs). The given environment and the A98, page 7 of 14 A&A 533, A98 (2011) 2.8 3.0 3.2 3.4 3.6 3.8 4.0 0.04 0.05 0.06 0.07 0.08 24 Themis 65 Cybele PAHs Orgueil 2.8 3.0 3.2 3.4 3.6 3.8 4.0 0.04 0.05 0.06 0.07 0.08 24 Themis 65 Cybele PAHs Orgueil Fig. 4. Reﬂectance spectra of 65 Cybele and 24 Themis from Licandro et al. (2011) show similarities in the band between 2.85 and 3.5 μm. The model shown for 24 Themis (Fig. 1, Rivkin & Emery 2010) and that for 65 Cybele, include water ice, but not organics. A spectrum of the insoluble organic material Orgueil (Orthous-Daunay et al. 2010b) and one showing an average of six polycyclic aromatic hydrocarbons (Colangeli et al. 1992) are included for comparison. Busemann et al. 2007), and short-duration thermal metamor- phism (Yabuta et al. 2010). IOM in interplanetary dust particules (IDPs) and antarctic micrometeorites (AMMs) has a fairly, but not strictly, similar polyaromatic structure to that of chondrites (Quirico et al. 2005; Bonal et al. 2006b; Dobrica et al. 2009). Thermal eﬀects occurring during the slowing down of the par- ticles in Earth’s atmosphere could partly account for these dif- ferences. The fact that IOM is ubiquitous in IDPs, AMMs and chondrites precludes its formation in a parent body through hy- drothermalism (Flynn et al. 2003). This latter process, in com- parison with thermal metamorphism, has generally weak eﬀects on the composition of IOM. p IOM chemical heterogeneity is reﬂected in the de- tailed structure of the 3.4-μm band (aliphatic hydrocarbons) (Orthous-Daunay et al. 2010b). Therefore, tracking of the 3.4-μm band is a potential observational test to infer the pres- ence or absence of IOM in astrophysical contexts. For exam- ple in the DISM, infrared spectra point to the presence of an amorphous hydrogenated carbon material, and lack of IOM as observed in chondrites (Dartois et al. 2005). 5.2. The nature of asteroids 24 Themis and 65 Cybele 5.2. The nature of asteroids 24 Themis and 65 Cybele While a number of volatile organic molecules have been identiﬁed in the gas phase in cometary comae, of particular interest for the present study is the more refractory solid car- bonaceous component of comets. Fomenkova (1999) deduced, from mass spectrometer observations obtained from the comet P/Halley ﬂyby mission, that 60% of the carbon on Halley is in the form of complex organic material. This material aﬀects the albedo and color of a comet’s nucleus when spatially iso- lated or mixed with ices. It is of additional interest because this richly organic molecular material can be dispersed in both the inner and outer solar system as a consequence of the dynami- cal properties of comets. Refractory components of comet dust (organic solids plus minerals) have been collected by aircraft in the stratosphere (a component of interplanetary dust particles, as described above), and by the Stardust spacecraft during its encounter with 81P/Wild 2 (Sandford et al. 2006; Sandford 2008; Clemett et al. 2010). Rotationally-resolved infrared (2−4 μm) spectra of 24 Themis and 65 Cybele indicate that ice and organics are present on the surfaces of these asteroids (Campins et al. 2010; Rivkin & Emery 2010; Licandro et al. 2011) as indicated by the absorp- tion bands in the range 2.85 to 3.5 μm (Fig. 4). A spectrum of the IOM Orgueil (Orthous-Daunay et al. 2010b), highlighting the position of the 3.4 μm, and one showing the average of six polycyclic aromatic hydrocarbons (Colangeli et al. 1992), are also shown for comparison. These are the ﬁrst detections of wa- ter and organics on asteroid surfaces. Spectral models for these objects include a mixture of sili- cates, amorphous carbon and water ice and are shown in Fig. 4 as solid lines. Complex organics are not included in the model: their absence is highlighted by the discrepancy between model and observations. The presence of any surface ice on these asteroids is surprising because of the instability of exposed water ice at their heliocentric distances (∼3.3 AU). The presence of H2O ice and absence of hydrated silicates indicate that 24 Themis and 65 Cybele have not undergone the widespread aqueous alteration or thermal metamorphism rec- ognized in most carbonaceous meteorites. This argues against parent-body processes (such as those described above for SOM in meteorites) as a source of these asteroidal organics. an indication of presolar material (“nature”) or irradiation prod- ucts (“nurture”). an indication of presolar material (“nature”) or irradiation prod- ucts (“nurture”). g p Finally, we should note that some grains in IDPs and organic meteorites appear to have intact presolar organics that are highly enriched in deuterium, suggesting that at least some cold molec- ular cloud particles experienced relatively non-violent condi- tions on arrival in the nebula preserving their original signature, which would be consistent with the “nature” idea. It is especially striking how the highly enriched grains are found adjacent to less enriched grains in meteorites (Duprat et al. 2010; Busemann et al. 2006; Messenger 2002), implying that particles with a wide variety of histories have coalesced, which would probably sup- port a scenario of mixing between “nature” and “nurture”. The importance of sample return from various primitive bodies, per- haps not so far represented in the meteorite collections, therefore becomes evident. 5.3. Organic molecules in cometary components Comets are highly relevant to the broad issue of the nature and distribution of complex organic solid material in the solar sys- tem. Recent investigations have shown a great compositional variety among comets. The prevalence of solid opaque materi- als on their surfaces, either as a consolidated crust or as a mantle of loose particles, may distinguish short-period comets from dy- namically new comets. The surfaces of comets 9P/Tempel and 103P/Hartley were imaged in 2005 and 2010, respectively, by the Deep Impact spacecraft as part of its original mission and its extended EPOXI mission (see Fig. 5). Both comets appear to have surfaces that are a combination of crusted regions and regions covered with ﬁne particles, having low albedos and red- dish colors characteristic of a combination of maﬁc silicates and carbonaceous materials. 5.1. Structural and chemical characterization of organic matter in meteoritic chondrites and interplanetary dust The 3.4-μm band could also help connect chondrite chemical classes to asteroids, still an appreciable challenge, considering the currently achiev- able signal precision (Licandro et al. 2011). Fig. 4. Reﬂectance spectra of 65 Cybele and 24 Themis from Licandro et al. (2011) show similarities in the band between 2.85 and 3.5 μm. The model shown for 24 Themis (Fig. 1, Rivkin & Emery 2010) and that for 65 Cybele, include water ice, but not organics. A spectrum of the insoluble organic material Orgueil (Orthous-Daunay et al. 2010b) and one showing an average of six polycyclic aromatic hydrocarbons (Colangeli et al. 1992) are included for comparison. Fig. 4. Reﬂectance spectra of 65 Cybele and 24 Themis from Licandro et al. (2011) show similarities in the band between 2.85 and 3.5 μm. The model shown for 24 Themis (Fig. 1, Rivkin & Emery 2010) and that for 65 Cybele, include water ice, but not organics. A spectrum of the insoluble organic material Orgueil (Orthous-Daunay et al. 2010b) and one showing an average of six polycyclic aromatic hydrocarbons (Colangeli et al. 1992) are included for comparison. A98, page 8 of 14 5.4. The surface composition of Jupiter Trojans A frac- tion of cometary PAHs containing N atoms in the form of aro- matic nitriles represent a signiﬁcant diﬀerence from meteorite organics (Clemett et al. 2010). Both O and N atoms are found in a variety of bonding states, while some particles contain abun- dant C atoms that are not in aromatic structures (Sandford 2008). Those particles lacking “aromatization” are instead more likely to support the “nature” situation. Initial compositional interpretations ascribed their low albe- dos and red, featureless visible spectral slopes at least partly to the presence of organics (Gradie & Veverka 1980). Comparisons with comets and other outer solar system bodies are not uncom- mon (e.g., Jewitt 2002), given the similarly dark, spectrally red surfaces, but many low-albedo Main Belt asteroids also exhibit many of the same spectral qualities. Detecting and characteriz- ing the possible organic component of JTs would move us a step closer to understanding the origin and evolutionary processes important for small bodies throughout the solar system. p g y Statistical analyses of the featureless reﬂectance spectra have provided some insights. Szabó et al. (2007) reported a corre- lation between visible colors (from Sloan Digital Sky Survey [SDSS] measurements) and orbital inclination for JTs that man- ifested itself as an apparent bimodal distribution of spectral slopes. Roig et al. (2008) conﬁrm the SDSS bimodal spec- tral slope distribution and ﬁnd a similar distribution in previ- ously published visible spectra. Emery et al. (2011) identify a much stronger bimodality in near-infrared spectral slopes of JTs. This bimodality is present in both the leading and trailing swarms and among the background (non-family member) JTs, and therefore appears to be a signature of the primordial pop- ulation rather than an artifact of collisional families or capture mechanisms. Spectral similarities between the redder group and some Centaurs and the less-red group and some outer Main Belt asteroids may indicate multiple source regions for JTs. Organic molecules such as CH4, C2H2, C2H6 and CH3OH have been identiﬁed in the gas phase of comets and in solid phases from IDPs and Stardust samples. It is therefore reason- able to infer that many short-period comet nuclei, the only ones we have been able to observe so far, have their low albedos and red colors, at least in part, due to the presence of a wide range of complex molecules. 5.4. The surface composition of Jupiter Trojans Jupiter Trojans (JTs) are one of the most intriguing populations of small bodies in the solar system. Currently residing in sta- ble orbits in the Lagrangian points of Jupiter, JTs are believed to have formed at relatively large heliocentric distances, where the solar nebula was rich in frozen volatiles and organics (Dotto et al. 2008; Marzari et al. 2002). Jupiter Trojans (JTs) are one of the most intriguing populations of small bodies in the solar system. Currently residing in sta- ble orbits in the Lagrangian points of Jupiter, JTs are believed to have formed at relatively large heliocentric distances, where the solar nebula was rich in frozen volatiles and organics (Dotto et al. 2008; Marzari et al. 2002). The origin of JTs and how they were trapped in librating or- bits around the Jupiter Lagrangian points are still a matter of de- bate. Several mechanisms have been so far proposed, including capture of fragments of Jovian satellites, trapping of planetesi- mals orbiting nearby a proto-Jupiter, mass growth of the planet or gas drag capture of short-period comets (Marzari et al. 2002). Morbidelli et al. (2005) suggested that JTs, like Kuiper belt ob- jects (KBOs) and the scattered disk, originated in the planetes- imal disk which drove the planetary migration. Therefore, the analysis of the physical properties, in particular the composition of JTs, can help us place important constraints on their origin and early evolution. Fig. 5. The surfaces of comets 9P/Tempel and 103P/Hartley imaged, in 2005 and 2010, respectively, by the Deep Impact spacecraft in its original mission and its extended EPOXI mission. P/Tempel is 7.6 km across and Hartley is 2.2 km in its long dimension. The ﬁgure shows them to the same scale. Image credit: NASA/JPL-Caltech/UMD. exactly. The Stardust mission particles show extreme hetero- geneity (Sandford et al. 2006); some show evidence of a high degree of heating expected from formation in the inner solar sys- tem, while others could not have survived such temperatures and must have originated further out. These data corroborate what was previously known from cometary IR spectra showing the existence of crystalline silicates in comets in contrast to their apparent scarcity in the ISM (Ehrenfreund et al. 2004, and ref- erences therein). This is also an argument in favor of mixing of “high-temperature” and “low-temperature” materials in the comet-forming region. 5.2. The nature of asteroids 24 Themis and 65 Cybele The presence of C-H bonds and possibly aromatic groups could be The organic molecules of cometary origin in the Stardust samples are similar, in many respects, to those found in the matrix material of some carbonaceous chondrite meteorites and IDPs. A detailed analysis of the X-ray absorption spectra of the Stardust particles by Wirick et al. (2009) has shown that these spectra are closer to the spectra of IDPs than those of carbonaceous chondrites, but do not match either category A98, page 8 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Fig. 5. The surfaces of comets 9P/Tempel and 103P/Hartley imaged, in 2005 and 2010, respectively, by the Deep Impact spacecraft in its original mission and its extended EPOXI mission. P/Tempel is 7.6 km across and Hartley is 2.2 km in its long dimension. The ﬁgure shows them to the same scale. Image credit: NASA/JPL-Caltech/UMD. comet nuclei may include complex molecules analogous to the soluble fraction in the meteorites (e.g., amino and hydroxylic acids, and nitrogen heterocycles). Therefore, comets are plausi- bly a mixture of native and irradiation products, with their initial composition diﬀering and being a function of their place of for- mation (Ehrenfreund et al. 2004, and references therein), and advocating for both the “nature” and “nurture” scenarios. 5.4. The surface composition of Jupiter Trojans The “high temperature” group supports the idea of a “nurture” scenario, while the “low-temperature” one could be presolar in origin and promote the “nature” alter- native. Aliphatic compounds appear to have withstood temper- atures of ∼550 C, probably because of protection by minerals such as sulﬁdes. Speciﬁc identiﬁcations include PAHs, with a predominance of single (benzene) and double ring (naphthalene) molecules, but also including much larger PAHs (extending up to ∼800 a.m.u.), as well as their alkylation series resulting from the successive addition of CH2- subunits. However, it is possible that PAHs formed as a result of the high temperature exposure, which would vouch for “nurture” rather than “nature”. A frac- tion of cometary PAHs containing N atoms in the form of aro- matic nitriles represent a signiﬁcant diﬀerence from meteorite organics (Clemett et al. 2010). Both O and N atoms are found in a variety of bonding states, while some particles contain abun- dant C atoms that are not in aromatic structures (Sandford 2008). Those particles lacking “aromatization” are instead more likely to support the “nature” situation. exactly. The Stardust mission particles show extreme hetero- geneity (Sandford et al. 2006); some show evidence of a high degree of heating expected from formation in the inner solar sys- tem, while others could not have survived such temperatures and must have originated further out. These data corroborate what was previously known from cometary IR spectra showing the existence of crystalline silicates in comets in contrast to their apparent scarcity in the ISM (Ehrenfreund et al. 2004, and ref- erences therein). This is also an argument in favor of mixing of “high-temperature” and “low-temperature” materials in the comet-forming region. The “high temperature” group supports the idea of a “nurture” scenario, while the “low-temperature” one could be presolar in origin and promote the “nature” alter- native. Aliphatic compounds appear to have withstood temper- atures of ∼550 C, probably because of protection by minerals such as sulﬁdes. Speciﬁc identiﬁcations include PAHs, with a predominance of single (benzene) and double ring (naphthalene) molecules, but also including much larger PAHs (extending up to ∼800 a.m.u.), as well as their alkylation series resulting from the successive addition of CH2- subunits. However, it is possible that PAHs formed as a result of the high temperature exposure, which would vouch for “nurture” rather than “nature”. 5.4. The surface composition of Jupiter Trojans Such molecular complexes can occur both in recognizable structures and in more complex aggregates sim- ilar to kerogen-like materials in the insoluble organic matter in carbonaceous meteorites. Similarly, the volatile fraction in Of particular interest in the present context are direct searches for organic materials. Strong fundamental vibrational bands of C-H, O-H, and N-H bonds in complex organics occur at wavelengths in the 2.7 to 3.6 μm region, and overtones and com- binations of these, though signiﬁcantly weaker, occur at shorter wavelengths in the reﬂectance spectra of organics. Several au- thors (e.g. Cruikshank et al. 2001; Emery & Brown 2003; Dotto et al. 2006; Fornasier et al. 2007; de Luise et al. 2010; Emery et al. 2011; Yang & Jewitt 2011) have published visible and near- infrared reﬂectance spectra of JTs. Despite the expectation that A98, page 9 of 14 A&A 533, A98 (2011) JTs formed with abundant volatiles and organics, as for KBOs, they do not appear to exhibit any of the spectral features of these materials on their surfaces. However, as there is more than an or- der of magnitude diﬀerence in the solar ﬂux on Trojans, at 5 AU, compared to the KBOs, the diﬀerence in spectral properties of the uppermost surfaces of Trojans and KBOs does not necessar- ily preclude a similar origin. A thermal model of the evolution of Iapetus’ surface (Spencer & Denk 2010), in which H2O ice is mobilized by the higher tem- peratures in the dark hemisphere and re-condensed elsewhere, convincingly reproduces the albedo pattern seen today. An absorption band at 3.28 μm, attributed to polycyclic aro- matic hydrocarbons, has been identiﬁed in the low-albedo mate- rial on Iapetus (Cruikshank et al. 2008). The data for that study came from the Visible- Infrared Mapping Spectrometer (VIMS) aboard the Cassini spacecraft that has been operating in orbit around Saturn since late 2004. The detection of PAHs is the ﬁrst clear identiﬁcation of a signiﬁcant chemical component. Current studies are attempting to discern which of the two low-albedo components presents the PAH signature. The characterization of the dark components of Iapetus is important as it sheds light on the origin of the materials and their evolution, as well as the “na- ture” versus “nurture” question regarding the satellites of Saturn. Furthermore, the featureless visible spectra seem to rule out a signiﬁcant quantity of hydrated silicates, crystalline an- hydrous silicates, organic materials and ices. 5.6. The surface composition of trans-neptunian objects and Centaurs The TNOs or KBOs are small icy bodies (the diameters of known objects range between about 50 and 3000 km) that orbit the Sun beyond Neptune. More than 1200 of them have been dis- covered so far. A number of spectroscopic and dynamical stud- ies (Barucci et al. 2008a, and references therein) have shown that TNOs are remnants of the formation of the solar system that have been subjected to minimal chemical evolution since their formation. Their upper surface layers have however been altered by various space weathering processes (solar and cosmic ion irradiation, collisions) and, for the largest of them, surface- atmosphere interaction and, possibly, cryovolcanism. A study of their surface compositions can therefore provide clues on solar nebula composition and solar system evolution as well as the origin of their reddening material. The detection of organic features in the 3.3 to 3.5 μm region on two outer Main Belt asteroids, 24 Themis and 65 Cybele, as noted in Sect. 5.2, is instructive. Only one of the JT spectra has high enough S/N in this wavelength range to have detected a Themis- or Cybele-like absorption feature. It therefore remains possible that, though not yet detected, organics may be a minor component on the surfaces of JTs. It is likely though, considering their relative proximity to the Sun, that their organic material, if present, could be partially the product of the irradiation of the original surface components. Based on the amount of processing that these surface have undergone the “nurture” scenario would likely be the prevalent one. The dynamical evolution of TNOs is believed to have been cataclysmic. A currently popular dynamical model (the Nice model, Levison & Morbidelli 2003) describes a series of events in the young solar system that caused the migration of the outer giant planets Neptune and Uranus, and the scattering of the plan- etesimals left over from their formation. TNOs are distinguished (Elliot et al. 2005; Gladman et al. 2008) into several diﬀerent classes that describe their current locations and orbital character- istics: the classical objects, the resonant objects (such as Pluto, which is in 2:3 orbital resonance with Neptune), the scattered disk objects and the detached objects (like Sedna). In addition, Centaurs (such as Pholus or Chiron) have orbits between those of Jupiter and Neptune. 5.6. The surface composition of trans-neptunian objects and Centaurs They may be escapees from the Kuiper belt (former TNOs), they are therefore studied in parallel with TNOs, but being closer to us, they are usually brighter. 5.4. The surface composition of Jupiter Trojans Amorphous sili- cates have been suggested to model the red slopes of JT spectra (Cruikshank et al. 2001; Emery & Brown 2004), but feature- less spectra provide at best a weak constraint on inferred com- positions. However, thermal emission spectra of several Trojans show strong spectral emissivity peaks near 10 and 20 μm, in- dicating the presence of ﬁne-grained, largely amorphous, anhy- drous silicates (Emery et al. 2006; Mueller et al. 2010). An absorption feature at ∼2.3 μm found in the spectra of the Centaur Pholus and the TNO 2002 VE95 has been attributed to organic materials (Cruikshank et al. 1998; Barucci et al. 2006), and speciﬁcally to frozen CH3OH or (less likely) hexamethylte- tramine. However, near-infrared spectra of ∼100 JTs have been published by various authors, and none of them show this fea- ture. Spectroscopic searches for the strong fundamental absorp- tions at longer wavelengths (2.8 to 4.0 μm) have been published for eight JTs. No absorption features have been found in these spectra, which strongly limits the type and abundance of organic material on the surfaces of JTs (Cruikshank et al. 2001; Emery & Brown 2004). C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Quaoar best ﬁtting model (solid thin line) includes about 30% H2O, 20% N2, and 13% CH4 with traces of C2H6 ice mixed in with organic materials (Dalle Ore et al. 2009). Hydrocarbon ices features are labeled in the ﬁgure. Quaoar is an RR object. The observations were scaled to Stansberry et al. (2008) visual geometric albedo. Fig. 6. Quaoar best ﬁtting model (solid thin line) includes about 30% H2O, 20% N2, and 13% CH4 with traces of C2H6 ice mixed in with organic materials (Dalle Ore et al. 2009). Hydrocarbon ices features are labeled in the ﬁgure. Quaoar is an RR object. The observations were scaled to Stansberry et al. (2008) visual geometric albedo. Table 1. Selected objects’ compositions. Table 1. Selected objects’ compositions. Object Dyn Taxon a∗ % % % class reddening darkening ices 5145 Pholusa C RR 20.3 34 61 15 8405 Asbolusb C BR 18.1 32 59 9 1999TC36c R RR 39.7 70 22 8 50000 Quaoard C RR 43.6 27 0 73 90377 Sednae D RR 518.6 76 0 24 90482 Orcusf R BB 39.2 1 58 40 Notes. (∗) Semi-major axis measurements (a) are from JPL Small-Body Database Browser at http://ssd.jpl.nasa.gov/sbdb.cgi References. (a) Cruikshank et al. (1998); (b) Poulet et al. (2002); (c) Dotto et al. (2003); (d) Dalle Ore et al. (2009); (e) Barucci et al. (2010); (f) Delsanti et al. (2010). p y μ When spectroscopic data are not available, colors have been obtained for a relatively large sample of objects (around 200). TNOs as well as Centaurs have very diverse colors, from neutral to very red. A taxonomic scheme based on multivariate statis- tics of the photometric color was proposed by Barucci et al. (2005): four classes were identiﬁed indicating diﬀerent composi- tion and/or evolutional history, with increasingly red colors from the BB (blue objects) to the RR class (reddest objects). The rela- tionship between taxonomical and dynamical classiﬁcations has been investigated in detail (Perna et al. 2010) yielding somewhat inconclusive results. No direct correlation between color and dy- namical parameters (in particular semi-major axis) was found. However, recent work by Romanishin et al. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems (2010) shows some evidence for a color gradient with distance from the Sun, if the Inner Classical KBOs (ICKBOs) are considered to be an exten- sion of the Cold Classical population: the Cold Classical objects are predominately red, and the Inner Classical belt objects are a mixture of neutral and red. function of distance. However, the measured semi-major axes describe the current position of these objects, after the migration of Neptune had scattered them: it therefore comes as no surprise that their compositions do not relate to their locations. Out to the Kuiper belt, we expect that current weathering from solar irradiation would aﬀect the surface compositions as a function of distance from the Sun, with the closest objects being most processed. This picture becomes more complicated at distances beyond 50 AU where cosmic rays become pre- dominant (Cooper et al. 2006). As seen in Sect. 5.4 close to the Sun weathering happens on very short timescales, imply- ing that some weathering eﬀects should be seen. In fact, we do see the eﬀects of weathering on the IKBOs, as mentioned above. However, for the TNOs, based on the complete lack of correlation between their composition, and their current semi- major axis, particularly for those that are within the 50 AU ra- dius, two possible explanations become apparent. The ﬁrst is that not a lot of weathering has happened since Neptune mi- gration repositioned objects to their current locations. The sec- ond that the collision rate is comparable to the weathering rate, keeping the surfaces fresh (Melita et al. 2009). In both cases the direct implication is that the weathering that we observe, at least in some of the objects (i.e., the presence of a population of objects with highly processed, dark materials, such as Orcus), must have preceded the migration and must have happened on a short timescale early on. When looking at the TNOs, a limited number of high resolu- tion TNO spectra have been modeled using a variety of com- positions that include several ices, carbonaceous, and organic materials varying in proportion from object to object. Figure 6 shows the best ﬁtting model of Quaoar, an RR object, orbit- ing at about 44 AU. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.2 0.4 0.6 0.8 1.0 Geometric Albedo Wavelength (μm) (50000) Quaoar Dalle Ore et al. (2009) N2 C2H6 CH4 Fig. 6. Quaoar best ﬁtting model (solid thin line) includes about 30% H2O, 20% N2, and 13% CH4 with traces of C2H6 ice mixed in with organic materials (Dalle Ore et al. 2009). Hydrocarbon ices features are labeled in the ﬁgure. Quaoar is an RR object. The observations were scaled to Stansberry et al. (2008) visual geometric albedo. 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 0.2 0.4 0.6 0.8 1.0 Geometric Albedo (50000) Quaoar Dalle Ore et al. (2009) N2 C2H6 CH4 simple distribution was apparently altered by giant planet migra- tion and planetesimal scattering like that described in the Nice model, yielding the much more complex picture that we observe today. Another complication in the unraveling of the chemical his- tory of solar system bodies lies with the processing due to so- lar and cosmic radiation that has weathered their surfaces, more eﬀectively for those closer to the Sun. The best candidates for our study are probably the smaller TNOs, which are possibly far enough from the Sun to be suﬃciently pristine to be useful in our investigation. Larger ones are not as reliable as internal melting and diﬀerentiation occurred in their history. Information about TNO and Centaur surface compositions or physical properties comes from albedo measurements, color measurements, polarimetric observations and spectroscopy. The measured albedos are low (between 0.04 and 0.20), except for a few objects that have albedos of about 0.6 (Pluto, Makemake, Haumea) or even higher (∼0.86 for Eris, Stansberry et al. 2008 and 2000 TX300; a member of the Haumea family, Elliot et al. 2010). Their generally low albedos, their distances, and their sizes make them very faint and therefore diﬃcult to observe. Even with the largest telescopes available (8 to 10 m class), only the brightest TNOs can be studied spectroscopically to obtain compositional information. Furthermore, the available TNO spectra extend only to about 2.5 μm (Barucci et al. 2008b), a limiting factor when trying to identify surface components whose fundamental spectral features often fall beyond 2.5 μm. Wavelength (μm) Wavelength (μm) Fig. 6. 5.5. Polycyclic aromatic and aliphatic hydrocarbons on Saturn’s satellite Iapetus Iapetus, Saturn’s third largest satellite, is an icy moon show- ing spectral signatures of H2O ice over more than half its sur- face. The low-albedo material on Iapetus’ surface is concen- trated primarily on the leading hemisphere in the sense of the satellite’s locked synchronous orbital motion around Saturn, but telescopic studies undertaken since the discovery of the albedo dichotomy by G. D. Cassini in 1671 have failed to convincingly establish the composition of the dark material. New studies, based on data from the Cassini spacecraft demonstrate that there are two low-albedo surface components, one very red in color and the other more nearly neutral (Denk et al. 2010), one native to Iapetus, the other exogenous in nature. The native material is probably ubiquitously present to some degree and was originally evenly distributed on the surface. Both low-albedo components contribute to the dark coloration of the leading hemisphere of Iapetus and are the cause of a temperature enhancement detected in this area of the satellite surface. They are ultimately respon- sible for the material segregation on the surface of the satellite. The chemical history of TNOs is unfortunately not as clearly understood. The concept of “snow lines” in the original neb- ula, i.e. the distance from the Sun at which materials can con- dense, provides us with some idea of what should have been the distribution of materials before the neptunian migration. In fact, we can envision that planetesimals, originally formed close to Jupiter, have little more than rock, metal, and water ice as part of their composition. On the other hand, planetesimals originating outward of Saturn would consist of more volatile ices as well as water ice and rock and metal depending on their distance from the Sun and size (Schaller & Brown 2007). As a result, we ex- pect that the original material distribution would have followed a composition pattern from simple to more complex in travel- ing from the inner to the outer solar system. Unfortunately this A98, page 10 of 14 C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Acknowledgements. This paper is based on the conclusions of the work- shop “Organic material in planetary system: “nature” or “nurture”?” held on May 27−29, 2010 at the Centre International d’Ateliers Scientiﬁques de l’Observatoire de Paris with the participation of all the authors. C.M.D.O. aknowledges support from the Centre International d’Ateliers Scientiﬁques de l’Observatoire de Paris. N.P.A. acknowledges support from the NASA postdoc- toral program at Ames Research Center administered by Oak Ridge Associated Universities through a contract with NASA. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems Its composition is listed among a few rep- resentative cases in Table 1, where the published quantities of the diﬀerent components have been grouped together into three categories: reddening components (mostly tholins), darkening components (mostly amorphous carbon) and ices (hydrocar- bons, H2O, CO2, CO, CH3OH ices). From a compilation of objects with spectra available in the literature (examples shown in Table 1) it appears that composition and semi-major axis (a) also do not correlate: ice abundance does not map with distance and likewise objects with a high abundance of processed dark materials do not orbit closest to the Sun. Grundy’s idea (i.e. the “nature” scenario) of the reddening material being primarily primordial in origin, would imply that the color of the objects vary with the quantity of ice and as a A98, page 11 of 14 A98, page 11 of 14 A&A 533, A98 (2011) Table 2. “Nature” vs. “nurture” summary. Evidence “Nature” “Nurture” ? Laboratory studies of refractory carbonaceous materials X X – Ion irradiation and colors of C-rich materials – X – The nature of interstellar organics X – – Hydrocarbonaceous dust in the ISM X – – Dense clouds X – – Evolution of organics from diﬀuse interstellar clouds to the solar system X – – Chemistry of protoplanetary disks – – X Evidence for organics in dusty disks – – X Refractory organic matter in chondrites and interplanetary dust X X – Asteroids 24 Themis & 65 Cybele – – X Comets X X – The surface composition of Jupiter Trojans – – X PAHs and aliphatic hydrocarbons on Iapetus X X – The surface composition of trans-Neptunian objects and centaurs X X – Some of the key factors outlined above are within reach with the current state-of-the-art telescopes, and observing instruments (e.g. d above), others are far from being solvable. We can therefore conclude, for the TNO case, that the “nature” reddening mechanism, whose traces are apparent in those objects with a lot of ices and red coloration, as well as the “weathering” component visible in the processed materials (“nurture”) could be interpreted, for some of the objects, as to date from a time previous to the neptunian migration. 6. Conclusions We present an examination of some recent evidence, encom- passing a number of laboratory experiments as well as obser- vational data, in an eﬀort to characterize the reddening material common to planetary systems. The available information, dis- cussed at length among the authors, leads us to infer that the red coloration of a variety of objects in the solar system could be due mainly to one of two mechanisms, or more likely to a com- bination of them: weathering and primordial composition, i.e. “nurture” and “nature”. Nevertheless, materials such as PAHs, indicative of the pres- ence of organics, are diﬃcult to interpret, as their origin could be due to “nurture” as well as “nature”. The spectroscopic ef- fects of the “nature” and “nurture” mechanisms on the icy ma- terial are practically indistinguishable: while intrinsically diﬀer- ent, macroscopically, they both reduce the ratio of H to C atoms. Further, with the current state of the art instruments we cannot yet detect the signature features of the organic material, whether primordial or resulting from irradiation, in most of the available observations. A few exceptions include Cassini VIMS data for the Saturn satellites, Triton, Pluto, 24 Themis and 65 Cybele data. Laboratory work has given, and is still in the process of giv- ing, us a few tools to aid us in discriminating between the diﬀer- ent choices. In this paper we explore in the observations avail- able in the literature the evidence for one process or the other, and view them in the frame of the knowledge acquired through the laboratory studies. Finally, a model of the dynamical evolution of the outer solar system has to be kept in the picture as the current location of the objects probably does not reﬂect their original position, therefore complicating the history of the weathering contributions. Table 2 summarizes the current status of the “nature” vs. “nurture” debate. In summary, the question at the basis of the “nature” vs. “nur- ture” debate, i.e. whether ice indeed magniﬁes the eﬀect of the reddening material, is still to be fully addressed. While some as- pects of the problem have been answered or will be in the near future, such as the determination of the amount of ice present in red objects, others, such as the ability to discriminate between reddening agents, are still far from being solvable. C. M. Dalle Ore et al.: Organic materials in planetary and protoplanetary systems At that time both mechanisms might have played a role, but their rela- tive importance is too diﬃcult to trace as their eﬀect on the spec- trum of the material is so macroscopically similar as to make it impossible, at this time, to discern the importance of one vs. the other. The identiﬁcation of the material dwells primarily on the ability to obtain spectroscopic data past the 2.5 μm current limit for a number of well chosen objects representing the main tax- onomic groups of TNOs, Centaurs, asteroids and Trojans in the hope that the behavior in the near-IR will shed light on their compositions, help map the reddening material(s) throughout the outer solar system, and distinguish minerals from complex organics. This information along with dynamical data might elucidate its origin and weathering level when coupled with lab- oratory experiments aimed at testing the eﬀects of radiation and other weathering mechanisms in various environments and guide us in the interpretation of the observations. (Tucson: Univ. Arizona Press), 725 F., Tsiganis, K., & Gomes, R. 2005, Nature, 435, 462 Moroz L Baratta G Strazzulla G et al 2004 Icarus 170 214 orbidelli, A., Levison, H. F., Tsiganis, K., & Gomes, R. 2005, Nature Cooper, J. F., Hill, M. E., Richardson, J. D., et al. 2006, Physics of the Inner Heliosheath, 858, 372 Moroz, L., Baratta, G., Strazzulla, G., et al. 2004, Icarus, 170, 214 Muñoz Caro, G. M., Matrajt, G., Dartois, E., et al. 2006, A&A, 459, 147 kshank, D. P., Roush, T. L., Bartholomew, M. J., et al. 1998, Icarus, Mueller, M., Marchis, F., Emery, J. P., et al. 2010, Icarus, 205, 505 Cruikshank, D. P., Dalle Ore, C. M., Roush, T. L., et al. 2001, Icarus, 153, 348 Najita, J. R., Carr, J. S., Strom, S. E., et al. 2010, ApJ, 712, 274 Cruikshank, D. P., Imanaka, H., & Dalle Ore, C. M. 2005, Adv. Space Res., 36, 178 Nakamura-Messenger, K., Messenger, S., Keller, L. P., et al. 2006, Science, 314, 1439 Cruikshank, D. P., Wegryn, E., Dalle Ore, C. M., et al. 2008, Icarus, 193, 334 Öberg, K. I., Qi, Ch., Fogel, J. K. J., et al. 2010, ApJ, 720, 480 Öberg, K. I., Qi, Ch., Fogel, J. K. J., et al. 2010, ApJ, 720, 480 Oro, J. 1961, Nature, 191, 1193 Cuzzi, J., Clark, R., Filacchione, G., et al. 2009, Saturn from Cassini-Huygens (Springer), 459 Oro, J. 1961, Nature, 191, 1193 Oro, J. 1961, Nature, 191, 1193 Orthous-Daunay, F.-R., Quirico, E., Lemelle, L., et al. 2010a, E&PSL, 300, 321 Orthous-Daunay, F.-R., Quirico, E., Beck, P., et al. 2010b, LPI, 1533, 1803 Dalle Ore, C. M., Barucci, M. A., Emery, J. P., et al. 2009, A&A, 501, 349 y Orthous-Daunay, F.-R., Quirico, E., Beck, P., et al. 2010b, LPI, 1533, 1803 y Dartois, E., & Muñoz-Caro, G. M. 2007, A&A, 476, 1235 Dartois, E., & Muñoz-Caro, G. M. 2007, A&A, 476, 1235 Owen, T. C., Cruikshank, D. P., Dalle Ore, C. M., et al. 2001, Icarus, 149, 160 Dartois, E., Muñoz-Caro, G. M., Deboﬄe, D., & d’Hendecourt, L. 2004, A&A, 423, L33 Pascucci, I., & Sterzik, M. 2009, ApJ, 702, 724 Pascucci, I., Gorti, U., Hollenbach, D., et al. 2006, ApJ, 651, 1177 Pascucci, I., Gorti, U., Hollenbach, D., et al. 200 rtois, E., Muñoz Caro, G. M., Deboﬄe, D., et al. 2005, A&A, 432, Pascucci, I., Hollenbach, D., Najita, J., et al. 6. Conclusions The picture emerging from the various experimental and ob- servational pieces of evidence is a lot more complicated than the simple question of “nature” vs. “nurture” originally posed. There are a few factors playing an important role in the identiﬁcation of the reddening process: a) the identiﬁcation of the reddening material(s) (minerals vs. complex organics); b) the determination of its origin, i.e. primordial vs. recent; c) the assessment of its condition with respect to its original state (degree of weathering); d) the measurement of its contamination with other materials (ices or other). A98, page 12 of 14 Solar System Beyond Neptune, 91 Poulet, F., Cuzzi, J. N., Cruikshank, D. P., et al. 2002, Icarus, 160, 313 tto, E., Barucci, M. A., Boehnhardt, H., et al. 2003, Icarus, 162, 40 Pontoppidan, K. M., Salyk, C., Blake, G. A., et al. 2010, ApJ, 720, 887 Dotto, E., Fornasier, S., Barucci, M. A., et al. 2006, Icarus, 183, 420 Quirico, E., Raynal, P. I., & Bourot-Denise, M. 2003, M&PS, 38, 795 Dotto, E., Emery, J. P., Barucci, M. A., et al. 2008, The S Dotto, E., Emery, J. P., Barucci, M. A., et al. 2008, The Neptune (Tucson: University of Arizona Press), 383 Quirico, E., Borg, J., Raynal, P.-I., et al. 2005, Planet. Space Sci., 53, 1443 Neptune (Tucson: University of Arizona Press), 383 Quirico, E., Montagnac, G., Rouzaud, J. N., et al. 2009, E&PSL, 287, 18 (Tucson: Univ. Arizona Press), 725 Melita, M. D., Strazzulla, G., & Bar-Nun, A. 2009, Icarus, 203, 134 Busemann, H., Alexander, C. M. O’D., & Nittler, L. R. 2007, M&PS, 42, 1387 Busemann, H., Alexander, C. M. O’D., & Nittler, L. R. 2007, M&PS, 42, 1387 Campins, H., Hargrove, K., & Pinilla-Alonso, N. 2010, Nature, 464, 1320 C J S & N jit J R 2008 S i 319 1504 Mennella, A., Bersanelli, M., Butler, R. C., et al. 1999, ApJ, 524 , , , , , , , , Campins, H., Hargrove, K., & Pinilla-Alonso, N. 2010, Nature, 464, 1320 C J S & N ji J R 2008 S i 319 1504 Mennella, V. 2008a, ApJ, 682, L101 Campins, H., Hargrove, K., & Pinilla-Alonso, N. 2010, Nature, 464, 1320 Carr, J. S., & Najita, J. R. 2008, Science, 319, 1504 p g Carr, J. S., & Najita, J. R. 2008, Science, 319, 1504 Carr, J. S., & Najita, J. R. 2008, Science, 319, 1504 p Mennella, V. 2008b, IAU Symp., 251, 459 Chiar, J. E., & Pendleton, Y. J. 2008, in Organic Matter in Space, ed. S. Kwok, Chiar, J. E., & Pendleton, Y. J. 2008, in Organic Matter in Space, ed. S. Kwok, & S. A. Sanford (Cambridge: Cambridge Univ. Press), IAU Symp., 251, 35 y p Mennella, V. 2010, ApJ, 718, 867 & S. A. Sanford (Cambridge: Cambridge Univ. Press), IAU Symp., 251, 35 Mennella, V., Brucato, J. R., Colangeli, L., & Palumbo, P. 2002, ApJ, 569, 531 Chiar, J. E., Pendleton, Y. J., Allamandola, L., et al. 2011, ApJ, submitted Mennella, V., Baratta, G. A., Esposito, A., et al. 2003, ApJ, 587, 727 Mennella, V., Baratta, G. A., Esposito, A., et al. 2003, ApJ, 587 Clark, R. N., Brown, R. H., Jaumann, R., et al. 2008, Icarus, 193, 372 Messenger, S. 2002. P&SS, 50, 1221 Clemett, S. J., Sandford, S. A., Nakamura-Messenger, K., et al. 2010, M&PS, 45, 701 g Micelotta, E. R., Jones, A. P., & Tielens, A. G. G. M. 2010a, A&A, 510, A37 Micelotta, E. R., Jones, A. P., & Tielens, A. G. G. M. 2010b, A&A, 510, A36 Cody, G. D., & Alexander, C. M. O’D. 2005, GeCoA, 69, 1085 Micelotta, E. R., Jones, A. P., & Tielens, A. G. G. M. 2011, A&A, 526, A52 Colangeli, L., Mennella, V., Baratta, G. A., et al. 1992, ApJ, 396, 369 Morbidelli, A., Levison, H. (Tucson: Univ. Arizona Press), 725 2007, ApJ, 663, 383 Dartois, E., Geballe, T. R., Pino, T., et al. 2007, A&A, 463, 635 Pascucci, I., Apai, D., Luhman, K., et al. 2009, ApJ, 696, 143 Debes, J. H., Weinberger, A. J., & Schneider, G. 2008, ApJ, 673, L191 Pendleton, Y. J., & Allamandola, L. J. 2002, ApJS, 138, 75 Delsanti, A., Merlin, F., Guilbert-Lepoutre, A., et al. 2010, A&A, 520, A40 Pendleton, Y. J., Sandford, S. A., Allamandola, L. J., et al. 1 Pendleton, Y. J., Sandford, S. A., Allamandola, L. J., et al. 1994, ApJ, 437, 683 de Luise, F., Dotto, E., Fornasier, S., et al. 2010, Icarus, 209, 586 Perna, D., Barucci, M. A., Fornasier, S., et al. 2010, A&A, 510, Denk, T., Neukum, G., Roatsch, T., et al. 2010, Science, 327, 435 Petrie, S., Stranger, R., & Duley, W. W. 2003, ApJ, 594, 869 Dobrica, E., Engrand, C., Quirico, E., et al. 2009, LPI, 40, 1688 g y p Pety, J., Teyssier, D., Foss’e, D., et al. 2005, A&A, 435, 885 Pety, J., Teyssier, D., Foss’e, D., et al. 2005, A&A, 435, 885 Doressoundiram, A., Boehnhardt, H., Tegler, S. C., & Trujillo Doressoundiram, A., Boehnhardt, H., Tegler, S. C., & Trujillo, C. 2008, The Solar System Beyond Neptune, 91 Pino, T., Dartois, E., Cao, A.-T., et al. 2008, A&A, 490, 665 References Gladman, B., Marsden, B. G., & Vanlaerhoven, C. 2008, The Solar System Beyond Neptune (Tucson: University of Arizona Press), 43 Gladman, B., Marsden, B. G., & Vanlaerhoven, C. 2008, The O’D. Alexander, C. M., Russell, S. S., Arden, J. W., et al. 1998, M&PS, 33, 603 O’D. Alexander, C. M., Russell, S. S., Arden, J. W., et al. 1998, M&PS, 33, 603 Alexander, C. M. O’D., Fogel, M., Yabuta, H., & Cody, G. D. 2007, GeCoA, 71, 4380 Gradie, J., & Veverka, J. 1980. Nature, 283, 840 Grigorieva, A., Thébault, P., Artymowicz, P., & Brandeker, A. 2007, A&A, 475, 755 Apai, D., Connolly, H. C., Jr., & Lauretta, D. S. 2010, Protoplanetary Dust: Astrophysical and Cosmochemical Perspectives, 230 Grundy, W. M. 2009, Icarus, 199, 560 y Grundy, W. M., & Stansberry, J. A. 2003, EM&P, 92, 331 Grundy, W. M., & Stansberry, J. A. 200 p y p Baratta, G. A., Brunetto, R., Leto, G., et al. 2008, JRSp, 39, 211 Güdel, M., Lahuis, F., Briggs, K. R., et al. 2010, A&A, 519, 113 p Barucci, A., Belskaya, I. N. Fulchignoni, M., & Birlan, M. 2005, Hoﬀmann, M., Fink, U., Grundy, W. M., & Hicks, M. 1993, JGR, 9 Hoﬀmann, M., Fink, U., Grundy, W. M., & Hicks, M. 1993, JGR, 98, 7403 Horner, J., & Lykawka, P. S. 2010, Internat. J. Astrobiol., 9, 227 Barucci, M. A., Merlin, F., Dotto, E., et al. 2006, A&A, 455, 725 Horner, J., & Lykawka, P. S. 2010, Internat. J. Astrobiol., 9, 227 Barucci, M. A., Boehnhardt, H., Cruikshank, D. P., et al. 2008a, The Solar System Beyond Neptune (Tucson: U. Arizona Press), 3 Imanaka, H., Khare, B. N., Elsila, J. E., et al. 2004, Icarus, 168, 344 Jäger, C., Krasnokutski, S., Staicu, A., et al. 2006, ApJS, 166, 55 Barucci, M. A., Brown, M. E., Emery, J. P., et al. 2008b Barucci, M. A., Brown, M. E., Emery, J. P., et al. 2008b, The Solar System Beyond Neptune (Tucson: U. Arizona Press), 143 Jewitt, D. 2002, AJ, 123, 1039 Beyond Neptune (Tucson: U. Arizona Press), 143 Jones, A. P. 1990, MNRAS, 247, 305 Barucci, M. A., Morea Dalle Ore, C., Alvarez-Candal, A., et al. 2010, AJ, 140, 2095 Jones, A. P. 2005, IAU Symp., 231, 34 Jones, A. P., & Nuth III, J. A. 2011, A&A, 530, A44 necchi, S. D., Noll, K. S., Grundy, W. M., et al. p y Duley, W. W. 2000, ApJ, 528, 841 Duley, W. W. 2000, ApJ, 528, 841 Remusat, L., Derenne, S., Robert, F., & Knicker, H. 200 Remusat, L., Derenne, S., Robert, F., & Knicker, H. 2005, GeCoA, 69, 3919 y, W. W., Jones, A. P., & Williams, D. A. 1989, MNRAS, 236, 709 Duley, W. W., Jones, A. P., & Williams, D. A. 1989, MNRAS, 236, 709 Duprat, J., Dobrica, E., Engrand, C., et al. 2010, Science, 328, 742 Rivkin, A. S., & Emery, J. P. 2010, Nature, 464, 1322 y Duprat, J., Dobrica, E., Engrand, C., et al. 2010, Science, 328, 742 , , y, , , , Roig, F., Ribeiro, A. O., & Gil-Hutton, R. 2008, A&A, 483, 911 Roig, F., Ribeiro, A. O., & Gil-Hutton, R. 2008, A&A, 483, 911 Ehrenfreund, P., Charnley, S. B., & Wooden, D. 2004, Comets II, 115 Romanishin, W., Tegler, S. C., & Consolmagno, G. J. 2010, AJ, 140, 29 Elliot, J. L., Kern, S. D., Clancy, K. B., et al. 2005, AJ, 129, 1117 Sandford, S. A. 2008, Ann. Rev. Anal. Chem., 1, 549 Elliot, J. L., Person, M. J., Zuluaga, C. A., et al. 2010 Elliot, J. L., Person, M. J., Zuluaga, C. A., et al. 2010, Nature, 465, 897 Sandford, S. A., Allamandola, L. J., Tielens, A. G. G. M., et al. 1991, ApJ, 371, 607 Sandford, S. A., Allamandola, L. J., Tielens, A. G. G. M., et al. 1991, ApJ, 371, 607 g Emery, J. P., & Brown, R. H. 2003, Icarus, 164, 104 Emery, J. P., & Brown, R. H. 2004, Icarus, 170, 131 Sandford, S. A., Aléon, J., Alexander, C. M. O.’D., et al. 2006, Science, 314, 1720 Emery, J. P., Cruikshank, D. P., & Van Cleve, J. 2006 Emery, J. P., Cruikshank, D. P., & Van Cleve, J. 2006, Icarus, 182, 496 Emery, J. P., Burr, D. M., & Cruikshank, D. P. 2011, AJ, 141, 25 Serra Díaz-Cano, L., & Jones, A. P. 2008, A&A, 492, 127 Fedele, D., Pascucci, I., Brittain, S., et al. 2011, ApJ, 732, 106 Scott, A., & Duley, W. W. 1996, ApJ, 472, L123 Feigelson, E. D., & Montmerle, T. 1999, ARA&A, 37, 363 Schaller, E. L., & Brown, M. E. 2007, ApJ, 659, L61 Flynn, G. J., Keller, L. P., Feser, M., Wirick, S., & Jacobsen, C. 2003, GeCoA, 67, 4791 Schneider, G., Weinberger, A. J., Becklin, E. E., et al. References 2009, Icarus, 200, 2 Bernard, J.-M., Quirico, E., Brissaud, O., et al. 2006, Icarus, 185, 301 Jones, A. P., Tielens, A. G. G. M., & Hollenbach, D. J. 1996, ApJ, 469, 7 Bonal, L., Quirico, E., Bourot-Denise, M., & Montagnac, G. 2006a, GeCoA, 70, 1849 Kanuchova, Z., Baratta, G. A., Garozzo, M., et al. 2010, A&A, 517, A60 Köhler, M., Mann, I., & Li, A. 2008, ApJ, 686, L95 Bonal, L., Quirico, E., Montagnac, G. & Reynard, B. 2006b, LPI, 37, 2271 Lamy, P., & Toth, I. 2009, Icarus, 201, 674 Lamy, P., & Toth, I. 2009, Icarus, 201, 674 Bonal, L., Bourot-Denise, M., Quirico, E., et al. 2007, GeCoA, 71, 1605 Licandro, J., Campins, H., Kelley, M., et al. 2011, A&A, 525, A34 Licandro, J., Campins, H., Kelley, M., et al. Marzari, F., Scholl, H., Murray, C., & Lagerkvist, C. 2002, In Asteroids III (Tucson: Univ. Arizona Press), 725 Marzari, F., Scholl, H., Murray, C., & Lagerk (Tucson: Univ. Arizona Press), 725 Busemann, H., Young, A. F., Alexander, C. M. O’D., et al. 2006, Science, 312, 727 (Tucson: Univ. Arizona Press), 725 Volk, K., & Malhotra, R. 2008, ApJ, 687, 714 Wirick, S., Flynn, G. J., Keller, L. P., et al. 2009, M&PS, 44, 1611 Yabuta, H., Naraoka, H., Sakanishi, K., & Kawashima, H. 2005, M&PS, 40, 779 Yabuta, H., Alexander, C. M. O.’D., Fogel, M. L., et al. 2010. M&PS, 45, 1446 Yang, B., & Jewitt, D. 2011, AJ, 141, 95 p y Duley, W. W. 2000, ApJ, 528, 841 2009, AJ, 137, 53 Sellgren, K., Smith, R. G., & Brooke, T. Y. 1994, ApJ, 433, 179 Fomenkova, M. N. 1999, Space Sci. Rev., 90, 109 Smith, R. G., Sellgren, K., & Brooke, T. Y. 1993, MNRAS, 263, 749 Smith, R. G., Sellgren, K., & Brooke, T. Y. 1993, M Fornasier, S., Dotto, E., Hainaut, O., et al. 2007, Icarus, 190, 622 Spencer, J., & Denk, T. 2010, Science, 327, 432 A98, page 13 of 14 A98, page 13 of 14 Stansberry, J., Grundy, W., Brown, M., et al. 2008, The Solar System Beyond Neptune (Tucson: U. Arizona Press), 161 Strazzulla, G., & Baratta, G. A. 1991, A&A, 241, 310 Szabó, G., Ivezi´c, ˇZ., Juri´c, M., & Lupton, R. 2007, MNRAS, 377, 1397 Thompson, W. R., Murray, B. G. J. P. T., Khare, B. N., & Sagan, C. 1987, J. Geophys. Res., 92, 14933 A&A 533, A98 (2011) A98, page 14 of 14
https://openalex.org/W3082531149	https://pure.iiasa.ac.at/id/eprint/16671/1/1-s2.0-S1364815220308963-main.pdf	English	null	Economy-wide impacts of behavioral climate change mitigation: Linking agent-based and computable general equilibrium models	Environmental modelling & software	2,020	cc-by-sa	26,951	Journal Pre-proof Economy-wide impacts of behavioral climate change mitigation: linking agent-based and computable general equilibrium models Leila Niamir, Olga Ivanova, Tatiana Filatova Journal Pre-proof Economy-wide impacts of behavioral climate change mitigation: linking agent-based and computable general equilibrium models Leila Niamir, Olga Ivanova, Tatiana Filatova © 2020 The Author(s). Published by Elsevier Ltd. Leila Niamir a,b,c, Olga Ivanova d, Tatiana Filatova c,e Leila Niamir a,b,c, Olga Ivanova d, Tatiana Filatova c,e a Mercator Research Institute on Global Commons and Climate Change (MCC) Torgauer Str. 12-15, 10829 Berlin, Germany (niamir@mcc-berlin.net) b International Institute for Applied Systems Analysis (IIASA) Schlossplatz 1, A-2361 Laxenburg, Austria c Department of Governance and Technology for Sustainability (CSTM), University of Twente Drienerlolaan 5, 7522NB Enschede, The Netherlands (t.filatova@utwente.nl) d Netherlands Environmental Assessment Agency (PBL) Bezuidenhoutseweg 30, 2594 AV, The Hague, The Netherlands (olga.ivanova@pbl.nl) e PERSWADE Research Center, School of Systems Management and Leadership, University of Technology Sydney Broadway 15, Ultimo NSW 2007, Australia of a Mercator Research Institute on Global Commons and Climate Change (MCC) Torgauer Str. 12-15, 10829 Berlin, Germany (niamir@mcc-berlin.net) b International Institute for Applied Systems Analysis (IIASA) Schlossplatz 1, A-2361 Laxenburg, Austria c Department of Governance and Technology for Sustainability (CSTM), University of Twente Drienerlolaan 5, 7522NB Enschede, The Netherlands (t.filatova@utwente.nl) d Netherlands Environmental Assessment Agency (PBL) Bezuidenhoutseweg 30, 2594 AV, The Hague, The Netherlands (olga.ivanova@pbl.nl) e PERSWADE Research Center, School of Systems Management and Leadership, University of Technology Sydney Broadway 15, Ultimo NSW 2007, Australia of Journal Pre-proof Economy-wide impacts of behavioral climate change mitigation: linking agent-based and computable general equilibrium models Accepted Date: 6 August 2020 Please cite this article as: Niamir, L., Ivanova, O., Filatova, T., Economy-wide impacts of behavioral climate change mitigation: linking agent-based and computable general equilibrium models, Environmental Modelling and Software, https://doi.org/10.1016/j.envsoft.2020.104839. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Author(s). Published by Elsevier Ltd. Abstract Households are responsible for a significant share of global greenhouse emissions. Hence, academic and policy discourses highlight behavioral changes among households as an essential strategy for combating climate change. However, formal models used to assess economic impacts of energy policies face limitations in tracing cumulative impacts of adaptive behavior of diverse households. The past decade has witnessed a proliferation of agent-based simulation models that quantify behavioral climate change mitigation relying on social science theories and micro-level survey data. Yet, these behaviorally-rich models usually operate on a small scale of neighborhoods, towns, and small regions, ignoring macro-scale social institutions such as international markets and rarely covering large areas relevant for climate change mitigation policy. This paper presents a methodology to scale up behavioral changes among heterogeneous individuals regarding energy choices while tracing their macroeconomic and cross-sectoral impacts. To achieve this goal, we combine the strengths of top-down computable general equilibrium models and bottom-up agent-based models. We illustrate the integration process of these two alien modeling approaches by linking data-rich macroeconomic with micro-behavioral models. Following a three-step approach, we investigate the dynamics of cumulative impacts of changes in individual energy use under three behavioral scenarios. Our findings demonstrate that the regional dimension is important in a low-carbon economy transition. Heterogeneity in individual socio-demographics (e.g. education and age), structural characteristics (e.g. type and size of dwellings), behavioral and social traits (e.g. awareness and personal norms), and social interactions amplify these differences, causing nonlinearities in diffusion of green investments among households and macro-economic dynamics. Journal Pre-pr 1 1 1 https://climatepolicyinfohub.eu/node/71/pdf 1. Introduction Energy consumption is the primary culprit behind anthropogenic global warming. Humanity’s demand for energy is satisfied by consuming fossil fuels as well as renewable energy sources, leading to varied greenhouse gas emission (GHGs) footprints. Households are responsible for 70% of global GHGs (Hertwich and Peters, 2009). In Europe, one quarter of direct total energy consumption and GHGs comes from households1. Academic and policy discourses highlight behavioral changes among households as an essential strategy for reducing GHG emissions and combating climate change (Dietz et al., 2013; Doppelt et al., 2009; Faber et al., 2012; McKinsey, 2009; Nielsen et al., 2020). Importantly, an individual’s decision-making is known to deviate from rational and perfectly informed optimization process, calling for a thorough understanding of behavioral aspects (Abrahamse and Steg, 2011; Bamberg et al., 2015, 2007; Poortinga et al., 2004; Stern, 2016; van Raaij, 2017). proof Policy-makers rely on decision support tools to assess future changes in energy markets and the economy as a whole. Macroeconomic Computable General Equilibrium (CGE) models serve as standard tools for quantitative policy assessments in climate change mitigation (Babatunde et al., 2017; Fujimori et al., 2017; IPCC, 2014; JRC, 2014; Rive et al., 2006; Vandyck et al., 2016). CGE models are popular among governments and academia for ex-ante policy analysis. They rely on advancements in micro-based macro-economic theory that represent the aggregate behavior of rational and fully-informed economic agents (households and firms) and their trade interactions via supply-chains. Behavioral changes, including behavioral climate change mitigation actions driven by the increased level of knowledge about climate change in society and shifts in preferences, are difficult to model directly with CGE models. This is one of the critics regarding their capacity to support climate change mitigation policy (Creutzig et al., 2018; Farmer et al., 2015; Farmer and Foley, 2009; Isley et al., 2015; Niamir et al., 2018b; Stern, 2016). Journal Pre-pr In contrast to this macroeconomic “top-down” approach, “bottom-up” agent-based models (ABMs) focus on behaviorally-rich representation of energy consumers, integrate technological learning, out-of-equilibrium dynamics and social interactions (Bhattacharyya, 2011; Farmer et al., 2015; Hunt and Evans, 2009; Niamir et al., 2018b; Tesfatsion, 2006). Agents in ABMs follow a set of if-else rules, sometimes combined with equations, that guide their actions, interactions with other actors or institutions (e.g. markets), and learning. 1. Introduction ABMs could compliment macro- economic models by accommodating heterogeneity, adaptive behavior and interactions, bounded 2 2 rationality, and imperfect information (Filatova and Niamir, 2019). However, their use for climate policy is hindered by high-data intensity for individual behavioral rules and interactions. When energy ABMs are grounded in empirical data, their upscaling remains limited (Humphreys and Imbert, 2013; Lamperti et al., 2019), preventing the assessment of economy-wide impacts, effects of national or EU policies and generalization of ABMs’ results. There is a long history in bridging top-down CGE models with bottom-up models (Krook- Riekkola et al., 2017), usually non-ABM. Specifically for energy, macroeconomic models are linked with engineering micro-simulation models focusing on the technological processes of electricity generation (Sue Wing, 2008). Scholars either establish a ‘soft-link’ between micro and macro models, or complement one by a reduced form of the other, or combine them directly through ‘hybrid’ modeling (Böhringer and Rutherford, 2009). Since engineering bottom-up models often rely on mathematical programming, the latter approach focuses on resolving mixed complementarity problems (Bohringer and Rutherford, 2008). Besides linking to engineering micro-simulations, national level CGEs rely on complimentary micro-simulation models for environmental analysis, taxation (Peichl and Schaefer, 2009), fiscal analyses (Debowicz, 2016) and labor market analysis (Benczúr et al., 2018). However, an integration of micro-macro approaches at the regional (sub-national) level is scarce (Verikios and Zhang, 2015). In parallel, as inequality and distributional impacts of climate change policies come into a spotlight internationally, introducing heterogeneity into CGE models becomes increasingly important (Bijl et al., 2017; Kulmer and Seebauer, 2019; Melnikov et al., 2017; Rao et al., 2017; van Ruijven et al., 2015). This is commonly done by disaggregating the representative agent in macro models with micro-level survey data (Rausch et al., 2011). Duarte et al. (2016) provide an excellent example on modelling of pro-environmental consumer behavior in a regional CGE model for Spain using micro-level data. This study evaluates the impact of improving environmental awareness by specifying drivers of behavioral changes – adoption of household appliances with different energy efficiency levels – for different income levels using household survey data (Duarte et al., 2016). While using survey data in CGEs is a major step in accommodating heterogeneity, the choices that economic agents pursue remain fixed and are still assumed to be taken under conditions of perfect information. 1. Introduction It hinders the representation of behavioral changes, bounded-rationality and social influences so prominent in understanding pro-environmental choices (Niamir et al., 2020a; Steg and Vlek, 2009). Journal Pre-proof There is a long history in bridging top-down CGE models with bottom-up models (Krook- Riekkola et al., 2017), usually non-ABM. Specifically for energy, macroeconomic models are linked with engineering micro-simulation models focusing on the technological processes of electricity generation (Sue Wing, 2008). Scholars either establish a ‘soft-link’ between micro and macro models, or complement one by a reduced form of the other, or combine them directly through ‘hybrid’ modeling (Böhringer and Rutherford, 2009). Since engineering bottom-up models often rely on mathematical programming, the latter approach focuses on resolving mixed complementarity problems (Bohringer and Rutherford, 2008). Besides linking to engineering micro-simulations, national level CGEs rely on complimentary micro-simulation models for environmental analysis, taxation (Peichl and Schaefer, 2009), fiscal analyses (Debowicz, 2016) and labor market analysis (Benczúr et al., 2018). However, an integration of micro-macro approaches at the regional (sub-national) level is scarce (Verikios and Zhang, 2015). In parallel, as inequality and distributional impacts of climate change policies come into a spotlight internationally, introducing heterogeneity into CGE models becomes increasingly important (Bijl et al., 2017; Kulmer and Seebauer, 2019; Melnikov et al., 2017; Rao et al., 2017; van Ruijven et al., 2015). This is commonly done by disaggregating the representative agent in macro models with micro-level survey data (Rausch et al., 2011). Duarte et al. (2016) provide an excellent example on modelling of pro-environmental consumer behavior in a regional CGE model for Spain using micro-level data. This study evaluates the impact of improving environmental awareness by specifying drivers of behavioral changes – adoption of household appliances with different energy efficiency levels – for different income levels using household survey data (Duarte et al., 2016). While using survey data in CGEs is a major step in accommodating heterogeneity, the choices that economic agents pursue remain fixed and are still assumed to be taken under conditions of perfect information. It hinders the representation of behavioral changes, bounded-rationality and social influences so prominent in understanding pro-environmental choices (Niamir et al., 2020a; Steg and Vlek, 2009). 1. Introduction Linking macroeconomic CGE models with micro level behaviorally rich ABMs can Journal Pre-proof Linking macroeconomic CGE models with micro-level behaviorally-rich ABMs can operationalize behavioral changes in formal policy analysis and open new synergies between 3 3 micro and macro approaches (Krook-Riekkola et al., 2017; Melnikov et al., 2017; Parris, 2005; Safarzyńska et al., 2013; Smajgl et al., 2009). Earlier attempts to integrate ABM and CGE models include the work of Safarzyńska et al. (2013) who propose an elegant way to integrate the evolutionary dynamics of ABMs into a CGE model. Yet, authors leave it at the conceptual level without an implementation. Smajgl et al. (2009) discuss a farm-level integration of ABM-CGE for fishery policy impact assessment, with no integration results. To the best of our knowledge, there is no empirical example of resolving the key methodological differences between ABM and CGE modelling while aligning with survey data on behavioral heterogeneity. The current paper addresses this methodological gap by demonstrating how aggregated impacts of household energy behavior changes emerging from an empirical ABM could be scaled up and linked to the macroeconomic dynamics of a CGE model. To demonstrate the feasibility of the method we employ a soft-linkage between the two empirical models; future work will focus on a hard-link integration following our earlier pilot on using software wrappers to assure a real-time data exchange between toy ABM and CGE models (Belete et al., 2019). Here we ensure models’ consistency by aligning functional forms and by using the same database and economic scenarios. The objective of this paper is twofold: (1) to investigate feasibility of an original approach to link empirical ABM and CGE models while targeting individuals’ heterogeneity, social interactions, and behavioral changes; and (2) to explore the impacts of climate change mitigation behavior across scales, from individuals to the EU regions. Towards this end, we propose a three-step upscaling approach that goes beyond our specific application and may serve as a systematic way to link ABM and CGE models (Section 2). Our results demonstrate that it permits tracing the macro-economic and cross-sectoral impacts and indirect effects of individual energy behavioral changes (Section 3). Section 4 concludes with a discussion and outlining future work. Journal Pre-proof 2. Methods To explore economy-wide impacts of behavioral changes and the role of social interactions the current paper employs the strengths of micro and macro socio-economic models. We use an empirical behavioral ABM (BENCH-v.3) originally developed to study cumulative impacts of individual changes in energy use (Niamir et al., 2020b, 2018a). To trace indirect effects and cross-sectoral impacts of shifts in residential energy demand and changes in households 4 consumption behavior, we employ an empirically-calibrated CGE model (EU-EMS) (Ivanova et al., 2019). consumption behavior, we employ an empirically-calibrated CGE model (EU-EMS) (Ivanova et al., 2019). entific challenge is in aligning the two models that differ in key assumptions. Namely: The scientific challenge is in aligning the two models that differ in key assumptions. Namely: The scientific challenge is in aligning the two models that differ in key assumptions. Namely: ● Representative vs. heterogeneous agents: CGE models work with a representative agent (group) while ABMs assume heterogeneity in attributes and behavior; ● Perfect vs. bounded rationality: agents in CGE are assumed to be fully rational while ABMs proliferate in tackling research problems where bounded rationality is relevant; oof ● Perfect vs. bounded rationality: agents in CGE are assumed to be fully rational while ABMs proliferate in tackling research problems where bounded rationality is relevant; oof ● Static vs. adaptive behavior: households in CGE have fixed preferences and perfect information, while ABM are designed to explicitly model adaptive expectations. Since ABM-agents do not have full information, they learn over the course of a simulation, either from their own experience, from their social network or through market signals; Pre-proo ● Unique one shot equilibrium, vs. out-of-equilibrium dynamics: CGE models are solved via the assumption of a unique equilibrium occurring in one shot when markets clear. In contrast, ABMs trace the process of out-of-equilibrium dynamics and transitions between multiple equilibria while eliciting path-dependencies. ournal P Jou 2 The Nomenclature of territorial units for statistics, abbreviated NUTS is a geographical nomenclature subdividing the economic territory of the European Union (EU) into regions at three different levels (NUTS 1, 2 and 3 respectively, moving from larger to smaller territorial units). 3 3 Photo sources: I1 by Tracey Nicholls (CC BY 3.0); I2 by Enrix-Knuth (CC BY-SA 4.0); I3 by Tommaso.sansone91(CC0). Availa https://commons.wikimedia.org 2 The Nomenclature of territorial units for statistics, abbreviated NUTS is a geographical nomenclature subdividing the economic territory of the European Union (EU) into regions at three different levels (NUTS 1, 2 and 3 respectively, moving from larger to smaller territorial units). 3 Photo sources: I1 by Tracey Nicholls (CC BY 3.0); I2 by Enrix-Knuth (CC BY-SA 4.0); I3 by Tommaso.sansone91(CC0). Available from: https://commons.wikimedia.org 2.1.1. The BENCH agent-based model 2.1.1. The BENCH agent-based model Originally, the BENCH ABM (Niamir et al., 2020b, 2018a; Niamir and Filatova, 2017) was developed to investigate the role of behavioral changes with respect to an individual energy use in the transition to a low-carbon economy. Households in BENCH ABM are heterogeneous in socio-demographic characteristics (e.g. income, age, education), dwelling characteristics (e.g. type, size, age), energy consumption patterns (e.g. electricity and gas consumption, energy provider), and behavioral factors (e.g. awareness, personal norms, social norms). BENCH is 5 spatially explicit, with behavioral rules of agents calibrated based on the survey data for two EU NUTS22 regions: Navarre, Spain and Overijssel, The Netherlands (Niamir et al., 2020a). We advance this ABM further to permit integration with the EU-EMS CGE both in terms of the theoretical consistency of functional forms used in ABM and CGE as well as the datasets and scenario assumptions. We start aligning the ABM model with its macro counterpart by including the empirically estimated discrete choice functions for the representation of households’ investment decisions. These functions stem from the utility optimization approach that is also used for the derivation of demand functions in the CGE model and are further relaxed in the ABM to accommodate bounded rationality. Namely, agents’ utility functions are modified to align with empirically-grounded energy decisions from the households’ survey (Niamir et al., 2020a), social interactions and learning – with macroeconomic dynamics in our data-driven CGE model. In particular, BENCHv.3 focuses on energy investments that households may decide to undertake: significant investments in house insulation (I1) or moderate investment in solar panels (I2), and modest investments in energy-efficient appliances (I3) (Figure 1). Pre-proof Figure 1: Households’ choices in the spatial BENCH agent-based model.3 Journal Pre r Figure 1: Households’ choices in the spatial BENCH agent-based model.3 Cognitive process behind individual behavioral changes: in accordance with the Theory of Planned Behavior and Norm Activation Theory from psychology, we assume that boundedly rational individuals in BENCH-v.3 make decisions following a number of cognitive steps: knowledge activation, motivation, and consideration (Niamir et al., 2020a, 2018a). Figure 2 shows heterogonous households in sociodemographic characteristics, dwelling conditions, 2 The Nomenclature of territorial units for statistics, abbreviated NUTS is a geographical nomenclature subdividing the economic territory of the European Union (EU) into regions at three different levels (NUTS 1, 2 and 3 respectively, moving from larger to smaller territorial units). 2.1.1. The BENCH agent-based model 3 Photo sources: I1 by Tracey Nicholls (CC BY 3.0); I2 by Enrix-Knuth (CC BY-SA 4.0); I3 by Tommaso.sansone91(CC0). Available from: https://commons.wikimedia.org 6 electricity and gas consumption follow a cognitive process to decide whether to pursue any energy investment (I1-I3). Niamir, et al. (2018a) describes how each individuals knowledge activation and motivation are measured and calculated at the model initialization stage based on the survey data. In summary, an individual knowledge activation level is calculated based on the average of three types of knowledge - person's climate-energy-environment knowledge (K), awareness about climate, environment and energy issues (AC), and energy decision (AE). If this average for an individual is above the empirical threshold, then the person is tagged as “feeling guilt” and proceeds to the next step to assess his/her motivation for actions I1-I3. Such individuals proceed to evaluate the motivational factors: personal and social norms (NP, NS) for each action (I1-I3). If individuals are highly motivated and “feel responsible”, the perceived behavior controls4 (PBC), and the dwelling ownership status (owner or renter) are evaluated to assess “intentions”. Individuals with a high level of intention proceed to estimate utilities, which are formulated as a discrete choice problem here. Household agents follow these stages for each action: when deciding whether to invest in insulation, solar panels or energy-efficient appliances. re-proof Figure 2: BENCH-v.3 ABM structure: cognitive process behind individual behavioral changes ( I1-I3) Journal Pre al Figure 2: BENCH-v.3 ABM structure: cognitive process behind individual behavioral changes ( I1-I3) 4 Own perception of their ability to perform an action or change behavior 7 Households in BENCH-v.3 make choices based on the indirect utility function (Eq.1). As the inverse of the expenditure function when prices are constant, it reflects individual preferences for different energy actions under budget constraints. Households in BENCH-v.3 make choices based on the indirect utility function (Eq.1). As the inverse of the expenditure function when prices are constant, it reflects individual preferences for different energy actions under budget constraints. (Eq.1) = + ɛ The utility of individual j associated with choice i () is calculated based on the vector of explanatory observed and latent variables ( ) – including socio-economic characteristics of the individuals, dwelling characteristics, and financial and ownership situation, as well as behavioral factors – and the parameter vector ( ) estimated using a probit regression (Niamir et al., 2020a). 2.1.1. The BENCH agent-based model Finally, ɛ is the vector of error terms. An individual chooses a particular sub-action (i) when their utility is non-negative: re-proof (Eq. 2) l Pre (Eq. 2) ≥0 = = al P ≥0 = = al P Social interactions and learning: The speed of green investments diffusion does not depend only on social interactions that affect updating of knowledge, awareness and norms. It depends also on the individual heterogeneity: socio-economic characteristics or dwelling characteristics, which affect utility of taking an action I1-I3 (i.e. serve as proxy for the perceived behavior control, PBC). In BENCH-v.3, agents exchange information following a simple opinion dynamics model (Moussaïd et al., 2015). When a neighbor takes an action (I1-I3), it may alter knowledge, awareness and the motivational factors regarding energy choices of others in this peer group. Namely, individuals compare own behavioral factors (K, AC, AE, NP, NS, PBC) with those of their closest neighbors, and gradually adjust them (Figure 3, Eq. 3). We run various scenarios of this social learning (see section 2.1.3). Journ 8 Figure 3: Social dynamics and learning in a neighborhood where an individual undertook an action at time t Figure 3: Social dynamics and learning in a neighborhood where an individual undertook an action at time t (Eq. 3) = , , ! , "# , "$ , %&'( , ) = 1, … 9.; 0 12) 1 34, 256) 1 344 ≥ 3 1 378 = 3 + 0.02 ⋅ 34 oof 0 12) 1 34, 256) 1 344 ≥ 3 1 378 = 3 + 0.02 ⋅ 34 oof Our ABM uses the same baseline scenario of regional demographic and economic development as the CGE model ensuring the consistency between the scenario analysis in two models. Further, the ABM takes as inputs data on the regional GDP projections estimated for 2015-2050 by the CGE model. The detailed description of the BENCH agent-based model is presented in Appendix Pre-proo 1. l Pre 5 http://themasites.pbl.nl/winnaars-verliezers-regionale-concurrentie/ 6 2.1.2. Computable General Equilibrium model rna EU-EMS (Ivanova et al., 2019) is a spatial CGE model developed by the PBL Netherlands Environmental Assessment Agency for policy impact assessments. The current version of EU- EMS covers 276 NUTS2 regions across the EU28 member states. Goods and services are produced by firms and consumed by households or other firms and exchanged on competitive markets. Spatial interactions between regions are captured through the trade in goods and services, factor mobility, and knowledge spill-overs. Journ Following the tradition of comprehensive empirical CGE models, EU-EMS uses large datasets of real economic data in combination with complex computational algorithms to assess how the economy reacts to changes in governmental policy, technology, availability of resources and other external macro-economic factors. The EU-EMS model consists of (a) the system of non- linear equations, which describes the behavior of various economic actors, and (b) a very detailed database of economic, trade, environmental and physical data. The core part of the model database is the Social Accounting Matrix, which represents in a consistent way all annual economic transactions. 9 The database5 of the model has been constructed by PBL using the combination of national, European and international data sources and represents a detailed regional level (NUTS2 for EU28 plus 34 non-EU countries) multi-regional input-output (MRIO) table for the world. The main datasets used for the construction of this MRIO include the 2013 OECD database, BACI trade data, Eurostat regional statistics, and national Supply and Use tables, as well as the detailed regional level transport database of DG MOVE called ETIS-Plus6. The later dataset allows us to estimate the inter-regional trade flows at the level of NUTS2 regions that are currently not available from official statistical sources. The aggregated groups of the sectors can be directly linked to the panel data econometric analysis and estimations that have been done for Total Factor Productivity (TFP) projections using the EU-KLEMS database7. We have used panel data techniques on EU-KLEMS data in order to model the development of TFP according to the technological catch-up theory. The detailed description of our CGE model is presented in Appendix 2. proof Measuring economic inequality: economists often measure regional disparities using Theil’s T inequality index (Eq.3), the absolute value of which indicates the distance from equality. Pre-p ) Eq. 3 ( al P ) Eq. http://viewer.etisplus.net/ 7 http://www.euklems.net/ p p 7 http://www.euklems.net/ p p 7 http://www.euklems.net/ p p 6 http://viewer.etisplus.net/ 7 8 As an ABM the BENCH model permits experimentation with numerous “what if” scenarios. Exploring the entire space of complex adaptive models, such as BENCH, is a massive research project on its own (Kwakkel and Pruyt, 2013). We tested different level of diffusion ranging from 1% to 4% and choose 2% since it captures the qualitative trend anticipated by experts. For example, the higher level of diffusion generate more active neighborhoods in earlier years converting all households to became energy-efficient between 2035-2040, but that does not resemble the narratives in the literature (Allen et al., 2018; Creutzig et al., 2016; Grubler et al., 2018; IPCC, 2014). Exploring the entire parameter space would be an interesting topic for future research. 2.1.2. Computable General Equilibrium model rna 3 ( 1 _ log N i i i i i Theil T θ γ θ µ =   =     ∑ ∑ urnal e oθ Where oθ is the GDP of each NUTS2 region, iγ is the GDP per capita in each region as a measure of regional income, and µ is the average GDP per capita across the EU28 NUTS2 regions. Jou The EU-EMS CGE model estimates the cross-sectoral aggregated impacts of individual behavioral changes produced by the ABM, and traces the consequent changes across the EU regions triggered by the macro economy. The CGE receives measures: a) the diffusion of each of the three types of actions (I1-I3) among heterogeneous households (classified in 12 age and education groups); b) the changes in electricity and gas consumption; c) saved CO2 emissions; and d) the amount of investment from BENCH model results. 10 2.1.3. Scenarios Micro-level end-user behavioral scenarios: besides being heterogeneous in terms of sociodemographic characteristics (e.g. age, income, education), housing they reside in (e.g. tenure status, size, energy label), and psychological factors (e.g. attitudes and beliefs, personal norms), agents in the BENCH-v.3 ABM exhibit heterogeneous behavioral characteristics, such knowledge and awareness, engage in social interactions and learn. BENCH.v3 ABM introduces three end-user behavioral scenarios (Baseline, FD, ID) by differentiating between the intensity of social interactions and the speed of learning (see Table 1). Based on the neighborhood size, this social learning may occur at either a slow or fast pace (see scenarios in Appendix 1). f Table 1: Micro-level End-user Behavioral Scenarios. Source: BENCH.v3 Behavioral scenarios Social dynamics Definition Pre-proof Table 1: Micro-level End-user Behavioral Scenarios. Source: BENCH.v3 - Individuals with the value of their behavioral attributes – components shaping awareness and motivation – lower than that of their neighbors adjust by increasing the value of by 2%8 (see Eq. 3). Pre In an active neighborhood: individuals interacts with a maximum of four neighbors rnal P FD (Fast Dynamics) Fast In an active neighborhood: individuals interacts with all available neighbors Journ Individuals with the value of their behavioral attributes – components shaping awareness and motivation – lower than that of their neighbors adjust by increasing the value of by 2% (see Eq. 3). FD (Fast Dynamics) This scenario represents a rapid bottom-up diffusion of pro-environmental social norms driven by households alone without any policy support. Dynamics) motivation across the entire population. Hence, at initialization all households agents start with 2% higher values of behavioral attributes, before engaging in any social learning. The ID scenario highlights the importance of information diffusion and information campaigns focusing on behavioral climate mitigation. It assumes that all individuals do update their knowledge and motivation when an information policy applies. Macro-level scenarios: in addition to these three behavioral scenarios, the EU-EMS CGE model relies on the demographic projections from Eurostat until 2050 and Total Factor Productivity (TFP) projections by economic sector based on our own econometric analysis. Hence, the macroeconomic and demographic scenarios are combined with the slow/fast/informative dynamics scenarios of micro-level behavior with respect to energy-related investments of heterogeneous households. Pre-proof 2.1.2. Computable General Equilibrium model rna ID (Informative Informative In an active neighborhood: individuals interacts with This scenario assumes an intense information policy – e.g. social advertising and the promotion of pro-environmental behavior – that raises the level of knowledge and ID In an active neighborhood: individuals interacts with In an active neighborhood: individuals interacts with 11 2.2. Upscaling behavioral changes al P ABM and CGE models each have their own assumptions, strength and weaknesses. We attempt to overcome the latter by linking the two models. To pursue this in a systematic manner, we take a step-wise approach to bridge the ABM with the CGE model (Figure 4). ournal Step 1: From individual households to regional shifts in energy use. BENCH-v.3 ABM calculates the extent of behavioral changes among heterogeneous household agents who evolve through a cognitive process (section 2.1.1, Figure 2) before reaching a more rational stage where the discrete-choice utility maximization is activated (section 2.1.1, Eq.1 and 2). Given the stochastic nature of ABMs, we use the mean values from 100 ABM simulations run for each scenario and case-study to feed them further into the CGE model. The main outcomes of the BENCH-v.3 ABM used in the EU-EMS CGE model are the relative changes in electricity and gas use and the total investments made by various individuals (I1-I3). The EU-EMS CGE model, however, operates at the level of all 276 EU28 NUTS2 regions, and needs regional changes in energy consumption and investments of the representative households as an input. Hence, the behavioral patterns emerging at the Overijssel and Navarre provinces for different households need to be scaled not only up to the national level, but up to the entire EU (see next steps and Figure 4). Jo 12 Step 2: Dynamic socio-demographic groups with similar behavioral patterns. We take an intermediate step to derive the changes in investments, gas and electricity consumption across households of different age and education levels for all 276 EU28 NUTS2 regions based on the outcomes of two regional ABMs. Economic theory suggests that investment choices depend on households’ incomes. However, our survey on behavioral changes regarding energy use (Niamir et al., 2020a) reveals that age and education are the most important factors explaining households preparedness to invest in low-carbon energy (I1-I3)9. Thus, we define behavioral patterns for a group of households in the Dutch and Spanish regional ABMs separately, aggregating by age and education level. Following the Eurostat classification, we work with 12 age-education groups (Table 2). roof Table 2: Socio-demographic groups, based on the Eurostat classification. e-p Table 2: Socio-demographic groups, based on the Eurostat classification. e- Table 2: Socio-demographic groups, based on the Eurostat classification. e- Table 2: Socio-demographic groups, based on the Eurostat classification. 9 With the help of our empirical data, we examined the impact socio-demographic factors, namely income, gender, education and age, on households energy bahavior changes in two provinces (Overijssel, NL and Navarre, ES). Particulary, our analysis shows the probability of households energy behavior increases with the level of eduction (95% confidential interval) (Niamir et al., 2020a). e-proo 2.2. Upscaling behavioral changes al P Group number Education level (1-3) Age group (1-4) G1 Low (ISCED 0-2) 1 (younger than 20) G2 Low (ISCED 0-2) 2 (20-40 years old) G3 Low (ISCED 0-2) 3 (40-60 years old) G4 Low (ISCED 0-2) 4 (older than 60) G5 Middle (ISCED 3-4) 1 (younger than 20) G6 Middle (ISCED 3-4) 2 (20-40 years old) G7 Middle (ISCED 3-4) 3 (40-60 years old) G8 Middle (ISCED 3-4) 4 (older than 60) G9 High (ISCED 5-8) 1 (younger than 20) G10 High (ISCED 5-8) 2 (20-40 years old) G11 High (ISCED 5-8) 3 (40-60 years old) G12 High (ISCED 5-8) 4 (older than 60) Journal Pre-p For all 12 groups, we estimate a number of households pursuing an action (I1-I3) and calculate the corresponding average gas and electricity savings and investments. The behavioral factors – awareness, motivations, intentions and likely actions– across 12 groups differ between the two For all 12 groups, we estimate a number of households pursuing an action (I1-I3) and calculate the corresponding average gas and electricity savings and investments. The behavioral factors – awareness, motivations, intentions and likely actions– across 12 groups differ between the two 13 countries in our survey sample, and so do the patterns of behavioral climate change mitigation emerging in the ABMs. To utilize the information regarding regional differences in patterns of behavioral change, we create the mapping between NUTS2 regions of the EU28 with the two ABM regions according to their perceived cultural distance. Social structure, wealth and lifestyle, religion, institutional and economic conditions, and natural environment play a role in assessing cultural distance (Gobel et al., 2018; Hofstede, 2011, 2001; Kaasa et al., 2016; Schwartz, 2014; Vignoles et al., 2018). Specifically, in the absence of more granular data, we use the Dutch case to approximate how the behavioral patterns may evolve in the North-West EU states, and the Spanish case – for the South-East EU states (see Table A3.1 in Appendix 3). We acknowledge that this approach does not fully capture all the cultural differences but it, for example, accounts for the role of social network (higher among the Spanish respondents compared to the Dutch) in behavioral climate change mitigation. Ideally, one should use native survey data regarding the modelled behavior or employ secondary data on revealed empirical differences on behavioral changes across regions. 10 https://ec.europa.eu/eurostat/documents/3433488/5564440/KS-SF-10-001-EN.PDF/d5b8bf54-6979-4834-998a-f7d1a61aa82d 11 http://www.iiasa.ac.at/web/home/research/researchPrograms/WorldPopulation/Projections_2014.html 2.2. Upscaling behavioral changes al P Furthermore, differences in policy, institutional, technological, and environmental conditions across EU countries are indirectly accounted for in our CGE model and the databases it relies upon. Pre-proof Since behavioral changes vary primarily among households with different age and education levels, the changes in these characteristics over time are crucial. Hence, we employ demographic projections for the period until 2050. The only regional NUTS2 level projections that have been done for the EU28 are EUROPOP200810 projections of Eurostat. Population projections of Eurostat provide information about the development of the population until 2050, detailed by age and gender groups. Furthermore, Eurostat population projections at NUTS2 level are combined with IIASA Global Education Trends scenario projections11 related to the share of high, medium and low-educated persons in each EU country. This allows us to construct population projections by age and education level for the period 2020-2050 for each NUTS2 region of the EU28. These NUTS2-level population projections till 2050 match with the scaled-up mapping of behavioral patterns of 12 groups in our ABM. Hence, now we use age and education information to linked it with the emerging behavioral patterns of the agent-based BENCH v.3 model when creating NUTS2 specific – that is, corresponding to the population structure of that region – inputs into the spatial EU-EMS CGE model. Journal P 10 https://ec.europa.eu/eurostat/documents/3433488/5564440/KS-SF-10-001-EN.PDF/d5b8bf54-6979-4834-998a-f7d1a61aa82d 11 http://www.iiasa.ac.at/web/home/research/researchPrograms/WorldPopulation/Projections_2014.html 14 Step 3: Cumulative economy-wide impacts of behavioral changes. Finally, we use the predicted population structure by age and education level for the period 2020-2050 to calculate aggregated changes in the residential use of gas and electricity for each NUTS2 regions of EU28 on the basis of calculated averages for each of the 12 individual groups. The EU-EMS CGE model estimates the cross-sectoral impacts of these shifts in the aggregated residential energy demand that impacts GDP projects. The linked ABM-CGE model quantifies the cumulative impacts of behavioral changes among heterogeneous households at the level of 276 EU28 NUTS2 regions. This allows us to understand the impacts of various behavioral scenarios within the CGE framework, including distributional effects across these EU regions. An important direction of future work would be to develop direct two-way linkages between the two models, with the CGE-generated GDP projections feeding back into the ABM. Data flows between two models are presented in Figure 4. 2.2. Upscaling behavioral changes al P proof Figure 4: Upscaling individuals behavioral change via linking ABM and CGE models Journal Pre-p rna our Figure 4: Upscaling individuals behavioral change via linking ABM and CGE models This step-wise approach to linking the ABM and CGE models allows us to address the key methodological challenges: ● From representative to heterogeneous agents: Heterogeneous households in the ABM are matched with representative households in the CGE model. Aggregation occurs along ● From representative to heterogeneous agents: Heterogeneous households in the ABM are matched with representative households in the CGE model. Aggregation occurs along 15 the two dimensions that impact relevant behavioral changes among households most: age and education levels. This is done using detailed information about the structure of the population by age and education in each NUTS2 region for the period 2020-2050 while keeping behavior heterogeneous across the 12 groups. ● From perfect to bounded rationality: Agents in our ABM are boundedly rational due to the presence of behavior factors (K, AC, AE, NP, NS, PBC) that precede discrete choice utility estimate: subjective knowledge and awareness, motivation, and intention to consider a change in behavior, which are all prone to social influence. The use of the ABM allows us to assess the impacts of pure behavioral changes in the CGE model and calculate their broader economic impacts. The rest of the economy in the CGE model – e.g. households’ decisions on a labor market, decisions of firms, clearing of the markets – still operates in line with the rationality principles, allowing for the coherent treatment of macro-economic processes in the CGE model. proof ● From static to adaptive agents: Agents in the ABM are prone to social influence and learn from their neighbors. As their behavior attributes – knowledge and awareness – evolve, they go through various cognitive stages of knowledge activation, motivation and consideration and may eventually decide to invest in low carbon energy. By scaling up these behavioral patterns through age-education groups, we are able to link to the architecture of a CGE. By default CGE models assume perfect information and rational expectations, omitting a variety of behavioral strategies through which adaptive behavior can be channeled into macro dynamics. Journal Pre-p ● From an equilibrium to adaptive dynamics with social learning: The CGE model is based on assumptions of market equilibrium and interlinkages between different agents, sectors and markets in the economy. 3. Results and discussion Given the stochastic nature of ABMs, we run BENCH multiple times under the same parameter settings for each scenario. The ABM results presented below plot the means across 100 random runs. Therefore, we use the mean values from each ABM scenario and case-study to scale up the observed behavioral patterns and to estimate their cross -sectoral impacts in the CGE model. of 2.2. Upscaling behavioral changes al P The ABM treats agents’ decisions as a cognitive process in the presence of social interactions and fast/slow/informative learning. Jo ● From an equilibrium to adaptive dynamics with social learning: The CGE model is based on assumptions of market equilibrium and interlinkages between different agents, sectors and markets in the economy. The ABM treats agents’ decisions as a cognitive process in the presence of social interactions and fast/slow/informative learning. Jo Before discussing the results, it may be useful to be explicit about the limitations of the current study. The presented CGE-to-ABM link is currently indirect, operationalized via the EU GDP growth rates scenarios (the dotted curve in Figure 4). Furthermore, to demonstrate the applicability of method, we work with two survey datasets; for a real policy analysis it is essential to work with a richer representation of regions that may also account for differences in climatic and institutional conditions across countries. While our ABM relies on households’ surveys (Niamir et al., 2020b, 2020a, 2018a) for micro-validation, macro-validation against regional-level panel data remains a subject of future work. We believe that micro-validation is sufficient for the Before discussing the results, it may be useful to be explicit about the limitations of the current study. The presented CGE-to-ABM link is currently indirect, operationalized via the EU GDP growth rates scenarios (the dotted curve in Figure 4). Furthermore, to demonstrate the applicability of method, we work with two survey datasets; for a real policy analysis it is essential to work with a richer representation of regions that may also account for differences in climatic and institutional conditions across countries. While our ABM relies on households’ surveys (Niamir et al., 2020b, 2020a, 2018a) for micro-validation, macro-validation against regional-level panel data remains a subject of future work. We believe that micro-validation is sufficient for the 16 methodological demonstration of the applicability of this approach for upscaling behavioral climate change mitigation. Complementing it with macro-validation would be essential when performing a real policy analysis. Step 1: From behavioral patterns in survey data to cumulative impacts in two provinces roof Firstly, we run the BENCH.v3 ABM for two EU provinces (Overijssel and Navarre) under the three behavioral scenarios (Baseline, FD and ID). We report the regional impacts of the energy behavior choices of heterogeneous households: the diffusion of each of the three types of behavioral actions among heterogeneous households over time, the changes in electricity and gas consumption, saved CO2 emissions, and the amount of investment. al Pre-pro (a) electricity (b) gas Figure 5: Saved energy (kWh) per household as a result of investment (I1-I3) under three behavioral scenarios in two EU provinces over 34 years (2017-2050). Source: BENCH-v.3 Journal (a) electricity Journa (a) electricity Figure 5: Saved energy (kWh) per household as a result of investment (I1-I3) under three behavioral scenarios in two EU provinces over 34 years (2017-2050). Source: BENCH-v.3 Figure 5 illustrates the dynamics of electricity and gas saving in the two EU provinces as a result of households’ energy investments. The general trend is as expected: faster learning boosted by an information campaign leads to more investments in solar panels (I2) and in appliances (I3), and consequently to higher electricity savings in both provinces. Intensive social learning boosts electricity savings by 40% and 100% in Overijssel and Navarre (FD vs Baseline, Figure 5.a and 17 Table 3). In addition, electricity savings increase by 14% and 22% in two provinces if pro- environmental awareness is raised through an information policy (ID vs FD, Figure 5.a and Table 3). However, these trends do not hold for investments in insulation (I1) and corresponding gas savings. Informative strategy (ID) has a mixed impact on insulation investments in Navarre (crossing of FD and ID curves in Figure 5.b) and the opposite effect in Overijssel (ID delivers 26% lower gas savings compared to FD, Figure 5.b). The difference between cases may be driven by initial conditions (climate, institutional settings, gas prices) in the two countries. In addition, comparing FD and ID scenarios shows that an information policy and social interactions among neighbors impact households’ insulation decisions in a non-linear way. f Table 3: Saved CO2 and household investment in two provinces (Overijssel and Navarre) under three micro-level behavioral scenarios over time. We report the mean value across 100 runs under each scenario. Source: BENCH-v.3 ABM e-pro Table 3: Saved CO2 and household investment in two provinces (Overijssel and Navarre) under three micro-leve scenarios over time. Step 1: From behavioral patterns in survey data to cumulative impacts in two provinces roof We report the mean value across 100 runs under each scenario. Source: BENCH-v.3 ABM e-pro Table 3: Saved CO2 and household investment in two provinces (Overijssel and Navarre) under three micro-level behavioral scenarios over time. We report the mean value across 100 runs under each scenario. Source: BENCH-v.3 ABM e-pro Scenarios Provinces 2030 2050 Saved CO2 emission (tons per household) Baseline Overijssel 0.50 1.09 Navarre 0.23 0.78 FD Overijssel 0.71 1.53 Navarre 0.47 1.59 ID Overijssel 0.75 1.93 Navarre 0.85 1.75 Total investments (in 2016 Euro per household) FD Overijssel 2,908 6,858 Navarre 2,198 8,020 ID Overijssel 2,578 5,430 Navarre 2,931 7,585 The share of preferred actions (in percentage) Overijssel I1:4.9% I2: 26.1% I3: 69% I1:4.0% I2: 20.1% I3: 75.9% Navarre I1:12.1% I2: 26.7% I1:9.4% I2: 22.5% Journal Pre-p 18 I3: 61.3% I3: 68.1% Total number of actions Overijssel 2,839 6,875 Navarre 1,239 3,690 Investments in 2016 Euro per action, % of total invested money in two provinces proof Table 3 shows the amount of CO2 emission savings that households’ energy behavior changes could deliver, and at what investment cost. Intensive social interaction (FD scenario) leads to 1.4 and 2 times more saved CO2 emissions in Overijssel and Navarre compared to the Baseline. As expected, information policy along with social interactions (ID scenario) amplify the impact 1.1 and 1.2 times more on top of the FD scenario in Overijssel and Navarre respectively. We observe a non-linear pattern in total investments (Euro per households) under behavioral scenarios over time. When information policy (ID scenario) is activated, Dutch households invest 17% more compared to the FD scenario in 2020 and this then drops in 2050 (20% less than the FD scenario). Spanish household investments in the ID scenario increases up to 33% in 2030 and then drops by 5% compared to the FD scenario. These nonlinearities emerge from households’ preferred actions (I1-I3) unequally distributed over time and space. These results are a pure effect of individual changes driven by behavioral factors: we do not include any price-based scenarios (subsidies for green or taxes on grey energy) or changes in technological costs in this article. Journal Pre-p Our analysis confirms that faster learning boosted by an information campaign (FD vs Baseline scenarios) leads to more investments (I2, I3), and consequently to higher electricity savings (40%-100%) in both provinces. Step 1: From behavioral patterns in survey data to cumulative impacts in two provinces roof In addition, electricity savings increase by 14%-22% in two provinces if pro-environmental awareness is raised through an information policy (ID vs FD scenarios). However, ID has a mixed impact on insulation investments (I1) and gas consumption in Navarre and the opposite effect in Overijssel (ID delivers 26% lower gas savings compared to FD). 19 Step 2: Scaling-up behavioral scenarios to national and EU level After analyzing the dynamics in households’ behavioral changes in two provinces over time, we switch to understanding how they change over space. Using the population projection scenarios for the EU28 (see section 2.2, step 2), we scale the dynamics in household energy behavioral changes in two provinces over time up to national and EU levels. Namely, we define behavioral patterns for a heterogeneous group of households in the Dutch and Spanish regional ABMs. For each of the 12 age-education groups (Table 2), a number of households perusing an action (I1-I3) is estimated together with the average investments, and gas and electricity savings. The analysis reveals that in the Netherlands and Spain that the majority of households – 75.9% and 68.1% – intend to invest in energy-efficient appliances (I3) by 2050. The minority – 4.9% and 9.4% – want to invest in insulation (I1); this trend is stable over time (2020-2050). Electricity consumption resulting from individual behavioral changes decreases between 51-71% (the Netherlands) and 51-66% (Spain) by 2050 (see Appendix 4, Table A4.1). Pre-proof Figure 6: Percentage change in electricity consumption in 2050 from the base 2015, calculated as a result of scaling up the outcomes of the ABM model with population changes in the “Fast dynamics” scenario. Source: scaled-up BENCH-v.3 results. Journal Pr Figure 6: Percentage change in electricity consumption in 2050 from the base 2015, calculated as a result of scaling up the outcomes of the ABM model with population changes in the “Fast dynamics” scenario. Source: scaled-up BENCH-v.3 results. Figure 6 shows percentage changes in residential electricity consumption as a result of scaling up the output of the empirical ABM with the population change scenario. Electricity consumption resulting from individual behavioral changes decreases between 56.2-69.5% and 13.8-63.8% by 20 2050 in the Netherlands and Spain correspondingly. Importantly, there is significant spatial heterogeneity in how behavioral changes diffuse and what regions emerge as laggers or pioneers in bottom-up investments in energy-efficiency. If behavioral patterns elicited through our survey hold in the next few decades, it could be expected that the Limburg, Drenthe, and Zeeland provinces in the Netherlands and the Castile-Leon and Asturias regions in Spain will be pioneers compared to others in respective countries. Journal Pre-pr ( , ) al Pre-proof Journal Pre Step 3: From regional to the national and EU28 economy Scaled-up outputs of the ABM are used as input to the simulation setup of the spatial CGE model. Namely, information from BENCH-v.3 on the decrease in households’ use of electricity and gas is used in order to exogenously modify the minimum subsistence level of households’ consumption of the respective services in EU-EMS (see Appendix 2). The ABM-CGE results indicate that households with higher education levels are more likely to change their behavior compared to less educated people. Importantly, among these higher educated households, younger people (20-40) are more active. In particular, Dutch youth saves up to 17% and 74% more electricity and gas compared to 40+ households under the FD scenario (Figure 7). Among the pioneers (g6-8, i.e. middle educated and 20+ age; see Table 2), Spanish households save 1.9- 2.8 and 1.0-1.4 times more gas and electricity compared to Dutch households depending on groups and behavioral scenarios. Intensive social dynamics (FD scenario) has a stronger impact on saving gas, while the informative ID scenario activates more households in saving electricity. Appendix 4 presents a more detailed ABM-CGE analysis on diffusion of households’ investment per capita per action among sociodemographic groups. Journal Pre-proof A reduction in the consumption of gas and electricity by households results in a higher budget share that becomes available for other types of consumption. Depending on households’ consumption patterns, such shifts in consumption might result in higher values of GDP over time. 21 (a) gas (b) electricity Figure 7: Saved energy per capita (electricity and gas) as a result of households’ energy investments among 12 sociodemographic groups (table 2) under behavioral scenarios (FD,ID) in two countries. Source: EU-EMS and BENCH-v.3 of (b) electricity (a) gas (b) electricity (a) gas Figure 7: Saved energy per capita (electricity and gas) as a result of households’ energy investments among 12 sociodemographic groups (table 2) under behavioral scenarios (FD,ID) in two countries. Source: EU-EMS and BENCH-v.3 of 22 The EU-EMS model operates at the level of NUTS2 regions of the EU28, and hence enables the calculation of the regional impacts of various behavioral scenarios on real GDP that is GDP that includes only quantity effects. We choose to use GDP in our analysis instead of welfare indicators such as equivalent variation measure because the monetary indicator such as GDP can be easily compared with the outcomes of the ABM model in terms of monetized energy savings and investments. Step 3: From regional to the national and EU28 economy The focus of the present study is in illustrating the added-value of the use of CGE model and the degree of the indirect and economy-wide effects calculated by the CGE which justifies the choice of monetary GDP indicator for our analysis. Figure 8 illustrates the difference in regional real GDP levels in 2050 between the Baseline and FD scenarios. Most of the EU28 regions benefit from the behavioral changes, which leads to a decrease in energy consumption, with a few regions affected negatively. The level of overall real GDP impacts depends on the size of the region in terms of population and its share of highly-educated youth. Appendix 4 presents the percentage changes on the level of regional GDP relative to the Baseline scenario (see Figures A4.2). e-proof Figure 8: Deviation in the levels of regional real GDP under the “Fast dynamics” scenario compared to Baseline in 2050 as an aggregated effect of households’ behavioral changes, in millions of Euros. Source: EU-EMS and BENCH-v.3 Journal Pre Figure 8: Deviation in the levels of regional real GDP under the “Fast dynamics” scenario compared to Baseline in 2050 as an aggregated effect of households’ behavioral changes, in millions of Euros. Source: EU-EMS and BENCH-v.3 Figure 9 presents the effects in relative terms (scenario as % of the baseline which already accounts for whether a region is rural or urban) and relate them to GDP per capita. It implies there is a statistical relationship between the two variables: the Baseline GDP per capita (which is also positively correlated with the share of highly educated persons) and the benefits in terms of 23 additional economic growth per capita from the modeled behavioral changes. Though the relationship is non-linear, the trend indicates that rich and economically well-developed regions receive higher benefits from promoting behavioral changes in the long-run compared to the lagging regions. This phenomena raises the question of whether the distribution of economic benefits skewed towards rich and well-developed regions increases the overall interregional inequality in Europe. To understand how behavioral changes under our scenarios impact EU28 regional disparities, we calculate economic inequality index for the period 2015-2050 (section 2.1.2, Eq. 3). The dynamics of Theil’s T inequality index demonstrate that the inequality between regions decreases in the period of large investments in energy savings (2025-2035) and then starts to increase again over time, indicating the non-linear nature of the process (Figure 10). Step 3: From regional to the national and EU28 economy However, the regional inequality in 2050 does not reach the level of 2015, indicating the positive overall impact of behavioral changes on equality. Despite this, changes in inequality due to the implementation of behavioral scenarios remain modest. re-proof Figure 9: Correlation between changes in GDP per capita under “Fast dynamics” scenario and the level of regional GDP per capita under “Baseline” scenario in 1000 Euros per individual in 2050. Source: EU-EMS and BENCH-v.3 Journal Pre Journal Pre Figure 9: Correlation between changes in GDP per capita under “Fast dynamics” scenario and the level of regional GDP per capita under “Baseline” scenario in 1000 Euros per individual in 2050. Source: EU-EMS and BENCH-v.3 24 Figure 10: Dynamics of the Theil-T income inequality index over time under “Fast dynamics”. Source: EU-EMS and BENCH- v.3. roof Figure 10: Dynamics of the Theil-T income inequality index over time under “Fast dynamics”. Source: EU-EMS and BENCH- v.3. roof Figure 10: Dynamics of the Theil-T income inequality index over time under “Fast dynamics”. Source: EU-EMS and BENCH- 3 oof 4. Conclusions and Outlook The potential of individual behavioral changes in reducing carbon emissions attracts considerable attention as one of the climate change mitigation strategies (Creutzig et al., 2016; IPCC, 2014; Niamir, 2019). Comprehensive empirical CGEs, which support quantitative climate change mitigation policy assessments, are strong in tracing cross-sectoral impacts, feedback in the economy as a whole and in linking to readily-available datasets. However, their econometrically- estimated equations reflect past behavior, making it difficult to integrate behavioral changes (Babatunde et al., 2017; Farmer and Foley, 2009). Moreover, while empirical evidence suggests that individual decision-making deviates from a rational and perfectly informed optimization process, the latter is the core of CGE models (Farmer et al., 2015; Stern, 2016; Wilkerson-Jerde and Wilensky, 2015). Journal Pre ABMs compliment macroeconomic models by accommodating heterogeneity, adaptive behavior and interactions, bounded rationality, and imperfect information (Rai and Henry, 2016). While there are few (largely non-empirical) ABMs in policy and institutional domain that take a macro, e.g. country and global scale perspective (Castro et al., 2020; Gerst et al., 2013), behaviorally- rich empirical ABMs mostly operate on small scales of neighborhoods, cities, and regions. Although these micro ABMs are strong in aggregating heterogeneous adaptive behavior, they omit feedbacks with the rest of the economy and cross-sectoral impacts. Survey data is increasingly used to specify individual agent’s rules, yet this behavioral data is not always compatible with the data used in macro models. Linking ABMs and CGE models could 25 ameliorate their weaknesses. Yet, the models should be aligned coherently conceptually and data- wise to benefit from their strengths (Voinov and Shugart, 2013). Methodologically, this article contributes to the ongoing debate (Krook-Riekkola et al., 2017; Parris, 2005; Safarzyńska et al., 2013; Smajgl et al., 2009) on linking these two alien approaches by presenting a method of systematic upscaling of individual heterogeneity and social dynamics to combine ABM and CGE models. The insights from this methodological exercise offer three conclusions. Firstly, we demonstrate the feasibility and importance of introducing heterogeneity and behavioral-rich dynamics in assessing climate change mitigation policies. We develop a transparent step-wise process to integrate an empirical behaviorally-rich ABM and a spatial CGE model. To the best of our knowledge, this is the first attempt to link empirical ABM and CGE models to estimate the macroeconomic impacts of individual energy behavioral changes. 4. Conclusions and Outlook In the absence of this integration, one should twist the CGE parameters and structure in an ad-hoc manner to permit some representation of a behavioral change. Instead, an ABM that relies strongly on the theoretical and empirical micro-foundations from surveys, quantifies the patterns of behavioral change across heterogeneous households in a transparent way accounting for non-monetary aspects of individual energy choices. al Pre-proof Secondly, this article demonstrates that scaling up behavioral change dynamics has policy- relevant consequences at large scales. Our ABM grounded in theory and survey data quantifies the patterns of behavioral change, which could further be channeled into the CGE models that traces macroeconomic and cross-sectoral dynamics. Specifically, here we find that the regional dimension is important in a low-carbon economy transition driven by individual behavioral change. Some regions lag behind while others are pioneers, due to the heterogeneity in individuals’ socio-demographics (e.g. education and age), structural characteristics (e.g. type and size of dwellings), behavioral and social traits, and spatial characteristics (e.g. urban vs. rural) which produce incremental differences at small scales. Yet, when aggregated, they cumulatively create disparities, which are amplified by macro-economic forces. Importantly, the inequality between regions decreases in the period of large investments (2015-2035) and starts to increase over time following it. Journa Finally, as behavioral barriers to climate change mitigation in designing policies gain attention, policy-makers would benefit from decision support tool that go beyond a stylized representation of households as perfectly-informed optimizers. Individual awareness, diversity in norms, and knowledge play a key role in a green economy transition and climate change mitigation policies 26 should ideally combine the conventional macroeconomic analysis with these behavioral barriers and rivers. Considering bottom-up behavioral patterns would not easily change over time. To see culpable changes, we need a mix of external intervention, from soft information policies aimed to raise awareness bottom-up, to financial incentives altering the macro landscape of energy markets and technological transitions. At times, information and price-based policies create a non-linear effect on cumulative behavioral changes regarding energy use (Niamir et al., 2020b). Our approach demonstrates that with computational ABM directly linked to survey data and macroeconomic CGE models, individual behavioral heterogeneity and social influences can now be considered when designing implementable and politically feasible policy options. f The future work can go in two main directions: advancing the modeling approach and improving the models dataset. 4. Conclusions and Outlook From the modeling perspective, future work could focus on introducing direct feedbacks between CGE-ABM, enabling the evaluation of price-based and information-policies jointly at multiple scales. The feedbacks between the two empirical models may be enabled through software wrappers and modern web interfaces for integration (Belete et al., 2019). In addition, due to the large number of parameters and multidimensionality of the generated data from any ABM (Lee et al., 2015), the global sensitivity and uncertainty analysis was out of scope of this article. Future work should focus on quantifying uncertainties that this integration of ABM and CGE models may impose, including for example exploratory analysis (Kwakkel and Pruyt, 2013) to understand the integrated model’s behavior and its sensitivity to initial configurations of its parameters. From the dataset perspective, running surveys in more EU countries would improve the model accuracy, especially vital when predicting policy impacts. Also, data-wise, the behaviorally rich demand-side modeling could benefit from endogenizing the dynamics of dwelling stock. Static and aging housing should be replaced by scenarios of structural and technological progress in new urban development (e.g., zero-carbon footprint buildings) and refurbishing old housing stock in cities. Journal Pre-proof Acknowledgments This work was supported by the EU FP7 COMPLEX (Knowledge Based Climate Mitigation Systems for a Low Carbon Economy) Project No. 308601, the SCALAR European Research Council (ERC) Project, grant agreement No. 758014 , and the IIASA-RITE distinguished young scientist award (recipient: Leila Niamir). We are thankful to Prof. Dr. Alexey Voinov (University of Technology Sydney, Australia) and Prof. Dr. Hans Bressers (University of Twente, the Netherlands) for their feedback and support. The authors would like to thank the TNS NIPO team 27 for their collaboration in conducting the household survey. We also appreciate the participation of the survey respondents and the constructive feedback we received from the three anonymous reviewers. for their collaboration in conducting the household survey. We also appreciate the participation of the survey respondents and the constructive feedback we received from the three anonymous reviewers. Data availability The extensive description of the models and data is presented in the Appendix of this manuscript. The BENCH model is calibrated based on the empirical dataset. We designed and conducted the survey in two provinces in Europe for the purpose of this research (Niamir et al., 2020a). The agent-based BENCH model is parameterized using the survey data on socio-demographic, economic, structural and behavioral attributes of households and their dwelling characteristic (Table A1.1). The BENCH agent-based model is open source and available on CoMSES . proof The main database of EU-EMS model is the PBL-JRC world-wide MRIO database documented in https://ec.europa.eu/jrc/sites/jrcsh/files/jrc115439.pdf and available to download from https://data.overheid.nl/dataset/pbl-euregio-database-2000-2010 . Besides this MRIO database we have also used the national accounts data from Eurostat (Research Project RPP 342/2016-CSIS- EU-SILC-HBS-LFS) and OECD for the construction of Social Accounting Matrices used to calibrate the model. According to the terms of use, authors are not allowed to redistribute the Eurostat micro-data. The derived intermediate result are available from the corresponding author upon reasonable request. ournal Pre-pr References Abrahamse, W., Steg, L., 2011. Factors Related to Household Energy Use and Intention to Reduce It: The Role of Psychological and Socio-Demographic Variables. Hum. Ecol. Rev. https://doi.org/10.2307/24707684 Abrahamse, W., Steg, L., 2011. Factors Related to Household Energy Use and Intention to Reduce It: The Role of Psychological and Socio-Demographic Variables. Hum. Ecol. Rev. https://doi.org/10.2307/24707684 Allen, M., Babiker, M., Chen, Yang, Taylor, M., Tschakert Australia, P., Waisman, H., Warren, R., Zhai, Panmao, Zickfeld, K., Zhai, P, Pörtner, H., Roberts, D., Skea, J., Shukla, P., Pirani, A., Moufouma- Okia, W., Péan, C., Pidcock, R., Connors, S., Matthews, J., Chen, Y, Zhou, X., Gomis, M., Lonnoy, E., Maycock, T., Tignor, M., Waterfield, T., 2018. Summary for Policymakers. In: Global Warming of 1.5°C. An IPCC Special Report on the impacts of global warming of 1.5°C above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate povertyummary for Policymakers SPM, Aromar Revi. Allen, M., Babiker, M., Chen, Yang, Taylor, M., Tschakert Australia, P., Waisman, H., Warren, R., Zhai, Panmao, Zickfeld, K., Zhai, P, Pörtner, H., Roberts, D., Skea, J., Shukla, P., Pirani, A., Moufouma- Okia, W., Péan, C., Pidcock, R., Connors, S., Matthews, J., Chen, Y, Zhou, X., Gomis, M., Lonnoy, E., Maycock, T., Tignor, M., Waterfield, T., 2018. Summary for Policymakers. In: Global Warming of 1.5°C. An IPCC Special Report on the impacts of global warming of 1.5°C above pre-industrial levels and related global greenhouse gas emission pathways, in the context of strengthening the global response to the threat of climate change, sustainable development, and efforts to eradicate povertyummary for Policymakers SPM, Aromar Revi. 28 Babatunde, K.A., Begum, R.A., Said, F.F., 2017. Application of computable general equilibrium (CGE) to climate change mitigation policy: A systematic review. Renew. Sustain. Energy Rev. 78, 61–71. https://doi.org/10.1016/J.RSER.2017.04.064 Bamberg, S., Hunecke, M., Blöbaum, A., 2007. Social context, personal norms and the use of public transportation: Two field studies. J. Environ. Psychol. 27, 190–203. https://doi.org/10.1016/j.jenvp.2007.04.001 Bamberg, S., Rees, J., Seebauer, S., 2015. Collective climate action: Determinants of participation intention in community-based pro-environmental initiatives. J. Environ. Psychol. 43, 155–165. https://doi.org/10.1016/J.JENVP.2015.06.006 f Belete, G.F., Voinov, A., Arto, I., Dhavala, K., Bulavskaya, T., Niamir, L., Moghayer, S., Filatova, T., 2019. Exploring Low-Carbon Futures: A Web Service Approach to Linking Diverse Climate- Energy-Economy Models. Energies 12, 2880. References https://doi.org/10.3390/en12152880 proof Benczúr, P., Kátay, G., Kiss, Á., 2018. Assessing the economic and social impact of tax and benefit reforms: A general-equilibrium microsimulation approach applied to Hungary. Econ. Model. 75, 441–457. https://doi.org/10.1016/j.econmod.2018.06.016 Pre-pr Bhattacharyya, S.C., 2011. Energy economics : concepts, issues, markets and governance. Springer. al P Bijl, D.L., Bogaart, P.W., Dekker, S.C., Stehfest, E., de Vries, B.J.M., van Vuuren, D.P., 2017. A physically-based model of long-term food demand. Glob. Environ. Chang. 45, 47–62. https://doi.org/https://doi.org/10.1016/j.gloenvcha.2017.04.003 urnal Bohringer, C., Rutherford, T., 2008. Combining bottom-up and top-down. Energy Econ. 30, 574–596. Jou Böhringer, C., Rutherford, T., 2009. Integrated assessment of energy policies: Decomposing top-down and bottom-up. J. Econ. Dyn. Control 33, 1648–1661. Jo Castro, J., Drews, S., Exadaktylos, F., Foramitti, J., Klein, F., Konc, T., Savin, I., van den Bergh, J., 2020. A review of agent-based modeling of climate-energy policy. WIREs Clim. Chang. 11, e647. https://doi.org/10.1002/wcc.647 Creutzig, F., Fernandez, B., Haberl, H., Khosla, R., Mulugetta, Y., Seto, K.C., 2016. Beyond Technology: Demand-Side Solutions for Climate Change Mitigation. https://doi.org/10.1146/annurev-environ- 110615-085428 Creutzig, F., Roy, J., Lamb, W.F., Azevedo, I.M.L., Bruine de Bruin, W., Dalkmann, H., Edelenbosch, O.Y., Geels, F.W., Grubler, A., Hepburn, C., Hertwich, E.G., Khosla, R., Mattauch, L., Minx, J.C., Ramakrishnan, A., Rao, N.D., Steinberger, J.K., Tavoni, M., Ürge-Vorsatz, D., Weber, E.U., 2018. Towards demand-side solutions for mitigating climate change, Nature Climate Change. Nature 29 Publishing Group. https://doi.org/10.1038/s41558-018-0121-1 Debowicz, D., 2016. Does the microsimulation approach used in macro–micro modelling matter? An application to the distributional effects of capital outflows during Argentina’s Currency Board regime. Econ. Model. 54, 591–599. Dietz, T., Stern, P.C., Weber, E.U., 2013. Reducing Carbon-Based Energy Consumption through Changes in Household Behavior. Daedalus 142, 78–89. https://doi.org/10.1162/DAED_a_00186 Doppelt, B., Markowitz, E.M., Climate Leadership Initiative, 2009. Reducing Greenhouse Gas Emissions through Behavioral Change : An Assessment of Past Research On Energy Use, Transportation and Water Consumption. f Duarte, R., Feng, K., Hubacek, K., Sánchez-Chóliz, J., Sarasa, C., Sun, L., 2016. Modeling the carbon consequences of pro-environmental consumer behavior. Appl. Energy 184, 1207–1216. https://doi.org/10.1016/J.APENERGY.2015.09.101 proof Faber, J., Schroten, A., Bles, M., Sevenster, M., Markowska, A., Smit, M., Rohde, C., Dütschke, E., Köhler, J., Gigli, M., Zimmermann, K., Soboh, R., van ’t Riet, J., 2012. Behavioural Climate Change Mitigation Options and Their Appropriate Inclusion in Quantitative Longer Term Policy Scenarios. CE Delft, Delft, Netherlands. l Pre-p Farmer, J., Hepburn, C., Mealy, P., Teytelboym, A., 2015. A Third Wave in the Economics of Climate Change. Environ. References & Resour. Econ. 62, 329–357. rnal Farmer, J.D., Foley, D., 2009. The economy needs agent-based modelling. Nat. 2009 4607256. ourn Filatova, T., Niamir, L., 2019. Changing climate – changing behavior: empirical agent based computational models for climate change economics, in: Lamperti, F., Monasterolo, I., Roventini, A. (Eds.), Complexity and Climate Change: An Evolutionary Political Economy Approach. Taylor & Francis. Jou Fujimori, S., Hasegawa, T., Masui, T., Takahashi, K., Herran, D.S., Dai, H., Hijioka, Y., Kainuma, M., 2017. SSP3: AIM implementation of Shared Socioeconomic Pathways. Glob. Environ. Chang. 42, 268–283. https://doi.org/10.1016/J.GLOENVCHA.2016.06.009 Gerst, M.D., Wang, P., Roventini, A., Fagiolo, G., Dosi, G., Howarth, R.B., Borsuk, M.E., 2013. Agent- based modeling of climate policy: An introduction to the ENGAGE multi-level model framework. Environ. Model. Softw. 44, 62–75. https://doi.org/https://doi.org/10.1016/j.envsoft.2012.09.002 Gobel, M.S., Benet-Martinez, V., Mesquita, B., Uskul, A.K., 2018. Europe’s Culture(s): Negotiating Cultural Meanings, Values, and Identities in the European Context. J. Cross. Cult. Psychol. 49, 858– 867. https://doi.org/10.1177/0022022118779144 30 Grubler, A., Wilson, C., Bento, N., Boza-Kiss, B., Krey, V., McCollum, D.L., Rao, N.D., Riahi, K., Rogelj, J., Stercke, S., 2018. A low energy demand scenario for meeting the 1.5° C target and sustainable development goals without negative emission technologies. Nat. Energy 3, 515–527. https://doi.org/10.1038/s41560-018-0172-6 Hertwich, E.G., Peters, G.P., 2009. Carbon footprint of nations: A global, trade-linked analysis. Environ. Sci. Technol. 43, 6414–6420. https://doi.org/10.1021/es803496a Hofstede, G., 2011. Dimensionalizing Cultures: The Hofstede Model in Context. Online Readings Psychol. Cult. 2. https://doi.org/10.9707/2307-0919.1014 Hofstede, G., 2001. Culture’s consequences: comparing values, behaviors, institutions, and organizations across nations, 2nd ed. Sage Publications, Thousand Oaks, California. of Humphreys, P., Imbert, C., 2013. Models simulations and representations, Models Simulations and Representations. Taylor and Francis. https://doi.org/10.4324/9780203808412 proo Hunt, L.C., Evans, J., 2009. International handbook on the economics of energy. Edward Elgar. e-p IPCC, 2014. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Geneva. l Pre Isley, S.C., Lempert, R.J., Popper, S.W., Vardavas, R., 2015. The effect of near-term policy choices on long-term greenhouse gas transformation pathways. Glob. Environ. Chang. 34, 147–158. https://doi.org/10.1016/J.GLOENVCHA.2015.06.008 urnal P Ivanova, O., Kancs, A., Thissen, M., 2019. EU Economic Modelling System: Assessment of the European Institute of Innovation and Technology (EIT) Investments in Innovation and Human Capital. Luxembourg. https://doi.org/10.2791/184008 Jour JRC, 2014. ETRI 2014 - Energy Technology Reference Indicator projections for 2010-2050. https://doi.org/10.2790/057687 Kaasa, A., Vadi, M., Varblane, U., 2016. References A new dataset of cultural distances for European countries and regions. Res. Int. Bus. Financ. 37, 231–241. https://doi.org/10.1016/J.RIBAF.2015.11.014 Krook-Riekkola, A., Berg, C., Ahlgren, E.O., Söderholm, P., 2017. Challenges in top-down and bottom- up soft-linking: Lessons from linking a Swedish energy system model with a CGE model. Energy 141, 803–817. https://doi.org/10.1016/J.ENERGY.2017.09.107 Kulmer, V., Seebauer, S., 2019. How robust are estimates of the rebound effect of energy efficiency improvements? A sensitivity analysis of consumer heterogeneity and elasticities. Energy Policy 132, 1–14. 31 Kwakkel, J.H., Pruyt, E., 2013. Exploratory Modeling and Analysis, an approach for model-based foresight under deep uncertainty. Technol. Forecast. Soc. Change 80, 419–431. https://doi.org/10.1016/J.TECHFORE.2012.10.005 Lamperti, F., Mandel, A., Napoletano, M., Sapio, A., Roventini, A., Balint, T., Khorenzhenko, I., 2019. Towards agent-based integrated assessment models: examples, challenges, and future developments. Reg. Environ. Chang. 19, 747–762. https://doi.org/10.1007/s10113-018-1287-9 Lee, J.-S., Filatova, T., Ligmann-Zielinska, A., Hassani-Mahmooei, B., Stonedahl, F., Lorscheid, I., Voinov, A., Polhill, J.G., Sun, Z., Parker, D.C., 2015. The Complexities of Agent-Based Modeling Output Analysis. J. Artif. Soc. Soc. Simul. 18, 4. https://doi.org/10.18564/jasss.2897 f McKinsey, 2009. Pathways to a Low-Carbon Economy. McKinsey, 2009. Pathways to a Low-Carbon Economy. of Melnikov, N.B., O’Neill, B.C., Dalton, M.G., van Ruijven, B.J., 2017. Downscaling heterogeneous household outcomes in dynamic CGE models for energy-economic analysis. Energy Econ. 65, 87– 97. https://doi.org/10.1016/J.ENECO.2017.04.023 e-proo Moussaïd, M., Brighton, H., Gaissmaier, W., 2015. The amplification of risk in experimental diffusion chains. Proc. Natl. Acad. Sci. 112, 5631 LP – 5636. https://doi.org/10.1073/pnas.1421883112 Pre Niamir, L., 2019. Behavioural Climate Change Mitigation. University of Twente, Enschede, The Netherlands. https://doi.org/10.3990/1.9789036547123 nal P Niamir, L., Filatova, T., 2017. Transition to low-carbon economy: Simulating nonlinearities in the electricity market, Navarre region, Spain, Advances in Intelligent Systems and Computing. https://doi.org/10.1007/978-3-319-47253-9_28 Journa Niamir, L., Filatova, T., Voinov, A., Bressers, H., 2018a. Transition to low-carbon economy: Assessing cumulative impacts of individual behavioral changes. Energy Policy 118, 325–345. https://doi.org/10.1016/J.ENPOL.2018.03.045 Jo Niamir, L., Ivanova, O., Filatova, T., Voinov, A., 2018b. Tracing Macroeconomic Impacts of Individual Behavioral Changes through Model Integration. IFAC-PapersOnLine 51, 96–101. Niamir, L., Ivanova, O., Filatova, T., Voinov, A., Bressers, H., 2020a. Demand-side solutions for climate mitigation: Bottom-up drivers of household energy behavior change in the Netherlands and Spain. Energy Res. Soc. Sci. 62, 101356. https://doi.org/10.1016/j.erss.2019.101356 Niamir, L., Kiesewetter, G., Wagner, F., Schöpp, W., Filatova, T., Voinov, A., Bressers, H., 2020b. Assessing the macroeconomic impacts of individual behavioral changes on carbon emissions. Clim. Change 158, 141–160. References https://doi.org/https://doi.org/10.1007/s10584-019-02566-8 Nielsen, K.S., Clayton, S., Stern, P.C., Dietz, T., Capstick, S., Whitmarsh, L., 2020. How psychology can 32 32 help limit climate change. Am. Psychol. No Pagination Specified-No Pagination Specified. https://doi.org/10.1037/amp0000624 Parris, B., 2005. Trade and Industrial Policy in Developing Countries: What Scope for Interfaces between Agent-Based Models and Computable-General Equilibrium Models?, in: ACEPOL05 : Proceedings of the International Workshop on Agent-Based Models for Economic Policy Design. pp. 1–25. Peichl, A., Schaefer, T., 2009. FiFoSiM - an integrated tax benefit microsimulation and CGE model for Germany. Int. J. Microsimulation 2, 1–15. Poortinga, W., Steg, L., Vlek, C., 2004. Values, Environmental Concern, and Environmental Behavior: A Study into Household Energy Use. Environ. Behav. 36, 70–93. https://doi.org/10.1177/0013916503251466 of Rai, V., Henry, A.D., 2016. Agent-based modelling of consumer energy choices. Nat. Clim. Chang. 6, 556–562. https://doi.org/10.1038/nclimate2967 proo Rao, N.D., van Ruijven, B.J., Riahi, K., Bosetti, V., 2017. Improving poverty and inequality modelling in climate research. Nat. Clim. Chang. 7, 857–862. https://doi.org/10.1038/s41558-017-0004-x Pre-p Rausch, S., Metcalf, G.E., Reilly, J.M., 2011. Distributional impacts of carbon pricing: A general equilibrium approach with micro-data for households. Energy Econ. 33, S20–S33. https://doi.org/10.1016/J.ENECO.2011.07.023 nal Pr Rive, N., Torvanger, A., Fuglestvedt, J., 2006. Climate agreements based on responsibility for global warming: Periodic updating, policy choices, and regional costs. Glob. Environ. Chang. 16. ourna Safarzyńska, K., Brouwer, R., Hofkes, M., 2013. Evolutionary modelling of the macro-economic impacts of catastrophic flood events. Ecol. Econ. 88, 108–118. https://doi.org/10.1016/J.ECOLECON.2013.01.016 Jou Schwartz, S.H., 2014. Chapter 20 - National Culture as Value Orientations: Consequences of Value Differences and Cultural Distance, in: Ginsburgh, V.A., Throsby, D.B.T.-H. of the E. of A. and C. (Eds.), Handbook of the Economics of Art and Culture. Elsevier, pp. 547–586. https://doi.org/https://doi.org/10.1016/B978-0-444-53776-8.00020-9 Smajgl, A., Morris, S., Heckbert, S., 2009. Water policy impact assessment - Combining modelling techniques in the Great Barrier Reef region. Water Policy 11, 191–202. https://doi.org/10.2166/wp.2009.022 Steg, L., Vlek, C., 2009. Encouraging pro-environmental behaviour: An integrative review and research agenda. J. Environ. Psychol. 29, 309–317. https://doi.org/10.1016/J.JENVP.2008.10.004 Stern, N., 2016. Economics: Current climate models are grossly misleading. Nature 530, 407–409. 33 https://doi.org/10.1038/530407a Sue Wing, I., 2008. The synthesis of bottom-up and top-down approaches to climate policy modeling: Electric power technology detail in a social accounting framework. Energy Econ. 30, 547–573. Tesfatsion, L., 2006. Agent-Based Computational Economics: A Constructive Approach to Economic Theory, in: Tesfatsion, L., Judd, K.L. (Eds.), . Elsevier, pp. 831-880 BT-Handbook of Computational Economics. van Raaij, W.F., 2017. References Social psychology and economic behaviour: Heuristics and biases in decision making and judgement., in: Steg, L., Keizer, K., Buunk, A.P., Rothengatter, T. (Eds.), Applied Social Psychology. Cambridge University Press, United Kingdom, pp. 149–170. f van Ruijven, B.J., O’Neill, B.C., Chateau, J., 2015. Methods for including income distribution in global CGE models for long-term climate change research. Energy Econ. 51, 530–543. https://doi.org/10.1016/J.ENECO.2015.08.017 proof Vandyck, T., Keramidas, K., Saveyn, B., Kitous, A., Vrontisi, Z., 2016. A global stocktake of the Paris pledges: Implications for energy systems and economy. Glob. Environ. Chang. 41, 46–63. https://doi.org/10.1016/J.GLOENVCHA.2016.08.006 Pre-p Verikios, G., Zhang, X., 2015. Reform of Australian urban transport: A CGE-microsimulation analysis of the effects on income distribution. Econ. Model. 44, 7–17. nal P Vignoles, V.L., Smith, P.B., Becker, M., Easterbrook, M.J., 2018. In Search of a Pan-European Culture: European Values, Beliefs, and Models of Selfhood in Global Perspective. J. Cross. Cult. Psychol. 49, 868–887. https://doi.org/10.1177/0022022117738751 Journ Voinov, A., Shugart, H.H., 2013. “Integronsters”, integral and integrated modeling. Environ. Model. Softw. 39, 149–158. https://doi.org/10.1016/j.envsoft.2012.05.014 Jo Wilkerson-Jerde, M.H., Wilensky, U.J., 2015. Patterns, Probabilities, and People: Making Sense of Quantitative Change in Complex Systems. J. Learn. Sci. 24, 204–251. https://doi.org/10.1080/10508406.2014.976647 34 Appendix 1: BENCH agent-based model The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. Main processes of the model (ODD protocol) Table A1.1: BENCH-v.3 ABM ODD protocol Guiding Protocol The BENCH-V.3 model A. Overview A.1. Purpose The BENCH-v.3 agent-based model is designe residential energy use and corresponding regional level driven by behavioral changes am individuals. This empirically grounded model is of environmental scientists interested in modellin and economic institutions, (ii) energy econo micro aspects, (iii) scholars integrating indi change in climate change mitigation modelling A.2. Entities, state variables and scales Agents (individuals) in BENCH-v.3 model are socio-demographic and dwelling characteristic consumption and patterns, source of energy and and behavioral factors. The BENCH-v.3 simulations 1035 and 755 ind in the Overijssel province, the Netherlands, and Spain over 34 years (2016-2050). One time step represents one round in the beha Each run consist of 34 time steps aligning to th behavioral experiments. A.3. Process overview One time step represents one-year. In each time goes through several processes: 1. Asses behavioral factors: ● Knowledge activation ● Motivation Journal Pre-proof The BENCH-v.3 agent-based model is designed to study shifts in residential energy use and corresponding emissions at the regional level driven by behavioral changes among heterogeneous individuals. roof This empirically grounded model is of interest to (i) environmental scientists interested in modelling human behavior and economic institutions, (ii) energy economists working on micro aspects, (iii) scholars integrating individuals behavioral change in climate change mitigation modelling. l Pre-pro Agents (individuals) in BENCH-v.3 model are heterogeneous in socio-demographic and dwelling characteristics, energy consumption and patterns, source of energy and energy provider, and behavioral factors. nal The BENCH-v.3 simulations 1035 and 755 individual households in the Overijssel province, the Netherlands, and Navarre province, Spain over 34 years (2016-2050). 35 ● Consideration 2. Calculate utilities 3. Pursue an action or not 4. Calculate saved energy and CO2 emission 5. Social dynamics and learning process 6. Satisfaction and regret 7. Updates See Figure 2 for algorithm and decision-making proccess in the BENCH-v.3 agent-based model. B. Design concept B.1 Theoretical and Empirical background In application to environmental- and energy-related choices, three behavioral change theories are commonly applied: theory of planned behavior (TPB), norm activation theory (NAT), and value–belief–norm (VBN) theory. ● TPB, formulated by Ajzen (1980) and based on the theory of reasoned action, is one of the most influential theories in social and health psychology and has been used in many environmental studies (Armitage and Conner, 2001; Onwezen et al., 2013). We introduce a framework that combines the strengths of the three key behavioral theories, see Figure A1.1. We introduce a framework that combines the strengths of the three key behavioral theories, see Figure A1.1. Lowest scale: Individuals B.5. Spatial scale The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. ● NAT, originally developed by Schwartz (1977), operates in the context of altruistic and environmentally friendly behavior. It is mostly focused on anticipating pride in doing the “right” thing and on studying the evolution of feelings of guilt. ● VBN theory (Stern et al., 1999; Stern, 2000) explains environmental behavior and “good intentions” such as willingness to change behavior (Nordlund and Garvill, 2003; Steg and Vlek, 2009; Stern et al., 1999), environmental citizenship (Stern et al., 1999), and policy acceptability (De Groot and Steg, 2009; Steg et al., 2005). B.2. Individual decision making We introduce a framework that combines the strengths of the three key behavioral theories, see Figure A1.1. Journal Pre-proof ● TPB, formulated by Ajzen (1980) and based on the theory of reasoned action, is one of the most influential theories in social and health psychology and has been used in many environmental studies (Armitage and Conner, 2001; Onwezen et al., 2013). nal Pr ● TPB, formulated by Ajzen (1980) and based on the theory of reasoned action, is one of the most influential theories in social and health psychology and has been used in many environmental studies (Armitage and Conner, 2001; Onwezen et al., 2013). nal P ● NAT, originally developed by Schwartz (1977), operates in the context of altruistic and environmentally friendly behavior. It is mostly focused on anticipating pride in doing the “right” thing and on studying the evolution of feelings of guilt. ● VBN theory (Stern et al., 1999; Stern, 2000) explains environmental behavior and “good intentions” such as willingness to change behavior (Nordlund and Garvill, 2003; Steg and Vlek, 2009; Stern et al., 1999), environmental citizenship (Stern et al., 1999), and policy acceptability (De Groot and Steg, 2009; Steg et al., 2005). 36 B.3. Heterogeneity Agents are heterogeneous in respect of the following variables, see Table A1.2: ● Socio-demographic ● Dwelling ● Energy consumption ● Energy provider ● Behavioral factors B.4. Interactions, social dynamics and learning Agents (heterogeneous individual households) engage in interactions and learn from each other. In particular, they can exchange information with neighbors, which may alter own knowledge, awareness, and motivation regarding energy-related behavior. We employ a simple opinion dynamics model (Acemoglu and Ozdaglar, 2011; Degroot, 1974; Hegselmann, 2002; Moussaïd et al., 2015) assuming that each agent interacts with a fixed set of nearby neighbors. B.4. Interactions, social dynamics and learning The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. The BENCH_v.3 model is a spatially explicit model that takes the raster maps of the two NUTS2 regions as an input. Hence, an agent who is in active neighborhood where at least one out of eight nearest spatial neighbors within 1 raster cell (Moor neighborhood concept) undertakes an energy-related action will interact and exchange opinions. The idea of the Moore neighborhood comes from cellular automata literature and used Journal Pre-proof Agents (heterogeneous individual households) engage in interactions and learn from each other. In particular, they can exchange information with neighbors, which may alter own knowledge, awareness, and motivation regarding energy-related behavior. We employ a simple opinion dynamics model (Acemoglu and Ozdaglar, 2011; Degroot, 1974; Hegselmann, 2002; Moussaïd et al., 2015) assuming that each agent interacts with a fixed set of nearby neighbors. l Pre-proof The BENCH_v.3 model is a spatially explicit model that takes the raster maps of the two NUTS2 regions as an input. Hence, an agent who is in active neighborhood where at least one out of eight nearest spatial neighbors within 1 raster cell (Moor neighborhood concept) undertakes an energy-related action will interact and exchange opinions. The idea of the Moore neighborhood comes from cellular automata literature and used only to enable opinion exchange between neighbors about climate and environmental awareness and compare norms. Agents compare values of their own behavioral factors – knowledge, awareness, and motivation – with those of their eight closest neighbors, and adjust their values for a closer match, see Figure 3 and Eq. 3. However, the agents’ heterogeneity beyond their spatial location (income, age, education) and economic factors affect individual choices of undertaking any of energy actions (I1- I3) or not. nal 37 Highest scale: NUTS212 The focus of this research is on Overijssel, the Netherlands (NL21) and Navarre, Spain (ES22) NUTS2 regions, which consist of 25 and 10 main cities/ municipalities respectively. B.6. Individual prediction Individuals do not predict future condition. B.7. Stochasticity There are various sources of stochasticity in the model: 1. Initial setting: Agents attributes (initialization are partly random) 2. During the process: Social dynamics and learning (process is partly random) B.8. Observation BENCH-v.3 estimates cumulative impacts of energy-related behavioral changes of individual households on electricity and gas consumption and CO2 emissions. 12 The Nomenclature of territorial units for statistics, abbreviated NUTS is a geographical nomenclature subdividing the economic territory of the European Union (EU) into regions at three different levels (NUTS 1, 2 and 3 respectively, moving from larger to smaller territorial units). The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. Reports: ● Number of energy-related actions per year: investment, conservation, switching ● Saved electricity and gas per action/year: investment, conservation, switching ● Avoided CO2 emission per action/year: investment, conservation, switching Across socioeconomic (age and education) groups (see Table 1) and cases (NL vs. ES). B.9. Implementation Details The model is coded in Netlogo 6.0.4, Open source and available on CoMSES (https://www.comses.net) R is used for the result visualizations. f i i l i f i i bb i d NUTS i hi l l bdi idi h i i f Journal Pre-proof Highest scale: NUTS212 12 The Nomenclature of territorial units for statistics, abbreviated NUTS is a geographical nomenclature subdividing the economic territory of the European Union (EU) into regions at three different levels (NUTS 1, 2 and 3 respectively, moving from larger to smaller territorial units). 38 C. Details C.1. Initialization The variations in socio-demographic, dwelling and psychological factors among our survey respondents are used to initialize a population of heterogeneous agents in the BENCH-v.3 model (see Table A1.1 and A1.3). C.2. Input data The data on the behavioral and economic factors affecting household energy choices were collected using an online questionnaire (N= 1790 households) and serve as empirical micro-foundation of agent rules in the BENCH-v.3 model. Figure A1.1: BENCH-v.3 conceptual behavioral framework. Source: (Niamir et al., 2020a) Table A1.2: Overview of main variables and parameters used in BENCH-v.3 Factors Variables Value range Journal Pre-proof The variations in socio-demographic, dwelling and psychological factors among our survey respondents are used to initialize a population of heterogeneous agents in the BENCH-v.3 model (see Table A1.1 and A1.3). The data on the behavioral and economic factors affecting household energy choices were collected using an online questionnaire (N= 1790 households) and serve as empirical micro-foundation of agent rules in the BENCH-v.3 model. Figure A1.1: BENCH-v.3 conceptual behavioral framework. Source: (Niamir et al., 2020a) Journal Pre-proof Jou Figure A1.1: BENCH-v.3 conceptual behavioral framework. Source: (Niamir et al., 2020a) J Table A1.2: Overview of main variables and parameters used in BENCH-v.3 Table A1.2: Overview of main variables and parameters used in BENCH-v.3 Factors Variables Value range Socio-demographic Income [1000 - 150,000] Education [primary - doctoral] Dwelling Energy label [a-f] Ownership status [owner - renter] 39 Energy Consumption [500 - 5000] Provider Grey, brown, green Energy saving habit [0-3] Behavioral Knowledge [1-7] Cee awareness [1-7] Ed awareness [1-7] Personal norms [1-7] Social norms [1-7] Intention a1 [1-7] Intention a2 [1-7] Intention a3 [1-7] Data al Pre-proof Data The BENCH-v.3 model is calibrated based on an empirical dataset. We designed and conducted the survey in two provinces in Europe for the purpose of this research. In 2016, 1035 households in the Overijssel province, the Netherlands, and 755 households in the Navarre province, Spain, filled out our online questionnaire (Niamir, 2019; Niamir et al., 2020a; Niamir and Filatova, 2017, 2016). The agent-based BENCH-v.3 model is parameterized using the survey data on socio-demographic, economic, structural and behavioral attributes of households and their dwelling characteristic (Table A1.3). Journa 40 40 41 havioral intentions. The data is used to parameterize households’ , 2018) Overijssel Navarre e: 46.4% 53.6% Female: 57.1% Male: 42.9% 41 nal_Standard_Classification_of_Education_(ISCED) Journal Pre-proof ons. The data is used to parameterize households’ erijssel Navarre Female: 57.1% Male: 42.9% 41 urnal Pre-proof eterize house proof oral in 18) 46.4% .6% Jo oral 18) 46.4 .6% Jo 42 t : 14.9% .1% Apartment : 77.8% House: 22.2% % % Owner: 80.3% Renter: 19.7% 5.0 (0.8) 5.4 (0.8) 5.3 (1.1) 5.4 (1.0) Journal Pre-proof Apartment : 77.8% House: 22.2% Owner: 80.3% Renter: 19.7% 5 0 (0 8) e-proof artme use: 2 ner: 8 nter: re-p 43 43 General description EU-EMS is a spatial computable general equilibrium (SCGE) model developed by PBL Netherlands Environmental Assessment Agency. The sectoral and geographical dimensions of the model are flexible and can be adjusted to the needs of a specific policy or research question. The model is used for policy impact assessment and provides sector-, region- and time-specific model-based support to Dutch and EU policy makers on structural reforms, growth, innovation, human capital and infrastructure policies. The current version of EU-EMS covers 276 NUTS2 regions of the EU28 Member States and each regional economy is disaggregated into 63 NACE Rev. 2 economic sectors14. Goods and services are consumed by households, government and firms, and are produced in markets that can be perfectly or imperfectly competitive. Spatial interactions between regions are captured through trade of goods and services, factor mobility and knowledge spill-overs. This makes EU-EMS particularly well suited for analyzing policies related to human capital, transport infrastructure, R&I and innovation. Journal Pre-pr In the current application of the model, we have aggregated the economic sectors to the following six large groups, following the Eurostat classification of the economic sectors according to their R&D intensity: (1) Traditional, (2) Low-tech industry, (3) Medium-tech industry, (4) High-tech industry, (5) Knowledge intensive services and (6) Other services. Outputs The agent-based BENCH-v.3 model tracks the individual and cumulative impacts of three energy behavioral changes (investments on insulation, PVs installation and energy-efficient appliances) among heterogeneous individuals in the Overijssel and Navarre provinces over 34 years (2016- 2050). We report the number of individuals pursuing a particular action (I1-I3), the cumulative electricity and gas consumption, and saved carbon emissions. Given the stochastic nature of ABMs, we perform multiple (N=100) repetitive runs of each simulation experiment (Lee et al., 2015). 14 https://ec.europa.eu/eurostat/documents/3859598/5902521/KS-RA-07-015-EN.PDF 14 https://ec.europa.eu/eurostat/documents/3859598/5902521/KS-RA-07-015-EN.PDF Main processes of the model EU-EMS accounts for the (a) feedback between price and demand/supply quantities, and (b) interactions between economic agents at the macro and sectorial level. Therefore, it gives the 44 economic relations between all industry sectors via their intermediate use. The EU-EMS model is a dynamic, recursive over time model, involving dynamics of capital accumulation and technology progress, stock and flow relationships and adaptive expectations. The model equations are neo-classical in spirit, assuming cost-minimizing behavior by producers, average- cost pricing and household demands based on optimizing behavior. The CGE model database consists of tables of transaction values and elasticities: dimensionless parameters that capture behavioral response. The database is presented as a Social Accounting Matrix, which covers an entire national economy, and distinguishes a number of sectors, commodities, primary factors and types of households. As a classical CGE model, EU-EMS represents the behavior of the whole population group or of the whole industrial sector as the behavior of one single aggregate agent. It is further assumed that the behavior of each such aggregate agent is driven by certain optimization criteria such as maximization of utility or minimization of costs. In following, detailed representation of the EU-EMS model and its main equations are presented. -proof Figure A2.1: Circular economic flow in the CGE EU-EMS model. Source: (Ivanova et al., 2019) Factors of production markets Firms Product markets Households Journal Pre Firms Figure A2.1: Circular economic flow in the CGE EU-EMS model. Source: (Ivanova et al., 2019) Household preferences and governmental sector The households’ and governmental demand for goods and services is represented by the Linear Expenditure System (LES) that is derived as a solution to the Stone-Geary utility maximization problem: (Eq. A2.1) (Eq. A2.1) (Eq. A2.1) (Eq. A2.1) ( ) ri r ri ri i U C γ µ = − ∏ f ( ) ri r ri ri i U C γ µ = − ∏ The resulting demand system, where rI denotes households’ disposable income and ri P are consumer prices of goods and services that include taxes, subsidies, transport and trade margins can be written as follows: proof (Eq. A2.2) 1 ri ri ri r rj rj j ri C I P P µ γ µ   = + ⋅ ⋅ − ⋅     ∑ al Pre-p (Eq. A2.2) 1 ri ri ri r rj rj j ri C I P P µ γ µ   = + ⋅ ⋅ − ⋅     ∑ al Pre Households always consume a certain minimum level of each good and services where this level reflects the necessity (or price elasticity) of the good or service. Necessities such as food have low price elasticity and hence a higher minimum level of consumption. The disposable income of the households consists of wages, return to capital and social transfers from the government minus the income taxes and households’ savings. Journal The government collects production, consumptions and income taxes. The tax revenue is further used to pay social transfers and buy goods and services for public consumption. The governmental savings can be either endogenous or exogenous in the model depending on the type of simulation and the type of chosen macro-economic closure. Regional structure of the model Regions differ by the type of production sectors which dominate overall production activities in the region. Some specialize in traditional sectors such as agriculture, whereas others specialize in modern sectors such as finance and industry. Those sectors are characterized by different levels of agglomeration and its importance. Traditional sectors do not experience any agglomeration effects, whereas modern sectors do; this allows some sectors to grow faster than other. The 45 prototype model will incorporate the regional difference in sectoral specialization and hence the difference of agglomeration economies between the regions. Table A2.1: Regions in EU-EMS CGE models. Source: (Ivanova et al., 2019) AUS Australia ARG Argentina AUT Austria BGR Bulgaria BEL Belgium BRA Brazil CAN Canada BRN Brunei Darussalam CHL Chile CHN China CZE Czech Republic CHN.DOM China Domestic sales only DNK Denmark CHN.PRO China Processing EST Estonia CHN.NPR China Non processing goods exporters FIN Finland COL Colombia FRA France CRI Costa Rica DEU Germany CYP Cyprus GRC Greece HKG Hong Kong SAR HUN Hungary HRV Croatia ISL Iceland IDN Indonesia IRL Ireland IND India ISR Israel KHM Cambodia ITA Italy LTU Lithuania JPN Japan LVA Latvia KOR Korea MLT Malta LUX Luxembourg MYS Malaysia MEX Mexico PHL Philippines MEX.GMF Mexico Global Manufacturing ROU Romania MEX.NGM Mexico Non-Global Manufacturing RUS Russian Federation NLD Netherlands SAU Saudi Arabia NZL New Zealand SGP Singapore NOR Norway THA Thailand POL Poland TUN Tunisia PRT Portugal TWN Chinese Taipei SVK Slovak Republic VNM Viet Nam SVN Slovenia ZAF South Africa ESP Spain RoW Rest of the world SWE Sweden CHE Switzerland TUR Turkey GBR United Kingdom USA United States Journal Pre-proof Table A2.1: Regions in EU-EMS CGE models. Source: (Ivanova et al., 2019) 46 Household preferences and governmental sector Firms production Domestic production D r i X is obtained using the nested-CES production technology of Capital- Labour-Energy-Materials (KLEM) type, where K is the capital, L is the labour, E is the energy and M is the materials. Figure II.2 represents the nests in the KLEM production function used in the model with services between used according to the fixed Leontief input coefficients in the production process. The energy in the model is differentiated between electricity and other types 47 of energy with some substitution possibilities between them. The labour is differentiated according to three education levels according to International Labour Organisation (ILO) classification. The domestic production is generated according to nested production CES function, which is described by the following set of composite CES functions that follow the production structure from top to the bottom nest (Eq. A2.3) (Eq. A2.3) ( ) ( ) , , , 1/ (1 ) M KLE M KLE M KLE D ri ri ri ri ri X a M a KLE ρ ρ ρ   = ⋅ + − ⋅   (Eq. A2.4) ( ) ( ) , , , 1/ (1 ) M KLE E KL E KL ri ri ri ri ri KLE b E b KL ρ ρ ρ   = ⋅ + − ⋅   (Eq. A2.5) ( ) ( ) , , , 1/ (1 ) K L K L K L ri ri ri ri ri KL c K c L ρ ρ ρ   = ⋅ + − ⋅   (Eq. A2.6) al Pre-proof (Eq. A2.4) ( ) ( ) , , , 1/ (1 ) M KLE E KL E KL ri ri ri ri ri KLE b E b KL ρ ρ ρ   = ⋅ + − ⋅   (Eq. A2.5) e-proof (Eq. A2.4) ( ) ( ) , , , 1/ (1 ) K L K L K L ri ri ri ri ri KL c K c L ρ ρ ρ   = ⋅ + − ⋅   (Eq. Firms production A2.6) al Pre p ( ) ( ) , , , 1/ (1 ) K L K L K L ri ri ri ri ri KL c K c L ρ ρ ρ   = ⋅ + − ⋅   Pre ( ) ( ) ( ) ri ri ri ri ri   (Eq. A2.6) al Pr ( ) ( ) 1/ (1 ) E E E NELEC ELEC ri ri ri ri ri E d E d E ρ ρ ρ   = ⋅ + − ⋅     (Eq. A2.7) ( ) 1/ L L ED ri rie rie e L f L ρ ρ   =     ∑ Journa (Eq. A2.7) ( ) 1/ L L ED ri rie rie e L f L ρ ρ   =     ∑ Where ri a , rib , ric , ri d and rie f are the share parameters of the corresponding production function nests and , M K L E ρ , , E K L ρ , , K L ρ , E ρ and L ρ represent the substitution possibilities for each of the production function nests. The inputs into the production are denoted as ri M input of materials, ri KLE composite capital-labor-energy nest, ri E energy inputs, ri KL composite capital- labor nest, ri K capital input, ri L labor input, N E L E C ri E input of non-electric energy, ELEC ri E input of electric energy and E D r ie L inputs of labor by type of education e. 48 Figure A2.2: Structure of KLEM production functions in the model. Source: (Ivanova et al., 2019) Pre-proof oo Figure A2.2: Structure of KLEM production functions in the model. Source: (Ivanova et al., 2019) Pr International and inter-regional trade rna The total sales ri X of tradable goods and services i in region r in the model is an Armington Constant Elasticity of Substitution (CES) [ref] composite between domestic output D r i X and imports M ri X such that Jour (Eq. A2.8) (Eq. A2.8) ( ) ( ) 1/ i i i D D M M ri ri ri ri ri X X X ρ ρ ρ α α   = ⋅ + ⋅     Where D r i α and M ri α are the calibrated share parameters of the CES function and i i i ρ σ = with Where D r i α and M ri α are the calibrated share parameters of the CES function and i i i ρ σ = with with iσ being the Armington elasticity of substitution between domestic and imported tradable goods and services. The elasticity of substitution varies between different types of goods and services depending on the available empirical estimates. In case of non-tradable, the composite is equal to the domestically produced product. 49 Imported goods can come from various regions and countries represented in the model and the composite imported goods and services are represented by the CES composite that uses a higher Armington elasticity of substitution as compared to the upper Armington nest. We assume, as in the GTAP model, that the elasticity of substitution between the same type of goods and services coming from different countries is twice as large as the elasticity of substitution between domestic and aggregate imported goods and services. The aggregate imported good is calculated according to the following CES composite function: (Eq. A2.9) (Eq. A2.9) ( ) 1/ T i T i M T T ri sri sri s X X ρ ρ α   =     ∑ proof Where T sri α is the calibrated share coefficient of the CES production function, T sr i X is the flow of e-p trade in commodity i from country sto country r . The coefficient 1 T i i T i σ ρ σ = where T i σ is the elasticity of substitution between commodities produced in different countries. al Pre Labour, capital and goods markets rna Market equilibrium in the economy results in equalization of both monetary values and quantities of supply and demand. Market equilibrium results in equilibrium prices that represent in the case of CGE models the solution to the system of nonlinear equations that include both intermediate and final demand equations as well as accounting constraints that calculate households’ and government incomes, savings and investments, as well as trade balance. EU-EMS model represents a closed economic system, meaning that nothing appears from nowhere or disappears into nowhere in it. This feature of the CGE model constitutes the core of the Walrasian equilibrium and ensures that even if one excludes any single equation of the model, it will still hold. This is the property of CGE models called Walras law that tells us that in the closed economic system, if n-1 markets are in equilibrium the last nth market will also be in equilibrium. In our EU-EMS model, the static equilibrium is described by the set of commodity and factor prices, total outputs, final demands of households and government, investments, savings and net transfers from abroad, such that (1) markets for goods and services clear, (2) total investments are equal to total savings, (3) total households’ consumption is equal to their disposable income minus savings, (4) total governmental consumption is equal to its net tax revenues minus Journ 50 transfers to households minus savings, (5) total revenue of each economic sector is equal to its total production costs and (6) difference between imports and exports is equal to the net transfers from abroad. transfers to households minus savings, (5) total revenue of each economic sector is equal to its total production costs and (6) difference between imports and exports is equal to the net transfers from abroad. Outputs The EU-EMS model produces detailed dynamics of regional GDP, production and value added by region and by economic sector, interregional trade flows by the type of commodity, electricity and gas consumption per region and sector, employment by regional and economic sector, household income and consumption, and governmental revenues and spending. For the purpose of this article we limit the presentation of the main CGE output to Gross Domestic Product (GDP), percentage change in the electricity consumption per NUTS2 region, country and the entire EU. Pre-proof Recursive dynamics A2.12) 1 1 1 ( ) t ri t ri r ri t r r WKR g PI δ − − − = ⋅ + 1 1 1 ( ) t ri t ri r ri t r r WKR g PI δ − − − = ⋅ + Where 1 t ri WKR− denotes the capital remuneration rate, rg the steady-state growth rate, ri B the calibrated gravity attraction parameter and ϑ the speed of investment adjustment. Where 1 t ri WKR− denotes the capital remuneration rate, rg the steady-state growth rate, ri B the calibrated gravity attraction parameter and ϑ the speed of investment adjustment. Recursive dynamics EU-EMS is a dynamic model and allows for the analysis of each period of the simulation time horizon. This horizon is currently set at 2050 but it can be extended to longer time periods. For each year of the time horizon, EU-EMS calculates a set of various economic, social and environmental indicators. The economic growth rate in EU-EMS depends positively on investments in R&D and education. By investing in R&D and education each region is able to catch up faster with the technological leader region and better adopt its technologies. oof Time periods in EU-EMS are linked by savings and investments. By the end of each time period, households, firms and government in the model save a certain amount of money. This money goes to the investment bank, distributing it as investments between the production sectors of the various regions. The allocation decisions of the investment bank sectors depend on the sector’s financial profitability. The model runs in time steps of five years for the period 2015-2050. l Pre-pro The capital stocks evolve according to the dynamic rule presented below, where the capital stock in period t is equal to the capital stock in period t-1 minus the depreciation plus the new investments into the capital stock urnal (Eq. A2.10) Jou (Eq. A2.10) 1 (1 ) tri t ri i tri K K I δ − = − + Jou (Eq. A2.10) 1 (1 ) tri t ri i tri K K I δ − = − + At the end of each period there is a pool of savings rS available for investments into additional capital stocks of the sectors. This pool of savings comes from households, firms and foreign investors. The sector investments tri I are derived as a share of the total savings in the economy according to the discrete choice formula (Eq. A2.11) (Eq. A2.11) 1 1 1 1 1 t ri t rj WKR t r ri t ri tri WKR rj t rj j ST B K e I B K e ϑ ϑ − − ⋅ − − ⋅ − = ∑ 1 1 1 1 1 t ri t rj WKR t r ri t ri tri WKR rj t rj j ST B K e I B K e ϑ ϑ − − ⋅ − − ⋅ − = ∑ 51 (Eq. A2.12) (Eq. e-proof Appendix 3: Upscaling rn Distance between countries is not only the geographical and therefore the regional economic integration should not happen regardless other local factors. Social structure, wealth and lifestyle, religion, institutional and economic conditions, and natural environment play a role in assessing cultural distance (Gobel et al., 2018; Hofstede, 2011, 2001; Kaasa et al., 2016; Schwartz, 2014; Vignoles et al., 2018). Table A3.1 summarized the value of cultural dimensions. In this study, due to the absence of more granular data, we use the Dutch case to approximate how the behavioral patterns may evolve in the North-West EU states, and the Spanish case for the South- East EU states, which is in line with the values presented below. Journ Table A3.4: Values of cultural dimensions for all EU countries, sources:(Čuhlová, 2018) Country PDI INV Austria 11 55 Belgium 65 75 Bulgaria 65 75 Table A3.4: Values of cultural dimensions for all EU countries, sources:(Čuhlová, 2018) Country PDI INV Austria 11 55 Belgium 65 75 Bulgaria 65 75 52 Croatia 73 33 Cyprus - - Czech Republic 57 58 Denmark 18 74 Estonia 40 60 Finland 33 63 France 68 71 Germany 35 67 Greece 60 35 Hungary 46 80 Ireland 28 70 Italy 50 76 Latvia 44 70 Lithuania 42 60 Luxembourg 40 60 Malta 56 59 Netherlands 38 80 Poland 68 60 Portugal 63 27 Romania 90 30 Slovakia 104 52 Slovenia 71 27 Spain 57 51 Sweden 31 71 UK 35 89 PDI – Power Distance Index, INV – Individualism Complete data for Cyprus are not available Journal Pre-p Complete data for Cyprus are not available J 53 Step 2: Scaling-up behavioral scenarios to national and EU level Using the population projection scenarios for the EU28, we scale the dynamics in household energy behavioral changes in two provinces over time up to national and EU levels (Table A4.1). Table A4.1: Share of actions in two countries over time. Source: scaled-up BENCH-v.3 results. able A4.1: Share of actions in two countries over time. Source: scaled-up BENCH-v.3 results. 2020 2030 2050 The share of preferred actions (in percentage) Insulation PVs installation EE appliances NL ES Total number of actions NL 3,291 22,026 50,322 ES 1,546 29,894 123,545 Step 3: From regional to the national and EU28 economy Journal Pre-proof 2020 2030 2050 The share of preferred actions (in percentage) Insulation PVs installation NL ES ournal Pre-proof 2020 2030 2050 The share of preferred actions (in percentage) Insulation PVs installation EE appliances NL ES Total number of actions NL 3,291 22,026 50,322 ES 1,546 29,894 123,545 Step 3: From regional to the national and EU28 economy Journal Pre-proof 2020 2030 2050 The share of preferred actions (in percentage) Insulation PVs installation NL ES ournal Pre-proof 2020 2030 2050 The share of preferred actions (in percentage) NL e-proof e-proo oof The share of preferred actions (in percentage) NL NL Insulation PVs installation EE appliances ES Total number of actions NL 3,291 22,026 50,322 ES 1,546 29,894 123,545 Step 3: From regional to the national and EU28 economy Journal Pre-p Insulation PVs installation EE appliances ES Journal Pre ES rnal Pre ES ES Total number of actions NL 3,291 22,026 50,322 ES 1,546 29,894 123,545 Step 3: From regional to the national and EU28 economy Step 3: From regional to the national and EU28 economy To estimate the macroeconomic and cross-sectoral impacts of individual energy behavioral changes, we link the up-scaled ABM output to the CGE EU-EMS model. The BENCH-v.3 behavioral patterns in each of the 12 age-education groups – changes in heterogeneous households’ electricity and gas consumption – exogenously modify the minimum subsistence level of households’ consumption of the respective services in EU-EMS. 54 The analysis of EU-EMS results indicates that most of the EU28 regions benefit from the behavioral changes and lead to the decrease in energy consumption, with a small number of regions being affected negatively. Importantly, regions with larger population as well as the regions with higher share of highly-educated people benefit more from the behavioral changes since they save more electricity and gas. (a) the Netherlands (b) Spain Figure A4.1: Diffusion of households investments per capita and per action (insulation, PVs installation, energy-efficient appliances) among 12 sociodemographic groups under the informative dynamics scenario in two province. Source: EU-EMS and BENCH-v.3 l Pre-proof (a) the Netherlands (b) Spain re-proof (b) Spain (a) the Netherlands (a) the Netherlands Figure A4.1: Diffusion of households investments per capita and per action (insulation, PVs installation, energy-efficient appliances) among 12 sociodemographic groups under the informative dynamics scenario in two province. Source: EU-EMS and BENCH-v.3 l Pre As expected, PVs get more of a share of the investments in both countries (Figure A4.1). Households in groups 6-8 invest 110-160 and 160-180 Euros per capita on PVs in Netherlands and Spain respectively, while insulation in Spain (82 Euros per capita) and EE appliances in Netherlands (37 Euros per capita) are second in household investments. Journal 55 Figure A4.2: Percentage changes in the levels of regional real GDP relative to the Baseline under the FD scenario in 2050 as an aggregated effect of households’ behavioral changes, in millions of Euros. Source: EU-EMS and BENCH-v03. e-proof Figure A4.2: Percentage changes in the levels of regional real GDP relative to the Baseline under the FD scenario in 2050 as an aggregated effect of households’ behavioral changes, in millions of Euros. Source: EU-EMS and BENCH-v03. -pro The EU-EMS model operates at the level of NUTS2 regions of the EU28, and hence enables the calculation of the regional impacts of various behavioral scenarios on changes in the GDP and income. Step 3: From regional to the national and EU28 economy The changes in income presents similar patterns as changes in real GDP (see Figure 6). However, it is interesting that different pattern in percentage changes in regional GDP levels from the absolute monetary changes in regional GDP is captured (see Figure A4.2). The majority of relatively large changes in GDP are located in Great Britain, Italy and Central Europe. This might be related to the assumed population and education level developments which influence the upscaling of the results of the BENCH ABM model. Journal Pre Appendix References Acemoglu, D., Ozdaglar, A., 2011. Opinion Dynamics and Learning in Social Networks. Dyn. Games Appl. 1, 3–49. https://doi.org/10.1007/s13235-010-0004-1 Armitage, C.J., Conner, M., 2001. Efficacy of the Theory of Planned Behaviour: A meta-analytic review. Br. J. Soc. Psychol. 40, 471–499. https://doi.org/10.1348/014466601164939 Armitage, C.J., Conner, M., 2001. Efficacy of the Theory of Planned Behaviour: A meta-analytic review. Br. J. Soc. Psychol. 40, 471–499. https://doi.org/10.1348/014466601164939 Armitage, C.J., Conner, M., 2001. Efficacy of the Theory of Planned Behaviour: A meta-analytic review. Br. J. Soc. Psychol. 40, 471–499. https://doi.org/10.1348/014466601164939 Čuhlová, R., 2018. Cultural distance in the context of european regional economic integration, in: Čuhlová, R., 2018. Cultural distance in the context of european regional economic integration, in: Klímová, V., Žítek, V. (Eds.), 21st International Colloquium on Regional Sciences. Masarykova univerzita, Brno, pp. 340–346. Čuhlová, R., 2018. Cultural distance in the context of european regional economic integration, in: Klímová, V., Žítek, V. (Eds.), 21st International Colloquium on Regional Sciences. Masarykova univerzita, Brno, pp. 340–346. Klímová, V., Žítek, V. (Eds.), 21st International Colloquium on Regional Sciences. Masarykova univerzita, Brno, pp. 340–346. De Groot, J.I.M., Steg, L., 2009. Morality and Prosocial Behavior: The Role of Awareness, De Groot, J.I.M., Steg, L., 2009. Morality and Prosocial Behavior: The Role of Awareness, De Groot, J.I.M., Steg, L., 2009. Morality and Prosocial Behavior: The Role of Awareness, 56 Responsibility, and Norms in the Norm Activation Model. J. Soc. Psychol. 149, 425–449. https://doi.org/10.3200/SOCP.149.4.425-449 Degroot, M.H., 1974. Reaching a Consensus. J. Am. Stat. Assoc. 69, 118–121. https://doi.org/10.1080/01621459.1974.10480137 Gobel, M.S., Benet-Martinez, V., Mesquita, B., Uskul, A.K., 2018. Europe’s Culture(s): Negotiating Cultural Meanings, Values, and Identities in the European Context. J. Cross. Cult. Psychol. 49, 858– 867. https://doi.org/10.1177/0022022118779144 Hegselmann, R.K.U., 2002. Opinion dynamics and bounded confidence models, analysis and simulation. J. Artif. Soc. Soc. Simul. 5. f Hofstede, G., 2011. Dimensionalizing Cultures: The Hofstede Model in Context. Online Readings Psychol. Cult. 2. https://doi.org/10.9707/2307-0919.1014 roof Hofstede, G., 2001. Culture’s consequences: comparing values, behaviors, institutions, and organizations across nations, 2nd ed. Sage Publications, Thousand Oaks, California. e-pro Ivanova, O., Kancs, A., Thissen, M., 2019. EU Economic Modelling System: Assessment of the European Institute of Innovation and Technology (EIT) Investments in Innovation and Human Capital. Luxembourg. https://doi.org/10.2791/184008 al Pre Kaasa, A., Vadi, M., Varblane, U., 2016. A new dataset of cultural distances for European countries and regions. Res. Int. Bus. Financ. 37, 231–241. Appendix References https://doi.org/10.1016/J.RIBAF.2015.11.014 urna Lee, J.-S., Filatova, T., Ligmann-Zielinska, A., Hassani-Mahmooei, B., Stonedahl, F., Lorscheid, I., Voinov, A., Polhill, J.G., Sun, Z., Parker, D.C., 2015. The Complexities of Agent-Based Modeling Output Analysis. J. Artif. Soc. Soc. Simul. 18, 4. https://doi.org/10.18564/jasss.2897 Jou Moussaïd, M., Brighton, H., Gaissmaier, W., 2015. The amplification of risk in experimental diffusion chains. Proc. Natl. Acad. Sci. 112, 5631 LP – 5636. https://doi.org/10.1073/pnas.1421883112 Niamir, L., 2019. Behavioural Climate Change Mitigation: from individual energy choices to demand-side potential. University of Twente, Enschede, The Netherlands. https://doi.org/10.3990/1.9789036547123 Niamir, L., Filatova, T., 2017. Transition to low-carbon economy: Simulating nonlinearities in the electricity market, Navarre region, Spain, Advances in Intelligent Systems and Computing. https://doi.org/10.1007/978-3-319-47253-9_28 Niamir, L., Filatova, T., 2016. From Climate Change Awareness to Energy Efficient Behaviour. Int. Congr. Environ. Model. Softw. 57 Niamir, L., Filatova, T., Voinov, A., Bressers, H., 2018. Transition to low-carbon economy: Assessing cumulative impacts of individual behavioral changes. Energy Policy 118, 325–345. https://doi.org/10.1016/J.ENPOL.2018.03.045 Niamir, L., Ivanova, O., Filatova, T., Voinov, A., Bressers, H., 2020a. Demand-side solutions for climate mitigation: Bottom-up drivers of household energy behavior change in the Netherlands and Spain. Energy Res. Soc. Sci. 62, 101356. https://doi.org/10.1016/j.erss.2019.101356 Niamir, L., Kiesewetter, G., Wagner, F., Schöpp, W., Filatova, T., Voinov, A., Bressers, H., 2020b. Assessing the macroeconomic impacts of individual behavioral changes on carbon emissions. Clim. Change 158, 141–160. https://doi.org/https://doi.org/10.1007/s10584-019-02566-8 f Nordlund, A.M., Garvill, J., 2003. Effects of values, problem awareness, and personal norm on willingness to reduce personal car use. J. Environ. Psychol. 23, 339–347. https://doi.org/10.1016/S0272-4944(03)00037-9 proof Onwezen, M.C., Antonides, G., Bartels, J., 2013. The Norm Activation Model: An exploration of the functions of anticipated pride and guilt in pro-environmental behaviour. J. Econ. Psychol. 39, 141– 153. https://doi.org/10.1016/J.JOEP.2013.07.005 Pre-pr Schwartz, S.H., 2014. Chapter 20 - National Culture as Value Orientations: Consequences of Value Differences and Cultural Distance, in: Ginsburgh, V.A., Throsby, D.B.T.-H. of the E. of A. and C. (Eds.), Handbook of the Economics of Art and Culture. Elsevier, pp. 547–586. https://doi.org/https://doi.org/10.1016/B978-0-444-53776-8.00020-9 urnal P Steg, L., Dreijerink, L., Abrahamse, W., 2005. Factors influencing the acceptability of energy policies: A test of VBN theory. J. Environ. Psychol. 25, 415–425. https://doi.org/10.1016/J.JENVP.2005.08.003 Jou Steg, L., Vlek, C., 2009. Encouraging pro-environmental behaviour: An integrative review and research agenda. J. Environ. Psychol. 29, 309–317. https://doi.org/10.1016/J.JENVP.2008.10.004 J Steg, L., Vlek, C., 2009. Encouraging pro-environmental behaviour: An integrative review and research agenda. J. Environ. Psychol. 29, 309–317. Journal Pre-pr nal Pre-proof Appendix References https://doi.org/10.1016/J.JENVP.2008.10.004 J Stern, P., Dietz, T., Abel, T., Guagnano, G., Kalof, L., 1999. A Value-Belief-Norm Theory of Support for Social Movements: The Case of Environmentalism. Hum. Ecol. Rev. 6. Stern, P., Dietz, T., Abel, T., Guagnano, G., Kalof, L., 1999. A Value-Belief-Norm Theory of Support for Social Movements: The Case of Environmentalism. Hum. Ecol. Rev. 6. Stern, P.C., 2000. New Environmental Theories: Toward a Coherent Theory of Environmentally Significant Behavior. J. Soc. Issues 56, 407–424. https://doi.org/10.1111/0022-4537.00175 Vignoles, V.L., Smith, P.B., Becker, M., Easterbrook, M.J., 2018. In Search of a Pan-European Culture: European Values, Beliefs, and Models of Selfhood in Global Perspective. J. Cross. Cult. Psychol. 49, 868–887. https://doi.org/10.1177/0022022117738751 58 59 Appendix 1: BENCH agent-based model Appendix 1: BENCH agent-based model The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. The BENCH ABM (Niamir et al., 2020b, 2018) is developed to study shifts in residential energy use and corresponding emissions driven by behavioral changes among individuals. Main processes of the model (ODD protocol) Table A1.1: BENCH-v.3 ABM ODD protocol Guiding Protocol The BENCH-V.3 model A. Overview A.1. Purpose The BENCH-v.3 agent-based model is designed to study shifts in residential energy use and corresponding emissions at the regional level driven by behavioral changes among heterogeneous individuals. This empirically grounded model is of interest to (i) environmental scientists interested in modelling human behavior and economic institutions, (ii) energy economists working on micro aspects, (iii) scholars integrating individuals behavioral change in climate change mitigation modelling. A.2. Entities, state variables and scales Agents (individuals) in BENCH-v.3 model are heterogeneous in socio-demographic and dwelling characteristics, energy consumption and patterns, source of energy and energy provider, and behavioral factors. The BENCH-v.3 simulations 1035 and 755 individual households in the Overijssel province, the Netherlands, and Navarre province, Spain over 34 years (2016-2050). One time step represents one round in the behavioral experiments. Each run consist of 34 time steps aligning to the 34 rounds in the behavioral experiments. A.3. Process overview One time step represents one-year. In each time step a household goes through several processes: 1. Asses behavioral factors: ● Knowledge activation ● Motivation Journal Pre-proof This empirically grounded model is of interest to (i) environmental scientists interested in modelling human behavior and economic institutions, (ii) energy economists working on micro aspects, (iii) scholars integrating individuals behavioral change in climate change mitigation modelling. l Pre-pro Agents (individuals) in BENCH-v.3 model are heterogeneous in socio-demographic and dwelling characteristics, energy consumption and patterns, source of energy and energy provider, and behavioral factors. nal 1 ● Consideration 2. Calculate utilities 3. Pursue an action or not 4. Calculate saved energy and CO2 emission 5. Social dynamics and learning process 6. Satisfaction and regret 7. Updates See Figure 2 for algorithm and decision-making proccess in the BENCH-v.3 agent-based model. B. Design concept B.1 Theoretical and Empirical background In application to environmental- and energy-related choices, three behavioral change theories are commonly applied: theory of planned behavior (TPB), norm activation theory (NAT), and value–belief–norm (VBN) theory. We introduce a framework that combines the strengths of the three key behavioral theories, see Figure A1.1. We introduce a framework that combines the strengths of the three key behavioral theories, see Figure A1.1. B.5. Spatial scale Appendix 1: BENCH agent-based model ● TPB, formulated by Ajzen (1980) and based on the theory of reasoned action, is one of the most influential theories in social and health psychology and has been used in many environmental studies (Armitage and Conner, 2001; Onwezen et al., 2013). ● NAT, originally developed by Schwartz (1977), operates in the context of altruistic and environmentally friendly behavior. It is mostly focused on anticipating pride in doing the “right” thing and on studying the evolution of feelings of guilt. ● VBN theory (Stern et al., 1999; Stern, 2000) explains environmental behavior and “good intentions” such as willingness to change behavior (Nordlund and Garvill, 2003; Steg and Vlek, 2009; Stern et al., 1999), environmental citizenship (Stern et al., 1999), and policy acceptability (De Groot and Steg, 2009; Steg et al., 2005). B.2. Individual decision making We introduce a framework that combines the strengths of the three key behavioral theories, see Figure A1.1. Journal Pre-proof ● TPB, formulated by Ajzen (1980) and based on the theory of reasoned action, is one of the most influential theories in social and health psychology and has been used in many environmental studies (Armitage and Conner, 2001; Onwezen et al., 2013). nal P ● NAT, originally developed by Schwartz (1977), operates in the context of altruistic and environmentally friendly behavior. It is mostly focused on anticipating pride in doing the “right” thing and on studying the evolution of feelings of guilt. ● VBN theory (Stern et al., 1999; Stern, 2000) explains environmental behavior and “good intentions” such as willingness to change behavior (Nordlund and Garvill, 2003; Steg and Vlek, 2009; Stern et al., 1999), environmental citizenship (Stern et al., 1999), and policy acceptability (De Groot and Steg, 2009; Steg et al., 2005). 2 Agents (heterogeneous individual households) engage in interactions and learn from each other. In particular, they can exchange information with neighbors, which may alter own knowledge, awareness, and motivation regarding energy-related behavior. We employ a simple opinion dynamics model (Acemoglu and Ozdaglar, 2011; Degroot, 1974; Hegselmann, 2002; Moussaïd et al., 2015) assuming that each agent interacts with a fixed set of nearby neighbors. l Pre-proof Agents (heterogeneous individual households) engage in interactions and learn from each other. In particular, they can exchange information with neighbors, which may alter own knowledge, awareness, and motivation regarding energy-related behavior. Lowest scale: Individuals B.4. Interactions, social dynamics and learning 1 The Nomenclature of territorial units for statistics, abbreviated NUTS is a geographical nomenclature subdividing the economic territory of the European Union (EU) into regions at three different levels (NUTS 1, 2 and 3 respectively, moving from larger to smaller territorial units). Appendix 1: BENCH agent-based model We employ a simple opinion dynamics model (Acemoglu and Ozdaglar, 2011; Degroot, 1974; Hegselmann, 2002; Moussaïd et al., 2015) assuming that each agent interacts with a fixed set of nearby neighbors. l Pre-proof The BENCH_v.3 model is a spatially explicit model that takes the raster maps of the two NUTS2 regions as an input. Hence, an agent who is in active neighborhood where at least one out of eight nearest spatial neighbors within 1 raster cell (Moor neighborhood concept) undertakes an energy-related action will interact and exchange opinions. The idea of the Moore neighborhood comes from cellular automata literature and used only to enable opinion exchange between neighbors about climate and environmental awareness and compare norms. Agents compare values of their own behavioral factors – knowledge, awareness, and motivation – with those of their eight closest neighbors, and adjust their values for a closer match, see Figure 3 and Eq. 3. However, the agents’ heterogeneity beyond their spatial location (income, age, education) and economic factors affect individual choices of undertaking any of energy actions (I1-I3) or not. nal 3 Highest scale: NUTS21 The focus of this research is on Overijssel, the Netherlands (NL21) and Navarre, Spain (ES22) NUTS2 regions, which consist of 25 and 10 main cities/ municipalities respectively. B.6. Individual prediction Individuals do not predict future condition. B.7. Stochasticity There are various sources of stochasticity in the model: 1. Initial setting: Agents attributes (initialization are partly random) 2. During the process: Social dynamics and learning (process is partly random) B.8. Observation BENCH-v.3 estimates cumulative impacts of energy-related behavioral changes of individual households on electricity and gas consumption and CO2 emissions. Reports: ● Number of energy-related actions per year: investment, conservation, switching ● Saved electricity and gas per action/year: investment, conservation, switching ● Avoided CO2 emission per action/year: investment, conservation, switching Across socioeconomic (age and education) groups (see Table 1) and cases (NL vs. ES). B.9. Implementation Details The model is coded in Netlogo 6.0.4, Open source and available on CoMSES (https://www.comses.net) R is used for the result visualizations. of territorial units for statistics abbreviated NUTS is a geographical nomenclature subdividing the economic territory of Journal Pre-proof Highest scale: NUTS21 The focus of this research is on Overijssel, the Netherlands (NL21) and Navarre, Spain (ES22) NUTS2 regions, which consist of 25 and 10 main cities/ municipalities respectively. B.6. Individual prediction Individuals do not predict future condition. B.7. Appendix 1: BENCH agent-based model Stochasticity There are various sources of stochasticity in the model: 1. Initial setting: Agents attributes (initialization are partly random) 2. During the process: Social dynamics and learning (process is partly random) B.8. Observation BENCH-v.3 estimates cumulative impacts of energy-related behavioral changes of individual households on electricity and gas consumption and CO2 emissions. Reports: ● Number of energy-related actions per year: investment, conservation, switching ● Saved electricity and gas per action/year: investment, conservation, switching ● Avoided CO2 emission per action/year: investment, conservation, switching Across socioeconomic (age and education) groups (see Table 1) and cases (NL vs. ES). B.9. Implementation Details The model is coded in Netlogo 6.0.4, Open source and available on CoMSES (https://www.comses.net) R is used for the result visualizations. Journal Pre-proof 1 The Nomenclature of territorial units for statistics, abbreviated NUTS is a geographical nomenclature subdividing the economic territory of the European Union (EU) into regions at three different levels (NUTS 1, 2 and 3 respectively, moving from larger to smaller territorial units). 4 C. Details C.1. Initialization The variations in socio-demographic, dwelling and psychological factors among our survey respondents are used to initialize a population of heterogeneous agents in the BENCH- v.3 model (see Table A1.1 and A1.3). C.2. Input data The data on the behavioral and economic factors affecting household energy choices were collected using an online questionnaire (N= 1790 households) and serve as empirical micro-foundation of agent rules in the BENCH-v.3 model. Figure A1.1: BENCH-v.3 conceptual behavioral framework. Source: (Niamir et al., 2020a) Table A1.2: Overview of main variables and parameters used in BENCH-v.3 Journal Pre-proof The data on the behavioral and economic factors affecting household energy choices were collected using an online questionnaire (N= 1790 households) and serve as empirical micro-foundation of agent rules in the BENCH-v.3 model. Figure A1.1: BENCH-v.3 conceptual behavioral framework. Source: (Niamir et al., 2020a) Journal Pre-proof Figure A1.1: BENCH-v.3 conceptual behavioral framework. Source: (Niamir et al., 2020a) Journal Pre-proof Jou Figure A1.1: BENCH-v.3 conceptual behavioral framework. Table A1.3). Journal Pre Appendix 1: BENCH agent-based model Source: (Niamir et al., 2020a) J Table A1.2: Overview of main variables and parameters used in BENCH-v.3 Table A1.2: Overview of main variables and parameters used in BENCH-v.3 Table A1.2: Overview of main variables and parameters used in BENCH-v.3 Factors Variables Value range Socio-demographic Income [1000 - 150,000] Education [primary - doctoral] Dwelling Energy label [a-f] Ownership status [owner - renter] Energy Consumption [500 - 5000] Provider Grey, brown, green Energy saving habit [0-3] Behavioral Knowledge [1-7] 5 Cee awareness [1-7] Ed awareness [1-7] Personal norms [1-7] Social norms [1-7] Intention a1 [1-7] Intention a2 [1-7] Intention a3 [1-7] Data The BENCH-v.3 model is calibrated based on an empirical dataset. We designed and conducted the survey in two provinces in Europe for the purpose of this research. In 2016, 1035 households in the Overijssel province, the Netherlands, and 755 households in the Navarre province, Spain, filled out our online questionnaire (Niamir, 2019; Niamir et al., 2020a; Niamir and Filatova, 2017, 2016). The agent-based BENCH-v.3 model is parameterized using the survey data on socio- demographic, economic, structural and behavioral attributes of households and their dwelling characteristic (Table A1.3). Pre-proof 6 6 Table A1.3: Survey data on households’ characteristics and behavioral intentions. The data is used to parameterize households’ behavior in the BENCH-v.3 ABM. Source: (Niamir et al., 2020a, 2018) Table A1.3: Survey data on households’ characteristics and behavioral intentions. The data is used to parameterize households’ behavior in the BENCH-v.3 ABM. Source: (Niamir et al., 2020a, 2018) Table A1.3: Survey data on households’ characteristics and behavioral intentions. The data is used to parameterize households’ behavior in the BENCH-v.3 ABM. Source: (Niamir et al., 2020a, 2018) behavior in the BENCH-v.3 ABM. Source: (Niamir et al., 2020a, 2018) Factors Overijssel Navarre Socio-demographic characteristics Gender Female: 46.4% Male: 53.6% Female: 57.1% Male: 42.9% Age, years 53 41 Education, ISCED2 Annual income, in thousand Euros per year Dwelling characteristics Type of residence Apartment : 14.9% House: 85.1% Apartment : 77.8% House: 22.2% Tenure status Owner: 71% Renter: 29% Owner: 80.3% Renter: 19.7% 2 https://ec europa eu/eurostat/statistics explained/index php/International Standard Classification of Education (ISCED) Journal Pre-proof Overijssel 2 https://ec.europa.eu/eurostat/statistics-explained/index.php/International_Standard_Classification_of_Education_(ISCED) General description EU-EMS is a spatial computable general equilibrium (SCGE) model developed by PBL Netherlands Environmental Assessment Agency. The sectoral and geographical dimensions of the model are flexible and can be adjusted to the needs of a specific policy or research question. The model is used for policy impact assessment and provides sector-, region- and time-specific model- based support to Dutch and EU policy makers on structural reforms, growth, innovation, human capital and infrastructure policies. The current version of EU-EMS covers 276 NUTS2 regions of the EU28 Member States and each regional economy is disaggregated into 63 NACE Rev. 2 economic sectors1. Goods and services are consumed by households, government and firms, and are produced in markets that can be perfectly or imperfectly competitive. Spatial interactions between regions are captured through trade of goods and services, factor mobility and knowledge spill-overs. This makes EU-EMS particularly well suited for analyzing policies related to human capital, transport infrastructure, R&I and innovation. Journal Pre-pro In the current application of the model, we have aggregated the economic sectors to the following six large groups, following the Eurostat classification of the economic sectors according to their R&D intensity: (1) Traditional, (2) Low-tech industry, (3) Medium-tech industry, (4) High-tech industry, (5) Knowledge intensive services and (6) Other services. 1 https://ec.europa.eu/eurostat/documents/3859598/5902521/KS-RA-07-015-EN.PDF Outputs Outputs The agent-based BENCH-v.3 model tracks the individual and cumulative impacts of three energy behavioral changes (investments on insulation, PVs installation and energy-efficient appliances) among heterogeneous individuals in the Overijssel and Navarre provinces over 34 years (2016- 2050). We report the number of individuals pursuing a particular action (I1-I3), the cumulative electricity and gas consumption, and saved carbon emissions. Given the stochastic nature of ABMs, we perform multiple (N=100) repetitive runs of each simulation experiment (Lee et al., 2015). The agent-based BENCH-v.3 model tracks the individual and cumulative impacts of three energy behavioral changes (investments on insulation, PVs installation and energy-efficient appliances) among heterogeneous individuals in the Overijssel and Navarre provinces over 34 years (2016- 2050). We report the number of individuals pursuing a particular action (I1-I3), the cumulative electricity and gas consumption, and saved carbon emissions. Given the stochastic nature of ABMs, we perform multiple (N=100) repetitive runs of each simulation experiment (Lee et al., 2015). Factors Navarre 41 7 7 Size of residence Age of residence Behavioral characteristics, value on the 1-7 scale Knowledge (K) 4.2 (0.7) 5.0 (0.8) Awareness, Climate (AC) 4.9 (0.8) 5.4 (0.8) Awareness, Energy decision (AE) 4.5 (1.0) 5.3 (1.1) Personal Norms(NP) 4.6 (0.9) 5.4 (1.0) Social Norms (NS) 3.3 (1.1) 4.5 (1.2) Perceived Behavior Control (PBC) 4.4 (1.1) 5.0 (1.3) Journal Pre-proof Size of residence Behavioral characteristics, value on the 1-7 scale 8 1 https://ec.europa.eu/eurostat/documents/3859598/5902521/KS-RA-07-015-EN.PDF Main processes of the model EU-EMS accounts for the (a) feedback between price and demand/supply quantities, and (b) interactions between economic agents at the macro and sectorial level. Therefore, it gives the economic relations between all industry sectors via their intermediate use. The EU-EMS model is 9 9 a dynamic, recursive over time model, involving dynamics of capital accumulation and technology progress, stock and flow relationships and adaptive expectations. The model equations are neo- classical in spirit, assuming cost-minimizing behavior by producers, average-cost pricing and household demands based on optimizing behavior. The CGE model database consists of tables of transaction values and elasticities: dimensionless parameters that capture behavioral response. The database is presented as a Social Accounting Matrix, which covers an entire national economy, and distinguishes a number of sectors, commodities, primary factors and types of households. As a classical CGE model, EU-EMS represents the behavior of the whole population group or of the whole industrial sector as the behavior of one single aggregate agent. It is further assumed that the behavior of each such aggregate agent is driven by certain optimization criteria such as maximization of utility or minimization of costs. In following, detailed representation of the EU- EMS model and its main equations are presented. proof Figure A2.1: Circular economic flow in the CGE EU-EMS model. Source: (Ivanova et al., 2019) Factors of production markets Production costs, Resources Firms Revenues, Demand for products Product markets Expenditures Supply of products Households Wages, Labor, Resources Journal Pre-pr Factors of production markets Pre Wages, Labor, Resources al Production costs, Resources Households ur Revenues, Demand for products Expenditures Supply of products o Product markets Figure A2.1: Circular economic flow in the CGE EU-EMS model. Source: (Ivanova et al., 2019) Regional structure of the model Regional structure of the model Regions differ by the type of production sectors which dominate overall production activities in the region. Some specialize in traditional sectors such as agriculture, whereas others specialize in modern sectors such as finance and industry. Those sectors are characterized by different levels of agglomeration and its importance. Traditional sectors do not experience any agglomeration effects, whereas modern sectors do; this allows some sectors to grow faster than other. The prototype model will incorporate the regional difference in sectoral specialization and hence the difference of agglomeration economies between the regions. 10 Table A2.1: Regions in EU-EMS CGE models. Source: (Ivanova et al., 2019) Table A2.1: Regions in EU-EMS CGE models. Source: (Ivanova et al., 2019) Table A2.1: Regions in EU-EMS CGE models. Source: (Ivanova et al., 2019) AUS Australia ARG Argentina AUT Austria BGR Bulgaria BEL Belgium BRA Brazil CAN Canada BRN Brunei Darussalam CHL Chile CHN China CZE Czech Republic CHN.DOM China Domestic sales only DNK Denmark CHN.PRO China Processing EST Estonia CHN.NPR China Non processing goods exporters FIN Finland COL Colombia FRA France CRI Costa Rica DEU Germany CYP Cyprus GRC Greece HKG Hong Kong SAR HUN Hungary HRV Croatia ISL Iceland IDN Indonesia IRL Ireland IND India ISR Israel KHM Cambodia ITA Italy LTU Lithuania JPN Japan LVA Latvia KOR Korea MLT Malta LUX Luxembourg MYS Malaysia MEX Mexico PHL Philippines MEX.GMF Mexico Global Manufacturing ROU Romania MEX.NGM Mexico Non-Global Manufacturing RUS Russian Federation NLD Netherlands SAU Saudi Arabia NZL New Zealand SGP Singapore NOR Norway THA Thailand POL Poland TUN Tunisia PRT Portugal TWN Chinese Taipei SVK Slovak Republic VNM Viet Nam SVN Slovenia ZAF South Africa ESP Spain RoW Rest of the world SWE Sweden CHE Switzerland TUR Turkey GBR United Kingdom USA United States Journal Pre-proof Household preferences and governmental sector The households’ and governmental demand for goods and services is represented by the Linear Household preferences and governmental sector Household preferences and governmental sector Household preferences and governmental sector The households’ and governmental demand for goods and services is represented by the Linear Expenditure System (LES) that is derived as a solution to the Stone-Geary utility maximization problem: (Eq. Regional structure of the model A2.1) 11   ri r ri ri i U C        ri r ri ri i U C      The resulting demand system, where rI denotes households’ disposable income and ri P are consumer prices of goods and services that include taxes, subsidies, transport and trade margins can be written as follows: (Eq. A2.2) (Eq. A2.2) (Eq. A2.2) 1 ri ri ri r rj rj j ri C I P P                 of 1 ri ri ri r rj rj j ri C I P P                 o Households always consume a certain minimum level of each good and services where this level reflects the necessity (or price elasticity) of the good or service. Necessities such as food have low price elasticity and hence a higher minimum level of consumption. The disposable income of the households consists of wages, return to capital and social transfers from the government minus the income taxes and households’ savings. Pre-proo The government collects production, consumptions and income taxes. The tax revenue is further used to pay social transfers and buy goods and services for public consumption. The governmental savings can be either endogenous or exogenous in the model depending on the type of simulation and the type of chosen macro-economic closure. ournal P Firms production Jo Firms production Jo A2.7)   1/ L L ED ri rie rie e L f L          al Pre-pro (Eq. A2.7)   1/ L L ED ri rie rie e L f L          al Pre-p (Eq. A2.7)   1/ L L ED ri rie rie e L f L          al Pr Where ri a , rib , ric , ri d and rie f are the share parameters of the corresponding production function nests and , M KLE  , , E KL  , , K L  , E  and L  represent the substitution possibilities for each of the production function nests. The inputs into the production are denoted as ri M input of materials, ri KLE composite capital-labor-energy nest, ri E energy inputs, ri KL composite capital- labor nest, ri K capital input, ri L labor input, NELEC ri E input of non-electric energy, ELEC ri E input of electric energy and ED rie L inputs of labor by type of education e. Journa Where ri a , rib , ric , ri d and rie f are the share parameters of the corresponding production function nests and , M KLE  , , E KL  , , K L  , E  and L  represent the substitution possibilities for each of the production function nests The inputs into the production are denoted as ri M input of Journa Where ri a , rib , ric , ri d and rie f are the share parameters of the corresponding production function nests and , M KLE  , , E KL  , , K L  , E  and L  represent the substitution possibilities for each of the production function nests. The inputs into the production are denoted as ri M input of materials, ri KLE composite capital-labor-energy nest, ri E energy inputs, ri KL composite capital- labor nest, ri K capital input, ri L labor input, NELEC ri E input of non-electric energy, ELEC ri E input of electric energy and ED rie L inputs of labor by type of education e. Firms production Jo Domestic production D ri X is obtained using the nested-CES production technology of Capital- Labour-Energy-Materials (KLEM) type, where K is the capital, L is the labour, E is the energy and M is the materials. Figure II.2 represents the nests in the KLEM production function used in the model with services between used according to the fixed Leontief input coefficients in the production process. The energy in the model is differentiated between electricity and other types of energy with some substitution possibilities between them. The labour is differentiated according to three education levels according to International Labour Organisation (ILO) classification. The domestic production is generated according to nested production CES function, which is described by the following set of composite CES functions that follow the production structure from top to the bottom nest J (Eq. A2.3) 12     , , , 1/ (1 ) M KLE M KLE M KLE D ri ri ri ri ri X a M a KLE             (Eq. A2.4) (Eq. A2.4)     , , , 1/ (1 ) M KLE E KL E KL ri ri ri ri ri KLE b E b KL             (Eq. A2.5)     , , , 1/ (1 ) K L K L K L ri ri ri ri ri KL c K c L             (Eq. A2.6)     1/ (1 ) E E E NELEC ELEC ri ri ri ri ri E d E d E               (Eq. A2.7)   1/ L L ED ri rie rie e L f L          nal Pre-proof     , , , 1/ (1 ) K L K L K L ri ri ri ri ri KL c K c L             f     1/ (1 ) E E E NELEC ELEC ri ri ri ri ri E d E d E               (Eq. Firms production Jo Journa Where ri a , rib , ric , ri d and rie f are the share parameters of the corresponding production function nests and , M KLE  , , E KL  , , K L  , E  and L  represent the substitution possibilities for each of the production function nests. The inputs into the production are denoted as ri M input of materials, ri KLE composite capital-labor-energy nest, ri E energy inputs, ri KL composite capital- Journa electric energy and ED rie L inputs of labor by type of education e. electric energy and ED rie L inputs of labor by type of education e. 13 Figure A2.2: Structure of KLEM production functions in the model. Source: (Ivanova et al., 2019) Pre-proof Figure A2.2: Structure of KLEM production functions in the model. Source: (Ivanova et al., 2019) Pre International and inter-regional trade rna The total sales ri X of tradable goods and services i in region r in the model is an Armington Constant Elasticity of Substitution (CES) [ref] composite between domestic output D ri X and imports M ri X such that Journ The total sales ri X of tradable goods and services i in region r in the model is an Armington Constant Elasticity of Substitution (CES) [ref] composite between domestic output D ri X and imports M ri X such that Journ (Eq. A2.8) (Eq. A2.8) (Eq. A2.8)     1/ i i i D D M M ri ri ri ri ri X X X                Where D ri  and M ri  are the calibrated share parameters of the CES function and i i    with Where D ri  and M ri  are the calibrated share parameters of the CES function and i i    with with ibeing the Armington elasticity of substitution between domestic and imported tradable goods and services. The elasticity of substitution varies between different types of goods and services 14 depending on the available empirical estimates. In case of non-tradable, the composite is equal to the domestically produced product. Firms production Jo Imported goods can come from various regions and countries represented in the model and the composite imported goods and services are represented by the CES composite that uses a higher Armington elasticity of substitution as compared to the upper Armington nest. We assume, as in the GTAP model, that the elasticity of substitution between the same type of goods and services coming from different countries is twice as large as the elasticity of substitution between domestic and aggregate imported goods and services. The aggregate imported good is calculated according to the following CES composite function: f (Eq. A2.9)   1/ T i T i M T T ri sri sri s X X           Pre-proof (Eq. A2.9) Where T sri  is the calibrated share coefficient of the CES production function, T sri X is the flow of al P trade in commodity i from country s to country r . The coefficient i i T i    where T i  is the elasticity of substitution between commodities produced in different countries. Journa elasticity of substitution between commodities produced in different countries. Jour Labour, capital and goods markets Jo Labour, capital and goods markets Jo Market equilibrium in the economy results in equalization of both monetary values and quantities of supply and demand. Market equilibrium results in equilibrium prices that represent in the case of CGE models the solution to the system of nonlinear equations that include both intermediate and final demand equations as well as accounting constraints that calculate households’ and government incomes, savings and investments, as well as trade balance. EU-EMS model represents a closed economic system, meaning that nothing appears from nowhere or disappears into nowhere in it. This feature of the CGE model constitutes the core of the Walrasian equilibrium and ensures that even if one excludes any single equation of the model, it will still hold. This is the property of CGE models called Walras law that tells us that in the closed economic system, if n-1 markets are in equilibrium the last nth market will also be in equilibrium. Firms production Jo In our EU-EMS model, the static equilibrium is described by the set of commodity and factor prices, total outputs, final demands of households and government, investments, savings and net transfers from abroad, such that (1) J 15 markets for goods and services clear, (2) total investments are equal to total savings, (3) total households’ consumption is equal to their disposable income minus savings, (4) total governmental consumption is equal to its net tax revenues minus transfers to households minus savings, (5) total revenue of each economic sector is equal to its total production costs and (6) difference between imports and exports is equal to the net transfers from abroad. Where calibrated gravity attraction parameter and the speed of investment adjustment. oof calibrated gravity attraction parameter and the speed of investment adjustment. oof Recursive dynamics EU-EMS is a dynamic model and allows for the analysis of each period of the simulation time horizon. This horizon is currently set at 2050 but it can be extended to longer time periods. For each year of the time horizon, EU-EMS calculates a set of various economic, social and environmental indicators. The economic growth rate in EU-EMS depends positively on investments in R&D and education. By investing in R&D and education each region is able to catch up faster with the technological leader region and better adopt its technologies. proof Time periods in EU-EMS are linked by savings and investments. By the end of each time period, households, firms and government in the model save a certain amount of money. This money goes to the investment bank, distributing it as investments between the production sectors of the various regions. The allocation decisions of the investment bank sectors depend on the sector’s financial profitability. The model runs in time steps of five years for the period 2015-2050. rnal Pre-p The capital stocks evolve according to the dynamic rule presented below, where the capital stock in period t is equal to the capital stock in period t-1 minus the depreciation plus the new investments into the capital stock Journ (Eq. A2.10) J (Eq. A2.10) (Eq. A2.10) (Eq. A2.10) 1 (1 ) tri t ri i tri K K I      At the end of each period there is a pool of savings rS available for investments into additional capital stocks of the sectors. This pool of savings comes from households, firms and foreign investors. The sector investments tri I are derived as a share of the total savings in the economy according to the discrete choice formula (Eq. A2.11) 16 16 1 1 1 1 1 t ri t rj WKR t r ri t ri tri WKR rj t rj j ST B K e I B K e           (Eq. A2.12) (Eq. Outputs The EU-EMS model produces detailed dynamics of regional GDP, production and value added by region and by economic sector, interregional trade flows by the type of commodity, electricity and gas consumption per region and sector, employment by regional and economic sector, household income and consumption, and governmental revenues and spending. For the purpose of this article we limit the presentation of the main CGE output to Gross Domestic Product (GDP), percentage change in the electricity consumption per NUTS2 region, country and the entire EU. urnal Pre-p Appendix 3: Upscaling Jo Appendix 3: Upscaling Jo Recursive dynamics A2.12) 1 1 1 ( ) t ri t ri r ri t r r WKR g PI        1 1 1 ( ) t ri t ri r ri t r r WKR g PI        Where 1 t ri WKR  denotes the capital remuneration rate, rg the steady-state growth rate, ri B the f Where 1 t ri WKR  denotes the capital remuneration rate, rg the steady-state growth rate, ri B the f e-proof Appendix 3: Upscaling Jo Distance between countries is not only the geographical and therefore the regional economic integration should not happen regardless other local factors. Social structure, wealth and lifestyle, religion, institutional and economic conditions, and natural environment play a role in assessing cultural distance (Gobel et al., 2018; Hofstede, 2011, 2001; Kaasa et al., 2016; Schwartz, 2014; Vignoles et al., 2018). Table A3.1 summarized the value of cultural dimensions. In this study, due to the absence of more granular data, we use the Dutch case to approximate how the behavioral patterns may evolve in the North-West EU states, and the Spanish case for the South-East EU states, which is in line with the values presented below. J 17 Table A3.1: Values of cultural dimensions for all EU countries, sources:(Čuhlová, 2018) Table A3.1: Values of cultural dimensions for all EU countries, sources:(Čuhlová, 2018) Country PDI INV Austria 11 55 Belgium 65 75 Bulgaria 65 75 Croatia 73 33 Cyprus* - - Czech Republic 57 58 Denmark 18 74 Estonia 40 60 Finland 33 63 France 68 71 Germany 35 67 Greece 60 35 Hungary 46 80 Ireland 28 70 Italy 50 76 Latvia 44 70 Lithuania 42 60 Luxembourg 40 60 Malta 56 59 Netherlands 38 80 Poland 68 60 Portugal 63 27 Romania 90 30 Slovakia 104 52 Slovenia 71 27 Spain 57 51 Sweden 31 71 UK 35 89 PDI – Power Distance Index, INV – Individualism Complete data for Cyprus are not available Journal Pre-proof Country PDI INV Austria 11 55 Belgium 65 75 Bulgaria 65 75 Croatia 73 33 Cyprus - - Czech Republic 57 58 Denmark 18 74 Estonia 40 60 Finland 33 63 France 68 71 Germany 35 67 Greece 60 35 Hungary 46 80 Ireland 28 70 Italy 50 76 Latvia 44 70 Lithuania 42 60 Luxembourg 40 60 Malta 56 59 Netherlands 38 80 Poland 68 60 Portugal 63 27 Romania 90 30 Slovakia 104 52 Slovenia 71 27 Spain 57 51 Sweden 31 71 UK 35 89 PDI – Power Distance Index, INV – Individualism Complete data for Cyprus are not available Journal Pre-p Complete data for Cyprus are not available 18 Appendix 4: Results and discussions Step 2: Scaling-up behavioral scenarios to national and EU level Using the population projection scenarios for the EU28, we scale the dynamics in household energy behavioral changes in two provinces over time up to national and EU levels (Table A4.1). Using the population projection scenarios for the EU28, we scale the dynamics in household energy behavioral changes in two provinces over time up to national and EU levels (Table A4.1). Table A4.1: Share of actions in two countries over time. Source: scaled-up BENCH-v.3 results. 2020 2030 2050 The share of preferred actions (in percentage) Insulation PVs installation EE appliances NL ES Total number of actions NL 3,291 22,026 50,322 ES 1,546 29,894 123,545 Step 3: From regional to the national and EU28 economy Journal Pre-proof 2020 2030 2050 The share of preferred actions (in percentage) Insulation PVs installation EE appliances NL ES Total number of actions NL 3,291 22,026 50,322 ES 1,546 29,894 123,545 Step 3: From regional to the national and EU28 economy Journal Pre-proof 2050 e-proof of S L 3,291 22,026 S 1,546 29,894 Journal Pre p ES Insulation PVs installation EE appliances ES Total number of actions NL 3,291 22,026 50,322 ES 1,546 29,894 123,545 Journal Pre p 50,322 123,545 Step 3: From regional to the national and EU28 economy J To estimate the macroeconomic and cross-sectoral impacts of individual energy behavioral changes, we link the up-scaled ABM output to the CGE EU-EMS model. The BENCH-v.3 behavioral patterns in each of the 12 age-education groups – changes in heterogeneous households’ electricity and gas consumption – exogenously modify the minimum subsistence level of households’ consumption of the respective services in EU-EMS. The analysis of EU-EMS results indicates that most of the EU28 regions benefit from the behavioral changes and lead to the decrease in energy consumption, with a small number of regions being affected negatively. Importantly, regions with larger population as well as the regions with higher share of highly-educated people benefit more from the behavioral changes since they save more electricity and gas. 19 (a) the Netherlands (a) the Netherlands (b) Spain Figure A4.1: Diffusion of households investments per capita and per action (insulation, PVs installation, energy-efficient appliances) among 12 sociodemographic groups under the informative dynamics scenario in two province. Step 2: Scaling-up behavioral scenarios to national and EU level Source: EU-EMS and BENCH-v.3 oof (b) Spain f (b) Spain (a) the Netherlands Figure A4.1: Diffusion of households investments per capita and per action (insulation, PVs installation, energy-efficient appliances) among 12 sociodemographic groups under the informative dynamics scenario in two province. Source: EU-EMS and BENCH-v.3 oof As expected, PVs get more of a share of the investments in both countries (Figure A4.1). Households in groups 6-8 invest 110-160 and 160-180 Euros per capita on PVs in Netherlands and Spain respectively, while insulation in Spain (82 Euros per capita) and EE appliances in Netherlands (37 Euros per capita) are second in household investments. Pre-pro Figure A4.2: Percentage changes in the levels of regional real GDP relative to the Baseline under the FD scenario in 2050 as an aggregated effect of households’ behavioral changes, in millions of Euros. Source: EU-EMS and BENCH-v03. Journal Figure A4.2: Percentage changes in the levels of regional real GDP relative to the Baseline under the FD scenario in 2050 as an aggregated effect of households’ behavioral changes, in millions of Euros. Source: EU-EMS and BENCH-v03. The EU-EMS model operates at the level of NUTS2 regions of the EU28, and hence enables the calculation of the regional impacts of various behavioral scenarios on changes in the GDP and 20 income. The changes in income presents similar patterns as changes in real GDP (see Figure 6). However, it is interesting that different pattern in percentage changes in regional GDP levels from the absolute monetary changes in regional GDP is captured (see Figure A4.2). The majority of relatively large changes in GDP are located in Great Britain, Italy and Central Europe. This might be related to the assumed population and education level developments which influence the upscaling of the results of the BENCH ABM model. Appendix References Acemoglu, D., Ozdaglar, A., 2011. Opinion Dynamics and Learning in Social Networks. Dyn. Games Appl. 1, 3–49. https://doi.org/10.1007/s13235-010-0004-1 oof Armitage, C.J., Conner, M., 2001. Efficacy of the Theory of Planned Behaviour: A meta-analytic review. Br. J. Soc. Psychol. 40, 471–499. https://doi.org/10.1348/014466601164939 -proo Čuhlová, R., 2018. Cultural distance in the context of european regional economic integration, in: Klímová, V., Žítek, V. (Eds.), 21st International Colloquium on Regional Sciences. Masarykova univerzita, Brno, pp. 340–346. l Pre-p De Groot, J.I.M., Steg, L., 2009. Morality and Prosocial Behavior: The Role of Awareness, Responsibility, and Norms in the Norm Activation Model. J. Soc. Psychol. 149, 425–449. https://doi.org/10.3200/SOCP.149.4.425-449 urnal Degroot, M.H., 1974. Reaching a Consensus. J. Am. Stat. Assoc. 69, 118–121. https://doi.org/10.1080/01621459.1974.10480137 Jou Gobel, M.S., Benet-Martinez, V., Mesquita, B., Uskul, A.K., 2018. Europe’s Culture(s): Negotiating Cultural Meanings, Values, and Identities in the European Context. J. Cross. Cult. Psychol. 49, 858– 867. https://doi.org/10.1177/0022022118779144 J Hegselmann, R.K.U., 2002. Opinion dynamics and bounded confidence models, analysis and simulation. J. Artif. Soc. Soc. Simul. 5. Hofstede, G., 2011. Dimensionalizing Cultures: The Hofstede Model in Context. Online Readings Psychol. Cult. 2. https://doi.org/10.9707/2307-0919.1014 Hofstede, G., 2001. Culture’s consequences: comparing values, behaviors, institutions, and organizations across nations, 2nd ed. Sage Publications, Thousand Oaks, California. Ivanova, O., Kancs, A., Thissen, M., 2019. EU Economic Modelling System: Assessment of the European Institute of Innovation and Technology (EIT) Investments in Innovation and Human Capital. 21 21 Luxembourg. https://doi.org/10.2791/184008 Kaasa, A., Vadi, M., Varblane, U., 2016. A new dataset of cultural distances for European countries and regions. Res. Int. Bus. Financ. 37, 231–241. https://doi.org/10.1016/J.RIBAF.2015.11.014 Lee, J.-S., Filatova, T., Ligmann-Zielinska, A., Hassani-Mahmooei, B., Stonedahl, F., Lorscheid, I., Voinov, A., Polhill, J.G., Sun, Z., Parker, D.C., 2015. The Complexities of Agent-Based Modeling Output Analysis. J. Artif. Soc. Soc. Simul. 18, 4. https://doi.org/10.18564/jasss.2897 Moussaïd, M., Brighton, H., Gaissmaier, W., 2015. The amplification of risk in experimental diffusion chains. Proc. Natl. Acad. Sci. 112, 5631 LP – 5636. https://doi.org/10.1073/pnas.1421883112 Niamir, L., 2019. Behavioural Climate Change Mitigation: from individual energy choices to demand-side potential. University of Twente, Enschede, The Netherlands. https://doi.org/10.3990/1.9789036547123 roof Niamir, L., Filatova, T., 2017. Transition to low-carbon economy: Simulating nonlinearities in the electricity market, Navarre region, Spain, Advances in Intelligent Systems and Computing. https://doi.org/10.1007/978-3-319-47253-9_28 Pre-pro Niamir, L., Filatova, T., 2016. From Climate Change Awareness to Energy Efficient Behaviour. Int. Congr. Environ. Model. Softw. al Pr Niamir, L., Filatova, T., Voinov, A., Bressers, H., 2018. p g Journal Pre Journal Pre-pr nal Pre-proof proof Appendix References Transition to low-carbon economy: Assessing cumulative impacts of individual behavioral changes. Energy Policy 118, 325–345. https://doi.org/10.1016/J.ENPOL.2018.03.045 ourna Niamir, L., Ivanova, O., Filatova, T., Voinov, A., Bressers, H., 2020a. Demand-side solutions for climate mitigation: Bottom-up drivers of household energy behavior change in the Netherlands and Spain. Energy Res. Soc. Sci. 62, 101356. https://doi.org/10.1016/j.erss.2019.101356 Jou Niamir, L., Kiesewetter, G., Wagner, F., Schöpp, W., Filatova, T., Voinov, A., Bressers, H., 2020b. Assessing the macroeconomic impacts of individual behavioral changes on carbon emissions. Clim. Change 158, 141–160. https://doi.org/https://doi.org/10.1007/s10584-019-02566-8 Nordlund, A.M., Garvill, J., 2003. Effects of values, problem awareness, and personal norm on willingness to reduce personal car use. J. Environ. Psychol. 23, 339–347. https://doi.org/10.1016/S0272-4944(03)00037-9 Onwezen, M.C., Antonides, G., Bartels, J., 2013. The Norm Activation Model: An exploration of the functions of anticipated pride and guilt in pro-environmental behaviour. J. Econ. Psychol. 39, 141– 153. https://doi.org/10.1016/J.JOEP.2013.07.005 Schwartz, S.H., 2014. Chapter 20 - National Culture as Value Orientations: Consequences of Value 22 Differences and Cultural Distance, in: Ginsburgh, V.A., Throsby, D.B.T.-H. of the E. of A. and C. (Eds.), Handbook of the Economics of Art and Culture. Elsevier, pp. 547–586. https://doi.org/https://doi.org/10.1016/B978-0-444-53776-8.00020-9 Steg, L., Dreijerink, L., Abrahamse, W., 2005. Factors influencing the acceptability of energy policies: A test of VBN theory. J. Environ. Psychol. 25, 415–425. https://doi.org/10.1016/J.JENVP.2005.08.003 Steg, L., Vlek, C., 2009. Encouraging pro-environmental behaviour: An integrative review and research agenda. J. Environ. Psychol. 29, 309–317. https://doi.org/10.1016/J.JENVP.2008.10.004 Stern, P., Dietz, T., Abel, T., Guagnano, G., Kalof, L., 1999. A Value-Belief-Norm Theory of Support for Social Movements: The Case of Environmentalism. Hum. Ecol. Rev. 6. f Stern, P.C., 2000. New Environmental Theories: Toward a Coherent Theory of Environmentally Significant Behavior. J. Soc. Issues 56, 407–424. https://doi.org/10.1111/0022-4537.00175 roof Vignoles, V.L., Smith, P.B., Becker, M., Easterbrook, M.J., 2018. In Search of a Pan-European Culture: European Values, Beliefs, and Models of Selfhood in Global Perspective. J. Cross. Cult. Psychol. 49, 868–887. https://doi.org/10.1177/0022022117738751 Pre-pro 23 − Macroeconomic effects of bounded rationality and social learning are quantified − Methodological differences in agent-based and equilibrium models are addressed − Diverse behavioral traits exacerbate inequality between EU regions over time − Climate policy models accommodate individual heterogeneity, behavioral change and social dynamics f − Climate policy models accommodate individual heterogeneity, behavioral change and social dynamics f Journal Pre al Pre-pr al Pre-p proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
https://openalex.org/W3159039542	https://link.springer.com/content/pdf/10.1007%2F978-3-030-63523-7_11.pdf	English	null	Place-Making by Cows in an Intensive Dairy Farm: A Sociolinguistic Approach to Nonhuman Animal Agency	The international library of environmental, agricultural and food ethics/The International library of environmental, agricultural and food ethics	2,021	cc-by	12,107	Chapter 11 Place-Making by Cows in an Intensive Dairy Farm: A Sociolinguistic Approach to Nonhuman Animal Agency Leonie Cornips and Louis van den Hengel Abstract Based on recent ethnographic ﬁeldwork at an intensive dairy farm, this chapter examines the usefulness of posthuman critical theory for developing a new sociolinguistic approach to nonhuman animal agency. We explore how dairy cows, as encaged sentient beings whose mobility is profoundly restricted by bars and fences, negotiate their environment as a material-semiotic resource in linguistic acts of place-making. Drawing on the ﬁelds of critical posthumanism, new mate- rialism and sociolinguistics, we explain how dairy cows imbue their physical space with meaning through materiality, the body and language. By developing a non- anthropocentric approach to language as a practice of more-than-human sociality, we argue for establishing egalitarian research perspectives beyond the assumptions of human exceptionalism and species hierarchy. The chapter thus aims to contribute towards a new understanding of nonhuman agency and interspecies relationships in the Anthropocene. © The Author(s) 2021 B. Bovenkerk and J. Keulartz (eds.), Animals in Our Midst: The Challenges of Co-existing with Animals in the Anthropocene, The International Library of Environmental, Agricultural and Food Ethics 33, https://doi.org/10.1007/978-3-030-63523-7_11 L. Cornips (B) NL-Lab, Humanities Cluster (KNAW), Amsterdam, The Netherlands e-mail: leonie.cornips@maastrichtuniversity.nl L. Cornips · L. van den Hengel Faculty of Arts and Social Sciences, Maastricht University, Maastricht, The Netherlands 11.1 Introduction Human thinkers in the western philosophical tradition have long relied upon the silencing of nonhuman animal others to conﬁrm the exceptionalism of their own species. Since Aristotle, philosophers and scientists have deﬁned “man” as a “rational animal” distinguished from other animals by his—and, more recently, her or their— capacity for a special kind of thinking, variously described as self-consciousness, reason, or representational thought (Cull 2015, 19). If, as Eva Meijer asserts (2016, 73), in this tradition “humans are viewed as radically different from other animals,” then language is commonly seen as “one of the main ways in which this difference is L. Cornips (B) NL-Lab, Humanities Cluster (KNAW), Amsterdam, The Netherlands e-mail: leonie.cornips@maastrichtuniversity.nl L. Cornips · L. van den Hengel Faculty of Arts and Social Sciences, Maastricht University, Maastricht, The Netherlands 177 © The Author(s) 2021 B. Bovenkerk and J. Keulartz (eds.), Animals in Our Midst: The Challenges of Co-existing with Animals in the Anthropocene, The International Library of Environmental, Agricultural and Food Ethics 33, https://doi.org/10.1007/978-3-030-63523-7_11 178 L. Cornips and L. van den Hengel expressed.” The idea that language is what makes us human, or more precisely, that the possession of language allows humans to separate themselves from nonhuman nature, including their own animality, is indeed a key component of philosophical humanism and its exclusionary conceptions of individual and collective person- hood. The philosopher Giorgio Agamben (2004, 33) uses the term “anthropological machine” to refer to the process by which the human is deﬁned over and against what is nonhuman or animal, thus dividing the human subject from more-than- human forms of sentience, sociality, intelligence and communication. The strict identiﬁcation of the human with language, or what Agamben (2004, 38) calls an articulation between “speaking being” and “living being,” is central to the func- tioning of the anthropological machine, that is, the ways in which humans ought to continually create themselves as speaking political beings by creating hierarchies between human, animal, vegetable and mineral species. The traditional humanist understanding of the human as a unique creature, one that rises above the natural world of animals, plants and the physical environment, thus rests on a fundamental denial that nonhuman animals might be capable of language and other forms of complex symbolic communication. 11.1 Introduction In this chapter, we wish to move beyond the assumptions of human exceptionalism and species hierarchy in order to advance an understanding of language that displaces the centrality of the human subject. Speciﬁcally, we will explore how dairy cows, as caged living beings or what sociologist Rhoda Wilkie (2010, 115) has called “sentient commodities,” negotiate their environment as a material-semiotic resource in the production of a meaningful world. While their physical mobility is profoundly restricted by bars and fences, we will examine how dairy cows enact social and linguistic agency through complex assemblages formed by human and nonhuman bodies, materials and environments. Starting from the assumption that, within the context of dairy farming, the subjectivities of cows and humans are continuously co- produced, we want to highlight how recognizing the linguistic agency of dairy cows may allow us to resist anthropocentric understandings of interspecies relationships and to formulate a new perspective on language as a social practice of human- nonhuman interaction. The central aim of this chapter, therefore, is to elaborate a radically post- or non-anthropocentric sociolinguistic approach that may help foster more egalitarian relationships to and between different species, or, as Agamben (2004, 83) phrases it, to bring to a “standstill” the anthropological machine that has historically articulated humanity and animality through their mutual exclusion. The chapter has four sections. The ﬁrst section examines how traditional humanist conceptionsoflanguagehavestructureddominantphilosophicalandlinguisticunder- standings of human-animal relationships. The second section discusses the cogni- tive, emotional and social capacities of cows as sentient and intelligent beings, and proceeds to argue for the usefulness of posthuman critical theory for expanding the linguistic research agenda to include the study of nonhuman animal languages. This sets the stage for the third section, which discusses recent ﬁeldwork at an inten- sive dairy farm in order to explore how cows, as social actors, engage in processes of linguistic place-making. Drawing, on the one hand, on recent work at the inter- section of critical posthumanism and applied linguistics (Pennycook 2018) and, on 11 Place-Making by Cows in an Intensive Dairy Farm … 179 11 the other, on new materialist conceptions of agency as a distributed phenomenon (Bennett 2010), we will elaborate a non-anthropocentric approach to human and nonhuman language practices. In the last and concluding section, we consider some of the implications of our ﬁndings for negotiating, or renegotiating, contemporary questions of nonhuman animal agency. 11.1 Introduction As a whole this chapter argues that acknowl- edging nonhuman linguistic agency is essential for thinking through and responding to the speciﬁc conditions and challenges of the Anthropocene, where the advent of the human as a global geophysical force has muddled conventional distinctions between culture and nature, human and nonhuman, self and other. If, as Donna Haraway suggests, nonhuman animals “are not here just to think with,” but rather they are here to “live with” (Haraway 2003, 5, emphasis added), then we must indeed embrace modes of inquiry suited to the task of confronting human and nonhuman acts of language, sociality and world-making. 1Aristotle, Politics, 1253a. Translation by Louis van den Hengel. 11.2 Language and the Politics of Human Exceptionalism Heidegger, despite his phenomenological critique of Descartes, also claimed that nonhuman animals are incapable of language and therefore lack access to what he called “world-formation”, that is, the ability to form true and conscious relationships with others and with their environment. According to Heidegger, nonhuman animals are unable to apprehend the world as such—that is to say, in the world-forming ways that language, understood as logos, allows for—because they are captivated by their instincts and bound to their environments. Animals do have access to the world—they are not, like stones and other inert objects, what Heidegger calls “world- less” (weltlos)—but their relationship to it is an impoverished one: Heidegger calls nonhuman animals “poor in world” (weltarm), whereas humans are deemed to be world-forming (weltbildend). Insofar as an animal is essentially absorbed in its envi- ronment (Umwelt), it cannot truly act in relation to the world (Welt) as such, or, as Heidegger puts it (1995, 239), an animal “behaves within an environment but never within a world.” Although Heidegger repeatedly observes that “the relation between poverty in world and world-formation does not entail hierarchical assessment” (1995, 192), he does nonetheless privilege human beings to the extent that, in his view, only language, by which he means human language, is capable of disclosing the world as an intelligible and meaningful place. “Language alone,” he writes, “brings what is, as something that is, into the Open for the ﬁrst time. Where there is no language, as in the being of stone, plant, and animal, there is also no openness of what is, and consequently no openness either of what is not” (Heidegger 1971, 73). In fore- grounding human language as central to the practice of world-formation, Heidegger not only seeks to demarcate human from nonhuman animals, but also postulates a distinction between language and communication that echoes Aristotle’s assumption of a fundamental difference between speech and sound, between logos and phonè, as well as Descartes’ view on animals as mindless machines. For Heidegger, language is “not only and not primarily an audible and written expression of what is to be communicated” (ibid.), but rather it serves to manifest the world as such as a ﬁeld of signiﬁcance: an open space of possibilities, as opposed to an animal’s instinctual captivation. 11.2 Language and the Politics of Human Exceptionalism The view that nonhuman animals have no speech, and therefore cannot establish themselves as ethical, juridical and political subjects, goes back at least to the ancient Greeks. In a well-known passage of his Politics, Aristotle associates the formation of political community with the supposedly unique human capacity for reasoned speech. Aristotle insists that the capacity for speech informed by reason (logos) is what separates “man”, as a political animal, from the mere beasts who do not speak but simply produce sound (phonè): And so the reason why man is a political animal more than any bee or any gregarious animal is clear. For nature, as we often say, does nothing in vain; and man alone of the animals possesses speech (logos). The mere voice (phonè), it is true, can indicate pain and pleasure, and therefore is available in the other animals as well (for their nature has been developed so far as to have feelings of pain and pleasure and to signify them to one another), but speech, for its part, is designed to express the useful and the harmful and therefore also the just and the unjust.1 Because nonhuman animals are incapable of speech, Aristotle argues, they cannot express the civic and moral virtues that he considered essential for the wellbeing of the household and the city-state. Aristotle, as Derrida explains in The Beast and the Sovereign (2009, 343–349), thus makes a categorical distinction between human and nonhuman animals to posit an inextricable link between language and the political sphere. His philosophy therefore not only delimits political agency to certain privi- leged human beings—the free adult male citizens that make up the polis—but also actively excludes nonhuman animal voices from the deﬁnition of language itself. Many later philosophers have followed Aristotle in rejecting the linguistic and cognitive abilities of nonhuman animals (Meijer 2016, 75–76). Descartes, for example, argued that animals do not think because they cannot speak: he regarded 180 L. Cornips and L. van den Hengel nonhuman animals as machines, governed by the laws of physical matter alone and hence devoid of mind and self-awareness.2 Although he did recognize that animals such as magpies and parrots can utter words, and that dogs make noises that might resemble speech, Descartes maintained that other animals “cannot speak as we do: that is, they cannot show that they are thinking what they are saying” (Descartes 1985, 140). 2Most commentators attribute to Descartes the concomitant view that animals, because they cannot think, have no feelings and do not suffer pain, yet some scholars (Harrison 1992; Cottingham 2008, 163–173) have sought to contest this interpretation. 11.2 Language and the Politics of Human Exceptionalism Language, in this view, is thus what separates human being-in-the-world, or Dasein, from the being of other animals, which, as Heidegger writes, “has nothing to do with the selfhood of the human being comporting him- or herself as a person” (Heidegger 1995, 238–239). Linguistics, no less than philosophy, has long reiterated the humanist under- standing of language as a fundamental dividing line between human and nonhuman 11 Place-Making by Cows in an Intensive Dairy Farm … 181 11 animals, thus reinforcing the predominant view of language as the essence of human personhood. Aristotle’s distinction between logos and phonè is still holding ground in conceptsoflanguageaseitheramentalorsocialconstructintwodominantcontempo- rarylinguistictheories,namelygenerativegrammar(Chomsky2002,2006)and(vari- ationist) sociolinguistics (Labov 1994, 2001). The generative framework, advanced since the late 1950 s by Noam Chomsky and others, theorizes language as a human mental construct where processes of thinking and knowledge about abstract symbols aregenerated:acognitivesystemor“innermentaltool”(BerwickandChomsky2016, 164) that works independently from phonetics or the speaking voice (phonè), which is assigned to language-in-use. This view consolidates the older assumption that the primary function of language is for human thought rather than for external commu- nication. Even though language can, of course, be used to communicate with others, most of speech is inner speech, or, as Chomsky (2002, 148) puts it: “almost all the use of language is to oneself.” In suggesting that language, deﬁned in the narrow sense of an abstract computational system for thought, does not occur beyond the human brain, Chomsky gives a new inﬂection to the Cartesian understanding of language as essentially disembodied and non-social, while at the same time reinforcing the anthropocentric idea that language constitutes “a yawning chasm between what we [humans] can do and what other animals cannot” (Berwick and Chomsky 2016, 110).3 Language, in generative linguistics, is thus seen as a species-speciﬁc ability that sets human beings apart from nonhuman animal others: “When we study human language, we are approaching what some might call the ‘human essence’, the distinc- tive qualities of mind that are, so far as we know, unique to man (sic)” (Chomsky 2006, 88). Even though, in a paper coauthored for Science, Chomsky has acknowl- edged that “available data suggest a much stronger continuity between animals and humans with respect to speech than previously believed” (Hauser et al. 4In this view, the linguistic ability to innovate—to form new statements that express new thoughts appropriate to but not directly caused by their immediate contexts—is considered a fundamental factor that distinguishes human language, seen as free from control by any detectable stimuli, from nonhuman animal communication, which is assumed to occur only in response to an external environment or to internal drives. 3In his Fundamental Concepts of Metaphysics, Heidegger in a similar manner suggests that nonhuman animals are “separated from man by an abyss” (1995, 26, emphasis added). Because nonhuman animals lack language, they cannot apprehend other beings conceptually, as beings: for Heidegger, only humans are capable of grasping that which is as such. 4In this view, the linguistic ability to innovate—to form new statements that express new thoughts appropriate to but not directly caused by their immediate contexts—is considered a fundamental factor that distinguishes human language, seen as free from control by any detectable stimuli, from nonhuman animal communication, which is assumed to occur only in response to an external environment or to internal drives. 3In his Fundamental Concepts of Metaphysics, Heidegger in a similar manner suggests that nonhuman animals are “separated from man by an abyss” (1995, 26, emphasis added). Because nonhuman animals lack language, they cannot apprehend other beings conceptually, as beings: for Heidegger, only humans are capable of grasping that which is as such. 11.2 Language and the Politics of Human Exceptionalism 2002, 1574), he nonetheless maintains his faith in a uniquely human property of language, located either in the capacity for recursion—the ability to “generate an inﬁnite range of expressions from a ﬁnite set of elements” (ibid.)—or in what he calls the “creative aspect” of language use, that is, the “distinctively human ability to express new thoughts and to understand entirely new expressions of thought” (Chomsky 2006, 6).4 And while Chomsky, as Donna Haraway notes, has been cautious enough to present the idea of linguistic uniqueness as “a testable hypothesis, not an assumption rooted in premises of human exceptionalism” (Haraway 2008, 373 note 44), there is no doubt that the tradition of anthropocentric thought assumed in the generative 182 L. Cornips and L. van den Hengel L. Cornips and L. van den Hengel framework has been a serious obstacle to investigating the linguistic, rather than communicative, abilities of nonhuman animals. In contrast to Chomsky’s non-social view of language, sociolinguistic research has built on the pioneering work of William Labov and others to theorize language as both dependent on cognition and interconnected with the workings of society and culture.5 In the sociolinguistic framework, language is understood as a social construct stemming from the need to contextualize how humans use language in interaction with others, aiming to ﬁnd out how and why languages vary and change, and how (groups of) speakers employ linguistic resources to shape individual and collective identities, communities and social hierarchies. This view converges with recent approaches to language as an embodied (Bucholtz and Hall 2016), multimodal (Müller et al. 2013) and multisensory (Pennycook and Otsuji 2015) phenomenon that includes not only verbal speech but, among others, bodily gestures and facial expres- sions, actions, movements, sensorial practices of meaning-making through tasting, touching, seeing and smelling, as well as the mediation of embodiment by material objects, spaces and environments. Encompassing a wide range of research areas, including the social meaning of different language varieties, the role of stylization in language use, the construction of social identity categories like class and gender through language practices, bi- and multilingualism, and social norms and attitudes towards linguistic diversity, sociolinguistics has opened valuable new avenues for researchers interested in the manifold relationships between language, identity and power. 5Neither generative nor sociolinguistic theory has questioned the legitimacy of each other’s discipline, yet attempts to integrate both have not been successful (Cornips and Gregersen 2016). 11.2 Language and the Politics of Human Exceptionalism Although much sociolinguistic research remains faithful to the human as the most important user of language—in fact, the very notion that humans may use certain linguistic skills and resources is in no small part dependent on liberal humanist conceptions of choice and agency—this framework is nevertheless promising for a linguistics that wishes to be inclusive of human and nonhuman actors (Cornips 2019). By approaching language as both embodied and embedded in a variety of interactive social practices, contexts and environments, sociolinguistic studies chal- lenge the anthropocentric understanding of a language as an exclusively verbal, decontextualized object that is completely autonomous, inaccessible from outside the mind, and therefore somehow ﬁxed in character. In this perspective, embodi- ment is central to the production and interpretation of language as a form of social practice, while bodies, in turn, are themselves part of the semiotic landscape as they are “imbricated in complex arrangements that include nonhuman as well as human participants, whether animals, epidemics, objects, or technologies” (Bucholtz and Hall 2016, 186). Broadening up the concept of language (grammar) to include multimodal and multisensory practices of meaning-making allows us to foreground nonhuman semi- otic capacities, including speciﬁc sensorial abilities such as olfactory ones for cows and dogs, as language-speciﬁc grammatical means. It thus provides a useful frame- work to analyze differences between and among human and nonhuman animals 11 Place-Making by Cows in an Intensive Dairy Farm … 183 11 in terms of grammatical possibilities and expressions instead of simply ascribing deﬁciencies to the latter (Kulick 2017, 373). For example, if a cow in an indoor dairy farm steps back and withdraws her face through the iron bars when humans approach her, this bodily movement combined with head positioning, gaze direc- tion and the sound of the moving iron bar may be analyzed equally to how human animals phrase negation as in the sentence do NOT approach me (Cornips and van Koppen 2019). Further below, we will demonstrate how recent scholarship produced at the intersection of sociolinguistic theory and critical posthumanism will allow us to take the study of language beyond the speaking human subject and into the more-than-human material world. But ﬁrst, let us discuss the linguistic abilities and communicative competence of cows in more detail. What can a non-anthropocentric approach to language contribute to our understanding of the ways in which cows speak to each other and to humans? 11.2 Language and the Politics of Human Exceptionalism And how can this understanding, in turn, help us confront, and respond to, the enabling and constraining conditions under which dairy cows, as speaking beings, participate in the formation of a meaningful world? 11.3 Cows as Social and Linguistic Beings Human thinkers, as we have seen, have produced the idea of language as a uniquely human trait by categorically marginalizing nonhuman animal speakers, denying them recognition as linguistic subjects. But, as Eva Meijer suggests, learning about how other animals use language “can help us understand them better, and build new rela- tions with them; challenging an anthropocentric view of language can help us see animals of other species, and their languages, differently” (Meijer 2016, 74). Recent research into how different animal species communicate, ranging from birds and bees to whales, apes and cephalopods, indeed suggests that there may not be a “sharp divide between human language and nonhuman communicative systems” (Evans 2014, 258; see also Meijer 2019). This does not mean that human and nonhuman forms of communication are the same, but, as Alastair Pennycook (2018, 82) notes, “it is an argument against human exceptionalism.” In this chapter, we take the view that both human and other animals create meaning through language conceptual- ized as a social, spatial and artefactual resource. By theorizing language in terms of material-semiotic assemblages and spatial repertoires (Pennycook 2017), we wish to avoid an anthropocentric deﬁnition of language that not only a priori excludes nonhuman animals, but also neglects all other aspects of language beyond the “dis- tinctive qualities of mind” so often privileged in philosophy, linguistics and cognitive science. Cows, including domestic dairy cows, have distinct personalities and stable personality characteristics and have a clear capacity to lead rich and socially complex lives. Measured assessments of cows’ cognitive, emotional and social abilities provide scientiﬁc support for what people familiar with cows already know, namely that cows demonstrate intelligence, experience a range of emotions and display a high level of social complexity, including social learning, in ways that human animals can 184 L. Cornips and L. van den Hengel recognize (Marino and Allen 2017; Colvin et al. 2017). When given the opportunity, cows form strongly bonded social groups, with mother cows and calves sharing an especially powerful emotional connection that, in part, depends upon the possibility for the mother to be able to lick her child for several hours after birth (Marino and Allen 2017, 484). 6In one study, cows have been demonstrated, within a few training sessions, an ability to discriminate photographs of different cows’ faces from faces of other species. A later study has shown that heifers can differentiate between two-dimensional facial images of familiar and unfamiliar cows, treating these images as mental representations of real individuals (Marino and Allen 2017, 478–479). So far, there is no knowledge yet on social learning from humans or the use of human-given cues in cattle (Nawroth et al. 2019, 5). 7Emotional reactions to learning in cows have to do with “the positive emotions and excitement that go with realizing one is controlling a situation” (Marino and Allen 2017, 482). This does not merely show that cows understand the causal relation between accomplishing a task and receiving a reward, but rather it suggests that they learn to experience task solving as intrinsically rewarding by adopting “an emotional perspective on their own agency” (Hagen and Broom 2004, 212). 11.3 Cows as Social and Linguistic Beings Cows are competent learners and possess both short- and long-term memories: they are capable of discriminating between different objects, colors and geometric shapes, and are able to learn and recognize individual differences among humans, as well as conspeciﬁcs, under a variety of circumstances.6 These abilities show that cows do not merely respond to external stimuli but engage in the forma- tion and categorization of mental concepts (Colvin et al. 2017, 7). Moreover, cows display emotional reactions to their own learning and in response to each other’s feelings, which has been suggested to reﬂect sophisticated levels of psychological capacities such as self-awareness and empathy (Hagen and Broom 2004; Marino and Allen 2017, 482–483).7 As social mammals, cows depend on each other for interaction and emotional support; social isolation therefore inﬂicts great stress on them, as does the imme- diate, and life-long, postpartum separation of mothers and calves in intensive dairy farming. In commercial settings, where human-cow relations are deeply instrumen- talized and commodiﬁed, the possibilities for cows to express species-appropriate behavior are severely compromised by periods of conﬁnement in indoor housing, health problems due to higher milk yields and distress caused by various forms of social separation. As caged living beings, with little or no opportunity to escape their exploitation by humans, cows raised for food in factory farms experience “unnat- ural conditions from birth to slaughter” (Marino and Allen 2017, 474), including procedures that cause severe pain and suffering such as dehorning and disbudding. In these circumstances, where young calves are raised individually and cows are killed before their time, social bonding formation is extremely difﬁcult to establish and maintain (McLennan 2013), which has devastating consequences for their well- being and welfare. It is decidedly problematic, then, that most research into the lives of cows is done within the framework of their use as “livestock” for human consump- tion. As the scientiﬁc literature on cow psychology and behavior is dominated by an applied science perspective mainly relevant to human practices of intensive farming (e.g. training cows to use automatic feeders) (Marino and Allen 2017, 475), there is a felt need to understand, and relate to, cows on their own terms. 7Emotional reactions to learning in cows have to do with “the positive emotions and excitement that go with realizing one is controlling a situation” (Marino and Allen 2017, 482). 11.3 Cows as Social and Linguistic Beings This does not merely show that cows understand the causal relation between accomplishing a task and receiving a reward, but rather it suggests that they learn to experience task solving as intrinsically rewarding by adopting “an emotional perspective on their own agency” (Hagen and Broom 2004, 212). 185 11 Place-Making by Cows in an Intensive Dairy Farm … While it is undeniable that dairy cows are always already caught in the anthro- pological machine of industrial animal production—an “apparatus” (Despret 2008) that essentially prevents them from experiencing a full quality of life—we do believe that an inquiry into how dairy cows make use of language, conceptualized in a non- anthropocentric manner, can help human animals to get to know cows better and to understand them as “the someones they actually are” (Colvin et al 2017, 3). This will, in turn, allow us to respond to the question of nonhuman animal agency in new ways that not only serve to challenge established structures of species hierarchy, but also entail a fundamental rethinking of how agency is enacted in and through language as a practice of human-nonhuman sociality. In the context of what has been termed the “cage age,” it is routinely assumed that the restrictive and monotonous captive envi- ronments in which domesticated animals usually live, will “limit the frequency and diversity with which [their] agency is expressed” (Špinka and Wemelsfelder 2011, 34). Yet, as we will demonstrate below, these same restrictive conditions can, para- doxically, also give rise to new modes of linguistic agency and resilience, revealing the copious ways in which dairy cows, as speaking beings, orient themselves towards the world. Dairy farmers bring their own perspectives on how cows, as social and sentient beings whose freedom of movement is nevertheless severely restricted, give meaning to their physical environment and negotiate their housing conditions. A female dairy farmer based in the south of the Netherlands recently provided this chapter’s ﬁrst author with a hand-written letter with some of her thoughts in preparation of an interview addressing how cows and farmers communicate with each other.8 She wrote: A true story: Cows are herd animals and they have a leader who will inform the others what to expect. Mientje was always the ﬁrst waiting by the fence for the farmer to collect them [from the meadow] to be milked. 8The interview anticipated in the letter took place at the second farm (Farm 2) in the south of the Netherlands where the ﬁrst author conducted her ﬁeld research which is further discussed below. This interview took place on 15 February 2019 and is not discussed further in the current chapter. 11.3 Cows as Social and Linguistic Beings The cows would ﬁrst be treated to snacks in the barn which is a feast. They might become so impatient as children, and Mientje always watched carefully how the farmer would unlock the fence. An iron slide bolt. For days she would be licking that bolt and the farmer assumed she liked the taste of it, but actually she was practicing how to accomplish that [unlocking the bolt] by sliding it across with her tongue bit by bit long enough, and yes the farmer stayed away for too long and she opened the fence by herself, moved a bit backwards so that the fence could open further and so she managed to steer all the cows to the barn where there were no snacks present since it was no milking time yet. The barn was an overshitted barn that ﬁrst had to be cleaned with very restless cows back in the meadow. The blacksmith made a new bolt. Mientje, in the narrative above, is clearly positioned as an actor, even though her actions arise from within a state of unfreedom that makes it difﬁcult, if not impos- sible, to draw sharp boundaries between action and passion, between doing and suffering. Seemingly functionless activities such as repetitive licking and/or biting of non-food objects, including bars and fences, are common stereotypic behaviors in captive ungulates and are caused by the frustration of natural behavior patterns 186 L. Cornips and L. van den Hengel L. Cornips and L. van den Hengel or by repeated attempts to deal with some problem (Bergeron et al. 2006). Tongue rolling, object licking and biting at fences—important indicators of compromised animal welfare—are especially prevalent among intensively housed cattle, as they are routinely deprived of the freedom to pursue natural patterns of grazing and rumi- nation (Moran and Doyle 2015, 47). Nevertheless, it is also clear that Mientje’s actions are not at all inconsequential or meaningless. On the contrary, by unlocking the bolt, leading the herd to the barn in the expectation of ﬁnding some snacks and by shitting the barn when they ﬁnd nothing there, Mientje and the other cows not only spur their humans into action (cleaning the barn, producing a new bolt) but also engage in linguistic acts of place-making by transforming their shared living space into a site for negotiating, or renegotiating, the semantics of power, resistance and belonging. 11.4 Linguistic Place-Making in an Intensive Dairy Farm In the previous section, we suggested that cows, as sentient and intelligent beings, engage their cognitive, emotional and social abilities in practices of linguistic place- making. Just as for people, we assume that the formation of meaningful bonds between cows and a place is “a powerful factor in social life… and is often based on the social relationships that are enacted in a place” (Schieffelin 2018, 35). In this section and the next, we will examine in more detail how intensively housed cows engage in place-making through language, understood as a distributed phenomenon emergingfromwithin“materialwebsofhumanandnonhumanassemblages”(Penny- cook 2017, 279). Drawing on recent ﬁeldwork at an intensive dairy farm, we seek to demonstrate how in this context linguistic place-making occurs through multi- modal and multilingual repertoires where human and nonhuman bodies, materials and environments come together in co-shaping motion. We will pay special atten- tion to the questions of material and nonhuman animal agency, not merely because “processes of place-making and place itself are always sensible to power dynamics and asymmetries” (Schieffelin 2018, 34), but also because these questions are crucial for thinking through the challenges of human-nonhuman coexistence in the current context of the Anthropocene. First, a cow becomes connected to her place as a “territory of knowledge” (Schief- felin 2018, 30, citing Århem 1998) through her verbal practices. While cattle vocal- izations are often proposed as indicators of animal welfare, scientiﬁc analysis of naturally occurring contact calls produced by crossbred beef cows and their calves have provided insight into the acoustic structure and information encoded in these vocalizations. One study showed that calf calls encode age, but not sex, and are produced (F0 = 142.8 ± 1.80 Hz) when separated from their mothers and preceded suckling (Padilla de la Torre et al. 2015, 58). Also, indoor housed calves produce individually recognizable calls to their mothers and vice versa whereas indoor housed cows signal verbally that they are hungry, sexually aroused, and experience milking delay in distinctive ways (Jahns 2013, 247). Thus, although cow sounds may be meaningless to most human animals, they constitute meaningful signs recognizable by mother cows and their calves, as well as by fellow cows as a sociolinguistic community of practice. Further,cowsestablishplace-makingthroughvisual,auditory,olfactory,gustatory and tactile practices, as well as through creative behavior such as play. Sight is a cow’s most dominant sense, with a ﬁeld of vision of at least 330° and a ﬁne eye for details. 11.3 Cows as Social and Linguistic Beings Once we acknowledge that, as Pennycook (2018, 51) notes, “linguistic and other semiotic resources are not contained in someone’s head, nor just choices available within a speech community, but are spatially distributed,” 1 Place-Making by Cows in an Intensive Dairy Farm … 187 11 we can begin to explore how dairy cows, such as Mientje, engage in linguistic place- making in relation to other cows, farmers, fences, iron bars and spaces such as barns and meadows, as well as through embodied acts of looking, smelling, licking, walking, eating, defecating, playing and listening. 11.3 Cows as Social and Linguistic Beings A place, in the sociolinguistic sense, is not simply a ﬁxed geographic location but rather a changeable site of symbolic meaning as well as a material assem- blage of objects or things that mediate social processes and relationships (Johnstone 2011; Cornips and de Rooij 2018a; Peck et al. 2019). Place-making, then, involves the assigning, through interaction and other forms of connectivity, of social mean- ings to physical (and, increasingly, digital) spaces, thereby “creating places that are perceived as the basis of belonging” (Cornips and de Rooij 2018b, 7–8). In contrast to other branches of linguistics where languages are seen as “naturally” anchored to speciﬁc spaces—a view that only holds if a language is conceptualized as a mono- lithic and identiﬁable object detached from real-time practices—a sociolinguistics of place takes a practice-based approach focused on speakers and their activities. This shifts the focus from the linguistic system or structure to a whole range of situated practices in which speech is produced, so that what is typically labelled as a language is reconceived as a linguistic resource that only becomes socially meaningful in combination with other material-semiotic resources distributed across people, places and environments (Pennycook 2017). Although previous sociolinguistic research has conceptualized place-making primarily or even exclusively in terms of human practices and institutions, we suggest that other animals, like cows, also engage their senses, thoughts and emotions in the material-semiotic production of the world as a meaningful place. This entails a clear break away from the previously discussed humanist conceptualizations of language as a computational system located exclusively within the human mind—a view on language which, as we have seen, is itself informed by a desire to place the human above all other animals—and steers us towards an understanding of language as a distributed phenomenon, an emergent property deriving from the interactions and interrelations between human and nonhuman actors, including spatial resources and things usually seen as inanimate (Cowley 2011; Pennycook 2017). This shift in thinking corresponds to the critical posthumanist “turn” that has been put on the linguistic research agenda recently by Alastair Pennycook, who urges us “not just to broaden an understanding of communication but to relocate where social semiosis occurs” (Pennycook 2016, 446). 9The ﬁeldwork took place on 25 July (observation), 26 July and 17 August 2018 (audio- and video recordings). A written and signed consent form by the owners of Farm 1 was obtained abiding by the guidelines for research as stated in the protocol of the Ethics Assessment Committee Humanities of the Radboud University Nijmegen and adopted by the Royal Academy of Arts and Sciences (KNAW). From the perspective of establishing egalitarian research methods for interspecies collab- oration, there is a need to examine how to receive permission from the nonhuman animals under study, while at the same time one should interrogate how the bioethical framework of “informed consent” is set up through human-centred discourses of rational agency and choice. 11.4 Linguistic Place-Making in an Intensive Dairy Farm Cows pay more attention to moving objects than ones that remain still, such as bars, and they are often “spooked” by sudden movements. A cow’s hearing 188 L. Cornips and L. van den Hengel is better than that of horses, but she is less able to locate sounds compared to goats, dogs and humans. She has an acute sense of touch, which enables her to enjoy some forms of tactile contact, such as scratching behind the ears, but it also means that the conditions of industrial farming cause her considerable pain. Olfaction plays an important role in cows’ social lives, and there is evidence that they can detect the scent of stress hormones present in the urine of fellow cows (Marino and Allen 2017, 475–476). Cows engage in all forms of play found in mammals, including gamboling and running, playing with objects such as balls and social play with members of other species. While play is an important indication of an animal’s pleasure, curiosity and capacity to innovate, and as such it “forms the basis for complex object-related and social abilities” (Marino and Allen 2017, 481), play behavior in captive animals is also dependent on their housing conditions; for example, being released from conﬁnement will increase the frequency of movement-based forms of play such as galloping and bucking. In what follows, we will discuss speciﬁc examples from ﬁeld research to demonstrate how dairy cows can mobilize these structural constraints to imbue their environment with linguistic meanings and thus negotiate their positioning within an anthropological machine that is, by and large, designed to deprive them from the opportunity to speak. 11.4.1 The Fieldwork Site From May 2018 through 15 February 2019, this chapter’s ﬁrst author conducted ﬁeldwork in three dairy farms in the south and west of the Netherlands and in one small dairy farm in Norway. The observations presented in this chapter are based on data collection at Farm 1 in the south of the Netherlands, where the ﬁrst author spent several weeks during her holidays in May and which subsequently became the site for three days of ethnographic observation, including two days of gathering audio- and video recordings.9 The dairy farm counts about 150 adult cows, heifers and calves. The dairy cows are milked by robots, while an automatic feeder takes care of pushing the food towards them, minimizing embodied practices between farmers and cows. A small camp site and some holiday apartments accompany the farm, as so often in the south. Many tourists, children in particular, seek contact with the newly born calves, as well as with the older calves and heifers to be discussed below, petting them and speaking to them. Feeding the newly born calves, who are housed individually in ﬁberglass cages outside the barn, is an especially popular activity among the human 1 Place-Making by Cows in an Intensive Dairy Farm … 189 11 visitors. During the on-site observations, the cows would often reach through the bars and fences to touch the farmers, tourists and ﬁeld worker through licking and nuzzling. They would also establish contact through nonverbal interactions such as eye gaze and body positioning, as well as by using language in the form of rumbling, calling, hooting, snifﬁng and coughing. These practices would happen frequently, even though neither humans nor cows were able to traverse to sharing space with each other directly. y Farmers usually assign dairy cows to ﬁxed places in artiﬁcial groupings based on their age without male peers, revealing extreme power asymmetries between cows and humans. In the farm under observation, cows are assigned to eight distinct places differentiated by age: new-born calves, older calves up until a few weeks of age, young heifers, older and oldest heifers, dry cows (pregnant cows), and dairy cows. As noted, new-born female calves are separated from their mothers immediately after birth and isolated in ﬁberglass enclosures, so-called “igloos,” for about three weeks. In Farm 1, these igloos are placed in the open air facing the dairy cows in the open barn. 10During her holidays in May, the ﬁrst author frequently visited the jongveestal since there was one heifer who was very much looking for contact with human animals. She was positioned near the main entrance and would nearly jump towards the author to put her head on her shoulder. This extravert expression of contact seeking behavior might be interpreted as indicative of a willingness to engage in interspecies collaboration. 11.4.1 The Fieldwork Site After this period, the somewhat older calves are housed together with their age mates in igloos holding up to four or ﬁve animals, positioned sideways to the open barn so that visual contact with the older cows is much more restricted. Growing older, the calves are placed in the so-called jongveestal (young cattle barn) in four different age groups (see Fig. 11.1). The dry cows are housed in a separate space and the dairy cows reside in the large open barn that also contains three milking robots. In spring and summer, the dry cows and dairy cows can graze in the meadow during the day and, when it is very hot, during the late evening and night. The assignment to speciﬁcphysicalplacesindistincthousingspreventsthecalves,heifersandcowsfrom forming a natural herd that would include a matrilineal social structure with strongly clustered networks and many non-random attachment and avoidance relationships (Marino and Allen 2017, 488). It also prevents the younger ones from engaging in processes of cognitive and social learning, and deprives them from being comforted by older conspeciﬁcs, including mother cows. The ﬁeldwork took place in the jongveestal, where audio and video recordings were made while observing the calves and heifers. The jongveestal is an oblong building, about twenty by ten meters, with half bowed windows, touching the house where the farmer’s family lives. It is the oldest barn on site with a main entrance in the middle of the long front side and a full opening at one of the short sides. The cattle stay indoors: their day includes some combinations of eating, lying and Fig. 11.1 Jongveestal (“young cattle barn”), housing 36 calves and heifers (Friday 17 August 2018, 2.30–6 p.m.) Fig. 11.1 Jongveestal (“young cattle barn”), housing 36 calves and heifers (Friday 17 August 2018, 2.30–6 p.m.) 190 L. Cornips and L. van den Hengel standing. The oldest heifers were about one year old during the ﬁeldwork.10 In May, swallows would ﬂy in and out to take care of their new-born in the many nests they had fabricated under the beams of this old building. The ﬂoor of the jongveestal consists of cubicle divisions for calves and heifers to lie down and stand up (see Picture 11.3 below), while in-between the cubicles, they can stand or walk on discrete beams where feces and urine pass. 11.4.1 The Fieldwork Site In the jongveestal, the calves and heifers (n = 36) were spatially positioned in four sections divided by iron bars, as illustrated in Fig. 11.1. The calves and heifers in the jongveestal are thus profoundly restricted in their mobility—much more so than the adult cows who are able to graze in the meadow, but less than the new-born calves conﬁned to the small igloos. Consequently, from birth onwards throughout their lives, calves, heifers and dairy cows—either individually or with same-sex and age mates—are conﬁned to human-made physical spaces. How, then, do they manage to assign their own meanings to the restricted environment in which they are placed? 11.4.2 Place-Making Through Practices of Sociality and Multilingualism The housing conditions of the jongveestal not only restrict mobility but also limit the visual, auditory, olfactory, gustatory and tactile practices that calves and heifers may display under less restrictive conditions. This signiﬁcantly affects their modes of sociality and processes of belonging: calves and heifers cannot touch and/or allogroom each other cross-sectionally; a lack of daylight hinders optimal vision and the walls obstruct a far vision; the sound of tractors may penetrate; calves and heifers are dependent on the farmer for how to lie down as well as for when, what and how to eat (with no attention for individual food preferences); ventilation is often not optimal so that calves and heifers, whose sense of smell is far superior to that of humans, deal with omnipresent scents of ammonia, carbon dioxide, methane and hydrogen sulﬁde (Vallez 2013, 12); and the beams on the ﬂoor, often slippery due to feces and urine, impede playing and running (see Picture 11.3). Limited space allowance furthermore makes it difﬁcult to maintain a preferred distance to neighbors with whom the individual likes to bond or not. The spatial distance that cows estab- lish between each other is affected by their relationship and proximity might indicate the existence of a social bond (McLennan 2013, 26). Under more natural conditions, cows seem to engage with particular individuals with whom they prefer to spend their time, creating voluntary bonds while grazing and lying together in close proximity (McLennan 2013, 49–50). In the captive environment of the jongveestal, however, 1 Place-Making by Cows in an Intensive Dairy Farm … 191 11 a calf or heifer who is a non-preferred partner may stand, lie down or eat in closer proximity than would naturally occur, which may lead to feelings of uneasiness and has been suggested to have negative consequences for animal welfare (McLennan 2013, 52). How, if at all, do calves and heifers in the jongveestal create sociality under these conditions? And how is this sociality mobilized in and through material-semiotic practices of place-making? Fieldwork observations show that the calves and heifers in their cubicle divisions (see Fig. 11.1) may not show any sign of interaction or connectivity, thus is it not self-evident for individuals who are placed in a restricted space to construe it together as a social place. Picture 11.1 shows an example: four older calves stand in the second section of the jongveestal. 11.4.2 Place-Making Through Practices of Sociality and Multilingualism Although they share a restricted physical space, their body positioning does not reveal any form of co- shaping the act of standing together. The calves position their bottoms to each other, taking diverging positions, avoiding eye contact and body contact. Although the calves in the right corner seem to align sideways, there is no form of interaction. Their bodies don’t touch and while the calf in the middle bows her head, the calf in the right corner is rubbing her chin at the iron bar and wooden demarcation while establishing eye contact with the ﬁeldworker, as shown in Picture 11.2. Both pictures also show that in the second section of the barn four cows are standing on their feet whereas two cows are lying down in cubicle divisions. The calf to the right in Picture 11.2 was headbutted by another for about two seconds when trying to move over to the most right-hand section of the barn (not visible in the picture). Picture 11.3, however, shows calves in section 2 mirroring each other’s body positions when lying down in the cubicles. Although their bodies do not touch and they are not able to lie down in a circle as less restrictive settings, they are able to choose to lie down all together in the same way at the same time. The two calves in the cubicles in the back are facing each other whereas the two shown in the foreground Picture 11.1 Standing in the jongveestal (Farm 1, 17 August 2018) Picture 11.1 Standing in the jongveestal (Farm 1, 17 August 2018) L. Cornips and L. van den Hengel 192 Picture11.2 Calfisrubbingherchinatthefencewhileestablishingeyecontactwiththeﬁeldworker (Farm 1, 17 August 2018) Picture11.2 Calfisrubbingherchinatthefencewhileestablishingeyecontactwiththeﬁeldworker (Farm 1, 17 August 2018) do not. Note that the younger calf in section 1 has decided to look out of the window instead of synchronizing with the others. As a material-semiotic resource, bodily synchronizing can be seen as part of what Frans de Waal has described as “identiﬁcation” with the other, a process of “bodily mapping the self onto the other (or the other onto the self)” which not only relates to a capacity for shared neural representation, but also forms “a precondition for imitation and empathy” (De Waal 2012, 123). 11Argent writes about synchronizing between horses and riders. 11.4.2 Place-Making Through Practices of Sociality and Multilingualism During the ﬁeldwork, a clear practice of bodily synchronizing—which we consider here as a social form of meaning-making typical for encaged dairy cows—emerged during the communicative event of feeding (by the farmer) and eating (by the cows). The farmer feeds the calves twice a day by putting upside down a wheelbarrow loaded with food on the ground before them. An iron feed fence separates the human and nonhuman animals during this event while at the same time it mediates the meanings that arise from their mutual interaction. The farmer provides the food from one side of the fence whereas the calves on the other side need to position themselves before individual openings and put their heads through the bars in order to reach the food below. Since there are only as many openings as calves, every individual has to touch her neighbor to secure a place (see Pictures 11.4a, b). As the calves put their heads through the iron bars they simultaneously bow their heads forward and downward to pick up the food from the ground, taking a slightly more upward head position in order to chew. Within this joint “embodiment of movement,” a collective form of sociality in which each calf will instantaneously “follow and lead” (Argent 2012, 120),11 the calves engage one another and the space around them in bodily acts of identiﬁcation that articulate the jongveestal as a shared social place. Thus, although for encaged calves feeding is clearly a habitual and 193 11 Place-Making by Cows in an Intensive Dairy Farm … Picture 11.3 Bodily synchronizing in cubicle divisions in the jongveestal (Farm 1, 1 2018) Picture 11.3 Bodily synchronizing in cubicle divisions in the jongveestal (Farm 1, 17 August 2018) L. Cornips and L. van den Hengel 194 194 L. Cornips and L. 11.4.2 Place-Making Through Practices of Sociality and Multilingualism van den Hengel Picture 11.4 a The calves positioning themselves at the feed fence (wheelbarrow on the right); b The calves are synchronizing during their eating practice Picture 11.4 a The calves positioning themselves at the feed fence (wheelbarrow on the right); b The calves are synchronizing during their eating practice Picture 11.4 a The calves positioning themselves at the feed fence (wheelbarrow on the right); b The calves are synchronizing during their eating practice Picture 11.4 a The calves positioning themselves at the feed fence (wheelbarrow on the right); b The calves are synchronizing during their eating practice routinized practice, from a sociolinguistic perspective it also entails creative acts of place-making through what Argent calls “kinesic, haptic, and proxemic communi- cation modes” (Argent 2012, 119). The synchrony of movement that occurs in and through the spacing of interactional distances not only orients calves to group living, but also enables them to imbue their restricted environment with meaning in the form of social bonding and thus constitute themselves as linguistic agents. Speciﬁcally, we argue that nonhuman practices of place-making in an intensive dairy farm can be seen as a form of bi- or multilingualism peculiar to the context of industrial animal production. The iron bars make sound when the synchronizing calves put their heads through them and move their faces up and down in co-motion 11 Place-Making by Cows in an Intensive Dairy Farm … 195 11 to reach for the food on the ground during eating practice. These sounds are not meaningless or arbitrary but constitute a semiotic resource for calves to reinforce social bonding, speciﬁcally since the sounds of the iron bars and bodies co-shape each other acoustically. Eating practices in the jongveestal, then, are acts of place- making where calves do not only use their vocal tract to produce language, but also establish themselves as linguistic beings through the rhythmic clattering of the iron bars that shapes the synchronizing bodies into socially meaningful sounds. 11.4.2 Place-Making Through Practices of Sociality and Multilingualism In other words, these calves engage in a process of nonhuman place-making not only by producing one language with their own bodies, that is, the words or vocaliza- tions for “greeting” and “hunger” which are inextricably combined with multimodal and multisensory ways of meaning-making through body positioning and visual, auditory, olfactory, gustatory and tactile practices, but also by producing a second language with the material-semiotic means that both compose and transcend their restricted environment. Being socialized into the environment of an industrial dairy farm, then, for cows implies being or becoming bilingual, where bilingualism is to be understood as a “complex set of practices” (Heller 2007, 15) which draw on linguistic resources that belong to two codes which are structurally maximally divergent (see Auer, forth- coming, 8), in this case one code produced by vocal tract and one code produced by synchronizing bodies and iron bars. The latter language is more context-dependent than the former because of its restriction to the practice of feeding in conditions of captivity. These two codes thus reveal structural constraints in the linguistic sense: they can combine together in a multimodal way but cannot be mixed. The obser- vations do not show that calves alternate between the two codes within one single discourse—the social practice of eating together—even if both codes are part of the broader material-semiotic assemblage through which place-making is established. In the sociolinguistic framework, the two codes might be said to correspond to different social functions and identities, since different languages, or language varieties, are associated with diverging “processes of construction of social difference and social inequality”(Heller2007, 15). The“bars andbodies”codewill beassociatedprimarily with encaged individuals, suggesting that this form of bilingualism is speciﬁc to the complex network of connections among human and nonhuman agents that constitutes daily life at an intensive dairy farm. Crucially, the material presence of the feed fence, which both enables and constrains the expression of linguistic agency in this context, should be understood not merely as a demarcation of physical space, but as belonging to the spatial reper- toire through which language is produced as a “distributed effect of a range of inter- acting objects, people and places” (Pennycook 2017, 278). 11.4.2 Place-Making Through Practices of Sociality and Multilingualism As an embodied and embedded practice, linguistic place-making in the jongveestal is thus not simply a conditioned response to an unresponsive environment, nor does it arise from the individual communicative competence of calves and heifers; rather, it emerges from within a complex assemblage of material-semiotic resources distributed across human and nonhuman subjects, artefacts and environments, including the means of conﬁnement by which humans seek to restrict the freedom of other animals. In other words, the conditions of captivity in an intensive dairy farm are not external but 196 L. Cornips and L. van den Hengel L. Cornips and L. van den Hengel intrinsic to how cows engage in acts of place-making that we, in Heideggerian terms, might understand as linguistic practices of world-formation. This view, as we will conclude, has signiﬁcant consequences for how we may conceptualize the expression of nonhuman animal agency, in particular linguistic agency, in the troubling context of the Anthropocene. 11.5 Conclusion From a traditional humanist perspective, domesticated captive animals are doubly barred from entering into a meaningful relationship with the world: not only are nonhuman animals, in this view, by nature captivated in their environment (as Heidegger and many others have suggested), it is also assumed that conﬁnement in cages does nothing but further limit their natural instincts and capabilities. This view effectively renders nonhuman animals, cows in particular, mute and dumb, while at the same time it reinforces a traditional mechanistic worldview where both nature and matter are considered to be passive and inert, available for manipulation by humans and exploitable for proﬁt (Merchant 1992, 48–55). The critically posthu- manist perspective developed in this chapter, by contrast, not only acknowledges cows as the social and intelligent speaking beings that they are, but also approaches their material encagement—the bars and fences meant as barriers to prevent calves and heifers from freely going wherever they want—as a social and spatial artefac- tual resource for building a meaningful world. Paradoxically, then, it is their state of unfreedom that allows dairy cows to open up the restricted environment of indus- trial animal farming, exempliﬁed here by the young cattle barn, as a linguistically meaningful place. In drawing attention to the linguistic agency of dairy cows, we do not wish to reit- erate the familiar observation that “agency is intrinsic to the way animals behave” (Špinka and Wemelsfelder 2011, 34), nor are we suggesting that the capacity of captiveanimalstoactissomehow“expanded”or“curtailed”throughactsoflinguistic place-making. In the context of industrial dairy farming, where categorical bound- aries between humans and other animals, as well as between organisms and machines, have collapsed—a condition exemplifying the “implosion of nature and culture” (Haraway 2003, 16) that marks the Anthropocene—neither agency nor language can be understood as a property of individual persons or collectivities. Rather, we must account for how different forms of agency, including linguistic agency, emerge from within what political theorist Jane Bennett (2010, 107) has called “agentic assemblages,” that is, networks of human and nonhuman actors living together in relations of systemic inequality. In this chapter, therefore, we have tried to show how a non-anthropocentric approach to linguistic place-making, understood as a practice of more-than-human sociality, can help us reckon with the question of nonhuman animal agency in new ways. 11.5 Conclusion Assemblages, as Pennycook (2017, 278) notes, “describe the way things are brought together and function in new ways” and as such they provide a way of 1 Place-Making by Cows in an Intensive Dairy Farm … 197 11 thinking about agency as a distributed force, much like we described language and cognition as spatially distributed. Bennett, indeed, suggests that we think of agency as “distributed across an ontologically heterogeneous ﬁeld, rather than being a capacity localized in a human body or in a collective produced (only) by human efforts” (Bennett 2010, 23). Linguistic agency, then, is not an individual or collec- tive competence that can be “mastered” or “possessed” but should rather be seen as a processually emergent quality arising from multiple assemblages of human and nonhuman elements, including material things, artefacts and spaces. This concep- tion of agency, rooted in what Bennett (2010) calls a “vital materialism” and what Pennycook (2018) describes as a “posthumanist applied linguistics,” disturbs the traditional understanding of agency as the capacity for self-willed action, linked espe- cially to human subjectivity and intentionality, as well as the corollary presumption that the more-than-human material world—including other animals and the physical environment—is essentially passive, inert and predetermined in its operations. Throughout this chapter, we have sought to demonstrate the usefulness of a non- anthropocentric approach to language and language practices in light of a long history of human exceptionalism that has routinely denied nonhuman animals the freedom and ability to speak. We have elaborated a posthumanist conception of language as a distributed effect of multiple interacting bodies in order to foreground the ﬂuidity throughwhichacow,acalf,calves,awheelbarrow,afarmer,anironfeedfence,alock, the clattering of bars, sounds of chewing, sounds of pufﬁng, sounds of urinating, the smell of food, urine, feces, other bodies in proximity or distance, movements up and down, become relationally entangled with one another and, crucially, with the anthro- pological machine of industrial animal production. Furthermore, we have shown how rethinking nonhuman animal agency in terms of material-semiotic assemblages, as an equally distributed effect of linguistic interactions and social processes, allows us to break away from the idea of lifeless matter, including the Cartesian under- standing of nonhuman animals as mindless machines, an idea which has shaped the pervasive modes of human exceptionalism and instrumentalism that have tradition- ally characterized the humanist agenda and which continue to inform ideas about the “muteness” and “bruteness” of nonhuman creatures today. Acknowledgements The ﬁrst author is very grateful to the participating farmer for all support, the opportunity to make recordings, and time. 11.5 Conclusion In this way, we hope to contribute to a greater recognition among humans of other animals, not only as sentient living beings, but as intelligent, social, speaking beings, linguistic agents who even under poor conditions form rich and complex relationships with the world to make it a meaningful place. 198 L. Cornips and L. van den Hengel References Agamben, G. 2004. The open: Man and animal. Stanford: Stanford University Press. Argent, G. 2012. Toward a privileging of the nonverbal: Communication, corporeal synchrony, and transcendence in humans and horses. In Experiencing animal minds: An anthology of animal- human encounters, ed. J.A. Smith and R.W. Mitchell, 111–128. New York: Columbia University Press. Århem, K. 1998. Powers of place: Landscape, territory and local belonging in Northwest Amazonia. In Locality and belonging, ed. N. Lovell, 78–102. London and New York: Routledge. Auer, P. forthcoming. “Translanguaging” or “doing languages”? Multilingual practices and the notion of “codes”. In Language(s): Multilingualism and it consequences, ed. J. MacSwann. Bristol: Multilingual Matters. Retrieved from https://www.researchgate.net/publication/332 593230_‘Translanguaging’_or_‘doing_languages’_Multilingual_practices_and_the_notion_of_ ‘codes’. Bennett,J.2010.Vibrantmatter:Apoliticalecologyofthings.DurhamandLondon:DukeUniversity Press. Bergeron, R., A.J. Badnell-Waters, S. Lambton, and G. Mason. 2006. Stereotypic oral behaviour in captive ungulates: Foraging, diet and gastrointestinal function. In Stereotypic animal behaviour: Fundamentals and applications to welfare, 2nd ed., ed. G. Mason and J. Rushen, 19–57. Wallingford and Cambridge: CABI. Berwick, R.C., and N. Chomsky. 2016. Why only us: Language and evolution. Cambridge and London: The MIT Press. Bucholtz, M., and K. Hall. 2016. Embodied sociolinguistics. In Sociolinguistics: Theoretical debates, ed. N. Coupland, 173–197. Cambridge: Cambridge University Press. Chomsky, N. 2002. On nature and language. Cambridge: Cambridge University Press. Chomsky, N. 2006. Language and mind, 3rd ed. Cambridge: Cambridge University Press. Colvin, C.M., K. Allen, and L. Marino. 2017. Thinking cows: A review of cognition, emotion, and the social lives of domestic cows. https://www.farmsanctuary.org/wp-content/uploads/2017/10/ TSP_COWS_WhitePaper_vF_web-v2.pdf. Cornips, L. 2019. The ﬁnal frontier: Non-human animals on the linguistic research agenda Linguistics in the Netherlands 36 (1): 13–19. Cornips, L., and F. Gregersen. 2016. The impact of Labov’s contribution to general linguistic theory. Journal of Sociolinguistics 20 (4): 498–524. https://doi.org/10.1111/josl.12197. Cornips, L., and V. de Rooij (eds.). 2018a. The sociolinguistics of place and belonging: Perspectives from the margins. Amsterdam: John Benjamins. Cornips, L., and V. de Rooij. 2018b. Introduction: Belonging through linguistic place-making in center-periphery constellations. In The sociolinguistics of place and belonging: Perspectives from the margins, ed. L. Cornips and V. de Rooij, 1–16. Amsterdam: John Benjamins. Cornips, L., and M. van Koppen. 2019. Embodied grammar in Dutch dairy cows. Poster presented at the workshop Animal Linguistics: Take the Leap!, L’École normale supérieure, Paris, June 17. C tti h J 2008 C i ﬂ i E D ’ hil h O f d O f d Cornips, L., and M. van Koppen. 2019. Derrida, J. 2009. The beast and the sovereign: Volume 1, trans. G. Bennington. Chicago and London University of Chicago Press. References Embodied grammar in Dutch dairy cows. Poster presented at the workshop Animal Linguistics: Take the Leap!, L’École normale supérieure, Paris, June 17. Cottingham, J. 2008. Cartesian reﬂections: Essays on Descartes’ philosophy. Oxford: Oxford University Press p pp g y p at the workshop Animal Linguistics: Take the Leap!, L’École normale supérieure, Paris, June 17. Cottingham, J. 2008. Cartesian reﬂections: Essays on Descartes’ philosophy. Oxford: Oxford University Press. p g p , p , , Cottingham, J. 2008. Cartesian reﬂections: Essays on Descartes’ philosophy. Oxford: Oxford University Press. y Cowley, S.J. 2011. Distributed language. Amsterdam: John Benjamins. Cull, L. 2015. From homo performans to interspecies collaboration: Expanding the concept of performance to include animals. In Performing animality: Animals in performance practices, ed. L. Orozco and J. Parker-Starbuck, 19–36. Basingstoke: Palgrave Macmillan. De Waal, F. 2012. A bottom-up view of empathy. In The primate mind: Built to connect with other minds, ed. F. de Waal and P.F. Ferrari, 121–138. Cambridge and London: Harvard University Press. Derrida, J. 2009. The beast and the sovereign: Volume 1, trans. G. Bennington. Chicago and London: University of Chicago Press. 11 Place-Making by Cows in an Intensive Dairy Farm … 199 Descartes, R. 1985. Discourse on the method. In The philosophical writings of Descartes: Volume 1, trans. J. Cottingham, R. Stoothoff, and D. Murdoch, 111–175. Cambridge: Cambridge University Press. Despret, V. 2008. The becomings of subjectivity in animal worlds. Subjectivity 23: 123–139. https:// doi.org/10.1057/sub.2008.15. Evans, V. 2014. The language myth: Why language is not an instinct. Cambridge: Cambridge University Press. Hagen, K., and D.M. Broom. 2004. Emotional reactions to learning in cattle. Applied Animal Behaviour Science 85: 203–213. Haraway, D. 2003. The companion species manifesto: Dogs, people, and signiﬁcant otherness Chicago: Prickly Paradigm Press. g y g Haraway, D. 2008. When species meet. Minneapolis and London: University of Minnesota Press. Haraway, D. 2008. When species meet. Minneapolis and London: University of Minnesota Press. Harrison, P. 1992. Descartes on animals. The Philosophical Quarterly 42 (167): 219–227. https:// doi.org/10.2307/2220217. y p p y Harrison, P. 1992. Descartes on animals. The Philosophical Quarterly 42 (167): 219–227. https:// doi.org/10.2307/2220217. Hauser, M.D., N. Chomsky, and W.T. Fitch. 2002. The faculty of language: What is it, who has it, and how did it evolve? Science 298 (5598): 1569–1579. http://dx.doi.org/10.1126/science.298. 5598.1569. Heidegger, M. 1971. Poetry, language, thought, trans. A. Hofstadter. New York: Harper & Row. Heidegger, M. 1995. References The fundamental concepts of metaphysics: World, ﬁnitude, solitude, tran McNeill and N. Walker. Bloomington and Indianapolis: Indiana University Press. gg , f p f p y , ﬁ , , McNeill and N. Walker. Bloomington and Indianapolis: Indiana University Press. Heller, M. 2007. Bilingualism as ideology and practice. In Bilingualism: A social approach, ed. M. H ll 1 22 B i t k P l M ill Heller, M. 2007. Bilingualism as ideology and practi Heller, 1–22. Basingstoke: Palgrave Macmillan. Heller, M. 2007. Bilingualism as ideology and practice. In Bilingualism: A social approach, ed. M. Heller, 1–22. Basingstoke: Palgrave Macmillan. Jahns, G. 2013. Computational intelligence to recognize animal vocalization and diagnose animal health status. In Computational intelligence in intelligent data analysis, ed. C. Moewes and A. Nürnberger, 239–249. Berlin: Springer. Johnstone, B. 2011. Language and place. In The Cambridge handbook of sociolinguistics, ed. R. Mesthrie, 203–217. Cambridge: Cambridge University Press. https://doi.org/10.1017/CBO978 0511997068.017. Kulick, D. 2017. Human-animal communication. Annual Review of Anthropology 46: 357–378 https://doi.org/10.1146/annurev-anthro-102116-041723. Labov, W. 1994. Principles of linguistic change. Volume 1: Internal factors. Oxford and Cambridge: Blackwell. Labov, W. 2001. Principles of linguistic change. Volume 2: Social factors. Oxford and Cambridge: Blackwell. Marino, L., and K. Allen 2017. The psychology of cows. Animal Behavior and Cognition 4 (4): 474–498. https://dx.doi.org/10.26451/abc.04.04.06.2017. McLennan, K.M. 2013. Social bonds in dairy cattle: The effect of dynamic group systems on welfare and productivity. Doctoral thesis, The University of Northampton. http://nectar.northampton.ac. uk/6466/. Meijer, E. 2016. Speaking with animals: Philosophical interspecies investigations. In Thinking about animals in the age of the Anthropocene, ed. M. Tønnessen, K. Armstrong Oma, and S. Rattasepp, 73–88. Lanham: Lexington Books. g Meijer, E. 2019. Animal languages: The secret conversations of the natural world. London: John Murray. Merchant, C. 1992. Radical ecology: The search for a livable world. London and New York: Routledge. Moran, J., and R. Doyle. 2015. Cow talk: Understanding dairy cow behavior to improve their welfare on Asian farms. Melbourne: CSIRO Publishing. http://www.publish.csiro.au/ebook/7929. Müller, C., J. Bressem, and S.H. Ladewig. 2013. Towards a grammar of gestures: A form-based view. In Body—Language—Communication: An international handbook on multimodality in human interaction, ed. C. Müller, A. Cienki, E. Fricke, S.H. Ladewig, D. McNeill, and S. Tessendorf, 707–733. Berlin and Boston: De Gruyter Mouton. L. Cornips and L. van den Hengel 200 Nawroth, C., J. Langbein, M. Coulon, V. Gabor, S. Oesterwind, J. Benz-Schwarzburg, and E. von Borell. 2019. References Farm animal cognition: Linking behavior, welfare and ethics. Frontiers in Veterinary Science 6 (24): 1–16. https://doi.org/10.3389/fvets.2019.00024. Padilla de la Torre, M., E.F. Briefer, T. Reader, and A.G. McElligott. 2015. Acoustic analysis of cattle (Bos taurus) mother-offspring contact calls from a source-ﬁlter theory perspective. Applied Animal Behaviour Science 163: 58–68. http://dx.doi.org/10.1016/j.applanim.2014.11.017. Animal Behaviour Science 163: 58–68. http://dx.doi.org/10.1016/j.applanim.2014.11.017. Peck, A., C. Stroud, and Q. Williams (eds.). 2019. Making sense of people and place in linguistic landscapes. London and New York: Bloomsbury. Pennycook, A. 2016. Posthumanist applied linguistics. Applied Linguistics 39 (4): 445–461. https:// doi.org/10.1093/applin/amw016. Pennycook, A. 2016. Posthumanist a doi.org/10.1093/applin/amw016. doi.org/10.1093/applin/amw016. Pennycook, A. 2017. Translanguaging and semiotic assemblages. International Journal of Multi lingualism 14 (3): 269–282. https://doi.org/10.1080/14790718.2017.1315810. g ( ) p g Pennycook, A. 2018. Posthumanist applied linguistics. London and New York: Routledge. Pennycook, A., and E. Otsuji. 2015. Making scents of the landscape. Linguistic Landscape 1 (3) 191–212. https://doi.org/10.1075/ll.1.3.01pen. Schieffelin, B.B. 2018. Language socialization and making sense of place. In The sociolinguistics of place and belonging: Perspectives from the margins, ed. L. Cornips and V. de Rooij, 27–54. Amsterdam: John Benjamins. Špinka, M., and F. Wemelsfelder 2011. Environmental challenge and animal agency. In Animal welfare, 2nd ed., ed. M.C. Appleby, J.A. Mench, I.A.S. Olsson, and B.O. Hughes, 27–43. Wallingford and Cambridge: CABI. g g Vallez, T. 2013. Huisvesting van jongvee op een melkveebedrijf. Master thesis, Ghent University. https://lib.ugent.be/fulltxt/RUG01/002/062/712/RUG01-002062712_2013_0001_AC.pdf. Vallez, T. 2013. Huisvesting van jongvee op een melkveebedrijf. Master thesis, Ghent University https://lib.ugent.be/fulltxt/RUG01/002/062/712/RUG01-002062712_2013_0001_AC.pdf. Wilkie, R.M. 2010. Livestock/deadstock: Working with farm animals from birth to slaughter Philadelphia: Temple University Press. Wilkie, R.M. 2010. Livestock/deadstock: Working with farm animals from birth to slaughter. Philadelphia: Temple University Press. Leonie Cornips is senior researcher at the NL-Lab, Humanities Cluster (KNAW) in Amsterdam and professor Languageculture in Limburg at Maastricht University, the Netherlands. Her research deals with sociolinguistic issues. Current topics are national and regional identity construction through language practices, bidialectal child acquisition, multilingualism and intra- and inter- species interactions, in particular, dairy cow language and communication. She received her Ph.D. from the University of Amsterdam. Her homepage is https://pure.knaw.nl/portal/en/persons/l-cor nips/publications/. Louis van den Hengel is Assistant Professor of Gender and Diversity Studies at Maastricht University, the Netherlands. His research deals with the potentiality of contemporary art perfor- mances for activating new socio-political imaginaries and for envisioning an afﬁrmative ethics of difference for the twenty-ﬁrst century. In other words, he is interested not only in how perfor- mance negotiates new relationships between contemporary art and the cultural politics of gender, sexuality, race, class, and species difference, but also in how it may help actualize new modal- ities of being in, and becoming with, the world. His most recent publications include contribu- tions to journals such as Criticism: An Interdisciplinary Quarterly and to edited volumes such as Doing Gender in Media, Art and Culture (Routledge, 2018) and Entanglements and Weavings: Diffractive Approaches to Gender and Love (Brill, 2021). doi.org/10.1093/applin/amw016. 11 Place-Making by Cows in an Intensive Dairy Farm … 201 Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. The images or other third party material in this chapter are included in the chapter’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
https://openalex.org/W2973504994	https://e-journal.usd.ac.id/index.php/IJHS/article/download/2058/1583	English	null	COMPLIMENTS IN THE EDUCATIONAL INSTITUTION: COMPLIMENTS AMONG INDONESIAN STUDENTS OF ENGLISH DEPARTMENT	International Journal of Humanity Studies (IJHS)	2,019	cc-by-sa	6,557	IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 International Journal of Humanity Studies http://e-journal.usd.ac.id/index.php/IJHS Sanata Dharma University, Yogyakarta, Indonesia IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 International Journal of Humanity Studies http://e-journal.usd.ac.id/index.php/IJHS Sanata Dharma University, Yogyakarta, Indonesia International Journal of Humanity Studies http://e-journal.usd.ac.id/index.php/IJHS Sanata Dharma University, Yogyakarta, Indonesia Nugraha Krisdiyanta STBA LIA Yogyakara nugrahakris@yahoo.com DOI: https://doi.org/10.24071/ijhs.2018.020110 received 2 August 2019; revised 31 August 2019; accepted 3 September 2019 Nugraha Krisdiyanta STBA LIA Yogyakara nugrahakris@yahoo.com DOI: https://doi.org/10.24071/ijhs.2018.020110 received 2 August 2019; revised 31 August 2019; accepted 3 September 2019 Abstract This paper investigated how Indonesian people give compliment to others in English to find the formula of the compliment the students use. The subject of the research is 27 Indonesian students of English Department from three universities. The data are collected by giving questionnaire to the students using the Discourse Completion Task. Students are asked to complete the sentences for giving compliment. The compliments requested are about appearance which consists of hairstyle, dress and figure; and ability and performance which consists of test result, getting the scholar and cooking ability. The result shows that for appearance, the most used formula is by using the word look then the use of be+adj. For ability and performance, students use the word congratulation and be+adj for cooking ability. However, some students give compliment based on Bahasa Indonesia form, not English form by giving some statements which are not related to the context. It is affirmed that compliment is influenced by the language that the people use and it is different from one culture to another. Keywords: dicsourse completion task, compliment, compliment formula Introduction Compliment is a remark that says something good about someone or something (Merriem-Webster dictionary). People tend to compliment other people in their daily conversation. Compliment is sometimes considered as a way to establish a communication. It is a speaker’s expression of a positive stance toward some referent attributable to her/his addressed recipient (Pillet-Shore, 2015). However, compliment is not always expressed in verbal communication. It can also be an action which expresses admiration or approval (Merriem-Webster dictionary). Holmes argues that ‘a compliment is a speech act which explicitly or implicitly attributes credit to someone other than the speaker, usually the person addressed, for some “good” (possession, characteristic, skill, etc.) which is positively valued by the speaker and the hearer’, and she also asserts that ‘compliments are generally regarded as the paradigm of a positive politeness strategy’ (Holmes,1986, cited in Holmes, 2015:117). It is a type of speech acts which maintain good feeling for the hearer. A genuine compliment always makes 107 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 others feel good. It can reduce the social distance between the speaker and hearer (Holmes, 2015). When conducting her research in New Zealand, Holmes even found that women do compliments more than men. They use compliments to maintain the solidity and solidarity between the speaker and hearer. Oppositely, amongst men, compliments are mostly face threatening. They use mock insulting and sparring to cement their friendships. Looking at this situation, Coates even says that complimenting is one of the women’s linguistic behaviour beside apologizing and it is a part of our communicative competence (2013:86). p p Manes and Wolfson (1981), cited by Aijmer (2014), show that compliments can be used to begin a conversation. It can function as a greeting. The response of the compliment can be used to continue the communication. Even when the interlocutors just meet for the first time. Compliment on the neutral subject can engage to a continuation when it is responded. It can even continue to come to an agreement or appointment. Further, Manes and Wolfson explain that compliments can be used as thanking. When a person says ‘You are such a good man.’, he sometimes says it as an expression of thanking. Compliment is mostly used for new things, such as new car, dress or even a hairdo. Introduction It can also be used to reestablish the friendship when two friends meet after long time of being separated. Complimenting, as other speech acts, is contextually dependent. It very much depends on the context. People sometimes find themselves different in expressing compliment. Some people, like Americans, use verbal compliment more often than other people, such as Japanese. On the contrary, Japanese sometimes feel more comfortable to express their feeling through action than verbal utterance including in complimenting others (Takahashi, 2009). People are using compliments more and more in their daily life. Compliments can be produced, in some extent, anywhere, anytime, and by anyone. It can in school or universities (Danziger, 2018), or in the workplace (Holmes and Stubbe, 2015), or even in the social media (Eslami et al., 2019). In some circumstances, compliment shows the respect to the addressee (Alfonzetti, 2013: 280). As it can be produced by anyone, therefore, compliment is considered as an interesting topic to conduct research. There are some researches on compliments conducted on different languages, such as Italian (Alfonzetti, 2013), Jordanian Arabic (Al-Batayneh, 2019), Indonesian (Indah and Rifana, 2018), Mexican Spanish and Irish English (Flores-Salgado and Witten, 2018), Iranian (Eslami et al., 2017, 2019), Javanese (Kumuda and Wrihatni, 2018), Israeli Hebrew (Danziger, 2018), American English (Placencia and Lower, 2018), and Chinese (Chen and Yang, 2010); or on non-native English learners (Valkova, 2013; Duan, 2011) to mention. Again, it is because compliment is a part of communication which people always do in their daily life, when they are getting along with other people. Compliments, then, are becoming routinized. Since it is routinized, it becomes formulaic especially in English. There are hundreds of compliment formula used by people as investigated by Manes and Wolfson. Eventhough Manes and Wolfson’s research is arguable since it is based on the field note, the fact that there are a lot of compliment formula used by people is still considerable as confirmed by Jucker (2009). They found that the majority of compliments 108 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 contain one of a highly restricted, set of adjectives and verbs. Adjectives nice and good are the most common. Others use adverbs and verbs like really and love (Manes and Wolfson cited by Aijmer, 2014). Introduction That is why it is common to see such compliment like: contain one of a highly restricted, set of adjectives and verbs. Adjectives nice and good are the most common. Others use adverbs and verbs like really and love (Manes and Wolfson cited by Aijmer, 2014). That is why it is common to see such compliment like: a. It is a nice book. b. Your car looks good. Wolfson and Manes further stated in their research that there are some semantic patterns for the compliments produced by people (Aijmer, 2013: 13-14). a. NP {is/looks} (really) ADJ Ex. Your belt looks nice This is really good. a. NP {is/looks} (really) ADJ Ex. Your belt looks nice This is really good. y g b. I really {like/love} NP Ex. I love your shoes. I really like those spectacles. c. PRO is {really} an ADJ NP Ex. That’s a nice piece of essay That was really a great performance. They also found some compliments which were rarely used like: a. You are driving well. b. Peter, you are so intelligent. Holmes (2015), on the other hand, also proposes some formulae of the compliment based on her research in New Zealand and America. There are some syntactic formulae that they use. That was really a great performance. a. You are driving well. b. Peter, you are so intelligent. Holmes (2015), on the other hand, also proposes some formulae of the compliment based on her research in New Zealand and America. There are some syntactic formulae that they use. syntactic formulae that they use. a. NP BE INT ADJ Ex. Your car is really great b. BE LOOKING Ex. You are stunning c. I (INT) LIKE NP Ex. I simply like that coat. d. PRO BE a (INT) ADJ NP Ex. That’s a very nice blouse d. PRO BE a (INT) ADJ NP Ex. That’s a very nice blouse e. PRO BE (INT) (a) ADJ NP e. PRO BE (INT) (a) AD Ex. That’s really great. Ex. That’s really great. Instrument The data are collected using a questionnaire based on the Discourse Completion Task (DCT). Discourse completion task (DCT) itself is a type of production questionnaire in which speech acts are elicited in the written form by some kind of situational description (Billmyer&Varghese, 2000 in Sweeney & Zhu, 2016). In this case, students are given a text complete with the context and they are asked to answer the answer based on the context provided. The questionnaire for this research is available in the appendix. As said, students were asked to complete the DCT based questionnaire. There are 2 situations in the DCT with 3 sub-situations in every situation. Table 1. Situation for DCT NO. SITUATION SUB-SITUATION 1 Appearance 1. Hairstyle 2. Dress 3. Figure 2 Ability and Performance 1. Test result 2. Getting the scholar 3. Cooking ability Table 1. Situation for DCT Table 1. Situation for DCT All the situations are about their friends in their institution. Therefore, the respondents can adapt their answer based on the reality that they face every day. They are not asked to pretend to be somebody else but themselves. When the DCT is related or close to the reality, it is hoped that the answers on the DCT is as natural as it can be. f. (INT) ADJ (NP) Ex. Really cool short. f. (INT) ADJ (NP) f. (INT) ADJ (NP) Ex. Really cool short. Ex. Really cool short. People over the world use compliment to begin a conversation. The response of the compliments is used as the means of engagement which sometimes can lead them to an appointment. It can also be used to establish a relationship when the interlocutors who meet for the first time talk about the neutral subject. When people talk about something new, like car, appearance, or even hairdo, they make a compliment. If it is produced for old thing, it can be inferred as an indication of jealousy (Holmes, 2015). Indonesians, as other people, also make a compliment for something new. They also produce a compliment for some achievement that people gain. In addition, they also make a compliment to begin a conversation. Compliment is a good way to create a good situation to begin a conversation because compliment 109 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 is believed as the best way to start a communication in which people can mitigate the face threatening. This research is aimed at finding the formula of English compliment produced by Indonesian learners (students) of English. It also tries to find the reasons why such pattern occurs in the compliment. Subjects of Research The subjects of the research are 37 students of University. They have been studying English since Elementary School. They are now studying English in the university. The length of their study in the university ranges from 1 year to 3 years. Since they are all studying in the English program, it is assumed that they are at least in the intermediate advanced level of English. Data Analysis The data are analyzed based on the formula of the compliments proposed by Manes and Wolfson (1980 in Coates 2013) and Holmes (1986 in Holmes 2015). They, then, are analyzed to find the reasons why such compliment or compliments occur, also to find the object and the interpretation of the compliment (Chen, 2010). Findings and Discussion Findings and Discussion 110 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 The results are managed based on each situation in the questionnaire. So, there are four situations with each sub-situations which are described in this session. a. Compliment on the hairdo Most, even all, of the compliments addressed to the hearer about the hairdo. Of course, since it is about the hairdo, all the compliments must be about it. However, in some sub-situations, Indonesians are giving compliments not only about the person but also on the thing while the compliment is supposed to be addressed to the hearer about his/her performance. Since it is about the thing, most of compliments use the word look. More than 50% of the respondents use that word. The next formula that is mostly used by the students is be + adj. The adjective mostly used by the speakers is beautiful which is used by 16 respondents then followed by nice. Not only using the above formula, students also use the wh + adj when expressing their compliments. Beside the general adjectives like beautiful and nice, they also use more specific adjectives like gorgeous and awesome. Indonesians, when asked to express their compliment on the new thing, sometimes express their feeling of being surprised. Therefore, the exclamation word is sometimes used when they give their compliment about the new thing. They are not used to express the compliment on a new thing without giving the exclamation. Exclamation is considered as an expression of intimacy or attention on someone’s appearance. The words wow and whoa are mostly used by the speakers who use the exclamation. Intensifiers, so and really, are also used to emphasize the compliment. The following are the examples of the compliment expressed by the students when they are asked to express their compliment on the hairdo of their friend’s. Example: 1. Whoa, you look so beautiful today. 2. Wow, how beautiful you are. 3. You are really awesome today. 4. Awesome. 4. Awesome. Some students not only give compliment but also give comments following their compliment. Even some of them give the comment before giving the compliment as seen in the example below. 1. Your hairstyle is so cool, and it makes you beautiful. 2. Wow, you are so beautiful today, you should keep this hairstyle. 3. New hairstyle, huh? It looks beautiful. y 4. Why do you change your hairstyle? But I like it. For Indonesians, especially Javanese, the society where almost all of the respondents come from, comments are considered as an expression of intimacy. The more comment they give, the more attention they pour to the hearer. Most of the speakers give short comments as stated above. However, there is one respondent who gives long comments which in reality nearly impossible to occur. Or, if it happens in the reality, the hearer will feel awkward to respond the compliment because the respondent, who is accidentally a male, gives a long and repeated compliment. It is strange since among Indonesians, women are 111 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 considered as the ones who always give more compliment than men. Most women are more talkative than men. This is the prejudice that occurs among Indonesians. Wow, how beautiful you are. You look so beautiful with your new hair style. I really love your hairstyle. It’s good. And it would be better if you always come to campus with this new hairstyle.” considered as the ones who always give more compliment than men. Most women are more talkative than men. This is the prejudice that occurs among Indonesians. p j g Wow, how beautiful you are. You look so beautiful with your new hair style. I really love your hairstyle. It’s good. And it would be better if you always come to campus with this new hairstyle.” The above compliment and the comment might happen when the speaker and hearer are having a conversation. Or, they stop for a while and chit-chat while stopping before continuing their activity. For some Indonesians, it is common to stop for a while and have a conversation especially when one of the interlocutors has something new. 4. Awesome. And, it is also common for Indonesians women, and sometimes men especially when they are close friends, to talk and give more comment on that new thing and also to suggest the hearer to g gg One of the respondents does not give compliment on the hairdo. When asked to give compliment, she writes as seen below. “Is this a special moment to celebrate?” Indonesians are familiar with such question while in some culture, the question can be considered as a privacy breaker. This such question is common to show the intimacy and it is accepted since among Indonesians this question shows that the speaker pays and gives attention on the new thing. b. Compliment on the dress b. Compliment on the dress Almost the same as the compliment on the hairdo, compliment on the dress mostly uses look as the verb which is mostly used by the speaker. More than 50% of the respondents use that verb. The formula be + adj is also used by few students, about 6 respondents. Formula Wh + adj N is also used by four respondents. The respondents give compliments not only on the thing (cloth/jacket) but also on the person wearing the cloth. Therefore, nice, which is a general adjective, and handsome, which is specifically used for specific term, are commonly used by the respondents. As stated, Indonesians are used to giving an exclamation when they give compliment to somebody or something. They use the exclamation word wow. Ten respondents are using the word wow. Below are examples of the compliment. Almost all respondents give compliment on the person. One respondent gives compliment on the thing. Only one respondent gives compliment on the thing, and followed by giving compliment on the person (thing, then person). Others are giving compliment on the person then followed by giving compliment on the thing. Below are examples of the compliment. 1. Wow, look at you now, you are very different from the old one. 2. I never thought you would be such a dashing while wearing a wedding dress. 3. Wow, nice suit dude. You have changed a lot. 4. Wow, how handsome you are with your dress. 5. You look cool. As stated, Indonesians like to give comments on the compliment. The comments are sometimes short, sometimes a little bit long. They put the comment 112 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 before or after the compliment. Even some of the respondents put their comment both before and after the compliment. before or after the compliment. Even some of the respondents put their comment both before and after the compliment. 1. You’re so perfect today. Do you enjoy your style? p y 2. Wow, this is the real side of you, huh? What a handsome man. 3. Hey, is it you? you look so different. I think it’s better for you to wear clothes tidily like this everyday. One of the respondents shows his compliment in the form of the expression of surprise as seen in the example below. “How come? I can’t believe my eyes.” Meanwhile, another respondent gives a unique compliment because it also shows how the speaker is feeling about the way the hearer dresses. The expression shows how close their relationship is and in some sense, it shows how much the speaker (a girl) shows her love to the hearer. “Why you make me feel happy to see you?” The above statement also expresses that the hearer rarely uses tidy cloth or rarely looks tidy. As it is stated, one respondent gives a compliment on the thing. However, his compliment seems awkward for English native since it is a long compliment. Nevertheless, for Indonesians, that such compliment is common for women and men who are close each other. They can even give compliment again and again on the same thing. “Nice. I absolutely like on what you are wearing especially your jacket. That jacket and tie are suit with you. It looks really nice as well as perfect on you.” Basically, when asked to give compliment on the dress, the respondents use either general or specific adjective. Some of the respondents use both general and specific adjectives on their compliments. However, one of the respondents gives a compliment in a strange way. 4. Wow, your body is good now. 4. Wow, your body is good now. As other Indonesians who like to give comment on compliment, the respondents also give comment on their compliment. Some of the comments are also the compliment when they are seen from the meaning. The comments which show their attention to the hearer are sometimes in the form of questions which do not need to answer. 1. Wow, your change is amazing. How often do you do sport in week? ften do you do exercise? You look so perfect 2. How often do you do exercise? You look so perfect. 2. How often do you do exercise? You look so perfect. 3. Wow, I don’t believe it’s you. You are so perfect, much better than before. 2. How often do you do exercise? You look so perfect. How often do you do exercise? You look so perfect. Wow, I don’t believe it’s you. You are so perfect, much better than before. 3. Wow, I don’t believe it’s you. You are so perfect, much better than before. 4. Glad to see you like that. One of the respondents gives a compliment and a comment at the same time. Her compliment is longer than the others and it shows how close her relationship to her friend. She is proud of him. Based on her compliment and comment, it seems that she does not expect him to change so much. She might have challenged him before and she is surprised to see how different he is now. “I’m really proud of you. You become different in the best way. Your body is proportional now and it is good for you.” However, if they do not have close relationship, this such compliment and comment might not be appropriate to utter. Another respondent also produces the similar comment. His comment seems to show that he challenged his friend in the past before they meet. “See, you look fantastic with your body now. It’s totally different since I haven’t seen you 5 years. For the first time I see you again, I almost don’t recognize that it is you. You look very handsome with your well-built body that you have now” In reality, those two comments rarely happen. Even if we have a close relationship, it is not appropriate to say how proud we are. It is not such a competition or an achievement. 4. Wow, your body is good now. We do not know what happens to him. When we meet after long time, it is not good to say ‘see’ as if we challenged him to lose his weight. When the word see is not used, the expression will be appropriate. c. Compliment on the figure When giving compliment on the body, most respondents (19 persons) use the word look, and 11 persons use be + adj. There is no dominant adjective which is used by the respondents. Most of them use general adjectives like good (by 5 persons), fantastic and perfect (by 4 persons each). Even the adjective beautiful which seems inappropriate since the hearer is a boy is also used by 4 persons. Only two persons use the specific adjective handsome. To start a compliment, the respondents use an exclamation word to show that they are surprised and also to show their attention to the hearer. Again, the word wow is used by the respondents to show their surprise. Instead of using exclamation word, the respondents also use intensifier to emphasize their 113 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 compliment. They use so, really and very to intensify their compliment. Examples of the compliment are exposed below. 1. Wow, you look so good now. 2. You look so different. You look fantastic. 3. You look more beautiful.* 3. Congratulations for your hard work. 3. Congratulations for your hard work. Since almost all respondents congratulate, the phrase good job and well done are used by the respondents. They appreciate the hard work that the hearer has done. 1. Good job. 2. Good job. You did it. 3. Well done, my friend. They give a compliment not only on the person as stated above but also on the thing or the result. The statements are common in Bahasa Indonesia, the language that all respondents use in their daily life. However, in some cases, the statements might express an underestimating expression that the hearer can do by himself. 1. Wow, that’s a great score. 2. That’s amazing result. 3. That’s gift from your effort. And from the compliments produced by the respondents, it seems that they do not use the semantic formula that most people do. They use various expressions which in some cases they seem not related to the compliment. 1. How can you do it? 2. Can you teach me how to do it? Can we study together? 2. Can you teach me how to do it? Can we study together? a. Compliment on the test result a. Compliment on the test result There is not any dominant formula produced by students in this case. Only PRO V in which the verbs are not stick to a particular verb. Most of the verb used is do which is variously used to show a congratulating action. The adjective that is mostly used is great and of course exclamation word is also used to show their surprise toward the achievement. They did not expect him to pass the test since he said that he did not have any hope to pass the test. Therefore, when he passes the 114 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 exam, they are surprised and they express their surprise together with their compliment. And of course, most of the respondents use congratulations for this case. 1. Wow, you did it well. 1. Wow, you did it well. 2. Wow, you get good score. Congratulations. 3. It’s miracle. One of the respondents even writes the expression which is widely used in the television. However, this expression does seem appropriate to be expressed to a friend. He writes ‘That’s my boy’. In many movies, this expression is used as a compliment but it is not used as a compliment to a friend who has the same status. This expression is patronizing, which is used by a father or a person who has higher status than the hearer. Some of the respondents even give comments which support the hearer to keep the achievement. They also say that they are proud of the achievement that the hearer gains. These expressions of support show that the speaker has a close relationship with the hearer. The expressions also show how big the attention that the speaker gives to the hearer. 1. Well, congratulations because you’ve got a satisfactory result. You’ve worked hard and kept trying. You also never give up, and here’s the result you got. Very impressive. 2. Excellent. I believe that you can pass on the final exam test. Your test result is good even you also got a satisfying score. I appreciate on you hard work. Good job and you are excellent. Keep your score. 2. Excellent. I believe that you can pass on the final exam test. Your test result is good even you also got a satisfying score. I appreciate on you hard work. 3. Congratulations. You do a good job. I am proud of you. As seen above, there is no dominant formula. Even some respondents use the Indonesian expressions but they express them in English. They still deliver Indonesian pragmatic expressions within the English language. 115 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 b. Compliment on the scholar b. Compliment on the scholar Almost similar to the previous situation, most of the respondents congratulate the hearer. The congratulating expression is followed by comment as most Indonesians do. 14 respondents use the word congratulation and followed by some comments. Surprisingly, not many respondents use exclamation. Only 1 respondent use the exclamation wow. For some respondents, being chosen as a scholar is a luck. Therefore, instead of congratulating, the respondents give a compliment on his luck. p 1. Congratulations. How lucky you are. 2. You’re so lucky, keep spirit to reach your dream. 3. What a lucky boy. 4. So lucky are you! However, being chosen a scholar is not only a luck. It needs a hard work. The respondents realize that getting a scholar is not easy. They have to study and work hard, even much harder than before. So, when they get the scholar, all the hard work is paid off. The scholar is like an award for given to them. It is an award for their hard work. 1. You have the right to get it. 2. That’s a good achievement. I know your effort, and this is the result. Good job. 2. That’s a good achievement. I know your effort, and this is the result. Good job. 3. Congratulations for your hard work. 4. Hard work always pays off. Getting a scholar is sometimes considered as an achievement. Those who get a scholar must be smart people. They must have worked hard. Therefore, they must be smart since they study hard to get the scholar. 1. You are so smart. The school is not wrong giving you scholarship. Congratulations. 1. You are so smart. The school is not wrong giving you scholarship. Congratulations. 2. How smart you are. I’m really proud of you. y y p y Getting a scholar can make everyone happy. Not only the getter who is happy, but also their friends. They also feel happy and proud when one of their friends gets it. The expression of being proud ‘proud of you’ is expressed again and again by ten respondents. Getting a scholar can make everyone happy. Not only the getter who is happy, but also their friends. They also feel happy and proud when one of their friends gets it. The expression of being proud ‘proud of you’ is expressed again and again by ten respondents. 1. Glad to hear that. 2. I’m proud of you. 3. As long as you work hard, nothing is impossible. c. Compliment on the cooking ability Actually, this situation is about to compliment the ability of a person in cooking. However, since it is also related to food, many respondents give a compliment on the food. Therefore, the construction be + adj is dominant in this situation, produced by 20 respondents. Consequently, the specific adjective which is related to food, delicious, is also used by 19 respondents. In this situation, the respondents also use exclamation word, either using wow or adjectives used as exclamation, like great, good, and terrific. Some respondents also use wh construction to show their surprise. Some examples can be seen below. 1. Wow, it’s delicious. 2. What a delicious food. 3. Love your cook so much. What a food. 4. Wow, your food is so delicious, yummy. Gi d li i f d d h k h h B id h l . Wow, your food is so delicious, yummy. Given delicious food, some respondents thank the hearer. Besides, they also give compliment on the food that they receive. Given delicious food, some respondents thank the hearer. Besides, they also give compliment on the food that they receive. Given delicious food, some respondents thank th give compliment on the food that they receive. Given delicious food, some respondents thank the hearer. Besides, they also give compliment on the food that they receive. 1. That was delicious food. Thanks for the delicious food. 2. It’s really delicious food. Thank you. 3. It’s good food. Thank you. Besides giving compliment merely on the food, some respondents give compliment on the ability of the person without giving compliment on the food. They do not know that their friend can cook. So, they are surprised when she gives some food to them, and they give the compliment to their friend without giving a compliment to her food. And dealing with the person, adjective talented is used by 10 respondents. 1. You’re talented. 1. You’re talented. 2. I’m surprised you can cook so good. You are so talented. 3. You’re a very talented cook. 4. You’re a great chef dude. The other respondents give compliment on the food and on the person. Some of the compliments on the person seem like a comment to show surprise. However, some show that they do not give comment, but they truly comment on the person. 1. It is very delicious food that I have tasted. b. Compliment on the scholar 3. Congratulations. I’m proud of you. 4. Amazing, man. Finally, you get the scholarship. I am proud of you. 4. Amazing, man. Finally, you get the scholarship. I am proud of you. However, they know that it is not the end of their study. Getting a scholar is just the beginning of their future study. They must study hard to keep the scholarship. Otherwise, the school will withdraw the scholarship. However, they know that it is not the end of their study. Getting a scholar is just the beginning of their future study. They must study hard to keep the scholarship. Otherwise, the school will withdraw the scholarship. 1. Congratulations for your scholarship. You must keep it and keep your study. 2. I am proud of you. congratulations, your effort is not useless. Good luck for your next education. 2. I am proud of you. congratulations, your effort is not useless. Good luck for your next education. Some of the respondents (7 person) do not give a compliment. They say something which is not directly connected to the compliment. They just give comment on the achievement. Some of the respondents (7 person) do not give a compliment. They say something which is not directly connected to the compliment. They just give comment on the achievement. 1. I never expected. 2. Well deserved. 3. As long as you work hard, nothing is impossible. 116 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 As stated before, Indonesians always give comment on something, even on the achievement like the scholarship. For them, giving comment and advice means that they get involved on somebody’s life. It, further, means that they are still having a relationship, close relation. He combines all the compliment stated above. You are very lucky. You can get a scholarship to continue your study. It is not easy. I am proud of you. congratulations. I say to you for on what you have got. I admire you as well.” c. Compliment on the cooking ability I think you are the good chef in your home. 2. I didn’t know you have a good hand to cook. It’s nice. 3. It tastes good. I think you can be a chef. Even one of the respondents shows his exaggerated feeling. He not only gives comment but also gives compliment and expresses his happiness. 117 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 Terrific! Evidently, you are great in cooking. I can’t believe this first but it’s real. You have great cooking ability. I am so happy to know it. Conclusion Compliments among Indonesian students of English are pragmatically based on Bahasa Indonesia, their language. It can be understood since they are all Indonesians and none has ever been in an English speaking country. However, some of the respondents imitate English natives in giving compliment with wrong target like in ‘That’s my boy’ which seems patronizing while they are complimenting their friends not their sub status. This phenomenon, complimenting using English with their native pragmatic context, confirms the theory that native language influences persons in complimenting and that complimenting differ from society to society even though they use the similar language (Chen, 2010). Therefore, there are some compliments that are not using the formula of compliments uttered by English natives. References Aijmer, K. (2014). Conversational routines in English: Convention and Creativity. New York: Routledge. Al-Batayneh, H. H. (2019). Implicit compliments in Jordanian Arabic. (A Thesis). doi: 10.13140/RG.2.2.31306.41927 Archer, D., Karin, A., & Anne, W. (2012). Pragmatics. An advanced resource book for students. New York: Routledge. Chen, R., & Yang, D. (2010). Responding to compliments in Chinese: Has it changed? Journal of Pragmatics, 42, 1951-1963. doi:10.1016/j.pragma.2009.12.006. Chen, R. (2010). Compliment and compliment response research. In Trasborg, A (ed.). Pragmatics across Languages and Cultures. Berlin: De Gruyter Mouton. Coates, J. (2013). Women, men, and languages. New York: Routledge. Danziger, R. (2018). Compliments and compliment responses in Israeli Hebrew: Hebrew university in Jerusalem students in interaction. Journal of Pragmatics, 124, 73-87. doi: 10.1016/j.pragma.2017.12.004. Duan, Y. (2011). A pragmatic research report on compliment speech act. Theory and Practice in Language Studies, 1(4), 356-360. doi:10.4304/tpls.1.4.356- 360. Eslami, Z. R., Jabbari, N., & Kuo, L. J. (2015). Compliment response behaviour on facebook: A study with Iranian facebook users. International Review of Pragmatics, 7, 244-277. doi: 10.1163/18773109-00702005. Eslami, Z. R., Jabbari, N., & Kuo, L. J. (2019). Online compliments of Iranian Facebook users. Retrieved on August 27, 2019 from https://doi.org/10.1017/9781108182119.004 Flores-Salgado, E., & Witten, M.T. (2019). A comparative study of Mexican and Irish compliment responses. Colombian. Applied Linguistic Journal, 21(1), 125-138. https://doi.org/10.14483/22487085.14670 p g Holmes, J. (2015). Women, men, and politeness. London: Longman 118 IJHS, e-ISSN 2597-4718, p-ISSN 2597-470X, Vol. 3, No. 1, September 2019, pp. 107-119 Holmes, J., & Stubbe, M. (2015). Power and politeness in the workplace. New York: Routledge. Holmes, J., & Stubbe, M. (2015). Power and politeness in the workplace. New York: Routledge. Indah, R. N., & Rifana, N. F. (2018). The patterns of compliments in instagram photo comments. International Journal of Research Studies in Language Learning, 7(3), 57-69. doi: 10.5861/ijrsll.2017.1830 Jucker, A. (2009). Speech act research between armchair, field and laboratory. The case of compliments. Journal of Pragmatics, 41, 1621-1625. Kumuda, P. D. N., & Wrihatni, N. S. (2018). Humor in Ludruk: Between insults and compliments. IOP Conf. Series: Earth and Environmental Science, 175. DOI :10.1088/1755-1315/175/1/012118. Ogiermann, E. (2018). Discourse completion tasks. In Jucker, A. H., Schneider, K. P. & Bublitz, W. (eds.). Methods in pragmatics, 229-255. Berlin/ Pillet-Shore, D. (2015). Compliments. In Tracy, K., Ilie, C., & Sandel, T. The International Encyclopedia of Language and Social Interaction. DOI: 10.1002/9781118611463/wbielsi127 Placencia, M. E., Lower, A., & Powell, H. (2016). References Complimenting behaviour on facebook responding to compliments in American English. Pragmatics and Society, 7(3), 339-365. DOI 10.1075/ps.7.3.01pla y p p Sweeney, E. & Zhu, H. (2016). Discourse completion tasks. In Hua Zhu (ed.), Research methods in intercultural coommunication: A practical guide. west sussex: John Wiley & Sons. Inc. Trasborg, A. (2010). Pragmatics across languages and cultures. Berlin: De Gruyter Mouton. Válková, S. (2013). Speech acts or speech act sets: apologies and compliments. Linguistica Pragensia, 2. Retrieved on August 26, 2019 from http://cejsh.icm.edu.pl/cejsh/element/bwmeta1.element.desklight-695bced8- 4b87-4e55-b106-7baf96a6add6/c/Silvie_Valkova_44-57.pdf 119
https://openalex.org/W3132525299	https://www.mdpi.com/1422-0067/22/4/2152/pdf?version=1613979101	English	null	Editorial for Special Issue “Genetic Basis and Epidemiology of Myopathies”	International journal of molecular sciences	2,021	cc-by	1,585	Editorial Editorial for Special Issue “Genetic Basis and Epidemiology of Myopathies” Eleni Peristeri * and Efthimios Dardiotis Department of Neurology, Faculty of Medicine, University of Thessaly, 41100 Larissa, Greece; edar@med.uth.gr * Correspondence: eperiste@uth.gr; Tel.: +30-241-350-2005 Abstract: We are pleased to announce a Special Issue on the Genetic Basis and Epidemiology of Myopathies. This Special Issue is collecting papers pertaining to various lines of research focusing on the genetic basis and the epidemiology of myopathies. The Guest Editors’ note combines the contributing authors’ reviews and ﬁndings of relevant research, and we hope that future studies on myopathies will attempt to conﬁrm these ﬁndings and, additionally, evaluate supplementary phenotypic and histological expressions of myopathies, as well as genetic factors in their pathogenesis. Keywords: myopathies; genetic basis; epidemiology 1. Introduction Congenital myopathies are a group of genetic muscle disorders clinically characterized by wide genetic and clinical heterogeneity. Identifying the candidate genes responsible for the various histological and phenotypic expressions of myopathies is of extreme importance for both highlighting the genetic basis of many forms of myopathies and improving treatment efﬁcacy. While the genetic basis of myopathies has been extensively investigated, the genes involved in defects, especially cognitive ones, are still unknown. Citation: Peristeri, E.; Dardiotis, E. Editorial for Special Issue “Genetic Basis and Epidemiology of Myopathies”. Int. J. Mol. Sci. 2021, 22, 2152. https://doi.org/10.3390/ ijms22042152 Academic Editor: Gail B. Mahady Received: 17 February 2021 Accepted: 19 February 2021 Published: 22 February 2021 International Journal of Molecular Sciences 2. Papers The fourth paper, titled “Preclinical Research in Glycogen Storage Diseases: A Comprehensive Review of Current Animal Models”, by [4] attempts a comprehensive review of animal models of glycogen storage diseases, including genetically modiﬁed mouse models, naturally occurring models and a genetically modiﬁed zebraﬁsh model. The review highlights both differences and commonalities across the animal models, which contributes to better understanding the models’ advantages and deﬁciencies. The ﬁfth paper, titled “Current Genetic Survey and Potential Gene-Targeting Therapeutics for Neuromuscular Diseases”, by [5] reviews studies that have focused on next-generation sequencing and genetic tools within the context of gene therapies for neuromuscular diseases. The review highlights both the interaction between mutations and the severity of the clinical phenotype, and the phenotype–genotype associations in neuromuscular diseases. The sixth paper, titled “Early-Onset Infantile Fa- cioscapulohumeral Muscular Dystrophy: A Timely Review”, by [6] presents recent ﬁndings on the pathomechanisms underlying early-onset infantile facioscapulohumeral muscular dystrophy. The paper reviews patterns of muscle, respiratory and systemic involvement in infantile facioscapulohumeral muscular dystrophy and biomarkers of the disease, as well as therapeutic approaches and standard care for individuals with infantile facioscapulo- humeral muscular dystrophy. The seventh paper, titled “Anti-Inﬂammatory and General Glucocorticoid Physiology in Skeletal Muscles Affected by Duchenne Muscular Dystrophy: Exploration of Steroid-Sparing Agents”, by [7] provides an overall presentation of the drugs that have been shown to stabilize the activation of proinﬂammatory and metabolic cellular pathways in skeletal muscle cells in Duchenne muscular dystrophy, as well as possible com- binations of drugs with glucocorticoid therapy. The eighth paper, titled “Cognitive Deﬁcits in Myopathies”, by [8] classiﬁes myopathies according to their clinical characteristics and reviews concomitant cognitive deﬁcits. The cognitive deﬁcits in myopathies range from executive function difﬁculties to language and intelligence deﬁciencies across a wide range of age groups. The ninth paper, titled “Update on Congenital Myopathies in Adulthood”, by [9] classiﬁes congenital myopathies following genetic-based approaches, with a special emphasis placed on late-onset congenital myopathies that ﬁrst manifest in adulthood. Fi- nally, the tenth paper, titled “New and Developing Therapies in Spinal Muscular Atrophy: From Genotype to Phenotype to Treatment and Where Do We Stand?”, by [10] presents an overview of the genotypes, phenotypes and recent therapeutic approaches for spinal muscular atrophy. The paper also highlights knowledge gaps in relevant research, as well as novel perspectives of recent therapeutics that are based on the identiﬁcation of the pathomechanisms underlying spinal muscular atrophy. 2. Papers This Special Issue of the International Journal of Molecular Sciences has attracted both research papers and reviews that contribute to our understanding of the genetic factors in the pathogenesis of myopathies and their epidemiology, their treatment, and their im- pact on the patients’ cognitive abilities. The ﬁrst paper, titled “Description of a Novel Mechanism Possibly Explaining the Antiproliferative Properties of Glucocorticoids in Duchenne Muscular Dystrophy Fibroblasts Based on Glucocorticoid Receptor GR and NFAT5”, by [1], examines the antiproliferative properties of glucocorticoids in ﬁbroblasts in Duchenne muscular dystrophy. Their results show the potential of glucocorticoids to slow down the process of ﬁbrosis in Duchenne muscular dystrophy via modulating T-cells 5 (NFAT5) localization in the cell. localization in the cell. The second paper, titled “Abnormal NFAT5 Physiology in Duchenne Muscular Dystrophy Fibroblasts as a Putative Explanation for the Permanent Fibrosis Formation in Duchenne Muscular Dystrophy”, by [2] investi- gates the localization and expression of NFAT5 in skeletal ﬁbroblasts in one patient with Duchenne muscular dystrophy and one healthy individual, after exposure to hyperosmolar or proinﬂammatory stress. Their ﬁndings show the unresponsiveness of NFAT5 to both hyperosmolar and proinﬂammatory stress in Duchenne muscular dystrophy, which may further explain the unchanged cell growth in ﬁbroblasts. The third research paper, titled “Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin”, by [3] explores the pathogenesis of skeletal muscle disease caused by the Gln147Pro substitution in β-tropomyosin through the technique of polarized microﬂuorimetry. According to their ﬁndings, a congenital myopathy-causing Q147P substitution in Tpm2.2 disrupts the myosin-induced displacement of tropomyosin over actin, which leads to the premature activation of actin monomers and increases the myosin Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2152. https://doi.org/10.3390/ijms22042152 Int. J. Mol. Sci. 2021, 22, 2152 2 of 3 cross-bridges in a state of strong binding with actin at low Ca2+. 2. Papers Author Contributions: Conceptualization, E.P. and E.D.; software, E.P. and E.D.; resources, E.P. and E.D.; writing—original draft preparation, E.P. and E.D.; writing—review and editing, E.P. and E.D.; supervision, E.D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We would like to thank all the authors who have contributed to this special issue. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 1. Herbelet, S.; De Paepe, B.; De Bleecker, J.L. Description of a Novel Mechanism Possibly Explaining the Antiproliferative Properties of Glucocorticoids in Duchenne Muscular Dystrophy Fibroblasts Based on Glucocorticoid Receptor GR and NFAT5. Int. J. Mol. Sci. 2020, 21, 9225. [CrossRef] [PubMed] References 1. Herbelet, S.; De Paepe, B.; De Bleecker, J.L. Description of a Novel Mechanism Possibly Explaining the Antiproliferative Properties of Glucocorticoids in Duchenne Muscular Dystrophy Fibroblasts Based on Glucocorticoid Receptor GR and NFAT5. Int. J. Mol. Sci. 2020, 21, 9225. [CrossRef] [PubMed] Int. J. Mol. Sci. 2021, 22, 2152 3 of 3 2. Herbelet, S.; De Paepe, B.; De Bleecker, J.L. Abnormal NFAT5 Physiology in Duchenne Muscular Dystrophy Fibroblasts as a Putative Explanation for the Permanent Fibrosis Formation in Duchenne Muscular Dystrophy. Int. J. Mol. Sci. 2020, 21, 7888. [CrossRef] [PubMed] [ ] [ ] 3. Karpicheva, O.E.; Simonyan, A.O.; Rysev, N.A.; Redwood, C.S.; Borovikov, Y.S. Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin. Int. J. Mol. Sci. 2020, 21, 7590. [CrossRef] [PubMed] 3. Karpicheva, O.E.; Simonyan, A.O.; Rysev, N.A.; Redwood, C.S.; Borovikov, Y.S. Looking for Targets to Restore the Contractile Function in Congenital Myopathy Caused by Gln147Pro Tropomyosin Int J Mol Sci 2020 21 7590 [CrossRef] [PubMed] 4. Almodóvar-Payá, A.; Villarreal-Salazar, M.; De Luna, N.; Nogales-Gadea, G.; Real-Martínez, A.; Andreu, A.L.; Martín, M.A.; Arenas, J.; Lucia, A.; Vissing, J.; et al. Preclinical Research in Glycogen Storage Diseases: A Comprehensive Review of Current Animal Models. Int. J. Mol. Sci. 2020, 21, 9621. [CrossRef] [PubMed] 5. Chiu, W.; Hsun, Y.-H.; Chang, K.-J.; Yarmishyn, A.A.; Hsiao, Y.-J.; Chien, Y.; Chien, C.S.; Ma, C.; Yang, Y.-P.; Tsai, P.-H.; et al. Current Genetic Survey and Potential Gene-Targeting Therapeutics for Neuromuscular Diseases. Int. J. Mol. Sci. 2020, 21, 9589. [CrossRef] [PubMed] [ ] [ ] Chen, T.-H.; Wu, Y.-Z.; Tseng, Y.-H. Early-Onset Infantile Facioscapulohumeral Muscular Dystrophy: A Mol. Sci. 2020, 21, 7783. [CrossRef] [PubMed] Z.; Tseng, Y.-H. Early-Onset Infantile Facioscapulohumeral Muscular Dystrophy: A Timely Review. Int. J 783. [CrossRef] [PubMed] 7. Herbelet, S.; Rodenbach, A.; De Paepe, B.; De Bleecker, J.L. Anti-Inﬂammatory and General Glucocorticoid Physiology in Skeletal Muscles Affected by Duchenne Muscular Dystrophy: Exploration of Steroid-Sparing Agents. Int. J. Mol. Sci. 2020, 21, 4596. [CrossRef] [PubMed] 8. Peristeri, E.; Aloizou, A.-M.; Keramida, P.; Tsouris, Z.; Siokas, V.; Mentis, A.-F.A.; Dardiotis, E. Cognitiv Int. J. Mol. Sci. 2020, 21, 3795. [CrossRef] [PubMed] u, A.-M.; Keramida, P.; Tsouris, Z.; Siokas, V.; Mentis, A.-F.A.; Dardiotis, E. Cognitive Deﬁcits in Myopathies 21, 3795. [CrossRef] [PubMed] 9. Papadimas, G.K.; Xirou, S.; Kararizou, E.; Papadopoulos, C. Update on Congenital Myopathies in Adulthood. Int. J. Mol. Sci. 2020, 21, 3694. [CrossRef] [PubMed] 10. References Chen, T.-H. New and Developing Therapies in Spinal Muscular Atrophy: From Genotype to Phenotype to Treatment and Where Do We Stand? Int. J. Mol. Sci. 2020, 21, 3297. [CrossRef] [PubMed]
https://openalex.org/W2091144635	https://www.scielo.br/j/eagri/a/bdkSh5vghwPttTZywDWWKkj/?lang=en&format=pdf	English	null	Vocalization data mining for estimating swine stress conditions	Engenharia agrícola	2,014	cc-by	2,847	1 Zootecnista, M.Sc. em Zootecnia, Faculdade de Ciências Agrárias-UFGD, Rod Dourados - Itahum, km 12, Dourados - MS, Brasil, Fone: (+55 67) 3410-2500, mar_moi@hotmail.com 2 Enga. Civil, Pesquisadora Visitante Sênior, Faculdade de Ciências Agrárias-UFGD, Dourados - MS, Brasil, irenilza@gmail.com 3 Zootecnista, Profa. Dra., Faculdade de Ciências Agrárias-UFGD, Dourados - MS, Brasil, fabianacaldara@ufgd.edu.br 4 Zootecnista, Profa. Dra., Faculdade de Ciências Agrárias-UFGD, Dourados - MS, Brasil, ibiarapaz@gmail.com 5 Zootecnista, Prof. Doutor, Faculdade de Ciências Agrárias-UFGD, Dourados - MS, Brasil, rodrigogarcia@ufgd.edu.br 6 Zootecnista, Dra. em Enga Agrícola, Faculdade de Enga Agrícola-UNICAMP, Campinas - SP, Brasil, Fone:(+55 19) 3251-1039 Recebido pelo Conselho Editorial em: 29-7-2013 Aprovado pelo Conselho Editorial em: 20-12-2013 VOCALIZATION DATA MINING FOR ESTIMATING SWINE STRESS CONDITIONS MARTA MOI1, IRENILZA DE A. NÄÄS2, FABIANA R. CALDARA3, IBIARA C. DE L. ALMEIDA PAZ4, RODRIGO G. GARCIA5, ALEXANDRA F. S. CORDEIRO6 ABSTRACT: This study aimed to identify differences in swine vocalization pattern according to animal gender and different stress conditions. A total of 150 barrow males and 150 females (Dalland® genetic strain), aged 100 days, were used in the experiment. Pigs were exposed to different stressful situations: thirst (no access to water), hunger (no access to food), and thermal stress (THI exceeding 74). For the control treatment, animals were kept under a comfort situation (animals with full access to food and water, with environmental THI lower than 70). Acoustic signals were recorded every 30 minutes, totaling six samples for each stress situation. Afterwards, the audios were analyzed by Praat® 5.1.19 software, generating a sound spectrum. For determination of stress conditions, data were processed by WEKA® 3.5 software, using the decision tree algorithm C4.5, known as J48 in the software environment, considering cross-validation with samples of 10% (10-fold cross-validation). According to the Decision Tree, the acoustic most important attribute for the classification of stress conditions was sound Intensity (root node). It was not possible to identify, using the tested attributes, the animal gender by vocal register. A decision tree was generated for recognition of situations of swine hunger, thirst, and heat stress from records of sound intensity, Pitch frequency, and Formant 1. KEYWORDS: sound intensity, sound attributes, animal welfare. MINERAÇÃO DE DADOS DE VOCALIZAÇÃO PARA ESTIMATIVA DE CONDIÇÕES DE ESTRESSE DE SUÍNOS RESUMO: Este trabalho teve o objetivo de identificar diferenças no padrão de vocalização em função do sexo dos animais e diferentes situações de estresse. Foram utilizados 150 animais machos castrados e 150 fêmeas (linhagem Dalland®), com 100 dias de idade. Os suínos foram submetidos a diferentes situações de estresse: sede (animais sem acesso à água), fome (suínos sem acesso ao alimento), estresse térmico (ITU superior a 74) e BEA (animais com alimento e água, com ITU abaixo de 70). Foram registrados os sinais acústicos a cada 30 minutos, totalizando seis coletas para cada situação de estresse. Posteriormente, os áudios foram analisados pelo software Praat® 5.1.19, gerando um espectro sonoro. Para a determinação das condições de estresse, os dados foram processados no programa computacional WEKA® 3.5, utilizando o algoritmo de árvore de decisão C4.5, conhecido como J48 no ambiente do programa computacional WEKA®, considerando validação cruzada com amostras de 10% (10-fold cross-validation). De acordo com a Árvore de Decisão, o atributo acústico mais importante para a classificação das condições de estresse foi a Intensidade do som (nó raiz). Não foi possível identificar o sexo dos animais pelo registro vocal, utilizando os atributos testados. Foi gerada uma árvore de decisão para reconhecimento de situação de fome, sede e estresse térmico em suínos, a partir de registros da intensidade do som, da frequência de Pitch e da Formante 1. PALAVRAS-CHAVE: intensidade do som, atributos sonoros, bem-estar animal Eng. Agríc., Jaboticabal, v.34, n.3, p.445-450, maio/jun. 2014 Marta Moi, Irenilza de A. Nääs, Fabiana R.Caldara et al. 446 INTRODUCTION Pork is the most consumed meat in the world, and the production systems have been searching for an automated way to estimate animal welfare in the livestock, since this is an important demand from the overseas market. Among ways to audit animal welfare, there is monitoring of their behavior and social interactions (BAPTISTA et al., 2011; SPINKA, 2012), observation of health and rearing conditions (BAPTISTA et al., 2011; CALDARA et al., 2012), and monitoring of their vocalization expressed during production phases (MARX et al., 2003; SAMPAIO et al., 2007; NÄÄS et al., 2008). Vocalization is an objective and noninvasive tool that may evidence lack of herd welfare based on the release of distinct vocal patterns (NÄÄS et al., 2008). MARX et al. (2003) characterized different types of swine sounds and identified diverse parameters of emitted energy, frequency, and duration. Animal vocalization is, thus, an expression of its current condition, and may occur spontaneously or, yet, be the result of an event, such as hunger and pain; for this reason, it became an important tool to assess animal welfare (DÜPJAN et al., 2008). There is evidence that each animal has individual vocalization characteristics. Animals use vocalization as a means of communication among individuals of the same species (GRANDIN, 1998). Female pigs express a composite frequency of grunts as identified by SCHRADER & TODT (1998). For sound data analysis, it is important to use intelligent techniques of data processing, which can help to identify stressful situations. These techniques are able to transform data into relevant information and knowledge, providing support for decision making. The techniques of data mining have shown promising results for knowledge increase on animal production (NÄÄS et al., 2008; CORDEIRO et al., 2012). This study aimed to identify differences in swine vocalization pattern according to gender and exposure to diverse stress conditions. MATERIAL AND METHODS The experiment was conducted at a commercial swine farm in April 2012, located in the northwest of Rio Grande do Sul State, Brazil (27°55'23'' S, 53°02'21'' W, mean altitude of 385 m). According to Köeppen classification, the climate in the region is humid subtropical. We used 150 surgically castrated males and 150 females (Dalland® genetic strain), aged 100 days (growth phase), distributed in 10 pens. Pigs were housed in sheds of east-west solar orientation, ceiling height of 3.0 m, side walls of 1 m height with curtains at side openings, and fiber cement tiles (0.006 m). Sheds had 10 pens of 30 m² each, with 30 animals allocated in each pen. The floor was built in concrete with a running water channel of 0.05 m at the end of each pen. Nipple drinkers were also placed at the end (three per pen). The automatic feeder had the capacity to feed six pigs. At the shed outside, there was a bush line with the aim to provide shade on the shed upper part (roof). Environmental data (temperature and relative humidity) were recorded every 30 min using a digital thermo-hygrometer. Values of wet bulb temperature (WBT, °C) were calculated using mean values of dry bulb temperature (DBT, °C) and relative humidity (RH, %), using Psicrom® software (RORIZ, 2003). The Temperature-Humidity Index (THI) was calculated using eq.(1), as described by ROLLER & GOLDMAN (1969): THI = 0.45 WBT + 1.35 DBT + 32 (1) Animals were exposed to different stressful managements, such as thirst (no access to water), hunger (no access to fodder), and thermal stress (TS, THI≥74); welfare conditions (WC, animals had continuous access to fodder and water, and were reared under THI<73). Stressful situations by hunger and thirst were determined after a period of 11 hours of fasting, for both cases; data recording began after this period. Eng. Agríc., Jaboticabal, v.34, n.3, p.445-450, maio/jun. 2014 Vocalization data mining for estimating swine stress conditions 447 Acoustic signals were recorded every 30 min, totaling six samples for each stress exposure. Acoustic recording was performed with a unidirectional microphone Marantz® PMD 660. The microphone was positioned at 1 m above the pigs, at the pen central area, and connected to a recorder. Recorded signals were digitalized using a frequency of up to 44,100 Hz, during 3 min. After finishing the swine vocalization recording, sound signals were downloaded to a computer. Each pen provided the record of a sequential range of "screams and grunts" (vocalizations). Subsequently, the audios were analyzed using Praat® 5.1.19 software, and the acoustic parameters were extracted using Fourier transform, generating a sound spectrum. The attributes generated by the software revealed the signal energy (Pa²s), maximum and minimum amplitudes (Pa), pitch frequency (Hz), sound intensity (dB), and four levels of formants (F1, F2, F3, and F4) that are also called harmonic (Hz). To determine stress conditions, data were processed using WEKA® (3.5) software, with J48 algorithm, and considering cross-validation with samples of 10% (10-fold cross-validation). The attributes used in data mining processing are described in Table 1, and the target attribute was the stress condition. TABLE 1. Attributes used for data mining. Attribute Unit Description Gender Time - Male or Female 1; 2; 3; 4; 5 and 6 Stress conditions - WC, TS, pain, thirst, and hunger Signal energy Pa²s Energy emitted by the sound wave Signal duration S Duration of the sound wave Maximum amplitude Pa Maximum amplitude of the sound wave Minimum amplitude Pa Minimum amplitude of the sound wave Intensity dB Intensity of the sound wave Pitch frequency Hertz Determine the sound wave dimension Formant 1 Hertz Frequency of formant 1 Formant 2 Hertz Frequency of formant 2 Formant 3 Hertz Frequency of formant 3 Formant 4 Hertz Frequency of formant 4 TABLE 1. Attributes used for data mining. RESULTS AND DISCUSSION The Decision Tree (Figure 1), obtained by using the J48 algorithm, had 77.66% accuracy and Kappa of 0.62. According to the Decision Tree result, the most important acoustic attribute for classification of the studied stress conditions was sound intensity (root node). For noise intensity lower than or equal to 73.87 dB, there was an indication that animals were under a welfare situation; on the other hand, for sound intensity ranging from more than 73.87 to 80.18 dB, we understood that animals were thirsty. For noise intensity higher than 80.18 dB, it was necessary to check the pitch frequency. Thus, for pitch frequency higher than 276.71 Hz, there was an indication that animals were under heat stress. For pitch frequency ranging from 276.71 to 212.87 Hz, we estimated that animals were hungry. From pitch frequency higher than 212.87 Hz, Formant 1 needed to be verified. In this case, when Formant 1 was greater than 1,066.4 Hz, there was an indication that animals were thirsty, while for Formant 1 lower than or equal to 1,066.4 Hz, animals may have been hungry. In a study on piglets during farrowing, RISI (2010) compared acoustic characteristics of piglet vocalizations between arthritis and health. Healthy piglets emitted vocalizations with greater Eng. Agríc., Jaboticabal, v.34, n.3, p.445-450, maio/jun. 2014 Marta Moi, Irenilza de A. Nääs, Fabiana R.Caldara et al. 448 intensity (79.76 dB) than arthritic animals (78.15 dB). This feature is interpreted by sound intensity (dB); the explanation for its occurrence is also related to the animal performance in stimulating air expulsion through the respiratory tract, promoting the vibration of vocal cords (RISI, 2010). The use of sound intensity as a determinant factor for welfare estimate was efficient in a study carried out by NÄÄS et al. (2008). FIGURE 1. Classification of the studied stress situations using the J48 algorithm. FIGURE 1. Classification of the studied stress situations using the J48 algorithm. Piglets respond with high sound intensity cries during pain (MARX et al., 2003; LEIDIG et al., 2009), what is easily identified by humans, facilitating the sound identification for only one attribute. Responses to hunger and cold sensations during extreme exposures that may cause pain are more difficult to identify, so more attributes are needed to be used in the identification process, as indicated by SCHRADER & TODT (1998). SAMPAIO et al. RESULTS AND DISCUSSION (2007) found that the noise emitted by pigs under thermal discomfort tends to increase with raised heat stress. This agrees with our findings and, also, with the pitch frequency, which determines the height of sound waves, enabling identification of heat stress. The identification of hunger and thirst by Formant 1 was also found by SCHRADER & TODT (1998) and DÜPJAN et al. (2008). The authors indicated that these vocal expressions are different from those related to other types of social stress, fear, and frustration. However, several authors highlight the difficulty of classifying these specific conditions, since these occur concomitantly and need more than one attribute, or combination of them, to more precisely identify the stress type (SCHRADER & TODT, 1998; MARX et al., 2003; CORDEIRO et al., 2012). It was not possible to identify gender by sound intensity, or to use the other studied attributes. This is different from studies on other animal species, which show that animal gender may be identified by sound signals (BALSBY & ADAMS, 2011; MATROSOVA et al., 2011; GOGOLEVA et al., 2012). REFERENCES BALSBY, J. S. T.; ADAMS, D. M. Vocal similarity and familiarity determine response to potential flock mates in orange-fronted conures (Psittacidae). Animal Behaviour, Amsterdam, v. 81, n. 5, p.983-991, 2011. BAPTISTA, R.I.A.; BERTANI, G.R.; BARBOSA, C.N. Indicadores do bem-estar em suínos. Ciência Rural, Santa Maria, v.41, n.10, p. 1823-1830, 2011. CALDARA, FABIANA R.; ROSA, P. S. G.; FERREIRA, R. A.; REIS, N. M. O.; NÄÄS, I. A.; PAZ, I. C. L. A.; GARCIA, R. G.; FERREIRA, V. M. O. S. Behavior, performance and physiological parameters of pigs reared in deep bedding. Engenharia Agrícola, Jaboticabal, v.32, n.1, p. 38-46, 2012. CORDEIRO, A. F.S.; NÄÄS, I. A.; OLIVEIRA, S. R. de M.; VIOLARO, F.; ALMEIDA, A. C. M. Efficiency of distinct data mining algorithms for classifying stress level in piglets from their vocalization. Engenharia Agrícola, Jaboticabal, v.32, n.2, p. 208-216, 2012. DÜPJAN, S.; SCHÖN, P. PUPPE, B.; TUCHSCHERER, A.; MANTEUFFEL, G. Differential vocal responses to physical and mental stressors in domestic pigs (Sus scrofa). Applied Animal Behaviour Science, Amsterdam, v. 114, p. 105-115, 2008. GEBERZAHN, N.; GOYMANN, W.; MUCK, C.; TEN CATE, C. Females alter their song when challenged in a sex-role reversed bird species. Behavioral Ecology and Sociobiology, Berlim, v. 64, n. 2, p 193-204, 2009. GOGOLEVA, S. S.; VOLODIN, I. A.; VOLODINA, E. V.; KHARLAMOVA A. V.; TRUT, L. N. Effects of selection for behavior, human approach mode and sex on vocalization in silver fox. Journal of Ethology, online, 2012. Disponível em: <http://link.springer.com/article/10.1007/s10164-012-0353-x/fulltext.html>. p. 1-6. DOI:10.1007/s10164-012-0353-x. Access: 7 dez 2012. GRANDIN, T. The feasibility of using vocalization scoring as an indicator of poor welfare during slaughter. Applied Animal Behaviour Science, Amsterdam, v.56, n.2, p.121-128, 1998. LEIDIG, M.S.; HERTRAMPF, B.; FAILING, K.; SCHUMANN, A.; REINER, G. Pain and discomfort in male piglets during surgical castration with and without local anesthesia as determined by vocalization and defence behaviour. Applied Animal Behaviour Science, Amsterdam, v.116, n. 2, p.174-178, 2009. MARX, G.; HORN, T.; THIELEBEIN, J.; KNUBEL, B.; BORELL, E. Analysis of pain-related vocalization in young pigs. Journal of. Sound Vibration, Amsterdam, v. 266, n. 4, p.687-698, 2003. MATROSOVA, V. A.; BLUMSTEIN, D. T.; VOLODIN, I. A.; VOLODINA, E. V. The potential to encode sex, age, and individual identity. Naturwissenschaften, Berlim, v.98, n.3, p.181-192, 2011. NÄÄS, I.A.; CAMPOS, L.S.L.; BARACHO, M.S.; TOLON, Y.B. Uso de redes neurais artificiais na identificação de vocalização de suínos. Engenharia Agrícola, Jaboticabal, v. 28, p. 204-216, 2008. RISI, N. CONCLUSIONS A decision tree was generated to identify situations of swine hunger, thirst, and heat stress, using records of sound intensity, pitch frequency, and formant 1. Eng. Agríc., Jaboticabal, v.34, n.3, p.445-450, maio/jun. 2014 Vocalization data mining for estimating swine stress conditions 449 Considering the studied attributes, it was not possible to identify animal gender by their vocalization. Eng. Agríc., Jaboticabal, v.34, n.3, p.445-450, maio/jun. 2014 REFERENCES Uso da vocalização como indicador patológico em leitões na fase de maternidade. 2010. 93f. Dissertação (Mestrado) - Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, 2010. ROLLER, W.L.; GOLDMAN, R.F. Response of swine to acute heat exposure. Transactions of the ASAE, St. Joseph, v.12, n.2, p.164-169, 174, 1969. Eng. Agríc., Jaboticabal, v.34, n.3, p.445-450, maio/jun. 2014 Marta Moi, Irenilza de A. Nääs, Fabiana R.Caldara et al. 450 RORIZ, M. Psicrom 1.0 - Relações Psicométricas. Universidade Federal de São Carlos, Departamento de Engenharia Civil. Programa de Pós-Graduação em Construção Civil. São Carlos, 2003. RORIZ, M. Psicrom 1.0 - Relações Psicométricas. Universidade Federal de São Carlos, Departamento de Engenharia Civil. Programa de Pós-Graduação em Construção Civil. São Carlos, 2003. SAMPAIO, C. A. P.; NÄÄS, I. A.; SALGADO, D. D.; MARCOS P. G.; QUEIRÓS, M. P. G. Avaliação do nível de ruído em instalações para suínos. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, v.11, n.4, p. 436-440, 2007. Avaliação do nível de ruído em instalações para suínos. Revista Brasileira de Engenharia Agrícola e Ambiental, Campina Grande, v.11, n.4, p. 436-440, 2007. SCHRADER, L.; TODT, D. Vocal quality is correlated with levels of stress hormones in domestic pigs. Ethology, Berlim, v.104, n. 10, 859-876, 1998. SPINKA, M. Social dimension of emotions and its implication for animal welfare. Applied Animal Behaviour Science, Amsterdam, v. 138, n.2, p. 170-181, 2012. SPINKA, M. Social dimension of emotions and its implication for animal welfare. Applied Animal Behaviour Science, Amsterdam, v. 138, n.2, p. 170-181, 2012.
https://openalex.org/W4235669752	https://www.frontiersin.org/articles/10.3389/fgene.2021.658796/pdf	English	null	Corrigendum: Genomic Prediction Accuracy Using Haplotypes Defined by Size and Hierarchical Clustering Based on Linkage Disequilibrium	Frontiers in genetics	2,021	cc-by	656	CORRECTION published: 16 March 2021 doi: 10.3389/fgene.2021.658796 CORRECTION Copyright © 2021 Won, Park, Son, Lee, Park, Park, Park, Chai, Kim, Lee and Lim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Corrigendum: Genomic Prediction Accuracy Using Haplotypes Deﬁned by Size and Hierarchical Clustering Based on Linkage Disequilibrium Sohyoung Won 1†, Jong-Eun Park 2†, Ju-Hwan Son 2, Seung-Hwan Lee 3, Byeong Ho Park 2, Mina Park 2, Won-Chul Park 2, Han-Ha Chai 2, Heebal Kim 1,4,5, Jungjae Lee 6* and Dajeong Lim 2* Sohyoung Won 1†, Jong-Eun Park 2†, Ju-Hwan Son 2, Seung-Hwan Lee 3, Byeong Ho Park 2, Mina Park 2, Won-Chul Park 2, Han-Ha Chai 2, Heebal Kim 1,4,5, Jungjae Lee 6* and Dajeong Lim 2* 1 Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, South Korea, 2 National Institute of Animal Science, RDA, Wanju, South Korea, 3 Department of Animal Science and Biotechnology, Chungnam National University, Daejeon, South Korea, 4 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea, 5 eGnome, Inc, Seoul, South Korea, 6 Jung P&C Institute, Inc., Yongin-si, South Korea Keywords: genomic prediction, haplotype, hierarchical clustering, linkage disequilibrium, best linear unbiased prediction, accuracy, Hanwoo Genomic Prediction Accuracy Using Haplotypes Deﬁned by Size and Hierarchical Clustering Based on Linkage Disequilibrium by Won, S., Park, J.-E., Son, J.-H., Lee. S.-H., Park, B. H., Park, M., et al. (2020). Front. Genet. 11:134. doi: 10.3389/fgene.2020.00134 Edited and reviewed by: Marco Milanesi, University of Tuscia, Italy Correspondence: Jungjae Lee jungjae.ansc@gmail.com Dajeong Lim lim.dj@korea.kr Edited and reviewed by: Marco Milanesi, University of Tuscia, Italy Correspondence: Jungjae Lee jungjae.ansc@gmail.com Dajeong Lim lim.dj@korea.kr In the published article, there were errors in aﬃliation for authors Sohyoung Won and Heebal Kim. For “Sohyoung Won”, instead of “Department of Agricultural Biotechnology and Research Institute of Population Genomics, Seoul National University, Seoul, South Korea,” it should be “Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, South Korea”. †These authors have contributed equally to this work For “Heebal Kim”, instead of “Department of Agricultural Biotechnology and Research Institute of Population Genomics, Seoul National University, Seoul, South Korea,” it should be “Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, South Korea”; “Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, South Korea” and “eGnome, Inc, Seoul, South Korea”. Specialty section: This article was submitted to Livestock Genomics, a section of the journal Frontiers in Genetics A correction has also been made in the conﬂict of interest section to declare competing interests of author Heebal Kim; “HK was employed by company eGnome, Inc.” The corrected statement has been added to the section. Received: 08 February 2021 Received: 08 February 2021 Accepted: 18 February 2021 Published: 16 March 2021 The authors apologize for these errors and state that this does not change the scientiﬁc conclusions of the article in any way. The original article has been updated. Accepted: 18 February 2021 Published: 16 March 2021 Citation: Conﬂict of Interest: JL was employed by company Jung P&C Institute, Inc and HK was employed by company eGnome, Inc. Won S, Park JE, Son JH, Lee SH, Park BH, Park M, Park WC, Chai HH, Kim H, Lee J and Lim D (2021) Corrigendum: Genomic Prediction Accuracy Using Haplotypes Deﬁned by Size and Hierarchical Clustering Based on Linkage Disequilibrium. Front. Genet. 12:658796. doi: 10.3389/fgene.2021.658796 The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. March 2021 \| Volume 12 \| Article 658796 Frontiers in Genetics \| www.frontiersin.org
https://openalex.org/W3022110845	https://europepmc.org/articles/pmc7205293?pdf=render	English	null	Revisiting the expression signature of pks15/1 unveils regulatory patterns controlling phenolphtiocerol and phenolglycolipid production in pathogenic mycobacteria	PloS one	2,020	cc-by	12,158	PLOS ONE PLOS ONE RESEARCH ARTICLE Revisiting the expression signature of pks15/1 unveils regulatory patterns controlling phenolphtiocerol and phenolglycolipid production in pathogenic mycobacteria Beatriz RamosID1,2, Stephen V. Gordon3, Mo´nica V. CunhaID1,2,4* 1 National Institute for Agrarian and Veterinary Research (INIAV, IP), Oeiras, Portugal, 2 Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal, 3 School of Veterinary Medicine and Conway Institute, University College Dublin, Dublin, Ireland, 4 Biosystems & Integrative Sciences Institute (BioISI), Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * monica.cunha@iniav.pt * monica.cunha@iniav.pt OPEN ACCESS One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthio- ceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phe- nolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of tran- scriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expres- sion approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtio- cerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron con- centration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level. Citation: Ramos B, Gordon SV, Cunha MV (2020) Revisiting the expression signature of pks15/1 unveils regulatory patterns controlling phenolphtiocerol and phenolglycolipid production in pathogenic mycobacteria. PLoS ONE 15(5): e0229700. https://doi.org/10.1371/journal. pone.0229700 Editor: Je´roˆme Nigou, Centre National de la Recherche Scientifique, FRANCE Received: February 6, 2020 Accepted: April 22, 2020 Published: May 7, 2020 Copyright: © 2020 Ramos et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Je´roˆme Nigou, Centre National de la Recherche Scientifique, FRANCE Received: February 6, 2020 Accepted: April 22, 2020 Published: May 7, 2020 Copyright: © 2020 Ramos et al. 1 Introduction Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that remains a major public health concern. In 2016, approximately 6.3 million new cases of TB were reported [1]. The mycobacterial cell wall, wherein Mycobacterium-specific components are located, is a crucial interface of Mtb and other pathogenic mycobacteria with the host [2]. Recently, Chiaradia and coworkers (2017) proposed a cell wall structure composed of three layers, namely the mycomembrane, arabinogalactan and peptidoglycan. This model proposes that the inner leaflet of the mycomembrane is composed of mycolic acids that are esterified to arabinogalactan, which in turn is covalently attached to peptidoglycan [3]. Amongst the Myco- bacterium-specific components are two related families of glycosylated lipids: diphthiocera- nates (DIP) and phthiocerol dimycocerosate (PDIM), along with its variant phenolic glycolipids (PGL) [2]. PGL are known to be associated with several cellular functions, namely impermeability of the cell wall, phagocytosis [4–6], defence mechanisms against nitrosative and oxidative stress [7] and to the ability of mycobacteria to form biofilms [8, 9]. Competing interests: The authors have declared that no competing interests exist. In this work, we focus on the transcriptional signature of genes comprising the biosynthetic pathway responsible for the synthesis of the phenolphthiocerol moiety of PGL, by investigating the expression of these genes when Mtb is grown under multiple stressors mimicking the host environment, namely pH variation, different carbon sources, limiting or excessive iron con- centration, hypoxia, dormancy, phosphate depletion and antibiotic exposure. The enzymes related to the biosynthesis of PGL belong to the class of polyketide synthases (PKS). There are three types of PKS, classified according to their structure and biosynthetic function. Type I PKS contain multiple catalytic domains and can be classified as modular or iterative. Modular type I PKS have distinct functional domains that are used only once during the formation of the product. On the other hand, iterative PKS have functional domains that intervene repeti- tively to produce the final polyketide. Type II PKS are composed of several enzymes, each one carrying a single and distinct catalytic domain that is used iteratively during formation of the polyketide product. Chalcone synthase-like PKS, the type III PKS, represents a more divergent group that, in contrast to types I and II PKS, do not require the involvement of acyl carrier proteins (ACP) [10]. OPEN ACCESS This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was funded by Programa Operacional de Competitividade e Internacionalizac¸ão (POCI) (FEDER component), Programa Operacional Regional de Lisboa, and Fundac¸ão para a Ciência e a Tecnologia (FCT) (www.fct.pt), Portugal, in the scope of project “Colossus: Control Of tubercuLOsiS at the wildlife/ 1 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE Revisiting the RNA signature of pks15/1 livestock interface uSing innovative natUre-based Solutions” (ref. POCI-01-0145- FEDER-029783) awarded to MVC, project “MyPATH- Insights into Mycobacterium tuberculosis complex PATHogenesis: assessing the benefit of confinement-induced biofilm strategies by comparative genomics” (ref. PTDC/CVT/117794/ 2010) awarded to MVC, and strategic funding to cE3c and BioISI Research Units (UID/BIA/00329/ 2020 and UID/Multi/04046/2020]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 1 Introduction Hence, transcriptional regulators previously described to act on gene expression under such conditions were introduced into our analyses so that a comparison between our own and previous work could be established, as well as to explore the potential regulation of our genes of interest by such regulators. Along with stress-induced genes, Mtb also contains a group of 13 σ subunits responsible for transcriptional regulation, namely the essential housekeeping sigma factor (σA), the stress- responsive factor (σB) and 11 other sigma factors that act as environmental responsive regula- tors (σC-M). Several studies have been performed to infer the role of each sigma factor and the condition that triggers their activation, initially by analysing expression levels and by the con- struction of deletion strains [18, 19]. The presence and articulation of this wide variety of sigma factors enables an adaptive transcriptional response to a large set of environmental con- ditions. Chauhan and co-workers (2016) performed a reconstruction of the sigma factor regu- latory network that enabled a clarification of the direct and indirect connections among the 13 factors [20]. This former study defined an hierarchical organization of sigma factors in Mtb, as well as the usage of multiple factors in response to specific stresses. Current knowledge advo- cates a hierarchical organization that comprises three regulation levels: (i) top level: sigA, sigB, sigH, sigM; (ii) middle level: sigE, sigF, sigG, sigJ, sigL; and (iii) bottom level: sigC, sigD, sigI, sigK. To get a view on which sigma factors are recruited upon the conditions that modulate pks15 and pks1 transcription, we therefore also introduced this breadth of sigma factors into our comparative transcriptome analyses. As well as host-induced stress, Mtb is frequently exposed to drug-induced stress via antibi- otic therapy. For treatment of drug-susceptible TB, a standard combination of isoniazid, eth- ambutol, rifampicin, and pyrazinamide is used [21]. Since TB treatment is a long-lasting process, Mtb’s transcriptional profile is expected to undergo defined changes along the chemo- therapeutic process. In vitro studies have shown that for each of the above-mentioned drugs, combined expression of a set of genes results in an antibiotic resistance phenotype in Mtb. Since some of these drugs act on cell surfaces, the genes responsible for the corresponding drug-resistance phenotype were also included in our analysis in order to assess co-regulation of these genes with genes involved in PGL production. 1 Introduction Among the genes required for PGL production are ppsA-E and pks15/1 encoding type I PKS, constituting the PDIM + PGL locus, which is known to be highly con- served in PDIM/PGL-producing strains [11]. Type I PKS modules are constituted by a mini- mal set of three domains, namely a ketoacyl synthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain. This module can also contain one or more of the following domains: keto reductase (KR), dehydratase (DH) and/or enoyl reductase (ER) [12]. The pks15 encodes a KS domain while pks1 encodes KR, DH, ER, AT and ACP domains. It has been reported that a 7 bp deletion in some Mtb strains, and a 1 bp deletion in a few Mycobacterium bovis (Mb) strains, leads to a frameshift that results in the split of pks15 and pks1 [13]. Constant and co-workers (2002) documented that, in PGL-producers, the pks1 and pks15 are a single gene, pks15/1, while they are separate (2 ORFs) in Mtb PGL-deficient strains such as H37Rv or Erdman. Production of PGL’s phenolphthiocerol moiety starts with the enzyme encoded by Rv2949c that catalyses the formation of p-hydroxybenzoic acid (p-HBA) that will later be activated by the fadD22 product, with p-hydroxybenzoyl-AMP ligase activity [4], and finally elongated with malonyl-CoA as extender unit by pks15/1, in a reaction that may comprise eight to nine elongation cycles [11]. The product of fadD29, a fatty acyl-AMP ligase, is then responsible for activation of p-hydroxyphenylalkanoates, later transferred onto the ppsA product and, finally, elongated with malonyl-CoA and mehtylmalonyl-CoA by PpsB-PpsE to yield the phenolphtiocerol moiety of PGL [14–16]. 2 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE Revisiting the RNA signature of pks15/1 As described [17], upon the entrance of Mtb into its target cells, a cascade of events is trig- gered by the immune system that, in an immunocompetent host, leads to granuloma forma- tion and bacterial confinement. This structure is beneficial for the host, since it confines infection to localized regions, preventing bacterial spread. Some of the stresses mycobacteria are exposed to during infection include: starvation; reactive oxygen and nitrogen intermedi- ates; hypoxia inside granulomas; iron limitation; scarcity of inorganic phosphate (Pi); and low pH [17]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 1 Introduction Understanding the transcriptional profiles and structure of pks15’ and pks1 under different stress conditions that mimic the host environment is of major importance given their role in the modulation of Mtb and Mb cell surfaces, acting as the interface with the host cell and affecting pathogenicity. As such, this work aimed to elucidate the regulatory patterns responsi- ble for controlling pks1 and pks15 transcription by exploring publicly available large datasets of transcriptome data (RNA-seq). This methodological approach of transcriptome profiling takes advantage of deep-sequencing technologies to get a precise measurement of transcripts at the whole genome level. Our differential expression approach enabled us to define sets of correlated genes according to their expression profiles under different stress conditions, and also to outline the transcriptional structure of the pks15/1 locus based on the available experi- mental data and in silico predictions. 3 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE Revisiting the RNA signature of pks15/1 2.1 In silico analysis of regulatory data of pks1 and pks15 Regulatory information on pks1 and pks15 were gathered from international databases such as: Mycobrowser [22], National Center for Biotechnology Information (NCBI) [23], TB Database [24, 25] and MTB Network Portal [26] (visited from 09/2018 to 01/2019). MTB Network Portal reports information produced by the cMonkey algorithm that demonstrates that pks1 and pks15 belong to the same two biclusters. Biclusters are sets of co-regulated genes defined by cMonkey according to mRNA-based expression levels, de novo identification of transcription factor binding motifs and pre-established association pathways. Location of putative ribosomal binding sites (RBS) was inferred for pks1 with Prokaryotic Dynamic Programming Gene finding Algorithm (PRODIGAL) [27]. Synteny analyses were performed for pks1, pks15 and fadD22 using SyntTax (Prokaryotic Synteny & Taxonomy Explorer)[28]. 2.2 RNA-seq data and differential expression analyses of a selected panel of g For expression analyses, 105 experiments (S1 Table) from Mtb strains (Mtb H37Rv and Mtb CDC1551), constituting a set of 40 experimental conditions in seven datasets (Accession codes at NCBI: GSE47863, GSE67035, GSE52020, GSE83814, GSE66408, GSE104599 and GSE107831) were considered. This analysis was performed for a set of 90 genes (S2 Table), including pks1, pks15, fadD22, fadD29 genes comprised in bicluster modules 0211 and 0490 from MTB Network Portal that represent co-regulated genes, genes encoding PKS and σ fac- tors, and genes encoding regulatory factors for each of the experimental conditions. Regard- ing M. bovis BCG, 21 experiments from M. bovis BCG str. Pasteur 1173P2 constituting a set of seven experimental conditions in two datasets (accession codes at NCBI: GSE66883 and GSM3160698) were analysed (S1 Table). For this analysis, a set of 50 genes were selected, including pks1, pks15, fadD22, fadD29 genes comprised in bicluster modules 0211 and 0490 from MTB Network Portal, that represent co-regulated genes, and genes encoding PKS and σ factors. For each experiment, reads were extracted in FASTQ format using NCBI SRA Toolkit v.2.8.1.3 [29]. Those FASTQ files were mapped against a reference genome, Mtb H37Rv (RefSeq code: NC_000962.3, version 3) with TopHat v.2.1.0.54, [30, 31], using default settings to produce a BAM file containing a list of read alignments. Transcript identification and counting was later performed with bias correction by Cufflinks v.2.2.1.0 [30, 32] using as ref- erence the annotation of the genomes listed above. Cufflinks was used to calculate the relative abundance of each gene in Reads Per Kilobase per Million mapped reads (RPKM). The RPKM values were transformed by log10 and values were normalized in relation to the housekeeping gene sigA expression level, heatmaps were plotted by GraphPad Prism 8 [33] and dendro- grams were computed using NTSYS v2.2, calculating Pearson correlation coefficient and the unweighted pair group method with arithmetic means (UPGMA) as the agglomerative cluster- ing algorithm. Pearson correlation coefficient was calculated using GraphPad Prism 8 and correlation network was plotted using Cytoscape v.3.7.2 [34, 35]. Node size of correlation net- works correspond to calculated betweenness-centrality [36]. For differential expression analy- sis, htseq-count v.0.9.1[37] was used to count reads mapped to each gene and DESeq v.2.11.40.1 [38] was used to determine differentially expressed genes from count tables using Wald statistic test with p-value adjusted for multiple testing with the Benjamini-Hochberg procedure (α = 0.05). 2.2 RNA-seq data and differential expression analyses of a selected panel of For evaluation of significance it was considered the following scale: sig- nificant (p-value = 0.01 to 0.05); very significant (p-value = 0.001 to 0.01); extremely signifi- cant (p-value = 0.0001 to 0.001); extremely significant (p-value< 0.0001). Data was plotted as For expression analyses, 105 experiments (S1 Table) from Mtb strains (Mtb H37Rv and Mtb CDC1551), constituting a set of 40 experimental conditions in seven datasets (Accession codes at NCBI: GSE47863, GSE67035, GSE52020, GSE83814, GSE66408, GSE104599 and GSE107831) were considered. This analysis was performed for a set of 90 genes (S2 Table), including pks1, pks15, fadD22, fadD29 genes comprised in bicluster modules 0211 and 0490 from MTB Network Portal that represent co-regulated genes, genes encoding PKS and σ fac- tors, and genes encoding regulatory factors for each of the experimental conditions. Regard- ing M. bovis BCG, 21 experiments from M. bovis BCG str. Pasteur 1173P2 constituting a set of seven experimental conditions in two datasets (accession codes at NCBI: GSE66883 and GSM3160698) were analysed (S1 Table). For this analysis, a set of 50 genes were selected, including pks1, pks15, fadD22, fadD29 genes comprised in bicluster modules 0211 and 0490 from MTB Network Portal, that represent co-regulated genes, and genes encoding PKS and σ factors. For each experiment, reads were extracted in FASTQ format using NCBI SRA Toolkit v.2.8.1.3 [29]. Those FASTQ files were mapped against a reference genome, Mtb H37Rv (RefSeq code: NC_000962.3, version 3) with TopHat v.2.1.0.54, [30, 31], using default settings to produce a BAM file containing a list of read alignments. Transcript identification and counting was later performed with bias correction by Cufflinks v.2.2.1.0 [30, 32] using as ref- erence the annotation of the genomes listed above. Cufflinks was used to calculate the relative abundance of each gene in Reads Per Kilobase per Million mapped reads (RPKM). The RPKM values were transformed by log10 and values were normalized in relation to the housekeeping gene sigA expression level, heatmaps were plotted by GraphPad Prism 8 [33] and dendro- grams were computed using NTSYS v2.2, calculating Pearson correlation coefficient and the unweighted pair group method with arithmetic means (UPGMA) as the agglomerative cluster- ing algorithm. Pearson correlation coefficient was calculated using GraphPad Prism 8 and correlation network was plotted using Cytoscape v.3.7.2 [34, 35]. Node size of correlation net- works correspond to calculated betweenness-centrality [36]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 3.1 Revisiting the organization of pks1 and pks15 genetic locus across Mtb genomes based on predicted regulatory data and homology searches 3.1 Revisiting the organization of pks1 and pks15 genetic locus across Mtb genomes based on predicted regulatory data and homology searches Encoded on the minus strand, from position 3291503 to 3296353 for pks1 and from position 3296350 to 3297840 for pks15, on the Mtb H37Rv genome, the pks1 and pks15 genes together encode a polyketide synthase with six identified domains involved in the synthesis of PGL. They appear to have a functional cooperation with fadD22, a bidomain initiation module. In the MTB Network Portal (retrieved on January, 2019), the pks1 and pks15 genes are placed together in bicluster modules 0211 and 0490, with residual values of 0.5 and 0.57, respectively, meaning that bicluster module 0211 presents a tight expression profile amongst its members, which indicates better bicluster quality and thus more certainty associated with co-expression. Furthermore, for the two genes upstream of pks15, fadD22 is included in the same two mod- ules, while Rv2949c is included in module 0490 (Fig 1). The mRNA-based expression levels, de novo identification of transcription factor binding motifs and pre-established association path- ways used by the Infelerator algorithm, according to data available at MTB Network Portal, all support that pks1 and pks15 may be regulated by the products of seven genes: positively by Rv0042c, sigK, Rv2258c and Rv3557c; negatively by sigB, Rv2745c and Rv3583c. Furthermore, according to ChIP-seq data, pks1 is bound by the transcription factor Rv3830c with no differ- ential expression reported. The operon structure is undetermined; the TB Database suggests four different combinations of six genes (fadD29, Rv2949c, fadD22, pks15, pks1 and lppX), while the MTB Network Portal suggests an operon composed by five genes, namely fadD29, Rv2949c, fadD22, pks15 and pks1. All these genes are involved in the biosynthesis of the phe- nolphthiocerol moiety of PGL, except lppX that was shown to be involved in the translocation of PDIM to the outer membrane [40]. To compare the conservation of pks15/1 locus across Mtb genomes, a synteny analysis was performed using the Pks1 sequence from Mtb H37Rv as the query. Among the 210 Mtb Fig 1. Genomic locus of pks1 and pks15, protein domains and their role in the biosynthetic pathway of PGL (as described in Mycobrowser [22]). A–Schematic representation of the location of pks1 and pks15 in the minus strand of M. tuberculosis H37Rv genome. In black: lipid metabolism. In green: cell wall and cell processes. In orange: intermediary metabolism and respiration. 2.2 RNA-seq data and differential expression analyses of a selected panel of For differential expression analy- sis, htseq-count v.0.9.1[37] was used to count reads mapped to each gene and DESeq v.2.11.40.1 [38] was used to determine differentially expressed genes from count tables using Wald statistic test with p-value adjusted for multiple testing with the Benjamini-Hochberg procedure (α = 0.05). For evaluation of significance it was considered the following scale: sig- nificant (p-value = 0.01 to 0.05); very significant (p-value = 0.001 to 0.01); extremely signifi- cant (p-value = 0.0001 to 0.001); extremely significant (p-value< 0.0001). Data was plotted as PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 4 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 heatmap using GraphPad Prism 8. The public server at usegalaxy.org [39] was used to analyse the data with NCBI SRA Toolkit, TopHat, Cufflinks, htseq-count and DESeq2. 3.1 Revisiting the organization of pks1 and pks15 genetic locus across Mtb genomes based on predicted regulatory data and homology searches B–Domain organization of Pks1 and Pks15. Abbreviations: KS, ketoacylsynthase; AT, acyltransferase; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; ACP, acylcarrier protein. https://doi.org/10.1371/journal.pone.0229700.g001 Fig 1. Genomic locus of pks1 and pks15, protein domains and their role in the biosynthetic pathway of PGL (as described in Mycobrowser [22]). A–Schematic representation of the location of pks1 and pks15 in the minus strand of M. tuberculosis H37Rv genome. In black: lipid metabolism. In green: cell wall and cell processes. In orange: intermediary metabolism and respiration. B–Domain organization of Pks1 and Pks15. Abbreviations: KS, ketoacylsynthase; AT, acyltransferase; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; ACP, acylcarrier protein. https://doi.org/10.1371/journal.pone.0229700.g001 https://doi.org/10.1371/journal.pone.0229700.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 5 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 accession codes available (S3 Table), those with synteny scores above 80 were 90.5% and 99.5% for pks15 and fadD22, respectively. For pks1, 89.5% of the accession codes used for analysis presented a score higher than 80 (Fig 2). These scores represent a normalization of the BLASTP homology score of the target protein in each genome against the reference Mtb H37Rv genome. When analysing local genomic conservation, an irregular pattern was noted for pks1 and pks15/1, while Rv2949c, fadD22, lppX, Rv2944 and Rv2943 presented a regular organization pattern across most genomes analysed. However, when comparing the top three scoring genomes, it was also possible to identify that, for Mtb 0B070XDR (GenBank code: CP008970.1), the element upstream of Rv2949c did not present homology with FadD29, whilst homology was found for a protein encoded on the opposite strand of pks1. Many parameters influence synteny analyses, such as the assembly quality of each genome which, in the case of inadequate accuracy, may introduce mismatches and thus not reflect true polymorphisms; this may greatly impact the final output of such analyses [41]. Despite this constraint, our data does enable the recognition of a synteny block constituted by the set of genes of interest. 3.2 Analysis of expression data for a selected panel of genes enclosing pks1 and pks15 To characterize the transcriptional signature of pks1 and of presumably correlated genes, RNA-seq based expression analyses focused on a set of 90 genes for scrutiny, including pks1, pks15, fadD22, fadD29, as well as other genes encoding polyketide synthases and σ factors. Transcriptional profiles were compared across a set of 40 experimental conditions. Data gath- ered were analysed by alignment against a reference genome [Mtb H37Rv (RefSeq code: NC_000962.3, version 3)], by read counting and through the calculation of RPKM as a proxy for gene expression in each condition. RPKM is a relative value, meaning that it varies accord- ing not only to read count, but also to the total number of reads obtained for each experiment. Fig 2. Representation of top three and bottom three scores from synteny analysis. Top 3 and bottom 3 synteny scores for pks1 as predicted by SyntTax. The colours associated with coding sequences (CDS) facilitate the schematic representation of each CDS across the genomes analysed. https://doi.org/10.1371/journal.pone.0229700.g002 Fig 2. Representation of top three and bottom three scores from synteny analysis. Top 3 and bottom 3 synteny scores for pks1 as predicted by SyntTax. The colours associated with coding sequences (CDS) facilitate the schematic representation of each CDS across the genomes analysed. https://doi.org/10.1371/journal.pone.0229700.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 6 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 In order to enable comparison across experiments, those values were normalized in relation to sigA housekeeping gene expression. Besides the presence of an hypothetical synteny block across Mtb strains, that we herein use as our main source of data, it is known that the Mtb H37Rv reference strain, as well as Mtb CDC1551, which are core to our dataset, both present a frameshift mutation in pks15/1, in comparison to Mb BCG Tokyo which retains an intact CDS and is capable of PGL production. To understand the similarity across profiles, a dendrogram was generated, using 80% similar- ity as a cut-off for cluster formation; this cut-off was established to include the top quarter of similarity values in the analysis. From this, we obtained a total of 53 clusters, 39 being single member clusters. Cluster I included some of our genes of interest (fadD22 and fadD29). Clus- ter II includes pks1 and pks6, which is a pks1 paralog, and pks12. (Fig 3). 3.2 Analysis of expression data for a selected panel of genes enclosing pks1 and pks15 Another relevant clus- ter (II) includes papA3 and pks4, both genes known to be upregulated at low pH (Fig 3). The remaining genes of interest were single member clusters. To obtain a more focused approach on direct interactions between genes, we constructed a correlation network with pairs of genes exhibiting correlation factors above 0.75. In this network, it becomes evident that pks1 is highly correlated with lppX (Pearson correlation coefficient of 0.777), fadD22 (0.811), fadD29 (0.757) and also with pks6 (0.807) and pks12 (0.803) (Fig 4A). A correlation across σ factors could also be confirmed: sigB is correlated with sigE (0.876) and sigH (0.833); sigE with sigH (0.783); sigG with sigJ (0.782); and sigJ with sigK (0.712) (Fig 4B). Both analyses provide evidence that pks1 expression is highly correlated with the expres- sion profiles of fadD22 and fadD29, in agreement with reports from microarray data [42]. The expression pattern of pks15 revealed by this analysis is strikingly different from the one found in pks1 in some of the stress conditions under examination, turning pks15 into a single mem- ber cluster and, consequently, absent from the correlation network (Fig 4A). In contrast with these results, available microarray data [49] suggests that pks15 is also highly correlated with pks1 and fadD22. Our analyses also suggest that pks4 is correlated with pks3, as their expression profiles share 71% similarity (Fig 3), thus agreeing with previously published data [43] report- ing that an Mtb H37Rv double mutant for pks4 and pks3 is not able to produce mycolipanoic, mycolipenic, and mycolipodienoic acids. Also, the fact that pks3 and pks4 form a polyketide structure similar to pks15 and pks1, respectively, wherein pks3 and pks15 both encode the ketoacyl synthase domain and pks4 and pks1 both encode the remaining polyketide synthase domains, would suggest that pks15 and pks1 could also be highly correlated. Naturally, we were expecting to confirm this correlation across the selected experimental datasets. Even though gene expression does not necessarily represent the activity of a specific σ fac- tor, we integrated our correlation network with sigma factor expression data to plot a repre- sentation of the putative regulation of selected genes by σ factors. Six of the 13 sigma genes under analysis are highly correlated. The sigA factor is known to regulate sigG, mostly induced during macrophage infection, which will thus regulate sigJ, known to be overexpressed in late stationary phase of dormant cultures. 3.2 Analysis of expression data for a selected panel of genes enclosing pks1 and pks15 Abbreviations: C—Mtb H37Rv grown in control conditions; DE—Mtb H37Rv grown in dextrose at exponential phase; DS–Mtb H37Rv grown in dextrose at stationary phase; FE—Mtb H37Rv grown in long fatty acids at exponential phase; FS—Mtb H37Rv grown in long fatty acids at stationary phase; HI—Mtb H37Rv grown in high iron concentration; LI-1day—Mtb H37Rv grown in low iron concentration for 1 day; LI-1week—Mtb H37Rv grown in low iron concentration for 1 week; HN878—Mtb HN878; Gly-pH 7—Mtb CDC1551 grown in glycerol at pH 7; Gly-pH 5.7—Mtb CDC1551 grown in glycerol at pH 5.7; Pyr-pH 7—Mtb CDC1551 grown in pyruvate at pH 7; Pyr-pH 5.7—Mtb CDC1551 grown in pyruvate at pH 5.7; Hyp—Mtb H37Rv grown in hypoxia; (1–4) day—Mtb H37Rv (1–4) day(s) after reaeration; ED—Mtb H37Rv in early dormancy phase; MD–Mtb H37Rv in medium dormancy phase; LD—Mtb H37Rv in late dormancy phase; CIP4 –Mtb H37Rv grown with CIP for 4h; INH4—Mtb H37Rv grown with INH for 4h; EMB4—Mtb H37Rv grown with EMB for 4h; STR4—Mtb H37Rv grown with STR for 4h; RIF4—Mtb H37Rv grown with RIF for 4h; CIP24—Mtb H37Rv grown with CIP for 24h; INH24—Mtb H37Rv grown with INH for 24h; EMB24—Mtb H37Rv grown with EMB for 24h; STR24—Mtb H37Rv grown with STR for 24h;; RIF24—Mtb H37Rv grown with RIF for 24h; PBS—Mtb H37Rv grown with PBS; and P-dep—Mtb H37Rv grown in phosphate depletion. https://doi org/10 1371/journal pone 0229700 g003 Fig 3. Expression profiling of selected genes from Mycobacterium tuberculosis, presented as log10 RPKM. Cut-off: 85% of similarity. 3.2 Analysis of expression data for a selected panel of genes enclosing pks1 and pks15 sigG and sigJ will further regulate sigL, known to be involved in PDIM biosynthesis, that in turn regulates sigK, whose precise functioning remains unclear [19]. Single correlations exist between discrete sigma factors and the selected panel of genes. The sigK factor, which is predicted by in silico analysis to positively regulate pks1 and pks15 [44], here shows a correlation with pks1 of 0.684 (Fig 4A). On the contrary, amongst the analyses focused on sigma factors, sigE was the factor that presented the lowest correlations with the established genes of interest (lppX, -0.567; pks1, -0.506; fadD22, -0.533; Rv2949c, -0.166; and fadD29, -0.521). For M. bovis BCG, we obtained a total of 8 clusters, with four being single member clusters using 85% similarity as a cut-off. Amongst these, it was possible to identify a cluster compris- ing Mb2973c, pks13, fadD22, pks15/1, pks12, rpmG and sigC (Fig 5). In M. bovis BCG, as PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 7 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 Fig 3. Expression profiling of selected genes from Mycobacterium tuberculosis, presented as log10 RPKM. Cut-off: 85% of similarity. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 3.2 Analysis of expression data for a selected panel of genes enclosing pks1 and pks15 Abbreviations: C—Mtb H37Rv grown in control conditions; DE—Mtb H37Rv grown in dextrose at exponential phase; DS–Mtb H37Rv grown in dextrose at stationary phase; FE—Mtb H37Rv grown in long fatty acids at exponential phase; FS—Mtb H37Rv grown in long fatty acids at stationary phase; HI—Mtb H37Rv grown in high iron concentration; LI-1day—Mtb H37Rv grown in low iron concentration for 1 day; LI-1week—Mtb H37Rv grown in low iron concentration for 1 week; HN878—Mtb HN878; Gly-pH 7—Mtb CDC1551 grown in glycerol at pH 7; Gly-pH 5.7—Mtb CDC1551 grown in glycerol at pH 5.7; Pyr-pH 7—Mtb CDC1551 grown in pyruvate at pH 7; Pyr-pH 5.7—Mtb CDC1551 grown in pyruvate at pH 5.7; Hyp—Mtb H37Rv grown in hypoxia; (1–4) day—Mtb H37Rv (1–4) day(s) after reaeration; ED—Mtb H37Rv in early dormancy phase; MD–Mtb H37Rv in medium dormancy phase; LD—Mtb H37Rv in late dormancy phase; CIP4 –Mtb H37Rv grown with CIP for 4h; INH4—Mtb H37Rv grown with INH for 4h; EMB4—Mtb H37Rv grown with EMB for 4h; STR4—Mtb H37Rv grown with STR for 4h; RIF4—Mtb H37Rv grown with RIF for 4h; CIP24—Mtb H37Rv grown with CIP for 24h; INH24—Mtb H37Rv grown with INH for 24h; EMB24—Mtb H37Rv grown with EMB for 24h; STR24—Mtb H37Rv grown with STR for 24h;; RIF24—Mtb H37Rv grown with RIF for 24h; PBS—Mtb H37Rv grown with PBS; and P-dep—Mtb H37Rv grown in phosphate depletion. https://doi.org/10.1371/journal.pone.0229700.g003 verified for M. tuberculosis, pks15/1 and fadD22 exhibit a correlation value of 0.913, with corre- lation values between pks15/1 and fadD22 and lppX and Mb2973c all above 0.9. In addition, lppX, pks15/1, fadD22 and Mb2973c were also shown to be correlated with sigC (above 0.79). Comparison of normalized expression levels across Mtb H37Rv and Mb for the set of selected genes gave a correlation of 0.78, suggesting that using Mtb H37Rv as a reference to infer the pks15/1 transcriptional profile across Mtb more broadly is a viable approach. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 8 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 0 13 1/j l 0229 00 M 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 9 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 Fig 4. Correlation network of expression data. A: Correlation threshold = 0.75. B: Correlation threshold = 0.7. Thicker connections represent stronger correlations. Node size represents centrality values. 3.3 Differential expression analyses As mentioned, pathogenic mycobacteria of the MTC are subjected to a set of different growth conditions while in the granuloma and also while exposed to antimicrobial therapy. In this context, we were able to validate our analyses, as well as to perform a comparison of the differ- ential expression of the selected genes with regulatory genes, using previously reported expres- sion analyses. Hence in our analysis we calculated the log2 fold change differential expression of 90 genes, encompassing our genes of interest plus 55 genes linked to regulatory networks in each experimental condition, employing the Wald statistical test and p-value adjusted for mul- tiple testing by the Benjamini-Hochberg procedure (α = 0.05),. When comparing regular growth conditions of Mtb CDC1551 with nutrient-depletion and phosphate-depletion [45], the selected set of genes did not present any significant fold-changes in expression. As for the regulatory genes, significant fold-changes were only found for sigB in phosphate-depletion conditions. With both glycerol and pyruvate as carbon sources for growth of Mtb CDC1551, pks1, With both glycerol and pyruvate as carbon sources for growth of Mtb CDC1551, pks1, pks15, fadD22, Rv2949c and fadD29 were significantly down-regulated in vitro at pH 7, in con- trast with the in vivo mimicking condition at pH 5.7. In the culture grown in pyruvate, log2 fold change values were found to be higher than in the sample grown in glycerol. When com- paring carbon sources, there is no significant difference in expression of the selected genes of interest at pH 7. However, at pH 5.7, a significant difference in lppX and fadD29 expression was seen, meaning that those genes are slightly downregulated in conditions where glycerol is the sole carbon source (Fig 6). As mentioned above, the conditions explored here allow com- parison between basal in vitro growth and in vivo growth inside phagosomes, where the pH is lower. It is known that the complex structure of the mycobacterial cell wall represents a major barrier to the entry of external protons [46]. Also, it is known that many acid-sensitive Mtb mutants present defects in genes involved in cell wall functions, and that several cell wall and lipid biosynthesis genes are differentially regulated by exposure to low pH [47]. Indeed, several of the regulatory genes reportedly responsive to acidic pH [48] were also found to be induced, namely pks2, pks3, pks4, papA1 and papA3 (Fig 6). 3.2 Analysis of expression data for a selected panel of genes enclosing pks1 and pks15 Expression analyses also enabled us to distinguish the expression signature of Mtb H37Rv from Mtb CDC1551 under control conditions, since the correlation factor between such strains was 0.2. This circumstance possibly reflects the impact of individual genomic differ- ences on the respective transcriptional signature, implying that from this point onward results relative to each of these strains should be treated individually. Nevertheless, when specifically comparing pks15 and pks1 CDSs between Mtb H37Rv and Mtb CDC1551, they show 99.93% Fig 5. Expression profiling of selected genes from Mycobacterium bovis, presented as log10 RPKM. Cut-off: 80% of similarity. Abbreviations: SRR1915476/7/8 –Mb grown in control conditions; SRR1915479/80/81 –Mb grown under starvation for 4 days; SRR1915482/3/4 –Mb grown under starvation for 10 days; SRR1915485/6/7 – Mb grown under starvation for 20 days; SRR1915488/89/90 –Mb after reintroduction of nutrients; SRR7221299/300/301 –Mb grown in control conditions; and SRR7221302/3/4 –Mb grown with addition of Vitamin B. https://doi.org/10.1371/journal.pone.0229700.g005 Fig 5. Expression profiling of selected genes from Mycobacterium bovis, presented as log10 RPKM. Cut-off: 80% of similarity. Abbreviations: SRR1915476/7/8 –Mb grown in control conditions; SRR1915479/80/81 –Mb grown under starvation for 4 days; SRR1915482/3/4 –Mb grown under starvation for 10 days; SRR1915485/6/7 – Mb grown under starvation for 20 days; SRR1915488/89/90 –Mb after reintroduction of nutrients; SRR7221299/300/301 –Mb grown in control conditions; and SRR7221302/3/4 –Mb grown with addition of Vitamin B. https://doi.org/10.1371/journal.pone.0229700.g005 https://doi.org/10.1371/journal.pone.0229700.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 10 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 sequence similarity, with only a single nucleotide polymorphism in pks15. Even so, further genomic differences may affect overall expression. sequence similarity, with only a single nucleotide polymorphism in pks15. Even so, further genomic differences may affect overall expression. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 3.3 Differential expression analyses Sig- nificant differential expression for ideR in high iron conditions was not detected, which is in conflict with reports describing its up-regulation under these conditions (Fig 6) [51, 52]. On the other hand, for whiB7 in low iron conditions, a strong up-regulation after 6 h of exposure has been previously described, a condition for which we do not have comparable data [53, 54]. However, after one week of exposure to low iron, we found that whiB7 was significantly down- regulated (Fig 6). while after 1 week of exposure, the six genes were extremely significantly down-regulated when compared to the culture grown in 0.4% glucose alone. These results are also supported by the direct comparison between the two cultures exposed to low iron concentration for dif- ferent periods of time, which show extremely significant up-regulation in the culture exposed for 1 day. Similar to the results from low iron concentration, exposure to high iron concentra- tion also showed that lppX, fadD22, Rv2949c and fadD29 were extremely significantly down- regulated and pks1 was significantly down-regulated (Fig 6). We noted that our selected set of genes was highly down-regulated under low iron concentrations, which could be related with the fact that iron takes part in several biological processes inside the cell, being required for cytochromes and other hemoproteins involved in oxygen metabolism. That means that iron deprivation can affect essential cellular processes, inducing a non-replicating state, thus reduc- ing synthesis of cell wall components [50]. Although these very interesting results were found, our data analysis does not support the currently described regulation of ideR and whiB7. Sig- nificant differential expression for ideR in high iron conditions was not detected, which is in conflict with reports describing its up-regulation under these conditions (Fig 6) [51, 52]. On the other hand, for whiB7 in low iron conditions, a strong up-regulation after 6 h of exposure has been previously described, a condition for which we do not have comparable data [53, 54]. However, after one week of exposure to low iron, we found that whiB7 was significantly down- regulated (Fig 6). Comparing results obtained when cells were grown in hypoxia with the first 4 days after reaeration [55], lppX, pks1, pks15, fadD22, Rv2949c and fadD29 were found to be extremely significantly down-regulated in hypoxia, with some of the highest log2 fold change values seen across all assays. 3.3 Differential expression analyses The comparisons between growth stages and carbon sources [49] indicated that, for both glycerol and pyruvate, lppX, pks1, pks15 and fadD29, are down-regulated in the stationary phase, when compared with the exponential phase. In the cells grown in long chain fatty acids, only pks1 and fadD29 display extremely significant down-regulation in the stationary phase, while lppX and pks15 also present significant fold changes (p-values are shown in S4 Table). By contrast, when bacteria were grown in dextrose, the complete set of our genes of interest was extremely significantly down-regulated in stationary phase, except for Rv2949c and fadD29 that presented lower levels of significance for down-regulation (Fig 6). Since we focused on genes that are part of the biosynthetic pathway of PGL, the significant down-regulation observed when the cultures entered stationary phase may be explained by the fact that synthe- sis of cell wall components is reduced at this time point. Comparing dextrose, the standard car- bon source used for in vitro growth, with long chain fatty acids mimicking the triacylglycerols available in human cells [49], we could only identify significant up-regulation of fadD29 dur- ing the exponential phase, and of pks15 and fadD22 in the stationary phase, for cell growth in long chain fatty acids. In the iron exposure assays, it was possible to observe that, after 1 day of growth under low iron concentration, only lppX, Rv2949c and fadD29 were significantly differentially expressed, PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 11 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 12 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 Fig 6. Differential gene expression represented in log2 fold change. Fold changes on the expression levels of genes previously described to be associated with the specific growth condition, along with our genes of interest, are identified by black outline. Blank squares represent non-significant fold-changes. 3.3 Differential expression analyses Abbreviations: C–Control condition; PBS–Mtb H37Rv grown with PBS; P-dep–Mtb H37Rv grown in phosphate depletion; INH4—Mtb H37Rv grown with INH for 4h; INH24—Mtb H37Rv grown with INH for 24h; STR4—Mtb H37Rv grown with STR for 4h; STR24—Mtb H37Rv grown with STR for 24h; EMB4—Mtb H37Rv grown with EMB for 4h; EMB24—Mtb H37Rv grown with EMB for 24h; RIF4—Mtb H37Rv grown with RIF for 4h; RIF24—Mtb H37Rv grown with RIF for 24h; Gly-pH 7—Mtb CDC1551 grown in glycerol at pH 7; Gly-pH 5.7—Mtb CDC1551 grown in glycerol at pH 5.7; Pyr-pH 7— Mtb CDC1551 grown in pyruvate at pH 7; Pyr-pH 5.7—Mtb CDC1551 grown in pyruvate at pH 5.7; HI—Mtb H37Rv—grown in high iron concentration; LI-1day–Mtb H37Rv grown in low iron concentration for 1 day; LI-1week- Mtb H37Rv grown in low iron concentration for 1 week; Hyp—Mtb H37Rv grown in hypoxia; (1–4) day—Mtb H37Rv (1–4) day(s) after reaeration; FS—Mtb H37Rv grown in long fatty acids at stationary phase; FE—Mtb H37Rv grown in long fatty acids at exponential phase; DS–Mtb H37Rv grown in dextrose at stationary phase; DE—Mtb H37Rv grown in dextrose at exponential phase; ED—Mtb H37Rv in early dormancy phase; MD–Mtb H37Rv in medium dormancy phase; LD—Mtb H37Rv in late dormancy phase. https://doi.org/10.1371/journal.pone.0229700.g006 while after 1 week of exposure, the six genes were extremely significantly down-regulated when compared to the culture grown in 0.4% glucose alone. These results are also supported by the direct comparison between the two cultures exposed to low iron concentration for dif- ferent periods of time, which show extremely significant up-regulation in the culture exposed for 1 day. Similar to the results from low iron concentration, exposure to high iron concentra- tion also showed that lppX, fadD22, Rv2949c and fadD29 were extremely significantly down- regulated and pks1 was significantly down-regulated (Fig 6). We noted that our selected set of genes was highly down-regulated under low iron concentrations, which could be related with the fact that iron takes part in several biological processes inside the cell, being required for cytochromes and other hemoproteins involved in oxygen metabolism. That means that iron deprivation can affect essential cellular processes, inducing a non-replicating state, thus reduc- ing synthesis of cell wall components [50]. Although these very interesting results were found, our data analysis does not support the currently described regulation of ideR and whiB7. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 3.3 Differential expression analyses Also, lppX and pks1 were found to be down-regulated with extremely signifi- cant differences from the first to the third and fourth days (Fig 6). Hypoxia induces many changes in mycobacteria. Both in microaerophilic and anaerobic cultures, Mtb is known to develop a thickened cell wall which may be important for adaptation to low oxygen conditions [56]. However, our selected set of genes was found to be extremely down-regulated under hyp- oxia, agreeing with previously published data [56], and suggesting that maybe the reported cell wall thickening does involve PGL production. On the contrary, some regulatory genes, such as members of the DosT regulon (namely devR, devS and dosT) were shown to be highly up-regu- lated in hypoxic conditions (Fig 6), which is also supported by previous studies [56–58]. In the publicly available experimental data used in our analyses, dormancy was induced by growing Mtb in K+-deficient medium and, after 14–15 days of culture, adding rifampicin (5 μg/ml) to eliminate dividing bacteria [59]. By comparing cells grown to three different states of dormancy with a culture grown to log phase in standard in vitro growth conditions, we obtained the highest fold changes across all assays. That comparison showed extremely signifi- cant down-regulation for all genes from our defined set in dormancy conditions. When PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 13 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 comparing between states of dormancy, it was possible to see that pks1 and pks15 were extremely significantly down-regulated in early dormancy when compared with mid-stage dormancy. Also, for all genes surveyed, extremely significant fold changes were found between medium and late dormancy (Fig 6). While in dormancy, mycobacteria enter a state of low met- abolic activity with alteration of gene regulation in order to accumulate triacylglycerols, loss of acid-fastness and a slower growth rate. These observations explain why our selected genes of interest showed a strong down-regulation under dormancy. In agreement with previous reports, and similarly to what happens under hypoxic conditions, devR, devS and dosT (mem- bers of the DosT regulon) were shown to be highly up-regulated during dormancy conditions (Fig 6) [56, 57, 60]. We analysed data from drug-induced stress assays that were performed by growing Mtb under exposure to 0.5 μg/ml of INH, 0.5 μg/ml of STR, 1 μg/ml of EMB, and 0.25 μg/ml of RIF, separately [61]. 3.3 Differential expression analyses For each drug, two time-points, 4h and 24h, were compared with refer- ence to the control (no drug). Under exposure to INH, lppX, pks1, pks15, fadD22, Rv2949c and fadD29 were significantly up-regulated at both 4h and 24h. Concerning target genes for INH exposure, inhA, fabG1, kasA and ahpC were up-regulated at both 4h and 24h; on the contrary, oxyR’ was found to be significantly down-regulated only at 4h of exposure. For katG and embB, the data showed significant but slight up-regulation (Fig 6). Concerning STR exposure, all genes under analysis were found to be up-regulated at both 4h and 24h (lppX, pks1, pks15, fadD22, Rv2949c and fadD29), by comparison with rpsL, rrs and gid, which showed significant differential expression but with small fold changes (Fig 6). When cells were exposed to EMB, lppX, pks1, pks15, fadD22, Rv2949c and fadD29 were significantly up-regulated at both 4h and 24h. The genes embA, embB and embC were also significantly up-regulated in the cultures exposed to EMB, which agrees with previous published data and validates our findings (Fig 6) [61]. The exposure to RIF leads to the up-regulation of the selected panel of genes, with signifi- cant fold changes at both 4h and 24h, with more pronounced fold changes after 24h of expo- sure (Fig 6). For M. bovis BCG, data from starvation assays were collected after 4, 10 and 20 days and after reintroduction of nutrients [62]. For the first three conditions, all genes of interest were up-regulated [lppX (p-value = 1.5462x10-42; p-value = 1.5347x10-17; p-value = 3.4984x10-18), pks15/1 (p-value = 3.2164x10-30; p-value = 1.3595x10-8; p-value = 5.5910x10-8), Mb2973c (p- value = 3.7632x10-12; p-value = 8.8716x10-5; p-value = 6.1298x10-10) and fadD29 (p-value = 2.6748x10-5; p-value = 0.0107; p-value = 3.3421x10-7)] when compared to the control. The dif- ferential expression of genes reported to play a regulatory role under starvation conditions in Mtb, such as Mb3614c (p-value = 2.7774x10-5), Mb2076 (p-value = 0.0034), relA (p-value = 0.0127), prrA (p-value = 0.0125), senX3 (p-value = 5.9579x10-5) and regX3 (p-value = 7.6639x10-12), was also evaluated, whereby after 20 days of starvation up-regulation of these genes was noted (Fig 7). In the assays involving the introduction of vitamin B [63], no differen- tial expression was evidenced. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 4 Concluding remarks Also, pks1 and pks15 both seem to be positively regulated by sigK and negatively regulated by sigE, based on algorithmic predictions [26]. By clustering the expression data from more than 100 RNA-seq datasets for Mtb, in a robust set of 40 growth conditions, it was possible to correlate pks1 expression with that of fadD22 and pks6. With a closer analysis, focused on the correlation coefficient values, we were able to confirm that all genes thought to belong to the putative polycistronic structure present similar expression profiles. Correlations between these genes were shown to be above 0.70, except for pks15. Also, we found that the pks1 correlation coefficient values were above 0.80 with pks6 and pks12. As noted, pks15 did not show such high correlation values, although this is mostly due to the presence of several null RPKM values and not to the dissimilarity of the expression profile, since the reads mapped to pks1 in strains without the pks1/15 frameshift (e.g. BCG). In this integrative analysis, it was also possible to link genes encoding σ factors with the selected genes of interest; e.g. we found a correlation coefficient of 0.8 between sigK and lppX. While these results must be treated with caution, since σ gene expression may not reflect effective fac- tor activity, they offer new insights into the potential function of sigma factors whose func- tional role remains unclear, such as SigK. As referred to previously, mycobacteria are subjected to several stress conditions while inside macrophages. By analysing the differential expression of lppX, pks1, pks15, fadD22, Rv2949c and fadD29, it was possible to define under which conditions these genes are posi- tively or negatively regulated. We analysed expression levels of strains grown under a diverse set of conditions, namely pH, carbon source, hypoxia and phosphate depletion for Mtb CDC1551, growth phase, exposure to limiting or excessive iron concentration, hypoxia, dor- mancy, and antibiotic exposure for Mtb H37Rv. This analysis revealed that our selected genes of interest are up-regulated at acidic pH (in Mtb CDC1551) and antibiotic exposure and down-regulated at stationary phase (in Mtb CDC1551), under hypoxia and dormancy, and at both low and high iron concentrations. The combination of two sets of data, i.e. clustering of genes by expression data and differential expression analysis, suggests that fadD29 may be set apart from the other genes in the set. 4 Concluding remarks Mtb virulence is related to its ability to survive inside macrophages. During infection, macro- phages engulf bacilli, constituting a hostile intracellular environment for bacterial replication. Yet Mtb can overcome these macrophage defences in a coordinated and complex process, allowing intracellular growth and persistence. Recent models of persistence inside the host point to bacterial subpopulations in a latent state that maintain their ability to reactivate upon host immunosuppression [17, 64, 65]. PGL is an important Mtb virulence factor and its pro- duction involves several PKS, such as pks1 and pks15, which have been shown to have a critical PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 14 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 al.pone.0229700 May 7, 2020 g/10.1371/journal.pone.0229700 May 7, 2020 15 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE Revisiting the RNA signature of pks15/1 Fig 7. Differential gene expression represented in log2 fold change. Blank squares represent non-significant fold- changes. C–Control condition; Starv4day–Mb grown under starvation for 4 days; Starv10day–Mb grown under starvation for 10 days; and Starv20day–Mb grown under starvation for 20 days. https://doi.org/10.1371/journal.pone.0229700.g007 Fig 7. Differential gene expression represented in log2 fold change. Blank squares represent non-significant fold- changes. C–Control condition; Starv4day–Mb grown under starvation for 4 days; Starv10day–Mb grown under starvation for 10 days; and Starv20day–Mb grown under starvation for 20 days. https://doi.org/10.1371/journal.pone.0229700.g007 role in PGL biosynthesis, since the presence of a frameshift mutation that disrupts the pks15/1 CDS was associated with the lack of PGL production in Mtb [13]. Also, it is known that the ref- erence strain for pathogenic mycobacteria, Mtb H37Rv, as well as the common Mtb CDC1551 strain, also contain this frameshift mutation, while other Mtb strains circulating across the world contain an intact pks1/15 locus. Inferring the regulatory pattern of pks1 and pks15, using a genome-wide approach by analysis of RNA-seq data, could unveil the regulatory patterns controlling phenolphtiocerol and phenolglycolipid production in pathogenic mycobacteria and, indirectly, shed light on the downstream processes in which these molecules participate. The analysis of expression data gathered from publicly available sources suggested that the target genes selected for this work, pks1 and pks15, may be transcribed as a polycistronic unit composed by three to six genes located both upstream of pks15 and downstream of pks1. All these gene products, except FadD29’, take part in the biosynthetic pathway of the phenolphtio- cerol moiety of PGL. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 4 Concluding remarks Also, in one of the conditions, fadD29 expression seemed to diverge from that of lppX, pks1, pks15 and Rv2949c, and in another, both Rv2949c and fadD29 expression profiles diverge from pks1 and pks15. Using differential expression analysis, we were also able to confirm that sigK shares the expression profile with the selected genes of interest in 88% of the exploited conditions with significant fold-changes; almost the same PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 16 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 Fig 8. Schematic representation of the proposed polycistronic structure model. The pks1, pks15 and fadD22 genes are represented with putative regulation from σD (positive), and σB and σE (negative). htt //d i /10 1371/j l 0229700 008 e proposed polycistronic structure model. The pks1, pks15 and fadD22 genes are represented with putative regulation from σ Fig 8. Schematic representation of the proposed polycistronic structure model. The pks1, pks15 and fadD22 (positive), and σB and σE (negative). Fig 8. Schematic representation of the proposed polycistronic structure model. The pks1, pks15 and fadD22 genes are represented with putative regulation from σD (positive), and σB and σE (negative). https://doi.org/10.1371/journal.pone.0229700.g008 https://doi.org/10.1371/journal.pone.0229700.g008 percentage is also verified for sigJ. On the contrary, sigE presents approximately 90% of expres- sion profile dissimilarity with the selected panel of genes in the conditions under analysis, with significant fold-changes, as well as sigB. While for Mtb we were able to gather a robust set of data from previous studies, for M. bovis BCG it was only possible to collect data from seven growth conditions, of which three represent regular in vitro growth, three represent growth under starvation at three time-points, and one represents the addition of vitamin B. This smaller data set led to a lower number of clusters. Interestingly, the members of the putative polycistronic structure, except for lppX, cluster in the same group. This analysis is in full agreement with the one performed for Mtb as most genes of interest are contained in the same cluster. Also, expression values of pks1 appear to be similar in control assays performed in Mb and in Mtb H37Rv for which relative gene expression has a correlation of 0.78, indicating that although PGL production may be abol- ished in Mtb H37Rv, the pks1 transcript is similarly expressed in both Mtb H37Rv and BCG, suggesting a secondary role for this transcript. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 4 Concluding remarks Building on the information from previously published reports and the transcriptome data we retrieved, compiled and analysed here, we propose a regulatory model for pks1 and pks15. In this model, we use a conservative approach selecting genes coherently sharing expression patterns and exhibiting functional similarity when considering a polycistronic structure model. For this model, we selected a set of four genes, pks1, pks15, fadD22 and Rv2949c, that fulfil the criteria stated above. Based on differential expression analysis, we also selected a set of three σ factors (σD, and σB and σE) that seem to be involved in the regulation of pks1, pks15 and fadD22 expression (Fig 8). Both σK and σE were previously computationally predicted to regulate the genes belonging to this polycistronic structure according to mRNA-based expres- sion levels [66], which is coherent with the expression data analyses reported herein. However, σD and σB exhibited similar expression patterns and thus are also included in our proposed model. The genes encoding factors σD and σK were shown to be down-regulated under hyp- oxia and dormancy, as well as in stationary phase. On the contrary, the genes encoding σB and PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 17 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 σE factors were up-regulated under the same conditions, with sigB being previously shown to be up-regulated under hypoxia [18]. While σD and σK, that appear to positively regulate the selected genes of interest, belong to the lower level of σ factors regulation, the σB and σE factors, which putatively regulate in a negative way the selected genes of interest, belong to a hierar- chically upper level of regulation. Also supporting the hypothesis that this putative polycis- tronic structure is regulated by σB and σD are some studies analysing the expression profile of knock-out mutants for those sigma factors. For σB it was reported by Lee and coworkers (2008) that genes encoding for proteins involved in cell wall processes are highly upregulated in complementation mutants, which is in contrast with our data analyses [67]. On the other hand, a ΔsigD mutant showed reduced expression of genes involved in the synthesis of phos- pholipids and fatty acids [68]. Author Contributions Writing – original draft: Beatriz Ramos, Mo´nica V. Cunha. Writing – review & editing: Stephen V. Gordon. Writing – review & editing: Stephen V. Gordon. 4 Concluding remarks Even though our data analyses suggests that σK is a regulator of this set of genes, previous analyses of an Mtb ΔsigK mutant reported no differential expression of our genes of interest [69], leading us to exclude this sigma factor as a hypothetical regulator in our model. Further experimental validation of our findings and proposed regulatory model could be achieved via ‘classical’ experiments such as Northern blots, construction of pks1 and pks15 knock-out mutants, and transcriptional fusions with reporter genes of the upstream regions of the constituent genes in this putative polycistronic unit in order to unveil the exact location and activity of the promoter. Analysis of pks1 and pks15 expression in mutant strains of their putative regulators under several growth conditions would further serve to validate the global networks that exert effects on pks1 and pks15 activities, genes that play a crucial role in PGL production and thus act at the interface of host-pathogen interaction. Supporting information S1 Table. (XLSX) S2 Table. (XLSX) S3 Table. (XLSX) S4 Table. (XLSX) 2. Onwueme KC, Vos CJ, Zurita J, Ferreras JA, Quadri LEN. The dimycocerosate ester polyketide viru- lence factors of mycobacteria. Progress in Lipid Research. 2005; 44(5):259–302. https://doi.org/10. 1016/j.plipres.2005.07.001 PMID: 16115688 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 References 1. Organization WH. Global Tuberculosis Report 2018. 2018. Organization WH. Global Tuberculosis Report 2018. 201 2. Onwueme KC, Vos CJ, Zurita J, Ferreras JA, Quadri LEN. The dimycocerosate ester polyketide viru- lence factors of mycobacteria. Progress in Lipid Research. 2005; 44(5):259–302. https://doi.org/10. 1016/j.plipres.2005.07.001 PMID: 16115688 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 18 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 3. Chiaradia L, Lefebvre C, Parra J, Marcoux J, Burlet-Schiltz O, Etienne G, et al. Dissecting the mycobac- terial cell envelope and defining the composition of the native mycomembrane. Scientific Reports. 2017; 7(1):12807. https://doi.org/10.1038/s41598-017-12718-4 PMID: 28993692 4. Ferreras JA, Stirrett KL, Lu X, Ryu J-S, Soll CE, Tan DS, et al. Mycobacterial PGL virulence factor bio- synthesis: mechanism and small-molecule inhibition of polyketide chain initiation. Chemistry & biology. 2008; 15(1):51–61. 5. Astarie-Dequeker C, Le Guyader L, Malaga W, Seaphanh F-K, Chalut C, Lopez A, et al. Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids. PLoS Pathog. 2009; 5(2):e1000289. https://doi.org/10.1371/ journal.ppat.1000289 PMID: 19197369 6. Chavadi SS, Edupuganti UR, Vergnolle O, Fatima I, Singh SM, Soll CE, et al. Inactivation of tesA reduces cell wall lipid production and increases drug susceptibility in mycobacteria. The Journal of bio- logical chemistry. 2011; 286(28):24616–25. https://doi.org/10.1074/jbc.M111.247601 PMID: 21592957 7. Rousseau C, Winter N, Pivert E, Bordat Y, Neyrolles O, Ave P, et al. Production of phthiocerol dimyco- cerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection. Cellular microbiol- ogy. 2004; 6(3):277–87. https://doi.org/10.1046/j.1462-5822.2004.00368.x PMID: 14764111 8. Pang JM, Layre E, Sweet L, Sherrid A, Moody DB, Ojha A, et al. The polyketide Pks1 contributes to bio- film formation in Mycobacterium tuberculosis. J Bacteriol. 2012; 194(3):715–21. https://doi.org/10.1128/ JB.06304-11 PMID: 22123254 9. Mohandas P, Budell WC, Mueller E, Au A, Bythrow GV, Quadri LEN. Pleiotropic consequences of gene knockouts in the phthiocerol dimycocerosate and phenolic glycolipid biosynthetic gene cluster of the opportunistic human pathogen Mycobacterium marinum. FEMS Microbiol Lett. 2016; 363(5):fnw016. https://doi.org/10.1093/femsle/fnw016 PMID: 26818253 10. Quadri LE. Biosynthesis of mycobacterial lipids by polyketide synthases and beyond. Crit Rev Biochem Mol Biol. 2014; 49(3):179–211. https://doi.org/10.3109/10409238.2014.896859 PMID: 24625105 11. He W, Soll CE, Chavadi SS, Zhang G, Warren JD, Quadri LEN. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 References The MycoBrowser portal: a comprehensive and manually annotated resource for mycobacterial genomes. Tuberculosis (Edinb). 2011; 91(1):8–13. 23. Database Resources of the National Center for Biotechnology Information. Nucleic Acids Research. 2017; 45(D1):D12–D7. https://doi.org/10.1093/nar/gkw1071 PMID: 27899561 24. Reddy TB, Riley R, Wymore F, Montgomery P, DeCaprio D, Engels R, et al. TB database: an integrated platform for tuberculosis research. Nucleic Acids Res. 2009; 37(Database issue):D499–508. https://doi. org/10.1093/nar/gkn652 PMID: 18835847 25. Galagan JE, Sisk P, Stolte C, Weiner B, Koehrsen M, Wymore F, et al. TB database 2010: overview and update. Tuberculosis (Edinb). 2010; 90(4):225–35. 26. Turkarslan S, Peterson EJ, Rustad TR, Minch KJ, Reiss DJ, Morrison R, et al. A comprehensive map of genome-wide gene regulation in Mycobacterium tuberculosis. Sci Data. 2015; 2:150010. https://doi.org/ 10.1038/sdata.2015.10 PMID: 25977815 27. Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recogni- tion and translation initiation site identification. BMC Bioinformatics. 2010; 11(1):119. 28. Oberto J. SyntTax: a web server linking synteny to prokaryotic taxonomy. BMC Bioinformatics. 2013; 14:4. https://doi.org/10.1186/1471-2105-14-4 PMID: 23323735 29. Leinonen R, Sugawara H, Shumway M. The Sequence Read Archive. Nucleic Acids Research. 2011; 39(suppl_1):D19–D21. 30. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expres- sion analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protocols. 2012; 7(3):562–78. https://doi.org/10.1038/nprot.2012.016 PMID: 22383036 31. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of tran- scriptomes in the presence of insertions, deletions and gene fusions. Genome Biology. 2013; 14(4): R36. https://doi.org/10.1186/gb-2013-14-4-r36 PMID: 23618408 32. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentia- tion. Nat Biotech. 2010; 28(5):511–5. 33. Metsalu T, Vilo J. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Com- ponent Analysis and heatmap. Nucleic Acids Res. 2015; 43(W1):W566–70. https://doi.org/10.1093/nar/ gkv468 PMID: 25969447 34. Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics. 2011; 27(3):431–2. https://doi.org/10.1093/bioinformatics/ btq675 PMID: 21149340 35. Saito R, Smoot ME, Ono K, Ruscheinski J, Wang PL, Lotia S, et al. A travel guide to Cytoscape plugins. Nat Methods. 2012; 9(11):1069–76. https://doi.org/10.1038/nmeth.2212 PMID: 23132118 36. Doncheva NT, Assenov Y, Domingues FS, Albrecht M. References Cooperation between a Coenzyme A- Independent Stand-Alone Initiation Module and an Iterative Type I Polyketide Synthase during Synthe- sis of Mycobacterial Phenolic Glycolipids. Journal of the American Chemical Society. 2009; 131 (46):16744–50. https://doi.org/10.1021/ja904792q PMID: 19799378 12. Ridley CP, Lee HY, Khosla C. Evolution of polyketide synthases in bacteria. Proc Natl Acad Sci U S A. 2008; 105(12):4595–600. https://doi.org/10.1073/pnas.0710107105 PMID: 18250311 13. Constant P, Perez E, Malaga W, Laneelle MA, Saurel O, Daffe M, et al. Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene. The Journal of biological chemistry. 2002; 277 (41):38148–58. https://doi.org/10.1074/jbc.M206538200 PMID: 12138124 14. Daffe M, Crick DC, Jackson M. Genetics of Capsular Polysaccharides and Cell Envelope (Glyco)lipids. Microbiol Spectr. 2014; 2(4):Mgm2-0021–2013. 15. Simeone R, Leger M, Constant P, Malaga W, Marrakchi H, Daffe M, et al. Delineation of the roles of FadD22, FadD26 and FadD29 in the biosynthesis of phthiocerol dimycocerosates and related com- pounds in Mycobacterium tuberculosis. Febs j. 2010; 277(12):2715–25. https://doi.org/10.1111/j.1742- 464X.2010.07688.x PMID: 20553505 16. Vergnolle O, Chavadi SS, Edupuganti UR, Mohandas P, Chan C, Zeng J, et al. Biosynthesis of Cell Envelope-Associated Phenolic Glycolipids in Mycobacterium marinum. J Bacteriol. 2015; 197(6):1040– 50. https://doi.org/10.1128/JB.02546-14 PMID: 25561717 17. Sia JK, Georgieva M, Rengarajan J. Innate Immune Defenses in Human Tuberculosis: An Overview of the Interactions between Mycobacterium tuberculosis and Innate Immune Cells. J Immunol Res. 2015; 2015:747543. https://doi.org/10.1155/2015/747543 PMID: 26258152 18. Flentie K, Garner AL, Stallings CL. The Mycobacterium tuberculosis transcription machinery: ready to respond to host attacks. J Bacteriol. 2016. 19. Sachdeva P, Misra R, Tyagi AK, Singh Y. The sigma factors of Mycobacterium tuberculosis: regula- tion of the regulators. FEBS Journal. 2010; 277(3):605–26. https://doi.org/10.1111/j.1742-4658.2009. 07479.x PMID: 19951358 20. Chauhan R, Ravi J, Datta P, Chen T, Schnappinger D, Bassler KE, et al. Reconstruction and topological characterization of the sigma factor regulatory network of Mycobacterium tuberculosis. Nat Commun. 2016; 7:11062. https://doi.org/10.1038/ncomms11062 PMID: 27029515 21. Smith T, Wolff KA, Nguyen L. Molecular Biology of Drug Resistance in Mycobacterium tuberculosis. Current topics in microbiology and immunology. 2013; 374:53–80. https://doi.org/10.1007/82_2012_ 279 PMID: 23179675 19 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE Revisiting the RNA signature of pks15/1 22. Kapopoulou A, Lew JM, Cole ST. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 References Topological analysis and interactive visualiza- tion of biological networks and protein structures. Nature Protocols. 2012; 7(4):670–85. https://doi.org/ 10.1038/nprot.2012.004 PMID: 22422314 37. Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31(2):166–9. https://doi.org/10.1093/bioinformatics/btu638 PMID: 25260700 38. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12):550. https://doi.org/10.1186/s13059-014-0550-8 PMID: 25516281 39. Afgan E, Baker D, van den Beek M, Blankenberg D, Bouvier D, Čech M, et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Research. 2016; 44(W1):W3–W10. https://doi.org/10.1093/nar/gkw343 PMID: 27137889 40. Sulzenbacher G, Canaan S, Bordat Y, Neyrolles O, Stadthagen G, Roig-Zamboni V, et al. LppX is a lipoprotein required for the translocation of phthiocerol dimycocerosates to the surface of Mycobacte- rium tuberculosis. The EMBO journal. 2006; 25(7):1436–44. https://doi.org/10.1038/sj.emboj.7601048 PMID: 16541102 41. Liu D, Hunt M, Tsai IJ. Inferring synteny between genome assemblies: a systematic evaluation. BMC Bioinformatics. 2018; 19(1):26. https://doi.org/10.1186/s12859-018-2026-4 PMID: 29382321 42. Dehal PS, Joachimiak MP, Price MN, Bates JT, Baumohl JK, Chivian D, et al. MicrobesOnline: an inte- grated portal for comparative and functional genomics. Nucleic Acids Research. 2010; 38(Database issue):D396–D400. https://doi.org/10.1093/nar/gkp919 PMID: 19906701 43. Dubey VS, Sirakova TD, Kolattukudy PE. Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 20 / 22 PLOS ONE Revisiting the RNA signature of pks15/1 and causes cell aggregation. Mol Microbiol. 2002; 45(5):1451–9. https://doi.org/10.1046/j.1365-2958. 2002.03119.x PMID: 12207710 and causes cell aggregation. Mol Microbiol. 2002; 45(5):1451–9. https://doi.org/10.1046/j.1365-2958 2002.03119.x PMID: 12207710 44. Bonneau R, Reiss DJ, Shannon P, Facciotti M, Hood L, Baliga NS, et al. The Inferelator: an algorithm for learning parsimonious regulatory networks from systems-biology data sets de novo. Genome Biol- ogy. 2006; 7(5):R36–R. https://doi.org/10.1186/gb-2006-7-5-r36 PMID: 16686963 45. Matern WM, Rifat D, Bader JS, Karakousis PC. Gene Enrichment Analysis Reveals Major Regulators of Mycobacterium tuberculosis Gene Expression in Two Models of Antibiotic Tolerance. Front Microbiol. 2018; 9:610. https://doi.org/10.3389/fmicb.2018.00610 PMID: 29670589 46. Ehrt S, Schnappinger D. Mycobacterial survival strategies in the phagosome: defence against host stresses. Cellular microbiology. 2009; 11(8):1170–8. https://doi.org/10.1111/j.1462-5822.2009.01335.x PMID: 19438516 47. Vandal OH, Nathan CF, Ehrt S. Acid Resistance in Mycobacterium tuberculosis. J Bacteriol. 2009; 191 (15):4714–21. https://doi.org/10.1128/JB.00305-09 PMID: 19465648 48. Baker JJ, Johnson BK, Abramovitch RB. References Slow growth of Mycobacterium tuberculosis at acidic pH is reg- ulated by phoPR and host-associated carbon sources. Molecular Microbiology. 2014; 94(1):56–69. https://doi.org/10.1111/mmi.12688 PMID: 24975990 49. Rodrı´guez JG, Herna´ndez AC, Helguera-Repetto C, Aguilar Ayala D, Guadarrama-Medina R, Anzo´la JM, et al. Global Adaptation to a Lipid Environment Triggers the Dormancy-Related Phenotype of Myco- bacterium tuberculosis. mBio. 2014; 5(3). 50. Sritharan M. Iron Homeostasis in Mycobacterium tuberculosis: Mechanistic Insights into Siderophore- Mediated Iron Uptake. J Bacteriol. 2016; 198(18):2399–409. https://doi.org/10.1128/JB.00359-16 PMID: 27402628 51. Pandey R, Rodriguez GM. IdeR is required for iron homeostasis and virulence in Mycobacterium tuber- culosis. Mol Microbiol. 2014; 91(1):98–109. https://doi.org/10.1111/mmi.12441 PMID: 24205844 52. Rodriguez GM, Voskuil MI, Gold B, Schoolnik GK, Smith I. ideR, An essential gene in mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect Immun. 2002; 70(7):3371–81. https://doi.org/10.1128/IAI.70.7.3371-3381.2002 PMID: 12065475 53. Geiman DE, Raghunand TR, Agarwal N, Bishai WR. Differential gene expression in response to expo- sure to antimycobacterial agents and other stress conditions among seven Mycobacterium tuberculosis whiB-like genes. Antimicrob Agents Chemother. 2006; 50(8):2836–41. https://doi.org/10.1128/AAC. 00295-06 PMID: 16870781 54. Burian J, Ramon-Garcia S, Sweet G, Gomez-Velasco A, Av-Gay Y, Thompson CJ. The mycobacterial transcriptional regulator whiB7 gene links redox homeostasis and intrinsic antibiotic resistance. The Journal of biological chemistry. 2012; 287(1):299–310. https://doi.org/10.1074/jbc.M111.302588 PMID: 22069311 55. Du P, Sohaskey CD, Shi L. Transcriptional and Physiological Changes during Mycobacterium tubercu- losis Reactivation from Non-replicating Persistence. Front Microbiol. 2016; 7:1346. https://doi.org/10. 3389/fmicb.2016.01346 PMID: 27630619 56. Alnimr AM. Dormancy models for Mycobacterium tuberculosis: A minireview. Braz J Microbiol. 2015; 46 (3):641–7. https://doi.org/10.1590/S1517-838246320140507 PMID: 26413043 57. Leistikow RL, Morton RA, Bartek IL, Frimpong I, Wagner K, Voskuil MI. The Mycobacterium tuberculo- sis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dor- mancy. J Bacteriol. 2010; 192(6):1662–70. https://doi.org/10.1128/JB.00926-09 PMID: 20023019 58. Rustad TR, Harrell MI, Liao R, Sherman DR. The Enduring Hypoxic Response of Mycobacterium tuber- culosis. PLoS ONE. 2008; 3(1):e1502. https://doi.org/10.1371/journal.pone.0001502 PMID: 18231589 59. Ignatov DV, Salina EG, Fursov MV, Skvortsov TA, Azhikina TL, Kaprelyants AS. Dormant non-cultur- able Mycobacterium tuberculosis retains stable low-abundant mRNA. BMC Genomics. 2015; 16:954. https://doi.org/10.1186/s12864-015-2197-6 PMID: 26573524 60. Peddireddy V, Doddam SN, Ahmed N. Mycobacterial Dormancy Systems and Host Responses in Tuberculosis. Front Immunol. 2017; 8:84. https://doi.org/10.3389/fimmu.2017.00084 PMID: 28261197 61. Boot M, Commandeur S, Subudhi AK, Bahira M, Smith TC 2nd, Abdallah AM, et al. PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 References Accelerating Early Antituberculosis Drug Discovery by Creating Mycobacterial Indicator Strains That Predict Mode of Action. Antimicrob Agents Chemother. 2018; 62(7). 62. BCC and BMC core MIT. Ketogenesis: An Achilles’ heel of starvation-selected persistent mycobacteria. NCBI SRA2015. 63. Song N, Li Z, Cui Z, Chen L, Cui Y, Dang G, et al. The prominent alteration in transcriptome and meta- bolome of Mycobacterium bovis BCG str. Tokyo 172 induced by vitamin B1. BMC Microbiol. 2019; 19 (1):104. https://doi.org/10.1186/s12866-019-1492-9 PMID: 31117936 21 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0229700 May 7, 2020 PLOS ONE Revisiting the RNA signature of pks15/1 64. Korbel DS, Schneider BE, Schaible UE. Innate immunity in tuberculosis: myths and truth. Microbes Infect. 2008; 10(9):995–1004. https://doi.org/10.1016/j.micinf.2008.07.039 PMID: 18762264 65. Sundaramurthy V, Pieters J. Interactions of pathogenic mycobacteria with host macrophages. Microbes Infect. 2007; 9(14):1671–9. 66. Reiss DJ, Plaisier CL, Wu W-J, Baliga NS. cMonkey2: Automated, systematic, integrated detection of co-regulated gene modules for any organism. Nucleic acids research. 2015; 43(13):e87–e. https://doi. org/10.1093/nar/gkv300 PMID: 25873626 67. Lee J-H, Karakousis PC, Bishai WR. Roles of SigB and SigF in the Mycobacterium tuberculosis Sigma Factor Network. Journal of Bacteriology. 2008; 190(2):699–707. https://doi.org/10.1128/JB.01273-07 PMID: 17993538 68. Calamita H, Ko C, Tyagi S, Yoshimatsu T, Morrison NE, Bishai WR. The Mycobacterium tuberculosis SigD sigma factor controls the expression of ribosome-associated gene products in stationary phase and is required for full virulence. Cellular microbiology. 2005; 7(2):233–44. https://doi.org/10.1111/j. 1462-5822.2004.00454.x PMID: 15659067 69. Saïd-Salim B, Mostowy S, Kristof AS, Behr MA. Mutations in Mycobacterium tuberculosis Rv0444c, the gene encoding anti-SigK, explain high level expression of MPB70 and MPB83 in Mycobacterium bovis. 2006; 62(5):1251–63. 22 / 22
https://openalex.org/W2058349512	https://ccforum.biomedcentral.com/track/pdf/10.1186/cc13297	English	null	A liberal strategy of red blood cell transfusion reduces cardiovascular complications in older patients undergoing cardiac surgery	Critical care	2,014	cc-by	247,269	References References 1. Vincent et al.: Adverse events in British hospitals. BMJ 2001, 322:517-519. 1. Vincent et al.: Adverse events in British hospitals. BMJ 2001, 322:517-519. p p Results We assessed 129 prescriptions in the fi rst round, and 111 after intervention, demonstrating a 70% improvement in safe prescribing. Only 24% of prescriptions initially fulfi lled best practice criteria, improving to 94% afterwards. We also reduced the number of infusions running without prescription, 7 (6%) versus 24 (19%). See Figures 1 and 2. Conclusion Our audit supports the need for standardised prescribing practices within critical care, especially when dealing with potentially harmful vasoactive/sedative drugs. With a small, cost-eff ective intervention (£20 for 6,200 stickers), we improved prescribing accuracy, and thus patient safety in intensive care. 2. Building a Safer NHS for Patients: Improving Medication Safety [http://webarchive.nationalarchives.gov.uk/+/www.dh.gov.uk/en/ Publicationsandstatistics/Publications/PublicationsPolicyAndGuidance/ DH_4071443] 3. Safe Prescribing [http://www.medicalprotection.org/uk/england-factsheets/ safe-prescribing] 3. Safe Prescribing [http://www.medicalprotection.org/uk/england-factsheets/ safe-prescribing] P2 The Limpet controlled drug cabinet alarm and camera M Mariyaselvam, D Pearson, P Moondi, P Young Queen Elizabeth Hospital NHS Trust, Kings Lynn, UK Critical Care 2014, 18(Suppl 1):P2 (doi: 10.1186/cc13192) P2 The Limpet controlled drug cabinet alarm and camera M Mariyaselvam, D Pearson, P Moondi, P Young Queen Elizabeth Hospital NHS Trust, Kings Lynn, UK Critical Care 2014, 18(Suppl 1):P2 (doi: 10.1186/cc13192) © 2010 BioMed Central Ltd Figure 1 (abstract P1). Accuracy of prescriptions before intervention. © 2014 BioMed Central Ltd Figure 1 (abstract P1). Accuracy of prescriptions before intervention. Introduction The theft and tampering of controlled drugs (CDs) remains a prevalent patient safety issue. Sadly there are numerous reports of critical care staff stealing CDs for personal use or fi nancial gain and notably there have been some cases where CDs have been substituted for other medications in order to delay detection of the theft. This creates both the hazard of medication errors and potentially exposes patients to opioid intoxicated healthcare workers. As most critical care staff have access to CDs, when drugs are found to be missing it can be diffi cult to identify the perpetrators. Therefore the implementation of a deterrent which also improves the methods of detection is warranted. Methods The Limpet, a device which incorporates a proximity sensor and a camera unit, was installed within the CD cupboard of the critical care unit. Whenever the cupboard was accessed the date and time were recorded and a photograph was taken to identify the staff member. MEETING ABSTRACTS MEETING ABSTRACTS Minimising prescribing errors in the ICU DJ M li S S h DJ Melia, S Saha Introduction We aimed to audit the prescribing practice on a busy 14-bedd general ICU, and develop standardised practices and tools to improve safety. Prescribing errors occur as commonly as in 10% of UK hospital admissions, costing 8.5 extra bed days per admission, and costing the National Health Service an estimated £1 billion per annum [1]. The majority of these mistakes are avoidable [2]. Methods We audited the daily infusion charts of all patients in three separate spot checks, over 1 week. We assessed all aspects of prescriptions that make them legal and valid, in accordance with national guidance [3]. New procedures were introduced, which included a standardised prescription sticker, with common, pre- printed, infusion prescriptions on (noradrenaline, propofol, and so forth), and education on using the new prescription stickers. A month later the audit process was repeated.i 34th International Symposium on Intensive Care and Emergency Medicine Brussels, Belgium, 18-21 March 2014 Publication of this supplement has been supported by ISICEM. Figure 2 (abstract P1). Accuracy of prescriptions after intervention. P1 Minimising prescribing errors in the ICU DJ Melia, S Saha Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P1 (doi: 10.1186/cc13191) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle J Barnes, R Darke, A Irving, S Wright Freeman Hospital, Newcastle upon Tyne, UK Critical Care 2014, 18(Suppl 1):P4 (doi: 10.1186/cc13194) Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle p Conclusion When CDs are missing it can be extremely stressful for the staff involved. Those who have access to CDs may feel unfairly scrutinised and the potential for false accusation exists. Investigating the theft of CDs is costly and usually involves pharmacists, managers and the police. Until the issue is resolved, potential suspects are usually suspended from work, leading to disruptions in patient care. The utility of the Limpet in modifying staff behaviour by reducing the number of occasions and the duration of time that open drug cupboards are left unattended, has previously been demonstrated [1]. By providing the facility to determine exactly who accessed each CD cupboard at which time, this initial study has shown the benefi ts of the Limpet as a tool for detecting theft. Therefore the installation of the Limpet mitigates the diffi culties of investigating CD theft and is likely to prove an eff ective deterrent. f Introduction The purpose of this study was to assess our practice in identifying and managing inadvertent hypothermia and whether this could be improved by education and introduction of a protocol. Hypothermia is associated with multiple physiological abnormalities including reduced myocardial contractility, peripheral vasoconstriction, increased infection risk and impaired coagulation [1]. Inadvertent hypothermia may therefore be an avoidable risk factor in the critically ill. The UK National Institute of Clinical Excellence has issued guidance for avoidance of inadvertent hypothermia (temperature <36°C) during the perioperative period. We audited our practice against three standards from these guidelines: all patients should have at least 4-hourly temperature observations; no patient should become inadvertently hypothermic; and all inadvertently hypothermic patients should be rewarmed. Reference 1. The Limpet drug cabinet alarm: technology for safer drug stewardship [abstract and poster presentation]. Presented at International Forum on Quality and Patient Safety; April 16-19 2013; London. 1. The Limpet drug cabinet alarm: technology for safer drug stewardship [abstract and poster presentation]. Presented at International Forum on Quality and Patient Safety; April 16-19 2013; London. Methods Data were collected prospectively. Body temperature was recorded routinely by nursing staff using a tympanic thermometer. Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle We noted any occasion where the body temperature dropped below 36°C along with any associated interventions – such as the use of additional bed sheets or a forced air warming device. After the fi rst audit period a simple education programme was delivered. We also introduced a departmental protocol for the prevention and management of inadvertent hypothermia. Six months later we re-audited our practice. Results Data were collected from 130 patients (2,931 patient-hours) in the fi rst audit period and from 87 patients (2,070 patient-hours) in the second audit period. In the fi rst period 29% of patients had at least 4-hourly temperature measurements compared with 40% in the second period (P <0.01). The average number of overdue temperature observations per day was 1.4 in the fi rst period and 0.9 in the second (P <0.01). Twenty-four per cent of patients became hypothermic in the fi rst period compared with 22% in the second (P = 0.07); however, the time these patients remained hypothermic reduced from an average of 7.9 hours to 6.1 hours (P <0.01). An intervention was made and documented in 15% of cases in the fi rst period and 46% in the second (P <0.001). P3 Role of pharmacist in multidisciplinary pediatric intensive care rounds: a retrospective descriptive study S Tripathi, K Graner, K Fryer, G Arteaga Mayo Clinic, Rochester, MN, USA Critical Care 2014, 18(Suppl 1):P3 (doi: 10.1186/cc13193) p p S Tripathi, K Graner, K Fryer, G Arteaga Introduction Multidisciplinary rounding practices in the ICU have now become the standard of care in most institutions. Few objective data are available, however, on the value each individual member of the team brings to the patient care. In our institution, pediatric pharmacists have been an integral part of the PICU rounds since 2002, although their role has evolved over the course of years. This study was undertaken with the primary aim of identifying the impact of pharmacist involvement in PICU rounds, with respect to changes in therapy. Conclusion We saw some improvement following an education programme and introduction of a clinical protocol although there remains room for further improvement. y Methods Since January 2003, pharmacists have recorded their clinical interventions and outcomes of those interventions in an institutionally developed Pharmaceutical Care database (P-Care). P5 P5 Incidence of adverse events in a Brazilian coronary ICU VF Paula1, CE Bosso1, OA Souza2, GE Valerio2, SV Ferreira2 1Instituto do Coração de Presidente Prudente, Brazil; 2UNOESTE – Universidade do Oeste Paulista, Presidente Prudente, Brazil Critical Care 2014, 18(Suppl 1):P5 (doi: 10.1186/cc13195) Incidence of adverse events in a Brazilian coronary ICU VF Paula1, CE Bosso1, OA Souza2, GE Valerio2, SV Ferreira2 1Instituto do Coração de Presidente Prudente, Brazil; 2UNOESTE – Universidade do Oeste Paulista, Presidente Prudente, Brazil Critical Care 2014, 18(Suppl 1):P5 (doi: 10.1186/cc13195) p y y Results From 1 January 2003 through 31 December 2012 pharmacists made 24,207 clinical interventions in the PICU and 19,252 of those interventions resulted in changes in medication therapy or therapy monitoring. Interventions that were accepted by the team involved 10,361 (53.8%) drug dosing regimen changes, 292 (1.5%) drug interactions or incompatibility, 969 (5%) drug monitoring suggestions, 1,665 (8.7%) drug routes/methods of administration, 3,895 (20.2%) drug selections, and 2,070 (10.8%) medication profi le/order clarifi cations. As the pharmacist role on the PICU multidisciplinary rounds has evolved, the number of interventions has increased from 1,643 in 2003 to 3,799 in 2012. Of the 19,252 interventions that were implemented, 304 were deemed to be of potentially life-threatening consequences, 10,767 had a moderate impact, and 8,181 had minimal impact on patient outcome. Conclusion To our knowledge this is the largest reported data on pharmacist’s involvement in pediatric intensive care. This can serve as background knowledge on implementing measures on novel methodologies to integrate pharmacists in intensive care practice. Critical Care 2014, 18(Suppl 1):P5 (doi: 10.1186/cc13195) Introduction Security policies for the patient are an interest to any health professional. The rates of adverse events in hospitals reach values ranging between 3.7 and 16.6%, being the highest range (40 to 70%) considered preventable or avoidable [1]. The objective of this study is to analyze the incidence of adverse events and their severity in a Brazilian coronary ICU (CICU). Methods Research conducted from database analysis regarding hospitalizations from 1 May 2012 until 31 October 2013 in a coronary care unit of the city of Presidente Prudente, Brazil. Statistical analysis was performed using EPI INFO, version 3.5.2 software, which was considered signifi cant at P <0.05 two-tailed and CIs at 95% (CI) were used for the odds ratios (OR) estimated in the sample. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Mock thefts were successfully accomplished on six occasions over a 4-week period. On each occasion the Limpet photographed the ‘thief’ and recorded the date and time of access. Therefore, in the event of a real theft it would be possible to quickly and easily indentify the culprit. Improvement in the identifi cation and management of inadvertent hypothermia in the critically ill: an audit cycle An intervention is defi ned as any recommendation the pharmacist makes to the patient care team regarding a change in the patient management or medication therapy. P-Care is designed to assist the pharmacist to optimize medication therapy, identify medication-related problems, decrease medication costs, improve pharmacist effi ciency and document pharmacist workload. Data concerning pharmacists’ interventions were extracted from the P-Care database in yearly increments via a reporting functionality that is available in the P-Care system. This study was exempted for review by the Mayo Clinic IRB. Reference 1. Andrzejowski et al.: Br J Anaesth 2008, 101:627-631. 1. Andrzejowski et al.: Br J Anaesth 2008, 101:627-631. References Mock thefts were subsequently undertaken by a designated staff member at random times. This allowed testing of the product to determine the number of times the ‘thief’ was correctly identifi ed. © 2010 BioMed Central Ltd © 2014 BioMed Central Ltd © 2014 BioMed Central Ltd S2 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1. Marra AR, et al.: Am J Infect Control 2009, 37:619-625. Organisational changes in service provision outside critical care impact on referral patterns A Tridente1, L Cecchini2, S Lobaz3, A Chick3, S Keep3, S Furmanova4, D Bryden3fi A Tridente1, L Cecchini2, S Lobaz3, A Chick3, S Keep3, S Furmanova4, D Bryden3fi Conclusion Post intervention, a statistically signifi cant improvement in overall bundle compliance was found. Thereby highlighting that through engaging all members of the multidisciplinary team in identifying barriers to noncompliance and delivering education, it is possible to improve compliance. While total compliance was suboptimal as the target was 95%, the bundle redesign has given a tool that records compliance with greater clarity due to the presence of clearly defi ned exclusion criteria. It has also been a signifi cant step in the right direction to improving the reliability of care delivered to patient and reinforces the concept that quality improvement is a continuous cycle. 1St Helens and Knowsley, Liverpool, UK; 2SMH, Manchester, UK; 3STH, Sheffi eld, UK; 4UHW, Cardiff , UK 1St Helens and Knowsley, Liverpool, UK; 2SMH, Manchester, UK; 3STH, Sheffi eld, UK; 4UHW, Cardiff , UK f Critical Care 2014, 18(Suppl 1):P8 (doi: 10.1186/cc13198) f Critical Care 2014, 18(Suppl 1):P8 (doi: 10.1186/cc13198) Introduction Demand for critical care (CC) resources is constantly increasing in the face of limited availability. Guidance for triage exists but may no longer refl ect current practice [1,2]. We previously identifi ed nonmedical and medical factors (comorbidities, physiological derangement and functional status) as predicting likelihood of admission of referred patients to CC [3-5]. Reduction in UK doctors’ working hours and numbers has resulted in new ways of multidisciplinary teams working the hospital at night (H@N), which may have an impact on CC referral. We aimed to establish any eff ect of Introduction Demand for critical care (CC) resources is constantly increasing in the face of limited availability. Guidance for triage exists but may no longer refl ect current practice [1,2]. We previously identifi ed nonmedical and medical factors (comorbidities, physiological derangement and functional status) as predicting likelihood of admission of referred patients to CC [3-5]. Reduction in UK doctors’ working hours and numbers has resulted in new ways of multidisciplinary teams working the hospital at night (H@N), which may have an impact on CC referral. We aimed to establish any eff ect of 1. Foster AJ, et al.: Adverse events among medical patients after discharge from hospital. CMAJ 2004, 170:345-349. Compliance of a ventilator-associated pneumonia care bundle in an adult intensive care setting G Wigmore, R Sethuraman The Princess Alexandra Hospital NHS Trust, Harlow, UK Critical Care 2014, 18(Suppl 1):P6 (doi: 10.1186/cc13196) Compliance of a ventilator-associated pneumonia care bundle in an adult intensive care setting G Wigmore, R Sethuraman The Princess Alexandra Hospital NHS Trust, Harlow, UK Critical Care 2014, 18(Suppl 1):P6 (doi: 10.1186/cc13196) Introduction Ventilator-associated pneumonia (VAP) is the leading cause of death amongst hospital-acquired infections and is also linked to an increased length of stay and cost of care. The Institute for Healthcare Improvement ventilator bundle is comprised of a series of interventions, which, when implemented together, have been shown to decrease the incidence of VAP. The aim of this study was to determine the compliance of the bundle and if <95% [1] devise strategies to improve compliance. Conclusion Acutely unwell patients require the expertise of the most senior clinicians regarding further management, including planning for end-of-life care. Our audit demonstrated poor adherence to NCEPOD [1] and Department of Health [2] recommendations. The majority of referrals to critical care were made by nonconsultants and for patients who had not been reviewed by a team consultant, prior to referral. The workload in critical care demonstrated that almost one- half of the referrals happen out of hours. These fi ndings have resulted in signifi cant changes to working practice, including the presence of an onsite consultant intensivist for a minimum of 13 hours daily. References p p Methods A retrospective review of the compliance of an existing VAP bundle was conducted for all adult patients ventilated in the ICU of a large district general hospital in 2011. The bundle comprised four elements: head up 30°, peptic ulcer prophylaxis, deep vein thrombosis (DVT) prophylaxis and sedation hold. The bundle was considered compliant if all four were performed. The fi ndings of the audit were presented to the department and, through subsequent discussions, barriers to noncompliance were identifi ed. Following a period of education, a revised and updated bundle was implemented. A repeat audit covering 3 months was subsequently conducted. P5 Demographics (gender and average age) and aspects related to adverse events were analyzed, the latter being under the impact and probability of death. S3 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results A total of 1,067 admissions were analyzed in the period, with 57.38% male and 42.63% female. The average age was 67.7 ± 13.2 years. The average CICU length of stay was 3.74 ± 5.51 days. A total of 211 adverse events occurring in 140 diff erent admissions were recorded. The most frequent were drug administration errors (24.3%), pressure ulcer (24.3%), phlebitis (22.1%), loss of enteric tube (13.6%) and central venous cannulation accident (7.1%). The statistical analysis shows that hospitalization time longer than 2 days are related to the occurrence of the events (OR: 8.08 CI: 4.95 to 13.2) and that the presence of the fi rst occurrence is signifi cant to increase the probability of death in the unit (OR = 5.33, CI: 3.49 to 8.12). Pressure ulcer (OR = 16.73, CI: 8.04 to 34.81), enteral tube loss (OR 3.64, CI: 1.36 to 9.75) and drug administration errors (OR = 2.87, CI: 1.31 to 6.31) were also related to higher mortality. Conclusion The research draws attention to a signifi cant incidence of adverse events and shows that their occurrence implies an increase of death in the unit. Therefore, security measures should be employed for the reduction, and thus enhance the quality of service provided by health professionals to patients admitted to a coronary care unit. Reference References 1. An Acute Problem? Report of the National Confi dential Enquiry into Patient Outcome and Death. London: National Confi dential Enquiry into Patient Outcome and Death; 2005. [http://www.ncepod.org.uk/2005report/] 1. An Acute Problem? Report of the National Confi dential Enquiry into Patient Outcome and Death. London: National Confi dential Enquiry into Patient Outcome and Death; 2005. [http://www.ncepod.org.uk/2005report/] 2. Guidelines on Admission to and Discharge from Intensive Care and High Dependency Units. London: Department of Health; 1996. Outcome and Death. London: National Confi dential Enquiry into Patient Outcome and Death; 2005. [http://www.ncepod.org.uk/2005report/] 2. Guidelines on Admission to and Discharge from Intensive Care and High Dependency Units. London: Department of Health; 1996. p p g p 2. Guidelines on Admission to and Discharge from Intensive Care and High Dependency Units. London: Department of Health; 1996. 2. Guidelines on Admission to and Discharge from Intensive Care and High Dependency Units. London: Department of Health; 1996. Results Pre-intervention, overall compliance of the bundle stood at 32% and subsequently increased to 63% post intervention (P <0.05). Compliance at the level of individual elements varied: head up 30°, 94%; ulcer prophylaxis, 91%; DVT prophylaxis, 85%; sedation hold, 37%. Post intervention, a statistically signifi cant increase in compliance with regard to sedation hold was observed; 72% (P <0.05). The other individual elements did not show a statistical change. However, the new elements that were introduced demonstrated high levels of compliance; cuff pressure 20 to 30 cm H2O 83% and oral hygiene with chlorhexidine 90%. Referrals to a critical care unit: compliance with the NCEPOD recommendations G Wigmore1, M Campbell2, P Walker2, K Raveendran2 1The Princess Alexandra Hospital NHS Trust, Harlow, UK; 2Barking Havering and Redbridge University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P7 (doi: 10.1186/cc13197) G Wigmore1, M Campbell2, P Walker2, K Raveendran2 1The Princess Alexandra Hospital NHS Trust, Harlow, UK; 2Barking Havering and Redbridge University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P7 (doi: 10.1186/cc13197) Introduction The report of the National Confi dential Enquiry into Patient Outcome and Death (NCEPOD) 2005 [1] provides recommendations to reduce morbidity and mortality among acutely ill patients. It highlights the importance for involvement of consultants in the referral process, clear physiological monitoring plans for each patient, and review by a consultant intensivist within 12 hours of admission to critical care. The objective of the audit was to assess compliance with these recommendations, ensuring safe management of acutely unwell patients and appropriate utilisation of scarce critical care resources. Methods Prospective data on referrals to adult critical care in a 20- bed unit in a teaching hospital were collected over 8 weeks. Collected data included: source, time, seniority of doctor referring and receiving referral, outcome of referral, involvement of team consultant prior to referral, documented management plan and review by a critical care consultant within 12 hours of referral. 1. Foster AJ, et al.: Adverse events among medical patients after discharge from hospital. CMAJ 2004, 170:345-349. 1. Foster AJ, et al.: Adverse events among medical patients after discharge from hospital. CMAJ 2004, 170:345-349. P6 Results Seventy-three referrals were analysed, the majority of which were medical. One-half of referrals came out of hours; 24% of referrals were made by a consultant; 51% were seen by a consultant prior to referral; 73% of referrals were admitted; likelihood of admission increased from 63% to 83% if the patient was reviewed by home consultant prior to referral. In 56% of cases the referral was received by a foundation doctor (all referrals were discussed with a consultant intensivist). Among the rejected referrals (maximal ward therapy not reached), 54% were from a trainee below registrar grade. Twenty-fi ve per cent of patients were not seen by a consultant intensivist within 12 hours of referral. Demands on a continuing education online-study program for physicians 1. ACCCM Guidelines for ICU admission, discharge, and triage. Crit Care Med 1999, 27:633-638. y g y Critical Care 2014, 18(Suppl 1):P10 (doi: 10.1186/cc13200) 2. ATS. Fair allocation of intensive care unit resources. Am J Respir Crit Care Med 1997, 156:1282-1301. Introduction Physicians have an intense and irregular workday life. Thus, they need to use their time for continuing education in the most effi cient way. This presents a major challenge to suppliers of continuing study programs. Our online master program Physico-Technical Medicine (PTM) addresses working physicians who want to acquire knowledge in the fi elds of biomedical engineering and medical physics [1]. We attach great importance to the special needs of our participants. Therefore, we investigated which general conditions are most important for the working physician regarding qualifying continuing education. 3. Tridente A, Chick A, Keep S, Furmanova S, Webber S, Bryden DC: Factors aff ecting critical care admission to a UK University Hospital [Poster P508]. Presented at 32nd International Symposium on Intensive Care and Emergency Medicine; March 19-23 2012; Brussels. 4. Tridente A, Chick A, Keep S, Furmanova S, Webber S, Bryden DC: Functional status as a predictor of admission to critical care in acutely unwell patients [Abstract 0424]. Presented at 25th Annual Congress of the European Society of Intensive Care Medicine; October 13-17 2012; Lisbon. 5. Tridente A, Chick A, Keep S, Furmanova S, Webber S, Bryden DC: Non medical factors infl uence likelihood of admission to critical care of acutely unwell patients [Abstract A-570-0025-00363]. Presented at 26th Annual Congress of the European Society of Intensive Care Medicine; October 5-7 2013; Paris. y y Methods The general conditions of our continuing education program (PTM) were examined on the basis of evaluation questionnaires. Additionally, students were asked in interviews about their demands, and they could give anonymous feedback on feedback cards.l y y Results Flexibility: students appreciated fl exibility regarding time and environment of their learning activities. This fl exibility enables the students to use even small time frames or traveling times for learning. Students can choose their preferred time and place for their learning activities. Security in planning: students have to organize their time frames for learning besides their daily work, their family life, and possibly their academic career. Especially phases of attendance should be planned and communicated as early as possible. 1. Guttmann J, et al.: Physikalisch-Technische Medizin (PTM) – ein neuer Master-Online Studiengang für Mediziner. GMS Z Med Ausbild 2011, 28(1):Doc01. Demands on a continuing education online-study program for physicians Transparency of structure: participants ask for transparency in terms of the expected workload and expenditure of time to plan their studies and get focused. Relation to previous knowledge: learning success is supported by relating new contents to previous knowledge from the student’s fi rst medical degree. Mainly clinical examples illustrate abstract topics, which also helps students to recognize the relevance of the content. Individual support: students need contact persons in terms of learning organization, technical support, and for questions regarding the course material. Demand versus supply in intensive care: an ever-growing problem M Tanaka Gutiez, R Ramaiah East Kent Hospitals University NHS Foundation Trust, Ashford, UK Critical Care 2014, 18(Suppl 1):P9 (doi: 10.1186/cc13199) Introduction The demand for intensive care has reached new heights, where medical advancement and aging populations have increased the proportion of patients with multi comorbidities. Ideally, bed occupancy on the ICU should be around 70% whereas beyond 80% has been associated with increased mortality. William Harvey Hospital is a district general hospital with nine ICU beds where the ICNARC national database suggested a consistently high occupancy. The purpose of this study was to assess bed occupancy and its impact on service delivery. Methods Data were collected between 2005 and 2013 to analyse trends in ICU bed occupancy, overnight discharges, surgical cancellations, and nonclinical transfers. Data were gathered from April 2012 to March 2013 using quality indicators (Intensive Care Society): readmissions; premature discharges; discharges at night; cancelled planned surgery; and nonclinical transfers. Regression analysis was conducted to assess: correlation between mortality and eff ects of excess occupancy; and causality of increasing demand for ICU beds. Conclusion We could show that an extra-occupational continuing study program for physicians should be adjusted to their special situation and therefore has to comply with particular requirements. As such we could identify fi ve main requirements which are continuously fulfi lled by the online master program PTM and regularly surveyed by evaluations. P10 P10 Demands on a continuing education online-study program f physicians J Seifried, V Titschen, J Guttmann, S Schumann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P10 (doi: 10.1186/cc13200) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (29%), surgical cancellations (90%), and nonclinical transfers (88%). Between 2012 and 2013, the calculated demand for beds was 174. There were 1.8 ICU to 100 hospital beds and 4.5 ICU beds per 100,000 population, which dropped signifi cantly when including regional specialty services. A persistent gap between ICU bed supply and demand was associated with increased unfavourable outcomes in all quality indicators. hospital changes in service provision outside CC on the referral pattern and admissions. hospital changes in service provision outside CC on the referral pattern and admissions. Methods Data from prospectively enrolled urgent patient referrals were analysed comparing two periods: before (period 1: 2011/12) and after (period 2: 2013) the introduction of H@N. We collected data on acute physiological parameters, hospital length of stay, demographic and functional status, dependency and comorbidities. STATA was used for these preliminary analyses. Conclusion A steady increase in occupancy over the last 8 years was due to multiple factors, including an increase in clinical services such as in the coronary care centre, head and neck unit, and trauma centre. Presentation of our results to managerial level facilitated increased bed capacity by 22%. f y y Results Comparing the two periods we found no signifi cant diff erences in age, gender distribution, degree of acute physiological derangement, comorbidities, specialty of origin, time spent in hospital prior to referral and grade of referrer. By contrast, the proportion of out- of-hours referrals greatly increased (from 49.3% to 69.7%) along with the proportion of referrals deemed inappropriate for CC (from 37.3% to 54.8%); the proportion of patients accepted increased only marginally (from 46.7 to 50%) in the second period, compared with the fi rst. Neither the number of beds available within the critical care department (P = 0.59) nor receiving the referral out of hours (P = 0.8) infl uenced the likelihood of admission. The factors predicting admission to CC (functional status, acute physiological derangement) did not diff er signifi cantly between the two periods examined. Housebound status was consistently found to be an independent predictor of admission refusal (OR 0.11, 95% CI 0.05 to 0.23, P <0.001). p y y References 1. Intensive Care Society: Standards for Intensive Care Units. London: ICS; 1997. 1. Intensive Care Society: Standards for Intensive Care Units. London: ICS; 1997. 2. Iapichino G, Gattinoni L, Radrizzani D, et al.: Volume of activity and occupancy rate in intensive care units. Association with mortality. Intensive Care Med 2004, 30:290-297. 3. Intensive Care Society: Standards, Safety and Quality Committee. Quality Indicators. London: Intensive Care Society; 2010. 3. Intensive Care Society: Standards, Safety and Quality Committee. Quality Indicators. London: Intensive Care Society; 2010. 3. Intensive Care Society: Standards, Safety and Quality Committee. Quality Indicators. London: Intensive Care Society; 2010. 4. Department of Health: Comprehensive Critical Care. A Review of Adult Critical Care Services. London: Department of Health; 2000. 4. Department of Health: Comprehensive Critical Care. A Review of Adult Critical Care Services. London: Department of Health; 2000. Conclusion Decision-making about admission to CC is based mainly on the assessment of patients’ functional status. Organisational changes in the provision of healthcare services outside CC, such as H@N, have had a signifi cant infl uence on CC workload and patterns of referrals. References Reference S4 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Convalescence via critical care collaboration C Shute, A Saaymanf y p Critical Care 2014, 18(Suppl 1):P12 (doi: 10.1186/cc13202) y p ,f , Critical Care 2014, 18(Suppl 1):P13 (doi: 10.1186/cc13203) Introduction The aim was to explore the HDU booking process, organ support requirements and fi nancial implications as the current approach to booking surgical cases for HDU has somewhat been based on a ‘just in case’ principle. Introduction A collaborative approach to patient management has been shown to improve patient outcomes. In a resource-limited NHS, critical care beds are at a premium, therefore preventing unnecessary admissions by optimising ward-based management is essential. The objective of this service quality improvement project was to improve collaboration between the critical care directorate and neurosurgical high care unit at a tertiary university teaching hospital. We proposed that the use of a simple ward round tool on collaborative rounds would facilitate systematic patient review, prompt early recognition of those critically unwell and improve patient outcomes. j p p Methods The booking and admission details from October 2012 to July 2013 were gathered from the HDU log-book and Ward Watcher software. The cost of HDU beds and staff were provided by the fi nance department. Data were analysed using MS Excel and SPSS software. Results There were 105 handwritten bookings over 10 months, several missing essential data. Only 89 admissions actually took place; 61 were elective, 66 colorectal, six urgent, 19 scheduled and three unspecifi ed (Figure 1). Most patients required basic cardiovascular and respiratory support (Figure 2). Data revealed 16 cancellations. Each booking requires allocated nursing staff which bears a basic cost of £23/hour (estimated annual loss >£10.000). If we add the daily cost of basic HDU care (approximately £1,000 for single organ support), the losses rise rapidly. We also registered 38 days in delayed discharges. This was predominantly due to ward bed shortages. In fi nancial terms there is at j p p Methods The booking and admission details from October 2012 to July 2013 were gathered from the HDU log-book and Ward Watcher software. The cost of HDU beds and staff were provided by the fi nance department. Data were analysed using MS Excel and SPSS software. Results There were 105 handwritten bookings over 10 months, several missing essential data. Only 89 admissions actually took place; 61 were elective, 66 colorectal, six urgent, 19 scheduled and three unspecifi ed (Figure 1). Most patients required basic cardiovascular and respiratory support (Figure 2). Reference Results Bed occupancy remained high between 2005 and 2013 with a 15% increase. This was associated with increased overnight discharges S5 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P12). Admissions per speciality. Figure 2 (abstract P12). Organ support requirements. Figure 1 (abstract P12). Admissions per speciality. P11 Do generic measures fully capture health-related quality of life in adult, general critical care survivors? W Lim1, N Black1, K Rowan2, N Mays1 1London School of Hygiene & Tropical Medicine, London, UK; 2Intensive Care National Audit & Research Centre, London, UK Critical Care 2014, 18(Suppl 1):P11 (doi: 10.1186/cc13201) Introduction We examined the extent to which the two generic health- related quality of life (HRQL) measures recommended for use in adult, general critical care [1] – the SF-36 and EQ-5D – captured survivors’ HRQL, which is important in assessing the eff ectiveness of critical care. Unlike other fi elds of healthcare that employ both generic and specifi c HRQL measures, most recent studies in critical care have used only generic measures, despite uncertainty as to their appropriateness. Methods A patient-based conceptual framework for survivors’ HRQL was built using: a systematic review of the literature; secondary analysis of 40 in-depth interviews with adult critical care survivors; and primary analysis of in-depth interviews with a maximum variation sample of 25 white critical care survivors in England. Two methods were then used to assess the extent to which the SF-36 and EQ-5D captured their HRQL, as detailed in the framework: the content of both instruments was examined, alongside data collected from the in-depth interviews; and the opinions that survivors expressed about how accurately the SF-36 and EQ-5D refl ected their ideas on health and HRQL were analysed and taken into account.i Introduction We examined the extent to which the two generic health- related quality of life (HRQL) measures recommended for use in adult, general critical care [1] – the SF-36 and EQ-5D – captured survivors’ HRQL, which is important in assessing the eff ectiveness of critical care. Unlike other fi elds of healthcare that employ both generic and specifi c HRQL measures, most recent studies in critical care have used only generic measures, despite uncertainty as to their appropriateness. Reference Methods A patient-based conceptual framework for survivors’ HRQL was built using: a systematic review of the literature; secondary analysis of 40 in-depth interviews with adult critical care survivors; and primary analysis of in-depth interviews with a maximum variation sample of 25 white critical care survivors in England. Two methods were then used to assess the extent to which the SF-36 and EQ-5D captured their HRQL, as detailed in the framework: the content of both instruments was examined, alongside data collected from the in-depth interviews; and the opinions that survivors expressed about how accurately the SF-36 and EQ-5D refl ected their ideas on health and HRQL were analysed and taken into account. Figure 1 (abstract P12). Admissions per speciality. Figure 2 (abstract P12). Organ support requirements. Figure 2 (abstract P12). Organ support requirements. Results The patient-based framework was in two parts: the fi rst covered survivors’ personal status which consisted of their physical status, emotional/psychological status and cognitive status; and the second comprised nine subdomains aff ected by their personal status. The latter were: activities and behaviours; physical zone of comfort and/or activity; interactions and relationships with others; perception of, interpretation of, and responses to life; personality; external appearance; suitability and availability of clothes; place of residence; and fi nances. The current generic measures recommended on the basis of expert consensus for use with survivors of adult, general critical care have substantial gaps in their coverage of this conceptual framework for survivors’ HRQL, particularly in relation to their cognitive status and its subsequent impact. Figure 2 (abstract P12). Organ support requirements. least £500/day diff erence between surgical and HDU (potential annual loss >£22,000). In addition there were 13 patients who spent a total of 37 days on the HDU but did not require an HDU level of care. Conclusion The currently limited resource is not being utilised eff ectively with implications on both staff deployment and fi nances. A more holistic approach is needed where all requests are reviewed by a consultant anaesthetist in conjunction with preadmission data. This could better identify patients for HDU care and potentially decrease cancellations. We have developed an improved booking form for this purpose. q p Conclusion The two most commonly used generic HRQL measures in adult, general critical care have signifi cant gaps in their coverage of survivors’ HRQL. Reference Any (new) critical care-specifi c HRQL measure should be designed specifi cally to capture the impact of critical illness on survivors’ cognitive status and its subsequent eff ects. Reference 1. Angus DC, et al.: Intensive Care Med 2003, 29:368-377. 1. Angus DC, et al.: Intensive Care Med 2003, 29:368-377. P12 Surgical HDU admissions: utilisation, organ support and fi nance G Brakmane, A Molokhia University Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P12 (doi: 10.1186/cc13202) Surgical HDU admissions: utilisation, organ support and fi nance G Brakmane, A Molokhia University Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P12 (doi: 10.1186/cc13202) Convalescence via critical care collaboration C Shute, A Saayman University Hospital Wales, Cardiff , UK Critical Care 2014, 18(Suppl 1):P13 (doi: 10.1186/cc13203) P15 time further data were collected to assess whether our interventions resulted in modifi ed behaviours and to document the number of changes made to patient management as a consequence of the collaborative approach to care. time further data were collected to assess whether our interventions resulted in modifi ed behaviours and to document the number of changes made to patient management as a consequence of the collaborative approach to care. Table 1 (abstract P15) Table 1 (abstract P15) Number of 28-day cost Annual cost Test inappropriate requests (€) (€) Full blood count 4 21.82 284.44 Urea and electrolytes 6 29.44 383.77 Liver function tests 95 583.18 7,602.1 Coagulation screen 117 1,276.86 16,644.78 C-reactive protein 45 245.55 3,200.91 Bone profi le 112 274.75 3,581.56 Magnesium 88 483.35 6,300.81 p J Jennings1, TD Thomsen2, JW Larsen2, J Markvardt3 1Sheffi eld Teaching Hospital, Sheffi eld, UK; 2Kolding Hospital, part of Lillebaelt Hospital, Kolding, Denmark; 3Danish Building Research Institute/Aalborg University, Copenhagen, Denmark Critical Care 2014, 18(Suppl 1):P14 (doi: 10.1186/cc13204) Introduction Hospital lighting may cause disruption in the circadian rhythm, partly due to suppression of melatonin production. This may create sleep diffi culties and delirium for ICU patients and health issues such as fatigue, poor sleep quality and chronic diseases for ICU staff . Dynamic RGB coloured light which changes colour and intensity in correlation with the time of day has been installed in a Danish ICU, aiming to create lighting conditions being close to daylight variations and supporting patient and staff rhythms. As the eff ect of dynamic light on patients may be biased by illness, organ failure, and so forth, the aim was to examine the infl uence of dynamic light on ICU nurses’ melatonin production, quality of sleep, and well-being. Conclusion In our ICU, unnecessary blood tests have a signifi cant cost burden of approximately €38,000 per year. To limit unnecessary costs, consultants now lead requesting by completing a blood test pro forma on the evening ICU ward round indicating which blood tests are clinically needed the following day. Reference Methods An intervention study examining the impact of dynamic light regarding the impact on the circadian rhythm (measured by melatonin profi les from saliva samples), quality of sleep (sleep effi ciency, number of awakenings and subjective assessment of sleep; measured by sleep monitors and sleep diary), and subjective experiences of well-being, health, and sleep quality (measured by a questionnaire survey). Results from the intervention group were compared with a control group of nurses from a similar ICU without dynamic light. Light conditions were documented by measurements. Data collection was from February to May 2013. 1. Seguin P, et al.: Intensive Care Med 2002, 28:332-335. P16 P14 P14 Can dynamic light improve melatonin production and quality of sleep? J Jennings1, TD Thomsen2, JW Larsen2, J Markvardt3 1Sheffi eld Teaching Hospital, Sheffi eld, UK; 2Kolding Hospital, part of Lillebaelt Hospital, Kolding, Denmark; 3Danish Building Research Institute/Aalborg University, Copenhagen, Denmark Critical Care 2014, 18(Suppl 1):P14 (doi: 10.1186/cc13204) Can dynamic light improve melatonin production and quality of sleep? Can dynamic light improve melatonin production and quality of sleep? Table 1 (abstract P15) Convalescence via critical care collaboration C Shute, A Saaymanf Data revealed 16 cancellations. Each booking requires allocated nursing staff which bears a basic cost of £23/hour (estimated annual loss >£10.000). If we add the daily cost of basic HDU care (approximately £1,000 for single organ support), the losses rise rapidly. We also registered 38 days in delayed discharges. This was predominantly due to ward bed shortages. In fi nancial terms there is at y Methods An initial observation period of behaviours and practice on the neurosurgical unit was conducted with qualitative and quantitative data collection. Following analysis of these outcome measures a simple ward round tool was constructed, with the mnemonic ‘DON’T FORGET’, and we devised a programme for collaborative ward rounds to take place three times per week over a 1-month period. During this S6 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Targeting blood tests in the ICU may lead to a signifi cant cost reduction R Gray, F Baldwin R Gray, F Baldwin y BSUH, Brighton, UK Results Our results showed that improvements were made in all assessed domains. Consultant-led ward rounds increased, attendance by members of the multidisciplinary team (MDT) dramatically improved and as a result MDT discussion was enhanced. In addition, documentation of structured plans improved and review of prescription charts signifi cantly increased. As a consequence of collaborative rounds, a total of 343 changes were made to patient management under the domains of the ward round tool. This was an average of 21.4 changes per collaborative round, with most changes being made to medication charts or decisions regarding thromboprophylaxis. BSUH, Brighton, UK Introduction Daily blood tests are an essential diagnostic tool and routine practice in the management of ICU patients, but are associated with cost and a risk of anaemia. There is no evidence for the frequency or breadth of blood testing but there is evidence that by rationalising blood tests, signifi cant cost saving can be made without a negative eff ect on mortality or length of ICU stay [1]. Blood tests in our ICU are requested by nurses, this is a unique practice to the ICU compared with other hospital departments. Conclusion The use of a simple ward round tool combined with a collaborative approach to ward rounds improved MDT involvement and discussion, promoted structured patient review and resulted in positive changes to multiple areas of patient management. In conclusion, structured collaborative rounds result in signifi cant changes to patient management that may prevent admission, or readmission, to critical care which has the potential to reduce healthcare costs and morbidity. Methods We had previously carried out a survey to assess blood requesting practise in UK ICUs. This demonstrated that routine tests were, as in our ICU, nurse led and the majority of staff underestimated the costs of tests. Using the results of this survey and a literature review we developed routine blood test guidelines for our general mixed 26- bed ICU/HDU. Following a period of staff education, we audited our blood requesting practice over 28 days. Results Our blood testing did not comply with our guidelines. Over the 28-day period, €12,849.96 was spent on all blood tests. According to our guidelines, €2,914.96 was spent on inappropriate tests (€37,998.63 per year) (Table 1). Over 40% of this cost was spent on the coagulation screen. P18 ICU nursing connectivity and the quality of care in an academic medical center: a network analysis Results Data were collected for 3 months for 106 patients. The 24-hour trends of acoustic parameters corresponded well to the daily routine events in the ICU. The analyses for the fi rst 4 days of the patients’ ICU stay showed that the average SPL varied depending on the day (P = 0.023) and on the time of the day (day/night) (P <0.001). The average noise level decreased from day 1 to day 2 with a signifi cant reduction at night (P = 0.008), but it was found to rebound from day 2 where the increase of the daytime noise level from day 2 to day 4 was signifi cant (P = 0.005). The results of the multiple linear regression showed that various patient conditions infl uenced diff erent types of noise-level parameters. For example, the location of the patient room, the usage of mechanical ventilators and the mortality rate were found to correlate to the 10th-percentile SPL (L90) in the fi rst 48 hours (adjusted R2 = 0.58; P <0.001). Also, the room location, gender and the usage of mechanical ventilators were found to be related to the 50th-percentile SPL (L50) in the same period (adjusted R2 = 0.54; P <0.001). y DM Kelly1, DC Angus1, D Krackhardt2, JM Kahn1 1University of Pittsburgh, PA USA; 2Carnegie Mellon University, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P18 (doi: 10.1186/cc13208) Introduction A collaborative nurse work environment is associated with ICU quality, yet collaborative interaction is diffi cult to measure. Network analysis may be an innovative tool to measure interactions. We sought to determine the feasibility of network analysis to measure ICU nurse connectivity and test whether key network measures were associated with the ICU quality of care. Methods We performed a network analysis in eight ICUs within an urban academic medical center in the United States during 2011. Using scheduling data, we defi ned network ties as instances when two ICU nurses worked together in the same ICU for 4 hours or more. We examined each ICU’s network by visualizing sociograms and by measuring two network properties: density and clustering. Density measures the cohesion within a network on a scale from 0 to 100, with a higher score indicating more cohesion. Clustering assesses the local neighborhoods on a scale from 0 to 100, with a higher score indicating a more decentralized network. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Use of the TM Program for the implementation of the Surviving Sepsis Campaign Protocol at a community Brazilian hospital improved compliance with recommended care bundles and signifi cantly decreased the hospital mortality of septic patients. out to investigate the eff ect of relevant factors (for example, the time of day). Furthermore, multiple linear regression models were established to relate the diff erent types of patient-related data (as independent variables) to each type of the acoustic data (as dependent variable), where independent variables were selected based on an exhaustive search method. P18 ICU nursing connectivity and the quality of care in an academic medical center: a network analysis We examined variation in network measures across ICUs and tested the correlation between network measures and the proportion of patients receiving daily interruption of sedation (DIS). Conclusion Noise-level parameters were found to vary depending on the day of ICU stay, the time of day, and other indicators of the patient’s status. For a rigorous analysis of the infl uence of noise on patients’ outcome, the eff ects of these factors must fi rst be controlled or corrected. f Results of the Telemedicine Program for implementation of the Surviving Sepsis Campaign Protocol in a community Brazilian hospital Conclusion We found substantial variation in ICU nursing networks across eight ICUs in one academic medical center. These patterns may have implications for evidence-based practice implementation. More work is needed to understand the role of network analysis as a reliable tool for improving and understanding ICU organization. Conclusion We found substantial variation in ICU nursing networks across eight ICUs in one academic medical center. These patterns may have implications for evidence-based practice implementation. More work is needed to understand the role of network analysis as a reliable tool for improving and understanding ICU organization. Reference 1. Van Rompaey et al.: Crit Care 2012, 16:R73. P19 P19 Compassion fatigue and burnout among healthcare professionals in the ICU M Van Mol1, E Kompanje1, J Bakker1, M Nijkamp2 1Erasmus MC, Rotterdam, the Netherlands; 2Open University, Heerlen, the Netherlands Critical Care 2014, 18(Suppl 1):P19 (doi: 10.1186/cc13209) Results of the Telemedicine Program for implementation of the Surviving Sepsis Campaign Protocol in a community Brazilian hospital Results There was wide variation in the networks, with density ranging from 79 to 96 and clustering ranging from 88 to 97. Two sample sociograms are shown in Figure 1: ICU A had a very high density (96) and clustering coeffi cient (97) suggesting a cohesive and decentralized network, contrasting with ICU H that had the lowest density (79) and clustering coeffi cient (88). Greater density and clustering was positively associated with DIS (B = 0.02 (–0.10, 0.14); B = 0.003 (–0.07, 0.07)) but did not achieve statistical signifi cance in our small sample. p C Abreu Filho1, M Steinman1, A Andrade2, F Piza1, A Caluza1, J Teixeira2, M Bracco2, R Abaladejo1, E Silva1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital Municipal Dr. Moysés Deutsch, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P17 (doi: 10.1186/cc13207) Introduction Sepsis has high incidence around the world, approximately 400,000 new cases occur annually in Brazil. In developing countries, it is still an important cause of death due to poor adherence to best- practice medicine protocols. The Brazilian mortality rate of septic shock is around 60%. The aim of this study is to describe the initial results for the implementation of the Brazilian initiative of a Telemedicine (TM) Project for therapeutic support of septic patients in a community hospital in São Paulo, Brazil. Figure 1 (abstract P18). Sociograms of ICU A and ICU H. p Methods Since May 2012, a TM Program has been implemented at two hospitals in São Paulo – Hospital Municipal Dr. Moysés Deutsch (HMMD), a public, secondary hospital, and Hospital Israelita Albert Einstein (HIAE), a tertiary private philanthropic entity – due to a partnership with Brazilian Health Ministry. A TM Central Command was located at HIAE with Endpoint 97 MXP Cisco® Solution and Medigraf Gowireless® technology. Mobile Intern MXP ISDN/IP Cisco® and Medigraf Gowireless® for the remote hospital (HMMD) via a dedicated connection. At HMMD, the sepsis protocol, based on the Surviving Sepsis Campaign, has been started for every recruited patient admitted to the emergency department (ED) or the ICU, and assessed by the Central Command through TM with an experienced consultant of HIAE. We compared the results of this group of patients with the group of patients with diagnosis of severe sepsis and septic shock, admitted to HMMD during 2011, who were not assisted with TM resources. Analysis of the acoustic environment in an ICU using patient information as a covariate y Results A total of 55 nurses (89%) from the intervention ICU (ICU1) and 58 nurses (88%) from the control ICU (ICU2) participated. No signifi cant diff erences were found between the two groups regarding personal characteristics. The nurses from ICU1 described their work light as comfortable, relaxing and natural compared with artifi cial, institutional and gloomy in ICU2. Preliminary analyses did not shown any signifi cant diff erences in melatonin level. During a 10-day period, the nurses from ICU2 assessed their actual sleep as less eff ective (OR 2.17; P = 0.03) and felt less rested (OR 1.89; P = 0.006) compared with nurses from ICU1. The nurses in ICU2 had 16% more awakenings (P = 0.05) during sleep, but there were no signifi cant diff erences in duration of awakenings or in total sleep effi ciency between the two groups. M Park1, P Vos2, A Kohlrausch1, B Vlaskamp1, AW Oldenbeuving2 1Philips Research Laboratories, Eindhoven, the Netherlands; 2St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P16 (doi: 10.1186/cc13206) M Park1, P Vos2, A Kohlrausch1, B Vlaskamp1, AW Oldenbeuving2 1Philips Research Laboratories, Eindhoven, the Netherlands; 2St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P16 (doi: 10.1186/cc13206) M Park1, P Vos2, A Kohlrausch1, B Vlaskamp1, AW Oldenbeuving2 1Philips Research Laboratories, Eindhoven, the Netherlands; 2St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P16 (doi: 10.1186/cc13206) Introduction Noise levels in ICUs are often very high, potentially aff ecting patient health outcome, which are also considered to be among the risk factors contributing to ICU delirium [1]. In the current study, multivariate linear regression models were established to relate the acoustic data to the indicators of patient status. Methods Acoustic measurements were taken in eight single-bed patient rooms in a level 3 ICU, while patient information was also collected, including APACHE IV score and mortality rate together with other potentially relevant variables; for example, the usage of mechanical ventilators, and so on. The hourly and daily trends of the acoustic data were obtained, and an analysis of variance was carried fi Conclusion Most participants from the intervention ICU found the dynamic light agreeable and assessed their sleep more positively than participants from the control ICU. No signifi cant diff erences were found between monitored sleep effi ciency and melatonin level. S7 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Prevalence, risk factors and consequences of intra-team confl icts in the ICU Prevalence, risk factors and consequences of intra-team confl icts in the ICU G Burghi1, F Verga1, M Acevedo2, V Martinez1, V Carrasco3, C Quiroga4, G Pittini5, G Fariña6, M Godino1, S Mareque7, P Alzugaray3, H Bagnulo1, E Azoulay8 Results All references, retrieved from electronically database search (n = 1,432) and manual search (n = 3), resulted in 100 relevant publications for full-text screening. Subsequently, articles with only an abstract available (n = 28), a language barrier (n = 27), or prevalence not shown in percentages (n = 23) were removed. Finally, a sample of 22 eligible articles were appraised as methodologically sound and systematically analysed for this review. The prevalence of BO in the ICU varied from 1.2 to 49% and diff ered in defi ning a severe BO. Two studies reported the prevalence of CF, 7.3% and 40%, and fi ve studies described the prevalence of STS in a range from 21 to 44%. 1Hospital Maciel, Montevideo, Uruguay; 2COMERO, Rocha, Uruguay; 3Sanatorio Americano, Montevideo, Uruguay; 4Hospital Español, Montevideo, Uruguay; 5CAAMEPA, Pando, Uruguay; 6Casa de Galicia, Montevideo, Uruguay; 7CAMS, Mercedes, Uruguay; 8Hopital Saint Louis, Paris, France Critical Care 2014, 18(Suppl 1):P21 (doi: 10.1186/cc13211) Introduction The ICU is a stressful place with several sources of confl icts. Among ICU workers, confl icts lead to burnout, loss of psychological well-being, and deterioration of the work performance. Intra-team confl icts (confl icts that occur among members of the intensive care team) are one of the most frequent types of confl icts in the ICU. The objectives of this study were to determine the prevalence, risk factors and consequences associated with intra-team confl icts in the ICU. Introduction The ICU is a stressful place with several sources of confl icts. Among ICU workers, confl icts lead to burnout, loss of psychological well-being, and deterioration of the work performance. Intra-team confl icts (confl icts that occur among members of the intensive care team) are one of the most frequent types of confl icts in the ICU. The objectives of this study were to determine the prevalence, risk factors and consequences associated with intra-team confl icts in the ICU. Methods A survey was conducted in 12 Uruguayan adult ICUs in 2009. ICUs and clinician’s characteristics were assessed for their association with the prevalence of confl icts. All factors associated with confl icts were introduced into a multivariable model. References 1. Multz AS, et al.: Am J Respir Crit Care Med 1998, 157(5 Pt 1):1468-1473. 2. Pronovost PJ, et al.: JAMA 2002 288:2151-2162. f g p g Methods A review of the literature between 1998 and December 2013 was conducted using eight databases, and included the keywords CF, STS, BO, ICU, nurses and physicians. The references were screened for relevancy at title/abstract using predefi ned inclusion and exclusion criteria. Studies were limited to original and review articles in the English language. Two independent researchers assessed the methodological soundness of each article. P19 Compassion fatigue and burnout among healthcare professionals in the ICU Results From January to December 2011, 283 septic patients were treated at HMMD, 161 (56.8%) patients were male, mean age was 53.7 years and the hospital mortality was 65.3%. After the implementation of the TM Program, 189 septic patients were admitted to the hospital and evaluated by skilled doctors of HIAE via TM, with the institution of the Surviving Sepsis Campaign protocol. In total, 68.9% of the consultations originated from the ICU and 31.1% from the ED; 111 patients were male (58.4%), mean age was 54.1 years and the hospital mortality was 32.9% (P <0.05). M Van Mol1, E Kompanje1, J Bakker1, M Nijkamp2 1Erasmus MC, Rotterdam, the Netherlands; 2Open University, Heerlen, the Netherlands Critical Care 2014, 18(Suppl 1):P19 (doi: 10.1186/cc13209) Critical Care 2014, 18(Suppl 1):P19 (doi: 10.1186/cc13209) Introduction This study comprises a systematic review of studies focusing on the prevalence of compassion fatigue (CF) and burnout Introduction This study comprises a systematic review of studies focusing on the prevalence of compassion fatigue (CF) and burnout S8 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (BO) among professionals in the ICU, to indicate the size of this problem. Both CF and BO have an important impact on daily quality of life of professionals and may threaten patient care. A growing body of research suggests that BO appears to be common among ICU nurses [1] and physicians [2] due to a highly stressful work environment. However, BO might be overestimated and seen as a fashionable diagnosis [3]. The overlap of BO with CF, secondary traumatic stress (STS) and vicarious traumatisation has recently been explored [4]; CF seems to apply more to ICU staff and might be a lead in future preventive strategies. compared with NDG. Discordance was associated with higher ICU and hospital mortality (35.5 vs. 11%; OR = 4.09; P <0.001 and 45.2 vs. 20.1%; OR = 2.77; P = 0.02 respectively).i Conclusion The occurrence of divergences, even during the fi rst days of ICU admission, about medical plans of care for critically ill patients is frequent and is associated with higher ICU and hospital mortality and more use of medical resources. Prevalence, risk factors and consequences of intra-team confl icts in the ICU Conclusion The emotional price of working at the ICU can become a burden in personal life, but the size of the problem in ICU staff remains unclear. Because the decreased well-being of the professionals might negatively infl uence the quality of care, preventive strategies should be developed in order to alleviate the distressed. Further exploration of CF might provide suffi cient starting points. ql Methods A survey was conducted in 12 Uruguayan adult ICUs in 2009. ICUs and clinician’s characteristics were assessed for their association with the prevalence of confl icts. All factors associated with confl icts were introduced into a multivariable model. g p References 1. Mealer et al.: Am J Respir Crit Care Med 2007, 175:693-697. 2. Embriaco et al.: Am J Respir Crit Care Med 2007, 175:686-682. 3. Kaschka et al.: Deutsch Arztebl Int 2011, 108: 781-787. 4. Meadors et al.: J Pediatr Health Care 2008, 22: 24-34. Results A total of 384 questionnaires were collected, 74.5% (n = 286) were nurses and 23.2% (n = 89) were intensivists. From the 384 forms, 45.8% perceived at least one confl ict in the past year. Confl icts were perceived more frequently by intensivists (61.8%) than nurses (40.9%) (P = 0.001). A worse relationship with intensivists (OR 1.3; 95% CI 1.09 to 1.59; P = 0.004) is independently associated with confl icts. However, lower grade of irritability (OR 0.8, 95% CI 0.73 to 0.97; P = 0.01) and a higher position in the ICU (OR 0.57 95% CI 0.36 to 0.92; P = 0.02) are factors independently associated with a lower incidence of intra-team confl icts. Interestingly, this study confi rms that workers with at least one confl ict had more frequently: libido disturbances (56% vs. 40%; P = 0.002), eating problems (52% vs. 40%; P = 0.02), as well as the wish to leave the ICU (53% vs. 39%; P = 0.005). P20 Eff ect of divergences about patient’s care plan on the outcome of critically ill patients EL Queiroz, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P20 (doi: 10.1186/cc13210) P20 Eff ect of divergences about patient’s care plan on the outcome of critically ill patients EL Queiroz, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P20 (doi: 10.1186/cc13210) P22 Do we spend less on older critically ill patients? Relationship among intensity of care, severity of illness and mortality MD Rosa, AC Pecanha Antonio, M Mattioni, L Tagliari, F Schaich, J Maccari, R Oliveira, C Teixeira, TF Tonietto, N Brandão da Silva Hospital Moinhos de Vento, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P22 (doi: 10.1186/cc13212) P20 Eff ect of divergences about patient’s care plan on the outcome of critically ill patients EL Queiroz, G Schettino Hospital Sírio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P20 (doi: 10.1186/cc13210) Introduction There is an actual discussion about the best way to provide medical care for critically ill patients, particularly between the classical opened versus closed ICU models [1,2]. In Brazil, all ICUs have to have a full-time and dedicated physician at the unit but the primary physician also can visit his patient every day and, most of the time, participates in the patient’s care plan. This hybrid model creates opportunity for patient’s care plan divergences between the ICU staff and the primary physician. The objective of this study is to evaluate the eff ect of divergences about patient’s care plan on the outcome of critically ill patients. Conclusion The prevalence of confl icts is very high among ICU workers in Uruguay. We have identifi ed diff erent risk factors associated with the development of confl icts. These results confi rm previous fi ndings and highlights that strategies to decrease intra-team confl icts in the ICU are urgently warranted. References g Methods A literature review was conducted. PubMed was searched. The following combinations of Mesh terms were used: [(decision- making) OR (communication)] AND (intensive care) OR (intensive care units)] AND [(cultural diversity) OR (multiculturalism) OR (migrants) OR (culture) OR (ethnicity)]. A total of 111 studies were retrieved. We excluded nonempirical studies, studies not written in English and articles published before 2003. After screening the titles and abstracts 12 articles were selected. The full text of these articles was analyzed and synthesized with a thematic-synthesis approach. During the process we performed additional searches based on the bibliographies of all the relevant articles.i 1. Cullen DJ, et al.: Therapeutic intervention scoring system: a method for quantitative comparison of patient care. Crit Care Med 1974, 2:57-60. 1. Cullen DJ, et al.: Therapeutic intervention scoring system: a method for quantitative comparison of patient care. Crit Care Med 1974, 2:57-60. 2. Hamel MB, et al.: Seriously ill hospitalized adults: do we spend less on older patients? Support investigators. Study to understand prognoses and preference for outcomes and risks of treatments. J Am Geriatr Soc 1996, 44:1043-1048. New policy for ICU visits: prospective study New policy for ICU visits: prospective study y y A Moughabghab, S Gemayel, N Azar, R Bousaba A Moughabghab, S Gemayel, N Azar, R Bousaba g g y Lebanese Canadian Hospital, Beyrouth, Lebanon Lebanese Canadian Hospital, Beyrouth, Lebanon C i i l C 2014 18(S l 1) P23 (d i 10 1186/ 13213) p y Critical Care 2014, 18(Suppl 1):P23 (doi: 10.1186/cc13213) Results Culturally specifi c expectations about communication and decision-making, diff erent views on gender roles, organization of the care and expression and resolving emotional and social crises may cause extra stress. The communication becomes more complex when the language of the care providers diff ers from the language of the patient and family. Intercultural communication can be improved by specifi c actions such as ethnic matching, using an interpreter and the formation of cultural-competent healthcare professionals. Introduction ICU visits are generally limited to 2 hours per day selected at any time during the 24 hours. At our institution we abide to the international regulations which were recently modifi ed allowing family members and signifi cant others to be present throughout the day accompanying their ICU-admitted relatives, participating in an uninterrupted timely fashion and no time limits with their daily activities. References Conclusion There is a lack of knowledge regarding the specifi c experiences of patients, families and care providers with communi- cation and especially decision-making in multicultural ICUs in Western Europe. Methods From January 2012 through April 2012, 200 questionnaires were fi lled by families of ICU-admitted patients; each questionnaire includes 13 questions concerning their overall degree of satisfaction at our unit and evaluating the quality. Accommodating spaces were off ered for families of ICU patients, off ering them a state of freedom within the ICU premises surrounding their beloved sick relatives; additionally they were constantly advised by a designated secretary to share their experience and any possible insight to improve the overall polity of our service. Symptoms of anxiety, depression and post-traumatic stress in pairs of patients and their family members during and following ICU stay: who suff ers most? R Fumis1, O Ranzani2, P Martins1, G Schettino1 1Hospital Sírio Libabês, São Paulo, Brazil; 2Hospital das Clínicas, Universidade de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P25 (doi: 10.1186/cc13215) Results Two hundred patients were included in the study, the average age of patients was 68.55 years and the average length of ICU stay was 5.2 days. Most family (85%) desired their presence accompanying their related patients within the ICU premises when meals were served. Those families expressed satisfaction while off ering psychological support to their beloved ill ones. Fifteen per cent of the families argued about the permitted number of visitors (one person) per visit and 12% addressed lack of communication between the medical staff and patient’s families. Introduction To compare the incidence of anxiety, depression and post-traumatic stress symptoms in pairs of patients and respective family members during the ICU stay and at 30 and 90 days post ICU discharge. According to the literature, both patients and family members suff er from psychological distress during and following an ICU stay [1]. Although these issues have been discussed, to date few studies have addressed the pairs at three times. p Conclusion Compared with the limited visit status within the ICU ward in the past, the majority of families expressed general acceptance and satisfaction toward the newly adopted policy. Additionally and by this new policy, patients displayed marked improvement at the psychological level, being more cooperative and experiencing less episodes of agitation. Dealing with cultural diversity during the process of communication and decision-making in the ICU: a literature review Dealing with cultural diversity during the process of communication and decision-making in the ICU: a literature review Dealing with cultural diversity during the process of communication and decision-making in the ICU: a literature review RV Van Keer, RD Deschepper, LH Huyghens, WD Distelmans, JB Bilsen Vrije Universiteit Brussel, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P24 (doi: 10.1186/cc13214) RV Van Keer, RD Deschepper, LH Huyghens, WD Distelmans, JB Bilsen Vrije Universiteit Brussel, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P24 (doi: 10.1186/cc13214) i Results TISS-28 scores at 24 and 72 hours of ICU admission were analyzed for 4,128 patients. Mean patient age was 68.3 (SD ± 17.6), 46.8% were female and 37% were surgically ill. The mean APACHE II score was 16 (SD ± 8) and 40% were submitted to mechanical ventilation at any time of the stay. Overall mortality was 14.4%. Neither APACHE II score adjusted for age nor TISS-28 in 24 and 72 hours of admission diff ers among age groups. However, mortality was signifi cantly higher in patients aged 70 years or older (P <0.001). Introduction Studies have shown that communication in the ICU is related to the cultural background of all involved actors. As a consequence, hospitals in multiethnic areas in western countries are currently forced to rethink their models of communication as they are increasingly transformed into spaces of cultural encounter. During this process one needs to be aware of the cultural diff erences in attitudes and experiences towards communication in critical medical situations. However, the growing academic interest in intercultural relations in the critical care has not yet led to the construction of cumulative knowledge. The aim of this study is to review the experiences of the involved actors, namely the care providers, the patients and their family members, with cultural diversity during the process of communication and decision-making in the ICU. Conclusion Mortality remained higher in older patients despite an absence of age-related diff erences in resource use at ICU. Comparing our results to a previous prospective cohort study [2], we emphasize the lower overall mortality of our population (14.4% vs. 50%). Limitations of our analysis include our unicenter design and lack of data for a closed model ICU. P24 P24 Methods During 7 years (2007 to 2013), TISS-28 was prospectively applied to all consecutive adult critically ill patients at 24 and 72 hours of ICU admission in a private hospital in Brazil. Demographic data, diagnoses on admission, comorbidities, ICU length of stay and mortality were recorded. Patients were stratifi ed according to their ages. 2. Bisaillon S, Li-james S : Family partnership in care: integrating families into the coronary intensive care unit. Can J Cardiovasc Nurs 1997, 8:43-46. 1. Derwick D, Kotagal M: Restricted visiting hours in ICU. J Am Med Assoc 2004, 292:736-737. Do we spend less on older critically ill patients? Relationship among intensity of care, severity of illness and mortality MD Rosa, AC Pecanha Antonio, M Mattioni, L Tagliari, F Schaich, J Maccari, R Oliveira, C Teixeira, TF Tonietto, N Brandão da Silva Hospital Moinhos de Vento, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P22 (doi: 10.1186/cc13212) Methods A prospective court study was conducted (from January to May 2013) to point out the patient’s care divergences (blood transfusion, diuretics, antibiotics, vasopressors, mechanical ventilation, and so forth) that happened in the fi rst 72 hours of ICU admission in a 30-bed adult Brazilian ICU. We enrolled only patients that stayed more than 48 hours in the ICU. Introduction The Therapeutic Intervention Scoring System (TISS-28) quantifi es the type and number of intensive care treatments; therefore, it indicates the workload of ICU and may be used for calculating costs [1]. A previous cohort study [2] demonstrated that seriously ill older patients receive fewer invasive procedures and less resource-intensive hospital care, showing a preferential allocation of hospital services to younger patients regardless of severity of illness. The objective of this study is also to compare resource utilization across diff erent ages of critically ill patients in an open model, mixed ICU. Results In a court of 357 patients at least one divergence between the ICU medical staff and the primary(s) physician(s) were identifi ed in 31 cases (8.6% – divergence group (DG)). The age (67.9 years), gender (55.2% of male), SAPS3 score (45.7) and reasons for ICU admission (emergency surgery 7.6%, nonemergency surgery 30%, clinical 62.4%) were similar in DG and nondivergence (NDG); however, the ICU length of stay (6.2 vs. 3.9 days, P = 0.023), use of mechanical ventilation (48.4% vs. 27%, P = 0.012), vasopressors (77.4% vs. 46%, P = 0.001) and blood transfusion (41.9% vs. 27.6%, P = 0.073) were higher in the DG S9 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References Even the medical team elaborated their interest towards this policy as being a more profound and comprehensive way of supporting the ICU patients. Further investigations are currently being employed to strengthen the outcome of the study. References Methods A prospective study conducted in a 22-bed adult general ICU including patients staying >48 hours. The Hospital Anxiety and Depression Scale (HADS) was completed by pairs (patients/respective family members). They were interviewed by telephone at 30 and 90 days after ICU discharge with the Impact of Event Scale (IES) and the HADS. We separated them into three diff erent groups (patients – group A, family member of patient who survived – group B and family member of patient who deceased – group C). P27 was 51.79 ± 13.47 and 64% were spouses. Patients had symptoms of anxiety (26.1%), depression (12%) or both (8.7%) during the ICU stay. Regarding family members, the incidence of anxiety, depression or both during the ICU was 33.2%, 9.8% and 8.2% respectively. Symptoms of PTSD within patients were found in 6.9% at 30 days and disappeared at 90 days. Diff erently, these symptoms in family members were found in 6.2% at 30 days and 9.0% at 90 days. There was a positive correlation between HADS at ICU and IES at 30 days for family members (r = 0.527, P <0.001). The HADS score over time between patients, family members of patients who deceased and who survived were diff erent (P = 0.002). In post-hoc analysis, group A presented less symptoms than group C at 30 days (P = 0.001) and less than groups B and C at 90 days (P <0.001 for both). At 90 days, group B showed less symptoms than group C (P = 0.039).f P27 Family satisfaction in the ICU: a 6-month experience M Zouka1, A Myrou2, I Soultati2, T Aslanidis2, A Euthymiou2, E Geka2, E Anastasiou2, V Ourailoglou2, M Giannakou2 1Papageorgiou General Hospital, Thessaloniki, Greece; 2AHEPA University Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P27 (doi: 10.1186/cc13217) Introduction The aim of the study was to prospectively assess family satisfaction in the intensive care setting and identify fi elds of possible improvement. Methods The study was performed over a 6-month period in a 10-bed ICU. A properly translated and validated Family Satisfaction in the ICU (FS-ICU 24) questionnaire was used. A total of 126 questionnaires were handed out to family members upon patient discharge. The number of questionnaires returned for evaluation was 120 (91.5% participation). Five-point Likert scale responses were transformed to percentage scores, higher values representing better satisfaction. Conclusion Family members of ICU patients suff er more than the patients principally when their loved one died. Patients’ symptoms of anxiety, depression and PTSD decrease with time but in family members these symptoms continue along the 90 days. R f Results Overall satisfaction with care was reported as very good or excellent by 81% of family members (32% and 49% respectively) both for ICU staff concern and caring as well as symptom management. Favorable scores were also noted in terms of decision-making, 89% of relatives reporting very good and excellent rates as regards information needs. Heart-focused anxiety in critically ill patients’ relatives Z Konstanti, M Gouva, G Nakos, V Koulouras University of Ioannina, Greece Critical Care 2014, 18(Suppl 1):P26 (doi: 10.1186/cc13216) Introduction A series of studies have shown an association between the ICU environment and symptoms of psychological distress in critically ill patients’ relatives [1-3]. The aim of this study was to investigate the risk of cardiophobia and panic attack crisis on ICU patients’ family members. Methods In the period from March 2012 to July 2013, we studied 223 family members (81 men and 142 women, mean age 41.5 ± 11.0 years) of critically ill patients. These patients were admitted to the intensive care department with various medical and surgical conditions (147 patients, 103 men and 44 women, mean age 58.3 ± 18.6 years). We used a questionnaire in which were included: social–demographic characteristics of the patients’ family environment, and the Cardiac Anxiety Questionnaire (CAQ). The results from the CAQ [4] were compared with those of the general population of Greece (GGP). Conclusion Most family members were highly satisfi ed with ICU care their patients received. Nevertheless, our study showed that the fi elds of communication with the nurses as well as the waiting room atmosphere defi nitely need further improvement. Concerning emotional support and decision-making, the study revealed that currently used measures in our ICU still do not apply to all families and probably require a few changes. P26 Heart-focused anxiety in critically ill patients’ relatives Z Konstanti, M Gouva, G Nakos, V Koulouras University of Ioannina, Greece Critical Care 2014, 18(Suppl 1):P26 (doi: 10.1186/cc13216) p y q References 1. Wall RJ et al.: Refi nement, scoring, and validation of the Family Satisfaction in the Intensive Care Unit (FS-ICU) survey. Crit Care Med 2007, 35:271-279. 2. Heyland DK et al.: Family satisfaction with care in the intensive care unit: results of a multiple center study. Crit Care Med 2002, 30:1413-1418. p g p p Results In comparison with the general Greek population, the relatives of critically ill patients demonstrate statistically signifi cant higher scores (P <0.001) on the scales of fear and anxiety in regards of thoracic and heart sensations (1.1 ± 0.9 vs. 0.9 ± 0.9 in GGP), avoidance of activities considered to reproduce cardiac symptoms (1.3 ± 1.0 vs. 1.1 ± 0.9 in GGP), heart-focused attention and self-monitoring of cardiac activity (0.9 ± 0.7 vs. 0.7 ± 0.6 in GGP), and at total CAQ score (1.1 ± 0.6 vs. 0.9 ± 0.6 in GGP). Analysis of variance among family members on heart- focused attention and self-monitoring of cardiac activity showed that hospitalization provoked heart-focused attention (that is, the main factor of perceived panic crisis risk). In accordance with the Bonferroni criterion, we found that patients’ siblings demonstrated statistically signifi cant diff erence compared with patients’ children (P = 0.015), with the latter showing lower levels of heart-focused attention and self- monitoring of cardiac activity. Further MANCOVA analysis showed a statistically signifi cant correlation between age and cardiophobia, and also between education level and heart-focused anxiety (P = 0.041 and P = 0.044 respectively). Qualitative analysis of a family satisfaction in an adult ICU C Ebm1, D Dawson2, M Cecconi2, A Rhodes2 1Wiener Privatklinik, Vienna, Austria; 2St George’s NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P28 (doi: 10.1186/cc13218) Introduction Patient satisfaction is a key determinant of the quality of care within the hospital environment. Critically ill patients often lack capacity. Under such circumstances, family members are often main decision-makers and their satisfaction about the ICU experience resembles an important surrogate in evaluating the quality of care. We aimed to measure family satisfaction with diff erent aspects of medical care. Methods In February 2013, surveys, including 24 qualitative questions, were sent out to relatives of 50 patients 1 month after their discharge from ICU St George’s, UK. Responses were graded 1 to 5, with higher values representing a greater degree of satisfaction.i Conclusion The hospitalization of a patient in the ICU is considered to be a factor that is aff ecting the mental health of patients’ relatives. Our results highlight a risk for psychologically induced symptoms on ICU relatives and underline the need for a psychosocial support system for them. The cardiophobia and self-monitoring of heart activity which infl icts the patients’ relatives appears to be more intense in the siblings, parents and partners of patients. Results Most respondents were satisfi ed with the overall performance and the respect received (mean ± SD score 4.7 ± 0.68). Skills and competency of doctors (4.38 ± 0.67) and nurses (4.57 ± 0.68) was also perceived very positive. However, family satisfaction with communication with doctors (3.52 ± 1.47) and nurses (3.71 ± 0.96), as well as inclusion in decision-making (3.39 ± 1.24), resulted in somewhat lower scores. In particular, ease, clarity, consistency, honesty, and completeness of information given by doctors resulted in inhomogeneous perceptions between relatives (Figure 1). Additionally, the majority of responders felt only moderately satisfi ed with time and support to make a decision. g p References 1. Derwick D, Kotagal M: Restricted visiting hours in ICU. J Am Med Assoc 2004, 292:736-737. p g p Results A total of 184 pairs were interviewed at the ICU. Mean patient age was 59.33 ± 15.5 years. The admission SAPS III was 47.6 ± 15.7 points. Median ICU LOS was 4 days (2 to 47). Family’s age 2. Bisaillon S, Li-james S : Family partnership in care: integrating families into the coronary intensive care unit. Can J Cardiovasc Nurs 1997, 8:43-46. S10 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P28 Qualitative analysis of a family satisfaction in an adult ICU C Ebm1, D Dawson2, M Cecconi2, A Rhodes2 1Wiener Privatklinik, Vienna, Austria; 2St George’s NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P28 (doi: 10.1186/cc13218) P27 On the other hand, most of the family members (73%) felt neither included nor excluded from the decision-making process. Moreover, a small number of them (6%) answered that they received poor emotional support. Finally, the frequency of communication with ICU nurses and the atmosphere in the waiting room were noted as fair by 28% and 47% of subjects respectively.i 1. Azoulay E, et al.: Intensive Care Med 2003, 29:1498-1504. 2. Garrouste-Orgeas M, et al.: Crit Care Med 2008, 36:30-35. 3. Stayt CL: J Adv Nursing 2007, 57:623-630. 4. Dragioti E, et al.: Psychol Rep 2011, 109:77-92. Reference 1. Myhren H, et al.: Patients’ memory and psychological distress after ICU stay compared with expectations of the relatives. Intensive Care Med 2009, 35:2078-2086. 1. Myhren H, et al.: Patients’ memory and psychological distress after ICU stay compared with expectations of the relatives. Intensive Care Med 2009, 35:2078-2086. Outcomes of ventilated surgical and medical ICU patients: do patients die from ARDS or with ARDS? Conclusion Only a small number of patients with HM that die in the ICU have documented ACP prior to admission. Since the majority of ICU patients lack personal decision-making capacity, ACP would ensure that care is consistent with patients’ wishes, EoL actions are congruent with their values, the burden on family and healthcare providers is alleviated and cost is decreased. 1Soroka University Medical Center, Beer Sheba, Israel; 2Beth Israel Deaconess Medical Center, Boston, USA Critical Care 2014, 18(Suppl 1):P29 (doi: 10.1186/cc13219) Introduction Patients with acute respiratory distress syndrome (ARDS) have a high mortality rate. Whether this excess mortality is contributed by ARDS or is a consequence of prolonged mechanical ventilation is unclear. References 1. Azoulay E, et al.: Intensive Care Med 2003, 29:1498-1504. 2. Garrouste-Orgeas M, et al.: Crit Care Med 2008, 36:30-35. 3. Stayt CL: J Adv Nursing 2007, 57:623-630. 4. Dragioti E, et al.: Psychol Rep 2011, 109:77-92. S11 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion In general, relatives felt very satisfi ed with the ICU, especially with the care of the patients and the professional workforce. The complete decision-making process was rated moderately good, which highlights some areas of improvement in involving relatives in the care of their beloved by providing regular, clear, easy to understand and consistent information. Figure 1 (abstract P28). Team performance. Figure 1 (abstract P28). Team performance. Advance care planning in critically ill haematology patients V Metaxa1, J Lambert2, A Barrow3, J De Vos4 1King’s College Hospital, London, UK; 2Royal London Hospital, London, UK; 3John Radcliff e Hospital, Oxford, UK; 4Royal Surrey County Hospital, Guildford, UK Critical Care 2014, 18(Suppl 1):P30 (doi: 10.1186/cc13220) Introduction Signifi cant improvements in chemotherapy, haemato- poietic stem cell transplantation and general intensive care mean that more patients are now living longer and often present to the ICU at various stages of their disease [1,2]. Encouraging patients with haematological malignancy (HM) to express their wishes and values on end of life (EoL) prior to ICU admission would ensure that their autonomy is respected. The aim of our study was to determine whether patients with HM that were admitted and died in the ICU had any form of advance care planning (ACP) documented prior to their admission. Methods Data were collected on all adult patients with HM that were admitted and died in the ICU of a tertiary haematology referral centre in London during the period of 1 year. g p y Results Information was collected for 34 patients and their charac- teristics are shown in Table 1. In 62% of the patients EoL decisions were made, and do-not-attempt-resuscitation documentation was found in 65% of the cases. Documentation on information exchange, and participation in the deliberation or the decision-making process was found in only 30% of the patients, even though more than 75% of the haematology physicians estimated the prognosis of these patients as moderate (17%) or poor (59%). In the vast majority of cases (31/34) the deaths occurred after withholding or withdrawal of treatment in ICU. A new questionnaire to determine the eff ect of team interaction in the ICU on perceived futility and intention to quit: results of a pilot study in two German hospitals A new questionnaire to determine the eff ect of team interaction in the ICU on perceived futility and intention to quit: results of a pilot study in two German hospitals D Schwarzkopf1, F Bloos1, A Meier-Hellmann2, C Icke2, N Riedemann1, CS Hartog1 1Jena University Hospital, Jena, Germany; 2Helios Hospital Erfurt, Germany Critical Care 2014, 18(Suppl 1):P31 (doi: 10.1186/cc13221) A new questionnaire to determine the eff ect of team interaction in the ICU on perceived futility and intention to quit: results of a pilot study in two German hospitals Results A total of 1,396 patients were enrolled between 2001 and 2008: 485 (34.7%) had ARDS on admission (early-onset ARDS), 219 (15.6%) developed ARDS during their ICU stay (late-onset ARDS) and 692 (49.5%) did not meet ARDS criteria prior to ICU discharge or death. Twenty-eight-day mortality rates were 23.7%, 25.6% and 25.7% for early, late and non-ARDS respectively. After adjusting for age, weight on admission, unit of admission (MICU vs. SICU), severity of disease score, comorbidity score, and primary diagnosis on admission, and mortality risk at 28 days, 1 year and 2 years were not signifi cantly diff erent between the three study groups. Neither severity of ARDS or timing of ARDS (early versus late) was associated with mortality. Sensitivity analysis, analyzing all ventilated patients, including those who were ventilated less than 48 hours, showed the same results. Conclusion Neither the presence of ARDS or the severity or timing contribute independently to the short and long mortality risk after adjustment for age, severity of disease, comorbidity score and diagnosis on presentation in patients hospitalized in a noncardiac ICU with acute respiratory failure. y p D Schwarzkopf1, F Bloos1, A Meier-Hellmann2, C Icke2, N Riedemann1, CS Hartog1 1Jena University Hospital, Jena, Germany; 2Helios Hospital Erfurt, Germany Critical Care 2014, 18(Suppl 1):P31 (doi: 10.1186/cc13221) Introduction Perceived futility of care may jeopardize patient quality of care and increase ICU staff turnover. It is related to workload and interdisciplinary collaboration. References 1. Bird GT, et al.: Br J Anaesth 2012, 108:452-459. 2. Howell DA, et al.: Palliat Med 2011, 25:630-641. Methods A longitudinal retrospective study focusing on noncardiac ICU patients who required mechanical ventilation. Patients were classifi ed as having ARDS on admission, late-onset ARDS or no ARDS. The analysis included patients ventilated for longer than 48 hours. Primary outcomes were mortality at 28 days, 1 year and 2 years from ICU admission. References Conclusion In general, relatives felt very satisfi ed with the ICU, especially with the care of the patients and the professional workforce. The complete decision-making process was rated moderately good, which highlights some areas of improvement in involving relatives in the care of their beloved by providing regular, clear, easy to understand and consistent information. Conclusion In general, relatives felt very satisfi ed with the ICU, especially with the care of the patients and the professional workforce. The complete decision-making process was rated moderately good, which highlights some areas of improvement in involving relatives in the care of their beloved by providing regular, clear, easy to understand and consistent information. Table 1 (abstract P30). Patient characteristics Mean age 30 Median APACHE II score 26 >3 organ support 60% Leukaemias >50% Table 1 (abstract P30). Patient characteristics P33 Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P33 (doi: 10.1186/cc13223) Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 qi y p g Results A total of 220 nurses and 55 physicians participated. Scales showed good reliability with all Cronbach’s alpha ≥0.8 and signifi cant between unit variability (all P ≤0.011). On a scale from 0 (‘never’) to 5 (‘very often’), ICU staff rated frequency of futile care as median 3.6 (IQR: 3 to 4). On a seven-point Likert scale with 7 denoting maximal values, intention to quit was rated as 2.3 (1 to 4). Nurses gave signifi cantly lower ratings of job satisfaction than physicians (4 (3 to 5) vs. 5 (4 to 6), P ≤0.001), perceived futility more often than physicians (P ≤0.001), and rated all aspects of nurse–physician collaboration worse than physicians, including attendings’ inclusive leadership, collaboration, decision-making and psychological safety (all P ≤0.001). Futility and intention to quit were each predicted by high workload and low psychological safety (all P ≤0.009). Among nurses, inclusive leadership by the head nurse prevented intention to quit (P = 0.001) and a composite score of good nurse–physician interactions predicted less perception of futile care (P ≤0.001). Introduction The aim of the ICU is to give the best life care to the admitted patients in order to preserve and restore the patients’ quality of life. In some critical patients, life-supporting therapies become actions to support the end stage of their life [1]. This is the paradox of the ICU. Patients aff ected by amyotrophic lateral sclerosis (ALS) belong to this category: for these patients, all medical actions do not improve the quality of life but just prolong it. ALS or Charcot’s disease is the most common motor neuron disease which causes severe motor disability and evolves in a few years to death [2]. We want to present our experience in patients aff ected by ALS in the ICU.f p pf y Methods We collected data about patients aff ected by ALS admitted to the ICU from 1 January 2010 to 31 October 2013. We considered entry diagnosis, mean age, need to perform tracheostomy and/or percutaneous endoscopic gastrostomy (PEG), and mortality in the ICU. Results Sixty patients were admitted for neuromuscular respiratory failure complicated by aspiration pneumonia. Mean age was 43 years. The male to female ratio was 3:1. P33 decision-making, psychological safety, workload, intention to quit and a newly developed scale for perceived futile care. English questions were translated into German by state-of-the art forward and backward translations. Reliability – that is, internal consistency and inter-rater reliability of scales – was evaluated. Predictors of perceived futile care and intention to quit were identifi ed by multiple regressions. P33 Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P33 (doi: 10.1186/cc13223) A new questionnaire to determine the eff ect of team interaction in the ICU on perceived futility and intention to quit: results of a pilot study in two German hospitals Our aim was to evaluate concepts from team science, namely inclusive leadership (a style that invites and appreciates others’ contributions) and psychological safety (a key antecedent of speaking up and learning behavior), and to determine their eff ect on perceived futility and intention to quit.f Conclusion Neither the presence of ARDS or the severity or timing contribute independently to the short and long mortality risk after adjustment for age, severity of disease, comorbidity score and diagnosis on presentation in patients hospitalized in a noncardiac ICU with acute respiratory failure. f Methods A staff survey in four interdisciplinary ICUs and two intermediate care units of two tertiary care hospitals. The questionnaire contained validated scales to assess inclusive leadership of head nurses and attending physicians, nurse–physician collaboration, collaborative S12 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Death rate of patients admitted to a Brazilian ICU on weekends and holidays y Methods We analyzed the records of 11,230 ICU admissions. ASA and elective/nonelective nature of the admission were combined into eight categories. We used the chi-square test and accepted the null hypothesis if P >0.05. Areas under the ROC curve using ASA and ASA/ admission categories were constructed. P Batista, R Passos, R Oliveira, M Ribeiro, F Alves Hospital Sao Rafael, Salvador Bahia, Brazil Critical Care 2014, 18(Suppl 1):P34 (doi: 10.1186/cc13224) p Critical Care 2014, 18(Suppl 1):P34 (doi: 10.1186/cc1322 g Results We included 5,998 patients admitted to the ICU immediately after surgery. Forty-one percent were older than 65 years and 51% were female. Length of stay previous to ICU admission was 2.5 ± 6.1 days and duration of ICU stay was 1.9 ± 2.4 days. Elective hospital admission occurred in 65% of the patients. Neurosurgery with 21% of the procedures was followed by abdominal surgery (20%), and orthopedic surgery (15%). The death rate at the hospital was 6.9% (2.8% in ASA I patients and 31% in ASA IV/E). Higher ASA classifi cation was signifi cantly associated with the death rate both in the ICU and at the hospital (P <0.00001). The nonelective nature of the hospital admission was also associated with higher risk of death at the ICU (P <0.0001) and in the hospital (P <0.0001). Gender and hour of admission was not associated with the death rate. The combination of ASA classifi cation with the nonelective nature of the hospital admission produced an eight- category index signifi cantly associated with mortality (P <0.00001). Analyzing hospital mortality, the area under the ROC curve was 0.77 (95% CI 0.74 to 0.79) for this index and was 0.72 (95% CI 0.69 to 0.75) for ASA. When analyzed for death at the ICU the AUROC was 0.71 (95% CI 0.69 to 0.75, P <0.0001) for ASA and 0.75 (95% CI 0.71 to 0.78, P <0.0001) for ASA nonelective admission index. Introduction Several studies conducted in a number of diff erent populations indicate that patients admitted to hospitals on weekends have a higher mortality rate. Many factors have been proposed to explain these observations and in most studies, however, this eff ect disappeared after controlling for illness severity. We undertook the present study to explore the eff ect that day of ICU admission may have on death rate. P33 Till death do us part: amyotrophic lateral sclerosis in the ICU Y Valzani1, A Marudi2, G De Grandis2, J Mandrioli2, S Baroni2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P33 (doi: 10.1186/cc13223) All patients underwent percutaneous tracheostomy and PEG. One of them died in the ICU because of septic shock. In every case we honestly communicated the worsening of the disease and we perceived the awareness of it by the patients and/or their family. In no case did patients ask us to withdraw the necessary cures such as percutaneous tracheostomy and PEG. In no case did they ask us to die. In Europe the ‘end of life care’ law is very dyshomogeneous because of diff erent cultures, traditions, religions, and beliefs. In Italy euthanasia is not allowed by law. It is the physician often by himself that has to make the fi nal decision according to the patient’s mind.f Conclusion The scales of the new questionnaire showed good reliability and diff erentiated between nurses and physicians. Psychological safety and inclusive leadership proved to be important concepts. P32 ASA helps prediction of the death rate in surgical ICU patients P Batista, R Passos, C Gomes, A Oliveira Hospital Sao Rafael, Salvador Bahia, Brazil Critical Care 2014, 18(Suppl 1):P32 (doi: 10.1186/cc13222) i g p Conclusion Our experience shows that patients aff ected by severe motor disability are not always willing to die. It is essential that decisions should be patient-centered and taken multidisciplinarily, based on open and emphatic communication, involving family and caregivers. References Introduction Many scoring systems have been designed to predict mortality in surgical patients; however, these systems require the collection of several parameters that may not be available at ICU admission. As a result, these classifi cation systems are not used as a routine part of clinical practice. The aim of this study was to evaluate the prognostic value of ASA classifi cation predicting ICU and hospital mortality. 1. Servillo G, Vargas M: Transl Med UniSa 2011, 1:237-242 2. Gordon PH: Aging Dis 2013, 4:295-310. 1. Servillo G, Vargas M: Transl Med UniSa 2011, 1:237-242 2. Gordon PH: Aging Dis 2013, 4:295-310. 3 Autopsy-detected diagnostic errors in critically ill patients with cirrhosis N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) Table 1 (abstract P35). Cluster 1 Cluster 2 Cluster 3 Cluster 4 (n = 353) (n = 755) (n = 667) (n = 272) ICU LOS, mean ± SD (days) 0.5 ± 0.2 2.0 ± 0.8 7.2 ± 2.8 25.6 ± 15.2 Mechanical ventilation 282 (79.9%) 631 (83.6%) 604 (90.6%) 269 (98.9%) Tracheostomy 3 (0.9%) 13 (1.7%) 14 (2.1%) 127 (46.7%) CPR 20 (5.7%) 19 (2.5%) 16 (2.4%) 8 (2.9%) Conclusion Cluster analysis can identify unique typologies of death within ICUs. This approach may be a novel method to more specifically Table 1 (abstract P35). Cluster 1 Cluster 2 Cluster 3 Cluster 4 (n = 353) (n = 755) (n = 667) (n = 272) ICU LOS, mean ± SD (days) 0.5 ± 0.2 2.0 ± 0.8 7.2 ± 2.8 25.6 ± 15.2 Mechanical ventilation 282 (79.9%) 631 (83.6%) 604 (90.6%) 269 (98.9%) Tracheostomy 3 (0.9%) 13 (1.7%) 14 (2.1%) 127 (46.7%) CPR 20 (5.7%) 19 (2.5%) 16 (2.4%) 8 (2.9%) Conclusion Cluster analysis can identify unique typologies of death within ICUs. This approach may be a novel method to more specifi cally target ICU eff orts to reduce mortality and improve the quality of death. P36 Independent risk factors associated with the decision to withhold therapeutic intervention in patients admitted to the emergency room L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P36 (doi: 10.1186/cc13226) Introduction The decision to withhold therapeutic intervention in a Introduction Even with the availability of new and more eff ective diagnostic procedures over the past decades, autopsy remains one of the most reliable methods to validate clinical diagnoses. The aim of this review was to determine whether autopsy plays a role in extending knowledge about the cause of death in patients with cirrhosis who died in the ICU. Methods We conducted a retrospective review of medical records and postmortem fi ndings in critically ill patients with cirrhosis who were admitted to a major university teaching medical ICU (MICU) between August 2007 and August 2013. Agreement between diagnoses before death and postmortem fi ndings were compared using the Goldman system [1]. Review was independently performed by a fellow in critical care medicine and a board-certifi ed attending. Autopsy diagnoses included histologic and microbiological fi ndings. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 role of the physician is to cure, treat and alleviate suff ering. When the fi rst two goals are not possible the medical role should be dedicated to end-of-life treatments adjusted to the patient’s benefi t [1,2]. Methods A retrospective cohort study including all adult patients with sepsis admitted to the emergency room (ER) at a tertiary care, university hospital between 1 July 2011 and 30 June 2012. Results During the study period 162 patients with sepsis were admitted to the ER, of which 40 (25%) had withheld therapeutic decisions. Comparing this group with the group without therapeutic limitations, patients in the fi rst group were older (81 ± 13 vs. 68 ± 14, P <0.001), with more comorbidities (90% vs. 66%, P = 0.004) and a higher proportion needing help in daily activities (Karnofsky performance status (KPS) <70% = 55% vs. 8%, P <0.001). The hospital mortality in patients with a decision to limit the therapeutic intervention was signifi cantly higher (83% vs. 43%, P <0.001). Variables independently associated with the withholding therapy decision were age (adjusted OR per year = 1.078, P <0.001), presence of comorbidities (adjusted OR = 4.632, P = 0.030), chronic wounds (adjusted OR = 5.965, P = 0.005) and patient’s needed of help in daily activities (KPS <70%, adjusted OR = 5.391, P = 0.012). In the fi rst group a lower proportion received antibiotics (70% vs. 99%, P <0.001) and when those were considered inadequate for the agent responsible for the sepsis episode it was less frequently changed (15% vs. 50%, P = 0.028). However, no diff erences were found regarding the elapsed time from admission to the ER until the fi rst medical contact or the time since the recognition of sepsis and antibiotic administration, although the group with withholding decisions had less specimens collected for microbiology: blood cultures (68% vs. 91%, P <0.001) or other specimens (58% vs. 96%, P <0.001). C l i I hi d h d i i i hh ld h role of the physician is to cure, treat and alleviate suff ering. When the fi rst two goals are not possible the medical role should be dedicated to end-of-life treatments adjusted to the patient’s benefi t [1,2]. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 patients admitted on weekends and on weekdays were found in the group admitted after surgery (respectively 10% and 30%, P <0.0001), originating from the emergency unit (respectively 30% and 16%; P <0.0001), originating from step-down units (respectively 31% and 17%; P <0.0001), patients from clinical teams compared with surgical teams (respectively 28% and 13%; P <0.0001), previous hospital length of stay lower versus higher than 2 days (respectively 15% and 25%; P <0.0001) and age ≥65 years (respectively 21% and 18%; P <0.0001). The mortality ratios were signifi cantly diff erent between these groups. Multiple logistic regression showed that the inclusion of other variables reduces the odds ratio associated with admission on weekends and holidays to 1.22 (95% CI 1.08 to 1.39, P <0.002) for ICU mortality and to 1.23 (95% CI 1.09 to 1.38, P <0.001). i Methods A retrospective cohort study including all adult patients with sepsis admitted to the emergency room (ER) at a tertiary care, university hospital between 1 July 2011 and 30 June 2012. y p y Results During the study period 162 patients with sepsis were admitted to the ER, of which 40 (25%) had withheld therapeutic decisions. Comparing this group with the group without therapeutic limitations, patients in the fi rst group were older (81 ± 13 vs. 68 ± 14, P <0.001), with more comorbidities (90% vs. 66%, P = 0.004) and a higher proportion needing help in daily activities (Karnofsky performance status (KPS) <70% = 55% vs. 8%, P <0.001). The hospital mortality in patients with a decision to limit the therapeutic intervention was signifi cantly higher (83% vs. 43%, P <0.001). Variables independently associated with the withholding therapy decision were age (adjusted OR per year = 1.078, P <0.001), presence of comorbidities (adjusted OR = 4.632, P = 0.030), chronic wounds (adjusted OR = 5.965, P = 0.005) and patient’s needed of help in daily activities (KPS <70%, adjusted OR = 5.391, P = 0.012). In the fi rst group a lower proportion received antibiotics (70% vs. 99%, P <0.001) and when those were considered inadequate for the agent responsible for the sepsis episode it was less frequently changed (15% vs. 50%, P = 0.028). References 1. Damghi et al.: BMC Emergency Med 2011, 11:12. 2. Conte et al.: Intensive Care Med 2010, 36:765-772. 1. Damghi et al.: BMC Emergency Med 2011, 11:12. 2. Conte et al.: Intensive Care Med 2010, 36:765-772. y Results Four clusters were identifi ed. Short ICU LOS and low life- sustaining therapy utilization but relatively frequent CPR characterized Cluster 1. Intermediate ICU LOS and moderate life-sustaining therapy utilization characterized Clusters 2 and 3. Prolonged ICU LOS and high life-sustaining therapy utilization (except CPR) characterized Cluster 4. Age and severity of illness decreased across clusters with the oldest, sickest patients in Cluster 1 and the youngest, least sick patients in Cluster 4. See Table 1. How many ways are there to die? Identifi cation of ICU death typologies using cluster analysis L Reineck, A Barnato, R Arnold, D Angus, J Kahn University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P35 (doi: 10.1186/cc13225) How many ways are there to die? Identifi cation of ICU death typologies using cluster analysis L Reineck, A Barnato, R Arnold, D Angus, J Kahn University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P35 (doi: 10.1186/cc13225) Introduction Although avoidance of death is a key goal of critical care, so too is provision of high-quality end-of-life care when life-prolonging therapy is not desired. One often-used measure of ICU performance is risk-adjusted mortality, yet this measure treats all deaths equally and thus is fl awed. In this work, we identify diff erent death typologies using cluster analysis, with the ultimate goal of more narrowly targeted ICU quality improvement measures and eff orts. Conclusion In this study the decision to withhold therapy was independently associated with increasing age, the presence of comorbidities and loss of functional autonomy. For the same level of intervention such as antibiotic administration, the decision to withhold therapy did not infl uence the effi cacy of therapeutic attitudes. References Methods We performed k-means cluster analysis in 2,047 ICU decedents admitted to a university medical center in 2011/12. Variables for the analysis included ICU length of stay (LOS), mechanical ventilation, tracheostomy, gastrostomy tube insertion, dialysis, enteral or parenteral nutrition, and cardiopulmonary resuscitation (CPR). Death rate of patients admitted to a Brazilian ICU on weekends and holidays Methods We analyzed 11,230 electronic records from patients admitted from 1 January 2006 to 30 June 2013. The ICU admission dates were categorized as normal weekdays and as weekends and holidays. The dependent variables were ICU mortality and hospital mortality. The chi- square test and Student t test were used as appropriate and signifi cant association was accepted when P <0.05. Multiple logistic regression (backward conditional) was used accepting a variable when P <0.05 and rejecting a variable with P >0.1. SPSS version 19.0 was used. Methods We analyzed 11,230 electronic records from patients admitted from 1 January 2006 to 30 June 2013. The ICU admission dates were categorized as normal weekdays and as weekends and holidays. The dependent variables were ICU mortality and hospital mortality. The chi- square test and Student t test were used as appropriate and signifi cant association was accepted when P <0.05. Multiple logistic regression (backward conditional) was used accepting a variable when P <0.05 and rejecting a variable with P >0.1. SPSS version 19.0 was used. Results Forty-nine percent of these patients were female, 53% was admitted immediately after surgery, 56% had <2 days of previous hospital admission, and 19.2% were admitted during weekends and holidays. Mortality in the ICU for admissions on weekends and holidays was 24% and was 13% for those admitted on weekdays (OR = 2.23, 95% CI 2.02 to 2.47; P <0.0001). The hospital mortality was respectively 35% and 20% (OR = 2.23, 95% CI 2.02 to 2.47; P <0.0001). Diff erences between Conclusion These results show that the ASA index can be used, preferably in combination with other data from electronic records, to predict mortality for surgical ICU patients. S13 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 However, no diff erences were found regarding the elapsed time from admission to the ER until the fi rst medical contact or the time since the recognition of sepsis and antibiotic administration, although the group with withholding decisions had less specimens collected for microbiology: blood cultures (68% vs. 91%, P <0.001) or other specimens (58% vs. 96%, P <0.001). Conclusion The higher death rate on weekends and holidays may be related to case mix. The interplay of other variables possibly related either to admission on weekends or higher death rate should be sought. 3 Autopsy-detected diagnostic errors in critically ill patients with cirrhosis N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) Conclusion In KSA, abdomino-pelvic traumas are serious injuries aff ecting mainly young male victims; however, with a lower mortality than predicted. Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. j Conclusion In KSA, abdomino-pelvic traumas are serious injuries aff ecting mainly young male victims; however, with a lower mortality than predicted. P39 Radiation exposure in trauma patients is aff ected by age M Wagner1, JV Vonk2, CW Wichman1, AH Hegde3, JO Oliveto3 1Creighton University, Omaha, NE, USA; 2University of Iowa, Iowa City, IA, USA; 3UNMC, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P39 (doi: 10.1186/cc13229) Introduction Trauma patients are subjected to higher radiation exposure (RE) as a function of Injury Severity Score (ISS) [1]. Analysis of this dataset was done to ascertain if RE varied with patient age. p g Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). RE as a function of the ISS and age was explored for 7,661 trauma patients. The ISS groupings were as follows: ISS 1 to 8, ISS 9 to 15, ISS 16 to 25, and ISS >25. Age groupings were 19 to 39, 40 to 59, 60 to 79, and >80 years. Conclusion Despite declining rates worldwide, autopsy remains an important tool for quality and safety assurance. In this retrospective study, autopsy showed that knowledge of the correct premortem diagnosis would have altered therapy in 10% of critically ill cirrhotic patients. y Results The data were analyzed in a hierarchical fashion: the mean exposure by age group was compared by one-sided, two-sample t tests. Diff erences were set up to test if mean exposure decreased with increasing age. Bonferroni adjustment was used for all multiple comparisons to maintain the overall error rate at 0.05. 3 Autopsy-detected diagnostic errors in critically ill patients with cirrhosis N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) Of the six independent diff erence tests conducted, fi ve were signifi cant: 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; 40 to 59 versus >79; and 60 to 79 versus >79 (P <0.01). Analysis was done for ISS categories: ISS 1 to 8, ISS 9 to 15, ISS 16 to 25, and ISS >25. For ISS 1 to 8, fi ve diff erences were signifi cant: 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; 40 to 59 versus >79; and 60 to 79 versus >79 (P <0.01). For ISS 9 to 15, all diff erences were signifi cant: 19 to 39 versus 40 to 59; 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; 40 to 59 versus >79; and 60 to 79 versus >79 (P <0.01). For ISS 16 to 25, four diff erences were signifi cant: 19 to 39 versus 60 to 79; 19 to 39 versus >79; 40 to 59 versus 60 to 79; and 40 to 59 versus >79 (P <0.01). For ISS > 25, none of the diff erences were signifi cant. 3 Autopsy-detected diagnostic errors in critically ill patients with cirrhosis N De Mey, J Wauters, A Wilmer, P Meersseman UZ Gasthuisberg, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P37 (doi: 10.1186/cc13227) The records were also reviewed for demographics, APACHE II score and all performed diagnostic procedures. P36 Independent risk factors associated with the decision to withhold therapeutic intervention in patients admitted to the emergency room L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P36 (doi: 10.1186/cc13226) Results Of 641 patients admitted with diagnosis of cirrhosis, 86 (13%) died in the MICU. Forty-fi ve (52%) patients underwent an autopsy. Forty-two autopsy reports were available for review, three histologic and microbiological reports were missing. Major missed diagnoses (principal underlying disease related to death and primary cause of death itself) were present in seven patients (17%), four in class I (10%) and three in class II (7%) (Table 1). Minor missed diagnoses were present in 13 patients (31%) (class III and IV). Postmortem fi ndings were in complete agreement (class V) with clinical cause of death in almost one-half of the patients (n = 19, 45%). Introduction The decision to withhold therapeutic intervention in a patient is a complex decision, wrapped in profound ethical debates. The S14 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P37). Major missed diagnosis Class I (n = 11) Class II (n = 3) 1. Metastatic linitis plastica 1. Acute pancreatitis 2. Oesophageal variceal bleeding 2. Necrotizing mucor mycosis pneumonia 3. Disseminated mucor mycosis 3. Focal aspergillosis (myocardial) 4,5,6. Invasive aspergillosis (n = 3) 7. Invasive candidiasis 8. Herpes simplex pneumonia 9. Pneumocystis pneumonia 10. Gastric rupture 11. Disseminated HCC, vs. porta thrombosis Class I = missed major diagnosis that would have changed management and might have resulted in cure or prolonged survival. Class II = missed major diagnosis that would not have modifi ed ongoing patient care. Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. Conclusion In KSA, abdomino-pelvic traumas are serious injuries aff ecting mainly young male victims; however, with a lower mortality than predicted. Compared with survivors, nonsurvivors were older in age; had higher ISS, APACHE II, BMI, lactic acid, INR and creatinine, and lower GCS, PaO2/ FiO2 and platelet count; were more likely to be mechanically ventilated and on vasopressors; and were associated with more head injuries. Reference 1. Goldman et al.: The value of autopsy in three medical eras. N Engl J Med 1983, 308:1000-1005. P38i Profi le, outcomes, and predictors of mortality of abdomino-pelvic trauma patients in a tertiary ICU in Saudi Arabia S Haddad, Z Youssef, S Azzam, A Aldawood, A Al-Zahrani, H Al-Zamel, H Tamim, A Deeb, Y Arabi King Abdulaziz Medical City, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P38 (doi: 10.1186/cc13228) Introduction Saudi Arabia (KSA) has the world’s highest number of deaths from motor vehicle accidents (MVAs). Numerous trauma victims sustain abdomino-pelvic injuries which are associated with considerable morbidity and mortality. The purpose of this study is to describe the profi le, outcomes and predictors of mortality of patients with abdomino-pelvic trauma admitted to the ICU in a tertiary care trauma center in Riyadh, KSA. gi Conclusion Previous studies show signifi cant RE in trauma patients [1]. We demonstrated the signifi cance of RE to trauma patients, that the amount of RE has gone up chronologically over time, and that patients with an increasing ISS have a higher RE. We sought to determine whether age played a factor in RE. Based on our statistical analyses, older patients receive less RE for a given ISS. Although this is a large assessment of RE and trauma patients broken down by ISS and patient age, data analysis by year was limited by the small number of patients in high ISS groups at higher ages. y Methods This is a retrospective analysis of a prospectively collected ICU database. All consecutive patients older than 14 years with abdomino-pelvic trauma from March 1999 to June 2013 were included. The followings were extracted: demographics, injury severity, mechanism and type of injury, associated injuries, use of vasopressors and mechanical ventilation, and worse laboratory results in the fi rst 24 hours. The primary outcome was hospital mortality. Secondary outcomes were ICU mortality, mechanical ventilation duration, need for tracheotomy, and ICU and hospital length of stay. We compared the profi le, outcomes, and the predictors of mortality between survivors and nonsurvivors. Reference Reference 1. Wagner M, et al.: 258 Increased Injury Severity Score is associated with increased radiation exposure in trauma patients. Crit Care Med 2013, 41:A59. Predictors of outcome in patients with haematological malignancies admitted to critical care 2 2 3 2 A Tridente1, K Browett2, J Hall 2, Y Sorour3, J Snowden2, S Webber2 1St Helens and Knowsley, Liverpool, UK; 2STH, Sheffi eld, UK; 3DBH, Doncaster, UK Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) p Results We included a total of 469 patients with oncological and hematological malignancies, of whom 8.9% (n = 42) were admitted to the ICU and 18.7% (n = 88) did not survive until the 30-day follow- up. There was a strong association of initial proADM levels and 30-day mortality risk (odds ratio (OR) per 10-fold increase 9.9, 95% CI 4.3, 22.9) with an AUC of 0.67 (95% CI 0.60, 0.74). This association remained signifi cant after multivariate adjustment for initial vital signs (blood pressure, pulse, temperature) and comorbidities (chronic heart failure, chronic obstructive pulmonary disease, diabetes, coronary heart disease) with an adjusted OR of 9.0 (95% CI 3.1, 26.4). There was also a signifi cant association of proADM and LOS (adjusted regression coeffi cient per 10-fold increase: 6.6, 95% CI 2.0, 11.2). Introduction Critical care (CC) admission has traditionally been viewed as likely to result in a poor outcome for immune-compromised haemato-oncological (HO) patients [1,2]. Recent studies have challenged such views [3,4]. We recently reported results from a cohort of HO patients admitted to CC, showing the pAO2/FiO2 (P/F) ratio to be the only independent predictor of mortality [5]. We report analyses of a larger cohort of HO patients admitted to CC. g p Methods We assessed outcome for HO patients at CC (primary outcome) and hospital discharge, and at 6-month and 1-year follow- up. Single variable logistic regression analyses, adjusted by age, gender and haematological diagnosis, and multivariate analyses were performed to identify independent predictors of outcome using STATA. Results A total of 225 HO patients were admitted to CC. Median age was 59 (interquartile range (IQR) 46 to 66) years. The most common haematological diagnoses on admission were acute myeloid leukaemia in 57 (25.3%) cases, non-Hodgkin lymphoma in 54 (24%) cases and multiple myeloma in 42 (18.7%) patients. Median APACHE II score was 21 (IQR 17 to 26). A total of 164 patients (72.9%) had at least one organ supported. Unit and hospital mortality rates were 34.7% (78 patients) and 49.3% (111 patients), respectively. At 6-month and 1-year follow-up, mortality increased to 63.1% (142 patients) and 70.5% (153 patients), respectively. P40 Survival rate and predictors of outcome in intubated patients with haematological malignancies in a Greek ICU V Tsolaki, M Karapetsa, G Ganeli, A Mpouzia, E Zakynthinos University Hospital of Larissa, Greece Critical Care 2014, 18(Suppl 1):P40 (doi: 10.1186/cc13230) Results Of 9,974 trauma patients during the study period, 702 patients with abdomino-pelvic trauma were admitted to the ICU. The average age was 30.7 ± 14.4 years and the majority was male (89.5%). MVA was the most common cause of abdomino-pelvic trauma (86%). Pelvis (46.2%), liver (25.8%), and spleen (23.1%) were the most frequent injuries; and chest (55.6%), head (41.9%), and lower extremities (27.5%) were the most commonly associated injuries. Mechanical ventilation was required in 89.6%, emergency surgery was performed in 45% and vasopressors were used in 46.6% of patients. The most commonly performed interventions during the ICU stay were surgery (39.5%) and chest tube insertion (33.3%). Of the 702 patients with abdomino-pelvic trauma, 115 (16.4%) patients did not survive. Associated head trauma and retroperitoneal hematoma, higher level of lactic acid on admission and ISS, and advanced age were independent risk factors for fatality. Introduction The admission of patients with haematological malignancies to the ICU is associated with high mortality, especially when mechanical ventilation is required [1,2]. The purpose of the present study was to explore survival rates and the predictors of survival in this group of patients. Methods A retrospective study of intubated patients with haemato- logical malignancies, admitted to a general ICU in central Greece, due to any cause. S15 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results During a 10-year period (2003 to 2013), 16 patients with haematological malignancies (nine with acute myelogenous leukaemia, four with non-Hodgkin lymphoma, one with Hodgkin lymphoma and two with multiple myeloma, mean age 50.75 ± 16.59) (male/ female 3/13, mean APACHE II score 23.18 ± 6.67, mean SOFA score 12.50 ± 2.82, CRP 16.00 ± 10.13, WBC 2.055 ± 30.053) were admitted to the ICU. The majority of patients were admitted due to ARDS (PO2/ FiO2 164 ± 109), one patient was admitted due to intestinal rupture and peritonitis and the other one due to intracerebral haemorrhage. P40 In the majority of the patients (13/16) diagnosis of the malignancy was made during the present admission and only three had the malignancy for a longer period (5 months to 3 years). The mean ICU length of stay was 10.56 ± 16.19 days. A total 68.75% (11/16) of the patients were intubated upon admission, whereas the mean time to intubation for the rest of the patients was 6.37 ± 4.16 hours. Neither intubation upon admission nor time to intubation was correlated with survival. Type of haematological malignancy, duration of immunosuppression and preceding length of stay in the general ward did not correlate with survival either. The survival rate was 18.7% and in linear regression analysis, duration of treatment with NIV in the general ward, increased SOFA score and the number of platelets (<50.000) upon admission to the ICU were independent predictors of survival (R2 = 0.77, P = 0.017). mortality, in agreement with previously published data [6]. The CC mortality rate was 34.7%; at 1 year, mortality had risen to 70.5%. Acknowledgement AT and KB are joint fi rst authors. References 1. Ewig: Eur Respir J 1998, 12:116-122. 2. Rubenfeld: Ann Int Med 1996, 125:625-633. 3. Kroschinsky: Intensive Care Med 2002, 28:1294-1300. 4. Cuthbertson: JICS 2008, 9:135-140. 5. Browett: Br J Aanaesth 2013, 110:860-885. 6. Bird: Br J Aanaesth 2012, 108:452-459. 1. Ewig: Eur Respir J 1998, 12:116-122. 2. Rubenfeld: Ann Int Med 1996, 125:625-633. 3. Kroschinsky: Intensive Care Med 2002, 28:1294-1300. 4. Cuthbertson: JICS 2008, 9:135-140. 5. Browett: Br J Aanaesth 2013, 110:860-885. 6. Bird: Br J Aanaesth 2012, 108:452-459. P42 Early risk stratifi cation in patients with oncological and hematological malignancies in the emergency department A Rast, D Steiner, A Kutz, M Bargetzi, B Mueller, P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P42 (doi: 10.1186/cc13232) Introduction There are no well-validated risk scores for patients with oncological and hematological malignancies presenting to the emergency department (ED). Previous research found the prognostic blood biomarker pro-adrenomedullin (proADM) to be associated with infection-related complications and mortality and thus may be helpful in managing febrile patients with malignancies. Yet the prognostic value of proADM in general oncological patients presenting to the ED remains unclear. Herein, the objective of this study is to evaluate the prognostic potential of proADM and clinical parameters in a consecutive cohort of patients with oncological and hematological malignancies with regard to ICU admission and 30-day mortality. P41 Predictors of outcome in patients with haematological malignancies admitted to critical care A Tridente1, K Browett2, J Hall 2, Y Sorour3, J Snowden2, S Webber2 1St Helens and Knowsley, Liverpool, UK; 2STH, Sheffi eld, UK; 3DBH, Doncaster, UK Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) P41 Predictors of outcome in patients with haematological malignancies admitted to critical care A Tridente1, K Browett2, J Hall 2, Y Sorour3, J Snowden2, S Webber2 1St Helens and Knowsley, Liverpool, UK; 2STH, Sheffi eld, UK; 3DBH, Doncaster, UK Critical Care 2014, 18(Suppl 1):P41 (doi: 10.1186/cc13231) yf References 1. Cherif H, et al.: Support Care Cancer 2007, 15:1393-1398. 2. Comet AD, et al.: Eur J Haematol 2005, 74:511-516. 1. Cherif H, et al.: Support Care Cancer 2007, 15:1393-1398. 1. Cherif H, et al.: Support Care Cancer 2007, 15:1393-1398. 2. Comet AD, et al.: Eur J Haematol 2005, 74:511-516. 1. Cherif H, et al.: Support Care Cancer 2007, 15:1393 1398. 2. Comet AD, et al.: Eur J Haematol 2005, 74:511-516. Methods We enrolled all consecutive patients with oncological and hematological malignancies seeking ED care at a tertiary care hospital from February 2013 to October 2013. We prospectively collected various clinical features, and measured blood parameters including proADM upon admission. To assess outcomes, data from electronic medical records – that is, ICU admission, length of stay (LOS), and post- acute care location – were used and we contacted all patients 30 days after hospital admission. Logistic regression models with area under the receiver operating curve (AUC) were used to assess association of baseline parameters and outcomes. Predictors of outcome in patients with haematological malignancies admitted to critical care 2 2 3 2 The APACHE II score (OR = 0.93, 95% CI = 0.89 to 0.97, P <0.001), number of organs supported (OR = 0.34, 95% CI = 0.23 to 0.48, P <0.001), P/F ratio (OR = 1.06, 95% CI = 1.03 to 1.09, P <0.001), inotropic requirement (OR = 0.27, 95% CI = 0.15 to 0.49, P <0.001), and IMV status (OR = 0.06, 95% CI = 0.03 to 0.14, P <0.001) infl uenced unit survival at single variable analyses. At multivariate analysis, the P/F ratio (OR = 1.05, 95% CI = 1.02 to 1.09, P = 0.002) and IMV status (OR = 0.12, 95% CI = 0.04 to 0.35, P <0.001) independently predicted outcome. Conclusion Organ failures and need for organ support correlated with outcome P/F ratio and need for IMV were independent predictors of fi Conclusion This study including consecutive patients with oncological and hematological malignancies found a moderate association of proADM with 30-day mortality and LOS. proADM in combination with clinical parameters may help to improve site-of-care decisions for these patients in the future. P40 Conclusion The present retrospective study indicates that patients with haematological malignancies have poor survival when they are admitted to the ICU. Longstanding treatment with NIV before ICU admission, high SOFA scores and low platelet levels upon admission negatively aff ect survival. References PP Dobesh, TR McGuire, DG Klepser, AL Himmelberg, DA Roberts, KM Olsen PP Dobesh, TR McGuire, DG Klepser, AL Himmelberg, DA Roberts, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P46 (doi: 10.1186/cc13236) University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P46 (doi: 10.1186/cc13236) Introduction The purpose of this study was to evaluate the impact of obesity on outcomes in patients with severe sepsis. Since obesity is considered an infl ammatory disease and is associated with elevations in several infl ammatory mediators important in the outcome of sepsis, the relationship between obesity and outcome in septic patients was studied. Methods This retrospective cohort study included all patients over the age of 40 with a confi rmed diagnosis of severe sepsis and an ICU stay at our academic medical center from 1 January 2005 to 31 March 2011. Obesity was defi ned as a body mass index of 30 or greater. Data on other patient demographics and APACHE II score at the time of sepsis were collected from patient charts. Outcomes measured included in- hospital mortality, development of acute respiratory distress syndrome (ARDS), days on mechanical ventilation, hospital cost, and length of stay.i Conclusion Regardless of the EWS, critical care teams are heavily involved in the management of patients outside critical care units. Fifty per cent of patients reviewed by the critical care team subsequently required admission to a critical care unit. The trigger threshold (7 and above) for referral to a critical care team currently recommended by the EWS escalation protocol is more likely to predict need for critical care admission. However, one in four patients referred below the threshold also required admission to a critical care environment. This study questions the safety of introducing such a protocol into acute hospitals. Will noncritical care staff be forced to wait until patients deteriorate further and reach the trigger threshold for referral or will the role of the critical care team expand further to look after all patients with abnormal EWS in hospital? Results We identifi ed 824 patients who met the inclusion criteria for this study. Of these patients, 257 (31.2%) were classifi ed as obese. The mean APACHE II score was similar between obese and nonobese patients (23.3 vs. 22.4; P = 0.068). Obese patients had a similar rate of in-hospital mortality (31.9% vs. Factors aff ecting the clinical response to National Early Warning score triggers g g Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). Group analysis was done for both ISS and NISS using the following severity score ranges (SSR) 1 to 8, 9 to 15, 16 to 25, and >25. I Kolic1, S McCartney1, S Crane1, Z Perkins2, A Taylor1 1South London Healthcare NHS Trust, London, UK; 2East Kent Hospitals University NHS Trust, Ashford, UK Critical Care 2014, 18(Suppl 1):P45 (doi: 10.1186/cc13235) Results The analysis conducted in previous research [1] was repeated for the data recategorized by NISS. The conclusions were identical; increased NISS is associated with increased radiation exposure. A total of 465 patients fell into diff erent SSR when classifi ed by NISS. The distribution of exposure for each SSR, NISS versus ISS, was compared using Wilcoxon’s test. For the range 1 to 8, there was no detectable diff erence. There was a signifi cant shift in the distribution of exposures for the remaining three ranges (P <0.02). The exposure shift was downward for NISS compared with ISS. Introduction We aimed to assess actions taken in response to variations in the National Early Warning (NEW) score and to identify factors associated with a poor response. The NEW score is a physiological score, which prescribes an appropriate response for the deteriorating patient in need of urgent medical care. This allows enhanced observation and clinical review of patients, identifying patients at risk of acute mortality. Methods We performed a prospective observational study of adult patients admitted to an acute medical ward in a London district general hospital over a 2-week period. Patient characteristics, NEW score, time of day, day of week and clinical response data were collected for the fi rst 24 hours of admission. Patients with less than a 12-hour hospital stay were excluded. The primary outcome measure was the quality of clinical response. Data were analysed with univariate and multivariate logistic regression. Conclusion In this analysis, with the exception of patients in groups 1 to 8 there was a signifi cant decrease in RE in the NISS groups when compared with the compared ISS group. Further analysis is needed to determine the cause for this change and whether this diff erence will be clinically signifi cant. P45 determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). Group analysis was done for both ISS and NISS using the following severity score ranges (SSR) 1 to 8, 9 to 15, 16 to 25, and >25. determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. Methods Data collection was as previously described [1]. RE was measured in milliSieverts (mSv). Group analysis was done for both ISS and NISS using the following severity score ranges (SSR) 1 to 8, 9 to 15, 16 to 25, and >25. determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. determine whether there is a diff erence in the calculated RE per group when the patients are grouped using either the NISS or ISS. Factors aff ecting the clinical response to National Early Warning score triggers I Kolic1, S McCartney1, S Crane1, Z Perkins2, A Taylor1 1South London Healthcare NHS Trust, London, UK; 2East Kent Hospitals University NHS Trust, Ashford, UK Critical Care 2014, 18(Suppl 1):P45 (doi: 10.1186/cc13235) P44 Early warning scores: breaking or building barriers to critical care E Dunne1, RO Leary1, K Srinivasan2, B Ahmed3, D Galvin4, R Ni Mhuircheartaigh1, B Marsh1 1Mater Misericordiae University Hospital, Dublin, Ireland; 2Tallaght Hospital, Dublin, Ireland; 3St Vincent’s University Hospital, Dublin, Ireland; 4University College Hospital, Galway, Ireland Critical Care 2014, 18(Suppl 1):P44 (doi: 10.1186/cc13234) j g Conclusion Clinical response to NEW score triggers is signifi cantly worse at weekends, highlighting an important patient safety concern. Reference Introduction A prospective multicentre observational study was carried out to assess the extent to which critical care teams manage patients in hospital who are cared for outside the critical care unit. The Health Service Executive (HSE) in Ireland is in the process of implementing a national Early Warning Score (EWS) and, at an EWS of 7 or above, a referral to critical care is recommended. This study recorded the EWS of patients referred to the critical care team and describes the subsequent interventions made by the critical care team and patient outcomes.fi 1. Royal College of Physicians: National Early Warning Score (NEWS): Standardising the Assessment of Acute Illness Severity in the NHS. Report of a Working Party. London: RCP; 2012. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Impact of obesity on outcomes in patients with sepsis Methods Six critical care departments in university-affi liated hospitals across Ireland collected data on all referrals to the critical care team over a 6-week period. Data were anonymised, coded and analysed centrally. Results A cumulative total of 399 calls were made to the critical care teams in the six hospitals. The most common reason for referral was to request a critical care review of a patient (n = 319, 79.9%). Other reasons for referral included cardiac arrest, request to transfer patients from other hospitals and requests for vascular access. The average duration spent by the critical care team reviewing patients on the wards was 57 minutes. This increased up to 67 minutes for cardiac arrest calls. Of the 319 critical care reviews, 160 (50.2%) patients were subsequently admitted to critical care. A total 118 of this 160 had EWS of 7 or above, while 42 scored less than 7 but were still deemed to require admission to critical care. Reference 1. Wagner M, et al.: 258 Increased Injury Severity Score is associated with increased radiation exposure in trauma patients. Crit Care Med 2013, 41:A59. Results During the study period 200 patients were included with a median age of 70 (20 to 102) years. NEW scores were evenly distributed between day and night (52% vs. 48%) with a greater proportion on weekdays compared with weekend days (82% vs. 18%). The majority of patients scored <5 (93% vs. 7%). Forty-seven (27%) patients received an inadequate clinical response. Univariate analysis showed no association with time of day (night 34% vs. day 38%, OR 0.83 (0.47 to 1.49), P = 0.556). However, day of the week (weekend 56% vs. weekday 32%, OR 2.8 (1.30 to 5.84), P = 0.01) and increasing score (NEWS ≥5 100% vs. NEWS <5 31%, OR 65 (3.8 to 1100), P < 0.0001) were signifi cantly associated with an inadequate response. Day of the week was independently associated with an inadequate response after adjusting for confounders (OR 3.08 (1.27 to 7.46), P = 0.013). P43 Calculated radiation exposure for trauma patients is lower when using the New Injury Severity Score versus the Injury Severity Score to calculate injury severity M Wagner1, J Vonk2, C Wichman1, A Hegde3, J Oliveto3 1Creighton University, Omaha, NE, USA; 2University of Iowa, Iowa City, IA, USA; 3UNMC, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P43 (doi: 10.1186/cc13233) Introduction We studied radiation exposure (RE) in trauma patients [1]. To adjust for injury severity the New Injury Severity Score (NISS) or the Injury Severity Score (ISS) can be used to group patients. We sought to y Conclusion Organ failures and need for organ support correlated with outcome. P/F ratio and need for IMV were independent predictors of S16 Reference 1. Schuetz P, et al.: Eff ect of procalcitonin-based guidelines vs standard guidelines on antibiotic use in lower respiratory tract infections: the ProHOSP randomized controlled trial. JAMA 2009, 302:1059-1066. y y y y Methods We used a platform of a negative randomized control trial in subjects (n = 51) with a diagnosis of severe sepsis with ≥1 organ failure. The cohort of severe septic subjects was stratifi ed by obesity status based on the body mass index (BMI >30). Primary outcomes: 30-day and 180-day mortality; secondary outcome: diff erence in median (IQR) of fi ve infl ammatory cytokines including tumor necrosis factor alpha (TNFα), TNFα-receptor 2, interleukin (IL)-6, IL-1-receptor-antagonist (IL- 1ra) and IL-10. The measurement of median baseline cytokine levels was done in serum by Luminex technology. Statistical signifi cance was defi ned as P <0.05. Obesity is not associated with poor outcomes in older patients with sepsis Conclusion Clinical parameters and discharge levels of proADM allow accurate long-term prognostication in COPD patients independent of initial type of exacerbation. The focus on the best use of long-term prognostic information to improve patient care and clinical outcomes seems promising/rational. MI Restrepo1, P Faverio2, LF Reyes3, A Anzueto1 1University of Texas Health Science Center, San Antonio, TX, USA; 2University of Milan-Bicocca, Monza, Italy; 3Universidad de La Sabana, Bogota, Colombia Critical Care 2014, 18(Suppl 1):P47 (doi: 10.1186/cc13237) Introduction Studies suggest that obesity may infl uence mortality in patients who develop sepsis. However, the mechanisms linked to improved outcomes are unclear. Our aim was to assess the impact of obesity on mortality at 30 and 180 days and cytokine expression. PP Dobesh, TR McGuire, DG Klepser, AL Himmelberg, DA Roberts, KM Olsen 33.7%; P = 0.810) compared with nonobese patients, but a signifi cantly higher rate of development of ARDS (49.4% vs. 34.4%; P <0.001). Obese patients also had signifi cantly S17 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 more days on mechanical ventilation (6.2 days vs. 5.0 days; P = 0.005). There was no relationship between mortality in obese patients on mechanical ventilation (34.4% vs. 39.5%; P = 0.26) or ARDS (33.9% vs. 42.6%; P = 0.13) compared with nonobese patients. Hospital costs and length of stay did not diff er between the groups. Results Overall mortality in the 469 included COPD patients was 55% (95% CI 0.5 to 0.6) with a 14% (95% CI 0.1 to 0.2) mortality incidence rate per year. Patients with pneumonic COPD exacerbation had a more pronounced infl ammatory response compared with patients with nonpneumonic exacerbation with regard to levels of initial C-reactive protein levels (median 158 mg/dl vs. 39 mg/dl, P <0.0001), procalctionin (median 0.4 μg/l vs. 0.1 μg/l, P <0.0001) and proADM (median 1.3 nmol/l vs. 0.9 nmol/l, P <0.0001), but long-term survival was similar (HR 1.0, 95% CI 0.8 to 1.2). In univariate regression models, proADM was signifi cantly associated with mortality after 1, 3 and 6 years (HR 16.1 (95% CI 6.9 to 37.7), 10.5 (95% CI 5.7 to 19.6) and 10.4 (95% CI 6.2 to 17.7), respectively). There was no eff ect modifi cation by type of exacerbation. A model including clinical parameters (age, coronary heart disease, heart failure, diabetes mellitus, chronic renal failure, neoplastic disease, pneumonia, smokers) and proADM showed good discrimination of long-term survivors from nonsurvivors with AUC of 0.74 (95% CI 0.6 to 0.7). g yf g Conclusion Obesity signifi cantly increased the incidence of ARDS and days on mechanical ventilation in patients with sepsis. Previous work has reported that obesity is associated with elevations in infl ammatory cytokines and adipokines, particularly IL-6, which is a known risk factor for ARDS. The higher rate of ARDS in obese patients with sepsis identifi es a high-risk group where new therapies may be most benefi cial and where new methods of preventing ARDS can be targeted. Frailty predicts need for medical review but not degree of organ support after complex orthopaedic surgery N Singatoullina1, A Panickar1, A Dennis1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Univeristy of Leicester Hospitals, Leicester, UK N Singatoullina1, A Panickar1, A Dennis1, R Porter2, D Bryden1 1Sheffi eld Teaching Hospitals, Sheffi eld, UK; 2Univeristy of Leicester Hospitals, Leicester, UK Critical Care 2014, 18(Suppl 1):P49 (doi: 10.1186/cc13239) Critical Care 2014, 18(Suppl 1):P49 (doi: 10.1186/cc13239) Introduction Based on expert opinion and case note review, the UK National Confi dential Enquiry into Peri-operative Outcome has recommended provision of perioperative level 2 and 3 care to support major surgery in older people, and particularly those with comorbidity [1]. We wished to identify whether we could predict if the need was uniform and whether any factors could predict the degree of organ supports needed. i Results Fifty-one subjects with severe sepsis were included in the study; 37% of the patients were obese (BMI >30). Paradoxically, obese severe septic patients had lower 30-day mortality (n = 1 (5%) vs. n = 9 (28%), P = 0.069) and 180-day mortality (n = 1 (5%) vs. n = 13 (41%), P = 0.008), when compared with nonobese. The expression of TNFα, TNFα-receptor 2, IL-6, IL-1ra and IL-10 was not statistically signifi cant diff erent among obese versus nonobese severe septic patients. f Conclusion Obesity is associated with lower mortality rates at 30 and 180 days in patients diagnosed with severe sepsis. This survival benefi t was not associated with lower cytokine production among obese patients. Further studies are needed to assess the mechanisms associated with the survival benefi t related to obesity in patients with severe sepsis. Methods A retrospective note review of all patients admitted to a level 2 critical care unit in the 12-month period from 1 January 2012 to 31 December 2012 undergoing revision hip surgery either as a two-stage or single-stage process. Surgery was undertaken at a national referral unit and chosen to represent an appropriate group of older, comorbid patients. Predefi ned preoperative and perioperative data were collected from chart review, along with postoperative physiological data whilst the patient was in critical care. This included frailty, comorbidities, operative blood loss, anaesthetic technique and level and duration or organ supports including the need for additional medical review whilst on the unit. Frailty was assessed preoperatively P48 Frail patients were signifi cantly more likely to need additional medical input in the postoperative period whilst on critical care (Figure 1, P = 0.002) but this was not signifi cantly linked to need for vasopressors, evidence of sepsis or choice of anaesthetic technique. Methods This study was conducted in a 20-bed mixed ICU of a teaching hospital. The study sample was extracted from a dataset of all ICU patients treated for more than 72 hours between 1 January 2007 and 1 October 2012. Demographic characteristics and clinical characteristics at admission and during the ICU stay were collected. Characteristics of patients alive 1 year after ICU discharge were compared with patients who died within the fi rst year after ICU discharge. Descriptive statistics were calculated. Multivariate analysis of 1-year mortality was performed using a logistic regression model with backward elimination. Survival was analysed by the Kaplan–Meier method using the time interval from day of ICU discharge until death. Conclusion In complex revision orthopaedic surgery, the need for postoperative level 2/3 support cannot be predicted from any preoperative or intraoperative factors but patient frailty does indicate the need for medical input in the postoperative period. References 1. Wilkinson K, et al.: An Age Old Problem. London: NCEPOD; 2010. 2. Rockwood K, et al.: CMAJ 2005, 173:489-495. 1. Wilkinson K, et al.: An Age Old Problem. London: NCEPOD; 2010. 2. Rockwood K, et al.: CMAJ 2005, 173:489-495. 1. Wilkinson K, et al.: An Age Old Problem. London: NCEPOD; 2010. 2. Rockwood K, et al.: CMAJ 2005, 173:489-495. y g Results During the study period, 740 patients were treated for more than 72 hours in the ICU. The ICU mortality was 106/740 (14%). The data of 617 ICU survivors were further analysed (17 patients were lost to follow up). One-year mortality was 175/617 (28%), of which 85/175 (49%) patients died during hospital stay after ICU discharge. Frailty measures in the critically ill: are we approaching a critical age? A systematic review R Pugh1, DL John1, C Thorpe2, C Subbe2 1Glan Clwyd Hospital, Bodelwyddan, UK; 2Ysbyty Gwynedd, Bangor, UK Critical Care 2014, 18(Suppl 1):P50 (doi: 10.1186/cc13240) g y R Pugh1, DL John1, C Thorpe2, C Subbe2 g , , p , 1Glan Clwyd Hospital, Bodelwyddan, UK; 2Ysbyty Gwynedd, Bangor, UK Critical Care 2014, 18(Suppl 1):P50 (doi: 10.1186/cc13240) Introduction As the general population ages, the proportion of critically ill patients in whom frailty may adversely aff ect outcome is likely to rise. The aim of this systematic review was to evaluate performance of frailty measures in predicting ICU, hospital and long-term outcomes following intensive care admission. Conclusion Of patients being treated for more than 72 hours in the ICU, 28% died within 1 year after ICU discharge. One-half of them within the hospital stay after ICU discharge. High age at ICU admission, high APACHE IV predicted mortality score, high number of comorbidities, readmission and an admission diagnosis within the categories ‘cardiovascular’ and ‘sepsis’ are associated with an increased 1-year mortality after ICU discharge in this population. The burden of patients dying after ICU discharge underlines the necessity for clear ICU discharge criteria and post-ICU care. Methods We performed a literature search for original studies in: EMBASE, MEDLINE, Web of Knowledge, Cochrane database of systematic reviews, and Database of Abstracts of Reviews of Eff ects, using the terms ‘frailty’, ‘frail elderly’, ‘critical care’, ‘critically ill’, ‘critical illness’ and ‘intensive care’. Our study inclusion criteria were that the study: included patients cared for in intensive care, captured data relating to premorbid frailty, and reported ICU, hospital and/or long- term outcome data. Results Initial searches identifi ed 606 reports, of which, following review of abstracts and removal of duplicates, 66 full-text papers were evaluated. Of these, 11 met inclusion criteria. A further 19 papers that met inclusion criteria were identifi ed from relevant review articles and reference lists. There was huge variation in populations studied, methodology, frailty measures utilised and reported outcome measures. Of the 30 included studies, 11 studies evaluated patients undergoing major (including cardiothoracic) surgery, two studies specifi cally assessed patients with pneumonia, one study investigated patients in a burns ICU and one study restricted its investigation to former nursing home residents. Long-term physical functioning and health-related outcomes in survivors of intensive care K Solverson, C Doig K Solverson, C Doig University of Calgary, Canada Critical Care 2014, 18(Suppl 1):P52 (doi: 10.1186/cc13242) Introduction The long-term impact of critical illness on survivors’ physical and mental health remains unknown. Our aim was to create a follow-up clinic for survivors of critical illness in order to quantitatively examine muscle weakness, physical functioning and mental health and relate these fi ndings to health-related quality of life (HRQL) and ICU risk factors. Methods Study patients were selected from a 24-bed multidisciplinary ICU. Fifty-six patients who were ≥18 years old, stayed longer than 4 days in the ICU and did not have an acute brain injury were followed-up at 2 and 4 months post hospital discharge. Peripheral muscle strength (grip, triceps, biceps, hamstrings, quadriceps and dorsifl exors) and physical functioning were objectively assessed using hand-held dynamometry and the six-minute walk test (6MWT); both were compared with age/ sex normative data. HRQL and mental health were assessed using the Short Form-36 (SF-36), EuroQol-5D (EQ-5D) and Hospital Anxiety and Depression Scale. p y p y Conclusion Measures of frailty appear to predict mortality and functional dependence following critical admission across a wide range of clinical conditions. However, comparative data regarding the relative accuracy and reliability of frailty measures in the intensive care population are currently defi cient. P48 Long-term outcome in COPD patients with pneumonic and nonpneumonic exacerbation: a 6-year prospective follow-up study EG Grolimund, AK Kutz, MA Alan, BM Mueller, PS Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P48 (doi: 10.1186/cc13238) Figure 1 (abstract P49). Proportion of patients requiring additional medical review on critical care by degree of frailty. Introduction Predicting long-term outcome in patients surviving a pneumonic or nonpneumonic COPD exacerbation remains challenging. This study investigates the association of clinical parameters and the prognostic blood marker pro-adrenomedullin (proADM) measured upon hospital discharge with 6-year mortality in well-defi ned cohort of COPD patients. Methods We prospectively followed consecutive COPD patients from a previous Swiss multicenter trial (2006 to 2008) [1] over a 6-year follow- up and investigated all-cause mortality following hospital discharge. Patients and/or treating general practitioners were contacted by telephone interview to assess the vital status of patients. We used Cox regression models and the area under the receiver operating characteristics curve (AUC) to investigate associations of baseline predictors and mortality. Figure 1 (abstract P49). Proportion of patients requiring additional medical review on critical care by degree of frailty. S18 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 determine the 1-year mortality after discharge from the ICU in patients who were treated in the ICU for more than 72 hours and to identify predictors for 1-year mortality. using the Rockwood assessment tool by trained staff [2]. Data were analysed using Microsoft Excel for Mac 2011 and Stata/IC 11.2 for Mac. Results A total of 182 patients with a mean age of 69.8 years (range 29 to 92) were identifi ed. Frail patients were signifi cantly more likely to need additional medical input in the postoperative period whilst on critical care (Figure 1, P = 0.002) but this was not signifi cantly linked to need for vasopressors, evidence of sepsis or choice of anaesthetic technique. using the Rockwood assessment tool by trained staff [2]. Data were analysed using Microsoft Excel for Mac 2011 and Stata/IC 11.2 for Mac. Results A total of 182 patients with a mean age of 69.8 years (range 29 to 92) were identifi ed. Frailty measures in the critically ill: are we approaching a critical age? A systematic review R Pugh1, DL John1, C Thorpe2, C Subbe2 1Glan Clwyd Hospital, Bodelwyddan, UK; 2Ysbyty Gwynedd, Bangor, UK Critical Care 2014, 18(Suppl 1):P50 (doi: 10.1186/cc13240) The most commonly used measures of frailty were: measures of Activities of Daily Living (n = 9), which predicted need for long-term institutional care, 30-day, 90-day, 6-month and 12-month mortality; Clinical Frailty Score [1] (n = 5), which predicted ICU mortality, hospital mortality, 12-month mortality, quality of life and functional dependence; and Knaus score [2] (n = 4), which predicted ICU mortality, hospital and 12-month mortality. P48 Independent predicting factors of 1-year mortality were: age at ICU admission (OR: 1.03; 95% CI: 1.01 to 1.05), APACHE IV predicted mortality score (OR: 1.02; 95% CI: 1.02 to 1.03), number of comorbidities (one or two co morbidities OR: 2.14; 95% CI: 1.42 to 3.23) (>3 comorbidities OR: 2.56; 95% CI: 1.16 to 5.62), readmission after ICU discharge within the same period of hospital stay (OR: 1.98; 95% CI: 1.13 to 3.46) and the diagnosis at admission (cardiovascular OR: 4.31; 95% CI: 1.73 to 10.76) (sepsis OR: 2.67; 95% CI: 1.50 to 4.77). p p References Referencesi 1. Rockwood et al.: A global clinical measure of fi tness and frailty in elderly people. CMAJ 2005, 173:489-495. 2. Knaus et al.: APACHE-acute physiology and chronic health evaluation: a physiologically based classifi cation system. Crit Care Med 1981, 9:591-597. Results The median age of patients was 60 years, 68% were admitted for respiratory illnesses, they were severely ill (median APACHE II, 19) and had long ICU lengths of stay (median, 11 days). Muscle strength was signifi cantly reduced when compared with normative data in all muscle groups at the 2-month and 4-month visits (2-month average indexed overall strength, 72%, P <0.05). The median distance walked during the 6MWT was 382 m at 2 months (median percent predicted, 72%) and it did not signifi cantly change at the 4-month visit. Reduced peripheral muscle strength was signifi cantly correlated with lower distance walked during the 6MWT. Reported HRQL by the SF-36 was below national averages at both 2-month and 4-month visits (2-month Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 physical composite score (PCS) 36.2, mental composite score 48.1; 50 = national average). Reduced muscle strength was associated with low scores on the SF-36 physical function and general health domains. Performance on the 6MWT correlated with the SF-36 including the PCS (P = 0.001). Screening positive for anxiety was associated with both poor 6MWT performance and reporting dysfunction on the EQ- 5D domains. ICU/hospital length of stay, number of days ventilated, severity of illness and organ dysfunction were not found to be predictive of muscle strength or physical functioning. had a signifi cantly higher logistic EuroSCORE (20.3 vs. 10.6; P <0.001). The logistic EuroSCORE was a reasonable predictor of prolonged ICU/ in-hospital mortality (OR 1.04, 95% CI 1.04 to 1.05, P <0.001) with a receiver operating characteristic (ROC) curve of 0.72. The relationship between a patient’s logistic EuroSCORE and predicted risk of prolonged ICU is shown in the fi gure; including low-risk, medium-risk and high risk groups. Around 50% of the entire cohort of patients had a logistic EuroSCORE of 10 or less and an associated risk of prolonged ICU stay of 5% or less. See Figure 1. physical composite score (PCS) 36.2, mental composite score 48.1; 50 = national average). Reduced muscle strength was associated with low scores on the SF-36 physical function and general health domains. Performance on the 6MWT correlated with the SF-36 including the PCS (P = 0.001). Screening positive for anxiety was associated with both poor 6MWT performance and reporting dysfunction on the EQ- 5D domains. ICU/hospital length of stay, number of days ventilated, severity of illness and organ dysfunction were not found to be predictive of muscle strength or physical functioning. had a signifi cantly higher logistic EuroSCORE (20.3 vs. 10.6; P <0.001). The logistic EuroSCORE was a reasonable predictor of prolonged ICU/ in-hospital mortality (OR 1.04, 95% CI 1.04 to 1.05, P <0.001) with a receiver operating characteristic (ROC) curve of 0.72. The relationship between a patient’s logistic EuroSCORE and predicted risk of prolonged ICU is shown in the fi gure; including low-risk, medium-risk and high risk groups. Around 50% of the entire cohort of patients had a logistic EuroSCORE of 10 or less and an associated risk of prolonged ICU stay of 5% or less. See Figure 1. y Conclusion Our study gives qualitative evidence that survivors of critical illness have reduced muscle strength, physical functioning and HRQL after hospital discharge. P53 M Soares1, JF Timsit2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, BF Almeida7, UV Silva8, LC Azevedo9, JI Salluh1, E Azoulay10 1DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil; 2Hôpital A. Michallon Chu de Grenoble, France; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton NHS Foundation Trust, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Fundação Pio XII – Hospital do Câncer de Barretos, Barretos, Brazil; 9Hospital Sírio Libanês, São Paulo, Brazil; 10Hôpital Saint Louis, Paris, France Critical Care 2014, 18(Suppl 1):P54 (doi: 10.1186/cc13244) Introduction The aim of this study was to determine an appropriate risk model to identify patients at high risk of prolonged ICU stay and to aid patient consent prior to cardiac surgery. Introduction The aim of this study was to determine an appropriate risk model to identify patients at high risk of prolonged ICU stay and to aid patient consent prior to cardiac surgery. Methods Data were prospectively collected on 5,440 consecutive cardiac surgery cases between April 2009 and March 2012. The primary outcome measure was the combined outcome of prolonged ICU stay (length of stay greater than 20 days) and/or in-hospital mortality. Logistic regression was performed to assess the predictability of logistic EuroSCORE against the primary outcome. Low-risk, medium- risk and high-risk groups were identifi ed and subsequent risk of 1-year mortality assessed. Survival status was determined at 1 year. Introduction Information about lung cancer patients surviving critical illnesses is very scarce. Our aim was to evaluate the outcomes and continuing of anticancer treatments in lung cancer patients surviving ICU admission. Methods Secondary analysis of a prospective multicenter study including patients admitted for >24 hours to 22 ICUs in six countries from Europe and South America during 2011. Readmissions and patients in cancer remission >5 years were excluded. Logistic regression was used to identify predictors for hospital mortality. Results A total of 192 (3.5%) patients had a prolonged ICU stay and 187 (3.4%) in-hospital deaths occurred, resulting in a combined primary outcome of 349 (6.4%). At 1 year, 371 (6.8%) deaths occurred. The risk of death in-hospital and at 1 year was signifi cantly higher in patients with prolonged ICU stay (in-hospital mortality, 15.6% vs. 3.0%; P <0.001/1 year, 27.6% vs. 6.1%; P <0.001). The mean logistic EuroSCORE for all patients was 10.9. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Also, we have shown muscle weakness is predictive of overall physical functioning, which in turn impacted HRQL and mental health. No ICU risk factors were identifi ed that predicted defi cits in muscle strength or physical functioning. Conclusion Using an existing risk prediction model, a patient’s risk of prolonged ICU stay can be calculated using contemporaneous data. This information could be relevant for aiding in providing informed consent for cardiac surgery patients. P54 Six-month outcomes in lung cancer patients surviving ICU admission: results from a multinational multicenter study M Soares1, JF Timsit2, G Burghi3, C Irrazabal4, N Pattison5, E Tobar6, BF Almeida7, UV Silva8, LC Azevedo9, JI Salluh1, E Azoulay10 1DOr Institute for Research and Education – IDOR, Rio de Janeiro, Brazil; 2Hôpital A. Michallon Chu de Grenoble, France; 3Hospital Maciel, Montevideo, Uruguay; 4Instituto Alexander Fleming, Buenos Aires, Argentina; 5Royal Brompton NHS Foundation Trust, London, UK; 6Hospital Clinico Universidad de Chile, Santiago, Chile; 7Hospital A.C. Camargo, São Paulo, Brazil; 8Fundação Pio XII – Hospital do Câncer de Barretos, Barretos, Brazil; 9Hospital Sírio Libanês, São Paulo, Brazil; 10Hôpital Saint Louis, Paris, France Critical Care 2014, 18(Suppl 1):P54 (doi: 10.1186/cc13244) Prediction of 1-year mortality of patients treated for more than 72 hours in an ICU S Steenbergen, S Rijkenberg, H Endeman S Steenbergen, S Rijkenberg, H Endeman Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P51 (doi: 10.1186/cc13241) S Steenbergen, S Rijkenberg, H Endeman , , Critical Care 2014, 18(Suppl 1):P51 (doi: 10.1186/cc13241) Introduction ICU or hospital mortality rates have been reported as the endpoint of ICU therapy for many years. The aim of this study was to S19 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Survival and quality of life in patients acquiring acute kidney injury in the fi rst 24 hours of ICU admission I Soliman, L Peelen, D De Lange, D Van Dijk University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P55 (doi: 10.1186/cc13245) Survival and quality of life in patients acquiring acute kidney injury in the fi rst 24 hours of ICU admission I Soliman, L Peelen, D De Lange, D Van Dijk University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P55 (doi: 10.1186/cc13245) Introduction The population of the UK is ageing, with the fastest increase in those ≥85 years. Increased age has been repeatedly associated with adverse outcome and it is uncertain to what extent this relates to the changes of ageing in themselves, or due to other considerations. Age is a key variable in the majority of scoring systems that relate patient characteristics to adverse outcome. We aimed to assess change in age distribution of patients admitted to our ICU over 20 years and examine the relationship between age of patient, mortality and length of stay (LOS). i I Soliman, L Peelen, D De Lange, D Van Dijk , , g , j University Medical Center, Utrecht, the Netherlands y , , Critical Care 2014, 18(Suppl 1):P55 (doi: 10.1186/cc13245) Introduction Survival and quality of life (QoL) in ICU patients with acute kidney injury (AKI) have been repeatedly reported as poor [1-3]. It is unknown whether early AKI, occurring during the fi rst 24 hours of ICU treatment, is also a strong predictor of poor long-term outcome. Our aim was to describe the long-term outcomes of this specifi c group of ICU patients. Methods Data were extracted from electronic records (WardWatcher) and analysed using SPSS 20, GraphPad Prism 5.0 and Excel 2007. Methods Data were extracted from electronic records (WardWatcher) and analysed using SPSS 20, GraphPad Prism 5.0 and Excel 2007. Results ICU patients have become older by 4.4 months/year. By 2013 the median age was 66 and 15% of all patients are now ≥80 years – a 36% increase since 1993. Compared with the reference group (61 to 70 years), those in the older deciles have increased risk of ICU and hospital mortality (P <0.01). Fifteen per cent of all those 81 to 90 years old and 20% of those >91 years old who do not die on the ICU go on to die on the ward. It is unknown what proportion of these post-ICU deaths was unexpected. Older patients had prolonged hospital LOS (P <0.01) but not ICU LOS. See Figures 1 and 2. P53 Patients with prolonged ICU stay Results A total of 449 patients (small-cell (SCLC) = 55; non-SCLC = 394)) were admitted to ICUs, and out of them 275 (SCLC = 29; NSCLC = 246) were discharged alive from the hospital. Among them, 200 (73%) patients were alive and 72 (26%) had died at 6 months; three Figure 1 (abstract P53). Risk of long stay versus 1-year mortality. Figure 1 (abstract P53). Risk of long stay versus 1-year mortality. S20 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (1%) patients were lost to follow-up. Mortality rates were far lower in the patient subset with nonrecurrent/progressive cancer and a good performance status (PS), even those with sepsis, multiple organ dysfunctions, and need for ventilatory support. Cancer recurrence or progression occurred in 53 (26%) hospital survivors. Anticancer treatments were recommended for 108 (39%) hospital survivors and administered to 102. Treatments used were variable combinations of surgical resection (7%), radiation therapy (34%), and chemotherapy (80%). The initial treatment plan required reduction or modifi cation in 35 (34%) patients. Post-hospital mortality was nonsignifi cantly lower in the patients given the initial treatment plan than in the other patients (17% vs. 32%, P = 0.065). Poor PS was the only factor associated with a lower probability of receiving the initial treatment plan (OR = 0.20; 95% CI, 0.05 to 0.87; P = 0.032). At 6 months, 71% patients were at home, 15% were hospitalized, and 7% were in hospice care; the location was unknown for 6% patients. PS at 6 months was 3 to 4 in 19 (9.5%) survivors. Results Out of 5,934 admissions, 269 patients were identifi ed with early AKI. After ICU discharge a large and signifi cant diff erence in survival between included patients and the Dutch population, matched for age and gender, was seen (P <0.001). The median QoL index in surviving patients was 0.65 (interquartile range (IQR) 0.45 to 1.00), versus 0.86 (IQR 0.84 to 0.89) in the Dutch population (P <0.001). Low QoL was found in 11/59 (18.6%) survivors. In total, poor ICU outcome was seen in 171/269 (63.6%) patients. See Figure 1. p g Conclusion Patients developing AKI in the fi rst 24 hours of ICU stay are prone to poor outcome. References References 1. Morgera S et al.: Crit Care Med 2008, 36(4 Suppl):S193-S197. 2. Nisula S et al.: Crit Care 2013, 17:R250. 3. Brinkman S et al.: Crit Care Med 2013, 41:1229-1236. 3. Brinkman S et al.: Crit Care Med 2013, 41:1229-1236. Conclusion Post-hospital mortality in critically ill lung cancer patients is relatively high and many patients require anticancer treatments after discharge. PS before ICU admission is a major determinant of both mortality and ability to receive optimal anticancer treatment in these patients. P53 Future research into prognostic factors for ICU patients should include early AKI in their models. Increasing age of patients admitted to intensive care, and association between increased age and greater risk of post-ICU death B Creagh-Brown, S Green B Creagh-Brown, S Green Royal Surrey County Hospital, Guildford, UK y y y p Critical Care 2014, 18(Suppl 1):P56 (doi: 10.1186/cc13246) Critical Care 2014, 18(Suppl 1):P56 (doi: 10.1186/cc13246) Outcomes of military patients treated at the UK Royal Centre for Defence Medicine 2007 to 2013 AM Johnston1, J Henning2, D Harrison3 1Royal Centre for Defence Medicine, Birmingham, UK; 2James Cook University Hospital, Middlesbrough, UK; 3ICNARC, London, UK Critical Care 2014, 18(Suppl 1):P57 (doi: 10.1186/cc13247) AM Johnston1, J Henning2, D Harrison3 1Royal Centre for Defence Medicine, Birmingham, UK; 2James Cook University Hospital, Middlesbrough, UK; 3ICNARC, London, UK Critical Care 2014, 18(Suppl 1):P57 (doi: 10.1186/cc13247) Introduction UK military personnel injured overseas are repatriated to the Royal Centre for Defence Medicine (RCDM) based at the Queen Elizabeth Hospital Birmingham (QEHB) in Birmingham UK. We report the demographics and outcomes of military patients treated on the ICU at RCDM using data from the Intensive Care National Audit and Research Centre over a 6.5-year period. Conclusion Very old patients with cancer present acceptable rates of survival after ICU admission. Similarly to younger patients, organ function evaluation in the fi rst hours of ICU can predict outcomes in this specifi c population. y p Methods Data on 570 admissions of 527 patients to the ICU at RCDM/ QEHB were analysed by ICNARC using standard methodology. y y g gy Results Some physiology and CCMDS data were missing for 175 patients. Age, sex and mortality are described in Table 1. A total of 90.9% of patients had traumatic injuries, 2.1% received CPR prior to ICU admission, 1.5% prehospital. A total of 20.6% had head, neck or spinal trauma. A total of 85.7% were transferred directly to the ICU from a military hospital overseas, others coming to the ICU following surgery at RCDM. Of the 382 patients with APACHE II score data the mean score was 11.0 (SD 4.9), probably refl ecting stabilisation in military hospitals overseas or during aeromedical critical care transfer. The mean number of ICU days was Level 3: 7.6 (SD 11.6); Level 2: 2.0 (SD 2.8). A total of 70.4% of patients required advanced respiratory support for a mean of 7.5 days, and 33% required advanced cardiovascular support for a mean of 3.7 days. Very old patients with cancer admitted to the ICU: outcome and predictive factors of mortality Introduction The rate of very old patients (≥80 years old) admitted to intensive care has increased in the last years. Older age is associated with a higher prevalence of chronic diseases, including cancer [1]. The aim of the present study was to describe characteristics, outcomes and predictive factors of mortality in very old patients admitted to the ICU. Methods We performed a retrospective analysis of all cancer patients who were 80 years or older admitted to the ICU between January 2009 and December 2012 in a tertiary reference cancer center. Data were collected from medical records. Results A total of 597 patients with cancer were included in the analysis. Hospital mortality was 28.5%. These patients were more likely to have a solid tumor, a localized disease and underwent surgery, chemotherapy or radiotherapy recently. Variables associated with hospital mortality in these patients were: lung cancer, metastatic cancer and at ICU admission vasopressor requirements, acute respiratory failure, low hemoglobin levels, elevated creatinine levels, elevated bilirubin levels, acidosis and hyperlactatemia. By the multivariate analysis, the following factors were independent factors associated with hospital mortality: lung cancer (odds ratio (OR) = 6.3, 95% CI = 2.6 to 15.0, P <0.001), lactate levels on ICU admission (OR = 1.03, 95% CI = 1.02 to 1.04), P <0.001), bilirubin levels on ICU admission (OR = 1.16, 95% CI = 1.04 to 1.30, P = 0.007) and creatinine levels on ICU admission (OR = 1.58, 95% CI = 1.35 to 1.85, P <0.001). Reference 1. Bagshaw SM, Webb SA, Delaney A, et al.: Very old patients admitted to intensive care in Australia and New Zealand: a multi-centre cohort analysis. Crit Care 2009, 13:R45. 1. Bagshaw SM, Webb SA, Delaney A, et al.: Very old patients admitted to intensive care in Australia and New Zealand: a multi-centre cohort analysis. Crit Care 2009, 13:R45. Survival and quality of life in patients acquiring acute kidney injury in the fi rst 24 hours of ICU admission I Soliman, L Peelen, D De Lange, D Van Dijk University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P55 (doi: 10.1186/cc13245) p Methods All patients admitted to our mixed ICU from July 2009 to May 2012 with early AKI were included. All survivors received the EuroQoL EQ-6D-3L questionnaire. Early AKI was defi ned as creatinine >1.5 mg/ dl and urine output <150 ml/8 hours. Poor ICU outcome was defi ned as either death or EuroQoL index <0.4 at 1-year follow-up. Figure 1 (abstract P55). Survival. Figure 1 (abstract P55). Survival. Conclusion There are increasing numbers of older patients on ICUs in the UK. In analyses uncorrected for severity of illness or comorbidities, older patients are more likely to die on the ICU, and on the ward after ICU. They also spend longer in hospital prior to discharge. the UK. In analyses uncorrected for severity of illness or comorbidities, older patients are more likely to die on the ICU, and on the ward after ICU. They also spend longer in hospital prior to discharge. Figure 1 (abstract P55). Survival. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Figure 1 (abstract P56). Proportion of patients admitted to ICU aged >80 years, over time. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S21 Figure 2 (abstract P56). Post-ICU in-hospital mortality, by age. Conclusion The data on resource utilisation for this group of patients may inform planning of critical care support for military operations overseas. Very old patients with cancer admitted to the ICU: outcome and predictive factors of mortality L Hajjar1, M Franca1, J Almeida1, J Fukushima1, F Bergamin1, C Zambolim1, P Camargo1, C Park1, E Osawa1, M Sundin2, R Nakamura1, F Galas1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Heart Institute, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P58 (doi: 10.1186/cc13248) Endpoint resuscitation-based prediction model for early mortality of severe sepsis and septic shock Endpoint resuscitation-based prediction model for early mortality of severe sepsis and septic shock R Sinto, S Suwarto, R Sedono, K Harimurti, A Sejati Faculty of Medicine, University of Indonesia, Jakarta, Indonesia Critical Care 2014, 18(Suppl 1):P62 (doi: 10.1186/cc13252) Introduction The fi rst description of SAPS II dates back to 1993 [1], but little is known about the scoring accuracy in daily practice and factors possibly aff ecting it. The purpose of this study was to evaluate accuracy of SAPS II scoring by means of a nationwide survey. Introduction There is an unknown role for macrocirculation and microcirculation endpoint resuscitation, which are combined as the component of a prediction model for early mortality of patients with severe sepsis and septic shock [1,2]. The aim of this study is to develop a prediction model for early mortality (fi rst 120 hours after onset [3]) of patients with severe sepsis and septic shock based on macrocirculation and microcirculation endpoint resuscitation. Methods Twenty clinical scenarios, covering a broad range of illness severity, were randomly assigned to a convenience sample of clinicians or nurses of Swiss adult ICUs. They were asked to assign a SAPS II score (including details for each item of the score) for one scenario. Results were compared with a reference, as defi ned by fi ve experienced researchers using a Delphi method, and cross-matched with data related to training and quality control for scoring, information on daily practice of scoring, and structural and organizational properties of each participating ICU. p Methods A retrospective cohort study was conducted in adult patients with severe sepsis and septic shock hospitalized in the ICU, Cipto Mangunkusumo Hospital, Indonesia. Patients’ outcome and time to outcome were observed during the fi rst 120 hours after severe sepsis and/or septic shock onset. Independent predictors for early mortality were identifi ed by Cox’s proportional hazard regression analysis and each was quantifi ed to develop an early mortality prediction model. The calibration and discrimination abilities of the model were determined. Results Subjects consisted of 268 patients. Early mortality developed in 70 patients. Two independent predictors for early mortality were: lactate clearance <10% (adjusted hazard ratio (HR) 11.81 (95% CI 6.50 to 21.46)) and number of organ dysfunctions (two organs, adjusted HR 1.47 (95% CI 0.58 to 3.72); >2 organs, adjusted HR 3.79 (95% CI 1.65 to 8.69)). 1. Moreno RP, et al.: Intensive Care Med 2010, 36:1207-1212. References 1. National Oesophago-cancer Audit Annual Report 2013. The NHS Information Centre [https://catalogue.ic.nhs.uk/publications/clinical/ oesophago-gastric/nati-clin-audi-supp-prog-oeso-gast-canc-2013/clin-audi- supp-prog-oeso-gast-2013-rep.pdf] g Results Forty-six patients were identifi ed in our hospital at the time of the snapshot as being high risk according to NEWS. After recategorising this cohort of patients using ViEWS, 36 were classifi ed as high risk (in this instance meaning a score of 5 or more). Subjectively the authors did not have any clinical concerns created by moving 10 patients out of the high-risk classifi cation. 2. Michelet P, et al.: Br J Surg 2009, 96:54-60. i Conclusion ViEWS is more specifi c without being less sensitive. We have replaced NEWS with ViEWS and feel that this is clinically safe. References Endpoint resuscitation-based prediction model for early mortality of severe sepsis and septic shock A scoring system as the predictive model was performed by assigning 1 point for two organ dysfunctions, 6.5 points for >2 organ dysfunctions, and 12 points for lactate clearance <10%. This scoring system was stratifi ed into two levels: low-risk (score <12, probability for early mortality 7.8%) and high-risk (score ≥12, probability for early mortality 72.3%) groups. The Hosmer–Lemeshow test revealed good precision (P = 0.745) and the area under the receiver operating characteristic curve showed very good discrimination ability (0.91 (95% CI 0.87 to 0.97)). Results Sixty-three (81%) of 78 adult ICUs participated in this survey. A perfect match with each single reference item was found in 27 (7.8%) of 345 scorings. The participants’ mean SAPS II scoring was 42.6 ± 23.4, with a bias of +5.7 (95% CI 2.0 to 9.5) as compared with the reference score. There was no evidence of variation of the bias according to case severity, number of beds in the unit, number of residents during workshifts, linguistic area, profession (physician vs. nurse), experience, initial SAPS II training, and presence of a quality control system. The items with highest scoring accuracy were bilirubin, temperature and chronic disease (93%, 93% and 91% respectively), whereas the lowest agreement was found for urinary output and for the Glasgow Coma Scale (63% and 64%). Conclusion This nationwide survey suggests a wide variability of SAPS II scoring results. On average, SAPS II was overestimated by more than 10%, irrespective of the profession or experience of the scorer or the structural characteristics of the ICUs. At least one person per unit involved in the scoring should be trained by the national society and should be responsible for the scoring quality. Reference P59 A retrospective review of mortality and complications following oesophagectomy in a large UK teaching hospital A retrospective review of mortality and complications following oesophagectomy in a large UK teaching hospital N Pawley, CM Ball, K Wickenden, B Riley, M Clapham, B Eltayeb, A Glossop, A Raithatha Sheffi eld Teaching Hospital, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P59 (doi: 10.1186/cc13249) oesophagectomy in a large UK teaching hospital N Pawley, CM Ball, K Wickenden, B Riley, M Clapham, B Eltayeb, A Glossop, A Raithatha Sheffi eld Teaching Hospital, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P59 (doi: 10.1186/cc13249) y N Pawley, CM Ball, K Wickenden, B Riley, M Clapham, B Eltayeb, A Glossop, A Raithathafifi Table 1 (abstract P57). Case mix, demographics and outcome Table 1 (abstract P57). Case mix, demographics and outcome Introduction Just over 1,200 curative oesophagectomies are carried out in the UK annually. Although in-hospital mortality rates have fallen (12 to 13% in 1998 to 2.5% 2013), complication rates remain high [1] with anastomotic failure and respiratory failure common postoperatively [2]. The aim of this retrospective review was to examine the outcomes in patients who underwent oesophagectomies in our unit between January 2010 and October 2012. Table 1 (abstract P57). Case mix, demographics and outcome n 570 Male (%) 97.9 Age 25.7 Trauma (%) 90.9 Died (%) 6.8 LOS 8.8 MV (%) 70.4 CRRT (%) 10.9 Neuro (%) 24.8 CV (%) 33.2 y Methods We examined demographic data, survival (30 day and 1 year) and length of ICU and hospital stay. Case notes were reviewed to identify postoperative complications including anastomotic breakdown, reintubation and respiratory failure. Data were analysed to examine the relationship between the use of postoperative non- invasive ventilation and intraoperative fl uid volume and the incidence of postoperative complications. p p p Results Seventy-two patients were identifi ed as having undergone an oesophagectomy between January 2010 and October 2012. Median age was 65 and 82% were male. One patient died within 30 days (1.39%) S22 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Abandoning the National Early Warning Score in our district general hospital Abandoning the National Early Warning Score in our district gene hospital B Mylrea Lowndes, M Mercer, H Robinson Torbay Hospital, Torquay, UK Critical Care 2014, 18(Suppl 1):P61 (doi: 10.1186/cc13251) B Mylrea Lowndes, M Mercer, H Robinson Torbay Hospital, Torquay, UK Critical Care 2014, 18(Suppl 1):P61 (doi: 10.1186/cc13251) Introduction Early Warning Scores (EWS) are used in UK hospitals to identify patients who are acutely unwell or needing urgent review. The National EWS (NEWS) [1] was implemented in our institution and led to a noticeable increase in the numbers of patients being triggered for escalation of medical care, although clinically this was thought unwarranted. This led to a potentially dangerous lack of faith in the NEWS by clinical staff . We assessed whether it was safe to move to VitalPAC™ EWS (ViEWS), a commercially available electronic EWS [2]. y Conclusion The overall mortality and morbidity rate was comparable with that seen nationally. Our data suggest that the use of non-invasive ventilation was not associated with anastomotic breakdown. A lower P/F ratio in the postoperative period was associated with prolonged ICU and hospital stay. y Methods All patients scored as ‘high risk’ by NEWS (with a score of 6 or more) in a snapshot audit of patients in our 500-bed acute district general hospital were identifi ed and reviewed clinically. All of these patients were then recategorised using ViEWS. The clinical safety of this recategorisation was then assessed. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 and nine patients had died by 1 year (12.5%). The median length of ICU and hospital stay was 4 days and 14 days respectively. Six patients had an anastomotic leak (of which two were chyle leaks). Use of non- invasive ventilation (in 23.1% of patients) was not associated with an anastomotic leak (chi-square P = 0.53), nor was the amount of fl uid given intraoperatively (Mann–Whitney U P = 0.410). Six patients had to be reintubated and this was associated with a signifi cantly increased length of both ICU and hospital stay (Mann–Whitney U P = 0.01 and 0.03 respectively). Lower P/F ratios were also associated with a signifi cant increase in length of both ICU and hospital stay (P = 0.007 and 0.043). P60 1. National Early Warning Score (NEWS). Standardising the Assessment of Acute- illness Severity in the NHS. London: Royal College of Physicians; July 2012. [http://wwwrcplondon ac uk/resources/national early warning score news] 1. National Early Warning Score (NEWS). Standardising the Assessment of Acute- illness Severity in the NHS London: Royal College of Physicians; July 2012 1. National Early Warning Score (NEWS). Standardising the Assessment of Acute- y illness Severity in the NHS. London: Royal College of Physicians; July 2012. [http://wwwrcplondon ac uk/resources/national-early-warning-score-news] SwissScoring: a nationwide survey about SAPS II assessing accuracy M Previsdomini1, B Cerutti2, P Merlani3, HU Rothen4, M Kaufmann5, A Perren1 1Ospedale Regionale Bellinzona e Valli, Bellinzona, Switzerland; 2Unit of Development and Research in Medical Education, Geneva, Switzerland; 3Ospedale Regionale, Lugano, Switzerland; 4Bern University Hospital, Bern, Switzerland; 5University Hospital Basel, Switzerland Critical Care 2014, 18(Suppl 1):P60 (doi: 10.1186/cc13250) SwissScoring: a nationwide survey about SAPS II assessing accuracy M Previsdomini1, B Cerutti2, P Merlani3, HU Rothen4, M Kaufmann5, A Perren1 1O d l R l B ll ll B ll S l d 2 f [http://www.rcplondon.ac.uk/resources/national-early-warning-score-news] 2. Prytherch D, et al.: Resuscitation 2010, 81:932-937. p p y g 2. Prytherch D, et al.: Resuscitation 2010, 81:932-937. 2. Prytherch D, et al.: Resuscitation 2010, 81:9 1Ospedale Regionale Bellinzona e Valli, Bellinzona, Switzerland; 2Unit of Development and Research in Medical Education, Geneva, Switzerland; 3Ospedale Regionale, Lugano, Switzerland; 4Bern University Hospital, Bern, Switzerland; 5University Hospital Basel, Switzerland Critical Care 2014, 18(Suppl 1):P60 (doi: 10.1186/cc13250) P63 Is the Golden hour important? Looking at disability and health-related quality of life in a Portuguese trauma registry J Estilita1,2, CC Dias3, A Costa-Pereira3,4, C Granja1,2,3,4, I Aragão4, L Orwelius3,4,5 1DEICM, Algarve Hospital Centre, Portugal; 2University of Algarve, Portugal; 3CINTESIS, FMUP, Porto, Portugal; 4FMUP, Porto, Portugal; 5Linköping University, Linköping, Sweden Critical Care 2014, 18(Suppl 1):P63 (doi: 10.1186/cc13253) P63 Is the Golden hour important? Looking at disability and health-related quality of life in a Portuguese trauma registry J Estilita1,2, CC Dias3, A Costa-Pereira3,4, C Granja1,2,3,4, I Aragão4, L Orwelius3,4,5 1DEICM, Algarve Hospital Centre, Portugal; 2University of Algarve, Portugal; 3CINTESIS, FMUP, Porto, Portugal; 4FMUP, Porto, Portugal; 5Linköping University, Linköping, Sweden Critical Care 2014, 18(Suppl 1):P63 (doi: 10.1186/cc13253) g Results Signifi cant predictors of the development of complications were age, number of rib fractures, chronic lung disease, use of pre- injury anticoagulants and oxygen saturation levels. The fi nal model demonstrated an excellent c-index of 0.97. Introduction The Golden hour (GH) complies the concept that defi nitive trauma care must be initiated within 60 minutes in order to improve outcome, but evidence is confl icting [1,2]. A variety of injury and health loss scores are used in post-impact care for assessing the injury severity, probability of survival and long-term loss of health [3]. The aim of this study is to establish a relationship between the concept of GH and disability or health-related quality of life (HRQoL) in trauma patients and to compare injury scores and health loss scores in this population. Conclusion In our two-phase study, we have developed and validated a prognostic model that can be used to assist in the management of blunt chest wall trauma patients. The fi nal risk score provides the clinician with the probability of the development of complications for each individual patient. References 1. Demirhan R, Onan B, Oz K, Halezeroglu S: Comprehensive analysis of 4205 patients with chest trauma: a 10-year experience. Interact Cardiovasc Thorac Surg 2009, 9:450-459. Methods We analyzed patients from a trauma registry of a 700-bed university hospital between January 2003 and December 2007 who were alive 6 months after injury. A follow-up interview with the EQ-5D questionnaire was conducted. Data included patient demographics, type of injury, ISS, RTS and TRISS scores, ICU and hospital length of stay (LOS), mortality and EQ-5D domains. g 2. Battle CE, Hutchings H, Evans PA: Risk factors that predict mortality in patients with blunt chest wall trauma: a systematic review and meta- analysis. Injury 2012, 43:8-17. 2. Battle CE, Hutchings H, Evans PA: Risk factors that predict mortality in patients with blunt chest wall trauma: a systematic review and meta- analysis. Injury 2012, 43:8-17. 2. Battle CE, Hutchings H, Evans PA: Risk factors that predict mortality in patients with blunt chest wall trauma: a systematic review and meta- analysis. Injury 2012, 43:8-17. g References 1. Orwelius L, et al.: J Trauma Acute Care Surg 2012, 72:504-512. 2. Aitken LM, et al.: J Trauma Acute Care Surg 2012, 72:1702-1708. 3. European Commission Mobility and Transport Road Safety [http:// ec.europa.eu/transport/road_safety/specialist/knowledge/postimpact/ data_and_information_systems/impairment_disability_and_loss_of_ function_scales_and_scores.htm] 3. European Commission Mobility and Transport Road Safety [http:// ec.europa.eu/transport/road_safety/specialist/knowledge/postimpact/ data_and_information_systems/impairment_disability_and_loss_of_ function_scales_and_scores.htm] j y Methods To develop crush syndrome, both hind limbs of rats were compressed for 6 hours under weights (3.0 kg each). Rats in the treated group were intravenously administrated BMMNCs (1×107 cells in 1 ml phosphate-buff ered saline (PBS)) immediately after weight removal (BMMNC group) and PBS alone was administered in the control group (CR group). The sham group underwent the same procedure without compression of hind limbs (SH group). The rats were observed over 7 days after the injury to evaluate survival. To estimate anti- infl ammatory eff ects of BMMNCs, sera were collected 3 hours, 6 hours and 24 hours after the injury. The levels of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) were measured by ELISA and statistically analyzed. P65 y Results There were 78% (n = 589) males and the average age was 44 ± 20 years. Most trauma cases originated from metropolitan areas and 91% (n = 688) of patients were managed within the GH. There was an association of mortality with older age and penetrating trauma. Mortality was higher for those with shorter length of stay. Those treated within the GH were less likely to die. In the HRQoL analysis, over one-half of the patients had moderate to severe problems in the Usual Activities domain. The median EQ-5D Index was 0.66 (0.47 to 0.98). There was no association of GH with HRQoL domains 6 months after discharge from the ICU. For the TRISS there was a lower probability of survival for the patients with severe problems in the physical health domains. The same results were obtained for association with age and LOS. Transplantation of bone marrow-derived mononuclear cells can improve the survival rate and suppress the infl ammatory response in a rat crush injury model J Nakagawa, N Matsumoto, K Yamakawa, T Yamada, H Matsumoto, T Muroya, H Ogura, T Shimazu Osaka University Graduate School of Medicine, Suita-shi, Osaka, Japan Critical Care 2014, 18(Suppl 1):P65 (doi: 10.1186/cc13255) Introduction Crush syndrome is often encountered in natural disasters. It is a critical condition leading to multiple organ failure. However, the mechanisms by which the local traumatic injuries aff ect distant organs remain unknown. We paid attention to bone marrow-derived mononuclear cells (BMMNCs) as therapeutic strategy against crush injury. Transplantation of BMMNCs can elicit protection and regeneration against damaged organs through their paracrine properties and its clinical application has been realized to treat ischemia reperfusion-related various diseases. We have previously reported that multiple organ damage based on systemic infl ammatory response is induced in crush injury and the pathogenesis is largely dependent on massive ischemia–reperfusion. We investigated whether BMMNCs could suppress systemic infl ammation and improve mortality in a rat model of crush injury. Conclusion The GH may be important as a survival outcome but not as a HRQoL outcome. Patients with severe problems in EQ-5D physical health domains showed lower probability of survival, were older and had a longer ICU LOS. R f Reference S23 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion A prediction model for early mortality of patients with severe sepsis and septic shock can be developed based on two parameters, lactate clearance and number of organ dysfunctions. A model has been developed to predict and classify mortality risk. References of this patient group is challenging as a result of the delayed onset of complications. The aim of this study was to develop and validate a prognostic model that can be used to assist in the management of blunt chest wall trauma. Methods There were two distinct phases to the overall study; the development and the validation phases. In the fi rst study phase, the prognostic model was developed through the retrospective analysis of all blunt chest wall trauma patients (n = 274) presenting to the emergency department of a regional trauma centre in Wales (2009 to 2011). Multivariable logistic regression was used to develop the model and identify the signifi cant predictors for the development of complications. The model’s accuracy and predictive capabilities were assessed. In the second study phase, external validation of the model was completed in a multicentre prospective study (n = 237) in 2012. The model’s accuracy and predictive capabilities were re-assessed for the validation sample. A risk score was developed for use in the clinical setting. 1. Trzeciak S, et al.: Acad Emerg Med 2008, 15:399-413. 2. Rivers EP, et al.: Minerva Anestesiol 2012, 78:712-724. 3. Macias WL, et al.: Crit Care Med 2004, 32(5 Suppl):S223-S228. 1. Trzeciak S, et al.: Acad Emerg Med 2008, 15:399-413. 2. Rivers EP, et al.: Minerva Anestesiol 2012, 78:712-724. 3. Macias WL, et al.: Crit Care Med 2004, 32(5 Suppl):S223-S228. Impact of a dedicated trauma desk in ambulance control on the identifi cation of major trauma in Scotland Impact of a dedicated trauma desk in ambulance control on the identifi cation of major trauma in Scotland J Bruce1, A Parker2, M Donald3, M Esposito2, L Cu J Bruce1, A Parker2, M Donald3, M Esposito2, L Curatolo2, A Kennedy2, K Simpson2, C Morton2, J Cormack2, M Austin2 1St Johns Hospital, Livingston, UK; 2Scottish Ambulance Service, Edinburgh, UK; 3Ninewells Hospital, Dundee, UK Critical Care 2014, 18(Suppl 1):P66 (doi: 10.1186/cc13256) Results We included a total of 1,452 patients (58% males, mean age 66.6 years). A total of 20.1% (n = 292) were classifi ed as high treatment priority, 5.4% (n = 79) were admitted to the ICU and 4.4% (n = 64) died within 30 days. The initial MTS showed a good prognostic accuracy to predict treatment priority (AUC 0.75) and ICU admission (AUC 0.76), but not for mortality prediction (AUC 0.58). Initial ProADM levels were independent predictors for all three outcomes and signifi cantly improved the MTS score to AUCs of 0.78 for treatment priority, 0.80 for ICU admission and 0.84 for mortality. Introduction In this study we determine the impact of a dedicated trauma desk on the identifi cation of patients suff ering from major trauma and on time to allocate critical care resources. Trauma is increasingly recognised as a serious global health problem and is the leading cause of death in those under the age of 45, accounting for 1,300 deaths in Scotland each year [1]. In addition, trauma causes considerable short-term and long-term morbidity and has personal, social and fi nancial impact on the population [2]. Evidence and expert opinion strongly support the presence of appropriately trained, clinically active, personnel integrated and fundamental to the command and control structure in order to optimise the right resource being dispatched to the right patient at the right time.f y Conclusion Within this large cohort of consecutive unselected medical patients seeking ED care, the MTS instrument in combination with a prognostic biomarker (ProADM) allowed accurate initial risk assessment in regard to treatment priority, ICU admission and mortality. A combined score has the potential to signifi cantly improve initial risk assessment in patients, which may translate into faster and more targeted care and better clinical patient outcomes. g g g Methods From October 2012 to April 2013 a trauma desk, staff ed by experienced paramedics, was created in ambulance control with access to all emergency calls across Scotland. Predicting outcomes after blunt chest wall trauma: development and external validation of a new prognostic model l 1 1 h 2 1 C Battle1, S Lovett1, H Hutchings2, PA Evans1 C Battle1, S Lovett1, H Hutchings2, PA Evans1 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK 1Morriston Hospital, ABMU Health Board, Swansea, UK; 2Swansea University, Swansea, UK Critical Care 2014, 18(Suppl 1):P64 (doi: 10.1186/cc13254) Critical Care 2014, 18(Suppl 1):P64 (doi: 10.1186/cc13254) Introduction Blunt chest wall trauma accounts for over 15% of all trauma admissions to emergency departments worldwide [1]. Reported mortality rates vary between 4 and 60% [2]. Management y Results The survival rate at day 7 in the BMMNC group was 80.0%, and that in the CR group was 47.6%, revealing that the 7-day survival S24 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 System (MTS), have not been well validated in unselected medical patients. Herein, we performed a prospective cohort study to assess the prognostic potential of the MTS and the prognostic biomarker pro- adrenomedullin (ProADM) to identify patients at high initial treatment priority, patients with admission to the ICU, and patients who die within a 30-day follow-up. was signifi cantly improved by the BMMNC injection (P <0.05). Crush injury-induced upregulation of serum IL-6 was signifi cantly reduced by the BMMNC treatment at all time points (P <0.05). The level of TNFα decreased signifi cantly in the BMMNC group compared with that in the CR group 24 hours after the compression release (P <0.05). These fi ndings suggest that transplantation of BMMNCs has an ability to evade the devastating condition following crush injury by suppressing systemic infl ammation. y p Methods This is a prospective, observational cohort study including all consecutive medical patients seeking ED care between June 2013 and October 2013, except nonadult and nonmedical patients. We collected detailed clinical information including the initial MTS and measured ProADM levels on admission in all patients. Initial treatment priority was adjudicated by two independent, blinded physicians based on all available results at the time of ED discharge to the medical ward. To assess outcomes, data from electronic medical records were used and all patients were contacted by telephone 30 days after hospital admission. The prognostic performance of MTS and ProADM was assessed in multivariate regression models with area under the receiver operating curve (AUC) as an overall measure of discrimination. P68 Since September 2013, we newly developed k-support to the Kainan Fire Department Mugi branch offi ce, Kainan Fire Department Hiwasa branch offi ce, Kaifu Fire-fi ghting Union Kainan fi re department, and Muroto Fire-Fighting Headquarters Touyou branch offi ce, and reviewed our experience of this approach in the use of k-support during the fi rst 3 months of the introduction. Results k-support was used for 62 patients, and 13 (21.0%) of them were categorized as neurosurgical diseases. The detail of neurosurgical diseases consisted of six patients (46.1%) with head injury, followed by fi ve patients (38.5%) with cerebral infarction, one patient (7.7%) with cerebral hemorrhage and one patient (7.7%) with miscellaneous diseases. We shared the information by ambulance services to tweet the patient information or transmit vital signs and electrocardiograms. We experienced a case that was diagnosed with cardiogenic embolism and tried the ‘drip and ship’ method of rt-PA. The outcomes were as follows: hospitalization, 28 (45.2%); discharge, 18 (29.0%); and transfer, 16 (25.8%). Conclusion Introduction of k-support is the fi rst trial in Japan. For emergency diseases such as ischemic heart diseases and stroke, we could perform responses earlier than ever by providing this system. Conclusion A clinician focusing on major trauma in the ambulance control room improves activation times of prehospital critical care teams. The number of patients receiving life-saving and limb-saving interventions has substantially increased. The stand-down rate of teams following activation by the trauma desk is considerably reduced. References 1. Major Trauma in Scotland. Edinburgh: Royal College of Surgeons; 2012. 2. Scottish Trauma Audit Group – Trauma Report, Edinburgh: NHS National Services Scotland, Information Services Division Publications; 2012. 1. Major Trauma in Scotland. Edinburgh: Royal College of Surgeons; 2012. 2. Scottish Trauma Audit Group – Trauma Report, Edinburgh: NHS National Services Scotland, Information Services Division Publications; 2012. pp gi Results k-support was used for 62 patients, and 13 (21.0%) of them were categorized as neurosurgical diseases. The detail of neurosurgical diseases consisted of six patients (46.1%) with head injury, followed by fi ve patients (38.5%) with cerebral infarction, one patient (7.7%) with cerebral hemorrhage and one patient (7.7%) with miscellaneous diseases. We shared the information by ambulance services to tweet the patient information or transmit vital signs and electrocardiograms. We experienced a case that was diagnosed with cardiogenic embolism and tried the ‘drip and ship’ method of rt-PA. P68 The outcomes were as follows: hospitalization, 28 (45.2%); discharge, 18 (29.0%); and transfer, 16 (25.8%). Conclusion Introduction of k-support is the fi rst trial in Japan. For emergency diseases such as ischemic heart diseases and stroke, we could perform responses earlier than ever by providing this system. Predicting outcomes after blunt chest wall trauma: development and external validation of a new prognostic model l 1 1 h 2 1 yl Conclusion The administration of BMMNCs reduced production of infl ammatory cytokines and improved survival rate in a rat model of crush injury. Cell therapy using BMMNCs might become a novel therapy against crush injury. P68 P68 Introduction of the Kaifu telemedicine system for emergency medicine to ambulance services with improvement of the survival rates F Obata1, R Tabata2, K Mori1, T Kageji3, K Tani2, H Bando1 1Tokushima Prefectural Kaifu Hospital, Tokushima, Japan; 2Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; 3Tokushima University Hospital, Tokushima, Japan Critical Care 2014, 18(Suppl 1):P68 (doi: 10.1186/cc13258) Results There were 190 activations of critical care teams during the study period compared with 73 from the historical data from the same time frame in the previous year, representing a 160% increase in activations. The mean time from emergency call to allocation of critical care resource was 6 minutes compared with 19 minutes from historical data. The stand-down rate for prehospital critical care resource from emergency calls identifi ed by trauma desk clinicians was 31.5% compared with 56% for those by nontrauma desk clinicians or dispatchers. Introduction Our hospital has introduced the telemedicine system for emergency medicine (k-support) using a smart device to rectify health disparity and reduce the burden to general physicians since February 2013. We have expanded to the ambulance services’ k-support toward further survival rate improvement. The mean time from emergency call to allocation of critical care resource was 6 minutes compared with 19 minutes from historical data. The stand-down rate for prehospital critical care resource from emergency calls identifi ed by trauma desk clinicians was 31.5% compared with 56% for those by nontrauma desk clinicians or dispatchers. p Methods The registration device, patient information and image information such as magnetic resonance imaging and computed tomography taken in the hospital were transferred in real time to k-support using the smart device. Since September 2013, we newly developed k-support to the Kainan Fire Department Mugi branch offi ce, Kainan Fire Department Hiwasa branch offi ce, Kaifu Fire-fi ghting Union Kainan fi re department, and Muroto Fire-Fighting Headquarters Touyou branch offi ce, and reviewed our experience of this approach in the use of k-support during the fi rst 3 months of the introduction. Methods The registration device, patient information and image information such as magnetic resonance imaging and computed tomography taken in the hospital were transferred in real time to k-support using the smart device. Impact of a dedicated trauma desk in ambulance control on the identifi cation of major trauma in Scotland The operational hours of the desk were 08:00 to 18:00, 7 days a week. A customised database provided a data entry portal. Retrospective data were collated for comparison. Primary outcome measures were the number of emergency calls identifi ed as potential major trauma and the time to allocation of critical care resources. Secondary outcome measures were the eff ects on stand-down rates of prehospital critical care teams. Training to achieve coordination of rescue and ambulance and medical teams Evaluation and prevention of violence in the emergency department in Lebanon p Z Fadel, N Hachem, C El Ramy, G Abi Nader, D Haykal Sainte Famille University, Batroun, Lebanon Critical Care 2014, 18(Suppl 1):P71 (doi: 10.1186/cc13261) T Yamashita1, K Yamazumi2, H Takayama3, Y Sakamoto1 1Saga University, Saga, Japan; 2Ureshino Medical Center, Ureshino, Saga, Japan; 3Nagasaki Medical Center, Omura, Nagasaki, Japan Critical Care 2014, 18(Suppl 1):P69 (doi: 10.1186/cc13259) Introduction At the hospital we are faced with situations of violence, whether verbal or physical, especially in the emergency department (ED) [1]. The objective of this study is to evaluate the phenomenon of violence at hospitals in Lebanon, especially in the ED, and to recommend techniques to prevent it. Introduction Japanese emergency medical technicians are not allowed to perform some advanced medical practices such as a tracheal intubation, establishment of an intravenous line, administration of a drug to a patient who retains their own circulation. So medical staff have to be dispatched to the accident scene to give a patient such medical practices. In Japan, we have developed and dispersed the educational course of the Disaster Medical Assistance Team since 2005. But the role of medical teams at the scene is not widely known among rescue workers. It is also distant for medical staff to coordinate rescue teams, because most of them do not work with rescue workers in their daily work. We have felt a need for practical training to achieve the coordination of rescue and medical teams. Methods A questionnaire consisting of 18 questions was sent to the caregivers in the ED of three randomly selected hospitals in Beirut, Lebanon in 2012. A total of 111 people (nurses, aides, doctors, interns, residents, social workers and security guards) responded to the survey questionnaire. Results The majority of the surveyed people are young women (62%) aged between 20 and 40 years (78%) with a nursing degree (74%) and professional experience <5 years (48%). In total, 59% of respondents have experienced violence in the ED during the night (58%) from the patients (31%) or their companions (68%). The caregivers most aff ected by the violence are nurses (54%) and employees of reception (46%). Violence can be verbal (threats 47%, insults 36%, criticism 18%) or physical (hitting 43%, slapping 40%, stabbing 17%). P72 P70 Complementary cooperation of an ambulance helicopter and car with medical doctors: meaning of simultaneous dispatch K Ishii, K Kurosawa, S Tanabe, T Noguchi Oita University Hospital, Oita, Japan Critical Care 2014, 18(Suppl 1):P70 (doi: 10.1186/cc13260) P72 Epidemiology and critical care management of patients admitted after intentional self-poisoning GP Misselbrook, N Sudhan Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P72 (doi: 10.1186/cc13262) Introduction Recently, the dispatch system with a medical doctor in pre-hospital care has made rapid progress in Japan. We usually use an ambulance helicopter or an ambulance car/rapid response car for dispatch. In this report, we analyzed the cases in which the medical doctors were dispatched using both means at the same time. Introduction Intentional self-poisoning is one of the most common presentations to acute medical units across the UK [1]. To our knowledge no studies have been published on incidence of admissions to critical care in England after overdose. Our aim was to investigate the epidemiology, clinical features and outcomes of patients admitted to critical care after intentional self-poisoning to establish patterns in our community. Introduction Intentional self-poisoning is one of the most common presentations to acute medical units across the UK [1]. To our knowledge no studies have been published on incidence of admissions to critical care in England after overdose. Our aim was to investigate the epidemiology, clinical features and outcomes of patients admitted to critical care after intentional self-poisoning to establish patterns in our community. Methods We analyzed 29 cases with simultaneous dispatch with the medical doctors using ambulance helicopter and car during October 2012 to September 2013. Methods We performed a retrospective data collection using our critical care database ‘Metavision’ to select all patients admitted with a diagnosis of ‘overdose’. Records were scrutinised to collect information on patient demographics, clinical features and medical management. Results Thirty-eight patients (male:female ratio 1:1.53) were admitted to critical care over a 1-year period (September 2011 to 2012). This represented 2.45% of the total admissions to critical care during the same period. The sample had a signifi cantly younger median age (45 years) than the standard patient population in critical care (68 years) during the same period (P <0.0001). The Manchester Triage System in optimizing triage in adult general medical emergency patients: the Triage Projectf Kantonsspital Aarau, Switzerland Introduction Some patients presenting to the emergency department (ED) currently face inacceptable delays in initial treatment due to suboptimal initial triage. Triage scores, such as the Manchester Triage S25 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P69 Training to achieve coordination of rescue and ambulance and medical teams T Yamashita1, K Yamazumi2, H Takayama3, Y Sakamoto1 1Saga University, Saga, Japan; 2Ureshino Medical Center, Ureshino, Saga, Japan; 3Nagasaki Medical Center, Omura, Nagasaki, Japan Critical Care 2014, 18(Suppl 1):P69 (doi: 10.1186/cc13259) P71 Training to achieve coordination of rescue and ambulance and medical teams The dissatisfaction of the patient in his care (42%) and his anxiety (33%) are the most important factors in the generation of violence that may have repercussions on the care workers and their psychological status. Methods We held a disaster-relief training workshop from 2010 to 2013. Prior to starting the drill, we gave two lectures about the role of a medical team in a rescue site and the basic knowledge of rescue skills. The drill was organized for 1 to 1.5 hours. The scenario was secret for participants. Before and after the workshop, we had a questionnaire survey for attitude changes about a collaborative work between a rescue team and a medical team. Results A total of 160 people participated in the workshop (63 rescue workers, 27 ambulance workers, 33 paramedics, 37 medical staff ). At fi rst, rescue workers tended to downplay the importance about the division of roles or the establishment of command and control system between rescue teams and medical teams, but their attitude changed after the drill (P <0.05). They also understood the need to know about basic trauma survey skills and some medical terms. Conclusion Violence in the ED may be due to the heavy workload of the caregivers causing a delay in care. Secondly, patients in the ED may feel insuffi ciently informed and heard by the nursing staff . The priority given to emergencies depending on the severity and not the order of arrival can be misunderstood [2]. Therefore, we recommend the following actions: encourage caregivers to improve their knowledge and training on the management of patients in emergency situations; train emergency caregivers to mediation, nonviolent communication and managing stressful situations; and increase the number of nurses and security guards in the ED and motivate them to ensure a better quality of care and minimize the delay in their care. y Conclusion To achieve mutual understanding, it is important to have drills in which both rescue teams and medical teams participate. 1. Prudhomme C: L’infi rmière et les urgences. Paris: Maloine; 2000:422. 2. Goddet B: La violence à l’hôpital, in Soins, Pratique et savoir infi rmier. Masson 1998, 624:3-23. P70 P72 Despite the young age and paucity of comorbidities, there was no diff erence in length of stay between overdose patients (2.0 days) and all other patients on critical care (1.58 days, P = 0.3). The median number of agents ingested was three Methods We performed a retrospective data collection using our critical care database ‘Metavision’ to select all patients admitted with a diagnosis of ‘overdose’. Records were scrutinised to collect information on patient demographics, clinical features and medical management. Results The average distance of dispatch was 25.6 km. The corres- pondence to the plural patients was three trauma cases. In nine cases, the ambulance car was fi rst selected for the dispatch vehicle and the ambulance helicopter was added. In three cases, both devices were canceled on the dispatch way to the scene. In seven cases, the ambulance car was canceled. In 10 cases, the patients were transferred by the ambulance helicopter. Results Thirty-eight patients (male:female ratio 1:1.53) were admitted to critical care over a 1-year period (September 2011 to 2012). This represented 2.45% of the total admissions to critical care during the same period. The sample had a signifi cantly younger median age (45 years) than the standard patient population in critical care (68 years) during the same period (P <0.0001). Despite the young age and paucity of comorbidities, there was no diff erence in length of stay between overdose patients (2.0 days) and all other patients on critical care (1.58 days, P = 0.3). The median number of agents ingested was three Conclusion In the selection of the dispatch means, the time to contact with the patient is the most important factor. However, the decision of the suitable means is occasionally quite diffi cult at the moment of fi rst information. The ambulance helicopter and car were complementary to each other in the dispatch mean of the medical doctors. S26 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (1 to 7) with 84.2% ingesting ≥2 agents. Hypnotics and antidepressants made up 45% of the agents ingested. A total 92.1% of the sample was admitted out-of-hours or at weekends. Fifty per cent had a past history of overdose, 25% had a history of alcohol misuse. Reference 1. Clark D, et al.: Epidemiology and outcomes of patients admitted to critical care after self-poisoning. JICS 2011, 12:268-273. J Liu1, G Zou1, Y Wu1, W Li2 J Liu1, G Zou1, Y Wu1, W Li2 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Critical Care 2014, 18(Suppl 1):P74 (doi: 10.1186/cc13264) P72 Eighty-nine per cent of patients with alcohol-related conditions required support for at least two organ failures, which subsequently equated to an overall cost to the unit of £725,308 and 12% of an approximate £6 million annual budget. pp g Conclusion The results of this study support the hypothesis that alcohol-related disease contributes considerably to admissions to this ICU. Furthermore, they have shown that the fi nancial impact was proportionally greater than the percentage number of admissions attributable to alcohol consumption, refl ecting the high frequency of multiorgan failure in this patient cohort. R f References 1. Geary T, et al.: Anaesthesia 2012, 67:1132-1137. 2. Jones L, et al.: Alcohol-attributable Fractions for England. Liverpool: Centre for Public Health, Liverpool John Moores University; June 2008. P75 Eff ect of low-dose hydrocortisone on gene expression profi les after b i j Eff ect of low-dose hydrocortisone on gene expression profi les after severe burn injury J Plassais1, MA Cazalis1, F Venet2, G Monneret2, A Pachot1, S Tissot2 1Joint Unit Hospices Civils de Lyon/bioMérieux, Lyon, France; 2Hospices Civils de Lyon, Hôpital Edouard Herriot, Lyon, France Critical Care 2014, 18(Suppl 1):P75 (doi: 10.1186/cc13265) Eff ect of low-dose hydrocortisone on gene expression profi les after severe burn injury J Plassais1, MA Cazalis1, F Venet2, G Monneret2, A Pachot1, S Tissot2 1Joint Unit Hospices Civils de Lyon/bioMérieux, Lyon, France; 2Hospices Civils de Lyon, Hôpital Edouard Herriot, Lyon, France Critical Care 2014, 18(Suppl 1):P75 (doi: 10.1186/cc13265) P73 Price per unit: the cost of alcohol-related admissions to a regional ICU Introduction The objective was to analyze the clinical features of multiple organ dysfunction syndrome (MODS) induced by severe heat stroke, and to investigate the pathogenesis, diagnosis and prevention strategies in patients with MODS caused by severe heat stroke. Introduction The objective was to analyze the clinical features of multiple organ dysfunction syndrome (MODS) induced by severe heat stroke, and to investigate the pathogenesis, diagnosis and prevention strategies in patients with MODS caused by severe heat stroke. Methods We retrospectively studied nine cases of MODS caused by severe heat stroke, and systemically reviewed the relevant literature. Results (1) Nine patients with MODS caused by severe heat stroke were all exposed in hot and humid environments. All nine patients are severe heat stroke, including seven cases of classic heat stroke (77.8%) and two cases of exertional heat stroke (22.2%). (2) Among nine cases of MODS caused by severe heat stroke, eight patients met the diagnostic criteria for SIRS. In addition, there was a signifi cant increase in blood PMN proportion and serum CRP of all nine patients. Of note, there was also a marked increase in serum IL-6 (average (36 ± 19) pg/ml; reference range (0 to 5.9 pg/ml)); and TNFα level (average (21 ± 10) pg/ml; reference range (0 to 8.1 pg/ml)), while IL-8 was normal (average (22 ± 25) pg/ ml; reference range (0 to 62 pg/ml)). (3) There were 34 organ damages involved in all nine patients with MODS induced by severe heat stroke. The kidney, circulatory and liver accounted for 58.8% of all these organ dysfunctions. The incidence of severe heatstroke-induced organ dysfunction in turn was the kidney, circulation, liver, blood coagulation, metabolism, brain, lungs, and gastrointestinal tract. (4) During hospitalization, the common complication was pulmonary infection in patients with MODS caused by heat stroke. (5) After early intensive care of organ supportive treatment, there was a quick improvement and recovery in most cases in several days. Seven patients survived, and the average length of stay in hospital was 9.5 days. Introduction A number of studies have demonstrated the signifi cant and increasing morbidity and mortality associated with alcohol-related disease in the UK, and the corresponding impact this has on critical care services [1]. P72 A total of 79% of patients were referred to critical care due to a low conscious level but only 50% required IPPV and 20% received vasopressors/inotropes. The mortality rate was 2.6%, with one further death 6 months after discharge due to alcoholic liver disease. Estimated fi nancial cost was £80,555 or £2,119 per patient (57 level 3 bed-days, 35 level 2 bed-days). Conclusion Intentional self-poisoning mortality rates are low in spite of the number of patients admitted. Despite the young age of patients and lack of comorbidities, their length of stay is similar to the average length of stay for all patients admitted to the unit, representing a signifi cant fi nancial cost. Self-poisoning requiring critical care support is more common out-of-hours when less senior expertise is available. Education of junior doctors into management of overdose is therefore vital to ensure the early identifi cation and appropriate treatment of these patients. number of admissions per month varied from zero in May to a peak of 14 in November, with the majority (40%) of admissions occurring over the autumn months (Figure 1). Eighty-nine per cent of patients with alcohol-related conditions required support for at least two organ failures, which subsequently equated to an overall cost to the unit of £725,308 and 12% of an approximate £6 million annual budget. Conclusion The results of this study support the hypothesis that alcohol-related disease contributes considerably to admissions to this ICU. Furthermore, they have shown that the fi nancial impact was proportionally greater than the percentage number of admissions attributable to alcohol consumption, refl ecting the high frequency of multiorgan failure in this patient cohort. R f number of admissions per month varied from zero in May to a peak of 14 in November, with the majority (40%) of admissions occurring over the autumn months (Figure 1). Eighty-nine per cent of patients with alcohol-related conditions required support for at least two organ failures, which subsequently equated to an overall cost to the unit of £725,308 and 12% of an approximate £6 million annual budget. number of admissions per month varied from zero in May to a peak of 14 in November, with the majority (40%) of admissions occurring over the autumn months (Figure 1). P73 Price per unit: the cost of alcohol-related admissions to a regional ICU This study was designed to similarly evaluate the current burden of alcohol-related disease on admission rates to a regional ICU, but also to calculate the fi nancial costs of such admissions.i g p y Methods We retrospectively studied nine cases of MODS caused by severe heat stroke, and systemically reviewed the relevant literature. i Methods Data-entry fi elds derived from recommendations in a public heath publication [2] were introduced to the local electronic records system to enable prospective data collection for all admissions that were either wholly or partially attributable to alcohol consumption. Using locally defi ned values for the cost per day of an admission to the ICU, depending on the maximum level of organ support required during the admission, it was possible to calculate the total expense incurred by the unit for each alcohol-related admission. y Results In 1 year from December 2012 to November 2013 inclusive, the ICU recorded 84 alcohol-related admissions, accounting for approximately 9% of annual unplanned admissions. With an average length of stay (5.8 days) similar to that of all other unplanned admissions, this totalled 534 ICU bed-days. A total of 86% of patients with alcohol-related conditions were male with an average age of 46.4 years (range 15 to 83 years), and the majority (42%) presented with chronic conditions partially attributable to alcohol consumption. The Figure 1 (abstract P73). Conclusion Severe heat stroke results in signifi cant abnormal changes in infl ammatory markers in patients with MODS induced by heat stroke. The types of organ dysfunction in heat stroke-induced MODS are usually distinct from those of infection and trauma. After active cooling and intensive organ function supportive treatment, most patients recovered in a relatively short period. Acknowledgements Supported by the Research Projects CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. P75 Eff ect of low-dose hydrocortisone on gene expression profi les after severe burn injury J Plassais1, MA Cazalis1, F Venet2, G Monneret2, A Pachot1, S Tissot2 1Joint Unit Hospices Civils de Lyon/bioMérieux, Lyon, France; 2Hospices Civils de Lyon, Hôpital Edouard Herriot, Lyon, France Critical Care 2014, 18(Suppl 1):P75 (doi: 10.1186/cc13265) P77 Low socioeconomic status, ethnicity and geographical location confers high risk of signifi cant accidental burns injuries in London JS Heng, O Clancy, I Jones, J Atkins, J Leon-Villapalos, A Williams, M Hayes, M Vizcaychipi Chelsea & Westminster Hospital, London, UK Critical Care 2014, 18(Suppl 1):P77 (doi: 10.1186/cc13267) Results Severe burn injury induced the deregulation of a considerable number of genes (n >2,200 at S1) in comparison with controls with an increased number of deregulated genes over time. Within burn patients, more than 300 genes were deregulated by hydrocortisone over time. The treatment had a rapid eff ect on gene expression, 339 and 627 genes were diff erentially expressed at S2 and S3 respectively. However, the number of these genes decreased drastically at S4 (only 24 genes signifi cant). The genes identifi ed at S2 were mostly related to the decrease of growth, development and quantity of leukocytes but these biological processes were not found signifi cant at S3, indicating that the action of glucocorticoid in the response to burn injury is short lived and time dependent. Introduction The majority of burns injuries are considered accidental, although previous studies have identifi ed demographic factors associated with higher risk of burns such as socioeconomic deprivation [1] and being from ethnic minority groups [2]. This study aims to identify population subgroups in London at high risk of burns injuries requiring admission to a burns centre through geographic mapping and socioeconomic statistics. Conclusion This study is an informative overview of the genomic responses after burn injuries. More importantly, it is the fi rst study providing information about mechanisms involved in glucocorticoid’s reduced shock duration after burn. Methods Records of all paediatric and adult inpatients admitted to the burns centre at Chelsea and Westminster Hospital were retrospectively reviewed for age, ethnic group and deprivation score of residence, as measured by the English Index of Multiple Deprivation 2010. Corresponding population data for London were obtained. Reference 1. Keck M, et al.: Wien Med Wochenschr 2009, 159:13-14. Results In total, 2,195 patients from London were admitted between January 2009 and August 2013, with 1,963 (89.4%) classifi ed as having accidental injuries. A total 1,725 (87.8%) of accidental burn injuries occurred in the patients’ own homes. Patients from ethnic minorities have the highest rate of burn injury at 7.1 per 100,000 population per annum (P <0.0001). Patients below the median for socioeconomic deprivation in London are more likely to suff er burn injuries (P <0.0001). Patients from the most deprived quartile are more likely to suff er burns injuries of >10% TBSA (P = 0.04), and have a trend towards higher rates of ICU admission (P = 0.144). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P76). Numbers of complex burns in Scotland 2010 to 2012 2010 2011 2012 Adult 304 392 399 Paediatric 195 185 138 Adult >15% 27 28 26 Paediatric >15% 0 2 4 Mortality 5 7 13 Table 1 (abstract P76). Numbers of complex burns in Scotland 2010 to 2012 shock. Patients need vasopressor infusion to maintain adequate delivery of oxygen but it could have deleterious eff ects on skin perfusion and worsen the burn depth. This shock could result from the interplay of the initial hypovolemia and the release of multiple infl ammatory mediators [1]. It has been shown that a low-dose of hydrocortisone could reduce the shock duration but the mechanisms involved remain unclear. We investigated the systemic genomic response after severe burn injuries and determine whether patterns of gene expression could be associated with a low dose of glucocorticoids. shock. Patients need vasopressor infusion to maintain adequate delivery of oxygen but it could have deleterious eff ects on skin perfusion and worsen the burn depth. This shock could result from the interplay of the initial hypovolemia and the release of multiple infl ammatory mediators [1]. It has been shown that a low-dose of hydrocortisone could reduce the shock duration but the mechanisms involved remain unclear. We investigated the systemic genomic response after severe burn injuries and determine whether patterns of gene expression could be associated with a low dose of glucocorticoids. Methods Thirty burn patients with over 30% of total body surface area were enrolled into a randomized double-blind clinical study. Fifteen patients were treated with a low dose of hydrocortisone and 15 patients were treated with placebo. Whole blood samples were collected after shock onset (S1) before any treatment, 1 day after treatment beginning (S2), and 120 hours and 168 hours after the burn injury (S3/S4). Blood samples of 13 healthy volunteers were collected. Pangenomic expression was evaluated with Aff ymetrix HG-U133plus 2.0 microarrays. Moderated t tests and F test were used to compare burn patients with controls and gene expression profi les between the two groups (B–H correction, P <0.05). sources. As data quality improves, detailed analysis of mortality data will allow COBIS to identify contributing issues aff ecting burns patients. Some issues identifi ed already are that patients with burns often die soon after their discharge from hospital of other related and unrelated causes. P77 Low socioeconomic status, ethnicity and geographical location confers high risk of signifi cant accidental burns injuries in London JS Heng, O Clancy, I Jones, J Atkins, J Leon-Villapalos, A Williams, M Hayes, M Vizcaychipi Chelsea & Westminster Hospital, London, UK Critical Care 2014, 18(Suppl 1):P77 (doi: 10.1186/cc13267) Domestic accidental burns were mapped to their respective administrative wards, and the rate of burns per 100,000 was calculated and divided into quintiles. The areas with the top quintile of burn injury rate, of up to 18.8 per 100,000 population per year, were almost four times the national average. Care of Burns in Scotland: 3-year data from the Managed Clinical Network National Registry CJ Gilhooly1, J Kinsella2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University of Glasgow, UK Critical Care 2014, 18(Suppl 1):P76 (doi: 10.1186/cc13266) Care of Burns in Scotland: 3-year data from the Managed Clinical Network National Registry CJ Gilhooly1, J Kinsella2 1Glasgow Royal Infi rmary, Glasgow, UK; 2University of Glasgow, UK Critical Care 2014, 18(Suppl 1):P76 (doi: 10.1186/cc13266) Introduction The Managed Clinical Network for Care of Burns in Scotland (COBIS) was launched in April 2007. Primary aims included establishing and maintaining a registry of complex burn injury in Scotland and setting mechanisms to regularly audit outcome of burn treatment against nationally agreed standards of care. On behalf of COBIS, we present 3-year incidence and mortality data of Scottish patients admitted with a complex burn injury in this abstract. y Conclusion Ethnicity, socioeconomic deprivation and geographical location appear to be risk factors for burn injuries. Identifying such groups may allow the development of targeted preventative strategies. References Methods From January 2010 onwards, data were prospectively collected for all patients in Scotland with complex burn injury admitted to Scottish burns units. Data collection was initially on a paper pro forma, but subsequently evolved into a web-based audit data capture system to securely link hospital sites involved in the delivery of care of complex burns. Data collected included extent and mechanism of burn, presence of airway burn or smoke inhalational injury, comorbidities, complications, length of stay, interventions and mortality. Quality, completeness and consistency of data collection are audited with feedback to the individual units. 1. Mistry RM, Pasisi L, Chong S, Stewart J, She RB: Socioeconomic deprivation and burns, Burns 2010, 36:403-408. 1. Mistry RM, Pasisi L, Chong S, Stewart J, She RB: Socioeconomic deprivation and burns, Burns 2010, 36:403-408.f 1. Mistry RM, Pasisi L, Chong S, Stewart J, She RB: Socioeconomic deprivation and burns, Burns 2010, 36:403-408. 2. Tan KT, Prowse PM, Falder S: Ethnic diff erences in burn mechanism and severity in a UK paediatric population, Burns 2012, 38:551-555. 2. Tan KT, Prowse PM, Falder S: Ethnic diff erences in burn mechanism and severity in a UK paediatric population, Burns 2012, 38:551-555. 2. Tan KT, Prowse PM, Falder S: Ethnic diff erences in burn mechanism and severity in a UK paediatric population, Burns 2012, 38:551-555. P78f Eff ect of low-dose hydrocortisone on gene expression profi les after severe burn injury Introduction The systemic eff ect of infl ammatory mediators released after severe burn is a severe cardiovascular dysfunction called burn Introduction The systemic eff ect of infl ammatory mediators released after severe burn is a severe cardiovascular dysfunction called burn S27 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Subsequent analysis of this will allow COBIS to identify and address issues that may be contributing to these statistics. P80 Almost all of the physicians used hydrocortisone (96.4%). The median daily dose of hydrocortisone was 300 mg (interquartile range, 200 to 300). Nearly 50% divided the dose every 8 hours and 31.8% infused continuously. The duration for tapering was varied (33.6% in 2 to 3 days). Central venous pressure (CVP) and fl uid challenge test were more frequently used for preload evaluation than new fl uid responsiveness methods. Less than 15% still used a pulmonary artery catheter in their routine practice. Antipyretics in the emergency department – intravenous paracetamol versus intramuscular diclofenac: a comparative study N Purayil, N Vamanjore, F Paramba, O Mohammad, P Chandra Hamad Medical Corporation, Doha, Qatar Critical Care 2014, 18(Suppl 1):P79 (doi: 10.1186/cc13269) Introduction Fever is a common problem in adults visiting the emergency department (ED) [1]. Although it is important to treat the cause of fever, symptomatic management of fever is also necessary. Multiple studies are available on the effi cacy of various antipyretics in children, but very little has been done in adults [1,2]. Hence we aimed to compare the antipyretic effi cacy of intravenous (i.v.) paracetamol and intramuscular (i.m.) diclofenac in adult patients presenting with fever to the ED. Methods In this parallel-group, open-label trial, participants aged 14 to 75 years who had a temperature more than 38.5°C were enrolled and treated in the ED, Alkhor, Hamad Medical Corporation, during the period June 2008 to December 2011. Patients were randomly assigned to receive either 1,000 mg i.v. paracetamol or 75 mg i.m. diclofenac. The primary outcome was degree of reduction in mean oral temperature at 90 minutes and the secondary outcomes were degree of reduction at 30, 60 and 120 minutes. The effi cacy of drugs was assessed by a superiority comparison. Analysis was done using intention-to-treat (ITT) principles. Conclusion Most physicians manage shock with protocols. Hemodynamic endpoints are preferred to tissue perfusion targets. Early antimicrobial therapy and de-escalation are routine practices without use of infective biomarkers. Crystalloid is preferred rather than colloid at initial resuscitation. CVP and fl uid challenge are still more popular than new fl uid responsiveness methods on preload assessment. Hydrocortisone is the most common steroid prescription in septic shock but the threshold of initiation, frequency and discontinuation are varied. Results In total, 139 patients received i.v. paracetamol and 150 received i.m. diclofenac. The mean age was 35.5 ± 14.24 and 36.4 ± 14.98 years in the i.m. P80 Survey of severe sepsis and septic shock management in Thailand: THAI-SHOCK SURVEY 2013 K Chittawatanarat1, B Patjanasoontorn2, S Rungruanghiranya3, Thai SCCM Study Group4 1Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 2Khon Khaen University, Khon Khaen, Thailand; 3Srinakharinwirot University, Nakornnayok, Thailand; 4Thai Society of Critical Care Medicine, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) Critical Care 2014, 18(Suppl 1):P80 (doi: 10.1186/cc13270) Introduction A pragmatic survey of shock management in Thai physicians is unavailable. The objective of this study is to identify shock management patterns for severe sepsis and septic shock in Thailand. Methods Two thousand questionnaires were sent to physicians involved in caring for shock patients across Thailand. The frequency scale was defi ned as fi ve levels by patient proportion estimation at routine practice. Introduction A pragmatic survey of shock management in Thai physicians is unavailable. The objective of this study is to identify shock management patterns for severe sepsis and septic shock in Thailand. Methods Two thousand questionnaires were sent to physicians involved in caring for shock patients across Thailand. The frequency scale was defi ned as fi ve levels by patient proportion estimation at routine practice. g p p p Methods Two thousand questionnaires were sent to physicians involved in caring for shock patients across Thailand. The frequency scale was defi ned as fi ve levels by patient proportion estimation at routine practice. Conclusion Performing noncontrast abdominal MDCT to the patient with renal insuffi ciency for evaluating acute abdominal symptoms, severe sepsis or septic shock in the emergency department was feasible and eff ective. Results Between April and August 2013, a total of 533 questionnaires (26.7%) were returned. For severe sepsis and septic shock management, a total of 406 physicians (76.2%) reported their routine usage of quantitative resuscitation protocols. Urine output, mean arterial pressure and central venous pressure are more frequently used than central venous oxygen saturation and lactate as the resuscitation endpoint. Nearly 80% of these had an ‘often and always’ resuscitation goal within 6 hours. Most physicians (65.3%) never used procalcitonin. The antimicrobial empirical treatments were started within 1 hour for 87.7% and these were continued less than 5 days in 67.3% before de- escalation. Crystalloid was the common initial fl uid therapy in 98.9%. The most common used vasopressor was norepinephrine (69.6%). The median of cortisol threshold level for steroid replacement therapy was 15 (interquartile range, 5 to 19) mg/dl. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ease of administration, i.m. diclofenac can be used as a fi rst-choice antipyretic in febrile adults in the ED. References ease of administration, i.m. diclofenac can be used as a fi rst-choice antipyretic in febrile adults in the ED. References symptoms or searching for an infection focus such as sepsis. But contrast dye can cause renal damage especially in critically ill patients. Noncontrast CT can be alternative options but few studies have shown the eff ectiveness of noncontrast MDCT. We want to evaluate the eff ectiveness of noncontrast abdominal MDCT in the emergency department. symptoms or searching for an infection focus such as sepsis. But contrast dye can cause renal damage especially in critically ill patients. Noncontrast CT can be alternative options but few studies have shown the eff ectiveness of noncontrast MDCT. We want to evaluate the eff ectiveness of noncontrast abdominal MDCT in the emergency department. 1. Karbasi SA, et al.: Comparison of antipyretic eff ectiveness of equal dose of rectal and oral acetamenophen in children. J Pediatr (Rio J) 2010, 86:228-233.fi Methods This is a retrospective chart review study conducted in a single tertiary academic hospital from January 2011 to April 2012. Patients were enrolled if they visited the emergency department with acute abdominal symptoms or infection signs, had elevated serum creatinine level, and received noncontrast 16-channel MDCT.fi 2. Pernerstofer et al.: Acetaminophen has a greaterantipyretic effi cancy thanaspirin in endotoxemia. A randomised double blind placebo controlled trail. Clin Pharmacol Ther 1999, 66:51-57. Results During study period, 78 patients with renal insuffi ciency received noncontrast abdominal CT. Causes of CT were infection focus (48, 61.5%), abdominal pain (21, 26.9%), GI bleeding (4, 5.2%), abnormal labs (3, 3.9%), ureter stone (1, 1.3%) and abdominal distension evaluation (1, 1.3%). Any abnormal CT fi ndings were detected in 61 (78.2%) patients. For 42 (53.8%) patients, noncontrast CT fi ndings showed diagnostic abnormal fi ndings. For excluding abdominal pathology, 35 (44.9%) were helpful. In one (1.6%) case with hematochezia, noncontrast CT showed no benefi t for patient diagnosis. Additional contrast-enhanced CTs were performed in 32 (41%) and additional fi ndings were found in fi ve cases (6.4%). There were 40 patients with severe sepsis or septic shock. Abnormal CT fi ndings were found in 30 (75%) cases. Additional contrast enhanced CT was done for 13 (32.5%) patients but no additional information was detected. Eff ectiveness of noncontrast abdominal multidetector CT for evaluating the patient with renal insuffi ciency in the emergency department Results In a population of approximately 5.3 million, the annual incidence of complex burn injury is 499 to 537 (9 to 10 per 100,000). The incidence of a major burn is 5% of burn admissions. The hospital mortality from a burn is 1 to 2.2%. See Table 1. HJ Lee, E Kang, JH Shin d HJ Lee, E Kang, JH Shin Conclusion From these data, Scotland now has comprehensive national fi gures for complex burn injury. This allows for benchmarking against other international indices, few of which provide comprehensive data. COBIS data can now also be correlated with other mortality data Introduction Contrast-enhanced abdominal multidetector CT (MDCT) is an important and accurate diagnostic approach for acute abdominal S28 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 shown that only 20 to 35% of patients trigger the clinical EWS prior to cardiac arrest. Jarvis and colleagues proposed that an EWS based on common laboratory fi ndings can predict patient mortality [3]. The aim of this study, as part of a wider review of cardiac arrests in our hospital, was to determine whether the laboratory early warning score (LEWS) might be of use identifying patients at risk of cardiac arrest in our trust. Methods Retrospective data collected identifi ed cardiac arrest calls that lead to CPR or defi brillation over 6 months. The LEWS was calculated according to the formula devised by Jarvis and colleagues [3]. LEWS ≥4 for males and ≥5 for females was taken as being a ‘trigger’ as suggested by Jarvis and colleagues [3]. References 1. Hillman K, et al.; MERIT study investigators. Introduction of the medical emergency team (MET) system: a cluster-randomised controlled trial. Lancet 2005, 365:2091-2097. 1. Hillman K, et al.; MERIT study investigators. Introduction of the medical emergency team (MET) system: a cluster-randomised controlled trial. Lancet 2005, 365:2091-2097. 2. Lee A, et al.: The Medical Emergency Team. Anaesth Intens Care 1995, 23:183-186. 2. Lee A, et al.: The Medical Emergency Team. Anaesth Intens Care 1995, 23:183-186. Results The mean age was 64 ± 19.8 years and 48% (n = 513) were male. There were 513 (48.9%) early calls and 537 (51.1%) late calls. The main reasons for RRT activation were respiratory failure in 18.2% (n = 191) and severe sepsis/septic shock in 13.1% (n = 138). The distribution of RRT activation was uniform over the 24-hour period, with 50.5% (n = 531) of calls during the day (7:00 a.m. through 7:00 p.m.) and 49.5% (n = 520) overnight (7:00 p.m. through 7:00 a.m.). A total of 460 patients (43.7%) were admitted to the ICU. The multivariate analysis showed the following variables as signifi cantly associated with hospital mortality: age (OR 1.03; 95% CI 1.01 to 1.04), late (>48 hours) RRT call (OR 2.73; 95% CI 1.79 to 4.71), acute change in oximetry saturation to <90% (OR 1.94; 95% CI 1.28 to 2.95) and acute change in respiratory rate to <8 or >28 breaths/minute (OR 1.79 95% CI 1.09 to 2.94). Hospital mortality predictive factors following Rapid Response Team activation H Palomba, F Piza, M Jaures, A Capone Hospital Israelita Albert Einstein, São Paulo, Brazil p Critical Care 2014, 18(Suppl 1):P82 (doi: 10.1186/cc13272) Introduction The Rapid Response Team (RRT) represents an important advance in the management of deteriorating ward patients and is recommended as a patient safety measure. Most studies on RRT evaluate the eff ects of its implementation on rate reduction of cardiopulmonary arrest outside the ICU and hospital mortality, with limited information on the criteria for RRT calls and predictive factors associated with hospital mortality [1,2]. Therefore, our objective was to determine what factors are associated with hospital mortality for patients seen by the RRT at the Hospital Israelita Albert Einstein (HIAE). Methods A total of 1,051 patients assessed by RRT between January and December 2012 at the HIAE, a general hospital with 650 beds, were included in this study. Multivariate analysis was used to evaluate what variables were associated with hospital mortality. Early RRT call was defi ned as RRT activation <48 hours from hospital admission and late RRT call if it happened >48 hours from hospital admission. Conclusion ERT system implementation was associated with pro gressive reduction in cardiac arrests over the 3-year period, especially statistically signifi cant in diff erence in the fourth year after implementation. We also found an inverse association between number of ERT use and the risk of occurrence of cardiac arrests, but we found no diff erence in overall hospital mortality rate. P82 Results Of a total 623 ERT cases from June 2009 until December 2012, there were 72 calls in 2009, 196 calls in 2010, 139 calls in 2011 and 245 calls in 2012.The number of ERT calls per 1,000 admissions in year 2009 to 2010 was 7.69, 5.61 in 2011 and 9.38 in 2013. The number of Code Blue calls per 1,000 admissions decreased signifi cantly from 2.28 to 0.99 per 1,000 admissions (P <0.001). The incidence of cardiac arrest decreased progressively from 1.19 to 0.34 per 1,000 admissions and was signifi cant in diff erence in year 2012 (P <0.001). The overall hospital mortality rate decreased by 8% from 15.43 to 14.43 per 1,000 admissions (P = 0.095). P80 diclofenac and i.v. paracetamol groups respectively. The majority of the patients were males in both groups. After 90 minutes both groups showed a signifi cant reduction in mean temperature, i.m. diclofenac showing a greater reduction (–1.44 ± 0.43(95% CI –1.4, –2.5)) than i.v. paracetamol (–1.35 ± 0.46 (95% CI –1.3, –3.1) P <0.0001). After 120 minutes, a signifi cant diff erence was observed in the mean change from the baseline temperature between the groups, –1.81 ± 0.46 (95% CI –1.7, –2.9) in i.m. diclofenac versus –1.63 ± 0.55 (95% CI –1.5, –4.1) in the i.v. paracetamol group (P <0.0001). Signifi cant changes in temperature were observed in favor of i.m. diclofenac over i.v. paracetamol at each time point from 60 minutes through 120 minutes inclusive.i P81 Laboratory early warning score versus clinical early warning score as a predictor of imminent cardiac arrest M Keil, S Hutchinson, T Leary Norfolk and Norwich University Hospital NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P81 (doi: 10.1186/cc13271) Introduction NCEPOD reported in 2012 that 75% of patients had warning signs for cardiac arrest present prior to their arrest [1]. NICE recommends a vital sign-based early warning score (EWS) to identify patients at risk of deterioration or death [2]. In our trust, audit has Conclusion Both i.m. diclofenac and i.v. paracetamol showed signifi cant antipyretic activity, with diclofenac having a greater eff ect. In view of S29 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P83 Long-term outcome of the Emergency Response Team system in in-hospital cardiac arrest P83 Long-term outcome of the Emergency Response Team system in in-hospital cardiac arrest J Suriyachaisawat, E Surakarn Bangkok Hospital Group, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P83 (doi: 10.1186/cc13273) y g Results Eighty-nine cardiac arrest calls lead to CPR and/or defi brillation in this time period. Of these, 65 patients had had blood tests within the last 24 hours. Median LEWS was 6 for females and 7 for males (range 1 to 12). Most patients (77%) had a LEWS trigger (≥4 for females and ≥5 for males). Introduction To improve early detection and the mortality rate of in- hospital cardiac arrest, the Emergency Response Team (ERT) system was planned and implemented since June 2009 to detect pre-arrest conditions and for any concerns. The ERT consisted of on-duty physicians and nurses from emergency department. ERT calling criteria [1,2] consisted of acute change of HR <40 or >130 beats per minute, systolic blood pressure <90 mmHg, respiratory rate <8 or >28 breaths per minute, O2 saturation <90%, acute change in conscious state, acute chest pain or worried about the patients. From the data on ERT system implementation in our hospital in the early phase (during June 2009 to 2011), there was no statistical signifi cance in diff erence in in-hospital cardiac arrest incidence and overall hospital mortality rate. Since the introduction of the ERT service in our hospital, we have conducted a continuous educational campaign to improve awareness in an attempt to increase use of the service. Conclusion The collected data suggest that more patients at risk of cardiac arrest in our hospital might be identifi ed using a LEWS rather than a clinical EWS. It is evident that for a clinical EWS regular observations need to be taken and are subject to user error. Clinical EWS could also not be sensitive enough or there is a failure to implement it successfully. LEWS can be generated automatically when bloods are taken. The downside is that it relies on bloods being done. It is beyond the scope of this study to examine the sensitivity/specifi city of LEWS or suggest that LEWS should replace EWS. We suggest that LEWS may compliment EWS by identifying a diff erent group of patients. The ongoing data collection aims to correlate the clinical EWS for each patient directly with their LEWS to confi rm the initial fi ndings. f 1. NCEPOD 2012 [http://www.ncepod.org.uk] 2. NICE 2007 [http://www.nice.org.uk] 3. P83 Long-term outcome of the Emergency Response Team system in in-hospital cardiac arrest Jarvis et al.: Resuscitation 2013, 84:1494-1499. 1. NCEPOD 2012 [http://www.ncepod.org.uk] Methods To investigate the outcome of the ERT system in in-hospital cardiac arrest and the overall hospital mortality rate, we conducted a prospective, controlled before and after examination of the long- term eff ect of an ERT system on the incidence of cardiac arrest. We performed chi-square analysis to fi nd statistical signifi cance. 2. NICE 2007 [http://www.nice.org.uk] 3. Jarvis et al.: Resuscitation 2013, 84:1494-1499. References References 1. Winters BD, et al.: Rapid response systems: going beyond cardiac arrest and mortality. Crit Care Med 2013, 41:911-912. 2. Leach LS, et al.: Rapid response teams: qualitative analysis of their eff ectiveness. Am J Crit Care 2013, 22:198-210. References 1. Winters BD, et al.: Rapid response systems: going beyond cardiac arrest and mortality. Crit Care Med 2013, 41:911-912. 2. Leach LS, et al.: Rapid response teams: qualitative analysis of their eff ectiveness. Am J Crit Care 2013, 22:198-210. References 1. Winters BD, et al.: Rapid response systems: going beyond cardiac arrest and mortality. Crit Care Med 2013, 41:911-912. 2 Leach LS et al : Rapid response teams: qualitative analysis of their Use of low-dose CT KUB: is it becoming the easy way out? P Shah, D Wilson Homerton University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P85 (doi: 10.1186/cc13275) Use of low-dose CT KUB: is it becoming the easy way out? P Shah, D Wilson Homerton University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P85 (doi: 10.1186/cc13275) Introduction In 1995, Smith and colleagues fi rst proposed the use of low-dose CT KUB for the diagnosis of ureteric colic [1]. Since then, popularity of this imaging modality has increased due to a number of reasons: a sensitivity of 94 to 97%, a specifi city of 96 to 99%, lack of intravenous contrast injection and speed of examination. The UK College of Emergency Medicine considers CT KUB as best practice for radiological investigation of renal colic. A review of the literature suggests that the true positive rate (number of patients diagnosed with an obstructing or symptom causing calculus) should be between 47.5 and 67% and alternative diagnoses should be confi rmed in approximately 10% of patients imaged [2]. Conclusion The results underline that the unit’s guidelines were not being followed. The re-audit does show an improvement in adherence. Patients are being exposed to unnecessary blood tests, which not only is implicated in iatrogenic anaemia, but also places a signifi cant fi nancial burden on the department. Continued staff education and encouragement are required in order to aid the transition from current to recommended practice. Decreasing central-line blood draws by consolidation of phlebotomy timing: results of a quality improvement project G Arteaga, S Tripathi, Y Ouellette, M Nemergut, G Rohlik, K Fryer, C Huskins Mayo Clinic, Rochester, MN, USA Critical Care 2014, 18(Suppl 1):P87 (doi: 10.1186/cc13277) pp y p g Methods All patients over the age of 18 years who had a CT KUB requested from the emergency department of the Homerton University Hospital, London over a period of 3 months, between May 2012 and July 2012, were identifi ed. Individual case notes were examined and data were collected on an Excel spreadsheet. Data were analysed and results were divided into positive for renal colic (true positive), positive for other pathology (other signifi cant diagnoses) and negative results. Results A similar previous audit carried out in 2009 at the same hospital demonstrated a true positive rate of 48.5%. The 2012 audit examined the outcome of 124 consecutive scans and a true positive rate of 29% (P = 0.0006) was identifi ed. Alternative diagnoses were 10% in 2009 compared with 17% in 2012 (P = 0.02). Bled dry? An audit of blood sampling practices on an adult intensive therapy unit Bled dry? An audit of blood sampling practices on an adult intensive therapy unit Methods A prospective observational evaluation of ICU unplanned admission through in-hospital ward referral over a 3-month duration. Anonymised data collection included baseline demographics, timing and grade of referral, Modifi ed Early Warning Score (MEWS) at time of referral, clinical reason for referral, senior review prior to referral, MEWS trend 24 hours prior to referral, if appropriate basic intervention commenced, time delay between referral and assessment by the ICU team, organ failure score, length of stay (LOS) and survival status. RK Vincent, P Temblett Introduction The aim of this audit was to evaluate whether guidelines produced in a local intensive therapy unit (ITU) with regard to blood sampling practices were being adhered to. The volume of blood taken and cost was also evaluated. g g y Results So far 22 patients have been enrolled, of which 60% of the unplanned admissions were ‘out of hours’, 60% of admissions were from medical wards and 33% from surgical wards. Pneumonia was the main reason for referral and admissions were due to respiratory failure requiring advanced ventilator care. Twenty per cent of the patients did not have senior medical review for at least 4 hours prior to the ICU referral. Basic interventions such as antibiotics and intravenous fl uids were not started in a few patients (13%) prior to admission. The mean ICU review time from referral was 70 minutes and the mean MEWS at the time of referral was 8. Methods A retrospective audit investigating the number of routine blood tests ordered on an ITU in January 2013 was performed. There were no exclusion criteria. Computer-based data collection systems were used to gather data with regard to patient details and when blood tests were processed. The collection bottles used were examined to see how much blood was needed to fi ll them. Thirty nurses were asked how much ‘dead space’ blood they discarded, and an average was recorded. The cost of the blood tests was also calculated. Following a period of education regarding the contents of the guidelines, this was re-audited in July 2013 retrospectively. Conclusion The study is due to be completed by mid-January 2014. With the limitation of incompleteness, the fi ndings so far have alluded that there is a necessitation for change and education for early identifi cation and intervention in deteriorating patients. P86 in order to identify the associative trends that led to the admission and hence to mitigate the processes of interventional strategies. in order to identify the associative trends that led to the admission and hence to mitigate the processes of interventional strategies. 1. Smith RC, et al.: Radiology 1995, 194:789-794. 2. Roth CS, et al.: Ann Emerg Med 1985, 14:311-31515. P84 Epidemiology of unplanned intensive care admissions through in- hospital referrals at a tertiary referral centre university hospital J Rigby, N Bradshaw, B Carr, R Matsa University Hospital of North Staff ordshire, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P84 (doi: 10.1186/cc13274) g y, , , University Hospital of North Staff ordshire, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P84 (doi: 10.1186/cc13274) Introduction The provision of intensive care has lagged behind demand [1]. Intensive care services in the UK have signifi cant resource restriction. Although rationing of beds has been a priority, equally important is to establish patient safety and also identify strategies to prevent admissions [2], which echoes the importance of early recognition of deteriorating patients and prompt intervention. This study evaluates the epidemiology of unplanned admissions to the ICU Conclusion In this study, hospital mortality predictive factors for patients seen by the RRT were: age, acute respiratory failure and late RRT call. S30 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References pf 2. McQuillan P et al.: Confi dential inquiry into quality of care before admission to intensive care. Br Med J 1998, 316:1853-1858. Bled dry? An audit of blood sampling practices on an adult intensive therapy unit The completed study with outcomes (that is, survival rates and ICU LOS) will help us identify whether delay or defi ciency in the early management had a causal relationship. Results The initial audit examined 901 patient-days. Urea and electrolytes (U&Es) and full blood counts (FBC) were requested in line with the guidelines. Liver function tests (LFTs), bone profi le, magnesium and a clotting screen were ordered approximately four times more than advocated. It was shown that many bone profi les and magnesium tests were probably inappropriate requests. Moreover, twice as much blood was taken from patients compared with that recommended by guidelines (almost 16 litres in total in January). The cost of the routine blood tests in January was €11,019. If guidelines had been followed, the estimated yearly saving would be €65,588. During the repeat audit, 731 patient-days were examined. The amount of times a U&E or FBC were requested was largely unchanged, but the amount of times a LFT, bone profi le, magnesium and clotting screen were ordered reduced by approximately 50%. Almost one-third more blood was taken from patients when compared with the suggested volume in the guidelines. The cost of the blood tests done in July was €5,423. Despite an improvement in the frequency of blood testing, an estimated €21,907 per year could still be saved. Reference 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. 1. Cartotto R, et al.: Minimizing blood loss in burn surgery. J Trauma 2000, 49:1034-1039. Results In the simulation study 10/15 clinicians (67%) injected atropine directly into arterial cannula via a three-way tap. Five of 15 doctors (34%) injected safely. In the laboratory study, swabbing of the arterial hubs showed bacterial contamination of all of the samples in the standard group (20/20) and no contamination in the NIC group (0/20), P <0.0001. Eighty-fi ve per cent (17/20) of samples in the standard arterial group showed onward transmission of pathogens to the patient circulation, and none of the patient samples taken from the NIC group were contaminated (0/20), P <0.0001. In the survey, 80% of the nurses found manual handling of a new device equally simple and 20% even simpler. Sampling of the blood was equally challenging for 87% participants. Ninety-four per cent found similar durability of the new device and better protection against accidental intra-arterial injection. Sixty-six per cent of the nursing staff would replace the standard system with a NIC. Should we avoid invasive treatment in cancer patients with pericardial tamponade? F Galas, J Fukushima, E Osawa, C Park, J Almeida, D Nagaoka, L Hajjar Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P90 (doi: 10.1186/cc13280) Introduction Patients with cancer now live longer due to advances in oncologic treatment. ICU admission is progressively more frequent in this population due to complications of both disease and therapy. Pericardial eff usion leading to tamponade and hemodynamic compromise is a common fi nding in the advanced stages of disease and results in death if not treated on an urgent basis. However, we do not know midterm outcomes of these patients after pericardial drainage. The aim of this study was to evaluate prospectively cancer patients with pericardial tamponade regarding mortality in 6 months. y Conclusion Most of the doctors would inject the drug intra-arterially. Currently, no other device is available to eliminate accidental intra- arterial injection or bacterial transmission. The nursing staff found the management of the NIC equally challenging. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 bleeding remains a challenge in tangential excision of the burn wound. Although various techniques to reduce intraoperative blood loss have been described, there is an absence of uniformity and consistency in their application. In the literature, the blood loss in burn surgery is estimated to be at least 123 ± 106 ml per percentage body surface area excised [1]. Recently we developed a novel hemostatic technique using a silicone gel dressing (SI-AID®; ALCARE Co., Ltd, Tokyo, Japan) to stop intraoperative bleeding. Briefl y, soon after tangential excision with the Humby knife, the wounds were sprayed with thrombin and with 1:100,000 adrenalin solution and wrapped tightly with SI-AID® for a full 10 minutes. Burn wounds on limbs were tangentially excised under tourniquet control and wrapped with SI-AID® before defl ation of the tourniquet. After defl ation of the tourniquets, we waited for a full 10 minutes. When the SI-AID® was removed, any major bleeders were cauterized, and the grafts were applied after rinsing the wounds with warm saline. 12.5% were by peripheral venipuncture. After policy implementation, these numbers were 10.9%, 49.7%, and 23.8%, respectively. The average central line unique entry after blood draw consolidation decreased from 10 to 6 line entries/central line-day. Consolidation of blood draws was associated with a cost saving of $7,200/year. Conclusion Consolidating time frames for blood draws in the PICU was associated with decreased central line entries, decreased utilization of vascular access teams, and decreased phlebotomy cost. We hypothesize that this policy will be associated with a decreased incidence of CLABSI when more patients are included for analysis. P89 Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery A Osuka, Y Kuroki, H Kojima, M Sekido, S Okuma, S Onishi, M Ueyama Social Insurance Chukyo Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) P89 Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery A Osuka, Y Kuroki, H Kojima, M Sekido, S Okuma, S Onishi, M Ueyama Social Insurance Chukyo Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) g Reference 1. Sen S, et al.: Complications after unintentional intraarterial injection of drugs: risks, outcomes, and management strategies. Mayo Clin Proc 2005, 80:783-795. Results Fifty-three patients (50%) were female. Most patients had a previous diagnosis of lung neoplasia (46%), followed by breast neoplasia (15%) and hematological neoplasia (15%). The mean Karnofsky performance status of patients was 70. All these patients underwent surgical drainage in a mean time of 1.5 hours since ICU admission until surgery. Length of ICU stay was 8 days (2 to 15) and of hospital stay was 16 days (8 to 31). ICU mortality was 75.2%, hospital mortality was 83% and at 3 months 92% of patients were dead. Reference Methods We evaluated consecutively 105 patients with cardiac tamponade consecutively admitted to the ICU of the Cancer Institute, a reference cancer hospital, during a 3-year period. Baseline characteristics and clinical data were collected prospectively. Patients were followed during a 6-month period. Introducing an arterial non-injectable connector into clinical practice Methods This is a prospective observational study. From 1 January to 31 October 2013 we collected preoperative and 24-hour postoperative hemoglobin levels from the patients who underwent tangential excision for burn injury, and calculated blood loss in the perioperative period. The data for amounts of blood transfusion, excised area, and harvest area were also collected. p g y Critical Care 2014, 18(Suppl 1):P88 (doi: 10.1186/cc13278) Introduction The standard arterial system for blood withdrawal provides no impediment to intra-arterial injection [1] and bacterial contamination. We assessed the probability of inadvertent intra-arterial injection, transmission of bacterial contamination and surveyed the nursing staff following introduction of a non-injectable connector (NIC). Results Nine patients, 13 operations were included. The mean excised area was 8.3 ± 4.6% body surface area. Estimated blood loss was 35.3 ± 38.3 ml per percentage body surface area excised. Intraoperative transfusion requirements were 86.2 ± 134.5 ml per case. The mean skin graft take rate was 4.5 ± 2.2%. Methods The simulation study data were descriptive. Fifteen junior doctors managed a case of bradycardia. A simulated patient had a peripheral intravenous cannula, central venous catheter and brachial arterial cannula. Atropine was available on request. A laboratory controlled trial compared a transmission of bacteria through standard arterial ports against the NIC when attached to an arterial sampling hub. The colonization rates were compared using a two-tailed Fisher’s exact test. A closed-circuit arterial sampling system was designed. A contaminated syringe tip was inserted into the conventional arterial hub or the NIC to take an arterial sample. Transducer fl ush fl uid fl owing to the patient bloodstream was cultured. In a survey the nursing staff were asked questions regarding the NIC system, including its manual handling, sampling of the blood and its durability. Conclusion The application of our new technique during burn excision and grafting resulted in a remarkable reduction in blood loss and transfusion requirements. Reference Use of low-dose CT KUB: is it becoming the easy way out? P Shah, D Wilson Homerton University Hospital, London, UK Critical Care 2014, 18(Suppl 1):P85 (doi: 10.1186/cc13275) Introduction The direct central line entry rate is believed to be a major contributor to the risk of central line infections. At Mayo Clinic there was historically no schedule for obtaining blood for analysis in the pediatric ICU. A policy was implemented in May 2013 to restrict blood draws to three times daily for nonemergent blood draws only. We subsequently conducted this study to determine whether implementation of this policy was associated with a reduction of blood draws as well as central-line unique entries. Methods Data from the laboratory as well as database for Central Line Unique Entry were analyzed at baseline and after implementation of the policy change for identifi cation of any decrease in line entry/blood draw rate. As per Mayo Clinic policy, IRB approval was not required for a QI project. Conclusion The true positive rate of CT KUB for renal colic has decreased signifi cantly. We are getting more negative scan results and more scans diagnosing other signifi cant pathology. The authors believe clinicians’ thresholds for imaging may have decreased due to the apparent low- dose radiation of CT KUBs. Furthermore, there is a perceived ease of access to CT KUB imaging and hence this modality appears to be being used to identify other signifi cant pathology. R f Results In the pre-implementation phase there were a total of 4,602 blood draws in 5,227 total patient-days, (0.88 blood draws/patient- days). After consolidation, there were 1,095 blood draws in 1,491 patient-days (0.73 blood draws/patient-day; 17% reduction). Of these line entries, 24.7% were arterial line entry, 50.5% central line entry and S31 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ROTEM: Multiplate monitoring in the ICU and outcome scores U Schött, A Larsson Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P93 (doi: 10.1186/cc13283) Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P93 (doi: 10.1186/cc13283) q p pp p Results Preoperatively, the HB group (n = 31) was characterized by lower blood fi brinogen and decreased clot amplitude at ROTEM compared with the LB group (n = 40). Intraoperatively, larger amounts of fi brinogen, fresh frozen plasma and platelets were deemed necessary to normalize the coagulation parameters in the HB group. Postoperatively, the incidence of major thromboembolic and ischemic events did not diff er between the two groups (<10%) and the observed in-hospital mortality was signifi cantly less than expected by the POSSUM score (22% vs. 35% in HB group and 5% vs. 13% in LB group). Conclusion ROTEM-derived information is helpful to detect early coagulation abnormalities and to monitor the response to hemostatic therapy. Early goal-directed management of coagulopathy may contribute to improve outcome after cardiovascular surgery. R f Introduction Hemostatic disorders are common in intensive care patients and can be used as prognostic markers. The aim of this prospective clinical study was to study platelet function in intensive care patients using point-of-care viscoelastic and platelet aggregometry instruments and platelet count (PLC). Our hypothesis was that measurement of platelet function would give more information about disseminated intravascular coagulation (DIC), morbidity and mortality than PLC alone. Methods Patients admitted to the ICU were monitored with ROTEM viscoelasticity and a new Multiplate platelet aggregometer; routine coagulation analyses; International Society of Thrombosis and Haemostasis and Japanese Association for Acute Medicine DIC calculated scores; Sequential Organ Failure Assessment scores, Simplifi ed Acute Physiology Score III – Expected Mortality Rate; and real in-hospital mortality. Nonparametric tests were chosen for all statistical evaluation. P <0.05 was considered signifi cant. 1. Ranucci M, et al.: The deadly triad of cardiac surgery. Ann Thorac Surg 2013, 96:478-485. Results A total of 128 patients with diff erent diagnoses were studied, with 330 sampling events. Multiplate analyses correlated signifi cantly with PLC and ROTEM. However, there were more test results below normal limits for Multiplate analyses than for ROTEM in patients with thrombocytopenia. Multiplate, ROTEM and PLC results were low in patients with high SOFA scores and in patients with overt DIC scores. These test results at admission to the ICU did not diff er between survivors and nonsurvivors, and did not correlate with the length of stay in the ICU. Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery y A Osuka, Y Kuroki, H Kojima, M Sekido, S Okuma, S Onishi, M Ueyama Social Insurance Chukyo Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) Conclusion Cardiac tamponade is a serious complication in cancer patients. Despite adequate treatment, high rates of mortality are observed. Prospective studies are needed to better defi ne whether in these patients end-of-life discussion should be implemented early in the diagnosis, avoiding futility. y p , g y , p Critical Care 2014, 18(Suppl 1):P89 (doi: 10.1186/cc13279) Introduction The purpose of this study is to demonstrate the effi cacy of our novel hemostatic technique for burn surgery. Signifi cant Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S32 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P91 Goal-directed hemostatic therapy using rotational thromboelastometry in patients requiring emergent cardiovascular surgery D Sartorius, JL Waeber, G Pavlovic, M Aldenkortt, MJ Licker Hôpital Cantonal Universitaire de Genève, Geneva, Switzerland Critical Care 2014, 18(Suppl 1):P91 (doi: 10.1186/cc13281) 1.2. The average measured value of INR in men was 1.50, and in women was 1.35. APTT normal values were 0.8 to 1.2. The average measured value of APTT in men was 1.09, and in women was 1.14. Platelet count was 150×109/l to 300×109/l level of platelets; in men was 252.57, in women was 226.78. ExTEM CT normal values at 41 to 74 seconds: measured value of CT in men was 62.58 and in women was 62.07. MCF normal values were 50 to 72 mm; the measured value of MCF in men was 69.88, and in women was 73.28. Considered administration of blood derivatives: fresh frozen plasma, 123 transfusion units (TU); platelets, 4 TU. In the examined cases no blood products were administered to avoid the risk of bleeding before the elective procedure. No periprocedural bleeding was observed. Introduction Massive bleeding remains a leading cause of potentially preventable death after cardiovascular surgery [1]. Conventional coagulation tests (CCT) fail to characterize the multiple hemostatic abnormalities observed in surgical patients and are further limited by their slow results and poor correlation with transfusion requirements. We assessed the clinical impact of goal-directed coagulation manage- ment based on rotational thromboelastometry (ROTEM) in patients undergoing an emergent cardiovascular surgical procedure. ROTEM: Multiplate monitoring in the ICU and outcome scores U Schött, A Larsson Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P93 (doi: 10.1186/cc13283) ROTEM: Multiplate monitoring in the ICU and outcome scores U Schött, A Larsson Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P93 (doi: 10.1186/cc13283) ROTEM: Multiplate monitoring in the ICU and outcome scores U Schött, A Larsson Only EMR diff ered between survivors and nonsurvivors and correlated with ICU length of stay. P92 P92 Thromboelastometric examination on the ICU before elective procedures P Lukas University Hospital Motol, Prague, Czech Republic Critical Care 2014, 18(Suppl 1):P92 (doi: 10.1186/cc13282) Thromboelastometric examination on the ICU before elective procedures P Lukas University Hospital Motol, Prague, Czech Republic Critical Care 2014, 18(Suppl 1):P92 (doi: 10.1186/cc13282) Introduction In critically ill patients a number of elective procedures with a potential risk of bleeding are performed. In this population, coagulation disorder is frequently observed according to the commonly used laboratory parameters of APTT (activated partial thromboplastin time), INR (international normalized ratio) or platelet count. Thromboelastometric examination (ROTEM) evaluates coagulation of whole blood and thus allows the testing of all components of secondary hemostasis. Conclusion In this study, Multiplate and ROTEM did not provide any more information than PLC about DIC, morbidity and mortality. Multiplate showed lower platelet function in more patients with low platelets than ROTEM. In patients without thrombocytopenia, all patients had normal ROTEM results, but 36% of Multiplate measurements were low. This higher sensitivity of the Multiplate to measure lowered platelet function needs to be linked to real bleeding in larger patient series to defi ne its clinical signifi cance. y Methods Identifi cation of patients with pathological values of INR, APTT and platelet count before the planned intervention. Through a questionnaire we found a potential correction of coagulopathy carried out by the physician. Performed ROTEM methodology: ExTEM (external pathway thromboelastometry). Inclusion criteria: normal curves and values, CT (clotting time), MCF (maximum clot fi rmness). In those patients with normal values according to ExTEM examination, no blood products were administered. Novel hemostatic technique using a silicone gel dressing for tangential excision in burn surgery p p p g Conclusion Examination EXTEM in these patients proved to be eff ective and effi cient in predicting bleeding complications in relation with interventions routinely performed on the ICU. Also, these specifi c cases proved not indicated and thus ineff ective administration of blood products. Methods Over a 2-year period, data from 71 patients were collected prospectively and blood samples were obtained for coagulation testing. Administration of packed red blood cells (PRBC) and hemostatic products was guided by an algorithm using ROTEM- derived information and hemoglobin level. Based on the amount of PRBC transfused, two groups were considered: high bleeders (≥5 PRBC; HB) and low bleeders (<5 PRBC; LB). Data were analyzed using the chi- square test, unpaired t test and ANOVA as appropriate. References References 1. Chee YL, et al.: Br J Haematol 2008, 140:496-504. 2. BCSH Blood Transfusion Task Force: Br J Haematol 2003, 122:10-23. References 1. Chee YL, et al.: Br J Haematol 2008, 140:496-504. 2. BCSH Blood Transfusion Task Force: Br J Haematol 2003, 122:10-23. Methods We prepared both types of bag: parenteral nutrition bags whose composition was defi ned in the unit, including sodium heparin (77 UI/ml); and bags containing only sodium heparin diluted in 50% dextrose (193 UI/ml). These bags (n = 6 per type) were infused over a period of 24 hours with and without in-line fi ltration. Heparin activity was measured using a chromogenic anti-Xa method in bags just being prepared (references for other measures) and after 24-hour infusion and in effl uents at the end of infusion line after 24 hours. P96 P96 Heparin stability in parenteral nutrition bags prepared in a neonatal ICU A Foinard1, M Perez1, B Décaudin2, C Barthélémy1, A Tournoys3, L Storme4, P Odou2 1Faculté de Pharmacie, Lille, France; 2Institut de Pharmacie, CHRU, Lille, France; 3Fédération de Biologie Pathologie du CHRU, Lille, France; 4Hôpital Jeanne de Flandre, CHRU, Lille, France Critical Care 2014, 18(Suppl 1):P96 (doi: 10.1186/cc13286) y Conclusion This study demonstrates inconsistent use of FFP, with no signifi cant diff erence in PT between patients who were transfused and those that were not. The lack of eff ect of FFP transfusion on PT and APTT creates additional confusion for its prophylactic usage. There is a need for further clarifi cation around coagulopathy and interventional radiology in the critical care setting. The low absolute incidence of bleeding complications and risk of complications from transfusion lends further support to the view that FFP should be used therapeutically rather than as prophylactic ‘cover’ [1]. Introduction Heparin is commonly given in our neonatal ICU (NICU) by continuous intravenous infusion. Heparin is diluted in parenteral nutrition bags and administered over a period of 24 hours with in-line fi ltration. However, there are no data on heparin stability in parenteral nutrition bags, especially on its compatibility with 50% dextrose mainly present in bags. The aim of our in vitro study was to determine heparin stability in parenteral nutrition bags prepared in a NICU after 24-hour infusion and to assess its interaction or not with 50% dextrose. P94 Retrospective observational study of interventional radiology and critical care coagulopathy SP Hibbs, S McKechnie, M Little, M Desborough John Radcliff e Hospital, Oxford, UK Critical Care 2014, 18(Suppl 1):P94 (doi: 10.1186/cc13284) Results During March 2013 to November 2013, 40 patients were identifi ed as relevant, 26 men of average age 53 years and 14 women of average age 59 years. Central venous cannulation, 12 cases; surgical revision, nine cases; thoracic drainage, six cases; percutaneous endoscopic gastrostomy, fi ve cases; nephrostomy, two cases; epidural catheter, two cases; tracheostomy, two cases; permanent pacemaker, one case; and bronchoscopy, one case. INR normal values were 0.8 to Introduction Estimation of bleeding risk in critical care patients undergoing interventional radiological procedures is often made on the basis of coagulation tests. If these tests are abnormal, fresh frozen plasma (FFP) is often given to reduce the risk of bleeding, despite Introduction Estimation of bleeding risk in critical care patients undergoing interventional radiological procedures is often made on the basis of coagulation tests. If these tests are abnormal, fresh frozen plasma (FFP) is often given to reduce the risk of bleeding, despite S33 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Clot initiation was prolonged with both drugs and detected by both ROTEM and ReoRox. Clot formation was more decreased with tinzaparin than enozaparin and only detected by ReoRox. Results Patients who received FFP had a mean PT of 18.5 seconds while those who did not receive FFP had a mean PT of 16.7 seconds (unpaired t test, P = 0.275). In the nine patients given FFP, the pre- transfusion mean PT was 18.5 seconds whereas the post-transfusion mean PT was 17.1 seconds (paired t test, P = 0.235). The pre-transfusion mean APTT was 41.6 seconds compared with a post-transfusion mean APTT of 38.1 seconds (paired t test, P = 0.127). No patient had platelet levels below the recommended transfusion threshold [2], but one patient nevertheless received a double-dose platelet transfusion. One patient had a recorded immediate bleeding complication. Their PT was 15.6 seconds and APTT was 40.9 seconds and they did not receive FFP. One patient had an anaphylactic reaction whilst receiving FFP. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods Enoxaparin (Klexane) and tinzaparin (Innohep) were added to 2 ml citrated blood from 10 intensive care patients to obtain plasma concentrations of 0, 0.5, 1.0 and 1.5 IU/ml enoxaparin and tinzaparin, respectively. The study was approved by the local ethics committee and with written consent (relatives). Clot formation and clot retraction was studied using ROTEM and ReoRox. a poor evidence base for this practice [1]. There is a relatively better evidence base for prophylactic platelet transfusion [2] but clinical practice is inconsistent. Through a retrospective study we aimed to establish the thresholds triggering use of FFP and platelet transfusion prior to percutaneous drain insertion in critical care patients.i Methods Enoxaparin (Klexane) and tinzaparin (Innohep) were added to 2 ml citrated blood from 10 intensive care patients to obtain plasma concentrations of 0, 0.5, 1.0 and 1.5 IU/ml enoxaparin and tinzaparin, respectively. The study was approved by the local ethics committee and with written consent (relatives). Clot formation and clot retraction was studied using ROTEM and ReoRox. Results ROTEM analysis showed prolonged clot formation (CT) with increasing concentrations of enoxaparin and tinzaparin (more so). ReoRox analysis showed that the initiation of clot formation (COT1) increased with increasing doses of enoxaparin and tinzaparin (more so), as did the progression of clot formation (COT2 – COT1), thus resulting in a prolongation until complete clot formation (COT2). See Table 1. Conclusion Clot initiation was prolonged with both drugs and detected by both ROTEM and ReoRox. Clot formation was more decreased with tinzaparin than enozaparin and only detected by ReoRox. Methods We identifi ed 68 consecutive chest, abdominal or pelvic drain insertions in 54 critical care patients between 1 January 2008 and 11 October 2012 at the John Radcliff e Hospital, Oxford. The prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet counts prior to each procedure were recorded to demonstrate triggers used for FFP and platelet transfusion. In patients who underwent transfusion, the next PT, APTT and platelet count post transfusion were recorded. Results ROTEM analysis showed prolonged clot formation (CT) with increasing concentrations of enoxaparin and tinzaparin (more so). ReoRox analysis showed that the initiation of clot formation (COT1) increased with increasing doses of enoxaparin and tinzaparin (more so), as did the progression of clot formation (COT2 – COT1), thus resulting in a prolongation until complete clot formation (COT2). See Table 1. Data presented as median (SD). P <0.05 compared with 0 IU/ml LMWH. P95 Conclusion We conclude that there is no loss of heparin activity when drug is infused over 24 hours for both types of bag prepared, with and without in-line fi ltration, showing heparin activity remains stable during this period and there is no interaction of drug with other nutrient components of bags, especially 50% dextrose. y y Methods Rabbits were treated with a high intravenous bolus dose of edoxaban (1,200 μg/kg) followed by the administration of Beriplex® (25 to 75 IU/kg). Bleeding was assessed based on the time to hemostasis and the total blood loss after induction of a standardized kidney injury. In parallel, the following biomarkers of hemostasis were determined: factor Xa inhibition, prothrombin time (PT), activated partial thrombo- plastin time (aPTT), whole blood clotting time (WBCT), and thrombin generation (TGA). Results The results confi rmed increased and prolonged bleeding of edoxaban-treated animals following standardized kidney injury compared with vehicle administration. Parallel monitoring of biomarkers of hemostasis showed a prolongation of PT, aPTT, WBCT, and changes in thrombin generation parameters. Subsequent administration of Beriplex® resulted in a dose-dependent reversal of edoxaban-induced bleeding as indicated by reduced time to hemostasis and total blood loss. Both parameters achieved statistical signifi cance compared with placebo at the Beriplex® dose of 50 IU/kg under fully blinded study conditions. The biomarkers correlating best with Beriplex®-mediated edoxaban anticoagulation reversal included PT, WBCT and endogenous thrombin potential.f P97 Bivalirudin or heparin: which anticoagulation strategy for critically ill cardiac surgery patients? F Pappalardo1, B Arnaez1, M Celinska-Spodar1, M Pieri1, F Isella1, S Silvetti1, A Montisci1, O Saleh1, A Koster2 1San Raff aele Scientifi c Institute, Milan, Italy; 2Heart and Diabetes Centre, North Rhine-Westphalia Ruhr-University Bochum, Bad Oeyenhausen, Germany Critical Care 2014, 18(Suppl 1):P97 (doi: 10.1186/cc13287) Introduction Anticoagulation with unfractionated heparin in cardiac surgery patients has several limitations, and above all the risk of heparin-induced thrombocytopenia. Bivalirudin is a direct thrombin inhibitor, use of which in cardiac surgery patients has expanded in recent years. The aim of the study was to analyze two strategies for introducing bivalirudin in this setting (as secondary drug switching from heparin or as primary anticoagulant) and to evaluate clinical outcomes. Conclusion In summary, Beriplex® treatment eff ectively reversed edoxaban-induced anticoagulation in an animal model of acute bleeding at clinically relevant dose levels. Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs g p g M Honickel1, O Grottke1, J Van Ryn2, H Ten Cate3, R Rossaint1, H Spronk3 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany; 3Maastricht University, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P99 (doi: 10.1186/cc13289) g g M Honickel1, O Grottke1, J Van Ryn2, H Ten Cate3, R Rossaint1, H Spronk3 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany; 3Maastricht University, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P99 (doi: 10.1186/cc13289) y p p Results Bivalirudin treatment was associated with a reduction of major bleeding (P = 0.05) compared with the control group. Interestingly, in an intention-to-treat analysis, patients receiving primary bivalirudin showed a signifi cant reduction in minor bleeding (P = 0.04), and mortality (P = 0.01) compared with the secondary bivalirudin group, and similarly if compared with UFH and secondary bivalirudin patients (P = 0.01 and P = 0.05 respectively). Predictors of hospital mortality at multivariate analysis included urgent admission (OR = 2.7; 95% CI, 1.03 to 7.2; P = 0.04), septic shock (OR = 8.0; 95% CI, 2.26 to 28.7; P <0.005) and primary therapy with UFH (OR = 19.2; 95% CI, 2.2 to 163.9; P = 0.007). Conclusion Novel anticoagulant strategies might play a crucial role in critically ill cardiac surgery patients. In a propensity-matched population, our study showed that primary bivalirudin anticoagulation may reduce bleeding complications and mortality. Further studies are therefore warranted. Results Bivalirudin treatment was associated with a reduction of major bleeding (P = 0.05) compared with the control group. Interestingly, in an intention-to-treat analysis, patients receiving primary bivalirudin showed a signifi cant reduction in minor bleeding (P = 0.04), and mortality (P = 0.01) compared with the secondary bivalirudin group, and similarly if compared with UFH and secondary bivalirudin patients (P = 0.01 and P = 0.05 respectively). Predictors of hospital mortality at multivariate analysis included urgent admission (OR = 2.7; 95% CI, 1.03 to 7.2; P = 0.04), septic shock (OR = 8.0; 95% CI, 2.26 to 28.7; P <0.005) and primary therapy with UFH (OR = 19.2; 95% CI, 2.2 to 163.9; P = 0.007). Introduction The reversal of the anticoagulant eff ects of the new oral anticoagulants in severely bleeding patients requires new therapeutic strategies. This study investigated the eff ectiveness of a specifi c antidote for idarucizumab to reverse bleeding in a dabigatran anticoagulated pig trauma model. P95 P95 Monitoring of treatment with low molecular weight heparins using viscoelastic devices U Schött1, A Larsson1, O Thomas1, N Tynngård2 1Lund University, Lund, Sweden; 2Linköping University, Linköping, Sweden Critical Care 2014, 18(Suppl 1):P95 (doi: 10.1186/cc13285) Introduction Few studies have investigated the use of viscoelastic devices for monitoring of treatment with LMWHs and to our knowledge there are no studies comparing diff erent LMWHs or diff erent visocoelastic methods. fl Results Our results show values of heparin activity measured in bags and effl uents with and without in-line fi ltration after 24-hour infusion for both types of bag assessed (Tables 1 and 2). Results are expressed as median values (minimum to maximum) in percent. Table 1 (abstract P95). ROTEM and FOR variables for enoxaparin and tinzaparin Enoxaparin Tinzaparin 0 IU/ml 0.5 IU/ml 1.0 IU/ml 1.5 IU/ml 0.5 IU/ml 1.0 IU/ml 1.5 IU/ml ROTEM CT (seconds) 175 ± 38 191 ± 89 214 ± 109 249 ± 94* 223 ± 53* 289 ± 84* 326 ± 125* CFT (seconds) 77 ± 23 87 ± 21 85 ± 16 83 ± 22 74 ± 27 84 ± 45 80 ± 21 Angle (°) 75 ± 4 74 ± 4 75 ± 3 74 ± 4 75 ± 5 73 ± 5 73 ± 4 MCF (mm) 62 ± 5 61 ± 7 63 ± 5 63 ± 6 68 ± 7* 64 ± 8 65 ± 6* MCL (%) 8 ± 4 6 ± 3+ 5 ± 4 2 ± 5* 5 ± 4* 6 ± 4 2 ± 4* ReoRox COT1 (seconds) 30 ± 5 35 ± 7* 39 ± 8* 38 ± 11* 36 ± 4* 45 ± 9* 48 ± 15* COT2 (seconds) 65 ± 11 76 ± 13* 82 ± 14* 85 ± 20* 74 ± 5* 91 ± 16* 108 ± 29* COT2 – COT1 (seconds) 34 ± 10 41 ± 8* 43 ± 9* 46 ± 10* 37 ± 4 46 ± 11* 51 ± 15* Data presented as median (SD). P <0.05 compared with 0 IU/ml LMWH. Table 1 (abstract P95). ROTEM and FOR variables for enoxaparin and tinzaparin S34 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P96). P95 Methods Data from 100 propensity matched patients who received heparin (Group H, n = 50) or bivalirudin (Group B, n = 50), from January 2009 to January 2012, in a cardiac surgery ICU of a university hospital were analyzed. Bivalirudin was administered as either fi rst-line or second-line drug after heparin discontinuation if heparin-induced thrombocytopenia was presumed. P99 Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs M Honickel1, O Grottke1, J Van Ryn2, H Ten Cate3, R Rossaint1, H Spronk3 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany; 3Maastricht University, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P99 (doi: 10.1186/cc13289) Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs P95 Results of heparin activity in parenteral nutrition bags Bags at T0 + Effl uent at T0 + Bags at T0 24 hours 24 hours With fi ltration 100 95.45 97.73 (84.09 to 109.09) (79.55 to 102.27) (75.00 to 104.55) Without fi ltration 100 97.62 97.62 (90.48 to 114.29) (83.33 to 107.14) (83.33 to 114.29) Table 2 (abstract P96). Results of heparin activity in bags with sodium heparin in 50% dextrose Bags at T0 + Effl uent at T0 + Bags at T0 24 hours 24 hours With fi ltration 100 95.10 98.04 (77.45 to 108.82) (90.20 to 109.80) (95.10 to 100.98) Without fi ltration 100 92.23 94.17 (85.44 to 107.77) (88.35 to 100.00) (86.41 to 108.74) Conclusion We conclude that there is no loss of heparin activity when drug is infused over 24 hours for both types of bag prepared, with and without in-line fi ltration, showing heparin activity remains stable during this period and there is no interaction of drug with other nutrient components of bags, especially 50% dextrose. Table 1 (abstract P96). Results of heparin activity in parenteral nutrition bags Table 1 (abstract P96). Results of heparin activity in parenteral nutrition b P98 Reversal of edoxaban-induced anticoagulation by the four-factor prothrombin complex concentrate Beriplex® in a rabbit model E Herzog1, F Kaspereit1, W Krege1, B Doerr1, J Mueller-Cohrs1, I Pragst1, Y Morishima2, G Dickneite1 1CSL Behring GmbH, Marburg, Germany; 2Daiichi Sankyo Co., Ltd, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P98 (doi: 10.1186/cc13288) Introduction The oral direct and selective factor Xa inhibitor edoxaban (Daiichi Sankyo) is currently available in Japan for the prophylaxis of venous thromboembolism (VTE) in patients undergoing major orthopedic surgery and is undergoing investigation in phase III trials for the prevention of stroke in patients with atrial fi brillation and the treatment and secondary prevention of VTE. The primary complication of any available anticoagulant therapy is the risk of bleeding. Rapid reversal of anticoagulation may be necessary in patients requiring emergency treatment due to uncontrolled bleeding. Prothrombin complex concentrates (PCC) are frequently used to reverse the eff ect of vitamin K antagonists such as warfarin and have also been suggested to be potentially eff ective in reversing the eff ects of the new oral anticoagulants. The present study was therefore designed to determine whether the four-factor PCC Beriplex® can eff ectively reverse bleeding and normalize coagulation following edoxaban administration in a rabbit kidney injury model. P101 dabigatran-treated animals were randomized (n = 6/group) to a single injection of idarucizumab at 30, 60 or 120 mg/kg i.v. or vehicle (control animals). Blood loss and hemodynamic variables were monitored over 4 hours or until time of death. Data were analyzed by ANOVA (± SD) and by the log-rank test. P101 Plasma-free hemoglobin and microvascular response to fresh or old blood transfusion in septic patients A Donati1, E Damiani1, R Domizi1, AT Colesnicenco1, E Montesi1, S Ciucani1, P Pelaia1, C Ince2 1Università Politecnica delle Marche, Ancona, Italy; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P101 (doi: 10.1186/cc13291) Plasma-free hemoglobin and microvascular response to fresh or old blood transfusion in septic patients Conclusion This study demonstrates for the fi rst time that anticoagulation with dabigatran can be reversed eff ectively and safely by idarucizumab. Even under supra-therapeutic dabigatran concentrations, the antidote decreased blood loss and mortality in this lethal pig animal model. Thus, data from clinical studies are warranted to confi rm the fi ndings of this study. Introduction Free hemoglobin (fHb) can scavenge nitric oxide and induce vasoconstriction [1]. The fHb content may be higher in older blood bags. We studied whether old red blood cell (RBC) transfusion increases plasma fHb in septic patients and if this aff ects the microvascular response. p Methods Twenty septic patients randomly received either fresh (<10 days storage) or old (>15 days) RBC transfusion. Plasma fHb was measured before and 1 hour after transfusion; the sublingual microcirculation was assessed with sidestream dark-fi eld imaging. The perfused boundary region (PBR) was measured as an index of glycocalyx damage [2]. The thenar Tissue Hb index (THI) was measured (near-infrared spectroscopy). g g Conclusion This study demonstrates for the fi rst time that anticoagulation with dabigatran can be reversed eff ectively and safely by idarucizumab. Even under supra-therapeutic dabigatran concentrations, the antidote decreased blood loss and mortality in this lethal pig animal model. Thus, data from clinical studies are warranted to confi rm the fi ndings of this study. p py Results fHb increased in the old RBC group (Figure 1). THI increased in both groups, while SDF parameters were unaltered. Negative correlations were found between ΔfHb and changes in total vessel density (r = –0.57, P <0.01; Figure 2) and THI (r = –0.71, P <0.001). These relations were lacking in patients with PBR <2.68 μm. Figure 1 (abstract P101). Changes in fHb. Figure 2 (abstract P101). Relation between changes in fHb and TVD. Figure 1 (abstract P101). Changes in fHb. Use of a specifi c antidote to dabigatran (idarucizumab) reduces blood loss and mortality in dabigatran-induced and trauma-induced bleeding in pigs Methods After ethical approval, male pigs (n = 30) were given dabigatran etexilate for 3 days (30 mg/kg bid p.o.), and the sham group (n = 6) received placebo. To achieve supra-therapeutic anticoagulation, dabigatran was infused prior to injury on day 4 in anesthetized pigs, and the sham group received placebo. A standardized blunt liver injury was infl icted and blood loss (BL) was recorded 10 minutes post trauma. The Conclusion Novel anticoagulant strategies might play a crucial role in critically ill cardiac surgery patients. In a propensity-matched population, our study showed that primary bivalirudin anticoagulation may reduce bleeding complications and mortality. Further studies are therefore warranted. therefore warranted. S35 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Plasma-free hemoglobin and microvascular response to fresh or old blood transfusion in septic patients A Donati1, E Damiani1, R Domizi1, AT Colesnicenco1, E Montesi1, S Ciucani1, P Pelaia1, C Ince2 , 1Università Politecnica delle Marche, Ancona, Italy; 2Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P101 (doi: 10.1186/cc13291) Results Dabigatran levels were 1,147 ± 370 ng/ml with no diff erences between groups prior to injury. BL in sham animals was 409 ± 53 ml 10 minutes after injury and 700 ± 107 ml after 4 hours (survival rate 100%). Anticoagulation with dabigatran (control animals) resulted in signifi cantly higher BL 10 minutes after injury (801 ± 66 ml, P <0.05). Mortality in these animals was 100%, with a mean survival time of 121 minutes (range: 90 to 153 minutes; P <0.05 vs. sham and idarucizumab-treated animals). Total BL in dabigatran-treated animals was 2,977 ± 316 ml. In contrast, treatment with idarucizumab was associated with a dose-dependent reduction in BL. The lowest dose of 30 mg/kg resulted in 17% mortality (1/6 animals) and BL was reduced by 50% (1,586 ± 619 ml). BL was further reduced in animals receiving 60 (1,077 ± 103 ml) or 120 mg/kg idarucizumab (1,137 ± 121 ml; both 100% survival). Hemodynamic parameters and markers of shock were similar to pre-trauma levels in latter groups receiving idarucizumab.i Results Dabigatran levels were 1,147 ± 370 ng/ml with no diff erences between groups prior to injury. BL in sham animals was 409 ± 53 ml 10 minutes after injury and 700 ± 107 ml after 4 hours (survival rate 100%). Anticoagulation with dabigatran (control animals) resulted in signifi cantly higher BL 10 minutes after injury (801 ± 66 ml, P <0.05). Mortality in these animals was 100%, with a mean survival time of 121 minutes (range: 90 to 153 minutes; P <0.05 vs. sham and idarucizumab-treated animals). Total BL in dabigatran-treated animals was 2,977 ± 316 ml. In contrast, treatment with idarucizumab was associated with a dose-dependent reduction in BL. The lowest dose of 30 mg/kg resulted in 17% mortality (1/6 animals) and BL was reduced by 50% (1,586 ± 619 ml). BL was further reduced in animals receiving 60 (1,077 ± 103 ml) or 120 mg/kg idarucizumab (1,137 ± 121 ml; both 100% survival). Hemodynamic parameters and markers of shock were similar to pre-trauma levels in latter groups receiving idarucizumab. Fatty acid composition of blood plasma in multiple organ dysfunction syndrome AN Osipenko1, AV Marochkov2 Introduction The aim of study was to assess the fatty acid (FA) composition of blood plasma in multiple organ dysfunction syndrome (MODS). Introduction The aim of study was to assess the fatty acid (FA) composition of blood plasma in multiple organ dysfunction syndrome (MODS). Table 1 (abstract P103). Univariate analysis of logistic regression for in-hospital mortality Table 1 (abstract P103). Univariate analysis of logistic regression for in-hospital mortality HR 95% CI P PAI-1 day 2 1.02 1.0 to 1.06 <0.01 TAT day 2 1.49 1.02 to 19 <0.05 PMG day 3 0.64 0.1 to 0.95 <0.01 SOFA day 1 1.2 0.9 to 1.69 0.21 IL-6 day 1 0.99 0.9 to 1.00 0.21 Methods The objects of the study were 18 people with multiple organ failure (35.6 ± 8.7 years) of various etiologies. The blood of 16 healthy volunteers aged 37.7 ± 3.2 years served as control. There were also analyzed blood plasma and fragments of artery luminal part from patients who died from diff erent causes. Analysis of FA was conducted using capillary gas–liquid chromatography. Quantitative evaluation of individual FA content was made as a mass percentage of their total. Statistical analysis was performed using the Mann–Whitney U test (P <0.05). Results Normalized contents of oleic and palmitoleic monounsaturated FA increase in patients with MODS, while the levels of stearic saturated FA and polyunsaturated FA decrease, as compared with the control. Considering these results, we conclude that blood plasma FA composition in MODS biased towards composition of FA characteristic of adipose and muscle tissue triglycerides, which quantitatively predominant monounsaturated FA, and stearic and polyunsaturated FA have a signifi cantly lower level [1,2]. Thus, the composition of the FA in MODS refl ects the degree of lipolysis activation in fat depots. As a result of these processes there should occur an imbalance in vascular endothelial cells between monounsaturated and polyunsaturated FA and, as a consequence, changes in metabolism of eicosanoids and other lipid vasoactive mediators should develop. One should note that in plasma of people who died in a hospital environment due to various reasons, similar changes in FA blood composition are observed. The degree of change of FA composition in postmortem blood samples presumably primarily depends on severity and duration of a previous critical and serious condition. Postmortem changes presumably exert some infl uence on blood plasma FA composition. References 1. Malcom G, et al.: Am J Clin Nur 1989; 50:288-291. 2. Andersson A, et al.: Am J Clin Nutr 2002; 76:1222-1229. Methods We retrieved data on 2,852 consecutive patients undergoing cardiac surgery at revascularization at Duke between 1 January 2004 and 31 December 2008. We compared patients receiving or not prophylatic ε-aminocaproic acid in coronary artery bypass graft surgery, or valve procedures or combined ones. We evaluated baseline characteristics, intraoperative data and severe clinical endpoints during 30 days. 1. Malcom G, et al.: Am J Clin Nur 1989; 50:288 291. 2. Andersson A, et al.: Am J Clin Nutr 2002; 76:1222-1229. Fatty acid composition of blood plasma in multiple organ dysfunction syndrome Moreover, the composition of FA in blood plasma in the case of MODS is similar to that of FA in the luminal part of the artery wall. This suggests a decrease in the infl uence of intertissue diff erences in lipid composition on metabolic processes. Conclusion In severe sepsis, older patients displayed a biomarker profi le suggestive of enhanced response of coagulation and fi brinolysis system compared with younger patients. Change of some markers depended on age and may contribute to the poor outcome in older patients. References 1. Donadee C, et al.: Circulation 2011, 124:465-476. 2. Donati A, et al.: Microvasc Res 2013, 90:86-89. 1. Donadee C, et al.: Circulation 2011, 124:465-476. 1. Donadee C, et al.: Circulation 2011, 124:465-476. 2. Donati A, et al.: Microvasc Res 2013, 90:86-89. Results For the in-hospital survival rate, that of older sepsis patients was signifi cantly lower than younger patients, 9/15 (60.0%) versus 27/28 (96.4%), P <0.05. Older patients had markedly higher total plasmin activator inhibitor-1 (TPAI-1) on day 1, thrombin–antithrombin complex (TAT) on days 2 and 3, and fi brin monomer complex on day 2, and markedly lower plasminogen (PMG) on day 3 and alpha-plasmin inhibitor (αPI) on days 2 and 3 compared with younger patients (all P <0.05). Age was an independent predictor of high TAT on days 2 and 3, high fi brin monomer complex on day 2, low PMG on day 3, and low αPI on days 2 and 3 after adjusting for some cofactors and covariables. TPAI-1 on day 2, TAT on day 2, and PMG on day 3 were risk factors of in- hospital mortality in older sepsis patients (Table 1). 2. Donati A, et al.: Microvasc Res 2013, 90:86-89. p References 1. Martin GS, et al.: N Engl J Med 2003, 348:1546-1554. 2. Gando S, et al.: Crit Care 2013, 17:R111. ε-Aminocaproic acid does not increase adverse eff ects in cardiac surgery: an analysis of 2,852 casesf Critical Care 2014, 18(Suppl 1):P104 (doi: 10.1186/cc13294 p Conclusion Given that the monounsaturated FAs in the case of MODS enter the bloodstream primarily from adipose and muscle tissues, one can assume that their blood plasma level refl ects the degree of hypermetabolism processes in the organism. Introduction Antifi brinolytic drugs recently have been associated with adverse outcomes in patients undergoing cardiac surgery. We reviewed our experience with prophylatic ε-aminocaproic acid in patients undergoing cardiac surgery at InCor – Heart Institute. P100 Primary bivalirudin anticoagulation for patients with an implantable ventricular assist device M Pieri, B Arnaez, AL Di Prima, M Celinska-Spodar, S Ajello, O Saleh, F Isella, A Montisci, F Pappalardo San Raff aele Scientifi c Institute, Milan, Italy Critical Care 2014, 18(Suppl 1):P100 (doi: 10.1186/cc13290) Introduction Bivalirudin is a direct thrombin inhibitor that is increasingly used in patients undergoing mechanical circulatory support as it presents many advantages compared with heparin. The aim of this study was to describe our experience with bivalirudin as primary anticoagulant in patients undergoing ventricular assist device (VAD) implantation. p Methods An observational study on the 12 consecutive patients undergoing VAD implantation from October 2011 at out institution. Five patients were implanted the Heart Mate II LVAD (Thoratec Corp., Pleasanton, CA, USA), six patients the Heartware HVAD (HeartWare Inc., Miramar, FL, USA), and one patient a Cardiowest Total Artifi cial Heart (Syncardia Systems Inc., Tucson, AZ, USA). Patients received a continuous infusion of bivalirudin, with a starting dose of 0.025 μg/ kg/hour. The target activated partial thromboplastin time (aPTT) was between 45 and 60 seconds. Figure 1 (abstract P101). Changes in fHb. Results Patients never received heparin during hospitalization nor had a prior diagnosis of HIT. Preoperative platelets count was 134,000 ± 64,000 platelets/mm3. The bivalirudin dose was 0.040 ± 0.026 mg/kg/hour, and the duration of therapy was 5 (5 to 12) days. The lowest platelet count during treatment was 73,000 ± 23,000 platelets/mm3. No thromboembolic complications occurred. Two episodes of minor bleeding from chest tubes which subsided after reduction or temporary suspension of bivalirudin infusion were observed. Six patients required blood red cell transfusions, and one patient had one fresh frozen plasma transfusion. No platelet transfusions were performed during treatment. The ICU stay was 8 (7 to 17) days, and the hospital stay 25 (21 to 33) days. y y y y Conclusion Bivalirudin is a valuable option for anticoagulation in patients with VAD, and can be easily monitored with aPTT. The use of a bivalirudin-based anticoagulation strategy in the early postoperative period may overcome many limitations of heparin, and above all the risk of HIT which is higher in patients undergoing cardiac surgery. Bivalirudin should no longer be regarded as a second-line therapy for anticoagulation in patients with VAD. Figure 2 (abstract P101). Relation between changes in fHb and TVD. 2. Andersson A, et al.: Am J Clin Nutr 2002; 76:1222-1229. P104 P104 ε-Aminocaproic acid does not increase adverse eff ects in cardiac surgery: an analysis of 2,852 cases S Zeff erino1, L Camara1, J Almeida2, F Galas1, L Camara1, J Auler Jr1, J Fukushima1, A Leme1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P104 (doi: 10.1186/cc13294) P100 S36 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Old RBC transfusion increased plasma fHb in septic patients. Increasing plasma fHb levels after transfusion were associated with decreased microvascular density and lower increase in tissue Hb content. This relation might be blunted when the glycocalyx is preserved. References Methods A prospective observational study of adult patients with severe sepsis was conducted in a single academic hospital. Plasma was analyzed for coagulation and fi brinolysis markers on days 1, 2, and 3. Patients were stratifi ed according to the age 67 years, which was the point of the Youden index (maximum sensitivity + specifi city – 1) in a receiver operation characteristics plot for a logistic regression model of in-hospital mortality. Eculizumab treatment of atypical haemolytic uraemic syndrome: results from the largest prospective clinical trial to date LE weekers1, JM Campistol2, M Espinosa3, AO Gaber4, J Menne5, E Minetti6, F Provot7, E Rondeau8, M Ogawa9, CL Bedrosian9, M hourmant10 1University of Liège Hospital, Liège, Belgium; 2Servicio de Nefrología, Hospital Clínic, Barcelona, Spain; 3Hospital Universitario Reina Sofía, Córdoba, Spain; 4Methodist Hospital, Houston, TX, USA; 5Hannover Medical School, Hannover, Germany; 6Careggi University Hospital, Florence, Italy; 7CHU Lille, France; 8Hôpital Tenon, Paris, France; 9Alexion Pharmaceuticals, Inc., Cheshire, CT, USA; 10Institut d’ImmunoTransplantation Urologie et Nephrologie, Nantes, France Critical Care 2014, 18(Suppl 1):P105 (doi: 10.1186/cc13295) Results As of March 2013, there were 1,288 patients randomized in the ABLE study. The median patient age was 63 years (IQR 50 to 74). The majority of patients were emergent admissions (97%) with nonoperative diagnoses (85%), including respiratory illness (27%) and sepsis (18%). Bleeding was infrequent (0.4%). At least one comorbidity was common (43%), frequently signifi cant cardiac disease (14%) and diabetes (12%). There is signifi cant variability in the median pre- transfusion hemoglobin across diff erent sites (P <0.05). The median pre-transfusion hemoglobin by site was 75 g/l (IQR 71 to 77). Six of 24 sites had a signifi cantly higher, and four had a signifi cantly lower, median transfusion threshold compared with the median. Signifi cant variability in the median pre-transfusion hemoglobin remained in multivariate analysis, after adjustment for age and type of ICU.f Introduction Atypical haemolytic uraemic syndrome (aHUS) is a rare, life-threatening disease in which patients experience un- controlled complement activation leading to systemic thrombotic microangiopathy (TMA). We report on the eff ect of eculizumab (Ecu), a terminal complement inhibitor approved for the treatment of aHUS, in the largest prospective clinical trial in aHUS. Introduction Atypical haemolytic uraemic syndrome (aHUS) is a rare, life-threatening disease in which patients experience un- controlled complement activation leading to systemic thrombotic microangiopathy (TMA). We report on the eff ect of eculizumab (Ecu), a terminal complement inhibitor approved for the treatment of aHUS, in the largest prospective clinical trial in aHUS. Methods Adult aHUS patients (≥18 years) with platelets <150×109/l and LDH ≥1.5 ULN were recruited into a 26-week single-arm, phase 2 study evaluating Ecu treatment. Patients with STEC-HUS (Shiga toxin and E. coli) and severe ADAMTS13 defi ciency (activity <5%) were excluded. Identifi cation of complement mutation was not required for enrolment. The primary endpoint was complete TMA response (platelet and LDH normalisation (>150×109/l and <ULN, respectively) and <25% increase in serum creatinine from baseline (BL)). Eculizumab treatment of atypical haemolytic uraemic syndrome: results from the largest prospective clinical trial to date Other effi cacy evaluations measured platelet counts and eGFR improvement (by MDRD). Conclusion There was a 16 g/l diff erence in median hemoglobin between the lowest and highest site and wide inter-site variation. These diff erences may be due to transfusion education, monitoring and blood-bank enforcement practices. Site, not country, remains a signifi cant predictor of transfusion. Future directions in transfusion best practices should focus on local transfusion culture. Signifi cant and nonevidence-based variability persists in transfusion thresholds for critically ill patients. p p y Results Forty-one patients were enrolled (mean (SD) age 40.3 (15.3) years; 28 (68%) females), 30 (73%) of whom with fi rst presentation of aHUS (median 2 weeks before Ecu). BL mean (SD) platelets and eGFR were 119 (66.1) ×109/l and 17.3 (12.1) ml/minute/1.73 m2, respectively. Thirty-eight (93%) patients received Ecu for 26 weeks. Table 1 shows improvements in outcomes. Dialysis was discontinued in 20 (83%) among the 24 patients requiring dialysis at inclusion. Ecu was generally well tolerated, with no deaths or unexpected safety concerns. Meningococcal infections occurred in two patients, one of whom continued treatment. A liberal strategy of red blood cell transfusion reduces cardiovascular complications in older patients undergoing cardiac surgery y R Nakamura1, JL Vincent2, J Fukushima1, J Almeida1, F Bergamin1, C Park1 E Osawa1, M Sundin1, A Muller1, F Galas1, L Hajjar1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P107 (doi: 10.1186/cc13297) Table 1 (abstract P105). Outcomes at 26 weeks in adult aHUS patients receiving Ecu Complete TMA response, n (%) patients 30 (73); 95% CI 57 to 86 Platelet count normalisation, n (%) patients 40 (98); 95% CI 87 to 100 Mean (SD) platelet count increase from baseline, ×109/l 135 (114); P <0.0001 Mean (SD) eGFR increase from baseline ml/minute/1.73 m2 29.3 (23.6); P <0.0001 Table 1 (abstract P105). Outcomes at 26 weeks in adult aHUS patients receiving Ecu Introduction Owing to their high risk in cardiac surgery, it is essential to defi ne which transfusion strategy results in a lower rate of cardio- vascular complications in older patients [1]. The aim of this study was to compare clinical outcomes after the implementation of either a restrictive or a liberal transfusion strategy in patients aged 60 years and above. Conclusion Results from this large prospective clinical trial in adult patients with aHUS confi rm prior trials with Ecu to inhibit complement- mediated TMA, improve renal and haematological outcomes and show the benefi t of early diagnosis and treatment. There were no unexpected safety concerns. The trial is ongoing. Methods This study was a substudy of the Transfusion Requirements After Cardiac Surgery study. In this subgroup analysis we included all patients aged 60 years and above randomized to a restrictive or a liberal strategy of RBC transfusion. A composite endpoint for cardiovascular complications was used and defi ned as a combination of 30-day all- cause mortality and severe cardiovascular morbidity. P106 Results The primary composite endpoint – all-cause 30-day mortality, cardiogenic shock, or myocardial infarction – occurred in 9.6% of patients in the liberal strategy group and in 18.4% in the restrictive strategy group (P = 0.041). The incidence of cardiogenic shock was 5.2% in the liberal group and 12.8% in the restrictive group (P = 0.031) and of myocardial infarction was 2.2% in the liberal group and 5.6% in the restrictive group (P = 0.203). There was no signifi cant diff erence between transfusion strategies in 30-day mortality rates (4.4% vs. 8%, respectively; P = 0.23). Response of coagulation and fi brinolysis system was diff erent between older and nonolder patients with severe sepsis Response of coagulation and fi brinolysis system was diff erent between older and nonolder patients with severe sepsis D Kudo, S Yamanouchi, T Sato, R Nomura, T Omura, N Miyagawa, S Kushimoto Tohoku University Hospital, Sendai, Japan Critical Care 2014, 18(Suppl 1):P103 (doi: 10.1186/cc13293) Response of coagulation and fi brinolysis system was diff erent between older and nonolder patients with severe sepsis D Kudo, S Yamanouchi, T Sato, R Nomura, T Omura, N Miyagawa, S Kushimoto Tohoku University Hospital, Sendai, Japan Critical Care 2014, 18(Suppl 1):P103 (doi: 10.1186/cc13293) y Results A total of 2,852 patients were included in the analysis and 1,389 (48.7%) received prophylactic ε-aminocaproic acid. The others did not receive any antifi brinolytic. In the risk-adjusted model, survival was similar among patients treated with aminocaproic acid or not treated (1.9 vs. 1.6%, P = 0.48). Bleeding in 24 hours was reduced in the treated group as compared with the nontreated (391 vs. 472 ml, P = 0.012). There were no diff erences regarding acute renal failure, stroke and infection in 30 days. y p , , p Critical Care 2014, 18(Suppl 1):P103 (doi: 10.1186/cc13293) Introduction The present study investigated the relationship between age [1] and biomarkers of coagulation and fi brinolysis [2] and their impact on outcome in severe sepsis patients. S37 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion In cardiac surgery, prophylactic use of aminocaproic acid reduces bleeding and does not result in higher incidence of acute complications. (RBC) transfusion practice remains variable and is infl uenced by patient characteristics and institutional transfusion culture. Methods We performed a multicenter retrospective analysis within the ongoing prospective randomized, Age of Blood Evaluation (ABLE) trial. The patient population included patients admitted to the ICU with an anticipated length of invasive or non-invasive mechanical ventilation >48 hours and who required a fi rst RBC transfusion during the fi rst 7 days of ICU admission. As of March 2013, completed and verifi ed data from 45 sites in Canada, France, and the UK were included. Sites with at least 12 patients were included in site analysis. The primary outcome is the association of enrolling centers on the median hemoglobin prior to transfusion. y References 1. Carson, et al.: Cochrane Database 2012, 4:CD002042. 2. Barr PJ, et al.: Transfusion 2011, 51:1684-1694. P107 A liberal strategy of red blood cell transfusion reduces cardiovascular complications in older patients undergoing cardiac surgery R Nakamura1, JL Vincent2, J Fukushima1, J Almeida1, F Bergamin1, C Park1, E Osawa1, M Sundin1, A Muller1, F Galas1, L Hajjar1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P107 (doi: 10.1186/cc13297) Evaluation of the implementation of a massive transfusion protocol K Balvers, M Coppens, JH Klinkspoor, SS Zeerleder, JC Goslings, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P110 (doi: 10.1186/cc13300) Introduction A massive transfusion protocol (MTP) aims to provide standardized and early delivery of blood products and prohemostatic agents by keeping pre-thawed fresh frozen plasma (FFP) available. Implementation of MTP is assumed to result in transfusion with higher ratios of FFPs and platelets to red blood cells (RBCs). Pre-thawing may also result in waste of FFPs. These MTP benefi ts or disadvantages have not yet been demonstrated. The aim of this study was to evaluate effi cacy of a MTP 1 year after implementation in our level I trauma center in an academic hospital. y j g Results A total of 4,952 patients were enrolled with an in-hospital mortality rate of 4% and a mean age of 58 ± 21 years. In total, 4,683 (95%) patients had their hematocrit measured: 3,857 (82%) patients had a normal hematocrit (30 to 44%), 413 (8.8%) patients had 25 to 29% hematocrit, and 66 (1.4%) patients ≤24% hematocrit. A total of 248 (5.3%) had a high hematocrit (≥45%). After adjusting for age, present or prior cancer, diabetes I and II, end-stage renal disease, AIDS and liver disease, anemia remained an independent predictor of in-hospital mortality with odds ratios of respectively 1.7 and 2.5 with worsening anemia as well as high hematocrit, with odds ratio 2.3 (Table 1). The AUC for the model was 0.78. Methods A retrospective analysis of an electronic blood bank transfusion database comparing massive transfusion before (January to December 2011) and after (January to December 2012) implementation of MTP. Activation of MTP consists of delivery of packages of 6 units of RBC, 6 units of pre-thawed FFP and 2 units of platelets collected from fi ve donors. Massive bleeding was defi ned as transfusion ≥10 units of RBCs. Statistics by t test and Mann–Whitney U test. Table 1 (abstract P108). Eff ect of severity of anemia or high hematocrit on in-hospital mortality Hematocrit Mortality OR P value ≥45% 2.3(1.3 to 4.1) 0.007 30 to 44%, ref. 1.0 25 to 29% 1.7(1.1 to 2.6) 0.019 < 20% 2.5(1.1 to 5.8) 0.032 Table 1 (abstract P108). Eff ect of severity of anemia or high hematocrit on in-hospital mortality Results In 2012, a total of 101 MTP activations was registered. Accurate prediction of massively bleeding patients (n = 30) was 29.7% of MTP activations. Of all massively bleeding patients in 2012, MTP was not activated in 55.2%. P109 P109 New simplifi ed criteria for predicting massive transfusion in trauma T Yumoto, A Iida, E Knaup, N Nosaka, S Morisada, T Hirayama, N Shiba, K Tsukahara, H Nosaka, Y Kinami, M Terado, H Yamanouchi, K Sato, T Ugawa, S Ichiba, Y Ujike Okayama University Hospital, Okayama, Japan Critical Care 2014, 18(Suppl 1):P109 (doi: 10.1186/cc13299) P109 New simplifi ed criteria for predicting massive transfusion in trauma T Yumoto, A Iida, E Knaup, N Nosaka, S Morisada, T Hirayama, N Shiba, K Tsukahara, H Nosaka, Y Kinami, M Terado, H Yamanouchi, K Sato, T Ugawa, S Ichiba, Y Ujike Okayama University Hospital, Okayama, Japan Critical Care 2014, 18(Suppl 1):P109 (doi: 10.1186/cc13299) , , p, , , y , ukahara, H Nosaka, Y Kinami, M Terado, H Yamanouchi, K Sato, P108 Anemia and high hematocrit are associated with in-hospital mortality in emergency department patients with suspected infection M Jessen1, S Skibsted1, N Shapiro2 1Aarhus University Hospital, Aarhus, Denmark; 2Beth Israel Deaconess Medical Center, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P108 (doi: 10.1186/cc13298) Variation in red blood cell transfusion thresholds in critically ill patients K Wilton, R Fowler, T Walsh, J Lacroix, J Callum Sunnybrook Hospital, Toronto, Canada Sunnybrook Hospital, Toronto, Canada Critical Care 2014, 18(Suppl 1):P106 (doi: 10.1186/cc13296) Introduction Restrictive transfusion practice in recent randomized trials and systematic reviews continues to have favorable outcomes [1]. Despite this, substantial variability in transfusion practice persists [2]. It is important to identify contemporary variability, and which patient, provider, and institutional factors drive this variability. Therefore, we performed a retrospective analysis hypothesizing that red blood cell Conclusion In this prospective, randomized clinical trial, older patients submitted to a restrictive strategy of RBC transfusion had a rate of cardiovascular complications in 30 days after cardiac surgery twice as high than a liberal strategy. In this group of patients, probably Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S38 Methods We retrospectively analyzed trauma patients transported to our center for the recent 2 years. Patients transferred from other hospitals with minor injuries or confi rmed cardiac arrest at the scene were excluded. untreated anemia would be more harmful than in a younger or healthier population undergoing cardiac surgery. Reference untreated anemia would be more harmful than in a younger or healthier population undergoing cardiac surgery. Reference 1. Hajjar LA, Vincent JL, Galas FR, Nakamura RE, Silva CM, Santos MH, et al.: Transfusion requirements after cardiac surgery: the TRACS randomized controlled trial. JAMA 2010, 304:1559-1567. Results A total of 297 trauma patients were included in this study. Thirty-one (10.4%) patients required MT. Sensitivity and specifi city for the Assessment of Blood Consumption (ABC) score were 48% and 99%, respectively. Because blunt injuries account for most trauma patients in Japan, we established new simple criteria using signifi cant factors that were derived from the examination on arrival. If trauma patients met any of the following conditions – that is, shock index (SI) >1, base excess (BE) <–3 mmol/l, and positive focused assessment of sonography for trauma (FAST) – sensitivity and specifi city was 97% and 80%, respectively. The area under the receiver operating characteristic curve of ABC and the new criteria was 0.889 (95% CI, 0.815 to 0.963) and 0.927 (95% CI, 0.881 to 0.974), respectively. Anemia and high hematocrit are associated with in-hospital mortality in emergency department patients with suspected infection M Jessen1, S Skibsted1, N Shapiro2 1Aarhus University Hospital, Aarhus, Denmark; 2Beth Israel Deaconess Medical Center, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P108 (doi: 10.1186/cc13298) Conclusion We suggest new criteria for early predicting MT in trauma using SI, BE, and FAST. This method may be valid especially in such areas because blunt injuries are the major cause of trauma in Japan. Reference Introduction Anemia and its association with mortality among emergency department (ED) patients with suspected infection has to our knowledge never been investigated. We hypothesize that anemia as well as high hematocrit increases the risk of in-hospital death among these patients. Nunez TC, et al.: J Trauma 2009, 66:346-352. P110 p Methods A prospective observational study of adult ED patients with suspected infection presenting to an urban, academic medical center ED at Beth Israel Deaconess Medical Center, Boston, MA, USA. Inclusion criterion was clinically suspected infection at ED presentation. Patients were enrolled over a 1-year period. Laboratory and clinical data were collected at enrollment. Primary outcome was in-hospital mortality. Logistic regression was performed determining the independent mortality odds adjusting for confounders. Evaluation of the implementation of a massive transfusion protocol K Balvers, M Coppens, JH Klinkspoor, SS Zeerleder, JC Goslings, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P110 (doi: 10.1186/cc13300) P111 P111 Blood product transfusions in septic patients are associated with mortality, ARDS, and more days on mechanical ventilation PP Dobesh, DG Klepser, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P111 (doi: 10.1186/cc13301) Blood product transfusions in septic patients are associated with mortality, ARDS, and more days on mechanical ventilation PP Dobesh, DG Klepser, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P111 (doi: 10.1186/cc13301) Evaluation of the implementation of a massive transfusion protocol K Balvers, M Coppens, JH Klinkspoor, SS Zeerleder, JC Goslings, NP Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P110 (doi: 10.1186/cc13300) In patients for whom MTP was activated, the RBC:FFP ratio was 1:0.9. In patients for whom MTP was activated and who were massively bleeding, the RBC:FFP ratio was 1:0.9, which was signifi cantly higher compared with 1:0.6 in massive bleeders in 2011 (n = 70) (P = 0.001). The median of blood products administered was 12.5 (6 to 21) in massive bleeding after MTP implementation, compared with 8 (3 to 13) in 2011 (P <0.001). In patients for whom MTP was activated, 9.7% of thawed FFPs were not transfused and wasted. When massive transfusion was accurately predicted, the waste of FFPs was 4.8% versus a waste of 16.2% in the group of unjustifi ed MTP activation. Conclusion Implementation of MTP is associated with an increase of blood products transfused and a signifi cant shift of RBC:FFP ratio to 1:1. However, MTP is also associated with a 9.7% waste of FFP. Improving prediction for massive bleeding patients may result in a decrease of wasted, costly blood products. P113 Infl ammatory properties of microparticles in stored red blood cell transfusion productsf Infl ammatory properties of microparticles in stored red blood cell transfusion products M Straat1, R Nieuwland1, R Van Bruggen2, N Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Sanquin, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P113 (doi: 10.1186/cc13303) Results We identifi ed 824 patients who met the inclusion criteria for this study. Of those patients, 543 (66%) received at least 1 unit of blood products during hospitalization. Patients receiving blood products had signifi cantly higher in-hospital mortality (36.1% vs. 26.9%; P = 0.003) and a higher rate of development of ARDS (45.3% vs. 27.1%; P <0.001) compared with patients not receiving blood products. Patients receiving packed red blood cells (PRBCs) (60%) did not demonstrate a signifi cant increase in mortality (35.1% vs. 28.8%; P = 0.058), while patients receiving platelets (20%) did have higher mortality (45.1% vs. 29.5%; P <0.001). Transfusions of PRBCs or platelets were both associated with a higher development of ARDS (P <0.001 for both). There was a signifi cant increase in days on mechanical ventilation (7.0 days vs. 2.3 days; P <0.001), hospital cost ($88,331 vs. $35,047; P <0.001), and length of stay (17.3 days vs. 9.7 days; P <0.001) for patients receiving blood products, regardless of the type. These diff erences were seen despite the mean APACHE II score being similar (22.8 vs. 22.5; P = 0.645). p M Straat1, R Nieuwland1, R Van Bruggen2, N Juff ermans1 1Academic Medical Center, Amsterdam, the Netherlands; 2Sanquin, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P113 (doi: 10.1186/cc13303) Introduction Blood transfusion is associated with increased morbidity and mortality in the critically ill [1]. Adverse eff ects of transfusion may be mediated by changes in the blood product that accumulate with storage time [2]. The mechanisms, however, are largely unknown. Erythrocyte-derived microparticles (MPs) have been found in transfusion bags [3,4] and their concentration increases with storage duration [5]. We hypothesize that accumulation of MPs during storage induces a proinfl ammatory state in the recipient. l Methods Microparticles were isolated by high-speed centrifugation from red blood cell transfusion bags stored for 41 or 42 days. Whole blood from healthy volunteers was incubated for 24 hours with supernatant from the bags either containing MPs or depleted from MPs. Controls were incubated with medium or LPS (10 ng/ml). TNFα, IL- 10 and IL-6 were measured in supernatant by ELISA. Data are expressed as median and range. References References 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. 2. Koch CG, et al.: N Engl J Med 2008, 358:1229-1239. 3. Rubin O, et al.: Transfusion 2012, 53:1744-1754. 4. Van Der Meijden PE, et al.: J Thromb Haemost 2012, 10:1355-1362. 5. Almizraq R, et al.: Transfusion 2013, 53:2258-2267. References 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. 2. Koch CG, et al.: N Engl J Med 2008, 358:1229-1239. 3. Rubin O, et al.: Transfusion 2012, 53:1744-1754. 4. Van Der Meijden PE, et al.: J Thromb Haemost 2012, 10:1355-1362. 5. Almizraq R, et al.: Transfusion 2013, 53:2258-2267. 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. 2. Koch CG, et al.: N Engl J Med 2008, 358:1229-1239. 3. Rubin O, et al.: Transfusion 2012, 53:1744-1754. 4. Van Der Meijden PE, et al.: J Thromb Haemost 2012, 10:1355-1362. 5. Almizraq R, et al.: Transfusion 2013, 53:2258-2267. 1. Marik PE, et al.: Crit Care Med 2008, 36:2667-2674. Methods The Transfusion Requirements After Cardiac Surgery (TRACS) study is a prospective, randomized, controlled clinical noninferiority trial conducted between February 2009 and February 2010 in an ICU at a university hospital cardiac surgery referral center in Brazil. Consecutive adult patients (n = 502) who underwent cardiac surgery with cardiopulmonary bypass were eligible; analysis was by intention to treat. P113 Infl ammatory properties of microparticles in stored red blood cell transfusion productsf Conclusion Patients with sepsis receiving blood products, particularly platelets, were signifi cantly more likely to develop ARDS, had more days on mechanical ventilation, and had higher mortality. The lack of an increase in mortality associated with PRBC transfusion may be due to the benefi t in oxygen delivery or sample size. g Results MP-depleted supernatant induced a modest increase in median levels of TNFα (9.2 (2.3 to 22.2) pg/ml) and IL-6 (2,140.7 (1,507 to 2,199) pg/ml) compared with the negative controls (2.3 (2.3 to 2.3) pg/ ml and 94.4 (64.7 to 124.1) pg/ml, respectively), while IL-10 levels were not aff ected. Addition of MP-containing supernatant resulted in highly increased levels of TNFα (699 (687 to 742) pg/ml), IL-6 (37,443 (26,493 to 40,967) pg/ml) and IL-10 (1,201 (1,178 to 1,533) pg/ml) compared with negative controls. This MP-induced increase in cytokine production was comparable with the increase observed after the addition of LPS. Transfusion requirements in septic shock patients: a randomized controlled trial Transfusion requirements in septic shock patients: a randomized controlled trial F Bergamin1, J Almeida1, C Park1, E Osawa1, J Silva1, F Galas1, D Nagaoka1, J Fukushima1, S Vieira1, L Candido1, CO Oshiro1, JL Vincent2, L Hajjar1 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Erasme Hospital, Université libre de Bruxelles, Brussels, Belgium Critical Care 2014, 18(Suppl 1):P112 (doi: 10.1186/cc13302) p Conclusion Erythrocyte-derived MPs from aged red blood cell transfusion bags induce a strong infl ammatory response in vitro, which is largely negated when MPs are removed. Whether MPs mediate adverse eff ects of blood transfusion in the critically ill remains to be determined. Introduction Perioperative red blood cell transfusion is commonly used to address anemia, an independent risk factor for morbidity and mortality in critically ill patients [1]; however, evidence regarding optimal blood transfusion practice in septic shock is lacking. The aim of this study was to defi ne which is the best transfusion strategy in septic shock patients regarding 28-day mortality and clinical outcomes: restrictive or liberal. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 (46.3%) were included in the liberal strategy and 73 patients (53.7%) in the restrictive strategy. Occurrence of 28-day mortality was similar between groups (54% in liberal group vs. 56.2% in restrictive group; P = 0.395). outcomes compared with those who do not require transfusions. It is not uncommon for patients with sepsis to require blood product transfusions. The need for transfusions may be indicative of infl ammatory consumption of blood cells, active blood loss, or impaired hematopoiesis. Regardless of the etiology, need for transfusion may be an indicator of a more severely ill patient and a valuable prognostic factor. Conclusion Among cancer patients with septic shock, the use of a restrictive perioperative transfusion strategy compared with a more liberal strategy resulted in similar rates of 28-day-mortality. R f Methods This retrospective cohort study included all patients over the age of 40 years with a confi rmed diagnosis of sepsis and an ICU stay at our academic medical center from 1 January 2005 to 31 March 2011. Use of blood product transfusion, patient demographics, and APACHE II score at the time of sepsis were collected from patient charts. Outcomes of interest were in-hospital mortality, development of acute respiratory distress syndrome (ARDS), days on mechanical ventilation, hospital cost, and length of stay.i 1. Hajjar LA, Vincent JL, Galas FR, Nakamura RE, Silva CM, Santos MH, et al.: JAMA 2010, 304:1559-1567. 1. Hajjar LA, Vincent JL, Galas FR, Nakamura RE, Silva CM, Santos MH, et al.: JAMA 2010, 304:1559-1567. Blood product transfusions in septic patients are associated with mortality, ARDS, and more days on mechanical ventilation PP Dobesh, DG Klepser, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P111 (doi: 10.1186/cc13301) Introduction Several predicting models have been described to identify the necessity for massive transfusion (MT) for trauma patients [1]. The purpose of this study is to validate the simplifi ed scoring systems reported previously and establish new criteria at emergency and in the ICU in Japan. Introduction The objective of this study was to determine whether septic patients requiring blood product transfusions have worse S39 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Application of damage control resuscitation strategies to patients with severe traumatic hemorrhage: review of plasma to packed red blood cell ratios at a single institution Introduction Prothrombin complex concentrate (PCC) has been suggested as a measure to terminate trauma and dabigatran-induced bleeding. Owing to the confl icting data concerning such therapy, we investigated the impact of a four-factor PCC to terminate massive bleeding following the infl iction of multiple trauma in dabigatran anticoagulated pigs. K Jung, K Lee, Y Kim K Jung, K Lee, Y Kim g Ajou University School of Medicine, Suwon, South Korea Ajou University School of Medicine, Suwon, South Korea Critical Care 2014, 18(Suppl 1):P117 (doi: 10.1186/cc133.07) Ajou University School of Medicine, Suwon, South Korea Critical Care 2014, 18(Suppl 1):P117 (doi: 10.1186/cc133.07) Critical Care 2014, 18(Suppl 1):P117 (doi: 10.1186/cc133.07) Introduction When treating trauma patients with severe hemorrhage, massive blood transfusions are often needed. Damage control resuscitation strategies can be used for such patients, but an adequate fresh frozen plasma:packed red blood cell (FFP:PRBC) administration ratio must be established. Introduction When treating trauma patients with severe hemorrhage, massive blood transfusions are often needed. Damage control resuscitation strategies can be used for such patients, but an adequate fresh frozen plasma:packed red blood cell (FFP:PRBC) administration ratio must be established. g p g Methods After ethical approval, 24 male pigs were administered dabigatran etexilate (30 mg/kg bid p.o.) for 3 days. On day 4, dabigatran in anaesthetised animals was infused prior to injury to achieve supra- therapeutic levels. Twelve minutes after infl iction of bilateral femur fractures and standardised blunt liver injury, animals randomly received PCC (25, 50 or 100 IU/kg; n = 6) or placebo (n = 6). Time-adjusted blood loss as primary endpoint (observation period 300 minutes) and a panel of coagulation variables were continually measured. Data were analysed by two-way ANOVA. Data are mean ± SEM.l Methods We retrospectively reviewed the medical records of 100 trauma patients treated with massive transfusions from March 2010 to October 2012. We divided the patients into two groups according to the FFP:PRBC ratio: a high-ratio group (≥0.5) and a low-ratio group (<0.5). The patient demographics, fl uid and transfusion quantities, laboratory values, complications, and outcomes of both groups were analyzed and compared. y y y Results Concentrations of dabigatran prior to infl iction of trauma was comparable between groups (590 ± 40 ng/ml). Anticoagulation with dabigatran and trauma caused severe coagulopathy as shown by prolonged TEM variables (CT, CFT), PT and aPTT. Following PCC application these eff ects were partially reversed. P116 Eff ect of a fi xed dose of fresh frozen plasma on systemic infl ammation and endothelial damage in nonbleeding critically ill patients M Straat, M Muller, J Meijers, M Schultz, N Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P116 (doi: 10.1186/cc13306) Methods We describe a fatal TRALI in a patient with infl uenza A (H1N1), suggesting a relationship between a fi rst-hit lung injury followed by the second lung impairment after blood transfusions. Results We report a 57-year-old female, without a previous medical record. She had an acute onset of fever, cough, muscle pain and progressive dyspnea leading to acute respiratory distress syndrome. The test for infl uenza A H1N1 was positive. She was recovering, but on day 12 of admission, after 1 hour of platelet transfusion, she started with intensive tachycardia, dyspnea and hypoxemia. Her mechanical ventilation parameters increased dramatically. She was in plan for extubation with FiO2 of 30% and positive end-expiratory pressure of 8, which became 100% and 14 respectively. The P:F ratio dropped to 62. Her leukocytes were 10.6×109/l a few hours earlier and went down to 1.5×109/l after the onset. Previous lactate was normal, but jumped to 42 mg/dl. She was free of vasopressors and after the off ending transfusion went through refractory shock and died approximately 24 hours after the blood transfusion. Introduction Fresh frozen plasma (FFP) is associated with onset of acute lung injury [1], the mechanism of which is largely unknown. On the other hand, FFP may be benefi cial, as a higher ratio of FFP to red blood cells decreases mortality in bleeding trauma patients [2] and is associated with an endothelial stabilizing eff ect in vitro [3]. We investigated the eff ect of transfusion with FFP on host response and markers of endothelial damage in nonbleeding critically ill patients. Methods This was a substudy of a multicenter trial in which nonbleeding critically ill patients with an increased International Normalized Ratio (1.5 to 3.0) were randomized to omitting or administering a prophylactic transfusion of FFP (12 ml/kg) prior to an invasive procedure. In 38 patients randomized to receive FFP transfusion, we measured levels of factor VIII, von Willebrand factor, and markers of proinfl ammatory response before and after transfusion. Data are presented as medians. Results FFP transfusion resulted in a signifi cant decrease of TNFα (from 12.3 to 3.1 pg/ml, P = 0.01), von Willebrand factor (from 475 to 424%, P <0.01) and factor VIII (from 246 to 244%, P <0.01). FFP did not alter levels of IL-1β, IL1-RA, IL-8, IL-10, MCP1, MIP1A or sCD40L. References 1. Toy P, et al.: Transfusion-related acute lung injury: defi nition and review. Crit Care Med 2005, 33:721-726. 1. Toy P, et al.: Transfusion-related acute lung injury: defi nition and review. Crit Care Med 2005, 33:721-726. 2. Kleinman S, et al.: Toward an understanding of transfusion-related acute lung injury: statement of a consensus panel. Transfusion 2004, 44:1774-1789. 2. Kleinman S, et al.: Toward an understanding of transfusion-related acute lung injury: statement of a consensus panel. Transfusion 2004, 44:1774-1789. Conclusion FFP transfusion is not associated with a proinfl ammatory response in the critically ill. Rather, FFP seemed to have an endothelial stabilizing eff ect. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion The use of high doses of PCC reversed the anticoagulant eff ects of dabigatran, which was associated with a signifi cant reduction of blood loss. However, the results of this study show that suffi cient concentrations of PCC are necessary to overcome thrombin inhibition. in capillary leakage and subsequent pulmonary edema [2]. TRALI is a clinical diagnosis with the following criteria: acute onset within 6 hours of blood transfusion, PaO2/FIO2 ratio <300 mmHg, or worsening of the P:F ratio, bilateral infi ltrative changes on chest radiograph, no sign of hydrostatic pulmonary edema (pulmonary arterial occlusion pressure ≤18 mmHg or central venous pressure ≤15 mmHg) and no other risk factor for acute lung injury [2].l in capillary leakage and subsequent pulmonary edema [2]. TRALI is a clinical diagnosis with the following criteria: acute onset within 6 hours of blood transfusion, PaO2/FIO2 ratio <300 mmHg, or worsening of the P:F ratio, bilateral infi ltrative changes on chest radiograph, no sign of hydrostatic pulmonary edema (pulmonary arterial occlusion pressure ≤18 mmHg or central venous pressure ≤15 mmHg) and no other risk factor for acute lung injury [2]. Conclusion The use of high doses of PCC reversed the anticoagulant eff ects of dabigatran, which was associated with a signifi cant reduction of blood loss. However, the results of this study show that suffi cient concentrations of PCC are necessary to overcome thrombin inhibition. P116 Eff ect of a fi xed dose of fresh frozen plasma on systemic infl ammation and endothelial damage in nonbleeding critically ill patients M Straat, M Muller, J Meijers, M Schultz, N Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P116 (doi: 10.1186/cc13306) f References 1. Vlaar AP, et al.: Crit Care Med 2010, 38:771-778. 2. Holcomb JB, et al.: Ann Surg 2008, 248:447-458. 3. Peng Z, et al.: Shock 2013, 40:195-202. 1. Vlaar AP, et al.: Crit Care Med 2010, 38:771-778. 1. Vlaar AP, et al.: Crit Care Med 2010, 38:771-778. 2. Holcomb JB, et al.: Ann Surg 2008, 248:447-458. 3. Peng Z, et al.: Shock 2013, 40:195-202. 2. Holcomb JB, et al.: Ann Surg 2008, 248:447-458. 3. Peng Z, et al.: Shock 2013, 40:195-202. 1RWTH Aachen University Hospital, Aachen, Germany; 2CardioMetabolic Diseases Research, Boehringer Ingelheim GmbH & Co. KG, Biberach, Germany; 3Maastricht University Medical Center, Maastricht, the Netherlands Critical Care 2014, 18(Suppl 1):P115 (doi: 10.1186/cc13305) P116 Eff ect of a fi xed dose of fresh frozen plasma on systemic infl ammation and endothelial damage in nonbleeding critically ill patients M Straat, M Muller, J Meijers, M Schultz, N Juff ermans Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P116 (doi: 10.1186/cc13306) Patients had some degree of lung injury at baseline as refl ected by a lung injury score of 2 (0.8 to 2.5), which did not change following transfusion. None of the patients developed TRALI.l Conclusion For our knowledge this is the fi rst case reported of TRALI in an infl uenza A (H1N1) patient. Although blood transfusion can be life saving, it also can be a life-threatening intervention. Prevention is still the best hit. Infl uenza A (H1N1): the fi rst hit for transfusion-related acute lung injury? ju y? F Piza1, F Carvalho1, H Li2, T Crochemore1, R Rodrigues1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital das Clinicas da Faculdade de Medicina da USP, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P114 (doi: 10.1186/cc13304) j y F Piza1, F Carvalho1, H Li2, T Crochemore1, R Rodrigues1 1Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Hospital das Clinicas da Faculdade de Medicina da USP, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P114 (doi: 10.1186/cc13304) This is a randomized controlled parallel-group trial, which included 300 patients admitted to a cancer ICU with diagnosis of septic shock. Patients were randomly assigned to a liberal strategy of blood transfusion (to maintain hemoglobin >9 g/dl) or to a restrictive strategy (hemoglobin >7 g/dl). Mortality in 28 days was the main outcome. Secondary outcomes were clinical complications days free of organ dysfunction, ICU and hospital length of stay, adverse eff ects of transfusion and 60-day mortality.i Introduction Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities [1]. A two-hit hypothesis has been proposed for TRALI. The fi rst hit is underlying patient factors, resulting in adherence of primed neutrophils to the pulmonary endothelium, such as severe pneumonia due to infl uenza A H1N1. The second hit is caused by mediators in the blood transfusion that activate the endothelial cells and pulmonary neutrophils, resulting Introduction Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities [1]. A two-hit hypothesis has been proposed for TRALI. The fi rst hit is underlying patient factors, resulting in adherence of primed neutrophils to the pulmonary endothelium, such as severe pneumonia due to infl uenza A H1N1. The second hit is caused by mediators in the blood transfusion that activate the endothelial cells and pulmonary neutrophils, resulting Results A total of 136 patients were included in the fi rst part of the trial. Mean age was 62 ± 14 years, SAPS 3 at admission was 65 ± 15 and all patients had the diagnosis of solid neoplasm. Sixty-three patients S40 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Application of damage control resuscitation strategies to patients with severe traumatic hemorrhage: review of plasma to packed red blood cell ratios at a single institution Due to ongoing blood loss both PT and TEM variables prolonged over time in PCC 25 IU/kg substituted animals. Accordingly, no-PCC (38.5 ± 4.7 ml/minute) and PCC 25 IU/kg (22.6 ± 5.5 ml/minute) animals showed highest blood loss (P <0.05 vs. PCC 50 IU/kg and PCC 100 IU/kg) with a mean survival time of 106 minutes (no-PCC animals) and 204 minutes for PCC 25 IU/kg animals, respectively. All animals of the PCC 50 IU/kg and PCC 100 IU/kg group survived. Blood loss in both groups was comparable (PCC 50 IU/ kg: 5.9 ± 0.2 ml/minute; PCC 100 IU/kg 6.0 ± 0.3 ml/minute). y Results There were 68 patients in the high-ratio group and 32 in the low-ratio group. There were statistically signifi cant diff erences between groups in the quantities of FFP, FFP:PRBC, platelets, and crystalloids administered, as well as the initial diastolic blood pressure. When comparing the incidence of complications, bloodstream infections were noted only in the high-ratio group, and the diff erence was statistically signifi cant (P = 0.028). Kaplan–Meier plots revealed that the 24-hour survival rate was signifi cantly higher in the high-ratio group (71.9% vs. 97.1%, P <0.001). The 30-day survival rate was also higher in the high-ratio group (56.2% vs. 67.6%), but the diff erence was not statistically signifi cant (P = 0.117). S41 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 , pp http://ccforum.com/supplements/18/S1 Conclusion For treating patients with severe hemorrhagic trauma, raising the FFP:PRBC ratio to 0.5 or higher may increase the chances of survival (especially the 24-hour survival rate). Eff orts to minimize bloodstream infections during the resuscitation process must be increased. References Studies suggest that low doses of both nitrite and carbon monoxide may protect tissues and organs from this reperfusion injury by limiting mitochondrial free radical production. We explored the eff ects of very small doses of nitrite and carbon monoxide on tissue injury in a porcine model of hemorrhagic shock. Studies suggest that low doses of both nitrite and carbon monoxide may protect tissues and organs from this reperfusion injury by limiting mitochondrial free radical production. We explored the eff ects of very small doses of nitrite and carbon monoxide on tissue injury in a porcine model of hemorrhagic shock. g Methods Fluid resuscitation of hemorrhagic shock is frequently associated with reperfusion injury and secondary organ damage. Validation of infl ationary non-invasive blood pressure monitoring in emergency room patients y p Results Oral DE prolonged clot formation (CFT: 159 ± 39 seconds) and PT (27 ± 9 seconds). Following the 90-minute infusion of dabigatran, the mean plasma levels of dabigatran increased to 1,423 ± 432 ng/ ml. This supra-therapeutic level was associated with a further prolongation of PT, aPTT, and the EXTEM variables CT and CFT. These changes in coagulation parameters were compounded by blood loss following trauma (total blood loss at 60 minutes: 1,978 ± 265 ml). Sixty minutes after trauma, four out of fi ve animals had no measurable clot formation (EXTEM CFT ≥4,000 seconds) and clot strength (EXTEM MCF) had reduced to 11 ± 7 mm. Both PCCs and aDabi-Fab, but not rFVIIa, reversed the eff ects of dabigatran on thromboelastometry parameters (clotting time and clot formation time) and PT at all time points. In contrast, aPTT was only normalised by idarucizumab. Plasma concentrations of dabigatran remained elevated after PCC therapy, but were not measureable after idarucizumab.f Introduction Currently, most non-invasive blood pressure (NIBP) monitoring is based on the oscillometric method and determines the blood pressure during cuff defl ation [1]. On the other hand, a measurement during cuff infl ation may be advantageous, as cuff infl ation requires lower cuff pressure and shorter duration than defl ation. In surgical patients during anesthesia, the infl ationary NIBP has reasonable accuracy compared with conventional defl ationary NIBP [2]. Few studies have reported NIBP monitoring using the infl ationary NIBP in ER patients with various unstable conditions. A purpose of this study is to verify the usefulness of the infl ationary NIBP monitoring in the emergency department. g y p Methods A total of 2,981 NIBP data were collected from 174 patients (age; 56.5 ± 22.2 (7 to 92) years) who have been accommodated in the resuscitation area of the ER at Keio University Hospital, using alternately two algorithms with a standard monitor (BSM-6000; Nihon Kohden Inc., Tokyo, Japan). One algorithm consists of continuous infl ationary and defl ationary measurement in a single cycle (dual algorithm, 1,502 data) performed in order to verify a success rate and a precision of data. The defl ationary algorithm (1,479 data) consists of only conventional defl ationary measurement performed in order to verify the duration of the measurement cycle. Conclusion Both PCC and aPCC are eff ective in reducing coagulopathy in a porcine trauma model with dabigatran anticoagulation. P119 Attenuation of ischemia–reperfusion injury in swine resuscitated for hemorrhagic shock by low-dose inhaled nitrite or carbon monoxide P119 Attenuation of ischemia–reperfusion injury in swine resuscitated for hemorrhagic shock by low-dose inhaled nitrite or carbon monoxide H Haugaa1, H Gomez2, D Maberry2, A Holder2, O Ogundele2, A Botero2, D Escobar2, L Gordon2, S Shiva2, C Dezfulian2, B Kenney2, TI Tønnessen1, B Zuckerbraun2, MR Pinsky2 1Oslo University Hospital, Oslo, Norway; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P119 (doi: 10.1186/cc13309) H Haugaa1, H Gomez2, D Maberry2, A Holder2, O Ogundele2, A Botero2, D Escobar2, L Gordon2, S Shiva2, C Dezfulian2, B Kenney2, TI Tønnessen1, B Zuckerbraun2, MR Pinsky2 1Oslo University Hospital, Oslo, Norway; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P119 (doi: 10.1186/cc13309) H Haugaa1, H Gomez2, D Maberry2, A Holder2, O Ogundele2, A Botero2, D Escobar2, L Gordon2, S Shiva2, C Dezfulian2, B Kenney2, TI Tønnessen1, B Zuckerbraun2, MR Pinsky2 1Oslo University Hospital, Oslo, Norway; 2University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P119 (doi: 10.1186/cc13309) Conclusion These data suggest that infl ationary NIBP has reasonable accuracy and suffi cient rapidity compared with defl ationary NIBP in emergency room patients. P119 y Results The success rate of the infl ationary NIBP (completed only by infl ationary method) was 69.0%. The bias and precision of systolic pressure and diastolic pressure (diff erence of systolic and diastolic pressure between infl ationary and defl ationary NIBP) were –0.6 ± 8.8 and 3.5 ± 7.5 mmHg, respectively (Figure 1). Infl ationary NIBP could also determine NIBP more quickly compared with defl ationary NIBP (16.8 vs. 29.1 seconds, median) (Figure 2).l Application of damage control resuscitation strategies to patients with severe traumatic hemorrhage: review of plasma to packed red blood cell ratios at a single institution Studies suggest that low doses of both nitrite and carbon monoxide may protect tissues and organs from this reperfusion injury by limiting mitochondrial free radical production. We explored the eff ects of very small doses of nitrite and carbon monoxide on tissue injury in a porcine model of hemorrhagic shock. 1. Duchesne JC, Hunt JP, Wahl G, et al.: Review of current blood transfusions strategies in a mature level I trauma center: were we wrong for the last 60 years? J Trauma 2008, 65:272-276. 1. Duchesne JC, Hunt JP, Wahl G, et al.: Review of current blood transfusions strategies in a mature level I trauma center: were we wrong for the last 60 years? J Trauma 2008, 65:272-276. 2. Duchesne JC, McSwain NE Jr, Cotton BA, et al.: Damage control resuscitation: the new face of damage control. J Trauma 2010, 69:976-990. 2. Duchesne JC, McSwain NE Jr, Cotton BA, et al.: Damage control resuscitation: the new face of damage control. J Trauma 2010, 69:976-990. Validation of infl ationary non-invasive blood pressure monitoring in emergency room patients The ex vivo addition of idarucizumab fully corrected all coagulation measures and signifi cantly decreased plasma concentrations of dabigatran. No signifi cant eff ects on haemostasis were observed after the application of rFVIIa. 1. van Montfrans GA: Blood Press Monit 2001, 6:287-290. 2. Onodera J, et al.: J Anesth 2011, 25:127-130. P120 Validation of infl ationary non-invasive blood pressure monitoring in emergency room patients J Sasaki, S Hori Keio University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P120 (doi: 10.1186/cc13310) Validation of infl ationary non-invasive blood pressure monitoring in emergency room patients J Sasaki, S Hori Keio University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P120 (doi: 10.1186/cc13310) P118 Results Although no increase in blood nitrite concentrations was observed after inhalation, nitrite was associated with signifi cant decreases in blood, muscle, and peritoneal fl uid lactate concentrations (P <0.05), whereas both nitrite and carbon monoxide were associated with signifi cant decreases in glycerol in peritoneal fl uid (P <0.05). Following resuscitation, the muscle mitochondrial respiratory control ratio was preserved in the nitrite and carbon monoxide groups and reduced in the control group. Adjuvant drugs had no eff ects on any gross hemodynamic parameters. In a trauma experimental pig model prothrombin complex concentrates and a specifi c antidote (idarucizumab) are eff ective to reverse the anticoagulant eff ects of dabigatran In a trauma experimental pig model prothrombin complex concentrates and a specifi c antidote (idarucizumab) are eff ective to reverse the anticoagulant eff ects of dabigatran O Grottke1, J Van Ryn2, R Rossaint1 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany Critical Care 2014, 18(Suppl 1):P118 (doi: 10.1186/cc13308) gf O Grottke1, J Van Ryn2, R Rossaint1 f O Grottke1, J Van Ryn2, R Rossaint1 1RWTH Aachen, Germany; 2Boehringer Ingelheim, Biberach, Germany Critical Care 2014, 18(Suppl 1):P118 (doi: 10.1186/cc13308) Introduction Strategies to reverse the anticoagulant eff ects of the new oral anticoagulants in severely bleeding patients remain challenging and confl icting results have been presented regarding the effi cacy of PCCs to alter dabigatran-induced coagulopathy. Thus, this study assessed the ability of PCC, activated PCC (aPCC), recombinant FVII (rFVIIa) and idarucizumab to reverse the anticoagulant eff ects of dabigatran in a porcine model of trauma. g y Conclusion We conclude that at low doses, nebulized sodium nitrite and inhaled carbon monoxide are associated with tissue protection during resuscitation from severe hemorrhagic shock. g g Acknowledgement This work was supported in part by DoD grant W81XWH-11-2-0049, NHLBI HL07820, and 2013121 from The South- Eastern Norwegian Health Authorities. Methods Studies were performed in fi ve pigs. Dabigatran etexilate (DE) was given orally for 3 days (30 mg/kg bid) and on the 4th day dabigatran was infused 90 minutes (30 minutes: 0.77 mg/kg/hour; 60 minutes 0.52 mg/kg/ minute) prior to blunt liver injury. Blood samples were taken before and after dabigatran infusion and 60 minutes post injury. Two doses of PCC (30, 60 IU/kg), aPCC (30, 60 IU/kg), rVIIa (90, 180 μg/kg) and idarucizumab (30, 60 mg/kg) were added to blood samples ex vivo. Coagulation was assessed by thromboelastometry, PT and diluted TT. One-way analysis of variance with the Dunnett post-hoc test for multiple comparisons was used for statistical analysis. Data are presented as mean ± SD. P121l Infl uence of the oscillometric calibration method on accuracy and precision of continuous non-invasive arterial pressure measurements using the CNAP™ device JY Wagner1, I Negulescu2, M Schöfthaler2, A Hapfelmaier2, AS Meidert2, W Huber2, RM Schmid2, B Saugel1 1University Medical Center Hamburg–Eppendorf, Hamburg, Germany; 2Klinikum rechts der Isar, Technical University Munich, Germany Critical Care 2014, 18(Suppl 1):P121 (doi: 10.1186/cc13311) Infl uence of the oscillometric calibration method on accuracy and precision of continuous non-invasive arterial pressure measurements using the CNAP™ device JY Wagner1, I Negulescu2, M Schöfthaler2, A Hapfelmaier2, AS Meidert2, W Huber2, RM Schmid2, B Saugel1 1University Medical Center Hamburg–Eppendorf, Hamburg, Germany; 2Klinikum rechts der Isar, Technical University Munich, Germany Critical Care 2014, 18(Suppl 1):P121 (doi: 10.1186/cc13311) Division of Anaesthesia, University of Cambridge, UK Critical Care 2014, 18(Suppl 1):P122 (doi: 10.1186/cc13312) Introduction The time profi le of the arterial pulse is known to have features such as the dicrotic notch and subsidiary peaks. Such features may provide useful information about the vascular system and are traditionally explained in terms of aortic valve closure or multiple refl ections from impedance mismatches within the arterial system. However, experimental evidence of such refl ections has been elusive. It has been proposed that arterial dynamics may obey a nonlinear equation [1]. This model predicts the existence of multipeaked solitons which can travel long distances without dissipation. We demonstrate that within the soliton model it is not necessary to model valve closure or wave refl ection: single or multiple notches arise de novo even from featureless theoretical LV pressure pulse profi les. We show that a number of clinically relevant features of the invasive blood pressure are reproduced by the soliton model and examine the role of LV pulse energy on pulse wave shape and progression. Introduction The CNAP™ system (CNSystems Medizintechnik AG, Graz, Austria) provides continuous non-invasive arterial pressure (AP) measurements based on the volume clamp method using a fi nger cuff . Finger AP values are calibrated to oscillometric upper-arm AP measurements. In the present study we investigated the infl uence of the calibration approach based on oscillometric upper-arm cuff measurements on the accuracy and precision of the CNAP™ device in comparison with invasively obtained AP measurements. p y Methods The datasets of simultaneously recorded invasive (via arterial catheter) and non-invasive (using the CNAP™ system) AP measurements in 43 patients treated in the medical ICU of a university hospital were analyzed in this study. P122 P122 Arterial pulse waveform as an n-soliton evolution of the left ventricular pressure pulse B Feix, A Ercole Division of Anaesthesia, University of Cambridge, UK Critical Care 2014, 18(Suppl 1):P122 (doi: 10.1186/cc13312) g y References Introduction Fluid resuscitation of hemorrhagic shock is frequently associated with reperfusion injury and secondary organ damage. 1. van Montfrans GA: Blood Press Monit 2001, 6:287-290. 2. Onodera J, et al.: J Anesth 2011, 25:127-130. 1. van Montfrans GA: Blood Press Monit 2001, 6:287-290. 2. Onodera J, et al.: J Anesth 2011, 25:127-130. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S42 Figure 1 (abstract P120). Precision of measurements between infl ationary and defl ationary NIBP. igure 1 (abstract P120). Precision of measurements between infl ationary and defl ationary NIBP. calibration of the CNAP™ device and the corresponding IAP values resulted in a mean diff erence (± standard deviation) of –0.8 mmHg (±8 mmHg), –5 mmHg (±14 mmHg), and +10 mmHg (±9 mmHg), respectively. (3) CAC of the CNAP™ fi nger AP values to IAP values instead of calibration to oscillometric upper-arm AP measurements resulted in a mean diff erence (± standard deviation) of +4 mmHg (±8 mmHg), +5.5 mmHg (±14 mmHg), and +3 mmHg (±7 mmHg), respectively. The accuracy of CAC-CNAP™-derived systolic and diastolic AP compared with the CNAP™-derived AP calibrated to oscillometric AP was signifi cantly higher (P = 0.004 and P <0.001, respectively). Conclusion When using the CNAP™ system, calibration to oscillometric upper-arm AP values integrated into the CNAP™ system is a relevant source of diff erence between CNAP™-derived continuous non-invasive AP measurements and invasively assessed AP values. calibration of the CNAP™ device and the corresponding IAP values resulted in a mean diff erence (± standard deviation) of –0.8 mmHg (±8 mmHg), –5 mmHg (±14 mmHg), and +10 mmHg (±9 mmHg), respectively. (3) CAC of the CNAP™ fi nger AP values to IAP values instead of calibration to oscillometric upper-arm AP measurements resulted in a mean diff erence (± standard deviation) of +4 mmHg (±8 mmHg), +5.5 mmHg (±14 mmHg), and +3 mmHg (±7 mmHg), respectively. The accuracy of CAC-CNAP™-derived systolic and diastolic AP compared with the CNAP™-derived AP calibrated to oscillometric AP was signifi cantly higher (P = 0.004 and P <0.001, respectively). Figure 2 (abstract P120). Frequency histogram of measurement time. P121l To confi rm this, we use the direct view of the tip by transesophageal echocardiography (TEE), a method which has the most recognized high sensitivity and specifi city [1]. antero-posterior radiograph of the chest (RX). New techniques have been developed to control the seat of the CVC, to avoid complications during the procedure and the RX control post insertion. The aim of this study is to demonstrate the ECG’s P-wave amplitude (PWA) increases as we approach the atrio-caval junction (ACJ) by recording the intra- cavity and show that this method is more sensitive and specifi c compared with RX control. To confi rm this, we use the direct view of the tip by transesophageal echocardiography (TEE), a method which has the most recognized high sensitivity and specifi city [1]. Figure 1 (abstract P122). i Methods In 55 adult patients, hospitalized in the ICU, a CVC was placed. We excluded patients with cardiac arrhythmias or pacemaker wearers. All CVCs were placed with an ultrasound-guided puncture technique of the internal jugular vein (IJV) or subclavian vein (SV). The CVC was introduced by the Seldinger technique. Introducing the CVC along the Seldinger guide links to the same terminal on the cable connection as the intra-cavity derivation ECG (set CVC Certofi x B; Braun), in turn connected to the adapter for ECG (Certodyn Universaladapter B; Braun). Then the detection mode of the adapter is converted by the ECG trace outside (through surface electrodes) to the ECG mode intra-cavity and an increase of PWA confi rmed the CVC in the vicinity of the ACJ. Through the TEE with esophageal average scan at 120° we measured the distance between the tip and the ACJ. The results were expressed as mean with standard deviation. Results Forty-fi ve CVCs were placed in the IJV while 10 were in the SV. No complications or arrhythmias were detected during the procedure. All CVCs produced an increase in PWA. Where the PWA increased by 25% compared with normal, the TEE scanning tip was 2.5 ± 1.3 cm from the ACJ. Where the PWA increased by 33%, the TEE scanning tip was 1.9 ± 1.1 cm from the ACJ. Where the PWA increased by 50%, the TEE scanning tip was 1.3 ± 0.6 cm from the ACJ. P121l The RX reports have described briefl y the presence of the catheter in the superior vena cava without making explicit the exact position relative to the ACJ. P121l The following comparative analyses between the two AP measurement techniques were performed: (1) comparison of CNAP™-derived AP values with invasive AP (IAP) measurements; (2) comparison of the CNAP™ oscillometric AP values used for the calibration of fi nger AP with IAP measurements; and (3) computer- aided calibration (CAC) of the CNAP™ fi nger AP values to IAP values instead of calibration to oscillometric upper-arm AP measurements with IAP measurements. gy p p p g Methods A model for the arterial pressure is given by solutions to a KdV equation with constants depending on the properties of the artery [1]. This can be solved with the initial condition of a parabolic left ventricular pressure pulse. Results The evolution of the arterial pulse along the arterial tree is shown in Figure 1. We also predict arterial pulses for increasing left ventricular ejection energies. Conclusion Our simple model explains many features of the arterial pulse observed in clinical practice such as the development of the dicrotic notch, the change in shape along the arterial tree and the steepening and acceleration with hypertension. Some phenomena that have traditionally been attributed to arterial wave refl ections or resonance of the invasive arterial pressure measurement can instead be explained by intrinsic properties of the arterial pulse. Results (1) The comparison of CNAP™-derived AP values with IAP measurements revealed a mean diff erence (± standard deviation) for mean AP, systolic AP, and diastolic AP of +0.6 mmHg (±10 mmHg), +11 mmHg (±17 mmHg), and –6 mmHg (±9 mmHg), respectively. (2) The comparison between the oscillometric AP values used for Critical Care 2014, Volume 18 Suppl 1 S43 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Reference 1. Laleg TM, et al.: Biomed Signal Process 2007, 2:163-170. P123 Tackling the burden of postsurgical complications in the USA: would perioperative goal-directed therapy help? G Manecke, A Asemota, F Michard UCSD Medical Center, San Diego, CA, USA Critical Care 2014, 18(Suppl 1):P123 (doi: 10.1186/cc13313) Figure 1 (abstract P122). antero-posterior radiograph of the chest (RX). New techniques have been developed to control the seat of the CVC, to avoid complications during the procedure and the RX control post insertion. The aim of this study is to demonstrate the ECG’s P-wave amplitude (PWA) increases as we approach the atrio-caval junction (ACJ) by recording the intra- cavity and show that this method is more sensitive and specifi c compared with RX control. Reference Laleg TM, et al.: Biomed Signal Process 2007, 2:163-170. 1. Laleg TM, et al.: Biomed Signal Process 2007, 2:163-170. P123 Tackling the burden of postsurgical complications in the USA: would perioperative goal-directed therapy help? G Manecke, A Asemota, F Michard UCSD Medical Center, San Diego, CA, USA Critical Care 2014, 18(Suppl 1):P123 (doi: 10.1186/cc13313) P123 Conclusion We have shown the increase in PWA by detecting that the endo-cavity, as we proceed with the insertion of the CVC, corresponds to higher proximity to the ACJ. These results canceled the need for RX control in the future. Introduction Pay-for-performance programs and economic constraints call for solutions improving the quality of healthcare without increasing costs. Many studies have shown decreased morbidity in major surgery when perioperative goal-directed therapy (PGDT) is used. We assessed the clinical and economic burden of postsurgical complications in the University HealthSystem Consortium (UHC) in order to predict potential savings with PGDT. P125 Impact of the neutral position and rotation of the head in ultrasound-guided internal jugular vein catheterization on duration of procedure and complications M Kurt, A Ozgultekin, G Turan, F Ormancı, S Batan, O Ekinci Haydarpasa Numune Teaching and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P125 (doi: 10.1186/cc13315) y Results A total of 75,140 patients met the search criteria. In total, 8,421 patients developed one or more postsurgical complications (morbidity rate 11.2%). In-hospital mortality was 12.42% and 1.39% (P <0.001), mean hospital length of stay was 20.48 ± 20.09 days and 8.05 ± 7.11 days (P <0.001), and mean direct cost was $47,284 ± 49,170 and $17,408 ± 15,612 (P <0.001) in patients with and without complications, respectively. With PGDT, morbidity rate was projected to decrease to 6.5 to 9.0%, yielding gross costs savings of $50 million to 151 million for the study population or $670 to $1,406 per patient. Introduction In internal jugular vein (IJV) catheterization, neck rotation may enhance the visibility of anatomical landmarks; however, it may increase the risk of carotid artery (CA) puncture by replacing the position of the IJV in relation to the CA. In our study, during US- guided IJV catheterizations, we investigated the eff ects of changing the position of the IJV by the CA depending on the neutral position and 45° rotation of the head on duration of procedure and complications. Methods After obtaining hospital ethics committee approval, 100 intensive care patients aged >18 years were included in the study. Patients were randomly selected and catheterization was performed in a neutral position of the head (n = 50) and by turning the head 45° to the opposite side (n = 50). The US-guided catheterization procedure was performed in accordance with general principles. Once the needle entered the IJV and blood was aspirated, the US probe was released from the hand, and the catheter was placed and fi xed according to the Seldinger technique. The data of the intervention side for each process, the count number of successful catheter insertion, whether there is arterial access or not and the duration of procedure (from skin contact of the needle to catheter insertion) were recorded. Conclusion Postsurgical complications have a dramatic impact on mortality, hospital length of stay and costs. Potential cost savings resulting from PGDT are substantial. PGDT may be recommended to improve quality of care and decrease costs. Reference 1. Pelegatti C, et al.: Endovascular ECG to guide placement of CVC. J Vasc Acces 2011, 12:348-353. Methods Data from adults who had 10 major surgical procedures in 2011 were screened in the UHC database. Thirteen postsurgical complications were tabulated. In-hospital mortality, hospital length of stay and costs from patients with and without complications were compared. The risk ratios reported by the most recent meta-analysis were used to estimate the potential reduction in postsurgical morbidity with PGDT. Potential cost savings were calculated from the actual and anticipated morbidity rates. P125 P124 P124 Radiological control of central venous catheter (CVC) versus electrocardiogram-guided control inserted CVC: confi rm with transesophageal echocardiography F Righetti, G Castellano St Boniface Hospital Verona, Italy Critical Care 2014, 18(Suppl 1):P124 (doi: 10.1186/cc13314) Radiological control of central venous catheter (CVC) versus electrocardiogram-guided control inserted CVC: confi rm with transesophageal echocardiography F Righetti, G Castellano St Boniface Hospital Verona, Italy Critical Care 2014, 18(Suppl 1):P124 (doi: 10.1186/cc13314) Development of a standardized method of peripherally inserted central catheter (PICC-line) bedside installation J Hobeika, K Serri, M Albert Hôpital Sacré-Coeur de Montréal, Montreal, Canada Critical Care 2014, 18(Suppl 1):P128 (doi: 10.1186/cc13318) Development of a standardized method of peripherally inserted central catheter (PICC-line) bedside installation J Hobeika, K Serri, M Albert Hôpital Sacré-Coeur de Montréal, Montreal, Canada Critical Care 2014, 18(Suppl 1):P128 (doi: 10.1186/cc13318) Introduction Bedside insertion of peripherally inserted central catheters (PICCs) results in tip malposition in up to 48% [1], with catheters frequently terminating in the internal jugular vein (IJV) or in peripheral veins [2]. In an attempt to reduce tip malposition, we developed a standardized approach to PICC installation. This study aims to validate that method. Results Sixty-fi ve CVCs were placed: 51 in the right internal jugular vein (IJV), 14 in the left IJV. The mean catheter length by intracavitary ECG was signifi cantly deeper than predicted by Lum’s formulas (18.2 ± 1.9 vs. 16.7 ± 1.7 cm, P <0.001) and not diff erent from Peres’ formulas (18.2 ± 1.9 vs. 18.2 ± 1.7 cm, P = 0.8). Methods From a 34-bed adult ICU, we retrospectively reviewed PICC insertions over a 6-month period before the intervention program (control group). We designed a prospective interventional pilot study on 40 consecutive patients (intervention group). Patients in the intervention group were positioned in a standardized fashion and the PICC length was measured from easily identifi ed anatomic landmarks. During PICC insertion, the patient’s head was either rotated ipsilaterally to the site of PICC insertion or the ipsilateral IJV was manually compressed, depending on the patient’s capacity to collaborate. Once the PICC was inserted, an ultrasound survey was conducted to identify the catheter in the subclavian vein (SC) and ensure its absence in the ipsilateral IJV. The primary endpoint was defi ned as PICC tip position, obtained from the post-procedural chest X-ray. A catheter was considered to be in an optimal position if the tip resided in the distal third of the superior vena cava (SVC), adequate if it resided between the subclavian vein (SC) and the distal third of the SVC, and aberrant if in any other location. At post-procedural CXR, 88% of the tips were in the target zone. In three cases (5%) the catheter went in the wrong direction but was immediately corrected during the procedure since the intracavitary P wave did not change its amplitude. Compared with the intracavitary ECG, the incidence of malposition would have been signifi cantly higher with Lum’s formulas (48% vs. 12%, P <0.001) and Peres’ formulas (51% vs. 12%, P <0.001). Development of a standardized method of peripherally inserted central catheter (PICC-line) bedside installation J Hobeika, K Serri, M Albert Hôpital Sacré-Coeur de Montréal, Montreal, Canada Critical Care 2014, 18(Suppl 1):P128 (doi: 10.1186/cc13318) Conclusion The intracavitary ECG was associated with a lower incidence of tip malposition than Peres’ and Lum’s formulas. It is also the only technique allowing one to immediately correct a primary malposition during catheter insertion. g References References 1. Peres PW: Anaesth Intensive Care 1990, 18:536-539. 2. Lum P: J Vasc Access 2004, 9:80-85. 3. Pittiruti M, et al.: J Vasc Access 2011, 12:280-291. 4. Schuster M, et al.: Br J Anaesth 2000, 85:192-194. 1. Peres PW: Anaesth Intensive Care 1990, 18:536-539. Results In the retrospective control arm, 105 PICCs were reviewed for tip position. Optimal, adequate, and aberrant positions were found in 22 (21%), 49 (47%), and 34 (32%) respectively, in comparison with 17 (43%), 15 (38%), and eight (20%) in the intervention group (P <0.05 between both groups). In the control arm, 11 (10%) PICCs terminated outside the central venous system, whereas none failed to achieve central venous access in the intervention arm. 2. Lum P: J Vasc Access 2004, 9:80-85. 3. Pittiruti M, et al.: J Vasc Access 2011, 12:280-291. 4. Schuster M, et al.: Br J Anaesth 2000, 85:192-194. Minkovich et al.: Can J Anaesth 2011, 58:709-713. Trerotola et al.: J Vasc Interv Radiol 2007, 18:513e8. Radiological control of central venous catheter (CVC) versus electrocardiogram-guided control inserted CVC: confi rm with transesophageal echocardiography g F Righetti, G Castellano Results The localization of the IJV in relation to the CA is 66% anterolateral, 4% anterior and 30% lateral in a neutral position, and 62% anterolateral, 28% anterior, 10% lateral position in 45° rotation. So while there is no change in a signifi cant proportion of patients in Introduction The placement of a central venous catheter (CVC) is now common practice. Control of the seat of the tip occurs through the S44 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P126 Anthropometric formulas versus intracavitary ECG for optimal tip position of central venous catheters MS Vallecoccia, M Biancone, F Cavallaro, D Settanni, C Marano, MG Annetta, M Pittiruti, M Antonelli Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P126 (doi: 10.1186/cc13316) Conclusion Residents who only learn the UG technique will not be immediately able to perform the LM technique, but require specifi c training based on at least 10 LM procedures. Whether the LM technique should still be taught when an ultrasound device is not available must therefore be addressed. Introduction Peres [1] and more recently Lum [2] developed some anthropometric formulas to correlate patient’s height (H) and ideal length of central venous catheter (CVC) in order to identify optimal tip position. The aim of this study is to compare the reliability of the anthropometric formulas with the method based on intracavitary ECG. Methods We enrolled patients admitted to our ICU candidate to elective CVC insertion, who had a detectable P wave on surface ECG. Intracavitary ECG was used to identify the optimum tip location since a maximal P wave indicates the cavo-atrial junction [3]. Post-insertion chest X-ray (CXR) was performed in all patients to verify the tip position. Assuming that the cavo-atrial junction is about 3 cm from the carina [4], the tip position was considered correct between 1 and 5 cm from the carina (between the lower 1/3 of the superior vena cava and the upper 1/3 of the right atrium). For each patient we retrospectively evaluated whether the catheter length calculated with Lum’s and Peres’ formulas on an estimated height would have been acceptable.i P127 Residents learning ultrasound-guided catheterization are not suffi ciently skilled to use landmarks J Maizel, L Guyomarc’h, P Henon, S Samy Modeliar, B De Cagny, G Choukroun, M Slama CHU Amiens, France Critical Care 2014, 18(Suppl 1):P127 (doi: 10.1186/cc13317) Residents learning ultrasound-guided catheterization are not suffi ciently skilled to use landmarks J Maizel, L Guyomarc’h, P Henon, S Samy Modeliar, B De Cagny, G Choukroun, M Slama CHU Amiens, France Critical Care 2014, 18(Suppl 1):P127 (doi: 10.1186/cc13317) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 the localization of the IJV in relation to the CA, the anterior placement rate increasing the risk of CA puncture was signifi cantly higher in a position of rotation compared with the neutral position (P = 0.001). No signifi cant diff erence was found between procedure durations. Complications were recorded when observed. may not be available in emergency situations, and therefore guidelines also propose that physicians remained skilled in landmark (LM) placement. We conducted this prospective observational study to determine the learning curve of the LM technique in residents only learning the UG technique. g q Methods During the fi rst 3 months of their rotation in our ICU, residents inexperienced in CVC used only the real-time UG technique. During the following 3 months, residents were allowed to place CVC by means of the LM technique when authorized by the attending physician. Conclusion In our study, it has been shown that anterior placement of the IJV in the neutral position is less but this has no advantage in avoiding arterial puncture. The smaller area of procedure in a neutral position can cause diffi culties in practice. However, the processing times between each head position were not diff erent. Nevertheless, further studies evaluating whether there are comparable complication rates and the same duration of procedure in emergency and trauma patients in whom head rotation cannot be possible are needed. q y g p y Results A total of 172 procedures (84 UG and 88 LM) were performed by the inexperienced residents during the study. The success rate was lower (72% vs. 84%; P = 0.05) and the complication rate was higher (22% vs. 10%; P = 0.04) for LM compared with UG procedures. Comparison between the fi ve last UG procedures and the fi rst fi ve LM procedures performed demonstrated that the transition between the two techniques was associated with a marked decrease of the success rate (65% vs. 93%; P = 0.01) and an increase of the complication rate (33% vs. 8%; P = 0.01). After 10 LM procedures, residents achieved a success rate and a complication rate of 81% and 6%, respectively. Residents learning ultrasound-guided catheterization are not suffi ciently skilled to use landmarks Conclusion Using the standardized method described above, PICC tip positioning can be greatly improved. In our results, 100% of catheters placed using the standardized method allowed for central venous access. This pilot study paves the way for a larger, multicentric evaluation of the bedside installation method of PICC. R f fi y J Maizel, L Guyomarc’h, P Henon, S Samy Modeliar, B De Cagny, G Choukroun, M Slama CHU Amiens, France Critical Care 2014, 18(Suppl 1):P127 (doi: 10.1186/cc13317) evaluation o References Hendrikse KA, et al.: Low value of routine chest radiographs in a mixed medical–surgical ICU. Chest 2007, 132:823-828. 2. Oba Y, Zaza T: Abandoning daily routine chest radiography in the intensive care unit: meta-analysis. Radiology 2010, 255:386-395. 2. Oba Y, Zaza T: Abandoning daily routine chest radiography in the intensive care unit: meta-analysis. Radiology 2010, 255:386-395. 2. Oba Y, Zaza T: Abandoning daily routine chest radiography in the intensive care unit: meta-analysis. Radiology 2010, 255:386-395. p Results Eighty CVCs were placed in 78 patients, either by residents in training or by attending doctors. The vein was selected by US scan: right internal jugular (IJ) 64%, left IJ 17%, right axillary 11%, left axillary 4%, right innominate 4%. One pre-existing PNX was identifi ed by US before the procedure. Accidental arterial puncture occurred in three cases (two by residents). In fi ve cases, the wrong direction of the wire was detected by US and corrected during the procedure. The mean number of attempts was 1.65 (1.35 for attending vs. 1.95 for residents, P <0.02) and mean insertion time was 10.4 minutes (8.72 for attending vs. 12.11 for residents, P <0.005). Pleural US scan ruled out puncture-related PNX in all patients. At post-procedural CXR: no PNX was detected, 90% of tips were in the target zone, 5% were in the middle 1/3 of SVC and 5% were in the middle 1/3 of RA (all CVC with tips out of the target zone were inserted by residents).f evaluation o References Introduction An ultrasound-guided (UG) technique is the recom men- ded procedure for central venous catheterization (CVC). But ultrasound Minkovich et al.: Can J Anaesth 2011, 58:709-713. Trerotola et al.: J Vasc Interv Radiol 2007, 18:513e8. S45 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P129 and pericardial eff usion with or without cardiac tamponade. Positions of the endotracheal tube and central venous catheter were also checked. P129 Is chest X-ray necessary after central venous catheter insertion? MS Vallecoccia, F Cavallaro, M Biancone, D Settanni, C Marano, MG Annetta, M Pittiruti, M Antonelli Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P129 (doi: 10.1186/cc13319) Results Ninety-four of the 151 patients included (62%) showed abnormalities on chest X-ray (AC 9%, AIS 25%, PLAPS 42%, PE 3.3%, PTX 2%). Compared with chest X-ray, chest ultrasound had a sensitivity of 86% and a specifi city of 99% for AC, a sensitivity of 95% and a specifi city of 100% for AIS, a sensitivity of 97% and a specifi city of 98% for PLAPS, a sensitivity of 99% and a specifi city of 100% for PE, and a sensitivity and specifi city of 100% for PTX. Furthermore, chest ultrasound detected all pericardial eff usions while neither chest X-ray nor chest auscultation were able to identify them. Chest ultrasound identifi ed all cases of endotracheal tube (two patients) and central venous catheter (two patients). There was a highly signifi cant correlation between abnormalities detected by chest ultrasound and X-ray (k = 0.90), but a poor correlation between chest auscultation and X-ray abnormalities (k = 0.15). Introduction According to the European Society of Parenteral and Enteral Nutrition guidelines [1], post-insertion chest X-ray (CXR) is not necessary if the location of the tip has been verifi ed during the procedure and if pleura-pulmonary damage has been ruled out by other methods. The aim of this study is to assess feasibility and safety of an echo-ECG-guided method of central venous catheter (CVC) insertion and to evaluate whether post-insertion CXR can be avoided. Methods We enrolled only patients admitted to our ICU and candidate to elective CVC insertion, who had a detectable P wave on surface ECG. evaluation o References Our insertion protocol included: preliminary ultrasound (US) scan of central veins and pleural space; US-guided puncture and US control of the correct direction of the guidewire; intracavitary ECG method for tip location (cavo-atrial junction (CAJ) = maximal P wave); and US scan of pleural space to rule out pneumothorax (PNX). Post-insertion CXR was performed in all patients to rule out PNX and verify tip location close to the CAJ (CAJ = 3 cm below the carina [2]). Tip location between 1 and 5 cm below the carina – in the lower 1/3 of the superior vena cava (SVC) or in the higher 1/3 of the right atrium (RA) – was considered acceptable [1]. Introduction According to the European Society of Parenteral and Enteral Nutrition guidelines [1], post-insertion chest X-ray (CXR) is not necessary if the location of the tip has been verifi ed during the procedure and if pleura-pulmonary damage has been ruled out by other methods. The aim of this study is to assess feasibility and safety of an echo-ECG-guided method of central venous catheter (CVC) insertion and to evaluate whether post-insertion CXR can be avoided. Methods We enrolled only patients admitted to our ICU and candidate to elective CVC insertion, who had a detectable P wave on surface ECG. Our insertion protocol included: preliminary ultrasound (US) scan of central veins and pleural space; US-guided puncture and US control of the correct direction of the guidewire; intracavitary ECG method for tip location (cavo-atrial junction (CAJ) = maximal P wave); and US scan of pleural space to rule out pneumothorax (PNX). Post-insertion CXR was performed in all patients to rule out PNX and verify tip location close to the CAJ (CAJ = 3 cm below the carina [2]). Tip location between 1 and 5 cm below the carina – in the lower 1/3 of the superior vena cava (SVC) or in the higher 1/3 of the right atrium (RA) – was considered acceptable [1]. Conclusion Chest auscultation may help identify endotracheal misplacement and tension pneumothorax but it may miss most of major abnormalities. Chest ultrasound represents a valid alternative to chest X-ray to detect all postoperative abnormalities and misplacements. References 1. Hendrikse KA, et al.: Low value of routine chest radiographs in a mixed medical–surgical ICU. Chest 2007, 132:823-828. 1. Hendrikse KA, et al.: Low value of routine chest radiographs in a mixed medical–surgical ICU. Chest 2007, 132:823-828. 1. Ultrasound measurement of carotid fl ow time changes with volume status DC Mackenzie1, NA Khan2, D Blehar3, CP Stowell2, VE Noble2, AS Liteplo2 1Maine Medical Center, Portland, ME, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3University of Massachusetts, Worcester, MA, USA Critical Care 2014, 18(Suppl 1):P131 (doi: 10.1186/cc13321) Introduction Assessment of volume status and responsiveness guides resuscitation strategy. Non-invasive techniques are desirable. Changes in carotid fl ow time have been proposed as a marker of volume status, but few data support their use. We sought to determine whether carotid fl ow time decreased in the volume-depleted state of acute blood loss, and whether volume-depleted individuals would demonstrate an increase in carotid fl ow time after a passive leg raise (PLR) maneuver. Methods Volunteers aged 18 to 55 presenting to the hospital’s blood donor center for whole blood donation were eligible to participate. Individuals with a history of aortic or carotid artery disease, atrial fi brillation, or a contraindication to blood donation were excluded. Prior to blood donation, an investigator performed an ultrasound of the right common carotid artery with a high-frequency linear transducer, obtaining a Doppler tracing of carotid artery fl ow. Measurements of peak velocity, systole time, and carotid fl ow time were obtained. A PLR was performed for 30 seconds, followed by repeat measurements of carotid velocity and fl ow time. Whole blood was then collected according to the blood donor center’s protocol. Immediately after blood donation, repeat measurements of carotid fl ow and velocity were obtained in the supine position and after a PLR. Carotid fl ow times corrected for heart rate (FTc) were analyzed with Student’s t test. The institutional review board approved the study. Conclusion Our insertion protocol was safe and eff ective, with minor diff erences between experienced versus nonexperienced operators. Our data suggest that immediate CXR is not necessary soon after CVC insertion, since PNX can be ruled out by US and since the intracavitary ECG method allows tip location in the SVC and RA in all cases. P131 Ultrasound measurement of carotid fl ow time changes with volume status DC Mackenzie1, NA Khan2, D Blehar3, CP Stowell2, VE Noble2, AS Liteplo2 1Maine Medical Center, Portland, ME, USA; 2Massachusetts General Hospital, Boston, MA, USA; 3University of Massachusetts, Worcester, MA, USA Critical Care 2014, 18(Suppl 1):P131 (doi: 10.1186/cc13321) References 1. Pittiruti M, et al.: Clin Nutr 2009, 28:365-377. 2. Schuster M, et al.: Br J Anaesth 2000, 85:192-194. P130 Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation A Vezzani1, T Manca1, F Benassi1, A Gallingani1, I Spaggiari1, C Brusasco2, F Corradi3, T Gherli1 1Azienda Ospedaliero Universitaria di Parma, Italy; 2Università degli studi di Genova, Italy; 3E.O. Ospedali Galiera, Genova, Italy Critical Care 2014, 18(Suppl 1):P130 (doi: 10.1186/cc13320) Transthoracic echocardiography used in conjunction with passive leg raising for assessment of fl uid responsiveness in severe sepsis or septic shock patients K Jaikriengkrai, K Chittawatanarat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2014, 18(Suppl 1):P133 (doi: 10.1186/cc13323) p p K Jaikriengkrai, K Chittawatanarat, A Limsukon g Chiang Mai University, Chiang Mai, Thailand lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93) (Figure 1). The Bland–Altman plot showed 95% limits of agreement from –8.96 to +8.83% and mean diff erence (bias) of –0.07% (Figure 2). g y g Critical Care 2014, 18(Suppl 1):P133 (doi: 10.1186/cc13323) Introduction During passive leg raising (PLR), we need a real-time device to demonstrate the hemodynamic change [1-3]. This study investigates the ability of transthoracic echocardiography (TTE) to predict fl uid responsiveness (FR) in terms of detecting change of stroke volume (ΔSV) after PLR compared with the transpulmonary thermodilution technique (TPTD) ΔSV after volume expansion (VE). f Conclusion We may use %ΔSV measured by TTE after PLR to predict FR, which is noninvasive and less time-consuming than other invasive techniques. Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Results The subclavian vein was successfully cannulated by ultrasound- guided techniques in all patients. Central venous cannulation failed in two and 10 cases respectively with short-axis and long-axis view and the other view was used successfully. The fi rst-pass success rate was signifi cantly higher in the short-axis group (73%) compared with the long-axis group (40%) (P = 0.005). The procedure time, number of attempts, needle redirection, and skin and vessel punctures were signifi cantly lower in the short-axis than long-axis group (P <0.05). The overall number of complications did not diff er signifi cantly between groups even if artery puncture and hematoma occurred more frequently in the long-axis group. Moreover, the need to change the ultrasound- guided insertion technique was more frequent in the long-axis group. Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Conclusion Ultrasound-guided subclavian vein cannulation by an experienced operator has a higher fi rst-pass success rate and lower access time using the short-axis than long-axis approach. q References References 1. Monnet X, et al.: Crit Care 2013, 17:217. 2. Lafanechere A, et al.: Crit Care 2006, 10:R132. 3. Biais M, et al.: Crit Care 2009, 13:R195. Methods A prospective study was carried out in a medical ICU. Eligible patients were age ≥18 years without necessarily full adaptation to the ventilator with hemodynamic instability who were considered for VE. SV assessment using the subaortic velocity–time measurement was obtained by TTE simultaneously with other hemodynamic parameters derived from TPTD at baseline, within 2 minutes of PLR and following VE (250 ml fl uid in 10 minutes). A fl uid responder was defi ned by ΔSV ≥15% after VE by TPTD. Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation y A Vezzani1, T Manca1, F Benassi1, A Gallingani1, I Spaggiari1, C Brusasco2, F Corradi3, T Gherli1 1Azienda Ospedaliero Universitaria di Parma, Italy; 2Università degli studi di Genova, Italy; 3E.O. Ospedali Galiera, Genova, Italy Critical Care 2014, 18(Suppl 1):P130 (doi: 10.1186/cc13320) Results Eighty donors were screened for participation by two investigators; 68 consented and completed donation (60.3% female, mean age 31). Donors had mean blood loss of 450 ml. The mean FTc supine after blood donation was 296 ms; this was signifi cantly diff erent from the FTc prior to donation (supine = 320 ms, PLR = 323 ms; P <0.0001). The mean FTc following blood donation and PLR was 321 ms, signifi cantly diff erent from the supine position after donation (P <0.0001), but not pre-donation measurements. Introduction Chest auscultation and chest X-ray are commonly used to detect postoperative abnormalities and complications in patients admitted to intensive care after cardiac surgery [1,2]. The aim of the study was to evaluate whether chest ultrasound represents an eff ective alternative to bedside chest X-ray to identify early postoperative abnormalities. Methods A total of 151 consecutive patients (103 male and 47 female) were studied by chest auscultation, ultrasound and X-ray upon admission to intensive care after cardiac surgery. Six pathologic entities were explored by each method: postero-lateral pleural eff usion and/ or alveolar consolidation (PLAPS), alveolar-interstitial syndrome (AIS), alveolar consolidation (AC), pneumothorax (PTX), pleural eff usion (PE), Conclusion Ultrasound measurement of carotid fl ow time was signifi cantly decreased in the setting of acute blood loss. An auto- bolus by PLR after blood loss restored FTc to pre-donation levels. Further investigation of FTc as a non-invasive predictor of volume responsiveness is warranted. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S46 lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93) (Figure 1). The Bland–Altman plot showed 95% limits of agreement from –8 96 to Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Figure 2 (abstract P133). P133 Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Figure 2 (abstract P133). Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Transthoracic echocardiography used in conjunction with passive leg raising for assessment of fl uid responsiveness in severe sepsis or septic shock patients K Jaikriengkrai, K Chittawatanarat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2014, 18(Suppl 1):P133 (doi: 10.1186/cc13323) Transthoracic echocardiography used in conjunction with passive leg raising for assessment of fl uid responsiveness in severe sepsis or septic shock patients K Jaikriengkrai, K Chittawatanarat, A Limsukon Chiang Mai University, Chiang Mai, Thailand Critical Care 2014, 18(Suppl 1):P133 (doi: 10.1186/cc13323) Reference 1. Fragou M, Gravvanis A, Dimitriou V, Papalois A, Kouraklis G, Karabinis A, Saranteas T, Poularas J, Papanikolaou J, Davlouros P, Labropoulos N, Karakitsos D: Real-time ultrasound-guided subclavian vein cannulation versus the landmark method in critical care patients: a prospective randomized study. Crit Care Med 2011, 39:1607-1612. Diagnostic value of chest ultrasound after cardiac surgery: a comparison with chest X-ray and auscultation Bland–Altman plot, comparing two methods, showing 95% limits of agreement from –8.96 to +8.83%ΔSV and the mean diff erence (bias) of measurement is –0.07%ΔSV. Dashed lines, upper and lower limits of agreement (95% CI for repeated measurements). Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). P132 P132 Real-time ultrasound-guided subclavian vein cannulation in cardiac surgery: comparison between short-axis and long-axis techniques F Corradi1, T Manca2, C Brusasco3, F Cocconcelli2, A Agostinelli2, F Benassi2, T Gherli2, A Vezzani2 1Ente Ospedaliero Ospedali Galliera Genova, Italy; 2Azienda Ospedaliero- Universitaria di Parma, Italy; 3Università degli Studi di Genova, Italy Critical Care 2014, 18(Suppl 1):P132 (doi: 10.1186/cc13322) Introduction Central venous catheters play an important role in patient care; however, their use is associated with various complications and more frequently through the subclavian vein (SCV) route. A previous study showed that ultrasound-guided cannulation of the SCV in critical care patients is superior to the landmark method and should be the method of choice in these patients [1]. The aim of this study was to compare short-axis and long-axis approaches for ultrasound-guided subclavian vein cannulation with respect to indicators of success. Methods Eighty-three patients undergoing cardiac surgery and requiring central venous cannulation were randomized to receive long- axis or short-axis ultrasound-guided cannulation of the subclavian vein by a skilled anesthesiologist. First-pass success, unsuccessful placement, number of attempts, number of needle passes, skin and vessel puncture, time to successful catheterization and complications were considered as outcomes. Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Figure 1 (abstract P133). ΔSV-TTE after PLR≥13.6% may predict fl uid responsiveness with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted fl uid responsiveness with sensitivity of 83.33%, lower specifi city of 60% and AUC of 0.64 (95% CI: 0.35 to 0.93). Figure 1 (abstract P133). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods Standard 12-lead and actual V3R to V5R, V7 to V9 lead ECGs at Tokyo Medical University Hospital were successfully recorded and compared with synthesized ECGs at the J point, M point that was defi ned for the point after 1/16 RR interval and T wave amplitude. ECGs of the complete right branch block, complete left branch block and pacing rhythm were excluded. (ECMO). In many circumstances, transoesophageal echocardiography (TOE) is the preferred monitoring tool. It can aid in cannula positioning, especially during double-lumen cannula placement for V-V ECMO, weaning of V-A ECMO and diagnose causes of high-access pressures and circuit fl ow problems. We use TOE as our preferred monitoring equipment before, during and after establishing ECMO. We sought to investigate how often information gained from TOE imaging had a major impact on management decisions. p g y Results A total of 1,216 ECGs were correctly recorded. The diff erences of actual and synthesized at the J point, M point and T wave amplitude were very small. Means of the diff erence ± 2SD were V3R/V4R/V5R/V7/ V8/V9: J point, 17 ± 1/14 ± 1/13 ± 1/12 ± 1/15 ± 1/18 ± 1 μV; M point, 15 ± 1/13 ± 1/12 ± 1/12 ± 1/12 ± 1/13 ± 1 μV; and T wave amplitude, 20 ± 3/32 ± 2/16 ± 2/37 ± 2/39 ± 2/43 ± 3 μV. There were positive correlations between all actual and synthesized ECGs of J point, M point and T wave amplitude (Figure 1). j p g Methods A single-centre observational study at a tertiary referral institution. All patients supported with V-A or V-V ECMO during an 18-month period were included. Routine procedures such as wire position checks during cannulation or information gained to assist weaning from V-A support were not included. g pp Results Twenty patients were supported with either V-A (all peripheral) or V-V ECMO during the observation period. In 12 patients (60%) TOE was instrumental in diagnosing potentially fatal complications or altered clinical management. In three patients on V-A support, afterload reduction and modulation of inotropic support was necessary due to extensive spontaneous echo contrast formation in the left ventricle and stagnant pulmonary blood fl ow; two out of these three patients, immediately after establishing support, required intra-aortic balloon counterpulsation to reverse clot formation around the aortic valve and root. Accuracy of synthesized right-sided/posterior chest lead electrocardiograms e ect oca d og a s Y Saitoh1, Y Goseki2, A Yamashina2 1Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium; 2Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P135 (doi: 10.1186/cc13325) Introduction Right-sided precordial leads (V3R to V5R) and posterior chest leads (V7 to V9) provide important information for the right ventricle and posterior wall. These additional lead electrocardiograms (ECGs) improve diagnostic value in acute coronary syndrome patients [1]. However, these additional electrocardiograms are not routinely recorded due to the time-consuming procedure involved. Recently these synthesized six additional lead ECGs using the standard 12-lead ECG system (Nihonkoden Co. Ltd) have been developed [2,3]. But the accuracy is not clear. The purpose of the present study was to evaluate the accuracy of synthesized ECGs at the ST part. Methods We recruited patients meeting criteria for severe sepsis and septic shock within 24 hours of admission to ICU. After haemodynamic stabilisation, PWV was recorded at inclusion and after 48 hours using dual-channel plethysmography. Severity of illness was assessed with APACHE II and serial SOFA scores, haemodynamic and infl ammatory parameters (CRP, procalcitonin and fi brinogen) recorded. A 28-day follow-up was performed to distinguish between survivors and nonsurvivors. Results Twenty consecutive general ICU patients (six with severe sepsis and 14 with septic shock) were enrolled in the study; median age 59 years (IQR 56.5 to 72), APACHE II score 17 (13 to 20.5), SOFA score 5 (IQR 4 to 9). At 28 days, six patients had died. Median initial PWV was 10.4 (IQR 6.9 to 12.1) m/second in patients with severe sepsis, and 6.8 (IQR 5.3 to 7.5) m/second in patients with septic shock (P = 0.13). After 48 hours, PWV in the severe sepsis and septic shock groups had become similar, 9.3 (IQR 7.3 to 11.1) m/second and 9.2 (IQR 7.8 to 13) m/second respectively (P = 0.96). PWV had signifi cantly increased in survivors (7.8 to 12.3 m/second) (P = 0.04) versus nonsurvivors (6 to 7.8 m/second) (P = 0.69). Higher PWV correlated with increasing systolic pressure and lower CRP levels (r = 0.73, P = 0.01).f Figure 1 (abstract P135). Correlation between actual and synthesized ECG at V7. Conclusion In early sepsis, aortic stiff ness is decreased in patients with greater disease severity, and in survivors increases to median levels within 48 hours. References 1. Zalenski RJ, et al.: Am J Cardiol 1997, 79:1579-1585. 2. Katoh T, et al.: J Nihon Med Sch 2011, 78:22-29. 3. Nakayama M, et al.: J Electrocardiol 2012, 32:221-228. 1. Zalenski RJ, et al.: Am J Cardiol 1997, 79:1579-1585. 1. Zalenski RJ, et al.: Am J Cardiol 1997, 79:1579-1585. 2. Katoh T, et al.: J Nihon Med Sch 2011, 78:22-29. 1. Zalenski RJ, et al.: Am J Cardiol 1997, 79:1579 158 2. Katoh T, et al.: J Nihon Med Sch 2011, 78:22-29. 2. Katoh T, et al.: J Nihon Med Sch 2011, 78:22-29. 3. Nakayama M, et al.: J Electrocardiol 2012, 32:221-228. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Further diagnosis were Avalon cannula misplacement in the blind end of a piggyback cava following liver transplant, collapsing right atrial free wall with ‘sucking down’ into the access cannula (n = 3), pericardial clot formation and tamponade (n = 1), persistent left superior vena cava making planned cannula placement into the right atrium impossible or potentially fatal, diagnosis of patent foraminae ovale providing left atrial decompression and appropriate drainage cannula positioning during liver transplantation on ECMO. Conclusion The ST part of synthesized V3R to V5R and V7 to V9 lead ECGs appears to be highly reliable. Synthesized additional lead ECGs might be useful to diagnose ischemic heart disease. References . Vlachopoulos C, et al.: Circulation 2005, 112:2193-2200. Aortic stiff ness in patients with early sepsis Aortic stiff ness in patients with early sepsis S a u e , abo s s , St ķe , a ags 1Hospital of Traumatology and Orthopaedics, Riga, Latvia; 2Institute of Atomic Physics and Spectroscopy, University of Latvia, Riga, Latvia; 3Riga Stradins University, Riga, Latvia Conclusion In our practice, TOE is an indispensable tool for safe ECMO practice, both during V-A and V-V support. y g Critical Care 2014, 18(Suppl 1):P136 (doi: 10.1186/cc13326) Introduction Acute and chronic systemic infl ammatory conditions are associated with aortic stiff ening. Carotid–femoral pulse wave velocity (PWV), a marker of aortic stiff ness, increases in patients with infl ammatory diseases and independently correlates to levels of C-reactive protein (CRP). The eff ects of massive infl ammatory response in early sepsis on mechanical properties of the aorta have not been investigated. The objective of the current study was to prospectively assess aortic stiff ness in patients with early severe sepsis and septic shock and relate it to infl ammatory and haemodynamic variables and outcome. P134 y xygenation: fancy for enthusiasts or indispensable tool? g g p , Critical Care 2014, 18(Suppl 1):P134 (doi: 10.1186/cc13324) y Results Preliminary reports on 16 patients with satisfactory cardiac windows were analyzed. ΔSV-TTE after PLR ≥13.6% predicts FR with sensitivity of 83.33%, specifi city of 70% and AUC of 0.78 (95% CI: 0.54 to 1.00). Initial PPV ≥11% predicted FR with sensitivity of 83.33% with Introduction Echocardiography is commonly used during both veno- arterial (V-A) and venovenous extracorporeal membrane oxygenation S47 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Accuracy of synthesized right-sided/posterior chest lead electrocardiograms The main factors associated with lower pulse wave velocity are lower systolic pressure and higher CRP levels. The association of high serum CRP levels with low aortic stiff ness in patients with sepsis does not match data described in the literature [1]. Reference Figure 1 (abstract P135). Correlation between actual and synthesized ECG at V7. P135 P135 Accuracy of synthesized right-sided/posterior chest lead electrocardiograms Y Saitoh1, Y Goseki2, A Yamashina2 1Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium; 2Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P135 (doi: 10.1186/cc13325) P136 Aortic stiff ness in patients with early sepsis S Kazune1, A Grabovskis2, E Strīķe3, I Vanags3 1Hospital of Traumatology and Orthopaedics, Riga, Latvia; 2Institute of Atomic Physics and Spectroscopy, University of Latvia, Riga, Latvia; 3Riga Stradins University, Riga, Latvia Critical Care 2014, 18(Suppl 1):P136 (doi: 10.1186/cc13326) Novel technology for non-invasive thoracic fl uid measurement: an animal model comparative study Methods Acute pulmonary edema was induced by intravenous infusion of noradrenaline and dextrane, with stepwise increased dosage. KYMA measurements of LWI were compared with PiCCO extravascular lung volume as the reference gold standard. The experiment continued until maximum increase of EVLW and complete heart failure were reached. Methods Acute pulmonary edema was induced by intravenous infusion of noradrenaline and dextrane, with stepwise increased dosage. KYMA measurements of LWI were compared with PiCCO extravascular lung volume as the reference gold standard. The experiment continued until maximum increase of EVLW and complete heart failure were reached. Results All seven sheep developed pulmonary edema, which was validated by increased LVEDP and EVLW. A linear correlation was found between invasive measurements of EVLW (PiCCO) and non-invasive KLWI. Results All seven sheep developed pulmonary edema, which was validated by increased LVEDP and EVLW. A linear correlation was found between invasive measurements of EVLW (PiCCO) and non-invasive KLWI.l Conclusion KYMA’s fl uid index demonstrated excellent correlation with invasive lung water measurement (R2 = 0.96; P <0.0001) and was able to detect dynamic accumulation of EVLW in a range of 40 to 50 cm3 increments (Figure 1). Since the change in fl uid content between a normal and congested lung ranges between 250 and 500 cm3, the demonstrated sensitivity as refl ected in this study, supports its use for high-resolution and accurate thoracic fl uid monitoring. Novel technology for non-invasive thoracic fl uid measurement: an animal model comparative study p y G Karp1, M Jonas1, T Mandelbaum2, E Kishinevsky1, N Adi1 1Kaplan Medical Center, Rehovot, Israel; 2Sheba Medical Center, Tel Aviv, Israel Critical Care 2014, 18(Suppl 1):P137 (doi: 10.1186/cc13327) Introduction Approximately 15 million people worldwide suff er from congestive heart failure (CHF). The most severe symptom of CHF is pulmonary congestion – an acute increase in extravascular lung fl uid due to acute decompensation of heart failure. To date, no direct, reliable, simple and non-invasive method is available for assessment of thoracic fl uids. Continuous dependable monitoring of pulmonary edema for hospital patients can improve management and reduce duration of hospitalization. KYMA’s solution is based on a matchbox- sized monitoring device, which monitors thoracic fl uid content and trends. The μCor monitor transmits and receives electromagnetic signals that are propagated through tissue layers. Conduction is highly related to the amount of accumulated fl uids in tissues. This trial investigated the correlation between KYMA’s lung water index (LWI) and directly assessed extravascular lung water (EVLW) in the scenario of acute pulmonary edema induced in seven sheep. p p Results A total 649 hours of hemodynamic record were analyzed in 40 patients chosen at random from 121 patients monitored in the last 2 years. We found 22 drops in nCI that were noticed by the nursing staff and led to volume challenge in accordance with the protocol. One-half of these interventions was carried out outside the 2-hour interval of protocol-required monitoring and documentation entry. We identifi ed nine drops in nCI that were not reacted to. None of them happened within the fi rst 4 hours postoperatively and all of them were outside the 2-hour interval of protocol-required monitoring. In fi ve of these episodes the pulse pressure also dropped, suggesting impairment of the reading of the waveform from the arterial line. Conclusion The need for hemodynamic intervention in postoperative care was quite rare in our patients. The drop in nCI was noticed in most of the cases and the patient was treated according to the protocol. However, isolated episodes of nCI drop were missed during the night after the operation. The results were pointed out to the nursing staff and a comparative survey will be conducted. As with any monitoring modality, (non)adherence to the protocol may become the limiting issue to the benefi t for the patient. Pulse wave transit time technique for perioperative non-invasive hemodynamic monitoring J Hruda, M Chobola, M Lukes, P Suk, J Klimes, V Sramek St Anna University Hospital and International Clinical Research Center, Brno, Czech Republic Figure 1 (abstract P137). Pooled correlation between EVLW and KYMA LWI. Figure 1 (abstract P137). Pooled correlation between EVLW and KYMA LWI. p Critical Care 2014, 18(Suppl 1):P139 (doi: 10.1186/cc13329) Critical Care 2014, 18(Suppl 1):P139 (doi: 10.1186/cc13329) Introduction The aim of this work is to evaluate perioperative hemodynamic monitoring for major abdominal surgery using the estimated continuous cardiac output (esCCO) technique available with the NIHON KOHDEN Vismo monitor as compared with the LiDCOrapid hemodynamic monitoring system. y g y Methods The esCCO technique is a novel noncalibrated non-invasive method of cardiac output monitoring. It is based on the estimation of pulse pressure from the speed of pulse wave propagation, which is measured as a time delay between the ECG R-wave and the corresponding peripheral pulse wave detected by pulse oximetry – the delay being called the pulse wave transit time (PWTT) [1]. LiDCOrapid estimates noncalibrated cardiac output (nCO) by pulse wave contour analysis from an arterial line and is a well-established monitoring modality in our department. The patients planned for elective major abdominal surgery with ASA score of III and expected operation duration over 90 minutes are routinely monitored by LiDCOrapid with a perioperative hemodynamic optimalization protocol. In accordance with the protocol, an arterial line was inserted and LiDCOrapid monitoring was started before the induction of anesthesia. At the same time, non-invasive esCCO monitoring was started. Figure 1 (abstract P137). Pooled correlation between EVLW and KYMA LWI. P138 Adherence to the nurse-driven hemodynamic protocol during postoperative care J Klimes, J Hruda, M Lukes, P Suk, V Sramek St Anna University Hospital and International Clinical Research Center, Brno, Czech Republic Critical Care 2014, 18(Suppl 1):P138 (doi: 10.1186/cc13328) Reference Vlachopoulos C, et al.: Circulation 2005, 112:2193-2200. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S48 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P137 Novel technology for non-invasive thoracic fl uid measurement: an animal model comparative study G Karp1, M Jonas1, T Mandelbaum2, E Kishinevsky1, N Adi1 1Kaplan Medical Center, Rehovot, Israel; 2Sheba Medical Center, Tel Aviv, Israel Critical Care 2014, 18(Suppl 1):P137 (doi: 10.1186/cc13327) P137 Methods High-risk patients planned to undergo elective major abdominal surgery were routinely monitored with LiDCOrapid; the monitoring continues overnight after surgery. The target nCI for the postoperative monitoring, as well as hemodynamic interventions in case of its drop, were prescribed by the anesthetist in the protocol upon handover of the patient to the postoperative ICU. The hemodynamic protocol in the ICU was then nurse driven with compulsory recording of patient’s hemodynamic data into the patient’s documentation every 2 hours and at the time of each intervention. Data from the memory of the LiDCOrapid monitor were analyzed using LiDCOview software and compared with patients’ documentation. P140 Validation of cardiac output from Mostcare compared with a pulmonary artery catheter in septic patients S Gopal1, J Pooni1, T Do2, A Karimi1, G Martinelli1 1New Cross Hospital, Wolverhampton, UK; 2University Hopsital of North Staff ordshire NHS Trust, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P140 (doi: 10.1186/cc13330) p p Methods Five pigs weighing 25 to 29 kg were used in the study approved by the local animal use and care committee. The animals were anesthetized, medically paralyzed, intubated and mechanically ventilated. Central venous and arterial catheters were placed for injection of the indicator and connection to the PiCCO system. The PA-S was placed in contact with the skin over the saphenous artery on the animal and adjusted to receive the maximum acoustic signal. The adjustment included the depth of ultrasonic penetration and the exact location on top of the blood vessel. Each indicator dilution was accomplished using three central venous injections of 15 ml normal saline at room temperature. Variation in CO was accomplished by removing up to 50% of the blood volume after splenectomy. The PA-S results were obtained using the empirical regression method. The resulting cardiac output readings from the PA-S were compared with the PiCCO readings. Introduction The Mostcare monitor is a non-invasive cardiac output monitor. It has been well validated in cardiac surgical patients but there is limited evidence on its use in patients with severe sepsis and septic shock [1].i Methods The fi rst 22 consecutive patients with severe sepsis and septic shock in whom the fl oatation of a pulmonary artery catheter was deemed necessary to guide clinical management were included. Cardiac output measurements were simultaneously calculated and recorded from a thermodilution pulmonary artery catheter and from the Mostcare monitor respectively. The two methods of measuring cardiac output were compared by Bland–Altman statistics and linear regression analysis. A percentage error less than 30% was defi ned as acceptable for this study. Results The correlation between the PA-S and the PiCCO system was (R2 = 0.746) over a range of CO from 1 to 6 l/minute. The Bland–Altman analysis demonstrated a bias of –0.05 l/minute and precision of 0.50 l/ minute. y Results Bland–Altman analysis for cardiac output showed a bias of 0.31 l/minute, precision (=SD) of 1.97 l/minute and a percentage error of 62.54%. Linear regression produced a correlation coeffi cient r2 for cardiac output of 0.403. See Figure 1. Conclusion This animal study demonstrated the feasibility of the new PA-S for determination of indicator dilution CO in a limited range. The system showed good correlation with the PiCCO system even in the case of vasoconstriction as a result of blood loss. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P140 P140 Validation of cardiac output from Mostcare compared with a pulmonary artery catheter in septic patients S Gopal1, J Pooni1, T Do2, A Karimi1, G Martinelli1 1New Cross Hospital, Wolverhampton, UK; 2University Hopsital of North Staff ordshire NHS Trust, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P140 (doi: 10.1186/cc13330) P140 Validation of cardiac output from Mostcare compared with a pulmonary artery catheter in septic patients S Gopal1, J Pooni1, T Do2, A Karimi1, G Martinelli1 1New Cross Hospital, Wolverhampton, UK; 2University Hopsital of North Staff ordshire NHS Trust, Stoke-on-Trent, UK Critical Care 2014, 18(Suppl 1):P140 (doi: 10.1186/cc13330) Since the PA-S utilizes transpulmonary indicator dilution, other variables such as intrathoracic blood volume, global end-diastolic volume and extravascular lung water can be calculated. Additionally, continuous CO can be obtained from the optical sensor signal utilizing arterial waveform analysis. References p g Conclusion Compared with thermodilution cardiac output, cardiac output studies obtained from the Mostcare monitor have an unacceptably high error rate. The Mostcare monitor is not a reliable monitoring device to measure cardiac output in patients with severe sepsis and septic shock on an ICU. P141 changes (as with any other noncalibrated cardiac output monitor), but it can still trace the trends of CO changes. Due to its non-invasiveness, esCCO might be the monitoring of choice for high-risk patients undergoing low-risk surgery. Reference Novel non-invasive technology for cardiac output determination U Borg, D Iyer, Q Huang, Y Li, C Baker Covidien, Longmont, CO, USA Critical Care 2014, 18(Suppl 1):P141 (doi: 10.1186/cc13331) 1. Sugo Y, Ukawa T, Takeda S, Ishihara H, Kazama T, Takeda J: A novel continuous cardiac output monitor based on pulse wave transit time. Conf Proc IEEE Eng Med Biol Soc 2010, 2010:2853-2856. Introduction The aim of this animal study was to determine the feasibility and accuracy of a new non-invasive system for determination of cardiac output (CO) compared with a known device (PiCCO; Pulsion Medical). In ICUs and ORs, hemodynamic management using goal- directed therapy has been shown to improve patient outcomes [1,2]. The invasiveness of current technology may prevent clinicians from obtaining hemodynamic information. We developed a non- invasive system for detection of hemodynamic variables utilizing a photo-acoustic sensor (PA-S) and indicator dilution. The PA-S utilizes a combination of a miniature ultrasound sensor, a laser diode and an optical detector to accomplish the measurement. P138 Adherence to the nurse-driven hemodynamic protocol during postoperative care Adherence to the nurse-driven hemodynamic protocol during postoperative care g Results Ten patients were monitored with a total of 141 esCCO–nCO measurement pairs. The correlation coeffi cient between esCCO and nCO was 0.65 and in the Bland–Altman diff erence analysis bias was +1.2 l/minute and 95% limits of agreement were ± 2.6 l/minute. The change of cardiac output between two consecutive measurements detected by LiDCOrapid was detected by esCCO in 80% of cases. Conclusion Hemodynamic monitoring with esCCO yields cardiac output values diff erent from those measured by LiDCOrapid. The reliability of PWTT is questionable when systemic vascular resistance Results Ten patients were monitored with a total of 141 esCCO–nCO measurement pairs. The correlation coeffi cient between esCCO and nCO was 0.65 and in the Bland–Altman diff erence analysis bias was +1.2 l/minute and 95% limits of agreement were ± 2.6 l/minute. The change of cardiac output between two consecutive measurements detected by LiDCOrapid was detected by esCCO in 80% of cases. Introduction The aim of this work is to verify that low incidence of hemodynamic interventions in hemodynamically monitored patients in our postoperative ICU is not caused by insuffi cient attention being paid to the drop of noncalibrated cardiac index (nCI). In the last 2 years there was a need for hemodynamic intervention in 92% of patients with perioperative cardiac output monitoring, while only 31% of these patients needed at least one hemodynamic intervention postoperatively in the ICU. Conclusion Hemodynamic monitoring with esCCO yields cardiac output values diff erent from those measured by LiDCOrapid. The reliability of PWTT is questionable when systemic vascular resistance S49 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P143 Comparison of PiCCO and VolumeView: simultaneous measurement in sepsis pig models H Imahase1, S Inoue1, Y Sakamoto1, T Miyasho2, K Yamashita2 1Saga University Hospital, Saga City, Japan; 2Rakuno Gakuen University, Ebetsu City, Japan Critical Care 2014, 18(Suppl 1):P143 (doi: 10.1186/cc13333) Introduction The aim was to evaluate the performance of arterial pressure-based cardiac output (APCO) [1] and pulse wave transit time- based cardiac output (esCCO) [2] monitors in Thai patients undergoing coronary artery bypass graft surgery (CABG) with cardiopulmonary bypass. Introduction Transpulmonary thermodilution is useful in the critical care. Following PiCCO, VolumeView is now available. Although previous studies concluded that signifi cant correlation was found between VolumeView and PiCCO [1,2], all experiments and data analysis were done by Edwards Lifescience. The aim of the present study was to compare the new VolumeView with PiCCO to clarify their compatibility and to give a neutral answer. y Methods We studied 50 Thai surgical patients undergoing CABG with cardiopulmonary bypass and requiring pulmonary artery catheter and radial artery catheter placement as a standard of clinical care. All patients were measured for APCO using the Vigileo/FloTrac and for esCCO using the esCCO monitoring system. The data were compared with thermodilution cardiac output (TDCO) monitoring as a reference method, simultaneously at pre-induction, post-induction, and every 30 minutes thereafter until the completion of the surgery. The bias and precision were assessed using Bland–Altman analysis. g Methods Six pigs (about 10 kg) were used and we made sepsis models by LPS administration. All pigs were instrumented with a right (Pulsio- Cath) and a left (VolumeView) thermomistor-tipped femoral arterial catheter. The central venous catheter was inserted through the right jugular vein. CO, GEDV and EVLW were measured by the two systems. Results We performed measurements at 57 points. There was a good correlation between the two devices regarding CO and GEDV, but VolumeView showed higher GEDV than PiCCO. Regarding EVLW, there was no signifi cant correlation between two systems. VolumeView showed signifi cantly higher EVLW than PiCCO (Figure 1 and Table 1). Methods Six pigs (about 10 kg) were used and we made sepsis models by LPS administration. All pigs were instrumented with a right (Pulsio- Cath) and a left (VolumeView) thermomistor-tipped femoral arterial catheter. The central venous catheter was inserted through the right jugular vein. CO, GEDV and EVLW were measured by the two systems. Results In total, 310 pairs of simultaneous measurements of APCO versus TDCO and 303 pairs of esCCO versus TDCO were obtained from 50 patients. Both APCO (R = 0.53, P <0.0001) and esCCO values (R = 0.56, P <0.0001) were correlated with TDCO values. p Reference 1. Franchi F, Silvestri R, Cubattoli L, et al.: Comparison between an uncalibrated pulse contour method and thermodilution technique for cardiac output estimation in septic patients. Br J Anaesth 2011, 107:202-208. 1. Franchi F, Silvestri R, Cubattoli L, et al.: Comparison between an uncalibrated pulse contour method and thermodilution technique for cardiac output estimation in septic patients. Br J Anaesth 2011, 107:202-208. 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-936. 2. Marik PE, et al.: Curr Pharm Des 2012, 18:6215-6224. 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-936. 2. Marik PE, et al.: Curr Pharm Des 2012, 18:6215-6224. Figure 1 (abstract P140). Cardiac output Bland–Altman analysis. Figure 1 (abstract P140). Cardiac output Bland–Altman analysis. S50 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P142 Referencesi References 1. Biancofi ore G, et al.: Br J Anaesth 2009, 102:47-54. 2. Ishihara H, et al.: J Clin Monit Comput 2004, 18:313-320. P142 Performance of pulse contour and pulse wave transit time-based continuous cardiac output analyses: clinical validation of two methods in Thai patients undergoing cardiac surgery P Wacharasint, P Kunakorn, P Pankongsap Phramongkutklao Hospital, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P142 (doi: 10.1186/cc13332) P143 P143 Comparison of PiCCO and VolumeView: simultaneous measurement in sepsis pig models H Imahase1, S Inoue1, Y Sakamoto1, T Miyasho2, K Yamashita2 1Saga University Hospital, Saga City, Japan; 2Rakuno Gakuen University, Ebetsu City, Japan Critical Care 2014, 18(Suppl 1):P143 (doi: 10.1186/cc13333) P144f P144 Eff ects of the restrictive fl uid strategy on postoperative pulmonary and renal function following pulmonary resection surgery M Arslantas1, H Batirel2, B Bilgili1, H Kara2, B Yıldızeli2, M Yuksel2, K Bostanci2, A Kararmaz1, I Cinel1 1Marmara University Pendik Education and Research Hospital, Istanbul, Turkey; 2Marmara University School of Medicine, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P144 (doi: 10.1186/cc13334) Introduction Goal-directed therapy used in the perioperative period of patients undergoing cardiac surgery shortens the length of ICU stay [1]. We aimed to compare the postoperative results of the liberal and restrictive fl uid strategy used in patients undergoing pulmonary resection surgery (PRC).l y Methods We have been using the restrictive fl uid strategy since March 2013 in our institute. Patients who were on the liberal fl uid regime were analyzed retrospectively. From March 2013 until today, patients who were on restrictive fl uid strategy were analyzed prospectively. A total of 125 patients were included in the study. Age, duration of anesthesia, type of fl uids given intraoperatively, fl uid index (ml/kg/hour), fl uid intake/output balance, creatinine and lactate levels were compared with pulmonary and renal morbidity, and length of stay in ICU, using multivariate analysis.i Methods We conducted a prospective audit of FB and renal function in patients undergoing major elective surgery admitted to critical care over a 28-day period. Fluid overload was defi ned as: (positive FB) / (weight × 0.6) × 100%. Results Thirty-two patients (56% female, median age 64) were studied over a median of 3 critical care days (range 1 to 7). Total FB was +3.9 l at discharge; however, 75% of this occurred intraoperatively so that positive FB in critical care was only +390 ml/day. Patients had median 9% fl uid overload at discharge. Two patients had transient AKI stage 1. Overall SCr decreased signifi cantly from preoperatively to discharge (P = 0.003), median 73 to 55 μmol/l, with decreases occurring both postoperatively and during critical care stay (Figure 1).l Results A signifi cant correlation (P <0.05) was established between the amount of crystalloid given intraoperatively, fl uid index and fl uid balance with pulmonary morbidity (n = 52). The fl uid index and inotropes usage were correlated with the postoperative creatinine levels (P <0.05). There was no correlation between perioperative lactate levels with fl uid balance and fl uid index. Reference Reference 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-836. 1. Goepfert MS, et al.: Anesthesiology 2013, 119:824-836. P144f Intraoperative blood loss, the amount of given crystalloid, colloid, blood and FFP, fl uid balance, duration of anesthesia and postoperative blood transfusion were found to be related (P <0.05) with the length of ICU stay. Four percent of the patients required renal replacement therapy and the overall mortality was 0.8%. y g y g Conclusion We achieved near-neutral fl uid balances after admission, but did not resolve intraoperative fl uid accumulation. SCr fell signifi cantly, even after there was no further net fl uid accumulation. This suggests a decrease in creatinine generation after major surgery, rather than fl uid expansion, may largely account for sCr decreases in these patients. Conclusion To reduce the morbidity of patients undergoing major surgery, the protocols of using the restrictive fl uid strategy in the perioperative period and simultaneous protection of end organs, especially the kidneys, is currently the subject of this discussion. We observed that the restrictive fl uid strategy did not lead to global organ hypoperfusion, which was monitored by lactate. Even though there was a negative correlation between the fl uid index with creatinine levels and renal failure, the need for renal replacement therapy was observed only in one case. As a conclusion, the postoperative pulmonary morbidity and length of ICU stay can be reduced in patients who undergo PRC by using the restrictive fl uid strategy (4.2 ± 0.3 ml/kg/hour), without causing any vital organ dysfunction. References 1. Bendjelid K, et al.: Crit Care 2010, 14:R209. 2. Kiefer N, et al.: Crit Care 2012, 16:R98. P143 Comparison of PiCCO and VolumeView: simultaneous measurement in sepsis pig models H Imahase1, S Inoue1, Y Sakamoto1, T Miyasho2, K Yamashita2 1Saga University Hospital, Saga City, Japan; 2Rakuno Gakuen University, Ebetsu City, Japan Critical Care 2014, 18(Suppl 1):P143 (doi: 10.1186/cc13333) Either of the changes in APCO (R = 0.63, P <0.0001) or any changes in esCCO (R = 0.60, P <0.0001) were correlated with changes in TDCO. For APCO relative to TDCO, the bias, precision, and the limits of agreement were 0.70, 1.63, and –2.5 to 3.9 l/minute, while those of esCCO were 1.20, 1.59, and –1.9 to 4.3 l/ minute, respectively. Comparisons of the bias of APCO and esCCO revealed a level of signifi cance of P <0.001. j g y y Results We performed measurements at 57 points. There was a good correlation between the two devices regarding CO and GEDV, but VolumeView showed higher GEDV than PiCCO. Regarding EVLW, there was no signifi cant correlation between two systems. VolumeView showed signifi cantly higher EVLW than PiCCO (Figure 1 and Table 1). Table 1 (abstract P143). Explanation of abbreviations Table 1 (abstract P143). Explanation of abbreviations CO GEDV EVLW PiCCO PCO PGEDV PEVLW VolumeView ECO EGEDV EEVLW Table 1 (abstract P143). Explanation of abbreviations i Conclusion Despite the overestimation of cardiac output measure- ments, APCO and esCCO calibrated with patient information has shown an acceptable trend as compared with TDCO in Thai patients undergoing CABG with cardiopulmonary bypass. Compared with esCCO, APCO demonstrated no signifi cant diff erences of precision; however, a lower mean bias was exhibited. Figure 1 (abstract P143). CO, GEDV, EVLW correlation and Bland–Altman analysis. Figure 1 (abstract P143). CO, GEDV, EVLW correlation and Bland–Altman analysis. S51 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P145). Serum creatinine changes after major surgery. Conclusion Our data analysis showed that PiCCO and VolumeView were not the same. Neither the normal values of two devices nor the rates of change were same. Careful attention should be paid to interpret the data obtained from two systems. P145 Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery R Rajendram, JR Prowle Barts Health NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P145 (doi: 10.1186/cc13335) P146 Very limited usefulness of pulse pressure variation as a predictor of volume responsiveness in critically ill septic patients GG Gavriilidis, IK Kostakou, VT Tsagari, AK Kyriakoudi, NK Koulouris, AK Koutsoukou University of Athens, Sotiria Chest Hospital, Athens, Greece Critical Care 2014, 18(Suppl 1):P146 (doi: 10.1186/cc13336) Introduction The aims of the study are to assess the usefulness of pulse pressure variation (PPV) as a predictive marker of fl uid responsiveness and to estimate the value of central venous–arterial diff erence of carbon dioxide (PCO2cv-a) to predict the outcome of critically ill septic patients. The question of whether a septic patient needs fl uids or not is crucial. Although PPV is a very reliable predictor of volume responsiveness, there are many limitations for its application. Cardiac arrhythmia, spontaneous breathing and low tidal volume ventilation prevent the extended use of this index. Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery Methods This is a post-hoc analysis of data from a prospective observational study [1], which included a population of patients with severe sepsis or septic shock as the main reason for ICU admittance. After an echo for the assessment of the cardiac systolic performance, we measured PPV, central venous oxygen saturation, blood gases from arterial and central venous lines, central venous pressure, systolic, diastolic, mean and pulse pressures, before and after volume challenge. We calculated changes in pulse pressure before and after volume challenge, and an increase of 9% was used as criterion to defi ne volume responsiveness [2]. R Rajendram, JR Prowle Barts Health NHS Trust, London, UK Introduction The rationale for perioperative fl uid therapy has been to preserve organ perfusion. However, conservative postoperative fl uid balance (FB) is now encouraged to avoid the eff ects of iatrogenic fl uid overload. In addition, fl uid expansion has been described as a confounder of acute kidney injury (AKI) diagnosis by dilution serum creatinine (SCr). S52 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 increased the RI in the right and left kidneys, from 0.6 ± 0.04 and 0.6 ± 0.05 to 0.7 ± 0.1 and 0.68 ± 0.15 (P <0.05), respectively. Results Among 72 patients (71% men, APACHE II score 23.2) included in this study, 41 (57%) were responders. Due to spontaneous breathing and cardiac arrhythmia we were able to calculate PPV only in 18 patients (25%). Moreover, only eight (11%) calculations of PPV proved useful because of the application of low tidal ventilation (4 to 6 ml/kg ideal body weight) in the remaining 10 patients. We did not fi nd any value of the PCO2cv-a to predict the outcome of the patients (mortality 26.3%), since the diff erence between survivors and nonsurvivors was not statistically signifi cant (7.7 vs. 7.9, P >0.05). Conclusion These preliminary results showed that Doppler renal RI was aff ected equally in both kidneys during HUT, suggesting an eff ect of hemodynamic alterations during our model of central hypovolemia. References 1. Schnell D, et al.: Renal perfusion assessment by renal Doppler during fl uid challenge in sepsis. Crit Care Med 2013, 41:1214. g p 2. Lerolle N, et al.: Renal failure in septic shock: predictive value of Doppler- based renal arterial resistive index. Intensive Care Med 2006, 32:1553. g p 2. Eff ects of central hypovolemia induced by tilt table on the Doppler- based renal resistive index in healthy volunteers A Sommese, A Lima, J Van Bommel, J Bakker Erasmus MC, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P147 (doi: 10.1186/cc13337) Methods We randomised 50 high-risk patients who underwent major abdominal surgery. Tissue oxygenation was monitored at the thenar eminence using near-infrared spectroscopy. All patients were treated according to a standard care algorithm. In addition, patients in the intervention group received dobutamine if necessary to keep tissue oxygenation ≥80%. Data were recorded continuously and complications were recorded during the hospital stay with a maximum of 28 days. A Sommese, A Lima, J Van Bommel, J Bakker Erasmus MC, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P147 (doi: 10.1186/cc13337) Introduction The renal resistive index (RI) determined by Doppler ultrasonography allows a semiquantitative evaluation of kidney vasculature at the bedside. Interpretation of the RI in clinical practice is diffi cult due to interaction with cardiac output, heart rate (HR) and blood pressure [1,2]. The impact of global hemodynamics on the RI remains to be evaluated. This study aims to investigate the relationship between the RI and changes in central hemodynamic during a central hypovolemia model in healthy volunteers (HV). Results The number of complications tended to be lower in the intervention group (11 vs. 20). Eleven patients in the intervention group had no complication, versus seven in the control group. There was no signifi cant diff erence between groups in length of stay in ICU or in hospital. Administration resulted in a 5% increase of tissue oxygenation. The cardiac index increased 0.3 (0.0 to 0.6) l/minute/m2 (Figure 1). The overall protocol adherence was 94%. yp y Methods Eleven healthy volunteers (27 ± 8 years; eight male) participated in this study. Two diff erent models were performed: the fi rst model was performed by applying the head-up tilt (HUT) test. The complete maneuver was done by a 10-minute step that consisted of tilting the table from a supine position (Sup) to an angle of 70° (HUT) and back to supine (Sup’). The second model was performed by applying three consecutive valsalva maneuvers. Global hemodynamics included stroke volume (SV), HR, and mean arterial pressure, which were continuously measured non-invasively with a Finometer. At least three RI readings were obtained and averaged from the right and left kidneys in all HV. g p Conclusion Intraoperative optimisation of tissue oxygenation will potentially result in better outcome after high-risk abdominal surgery. The protocol used may be considered feasible for clinical practice. Reference 8 Tissue oxygenation as a target for goal-directed therapy in high-risk surgery l h 1. Gavriilidis G, et al.: Central venous oxygen saturation does not predict fl uid responsiveness in critically ill septic patients Intensive Care Med 2013, S2:0949. PA Van Beest, JJ Vos, M Poterman, AF Kalmar, TW Scheeren University Medical Center Groningen, the Netherlands PA Van Beest, JJ Vos, M Poterman, AF Kalmar, TW Scheeren University Medical Center Groningen, the Netherlands Critical Care 2014, 18(Suppl 1):P148 (doi: 10.1186/cc13338) University Medical Center Groningen, the Netherlands 2. Preau S, et al.: Passive leg raising is predictive of fl uid responsiveness in spontaneously breathing patients with severe sepsis or acute pancreatitis Crit Care Med 2010, 38:819-825. 2. Preau S, et al.: Passive leg raising is predictive of fl uid responsiveness in spontaneously breathing patients with severe sepsis or acute pancreatitis Crit Care Med 2010, 38:819-825. y g Critical Care 2014, 18(Suppl 1):P148 (doi: 10.1186/cc13338) Introduction Tissue hypoxia occurs frequently during surgery and may contribute to postoperative organ dysfunction [1]. We hypothesised that intraoperative optimisation of tissue oxygenation reduces postoperative complications and evaluated the feasibility of the optimisation protocol used. Perioperative fl uid balance and postoperative changes in serum creatinine in patients admitted to critical care after elective major surgery Lerolle N, et al.: Renal failure in septic shock: predictive value of Doppler- based renal arterial resistive index. Intensive Care Med 2006, 32:1553. i Conclusion The usefulness of PPV, the marker with the best performance in the prediction of volume responsiveness, due to its limitations, is very limited. Eff ects of central hypovolemia induced by tilt table on the Doppler- based renal resistive index in healthy volunteers Eff ects of central hypovolemia induced by tilt table on the Doppler- based renal resistive index in healthy volunteers A Sommese, A Lima, J Van Bommel, J Bakker Erasmus MC, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P147 (doi: 10.1186/cc13337) p Reference 1. Scheeren TW, et al.: Goal-directed intraoperative fl uid therapy guided by stroke volume and its variation in high-risk surgical patients: a prospective multicentre study. J Clin Monit Comput 2013, 27:225-233. y Results All HV had a signifi cant decrease of SV from 83 ± 17 ml to 63 ± 14 ml and an increase in HR from 67 ± 10 bpm to 88 ± 13 bpm during the HUT. Figure 1 shows the temporal changes of mean RI in both kidneys. A signifi cant decrease in the RI in both kidneys was seen during HUT. After the move back to supine, RI returned to baseline values, with a signifi cant variation of RI in the early measurements on the right kidney compared with late measurements on the left kidney (0.67 ± 0.05 vs. 0.61 ± 0.05, P <0.05). Valsalva maneuvers signifi cantly Figure 1 (abstract P148). Evolution of individual patient values (thin lines) and the average values (thick lines) of the tissue oxygenation (StO2) and the relative change of cardiac index (CI), delta CI. All graphs are synchronised at the moment of the fi rst dobutamine administration (time = 0; arrow). Values are shown from 10 minutes before dobutamine administration until 45 minutes after. Figure 1 (abstract P147). Doppler-based renal resistive index in both kidneys during HUT. Figure 1 (abstract P147). Doppler-based renal resistive index in both kidneys during HUT. Figure 1 (abstract P148). Evolution of individual patient values (thin lines) and the average values (thick lines) of the tissue oxygenation (StO2) and the relative change of cardiac index (CI), delta CI. All graphs are synchronised at the moment of the fi rst dobutamine administration (time = 0; arrow). Values are shown from 10 minutes before dobutamine administration until 45 minutes after. Figure 1 (abstract P147). Doppler-based renal resistive index in both kidneys during HUT. Figure 1 (abstract P147). Doppler-based renal resistive index in both kidneys during HUT. S53 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P149 Methods In a period of 1 year, 42 adult patients were admitted to the ICU in septic shock. All patients were treated according to the guidelines of the Surviving Sepsis Campaign International Guidelines for Management of Severe Sepsis and Septic Shock 2012 [2]. p Reference p p g Results Sixteen patients (38%) had a change in DIIVC <18%, on average 11 ± 5%, and when subjected to fl uid challenge did not have an increase in CI >15%, average 8 ± 3%, not proving to be a fl uid responder. Twenty- six patients (62%) had a change in DIIVC >18%, on average 45 ± 22%, and when subjected to fl uid challenge had an increase of the CI >15%, on average 31 ± 8%, proving to be a fl uid responder. g p gl p Conclusion We have shown that the DIIVC can predict the response of patients to fl uid challenge regardless of the values of CVP and then distinguish fl uid responders from nonresponder patients. This can help us in the choice of treatment for patients in septic shock. Referencesf y Results We included 21 patients in each group. There was no signifi cant diff erences in the fl uid balance between the control and study group after 24 hours (5.0 ± 2.9 l vs. 3.6 ± 2.7 l, P = 0.11) and 48 hours (5.7 ± 3.5 l vs. 4.8 ± 3.7 l, P = 0.39). However, compliance with the protocol was poor (56%). After 2/11 positive tests, fl uid was not administered; and after 21/39 tests, fl uid was administered despite a negative test result (Figure 1). 1. Griff ee J et al.: The role of echocardiography in hemodynamic assessment of septic shock. Crit Care Clin 2010, 26:365-382. 1. Griff ee J et al.: The role of echocardiography in hemodynamic assessment of septic shock. Crit Care Clin 2010, 26:365-382. 2. Dellinger RP et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013, 41:580-637. 2. Dellinger RP et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013, 41:580-637. Figure 1 (abstract P149). SVI, stroke volume index; –, negative PLR; +, positive PLR; fl uid 250 cm3 RL solution. Use of pulse pressure variation and stroke volume variation in spontaneously breathing patients to assess dynamic arterial elastance and to predict arterial pressure response to fl uid administration M Cecconi1, MI Monge García2, M Gracia Romero2, J Mellinghoff 2, F Caliandro2, RM Grounds2, A Rhodes2 1St George’s Hospital, London, UK; 2St George’s Healthcare NHS Trust and St George’s University of London, UK Critical Care 2014, 18(Suppl 1):P151 (doi: 10.1186/cc13341) M Cecconi1, MI Monge García2, M Gracia Romero2, J Mellinghoff 2, F Caliandro2, RM Grounds2, A Rhodes2 1St George’s Hospital, London, UK; 2St George’s Healthcare NHS Trust and St George’s University of London, UK Critical Care 2014, 18(Suppl 1):P151 (doi: 10.1186/cc13341) Figure 1 (abstract P149). SVI, stroke volume index; –, negative PLR; +, positive PLR; fl uid 250 cm3 RL solution. Introduction Dynamic arterial elastance (Eadyn), defi ned as the pulse pressure variation (PPV) to stroke volume variation (SVV) ratio, has been suggested as a predictor of the arterial pressure response to fl uid administration. Rather than a steady-state assessment, Eadyn depicts the actual slope of the pressure–volume relationship providing a dynamic evaluation of the arterial load. So the higher the Eadyn value, the more likely arterial blood pressure is to improve after fl uid challenge (FC). The aim of this study was to assess the eff ectiveness of Eadyn, measured non-invasively in preload-dependent, spontaneously breathing patients. Conclusion The implementation of a PLR-driven protocol of fl uid administration did not change mean fl uid balances after the fi rst 48 hours. Noncompliance with the protocol was an important confounder. Reference 1. Michard F, et al.: Predicting fl uid responsiveness in ICU patients. A critical analysis of the evidence. Chest 2000, 121:2000-2008. Methods Patients admitted postoperatively and monitored with the Nexfi n monitor were enrolled in the study. Patients were included if they were spontaneously breathing and had an increase in cardiac output ≥10% after a FC. They were classifi ed according to the increase in mean arterial pressure (MAP) after FC into MAP-responders (MAP increase ≥10%) and MAP-nonresponders (MAP increase <10%). Eadyn was calculated from the PPV and SVV values obtained from the monitor. Results A total of 34 FCs from 26 patients were studied. Seventeen FCs (50%) induced a positive MAP response. Preinfusion Eadyn was signifi cantly higher in MAP-responders (1.39 ± 0.41 vs. 0.85 ± 0.23; P = 0.0001) (Figure 1). Preinfusion Eadyn predicted a positive MAP response to FC with an area under the ROC curve of 0.92 ± 0.04 of SE (95% CI: 0.78 to 0.99; P <0.0001). A preinfusion Eadyn value ≥1.06 (grey zone: 0.9 to 1.15) discriminated MAP-responders with a sensitivity and specifi city of 88.2% (95% CI: 64 to 99%).i P151 Use of pulse pressure variation and stroke volume variation in spontaneously breathing patients to assess dynamic arterial elastance and to predict arterial pressure response to fl uid administrationf p Reference All patients were mechanically ventilated and subjected to transesophageal echocardiography in bicavale projection, calculating the DIIVC through the M-mode by measuring the change in diameter with the acts of positive pressure ventilation and using the formula: (100 × (maximum diameter – minimum diameter)) / minimum diameter. A value >18% was considered a predictor of an increase in CI >15%. The CI was measured continuously through use of the pressure recording analytical method on a Mostcare Vytech monitor. All patients were subjected to a bolus of 500 ml crystalloid through a central venous catheter only after reaching central venous pressure (CVP) ≥10 mmHg during treatment. The results were expressed as the mean with standard deviation and percentage. Results Sixteen patients (38%) had a change in DIIVC <18%, on average 11 ± 5%, and when subjected to fl uid challenge did not have an increase in CI >15%, average 8 ± 3%, not proving to be a fl uid responder. Twenty- six patients (62%) had a change in DIIVC >18%, on average 45 ± 22%, and when subjected to fl uid challenge had an increase of the CI >15%, on average 31 ± 8%, proving to be a fl uid responder. Why measurements do (not) work: the human factor A Rameau, NA Bruins, P Egbers, MA Kuiper, EC Boerma Medical Center Leeuwarden, the Netherlands Critical Care 2014, 18(Suppl 1):P149 (doi: 10.1186/cc13339) Introduction Passive leg raising (PLR) has been suggested as a simple diagnostic tool to guide fl uid administration in critically ill patients [1]. Although the basic principles of PLR have been well studied, little is known about the impact of the introduction of this technique in daily clinical practice. The aim of the study was to describe the changes in fl uid balance of ICU patients before and after the introduction of PLR, and to determine the compliance of medical personnel with a PLR- driven protocol of fl uid administration. pl Methods In this single-centre prospective study, mixed ICU patients equipped with a PiCCO system received fl uid therapy on the basis of PLR test results in the fi rst 48 hours of treatment, after careful introduction of a new PLR-driven protocol. Exclusion criteria were increased abdominal pressure, fracture of leg or pelvis, deep vein thrombosis, head trauma and pregnancy. The control group existed of patients admitted to the ICU 1 year prior to the introduction of the protocol. P152 Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery 2 2 P153 Kinetics of volume expansion during a fl uid challenge H Aya, A Rhodes, M Grounds, M Cecconi St George’s Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P153 (doi: 10.1186/cc13343) Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery H Merdji1, P Biston2, M Piagnerelli2 1CHU de Reims, France; 2CHU de Charleroi, Belgium Critical Care 2014, 18(Suppl 1):P152 (doi: 10.1186/cc13342) l p H Merdji1, P Biston2, M Piagnerelli2 j , , g 1CHU de Reims, France; 2CHU de Charleroi, Belgium g Critical Care 2014, 18(Suppl 1):P152 (doi: 10.1186/cc13342) Introduction According to Guyton and colleagues [1], expansion of blood volume can increase cardiac output (CO) in so far as the change in volume aff ects the mean systemic fi lling pressure (Pmsf). However, rapid stress relaxation occurs after acute intravascular volume expansion so that the eff ect of volume on Pmsf and CO may disappear after a few minutes. The objective of the present study is to describe the extent of the haemodynamic changes generated by a fl uid challenge during 10 minutes on critically ill patients. Introduction Recent studies showed that the plethysmographic variability index (PVI), a dynamic index resulting from cardiopulmonary interactions, could predict fl uid responsiveness (FR) in mechanically ventilated patients during general anesthesia and in the ICU [1,2]. In this study, we aimed to compare, in patients after cardiac surgery, the clinical utility of the PVI versus traditional statics and dynamics indices to predict FR. p Methods We prospectively enrolled 52 consecutive adult patients in sinus rhythm and mechanically ventilated (tidal volume of 8 (7.5 to 8.5) ml/kg ideal body weight) admitted to the ICU of CHU de Charleroi (Belgium) after scheduled cardiac surgery. Before and after fl uid challenge (FC), we measured: heart rate (HR), BP, PVI (Masimo Corporation, Irvine, CA, USA) and PPV. PAOP and cardiac index (CI) were estimated by Swan–Ganz catheter (Edwards Lifesciences LLC, Irvine, CA, USA). Patients with an increase in CI of at least 15% after FC and subsequently were considered responders (R). Continuous variables were analyzed with Mann–Whitney U tests. Categoric variables were analyzed with the Fisher’s exact test. Correlations were assessed by Spearman’s correlation. The discriminatory ability of each haemodynamic parameter to determine FR was assessed by area under the receiver operating characteristic curve (AUC), between R and nonresponders (NR). P152 Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery 2 2 P <0.05 was considered statistically signifi cant.ii g y Methods Patients admitted to the ICU were monitored with a calibrated LiDCOplus (LiDCO, UK) and Navigator (Applied Physiology, Australia) to estimate Pmsf analogue (Pmsa). Then 250 ml Hartmann’s solution or Volplex was infused over 5 minutes. Data were recorded automatically from baseline to 10 minutes after the end of fl uid infusion. The time to maximum response and percentage change between baseline and last value of diff erent haemodynamic parameters are also reported. f Results Sixty fl uid challenges were studied in 34 patients, with a mean duration of 5.3 minutes (± 2.5). In 22 events (37%), CO increased more than 10% (responders). The change between baseline and last value was greater in nonresponders for heart effi ciency (Eh) (–9.2% ± 9.7 vs. –3.1% ± 13.9, P = 0.05) but not in other haemodynamic variables. Time to maximal response on CO was 2 minutes after the end of the infusion (Figure 1).fl g Conclusion Stress relaxation damps down the eff ect of a fl uid challenge after 10 minutes except in terms of heart effi ciency. The eff ect of a fl uid challenge should be assessed up to 2 minutes after the end of fl uid infusion. Failure to do so may mislead clinicians about the patient’s fl uid responsiveness. i Results Twenty-fi ve (48%) patients were classifi ed as R. Before FC, R and NR patients were comparable except for HR (91 (83 to 108) for R vs. 80 beats/minute (72 to 89) for NR; P = 0.009), PVI (18 (12 to 26) for R vs. 12% (9 to 15) for NR; P = 0.003), PAOP (8 (6 to 9) for R vs. 11 mmHg (8 to 12) for NR; P = 0.001), and PPV (13 (8 to 19) for R vs. 10% (4 to 13) for NR; P = 0.086). PVI and delta PP before FC were correlated (r = 0.53, P <0.0001). AUC showed a better prediction of FR for PVI (AUC = 0.74, P <0.001) than for PPV (AUC = 0.64, P = 0.05). Nevertheless, the sensitivity (Se) and specifi city (Sp) of both indices were low: for a PPV over 13%, Se = 48% (0.301 to 0.665),and Sp = 82% (0.627 to 0.921); for a PVI over 12%, Se = 68% (0.482 to 0.828) and Sp = 69% (0.498 to 0.835). 1. Cannesson et al.: Br J Anaesth 2008, 101:200-206. 2. Loupec et al.: Crit Care Med 2011, 39:294-299. 1. Cannesson et al.: Br J Anaesth 2008, 101:200-206. 2. Loupec et al.: Crit Care Med 2011, 39:294-299. Fluid responsiveness in septic shock F Righetti, G Castellano St Boniface Hospital Verona, Italy p , y Critical Care 2014, 18(Suppl 1):P150 (doi: 10.1186/cc13340) y Critical Care 2014, 18(Suppl 1):P150 (doi: 10.1186/cc13340) Introduction The correct assessment of volume status and prediction of response to fi lling fl uid challenge is of crucial importance in the patient with septic shock. A patient is a fl uid responder if there is an increase in cardiac index (CI) >15% in response to the fl uid challenge. Identifying which patient will be a fl uid responder is crucial. The aim of this prospective study is to evaluate which patient is a fl uid responder in septic shock using dynamics echocardiography with the distensibility index of the inferior vena cava (DIIVC), which has been shown to be predictive of an increase in CI [1]. i Conclusion Non-invasive dynamic arterial elastance, defi ned as the PPV to SVV ratio, predicted the arterial pressure increase to fl uid administration in spontaneously, preload-dependent patients. S54 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P151). Distribution of individual arterial load parameters at preinfusion time. Figure 1 (abstract P151). Distribution of individual arterial load parameters at preinfusion time. 1. Cannesson et al.: Br J Anaesth 2008, 101:200-206. 2. Loupec et al.: Crit Care Med 2011, 39:294-299. P152 Accuracy of the plethysmographic variation index as a predictor of fl uid responsiveness after cardiac surgery 2 2 As for PAOP, increasing values are associated with NR (AUC = 0.22, P <0.0001). Figure 1 (abstract P153). Change of CO during and after a fl uid challenge. BL, baseline; end, end of fl uid infusion. Figure 1 (abstract P153). Change of CO during and after a fl uid challenge. BL, baseline; end, end of fl uid infusion. Conclusion The PVI appears to be useful to predict FR in mechanically ventilated patients after cardiac surgery. However, Se and Sp remain low in the type of patients with relative euvolemia and cardiac dysfunction. References Conclusion The PVI appears to be useful to predict FR in mechanically ventilated patients after cardiac surgery. However, Se and Sp remain low in the type of patients with relative euvolemia and cardiac dysfunction. References Figure 1 (abstract P153). Change of CO during and after a fl uid challenge. BL, baseline; end, end of fl uid infusion. S55 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P156 P156 BXL 628 ameliorates toxicity of lactated Ringer in HK-2 human renal proximal tubule cells in a hypovolemia mimicking model YT Huang, CC Cheng, TC Lin, PC Lai Buddhist Tzu Chi General Hospital, Hualien, Taiwan Critical Care 2014, 18(Suppl 1):P156 (doi: 10.1186/cc13346) p Methods Patient records (APACHE II, length of stay (LOS), CVP, DFB, vasopressor and ventilator needs) were retrospectively collaborated, and a randomly-assigned 100 (63 men and 37 women, age 64.2 ± 15.5 years) were statistically analyzed for survival function. Methods Patient records (APACHE II, length of stay (LOS), CVP, DFB, vasopressor and ventilator needs) were retrospectively collaborated, and a randomly-assigned 100 (63 men and 37 women, age 64.2 ± 15.5 years) were statistically analyzed for survival function. Results The mean APACHE II score was 23.6 ± 7.7, LOS was 9.7 ± 10.0 days, intubated period was 6.4 ± 8.6 days, vasopressor period was 4.7 ± 5.5 days, CVP was 10.5 ± 5.5 mmHg, DFB was 1,147.9 ± 1,157.6 ml, and 42 survived. Kaplan–Meier survival and COX regression analysis showed that CVP levels of 6 to 9 mmHg and DFB +800 to +900 ml, but not above, signifi cantly predicted survival, and also shorter LOS, intubated days and lower vasopressor needs with earlier discharge possibility. On the other hand, over-increased DFB and CVP levels strictly correlated with longer LOS and higher mortality rates, and the fi rst 24-hour mean fl uid balance alone was surprisingly not predictive. Conclusion Fluid resuscitation therapy is a double-edge-sword. (1) Despite lower volumes, higher volumes also increase mortality. (2) Overall, DFB seemed more important than the fi rst 24-hour DFB. References Results The mean APACHE II score was 23.6 ± 7.7, LOS was 9.7 ± 10.0 days, intubated period was 6.4 ± 8.6 days, vasopressor period was 4.7 ± 5.5 days, CVP was 10.5 ± 5.5 mmHg, DFB was 1,147.9 ± 1,157.6 ml, and 42 survived. Kaplan–Meier survival and COX regression analysis showed that CVP levels of 6 to 9 mmHg and DFB +800 to +900 ml, but not above, signifi cantly predicted survival, and also shorter LOS, intubated days and lower vasopressor needs with earlier discharge possibility. On the other hand, over-increased DFB and CVP levels strictly correlated with longer LOS and higher mortality rates, and the fi rst 24-hour mean fl uid balance alone was surprisingly not predictive. Fluid challenge with shock V I l O M itical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) Introduction The latest sepsis guideline has emphasized early resuscitative fl uid management [1]. Early goal-directed therapy (EGDT) has been shown to improve 28-day mortality in recent studies [2,3]. This strategy was based on improving tissue perfusion and oxygenation in spite of other supportive and therapeutic measures. Technically and historically, central venous pressure (CVP) measurement is one of the most dependent methods to estimate fl uid responsiveness and intravascular volume status on resuscitation. The Surviving Sepsis Campaign (SSC) guidelines recommended goal levels of CVP 8 to 12 mmHg in order to obtain appropriate tissue perfusion [1]. In this study, we objected to re-evaluate eff ectiveness of a fl uid resuscitation strategy in sepsis, comparing the eff ect of patients’ daily fl uid balances (DFB) and CVP on patients’ survival. may lead to undercorrection of fi brinogen in critical bleeding situations. The aggravated response on the FibTEM-MCF may better refl ect HES eff ects on clot structure. P156 Concl sion Fl id res scitation therap is a do ble edge s ord (1) Introduction Lactated Ringer (L/R) for resuscitation of hemorrhagic shock is suggested by the ATLS program. However, prior studies showed that resuscitation with L/R was associated with more kidney damage in rats with severe hemorrhagic shock. The direct eff ects of L/R on human renal tubule cells have not been reported. p Methods Human proximal renal tubular cell line HK-2 was used. Viability was examined by MTT assay. An additional equal volume of phosphate- buff ered saline (PBS) served as control. Addition of 200 nM dipyridyl and 1 mM H2O2 served as conditions of hypoxia and oxidative stress, respectively. Patterns of cell death were observed by fl ow cytometry. Results To imitate early resuscitation in hypovolemic shock, medium was replaced with 40% v/v of L/R with dipyridyl plus H2O2 for 4 hours, followed by replacing with complete medium for 44 hours. In such Methods Human proximal renal tubular cell line HK-2 was used. Viability was examined by MTT assay. An additional equal volume of phosphate- buff ered saline (PBS) served as control. Addition of 200 nM dipyridyl and 1 mM H2O2 served as conditions of hypoxia and oxidative stress, respectively. Patterns of cell death were observed by fl ow cytometry. R lt T i it t l it ti i h l i h k di il Conclusion Fluid resuscitation therapy is a double-edge-sword. (1) Despite lower volumes, higher volumes also increase mortality. (2) Overall, DFB seemed more important than the fi rst 24-hour DFB. References 1. Dellinger RP, Levy MM, et al.: Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit Care Med 2013, 41:580-637. Figure 1 (abstract P156). Cell death of HK-2 cells after lactated Ringer administration in a hypovolemia mimic model. 2. Cannon CM, Holthaus CV, et al.: The GENESIS Project. J Intensive Care Med 2013, 28:355-368. 3. Early Goal-Directed Therapy Collaborative Group of Zhejiang P: Chin Crit Care Med 2010, 22:331-334. 3. Early Goal-Directed Therapy Collaborative Group of Zhejiang P: Chin Crit Care Med 2010, 22:331-334. P154 Fluid challenge with shock V Inal, O Mert Trakya University Medical Faculty, Edirne, Turkey Critical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) P154 Fluid challenge with shock V Inal, O Mert Trakya University Medical Faculty, Edirne, Turkey Critical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) Reference Reference 1. Prather JW, et al.: Eff ect of blood volume, mean circulatory pressure, and stress relaxation on cardiac output. Am J Physiol 1969, 216:467-472. may lead to undercorrection of fi brinogen in critical bleeding situations. The aggravated response on the FibTEM-MCF may better refl ect HES eff ects on clot structure. Figure 1 (abstract P155). Relative change of P-fi brinogen compared with preoperative values. P154 Fluid challenge with shock V Inal, O Mert Trakya University Medical Faculty, Edirne, Turkey Critical Care 2014, 18(Suppl 1):P154 (doi: 10.1186/cc13344) P155 P155 In vivo eff ect of hydroxyethyl starch solution (HES 130/0.4) on diff erent fi brinogen assays U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) P157 P157 Hypotonic fl uids after liver transplantation may be associated with prolonged ICU stay A Nadeem, N Salahuddin, A ElHazmi, M Joseph, B Bohlega, H Sallam, Y Elsheikh, D Broering King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P157 (doi: 10.1186/cc13347) Introduction Morbidity after liver transplantation has been linked with preoperative MELD scores and transfusion volumes [1]. We hypothesized that in the immediate post-transplant period there may be modifi able risk factors associated with prolonged ICU stays. y y p y Conclusion Early application of vasopressin reduced the time of vasopressor use, progression to multiple organ dysfunction, length of stay in the ICU, and mortality rates at 14 and 28 days as compared with norepinephrine only. This observed diff erence can be attributed to early restoration of tissue perfusion in the control group making the state of septic shock shorter and reducing the potential for multiple organ dysfunction, which directly infl uenced patient survival. Reference i Methods In a retrospective, case–control study, we reviewed all liver transplant adult recipients over a 33-month period, January 2010 to September 2013. Recipients were divided into two groups based on a priori determined cutoff value of 8 days after transplantation. Signifi cant associations for prolonged ICU admission were identifi ed using chi-square analysis for categorical and ANOVA for continuous variables. P <0.05 was considered signifi cant. SPSS version 22.0 was used for all analyses. 1. Dellinger RP, Levy MM,Carlet JM, et al.: Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock. Crit Care Med 2008, 36:296-327. Results Total numbers of transplants performed were 162. Mean pre- transplant MELD score was 19.5 ± 7.7; viral hepatitis was the most common indication for transplant, 44 (27%). Living-related donor transplants were carried out in 87 (54%) cases. Median ICU length of stay was 4 ± 11.9 days and ICU mortality was 15 (9%). Acute kidney injury developed in 30 (19%) with 5% needing renal replacement therapy. Early complications developed in 33%. Prolonged ICU stay was related to: a higher pre-transplant INR (P = 0.000), larger volumes of RBC (2,064 ± 1,701 vs. 1,176 ± 1,454 ml, P = 0.000), platelets (453 ± 271 vs. 365 ± 276 ml, P = 0.046) and cryoprecipitate transfusions (47 ± 98 vs. Reference 1. Bennett-Guerrero E., et al.: Preoperative and intraoperative predictors of postoperative morbidity, poor graft function, and early rejection in 190 patients undergoing liver transplantation. Arch Surg 2001, 136:1177-1183. 1. Bennett-Guerrero E., et al.: Preoperative and intraoperative predictors of postoperative morbidity, poor graft function, and early rejection in 190 patients undergoing liver transplantation. Arch Surg 2001, 136:1177-1183. Early Vasopressin Application in Shock study S Oliveira, F Dessa, C Rocha, F Oliveira Hospital Bangu, Rio de Janeiro, Brazil Critical Care 2014, 18(Suppl 1):P158 (doi: 10.1186/cc13348) Introduction Vasopressin is frequently used to maintain blood pressure in refractory septic shock [1]. This study is looking into the hypothesis that vasopressin compared with norepinephrine would decrease the severity of septic status, evolution to multiple organ dysfunction, length of hospitalization, and mortality among patients with septic shock. Methods In this randomized, double-blind study, we assigned patients who still needed vasopressor to restore tissue perfusion after fl uid resuscitation to receive norepinephrine (0.05 to 2.0 μg/kg/minute) or vasopressin (0.01 to 0.03 U/minute) with low doses of norepinephrine. Both groups had the vasoactive drug infusions titrated and tapered to maintain a target mean blood pressure. The analysis included the total time of use and dosage of vasopressors every 6 hours, the move to single organ dysfunction and multiple organ failure, length of ICU stay and hospitalization, and mortality 7, 14 and 28 days after the start of infusions. Figure 2 (abstract P156). Elocalcitol (BXL 628) ameliorates toxicity of lactated Ringer in HK-2 human renal tubule cells. conditions, L/R augmented cytotoxicity, and more annexin V (+) cells were observed. Co-treatment or post-treatment with BXL 628, a novel VDR agonist, reversed L/R-induced cytotoxicity. See Figures 1 and 2. Conclusion BXL 628 rescued L/R-induced apoptosis in human renal proximal tubule cells in a hypovolemia mimicking model. Results A total of 407 patients underwent randomization but 387 patients were included in this study (191 patients received vasopressin, and 196 received norepinephrine only). The total time for vasopressors was 37 hours and 68 hours in the vasopressin and norepinephrine groups with P = 0.02. Single organ dysfunction and multiple organ dysfunction using vasopressin and norepinephrine were respectively: 37.7% vs. 49.2%, P = 0.02; and 17.8% vs. 26%; P = 0.05. Length of stay in the ICU was 14 and 17 days (P = 0.29) and the time of hospitalization was 23 and 28 days (P = 0.11) respectively. There was a signifi cant diff erence between the vasopressin and norepinephrine groups in the mortality rate at 14 and 28 days (29.3% vs. 36.7%, P = 0.05; 34% and 42.3%, P = 0.03), but there were no signifi cant diff erences in the overall rates for 7-day mortality (21.2% vs. 23.9%, respectively; P = 1.1). P157 23 ± 85 ml, P = 0.037) received in the OR and mean volumes of hypotonic crystalloids administered in the fi rst 24 hours (P = 0.002), 48 hours (P = 0.010) and 72 hours (P = 0.007) of ICU admission. Serum P159 1. Dellinger RP, Levy MM,Carlet JM, et al.: Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock. Crit Care Med 2008, 36:296-327. Early Vasopressin Application in Shock study Early Vasopressin Application in Shock study S Oliveira, F Dessa, C Rocha, F Oliveira Hospital Bangu, Rio de Janeiro, Brazil Critical Care 2014, 18(Suppl 1):P158 (doi: 10.1186/cc13348) P155 In vivo eff ect of hydroxyethyl starch solution (HES 130/0.4) on diff erent fi brinogen assays U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) In vivo eff ect of hydroxyethyl starch solution (HES 130/0.4) on diff erent fi brinogen assays U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) fi y U Schött, DW Winstedt, AH Hillarp Lund University, Lund, Sweden y Critical Care 2014, 18(Suppl 1):P155 (doi: 10.1186/cc13345) Introduction Previous in vitro studies have shown that photometric assays may overestimate fi brinogen levels after hemodilution with HES. The in vivo eff ect of HES on fi brinogen assays was therefore studied. fi Methods Forty patients with intracranial tumor gave their consent to participate in this ethical approved study. Plasma fi brinogen levels were analyzed with ELISA, two photometric assays (Dade and Multifi bren) and one mechanical (Hook). In addition, ROTEM FibTEM- MCF was analyzed.i y Results Twenty-fi ve of the 40 patients received 1 l HES. Mean reduction of hematocrit was 17%. ELISA was lower than Hook and Multifi bren. The FibTEM relative decrease of 43% diff ered signifi cantly from the other assays. See Figure 1. Figure 1 (abstract P156). Cell death of HK-2 cells after lactated Ringer administration in a hypovolemia mimic model. y g Conclusion After in vivo hemodilution with HES, some assays overestimated fi brinogen levels. An overestimation of around 0.3 g/l Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S56 Figure 2 (abstract P156). Elocalcitol (BXL 628) ameliorates toxicity of lactated Ringer in HK-2 human renal tubule cells. sodium, chloride, lactate or creatinine levels were not signifi cantly diff erent between the two groups.li f Conclusion The administration of hypotonic fl uids in the fi rst 72 hours of liver transplantation may be a risk factor for prolonged ICU admission. This eff ect appears independent of serum electrolyte levels or renal dysfunction. P160 Angiotensin II may be useful for the treatment of hypotension in distributive shock, but a safe and effi cacious dose is unknown LW Busse1, E Brasha2, DL Davison2, MG Seneff 2, J Honig2, ZK Alotaibi3, LS Chawla2 1Inova Fairfax Health System, Falls Church, VA, USA; 2George Washington University, Washington, DC, USA; 3Prince Sultan Medical Military City, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P160 (doi: 10.1186/cc13350) Conclusion Vasopressin did not reduce mortality rates as compared with norepinephrine among patients with cancer and septic shock who were treated with catecholamine vasopressors. Critical Care 2014, 18(Suppl 1):P160 (doi: 10.1186/cc13350) Introduction Patients with distributive shock who require high-dose vasopressors have a high mortality. Vasopressors belong to two classes: catecholamines and vasopressin analogs. Each class has limitations. Patients receiving catecholamines often develop tachyphylaxis and metabolic complications (for example, lactic acidosis). Vasopressin analogs can cause mesenteric or myocardial ischemia and oliguria. Angiotensin II (ATII) is an endogenous peptide that increases blood pressure and aldosterone production. Preclinical data suggest a role for ATII in the treatment of sepsis-associated acute kidney injury. ATII may prove useful in patients who remain hypotensive despite catecholamine and vasopressin therapy. This is the fi rst randomized clinical trial to date which seeks to evaluate ATII for use in distributive shock. Acknowledgement Clinical Trials number: NCT 01718613. Heart rate reduction with esmolol in septic shock: eff ects on myocardial performance A Morelli1, A D’Egidio1, A Orecchioni1, E Maraff a1, S Romano2 1University of Rome La Sapienza, Rome, Italy; 2University of Florence, Italy Critical Care 2014, 18(Suppl 1):P162 (doi: 10.1186/cc13352) Introduction Clinical study suggests that beta-blockers, by decreasing the heart rate (HR) together with an increase in stroke volume (SV), do not negatively aff ect cardiac output (CO), allowing an economization of cardiac work and oxygen consumption in patients with septic shock [1]. Whether this hemodynamic profi le leads to an amelioration of myocardial performance is still unclear. The objective of the present study was therefore to elucidate whether a reduction in HR with esmolol is associated with an improvement of cardiac effi ciency in patients with septic shock who remained tachycardic after hemodynamic optimization. Methods Twenty patients with distributive shock and a cardiovascular Sequential Organ Failure Assessment score ≥4 were randomized to either ATII infusion (n = 10) or placebo (n = 10) plus standard of care. Vasopressin versus norepinephrine for the management of septic shock in cancer patients Conclusion There are few case reports of terlipressin-induced hyponatraemia [1]. Hyponatraemia is considered a rare side eff ect brought about by the partial agonist eff ect of terlipressin on renal vasopressin V2 receptors. Sola and colleagues monitored serum sodium concentrations in patients with acute variceal bleeding. They found that rapid reduction in serum sodium was common (up to 36%) and one patient developed osmotic demyelination syndrome. Hyponatraemia resolved on cessation of terlipressin [2]. Close monitoring of serum sodium levels is essential in patients on terlipressin therapy so rapid drops in sodium can be identifi ed and managed to stop associated neurological complications. Introduction Patients with septic shock die mainly due to refractory shock. Vasopressin is commonly used as an adjunct to catecholamines to support blood pressure in refractory septic shock, but its eff ect on mortality is unknown. We hypothesized that vasopressin as compared with norepinephrine would decrease mortality among cancer patients with septic shock. g References References p Methods In this, randomized, double-blind trial, we assigned patients who had cancer and septic shock and needed a vasopressor to receive norepinephrine or vasopressin in addition to open-label vasopressors. All vasopressor infusions were titrated and tapered according to protocols to maintain a target blood pressure. The primary endpoint was the mortality rate 28 days after the start of infusions.i 1. Hyun J, et al.: Terlipressin induced hyponatremic seizure. Scand J Gastroenterol 2010, 45:501-504. 1. Hyun J, et al.: Terlipressin induced hyponatremic seizure. Scand J Gastroenterol 2010, 45:501-504. 2. Sola E, et al.: Hyponatremia in patients treated for severe gastrointestinal bleeding due to portal hypertension. Hepatology 2010, 52:1783-1790. Results A total of 107 patients underwent randomization in this fi rst part of trial, and were infused with the study drug (53 patients received vasopressin, and 54 norepinephrine), and were included in the analysis. There was no signifi cant diff erence between the vasopressin and norepinephrine groups in the 28-day mortality rate (67.9 and 58.5%, respectively; P = 0.31). There were no signifi cant diff erences in the overall rates of serious adverse events (5.3% and 5.5%, respectively; P = 1.00). Terlipressin-induced hyponatraemia Introduction Terlipressin is a vasopressin (V1 receptor) agonist that causes splanchnic constriction and is used in the management of variceal bleeding. This case report demonstrates the profound hyponatraemia, suffi cient to cause a fall in conscious level, developing following terlipressin administration for variceal gastrointestinal bleeding. S57 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A 28-year-old man was admitted to the ICU following intubation and sedation for seizures due to acute alcohol withdrawal. A CT scan of the brain was normal. In the ICU the patient became haemodynamically unstable and fresh blood was aspirated from the nasogastric tube. Gastroscopy showed bleeding oesphageal varicies and hypertensive portal gastropathy. The patient was treated with variceal banding, tazobactam/pipacillin (4.5 g three times daily) and terlipressin (2 g four times daily) as per protocol for variceal bleeding. He was successfully extubated 48 hours later. Results The mean age was 62.9 ± 15.8 years. Of the patients, 75% were male, 45% were Caucasian and 40% were African American. The 30-day mortality for the two groups were similar for the ATII cohort and the placebo cohort (50% vs. 60%, P = 1.00). ATII resulted in marked reduction in norepinephrine dosing in all patients. The mean norepinephrine dose for the placebo cohort was 20.1 ± 16.8 μg/ minute vs. 7.3 ± 11.9 μg/minute for the ATII cohort (P = 0.022). The most common adverse event was hypertension, which occurred in 20% of patients receiving ATII.f Conclusion ATII is an eff ective vasopressor agent in patients with distributive shock requiring multiple vasopressors. The initial dose range of ATII that appears to be appropriate for patients with distributive shock is 2 to 10 ng/kg/minute. Further studies to assess the use of ATII in patients with distributive shock are warranted. Results On admission the patient’s serum sodium was 139 mmol/l. The patient received terlipressin for 4 days in the following regimen: 2 g four times daily for 48 hours, then 1 mg four times daily for 24 hours and then 0.5 mg four times daily for a further 24 hours before stopping. On the last day of his terlipressin therapy, the patient’s GCS dropped from 15 to 11. Serum sodium had fallen acutely to 116 mmol/l. The last two doses of terlipressin were cancelled and no other treatment for hyponatraemia was administered. His serum sodium and GCS returned to normal limits within 13 hours of terlipressin cessation with no neurological consequences. P161 P161 Vasopressin versus norepinephrine for the management of septic shock in cancer patients C Zambolim, D Nagaoka, J Fukushima, C Park, J Carneiro, E Osawa, J Almeida, S Zeff erino, F Galas, L Hajjar Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P161 (doi: 10.1186/cc13351) Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery g y H Araujo1, R Campbell2, D Green2, E Mills3 1King’s College London, UK; 2King’s College Hospital NHS Foundation Trust, London, UK; 3LiDCO Ltd, London, UK Critical Care 2014, 18(Suppl 1):P164 (doi: 10.1186/cc13354) g y H Araujo1, R Campbell2, D Green2, E Mills3 1King’s College London, UK; 2King’s College Hospital NHS Foundation Trust, London, UK; 3LiDCO Ltd, London, UK Critical Care 2014, 18(Suppl 1):P164 (doi: 10.1186/cc13354) g pi Results For a MAP between 65 and 75 mmHg, esmolol administration signifi cantly decreased HR (115 ± 10 to 91 ± 7 bpm), NE (0.7 ± 0.4 to 0.5 ± 0.3 μg/kg/minute), and dP/dt MAX (1.1 ± 0.3 to 0.8 ± 0.3 ms/ mmHg). Conversely, TAPSE (15 ± 3 to 20 ± 3 mm), CCE (–0.2 ± 0.4 to –0.03 ± 0.4 units) and SV (37 ± 8 to 42 ± 10 ml) signifi cantly increased at the end of the study period (all P <0.05). CO (4.1 ± 0.8 to 3.9 ± 0.8 l/ minute) and LVEF (46 ± 10 to 48 ± 10%) did not change. Introduction Fall in mean arterial blood pressure (MAP) during anaesthetic induction is common and may result in profound hypo- tension. Using the LiDCORapid (LiDCO Ltd, UK), we previously demonstrated that the fall in MAP is usually driven by a reduction in stroke volume (SV) that was presumed due to venodilation. Low- dose phenylephrine (PE), a venoconstrictor, commenced immediately prior to induction ameliorated these eff ects to a signifi cant degree [1]. However, it is important that the pre-induction administration of PE should not cause any marked changes in MAP and CO. We retrospectively studied a group of 40 high-risk patients about to undergo major peripheral vascular surgery where PE was commenced immediately pre-induction. The haemodynamic eff ects of this dose of PE on MAP, SV and CO were analysed. g Conclusion In patients with established septic shock who remained tachycardic after hemodynamic optimization, titration of esmolol to reduce the HR to a predefi ned threshold economized cardiac function, resulting in a maintained CO with a lower HR and a higher stroke volume. Such a hemodynamic profi le was characterized by an improved cardiac effi ciency, as indicated by the decrease in dP/dt MAX associated with an increase in CCE. P163 Presented at American Society of Anesthesiologists 2012 Annual Meeting; October 13-17 2012; Washington, DC. [http://www.asaabstracts.com/strands/asaabstracts/search.htm] 1. Bidd H, Tan A, Mills E, Araujo H, Green D, O’Brien T: Phenylephrine commenced prior to anaesthesia reduced the haemodynamic changes associated with induction of anaesthesia in patients undergoing high risk vascular surgery [Abstract A378]. Presented at American Society of Anesthesiologists 2012 Annual Meeting; October 13-17 2012; Washington, DC. [http://www.asaabstracts.com/strands/asaabstracts/search.htm] g Reference 1. Morelli A, Ertmer C, Westphal M, et al.: Eff ect of heart rate control with esmolol on hemodynamic and clinical outcomes in patients with septic shock: a randomized clinical trial. JAMA 2013, 310:1683-1691. P164 despite adequate volume resuscitation received a continuous esmolol infusion to maintain the HR between 94 and 80 bpm. NE was titrated to achieve a MAP between 65 and 75 mmHg. To investigate myocardial performance, we simultaneously assessed LV ejection fraction (LVEF), tricuspidal annular plane solid excursion (TAPSE) by echocardiography, the dP/dt MAX and the cardiac cycle effi ciency (CCE) both estimated from the arterial pressure waveform. Data were obtained at baseline and after achieving the predefi ned HR threshold (T1). Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery H Araujo1, R Campbell2, D Green2, E Mills3 1King’s College London, UK; 2King’s College Hospital NHS Foundation Trust, London, UK; 3LiDCO Ltd, London, UK Critical Care 2014, 18(Suppl 1):P164 (doi: 10.1186/cc13354) Haemodynamic eff ects of phenylephrine commenced prior to induction of anaesthesia in older patients undergoing high-risk vascular surgery Finally, echocardiographic data suggest that reducing HR with esmolol positively aff ects right ventricular function. Reference Methods A radial artery line was inserted prior to induction of anaesthesia, and baseline MAP, CO and SV were obtained using the LiDCORapid. The PE infusion was commenced at a rate of 1 to 2 mg/ hour. Remifentanil was then administered using a target-controlled infusion pump until a 2 ng/ml predicted eff ect site concentration (Ceff ) was reached. Propofol was then administered to induce anaesthesia. Haemodynamic changes from the pre-PE baseline to commencement of propofol were recorded at 5-second intervals. The changes in MAP, SV and CO were assessed as the percent change in values obtained immediately prior to PE and followed for at least 5 minutes until commencement of propofol. Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P165 (doi: 10.1186/cc13355) P160 ATII was started at a dose of 20 ng/kg/minute, and titrated per a protocolized schedule for a goal of maintaining a mean arterial pressure (MAP) of 65 mmHg. The infusion (either ATII or placebo) was continued for 6 hours and then titrated off . The primary endpoint was the eff ect of ATII on the standing dose of norepinephrine required to maintain a MAP of 65 mmHg. Secondary endpoints included the eff ect of ATII on urine output, serum lactate and creatinine clearance, as well as 30-day mortality. Methods After 24 to 36 hours of initial hemodynamic stabilization, 24 septic shock patients with HR >95 bpm and requiring norepinephrine (NE) to maintain mean arterial pressure (MAP) between 65 and 75 mmHg S58 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P163 A new automatic urinometer shows lower bias, no loss of precision due to temporal deviation and higher user evaluation when compared with a manual standard urinometer in an ICU setting A Eklund, J Van der Linden Karolinska University Hospital, Stockholm, Sweden Critical Care 2014, 18(Suppl 1):P163 (doi: 10.1186/cc13353) Results Patient demographics, mean (range): age 71 (46 to 89) years, 31 male, weight 80 (55 to 115) kg, ASA 3 (2 to 4). The changes in MAP and CO were not clinically or statistically signifi cant. MAP increased by an average of only 2%, range –25 to +12; CO by 0%, range –23 to +16 (P = 0.35 and P = 0.36 respectively). Karolinska University Hospital, Stockholm, Sweden y p Critical Care 2014, 18(Suppl 1):P163 (doi: 10.1186/cc13353) y Conclusion In a previous study [1], PE administered prospectively prior to induction markedly reduced the haemodynamic changes following induction of anaesthesia in high-risk patients using remifentanil and propofol. This eff ect was achieved without signifi cant changes in MAP and CO in the 5-minute to 10-minute period between commencement of PE and induction of anaesthesia. Introduction In the intensive care setting, most physiologic parameters are monitored automatically. However, urine output (UO) is still monitored hourly by manually handled urinometers. This study evaluated an automatic urinometer (AU) and compared it with a manual urinometer (MU). Methods This was a prospective study in the ICU of a cardio-thoracic surgical clinic. In postoperative patients (n = 36) with indwelling urinary catheters and an expected stay of 24 hours or more, hourly UO samples were measured with an AU (n = 220, Sippi®; Observe Medical, Gothenburg, Sweden) or a MU (n = 188, UnoMeter™ 500; Unomedical a/s, Birkeroed, Denmark), and thereafter validated by cylinder measurements. Malposition of instrument at reading excluded measurement. Data were analyzed with the Bland–Altman method. The performance of the MU was used as minimum criteria of acceptance when the AU was evaluated. The loss of precision with the MU due to temporal deviation from fi xed hourly measurements was recorded (n = 108). A questionnaire, fi lled out by the ward staff (n = 28), evaluated the ease of use of the AU compared with the MU. . Bidd H, Tan A, Mills E, Araujo H, Green D, O’Brien T: Phenylephrine commenced prior to anaesthesia reduced the haemodynamic changes associated with induction of anaesthesia in patients undergoing high risk vascular surgery [Abstract A378]. 6 Potential use of veno-arterial extracorporeal membrane oxygenation for cardiogenic shock refractory to mechanical assist devices: baseline physiology and mortality data C Vimalanathan, N Barrett, N Ioannou, C Langrish, C Meadows, G Salt, G Glover Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P167 (doi: 10.1186/cc13357) Potential use of veno-arterial extracorporeal membrane oxygenation for cardiogenic shock refractory to mechanical assist devices: baseline physiology and mortality data C Vimalanathan, N Barrett, N Ioannou, C Langrish, C Meadows, G Salt, G Glover Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P167 (doi: 10.1186/cc13357) g Results Of the 11 patients included, six had spontaneous intracranial hemorrhage (ICH), four had traumatic ICH and one was free of neurologic injury. Following administration of 393 doses of APAP, the average change in MAP for all patients was –7.52 mmHg and SBP was –12.04 mmHg. When evaluated on an individual basis, patients with ICH had an average change in MAP of –10.64 (–5.2 to –19.08) and SBP of –17.81 (–7.54 to –35.75). The patient without neurologic injury had an average change in MAP and SBP of –3.01 and –2.42. The average change in MAP and SBP following administration of oral APAP solution (n = 357) was –7.84 and –12.39 while the change following rectal administration (n = 3) was –4.22 and –8 and tablet administration (n = 33) was –4.4 and –8.61. Introduction Mortality from cardiogenic shock remains high [1] and, despite a physiological rationale, intra-aortic balloon counterpulsation (IABP) has recently been shown to be ineff ective in reducing mortality [2,3]. Veno-arterial extracorporeal membrane oxygenation (V-A ECMO) may off er a survival advantage over IABP. The objective of this study was to describe the characteristics and outcomes of patients supported with IABP or Impella and to identify the characteristics of patients who die, despite mechanical assistance, for whom a proposed V-A ECMO programme may be benefi cial. p g yi Methods A retrospective cohort study in a 30-bed, medical–surgical ICU. All adult patients supported with IABP or Impella over 2 years to March 2013 were identifi ed and data were extracted by case-note review. Subgroup analysis was carried out for patients aged ≤65 and for those who fulfi lled the modifi ed Melbourne criteria for V-A ECMO [4]. Data collected included demographic data, physiology and organ support at baseline and at 6, 12, and 24 hours, ICU and hospital outcomes and cause of death. Comparisons between survivors and nonsurvivors were made with t test/chi-squared tests as appropriate. Results A total of 129 patients were identifi ed: 78% were male, mean age was 70 years (SD ±11.8), mean APACHE II score was 20 (±5) and ICU mortality was 44%. 6 Potential use of veno-arterial extracorporeal membrane oxygenation for cardiogenic shock refractory to mechanical assist devices: baseline physiology and mortality data C Vimalanathan, N Barrett, N Ioannou, C Langrish, C Meadows, G Salt, G Glover Guy’s & St Thomas’ NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P167 (doi: 10.1186/cc13357) Comparing survivors with nonsurvivors the only statistically signifi cant diff erence was metabolic acidosis (–6.8 ± 5.3 vs. –10.9 ± 7.0 mEq/l; P <0.05). Heart rate, mean arterial pressure, lactate, central venous oxygen saturation, cardiac index, arterial blood pH and mechanical ventilation failed to show a signifi cant diff erence. Eleven of these patients would have fulfi lled the proposed criteria for V-A ECMO, with an ICU mortality of 36%. i Methods A retrospective cohort study in a 30-bed, medical–surgical ICU. All adult patients supported with IABP or Impella over 2 years to March 2013 were identifi ed and data were extracted by case-note review. Subgroup analysis was carried out for patients aged ≤65 and for those who fulfi lled the modifi ed Melbourne criteria for V-A ECMO [4]. Data collected included demographic data, physiology and organ support at baseline and at 6, 12, and 24 hours, ICU and hospital outcomes and cause of death. Comparisons between survivors and nonsurvivors were made with t test/chi-squared tests as appropriate.i Conclusion It appears from this small case series that patients with ICH may experience more isolated hypotension following administration of APAP when compared with others. These changes seemed to be greater than previously reported for intravenous administration. It may also be possible that APAP solution exudes hypotensive eff ects more commonly when compared with tablet or rectal formulations. References 1. Brown G: Acetaminophen-induced hypotension. Heart Lung 1996, 25:137-140. q pp p Results A total of 129 patients were identifi ed: 78% were male, mean age was 70 years (SD ±11.8), mean APACHE II score was 20 (±5) and ICU mortality was 44%. Comparing survivors with nonsurvivors the only statistically signifi cant diff erence was metabolic acidosis (–6.8 ± 5.3 vs. –10.9 ± 7.0 mEq/l; P <0.05). Heart rate, mean arterial pressure, lactate, central venous oxygen saturation, cardiac index, arterial blood pH and mechanical ventilation failed to show a signifi cant diff erence. Eleven of these patients would have fulfi lled the proposed criteria for V-A ECMO, with an ICU mortality of 36%. 2. Mrozek, S, et al.: [Acetaminophene-induced hypotension in intensive care unit: a prospective study]. Ann Fr Anesth Reanim 2009, 28:448-453. P167 Methods Patients admitted to the SICU over a 7-month time frame who were reported by the nursing staff to have experienced hypotension following the oral or rectal administration of APAP were included. Their electronic medical records were retrospectively reviewed to describe the change in systolic blood pressure (SBP) and mean arterial pressure (MAP) within the fi rst hour following all administrations of APAP. Additional data collected consisted of patient age, sex, admission diagnoses and formulation/route of APAP administration. i References 1. Jeger RV, et al.: Ann Intern Med 2008, 149:618-626. 2. Thiele H, et al.: N Engl J Med 2012, 367:1287-1296. 3. Werdan K, et al.: Eur Heart J 2013. [Epub ahead of print] 4. Aubron C, et al.: Crit Care 2013, 17:R73. 1. Jeger RV, et al.: Ann Intern Med 2008, 149:618-626. 2. Thiele H, et al.: N Engl J Med 2012, 367:1287-1296. 3. Werdan K, et al.: Eur Heart J 2013. [Epub ahead of print] 4. Aubron C, et al.: Crit Care 2013, 17:R73. 2. Thiele H, et al.: N Engl J Med 2012, 367:1287 1296. 3. Werdan K, et al.: Eur Heart J 2013. [Epub ahead of print] 4. Aubron C, et al.: Crit Care 2013, 17:R73. Introduction Profound myocardial depression can occur after cardiac surgery. Use of ventricular assist support through venoarterial extracorporeal membrane oxygenation (ECMO) has been positively reported. This study will focus on the outcomes of patients who, upon suff ering hemodynamic failure after cardiac surgery, were supported by the use of ECMO during their stay in the surgical ICU of Incor FMUSP. Methods This was a retrospective, single-center and observational study. The records of 48 patients who underwent cardiac surgery and, subsequently, needed percutaneous or surgical implantation of ventricular assist devices were evaluated. The evaluation considered the following criteria: basal characteristics, indications for ventricular assistance, duration, length of ICU and hospital stay, and hospital mortality, through data collection forms. P166 y Conclusion Only metabolic acidosis was associated with mortality in patients supported with mechanical assist devices. Our data do not allow discrimination of survivors from nonsurvivors. Patients who fulfi lled the proposed criteria for V-A ECMO showed a similar mortality to a recent series treated with V-A ECMO [4]. The proposed criteria do not identify a cohort, in this population, that would expect a mortality benefi t from V-A ECMO. R f P166 Experiences of a tertiary center with use of extracorporeal membrane oxygenation support in patients with cardiogenic shock after cardiac surgery L Galas1, V Guimaraes2, M Sundin2, L Caneo2, M Jatene2, F Jatene2, L Hajjar2, J Almeida1, F Galas2 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Heart Institute, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P166 (doi: 10.1186/cc13356) P166 Experiences of a tertiary center with use of extracorporeal membrane oxygenation support in patients with cardiogenic shock after cardiac surgery L Galas1, V Guimaraes2, M Sundin2, L Caneo2, M Jatene2, F Jatene2, L Hajjar2, J Almeida1, F Galas2 1Instituto do Cancer do Estado de São Paulo, Brazil; 2Heart Institute, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P166 (doi: 10.1186/cc13356) Acetaminophen-induced hypotension in the surgical ICU Z Stoecker Acetaminophen-induced hypotension in the surgical ICU Z Stoecker Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P165 (doi: 10.1186/cc13355) Results Analysis according to Bland–Altman showed a smaller mean bias for the AU, +1.9 ml, compared with the MU, +5.3 ml (P <0.001). There was no statistical diff erence in measurement precision between the two urinometers, defi ned by their limits of agreement (±15.2 ml vs. ±16.6 ml, P = 0.11). The mean temporal variation with the MU was ±7.4 minutes (±12.4%), limits of agreement ±23.9 minutes (±39.8%), compared with no temporal variation with the AU (P <0.001). A total 86% of the ward staff considered the AU superior to the MU (P <0.001). Conclusion The AU had a signifi cantly lower bias than the MU and the loss of precision of hourly UO due to temporal deviations using the MU was avoided with the AU. The AU was also evaluated higher by the ward staff , refl ecting perception of higher reliability, easier use, less contact with urine bags and less time usage for measurements. The features of the AU may also indicate a favorable clinical impact in the normal ward, when staffi ng does not allow hourly measurements with a MU. Hennepin County Medical Center, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P165 (doi: 10.1186/cc13355) Introduction Acetaminophen (APAP) is commonly administered in the surgical ICU (SICU) for its analgesic and antipyretic eff ects. Case reports have described the potential for APAP to cause allergic reactions with and without hypotension. Furthermore, there have been case reports of APAP causing isolated hypotension in the absence of other allergic responses [1]. This has been well described following intravenous administration [2]. However, other routes of administration causing hypotension and associated diagnoses remain to be elucidated. The present case series describes 11 patients with APAP-induced hypotension in the SICU at a Level I trauma center. S59 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P168 Normobaric oxygen paradox and the microcirculation in the critically ill patient: a prospective observational study A Donati, E Damiani, AT Colesnicenco, E Montesi, S Ciucani, A Carsetti, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2014, 18(Suppl 1):P168 (doi: 10.1186/cc13358) Normobaric oxygen paradox and the microcirculation in the critically ill patient: a prospective observational study A Donati, E Damiani, AT Colesnicenco, E Montesi, S Ciucani, A Carsetti, P Pelaia Università Politecnica delle Marche, Ancona, Italy Critical Care 2014, 18(Suppl 1):P168 (doi: 10.1186/cc13358) Università Politecnica delle Marche, Ancona, Italy y Critical Care 2014, 18(Suppl 1):P168 (doi: 10.1186/cc13358) Introduction The normobaric oxygen paradox (NOP) is a recent concept that postulates the use of intermittent hyperoxia to stimulate erythropoietin (EPO) production [1]. Hyperoxia increases oxygen free radicals and may lead to endothelial damage and vasoconstriction [2]. We evaluated the microvascular response to transient hyperoxia and its eff ects on EPO production. y g Results Of the 48 patients included on the study, 26 (54%) were males, and 31 (64%) were younger than 18 years old. These patients developed cardiogenic shock during 72 hours after cardiac surgery. In all cases, ECMO was inserted after cardiac surgery. Of all patients, 32 (66%) were central ECMO, inserted in the operative room, and 16 were percutaneous, inserted in the ICU. The median duration of ventricular assistance was 6 days (IQR 0 to 41), the length of ICU stay was 16 days (IQR 1 to 111), and hospital stay was 29 days (IQR 1 to 198). Twenty patients survived (41%) and were discharged from our hospital. f Methods Six patients with hemodynamic stability and mechanically ventilated with FiO2 <50% were included in this prospective observational study. Patients underwent a 2-hour period of hyperoxia (FiO2 100%). The sublingual microcirculation (sidestream dark-fi eld imaging (SDF)) was evaluated at baseline (t0), 2 hours after hyperoxia (t1), and 2 hours after return to basal FiO2 (t2). SDF monitoring was continuously performed also during the variation of FiO2 for 2 minutes. EPO levels were assayed at baseline and for 2 days. Conclusion The use of mechanical circulatory assists devices is an effi cient tool to manage seriously ill patients after cardiac surgery. This tool should be considered early in the diagnosis of cardiogenic shock after cardiac surgery. Predictive criteria for the development of intra-abdominal hypertension and abdominal compartment syndrome D Lyer, P Rastogi, A Aneman, S D’Amours Liverpool Hospital, Sydney, Australia Critical Care 2014, 18(Suppl 1):P169 (doi: 10.1186/cc13359) Predictive criteria for the development of intra-abdominal hypertension and abdominal compartment syndrome D Lyer, P Rastogi, A Aneman, S D’Amours Liverpool Hospital, Sydney, Australia Critical Care 2014, 18(Suppl 1):P169 (doi: 10.1186/cc13359) Conclusion In patients with hyperlactatemia on ICU admission, lactate-guided therapy did not reduce complications and was related to a longer ICU length of stay. This study suggests that goal-directed therapy aiming to decrease initial lactate levels does not result in clinical benefi t. Introduction This study aims to develop predictive criteria to identify which patients are at risk of developing intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) upon admission to the ICU. Early lactate-guided therapy in cardiac surgery patients: a randomized controlled trial M Sundin1, J Almeida2, E Osawa1, F Galas1, M Gaiane1, S Zeff erino1, L Camara1, LG Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P170 (doi: 10.1186/cc13360) Figure 1 (abstract P168). TVD and NOP. Results An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure 1). The TVD tended to increase without returning to baseline levels at t2. EPO increased in four patients out of 6 (P = NS). A negative correlation was found between the change in TVD after hyperoxia (t1 – t0) and the change in EPO (r = –0.88, P = 0.03). Results An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure 1). The TVD tended to increase without returning to baseline levels at t2. EPO increased in four patients out of 6 (P = NS). A negative correlation was found between the change in TVD after hyperoxia (t1 – t0) and the change in EPO (r = –0.88, P = 0.03). Introduction It is unknown whether lactate monitoring aimed to decrease levels during the fi rst hours in patients undergoing cardiac surgery improves outcome. The aim of this study was to evaluate the eff ect of lactate monitoring and resuscitation directed at decreasing lactate levels in patients admitted to the ICU in the fi rst 8 hours with lactate level ≥3.0 mEq/l. Conclusion Hyperoxia leads to vasoconstriction that seems to be reversible at hyperoxia cessation. Further data are needed to verify the effi cacy of the NOP in stimulating erythropoiesis in the critically ill. There might be a relation between hyperoxia-induced reduction in vessel density and the EPO increase. P168 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S60 Results An early vasoconstriction and a trend towards total vessel density (TVD) reduction were observed at t1 (Figure 1). The TVD tended to increase without returning to baseline levels at t2. EPO increased Figure 1 (abstract P168). TVD and NOP. Figure 1 (abstract P168). TVD and NOP. (odds ratio, 4.95; 95% CI, 1.19 to 7.24; P <0.001), hemoperitoneum/ pneumoperitoneum/intraperitoneal fl uid collection (odds ratio, 3.61; 95% CI, 1.29 to 10.12; P = 0.014), obesity (odds ratio, 3.41; 95% CI 1.97 to 5.90; P <0.001), fl uid received >2.3 l (odds ratio, 2.68; 95% CI, 1.48 to 4.84; P = 0.001), abbreviated SOFA score >4 points (odds ratio, 2.49; 95% CI, 1.49 to 4.15; P <0.001) and lactate >1.4 mmol/l (odds ratio, 2.28; 95% CI, 1.33 to 3.91; P <0.001) were identifi ed as independent predictors of IAH upon admission to ICU. The presence of three or more of these risk factors at admission predicted the onset of IAH with sensitivity of 75% and a specifi city of 76%, and the onset of grade II, III and IV IAH with a sensitivity of 91% and a specifi city of 62%. (odds ratio, 4.95; 95% CI, 1.19 to 7.24; P <0.001), hemoperitoneum/ pneumoperitoneum/intraperitoneal fl uid collection (odds ratio, 3.61; 95% CI, 1.29 to 10.12; P = 0.014), obesity (odds ratio, 3.41; 95% CI 1.97 to 5.90; P <0.001), fl uid received >2.3 l (odds ratio, 2.68; 95% CI, 1.48 to 4.84; P = 0.001), abbreviated SOFA score >4 points (odds ratio, 2.49; 95% CI, 1.49 to 4.15; P <0.001) and lactate >1.4 mmol/l (odds ratio, 2.28; 95% CI, 1.33 to 3.91; P <0.001) were identifi ed as independent predictors of IAH upon admission to ICU. The presence of three or more of these risk factors at admission predicted the onset of IAH with sensitivity of 75% and a specifi city of 76%, and the onset of grade II, III and IV IAH with a sensitivity of 91% and a specifi city of 62%. yi y Conclusion IAH is a common clinical entity in the intensive care setting. Predictive criteria, based on data readily available upon a patient’s admission to ICU, were developed and eff ectively predicted the risk of developing IAH. Lactate as a predictor of deterioration in emergency department patients with and without infection K Oedorf1, D Day1, Y Lior2, V Novack2, N Shapiro1, D Henning1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Soroka University Medical Center, Beersheba, Israel Critical Care 2014, 18(Suppl 1):P171 (doi: 10.1186/cc13361) y Results Thirty-nine and 2% of patients developed IAH and ACS as per consensus defi nitions. Upon ICU admission, patients that would go on to develop IAH had a signifi cantly higher APACHE III score (62 (44 to 81) vs. 50 (37 to 68); P <0.001), abbreviated SOFA score (6 (4 to 8) vs. 4 (2 to 6); P <0.001), CVP (17 (13 to 20) vs. 15 (12 to 17) mmHg; P <0.001), lactate (2.2 (1.4 to 3.8) vs. 1.5 (1.1 to 2.4) mmol/l; P <0.001), INR (1.3 (1.1 to 1.5) vs. 1.2 (1.1 to 1.3); P <0.001), bilirubin (11 (7 to 19) vs. 10 (6 to 15) μmol/l; P = 0.027), creatinine (108 (76.8 to 175) vs. 84.0 (66.0 to 118) μmol/l; P <0.001), 24-hour fl uid balance (2.47 (0.95 to 4.05) vs. 1.23 (0.29 to 2.27) l; P <0.001), vasopressor requirement (60 vs. 39%; P <0.001), mechanical ventilation requirement (71 vs. 58%; P = 0.011), PEEP (8.00 (5.00 to 10.00) vs. 5.50 (5.00 to 8.00) cmH2O; P <0.001), and peak airway pressure (24 (21 to 30) vs. 22 (19 to 24) cmH2O; P <0.001). These patients also had a signifi cantly lower abdominal perfusion pressure (61 (55 to 71) vs. 69 (60 to 77) mmHg; P <0.001), pH (7.29 (7.22 to 7.35) vs. 7.33 (7.28 to 7.37); P <0.001) and PaO2:FiO2 ratio (182 (100 to 263) vs. (264 (168 to 371); P <0.001) upon ICU admission. Abdominal distension Introduction The use of serum lactate level to risk-stratify emergency department (ED) patients with sepsis is widely used. Studies in nonsepsis populations also suggest its utility in predicting adverse outcomes. Whether lactate prognosticates equally across disease states is not clearly defi ned. This study compares the ability of lactate to identify patients at risk for deterioration (intubation, acute renal dysfunction, vasopressor use, or death) and mortality during hospitalization in infected and non-infected populations. Introduction The use of serum lactate level to risk-stratify emergency department (ED) patients with sepsis is widely used. Studies in nonsepsis populations also suggest its utility in predicting adverse outcomes. Whether lactate prognosticates equally across disease states is not clearly defi ned. P170 Early lactate-guided therapy in cardiac surgery patients: a randomized controlled trial M Sundin1, J Almeida2, E Osawa1, F Galas1, M Gaiane1, S Zeff erino1, L Camara1, LG Galas1, L Hajjar1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P170 (doi: 10.1186/cc13360) References Methods Patients were randomly allocated to two groups. In the lactate group, treatment was guided by lactate levels with the objective to decrease lactate by 20% or more per 2 hours for the initial 8 hours of ICU stay. In the control group, the treatment team had no knowledge of lactate levels (except for the admission value) during this period. The primary outcome measure was the incidence of complications in 28 days. 1. Balestra C, Germonpré P, Poortmans JR, Marroni A: J Appl Physiol 2006, 100:512 518 1. Balestra C, Germonpré P, Poortmans JR, Marroni A: J Appl Physiol 2006, 100:512-518. 2. Tsai AG, Cabrales P, Winslow RM, Intaglietta M: Am J Physiol Heart Circ Physiol 2003, 285:H1537-H1545. 2. Tsai AG, Cabrales P, Winslow RM, Intaglietta M: Am J Physiol Heart Circ Physiol 2003, 285:H1537-H1545. Results The lactate group received more fl uids and dobutamine. However, there were no signifi cant diff erences in lactate levels between the groups. The rate of complications was similar between groups (11% vs. 7%, P = 0.087). Length of ICU stay was higher in the lactate group (3.5 vs. 2.4 days, P = 0.047) when compared with the control group. Results The lactate group received more fl uids and dobutamine. However, there were no signifi cant diff erences in lactate levels between the groups. The rate of complications was similar between groups (11% vs. 7%, P = 0.087). Length of ICU stay was higher in the lactate group (3.5 vs. 2.4 days, P = 0.047) when compared with the control group. Conclusion In patients with hyperlactatemia on ICU admission, lactate-guided therapy did not reduce complications and was related to a longer ICU length of stay. This study suggests that goal-directed therapy aiming to decrease initial lactate levels does not result in clinical benefi t. P171 Methods This is a prospective, observational study of 403 consecutively admitted ICU patients in a 3-month period, requiring the insertion of an indwelling urinary catheter. Intra-abdominal pressure was measured at least twice daily in all patients. Lactate as a predictor of deterioration in emergency department patients with and without infection K Oedorf1, D Day1, Y Lior2, V Novack2, N Shapiro1, D Henning1 1Beth Israel Deaconess Medical Center, Boston, MA, USA; 2Soroka University Medical Center, Beersheba, Israel Critical Care 2014, 18(Suppl 1):P171 (doi: 10.1186/cc13361) 1. Balestra C, Germonpré P, Poortmans JR, Marroni A: J Appl Physiol 2006, 100:512-518. Lactate as a predictor of deterioration in emergency department patients with and without infection This study compares the ability of lactate to identify patients at risk for deterioration (intubation, acute renal dysfunction, vasopressor use, or death) and mortality during hospitalization in infected and non-infected populations. Methods A prospective, observational cohort study of ED adult patients presenting from 11 November 2012 to 1 February 2013 who had lactate measured and abnormal vital signs (hearth rate ≥130, respiratory rate ≥24, shock index ≥1, systolic blood pressure <90 mmHg). Patients with isolated atrial tachycardia, seizure, intoxication, or psychiatric agitation S61 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 lactate (initial)) × 100%. Lactate (initial) is blood or tissue lactate within the fi rst 24 hours after ICU admission (H0). Lactate (delayed) is blood or tissue lactate at H4, H8, H12, H16, H20, H24 and H48 (H = hours). were excluded. Patients were stratifi ed into three groups by lactate <2.5 (low), 2.5 to 4 (intermediate), and >4 mmol/l (high). Chi-square test for trend was used to compare outcome rates between lactate levels for each diagnostic category.i were excluded. Patients were stratifi ed into three groups by lactate <2.5 (low), 2.5 to 4 (intermediate), and >4 mmol/l (high). Chi-square test for trend was used to compare outcome rates between lactate levels for each diagnostic category.i Results A total of 112 patients having septic shock (n = 79) or severe sepsis (n = 33) were examined. Tissue lactate clearance was higher compared with blood lactate clearance at H0 to H8 (P = 0.02), H0 to H12 (P = 008), H0 to H16 (P = 0.01), H0 to H20 (P = 0.01), and H0 to H24 (P = 0.02). Tissue lactate clearance was higher in survivors compared with nonsurvivors at H0 to H12, H0 to H20 and H0 to H24 (P = 0.02, for all). Multivariate analysis showed that ARACHE II along with tissue clearances at H0 to H12, H0 to H20 and H0 to H24 <30% were independent outcome predictors. Blood lactate clearance was not related to survival. g g y Results Of 1,152 patients identifi ed, 366 were excluded and 298 did not have lactate measurements, leaving 488 for the analysis: 289 sepsis patients and 202 nonsepsis patients. Of these, 168 (34.4%) met the deterioration outcome, and there were 61 (12.5%) deaths. Lactate as a predictor of deterioration in emergency department patients with and without infection For infected patients, 46/342 (13.5%; 95% CI 10.2 to 17.5) low, 34/100 (34.0%; 95% CI 25.4 to 43.7) intermediate, and 20/46 (43.5%; 95% CI 30.2 to 57.8) high lactate patients suff ered deterioration (P <0.01). Likewise, 6/342 (1.6%; 95% CI 0.7 to 3.9) low, 19/100 (19.0%; 95% CI 12.4 to 27.9) intermediate, and 11/46 (23.9%; 95% CI 13.8 to 38.0) high lactate patients died during hospitalization (P <0.01). For non-infected patients, 42/342 (12.3%; 95% CI 9.2 to 16.2) low, 13/100 (15.1%; 95% CI 7.6 to 21.1) intermediate, and 13/46 (28.2%; 95% CI 17.2 to 42.7) high lactate patients suff ered deterioration (P = 0.01). In the regression models, lactate was strongly associated with deterioration for both infected (odds ratio (OR) = 1.94; 95% CI 1.4 to 2.3) and non-infected (OR = 1.48; 95% CI 1.14 to 1.91) groups. In regression models for mortality, lactate had better discrimination ability in infected (OR = 1.8; 95% CI 1.4 to 2.3) patients than in the non-infected (OR = 1.11; 95% CI 0.85 to 1.47) patients. Conclusion In critically ill septic patients, after the initial resuscitation phase, adipose tissue clears lactate earlier than blood. High tissue lactate clearance, but not blood lactate clearance, is associated with a favorable clinical outcome. P173 P172 Introduction The aim of this study was to determine the correlation between levels of serum lactate (SL) and conventional hemodynamic parameters (HPs) (mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), urinary output (UP)) in patients with severe sepsis. In a subgroup, advanced HPs (central venous saturation (SvO2), peripheral temperature (PT), cardiac index (CI), global end-diastolic volume index (GEDI) and extravascular lung water (ELWI)) were compared with levels of SL. Adipose tissue lactate clearance but not blood lactate clearance is associated with clinical outcome in severe sepsis or septic shock during the post-resuscitation period M Theodorakopoulou, S Orfanos, N Nikitas, D Vasilliadi, S Apolonatou, A Diamantakis, G Karkouli, C Diakaki, A Armaganidis, I Dimopoulou University Hospital of Athens Greece ‘ATTIKON’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P172 (doi: 10.1186/cc13362) P173 P173 Correlation between conventional and advanced hemodynamic parameters versus serum lactate in patients with severe sepsis M Kok1, A Oei2, A Karakus3, H Endeman4 1UMC Utrecht, the Netherlands; 2AMC, Amsterdam, the Netherlands; 3Diakonessenhuis, Utrecht, the Netherlands; 4OLVG, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P173 (doi: 10.1186/cc13363) Conclusion Lactate levels can be used to identify patients who are at increased risk of deterioration regardless of infection status. Lactate identifi es high-risk patients for mortality in infected patients more strongly than in non-infected patients. Critical Care 2014, 18(Suppl 1):P173 (doi: 10.1186/cc13363) Delayed assessment of serum lactate in sepsis is associated with an increased mortality rate Delayed assessment of serum lactate in sepsis is associated with an increased mortality rate R Arnold1, Z Zhang1, S Patel2, S Smola1, J Isserman1, E Jackson1 1Christiana Care Health System, Newark, DE, USA; 2Cooper University Hospital, Camden, NJ, USA Critical Care 2014, 18(Suppl 1):P174 (doi: 10.1186/cc13364) Results Of the 61 patients identifi ed, fi ve were excluded as their ICU stay was <24 hours. The overall mortality in the remaining 56 patients (mean age of 76.7 ± 10.4 (SD) years) was 21.4%. In univariate analysis, mSOFA and several other variables correlated signifi cantly (P <0.05) with mortality. The area under the ROC curve for LC at 6, 12, and 24 hours was 0.601, 0.719, and 0.731, respectively. LC at 24 hours was the most accurate, and its optimum cutoff value was 37.5%. LC, lactate level, and BE at 24 hours, as well as the signifi cant factors in univariate analysis, were entered into a stepwise logistic regression model, which revealed 24-hour LC ≤37.5% (odds ratio (OR), 23.0) and mSOFA score (OR, 2.1) as independent predictive values of mortality. Introduction Lactate assessment early in the resuscitation of sepsis has been recommended as a diagnostic biomarker. An abnormal lactate, independent of blood pressure, is an indication for aggressive fl uid resuscitation and its normalization is a recommended endpoint of resuscitation. The objective of this study was to evaluate the eff ect of the timing of lactate assessment on patient outcomes in sepsis. Conclusion In patients with colorectal perforation, 24-hour LC is more accurate than LC measured at earlier time points. Patients with 24- hour LC ≤37.5% and a high mSOFA score have a high risk of in-hospital mortality. R f g p p Methods Data were compiled using the Clinical Vigilance for Sepsis electronic health record (EHR) screening tool, which identifi ed consecutive patients from two hospital systems over 12 months at a 300-bed community hospital and over 24 months from a 500-bed academic tertiary care center. CV Sepsis alert screens the EHR to identify the presence of infection based on a multifactor alert system including labs, vital signs, and treatment team documentation. A physician order for intravenous antibiotics was used as a surrogate for suspected infection. The database identifi ed 37,160 consecutive patients treated for infection from a total of 216,550. Lactate quartile concentration and prognosis in severe sepsis and septic shock M De La Torre-Prados1, A Garcia-de la Torre2, C Trujillano-Fernández1, J Perez-Vacas1, A Puerto-Morlan1, E Camara-Sola1, A Garcia-Alcantara1, A Garcia-Alcantara1 M De La Torre-Prados1, A Garcia-de la Torre2, C Trujillano-Fernández1, J Perez-Vacas1, A Puerto-Morlan1, E Camara-Sola1, A Garcia-Alcantara1, A Garcia-Alcantara1 1Hospital Virgen de la Victoria, Málaga, Spain; 2Puerto Real University Hospital, Cadiz, Spain Critical Care 2014 18(Suppl 1):P176 (doi: 10 1186/cc13366) p y p y Results A total 5,072 of 37,160 consecutive patients (13%) had a measured lactate. Sepsis patients experienced an overall 3% (1,186/37,160) mortality rate. In total, 4,153 (82%) patients had measured lactate within 3 hours, and 919 (18%) were delayed, with a decreased morality rate (eLac 6.8 vs. dLac 24.7, P <0.0001). There was no diff erence in average lactate levels between the groups (eLac: 2.1 ± 2.6, dLac: 2.3 ± 3.0, P = NS). A larger ratio of delayed-lactate patients had a lactate ≥4 mmol/l (dLac 12.6% vs. eLac 8.7%). Introduction The Surviving Sepsis Campaign (SSC) indicates that a lactate (LT) concentration greater than 4 mmol/l indicates early resuscitation bundles. However, several recent studies have suggested that LT values lower than 4 mmol/l may be a prognostic marker of adverse outcome. The aim of this study was to identify clinical and analytical prognostic parameters in severe sepsis (SS) or septic shock (ShS) according to quartiles of blood LT concentration. Conclusion The delay in lactate assessment relative to clinical evidence of infection was associated with an increased mortality rate. The average lactate level in each group did not account for this eff ect. The timing of the assessment, not the lactate level, was prognostic of outcome. The mortality benefi t associated with lactate assessment within the 3-hour guideline suggests that an increased clinical awareness may lead to early initiation of time-sensitive interventions known to improve outcomes. Methods A cohort study was designed in a polyvalent ICU. We studied demographic, clinical and analytical parameters in 148 critically ill adults, within 24 hours from SS or ShS onset according to SSC criteria. We tested for diff erences in baseline characteristics by lactate interval using a Kruskal–Wallis test for continuous data or a chi-square test for categorical data and reported the median and interquartile ranges; SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 148 consecutive episodes of SS (16%) or ShS (84%). The median age was 64 (interquartile range, 48.7 to 71) years; male: 60%. y Reference 1. Nates JL, et al.: Automating and simplifying the SOFA score in critically ill patients with cancer. Health Informatics J 2010, 16:35-47. P176 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 MAP 73 mmHg (13, 37 to 120), HR 101 beats/minute (22, 51 to 172), CVP 12 mmHg (5, 1 to 29), UP 55 ml/hour (62, 0 to 500), SL 3.2 mmol/l (3, 1 to 18.6), CI 4.1 ml/kg/minute (1.1, 1.6 to 6.7), GEDI 871 ml/m2 (210, 500 to 1,691), ELWI 11 ml/kg (5, 4 to 23), PT 32.1 C (2.8, 26.4 to 38), and SvO2 75% (8, 39 to 93). Relevant and signifi cant (P <0.005) PCCs between HPs and SL were respectively: MAP –0.417, HR 0.195, UP –0.237, SvO2 –0.204 and PT –0.569. Figure 1 shows the relation between two HPs with the highest correlation with SL. Methods We retrospectively analyzed the clinical data of patients who underwent emergency surgery for colorectal perforation and were admitted to the ICU of our hospital from January 2003 to August 2013. Patients with traumatic, iatrogenic, and appendicitis perforations were excluded. The primary endpoint was survival to hospital discharge. The modifi ed Sequential Organ Failure Assessment (mSOFA) score, a customized SOFA score excluding the central nervous system component [1], was used for prognostic scoring. The mSOFA score and several clinical factors were analyzed by univariate analysis as possible predictors of survival. We collected lactate levels and base excess (BE) measured during surgery and at 6, 12, and 24 hours after the fi rst measurement and calculated the respective LC values. The associations of initial blood lactate level, LC, and BE with mortality were assessed by receiver operating characteristics (ROC) curve and logistic regression analyses. g Conclusion The conventional HPs MAP, HR, UP and SvO2 are signifi cantly correlated to levels of SL, but clinical value might be limited due to the relatively low correlation coeffi cients. In a small subgroup, PT is better correlated to the level of SL. Lactate quartile concentration and prognosis in severe sepsis and septic shock The main sources of infection were respiratory tract 38% and intra-abdomen 45%; 70.7% had medical pathology. Mortality at 28 days was 22.7%. Quartiles of blood LT concentration were quartile 1 (Q1): 1.87 mmol/l or less, quartile 2 (Q2): 1.88 to 2.69 mmol/l, quartile 3 (Q3): 2.7 to 4.06 mmol/l, and quartile 4 (Q4): 4.07 mmol/l or greater (Table 1). The median LT concentrations of each quartile were 1.43 (Q1), 2.2 (Q2), 3.34 (Q3), and 5.1 (Q4) mmol/l (P <0.001). The diff erences between these quartiles were that the patients in Q1 had signifi cantly lower APACHE II scores (P = 0.04), SOFA score (P = 0.024), number of organ failures (NOF) (P <0.001) and ICU mortality (P = 0.028), compared with patients in Q2, Q3 and Q4. Patients in Q1 had signifi cantly higher Delayed assessment of serum lactate in sepsis is associated with an increased mortality rate Patients with a measured lactate were divided relative to its measurement within 3 hours (eLac) or greater than 3 hours (dLac) of sepsis identifi cation as recommended by the Surviving Sepsis Campaign. The CV Sepsis alert was the reference standard for time zero. Patients were compared in each group for the occurrence of the primary outcome of in-hospital mortality. y Reference Adipose tissue lactate clearance but not blood lactate clearance is associated with clinical outcome in severe sepsis or septic shock during the post-resuscitation period g p p M Theodorakopoulou, S Orfanos, N Nikitas, D Vasilliadi, S Apolonatou, A Diamantakis, G Karkouli, C Diakaki, A Armaganidis, I Dimopoulou University Hospital of Athens Greece ‘ATTIKON’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P172 (doi: 10.1186/cc13362) Methods An observational prospective, single-center, pilot study was performed in intensive care (IC) of a medium-sized teaching hospital. Adult patients with severe sepsis were included and received standard goal-directed therapy (Surviving Sepsis Guidelines). Every patient received an arterial line and a central venous line in the upper diaphragm position. A subgroup received pulse contour cardiac output (PiCCO)-guided resuscitation and PT measurements. Pearson correlation coeffi cients (PCCs) were calculated between HPs and SL, which were measured every 4 hours for the fi rst 48 hours after inclusion. P <0.05 was considered statistically signifi cant.i Introduction Blood lactate clearance, a surrogate of tissue hypoxia, is associated with increased mortality in septic patients. However, no study has directly measured lactate clearance at the tissue level in the post-resuscitation period of sepsis. This study aimed to examine the relative kinetics of blood and tissue lactate clearances and to investigate whether these are associated with outcome in ICU patients having severe sepsis or septic shock during the post-resuscitation phase. p Methods A microdialysis catheter was inserted in the subcutaneous adipose tissue of the upper thigh and interstitial fl uid samples were collected. Serial measurements of blood and interstitial fl uid lactate levels were performed over a 48-hour period. Lactate clearance was calculated according to the formula: (lactate (initial) – lactate (delayed) / y gi Results Twenty-fi ve patients (12 men) were included. Mean age was 68 years (30 to 93), mean APACHE II score 31 (20 to 42). The most frequent reasons for IC admission were abdominal sepsis (n = 11) and pneumosepsis (n = 7). Mean HPs (with SD and range) were respectively: Figure 1 (abstract P173). Scatterplot of (a) MAP and SL and (b) PT and SL. Figure 1 (abstract P173). Scatterplot of (a) MAP and SL and (b) PT and SL. S62 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P175 It may be useful to revise the cutoff value of lactate according to the SSC (4 mmol/l). P177 Correlation between arterial lactate and venous lactate in patients with sepsis and septic shock P Theerawit, C Na Petvicharn Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P177 (doi: 10.1186/cc13367) Introduction Measurement of arterial lactate (A-LACT) levels has been used to monitor poor tissue perfusion, predicting mortality and guiding resuscitation. Peripheral venous lactate (V-LACT) has been regarded as an unreliable test, but a less invasive approach. We aimed to determine correlation between A-LACT and V-LACT and agreement of both in order to determine the usefulness of V-LACT as a biomarker for assessment in sepsis. Methods We conduct a prospective, cross-sectional study during June Table 1 (abstract P176). Baseline characteristics in SS and ShS patients by quartiles of blood LT Lactate <1.87 (n = 33) Lactate 1.88 to 2.69 (n = 41) Lactate 2.7 to 4.06 (n = 34) Lactate>4.07 (n = 37) P value Age (years) 57 (45 to 71) 64 (51.5 to 74.5) 65 (48 to 69) 60 (48.5 to 71) NS APACHE II 25 (18.5 to 30) 25 (19.5 to 27) 25 (21.5 to 29.5) 27 (22 to 33) 0.04 SOFA 9 (7 to 10.5) 9 (7 to 11) 9 (8 to 11) 11(8 to 13) 0.024 NOF 3 (3 to 4) 3 (3 to 4) 4 (3 to 5) 5 (3.5 to 5) <0.001 LT (mmol/l) 1.43 (1.16 to 1.56) 2.2 (1.99 to 2.47) 3.34 (3 to 3.72) 5.1 (4.4 to 7.34) <0.001 Procalcitonin (ng/ml) 2.81 (0.76 to 20.7) 11.5 (2.88 to 37.15) 13.47 (1.91 to 42.1) 21.6 (5.2 to 5.8) 0.05 Cholesterol (mg/dl) 127 (97.5 to 165) 130 (95.5 to 152.5) 100 (72 to 128) 91 (79 to 116.7) 0.06 28-day mortality (%) 10.8 21.2 24.4 35.1 0.029 ShS (%) 83.8 85.4 81.1 87.9 NS Figure 1 (abstract P177). Conclusion The arterial lactate and venous lactate levels were strongly correlated in the condition of sepsis or septic shock. Consequently, V-LACT may be used in substitution for A-LACT particularly in lactate levels not higher than 4 mmol/l. However, trending should be generally applied instead of the absolute value. P175 Lactate clearance as a predictor of mortality in colonic perforation R Egashira, T Kobayashi Osaki Citizen Hospital, Miyagi, Japan Critical Care 2014, 18(Suppl 1):P175 (doi: 10.1186/cc13365) Introduction The objective of this study was to determine whether lactate clearance (LC) is a signifi cant indicator of mortality in patients with colorectal perforation. LC has been associated with mortality in heterogeneous critically ill patients, but its role as a predictor of mortality in homogeneous patients with colorectal perforation is unclear. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S63 Table 1 (abstract P176). Baseline characteristics in SS and ShS patients by quartiles of blood LT Lactate <1.87 (n = 33) Lactate 1.88 to 2.69 (n = 41) Lactate 2.7 to 4.06 (n = 34) Lactate>4.07 (n = 37) P value Age (years) 57 (45 to 71) 64 (51.5 to 74.5) 65 (48 to 69) 60 (48.5 to 71) NS APACHE II 25 (18.5 to 30) 25 (19.5 to 27) 25 (21.5 to 29.5) 27 (22 to 33) 0.04 SOFA 9 (7 to 10.5) 9 (7 to 11) 9 (8 to 11) 11(8 to 13) 0.024 NOF 3 (3 to 4) 3 (3 to 4) 4 (3 to 5) 5 (3.5 to 5) <0.001 LT (mmol/l) 1.43 (1.16 to 1.56) 2.2 (1.99 to 2.47) 3.34 (3 to 3.72) 5.1 (4.4 to 7.34) <0.001 Procalcitonin (ng/ml) 2.81 (0.76 to 20.7) 11.5 (2.88 to 37.15) 13.47 (1.91 to 42.1) 21.6 (5.2 to 5.8) 0.05 Cholesterol (mg/dl) 127 (97.5 to 165) 130 (95.5 to 152.5) 100 (72 to 128) 91 (79 to 116.7) 0.06 28-day mortality (%) 10.8 21.2 24.4 35.1 0.029 ShS (%) 83.8 85.4 81.1 87.9 NS Table 1 (abstract P176). Baseline characteristics in SS and ShS patients by quartiles of blood LT cholesterol (P = 0.06) and lower procalcitonin (P = 0.05) at enrolment. At the extremes, patients in Q1 had decreased 28-day mortality (P = 0.023) and, patients in Q4 had increased 28-day mortality, compared with the other quartiles of patients (P = 0.009). Interestingly, patients in Q2 had signifi cant increased mortality compared with patients in Q1 (P = 0.043), whereas the patients in Q2 had no signifi cant diff erence in 28-day mortality compared with patients in Q3. Conclusion Adverse outcomes and several potential risk factors, including organ failure, are signifi cantly associated with higher quartiles of LT concentrations. P175 41) Lactate 2.7 to 4.06 (n = 34) Lactate>4.07 (n = 37) P 65 (48 to 69) 60 (48.5 to 71) NS 25 (21.5 to 29.5) 27 (22 to 33) 0.04 9 (8 to 11) 11(8 to 13) 0.024 4 (3 to 5) 5 (3.5 to 5) <0.001 3.34 (3 to 3.72) 5.1 (4.4 to 7.34) <0.001 13.47 (1.91 to 42.1) 21.6 (5.2 to 5.8) 0.05 100 (72 to 128) 91 (79 to 116.7) 0.06 24.4 35.1 0.029 81.1 87.9 NS Figure 1 (abstract P177). Figure 2 (abstract P177). cholesterol (P = 0.06) and lower procalcitonin (P = 0.05) at enrolment. At the extremes, patients in Q1 had decreased 28-day mortality (P = 0.023) and, patients in Q4 had increased 28-day mortality, compared with the other quartiles of patients (P = 0.009). Interestingly, patients in Q2 had signifi cant increased mortality compared with patients in Q1 (P = 0.043), whereas the patients in Q2 had no signifi cant diff erence in 28-day mortality compared with patients in Q3. Figure 1 (abstract P177). Conclusion Adverse outcomes and several potential risk factors, including organ failure, are signifi cantly associated with higher quartiles of LT concentrations. It may be useful to revise the cutoff value of lactate according to the SSC (4 mmol/l). P180 Impact of nitric oxide on pulmonary regurgitation and cardiac function in the acute stage after right ventricular outfl ow surgery Y Ko, K Morita, R Nagahori, T Abe, K Hashimoto Jikei University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P180 (doi: 10.1186/cc13370) y , y , p Critical Care 2014, 18(Suppl 1):P180 (doi: 10.1186/cc13370) Hyperdynamic ejection fraction in the critically ill patient JR Paonessa1, TP Brennan2, LA Celi1 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Massachusetts Institute of Technology, Cambridge, MA, USA Critical Care 2014, 18(Suppl 1):P179 (doi: 10.1186/cc13369) Introduction Pulmonary regurgitation (PR) that develops after right ventricular (RV) outfl ow reconstruction including the Rastelli and Norwood procedure may often result in serious cardiac events early after surgery. We hypothesized that PR may be associated with pulmonary vascular resistance (PVR) and RV contraction. Accordingly, we assessed the impact of PVR on PR and RV function using a swine model. 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Massachusetts Institute of Technology, Cambridge, MA, USA Critical Care 2014, 18(Suppl 1):P179 (doi: 10.1186/cc13369) Introduction The hyperdynamic left ventricular ejection fraction (HDLVEF) in the ICU is a common fi nding thought to be associated with critical illness and possibly sepsis. The exact etiology of hyperdynamic ejection fraction has yet to be determined, and the prognosis of these patients has not been well defi ned. Methods Eight pigs (14 ± 2 kg) underwent total resection of the pulmonary valve cusps under cardiopulmonary bypass (PR group). This was compared with a control group (n = 6) that underwent only bypass. In both groups, the pulmonary regurgitant fraction (PRF) and cardiac output were measured by a pulsed Doppler fl ow meter, and the percent segmental shortening of RV (%RVSS) and RV end-diastolic dimension (RVDd) were measured by sonomicrometry. We also performed dobutamine stress evaluation as well as changing the PVR by carbon dioxide (PaCO2) and inhaled nitric oxide (NO). pi Methods The cohort consisted of 2,632 adults admitted to the ICU with echocardiogram reports using the MIMIC-II database, and was divided into those with HDLVEF and those with normal left ventricular ejection fraction (NLVEF). Those with impaired ejection fraction were excluded from the analysis. Baseline comparisons were performed using chi- squared tests for equal proportion with results reported as numbers, percentages, and 95% CIs. Continuous variables were compared using t tests and reported as means with 95% CIs, while non-normally distributed data were compared using Wilcoxon rank-sum tests and reported as medians. 2 Results All bypass time was 18 ± 3 minutes. In the PR group, the PRF was 40 ± 4% and the RVDd was 53 ± 9 mm (vs. P177 S64 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P179) P178 P178 Comparison of the eff ects of histidine–triptophan–ketoglutarate solution and crystalloid cardioplegia on myocardial protection during pediatric cardiac surgery S Kuslu, P Zeyneloglu, A Pirat, A Camkiran, M Ozkan, G Arslan Baskent University Faculty of Medicine, Ankara, Turkey Critical Care 2014, 18(Suppl 1):P178 (doi: 10.1186/cc13368) ( ) Normal Hyperdynamic (n = 2,373, 90%) (n = 259, 10%) P value Male 1,159 (49) 98 (38) <0.001 Service type MICU 1,234 (52) 138 (53) 0.695 CCU 364 (15) 28 (11) 0.052 SICU 572 (24) 69 (27) 0.367 CSRU 188 (8) 23 (9) 0.590 Primary outcome Twenty-eight-day mortality 458 (19) 79 (31) <0.001 One-year mortality 892 (38) 129 (50) <0.001 ICU mortality 296 (12) 60 (23) <0.001 Hospital mortality 433 (18) 80 (31) <0.001 Treatment RRT 363 (15) 55 (21) 0.013 Vasopressor 1,063 (45) 139 (54) 0.006 Ventilated 1,453 (61) 186 (72) <0.001 Introduction The major components of myocardial protection during cardiac surgery are the combination of cardioplegia solutions with hypothermia. The primary endpoint of this study is to compare the eff ects of histidine–triptophan–ketoglutarate (HTK) solution and crystalloid cardioplegia on release of cardiac troponin-I (cTn-I) and creatine kinase-myocardial band (CK-MB), which are perioperative determinants of myocardial protection; the secondary endpoint is to evaluate the intraoperative and postoperative hemodynamic variables and clinical outcome parameters. p Methods A total of 66 children aged 1 month to 6 years undergoing elective congenital heart surgery were randomly allocated to HTK solution (Group H, n = 32) or crystalloid cardioplegia (Group C, n = 34) after aortic cross-clamping. Blood samples for cTn-I and CK-MB levels were measured before the surgical incision, at the end of surgery and at 4, 16, 24 and 48 hours postoperatively. Results Demographic features were similar in both groups. Duration of surgery, aortic clamp and cardiopulmonary bypass times, amounts of intraoperative fl uids used and urine outputs were similar between the groups. The groups were not signifi cantly diff erent in terms of cTn-I and CK-MB levels at the intraoperative and postoperative period (P >0.05 for all). The dose of positive inotropic drug at the end of surgery was signifi cantly high in Group H (P = 0.01). The requirements for defi brillation were similar in both groups. P177 There were no signifi cant diff erences between the groups regarding postoperative hemodynamic parameters, positive inotropic requirements, amounts of fl uids and blood given and pacemaker requirements (P >0.05 for all). Duration of mechanical ventilation, lengths of ICU and hospital stay were similar in both groups. Conclusion Patients with hyperdynamic LVEF in the ICU clearly have increased mortality. Hyperdynamic LVEF may be a result of increased catecholamines during cytokine storm. It is unclear whether hyperdynamic LVEF itself worsens outcomes. Further investigation is needed. g p Conclusion The present study demonstrated that there is no superiority of HTK solution and crystalloid cardioplegia to each other for myocardial protection during pediatric cardiac surgery. P177 P177 Correlation between arterial lactate and venous lactate in patients with sepsis and septic shock P Theerawit, C Na Petvicharn Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P177 (doi: 10.1186/cc13367) Figure 1 (abstract P177). Figure 2 (abstract P177). Introduction Measurement of arterial lactate (A-LACT) levels has been used to monitor poor tissue perfusion, predicting mortality and guiding resuscitation. Peripheral venous lactate (V-LACT) has been regarded as an unreliable test, but a less invasive approach. We aimed to determine correlation between A-LACT and V-LACT and agreement of both in order to determine the usefulness of V-LACT as a biomarker for assessment in sepsis. p Methods We conduct a prospective, cross-sectional study during June to December 2011 at a university hospital. Septic patients in the ICU were enrolled in this research. Sepsis was defi ned according to the Surviving Sepsis Campaign: International Guidelines for Management of Severe Sepsis and Septic Shock: 2008. The exclusion criteria were: contraindication for arterial puncture; and denying inform consent. The venous lactate would be sampled at the same point in time as arterial lactate measurement. The correlation and agreement between arterial and venous lactate was the primary outcome. p y Results A total of 73 pair-samples in 45 intensive care patients were collected. Mean age was 68.33 ± 14.5 years. Fifty percent of all patients received the vasopressors to stabilize hemodynamics. The mean serum creatinine level was 2.78 mg/dl and the mean anion gap was 13.55 mmol/l. The mean arterial lactate (A-LACT) level was 3.73 ± 4.0 mmol/l, and the mean venous lactate (V-LACT) level was 4.6 ± 4.2 mmol/l. The A-LACT and V-LACT were strongly correlated as shown in Figure 1 (r = 0.927, P <0.0001, r2 = 0.859). The mean diff erence between V-LACT and A-LACT was 0.889 mmol/l. The 95% limits of the V-A diff erence in the individual patients were between –2.3 and 4.1 mmol/l. However, the agreement looks very good at lactate levels not higher than 4 mmol/l (Figure 2). The regression equation was: A-LACT = (0.877 × V-LACT) – 0.320. Figure 2 (abstract P177). Figure 2 (abstract P177). Conclusion The arterial lactate and venous lactate levels were strongly correlated in the condition of sepsis or septic shock. Consequently, V-LACT may be used in substitution for A-LACT particularly in lactate levels not higher than 4 mmol/l. However, trending should be generally applied instead of the absolute value. P180 Impact of nitric oxide on pulmonary regurgitation and cardiac function in the acute stage after right ventricular outfl ow surgery Y Ko, K Morita, R Nagahori, T Abe, K Hashimoto Jikei University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P180 (doi: 10.1186/cc13370) The infl ammatory response due to surgery and cardiopulmonary bypass (CPB) may contribute to PNAF by inducing atrial dysfunction [1]. Corticosteroids reduce the infl ammatory response and may thus reduce atrial dysfunction and PNAF [2]. The aim of this study was to determine whether dexamethasone protects from left atrial dysfunction and PNAF in cardiac surgical patients. same as catecholamine during the acute stage after RV outfl ow surgery with PR. P181 Cardiogenic oscillation in pediatric patients after cardiac surgery H Imanaka, N Okuda, T Itagaki, M Onodera, M Nishimura Tokushima University Hospital, Tokushima, Japan Critical Care 2014, 18(Suppl 1):P181 (doi: 10.1186/cc13371) Methods Patients undergoing cardiac surgery were randomized to a single dose of dexamethasone (1 mg/kg) or placebo after inducing anesthesia. Transesophageal echocardiography was performed in patients after CPB. The primary outcome was left atrial total ejection fraction (LA-TEF) after sternal closure; secondary outcomes included left atrial diameter and PNAF, detected by Holter monitoring. Introduction Cardiogenic oscillation is the fl uctuation in fl ow tracing in mechanically ventilated patients. Large cardiogenic oscillation may cause autotriggering in adult patients after cardiac surgery [1] and inaccurate volume monitoring [2]. However, it is unknown how cardiogenic oscillation is problematic in pediatric patients. Therefore, we prospectively surveyed cardiogenic oscillation in pediatric patients after cardiac surgery. y g Results Sixty-two patients were included. Baseline characteristics were well balanced. Postoperative LA-TEF was 36.4% in the dexamethasone group and 40.2% in the placebo group (P = 0.15) (Figure 1). Secondary echocardiographic outcomes were also insignifi cant (Table 1). The incidence of PNAF was 30% in the dexamethasone group and 39% in the placebo group (P = 0.47). g y Methods We enrolled 17 pediatric patients who underwent cardiac surgery using cardiopulmonary bypass. They were mechanically ventilated with pressure-controlled ventilation. We measured the amplitude in cardiogenic oscillation and compared them between their admission to the ICU and before extubation. We performed statistical analysis with the t test and considered P <0.05 signifi cant. Table 1 (abstract P182). Secondary postoperative echocardiographic parameters in both groups Parameter Dexamethasone Placebo P value LA-TEF 36.4 40.2 0.15 LA diameter 4.6 4.3 0.19 LA area 16.0 16.4 0.81 y gi Results Cardiogenic oscillation was 2.1 ± 0.6 l/minute just after the surgery (Figure 1). Autotriggering occurred in seven of 17 patients when triggering sensitivity was set at 1 l/minute. P180 Impact of nitric oxide on pulmonary regurgitation and cardiac function in the acute stage after right ventricular outfl ow surgery Y Ko, K Morita, R Nagahori, T Abe, K Hashimoto Jikei University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P180 (doi: 10.1186/cc13370) control 34 ± 6 mm). P <0.05. A signifi cant reduction in the %RVSS (18 ± 1% vs. control 22 ± 1%) and the cardiac output (2.1 ± 0.2 l/minute* vs. control 2.5 ± 0.3 l/minute) were observed. The PRFs were 60 ± 5% (PaCO2 >80 mmHg), 37 ± 2% (PaCO2 <20 mmHg), 24 ± 2% (NO 20 ppm; PaCO2 40 mmHg), and were positively correlated with the PVR (Figure 1A). During the dobutamine stress, the %RVSS was increased (baseline 18 ± 1%, 5γ 21 ± 2%, 10γ 26 ± 3%), and was negatively correlated with the PRFs (Figure 1B). Results Patients with HDLVEF had increased mortality in hospital, at 28 days and at 1 year when compared with patients with NLVEF. HDLVEF patients more frequently required renal replacement therapy (RRT), vasopressors and mechanical ventilation. Of the 2,632 patients, 1,220 were septic. There was an increased proportion of HDLVEF in the septic compared with the nonseptic groups (11.2% vs. 8.6%, P = 0.026). Interestingly, other statistically signifi cant associated comorbidities were cancer, CHF, arrhythmias, and hypertension, which were more commonly seen in the HDLVEF group. See Table 1. Conclusion These results indicated that massive PR resulted in marked deterioration of RV performance; however, low PVR and high RV contractility may contribute to reduce the severity of PR and improve cardiac function. Nitric oxide may be a useful treatment modality the S65 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 l Figure 1 (abstract P180). of inotropes and intravascular volume might have contributed to the decrease in cardiogenic oscillation. of inotropes and intravascular volume might have contributed to the decrease in cardiogenic oscillation. Figure 1 (abstract P180). Conclusion In pediatric patients after cardiac surgery, cardiogenic oscillation was initially large but was decreasing at the extubation. References e e e ces 1. Crit Care Med 2000, 28:402. 2. Crit Care Med 2004, 32:1546. P182 Intraoperative dexamethasone on left atrial function and postoperative atrial fi brillation in cardiac surgical patients K Jacob, S Dieleman, H Nathoe, D Van Osch, E De Waal, M Cramer, J Kluin, D Van Dijk Utrecht University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P182 (doi: 10.1186/cc13372) Figure 1 (abstract P180). same as catecholamine during the acute stage after RV outfl ow surgery with PR. Introduction Postoperative new-onset atrial fi brillation (PNAF) is very common after cardiac surgery. P183 White blood cell count and new-onset atrial fi brillation after cardiac surgery S Dieleman, K Jacob, H Nathoe, M Ten Berg, D Van Osch, J Frencken, D Van Dijk Utrecht University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P183 (doi: 10.1186/cc13373) Introduction Ranolazine, a piperazine derivative, is used as an anti- anginal drug to treat patients with chronic angina in clinical practice [1] and may improve coronary blood fl ow by reducing compression eff ects of ischemic contracture, and by improving endothelial function [2,3]. In the present study we investigate the vascular eff ects of ranolazine on the endothelium, adrenergic system and Ca2+ in isolated rat aorta. Introduction Postoperative new-onset atrial fi brillation (PNAF) is the most common complication after cardiac surgery. Infl ammation as an underlying mechanism has been studied by various infl ammatory markers, and white blood cell count (WBC) is the only present consequent infl ammatory marker predicting PNAF [1]. This study aimed to determine the association between perioperative WBC and PNAF. Methods Rat aortic segments (3 mm long) with and without endothelium were mounted for isometric tension recording in organ baths containing Krebs–Henseleit solution. Electrical fi eld stimulation (2, 4 and 8 Hz, 20 V, 0.25 ms duration for 30 seconds) was provided by a Grass S88 stimulator via two platinum electrodes positioned on each side and parallel to the axis of the aortic segment. Concentration– response curves of ranolazine (10–7 to 10–4 M) were obtained in a cumulative manner using endothelin-1, noradrenaline, thromboxane A2 and KCl as constrictor agents. Methods Patients >18 years undergoing elective cardiac surgery with a sinus rhythm preoperatively were recruited from the Dexamethasone for Cardiac Surgery-PNAF trial for this post-hoc cohort study. The WBC was prospectively measured preoperatively and once during each of the fi rst four postoperative days. Development of PNAF was evaluated with continuous 12-lead ECG monitoring the fi rst 5 days postoperatively. Results The contractile responses to electrical fi eld stimulation were abolished by tetrodotoxin, guanethidine and prazosin, indicating that the contractile eff ect is due to the action of noradrenaline on alpha adrenoreceptors. Ranolazine diminished (P <0.05) neurogenic adrenergic contractions induced by electrical fi eld stimulation in aortic rings with and without endothelium. Ranolazine produced concentration-dependent relaxation in rings precontracted with noradrenaline (Emax 86 ± 6%, n = 10; P <0.05) but not in rings precontracted with endothelin-1, thromboxane A2 and KCl. Neither L-NAME (10–4 M), an inhibitor of nitric oxide synthase, nor indomethacin (10–5 M), an inhibitor of cyclooxygenase, modifi ed the relaxation induced by ranolazine. The calcium antagonist nifedipine (10–6 M) reduced the relaxation induced by ranolazine. P184 References 1. Haff ajee et al.: JACC Cardiovasc Imaging 2011, 4:833-840. 2. Chaney et al.: Chest 2002, 121:921-923. P184 Anti-adrenergic eff ects of ranolazine in isolated rat aorta P Marchio, MD Mauricio, FB El Amrani, S Guerra, D Aguirre-Rueda, SL Vallés, JM Vila, M Aldasoro University of Valencia, Spain Critical Care 2014, 18(Suppl 1):P184 (doi: 10.1186/cc13374) P180 Impact of nitric oxide on pulmonary regurgitation and cardiac function in the acute stage after right ventricular outfl ow surgery Y Ko, K Morita, R Nagahori, T Abe, K Hashimoto Jikei University School of Medicine, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P180 (doi: 10.1186/cc13370) Before the extubation, cardiogenic oscillation signifi cantly decreased to 1.4 ± 0.4 l/minute when autotriggering disappeared. Intensive care including adjustment Conclusion Intraoperative high-dose dexamethasone did not have any protective eff ect on postoperative LA-TEF or dimension and did not reduce the risk of PNAF in cardiac surgical patients. Conclusion Intraoperative high-dose dexamethasone did not have any protective eff ect on postoperative LA-TEF or dimension and did not reduce the risk of PNAF in cardiac surgical patients. Figure 1 (abstract P181). Cardiogenic oscillation. Figure 1 (abstract P182). Primary outcome preoperatively and postoperatively in dexamethasone and placebo groups. S66 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References 1. Haff ajee et al.: JACC Cardiovasc Imaging 2011, 4:833-840. 2. Chaney et al.: Chest 2002, 121:921-923. P183 White blood cell count and new-onset atrial fi brillation after cardiac surgery S Dieleman, K Jacob, H Nathoe, M Ten Berg, D Van Osch, J Frencken, D Van Dijk Utrecht University Medical Center, Utrecht, the Netherlands Critical Care 2014, 18(Suppl 1):P183 (doi: 10.1186/cc13373) Results A total of 657 patients were included in this trial, 277 developed PNAF. The WBC was signifi cantly higher in the PNAF group on day 2 and day 4 (Figure 1). However, multivariate analysis showed that preoperative and postoperative WBC, days 1 to 3, were not associated with PNAF (Table 1). Older age (OR: 1.05; CI: 1.03 to 1.07; P <0.001), CABG plus valve surgery (OR: 2.95; CI: 1.78 to 4.88), single valve surgery (OR: 3.09; CI: 2.03 to 4.69; P <0.001) and other surgery (OR: 2.21; CI: 1.23 to 3.97; P <0.001) were correlated with the occurrence of PNAF. Table 1 (abstract P183). Multiple regression analysis of association between high WBC and developing PNAF Time point OR 95% CI Baseline 1.04 0.96 to 1.13 Day 1 1.03 0.98 to 1.08 Day 2 1.03 0.99 to 1.08 Day 3 1.03 0.96 to 1.11 Day 4 1.09 1.01 to 1.16 Table 1 (abstract P183). Multiple regression analysis of association between high WBC and developing PNAF Conclusion These results indicate that ranolazine diminished the contractile response induced by adrenergic stimulation, suggesting an eff ect as an adrenergic blocker. The relaxant eff ects of ranolazine on rat aortic vessels is not dependent on the endothelium-derived factors (nitric oxide or dilator prostanoids) but involves an interference with the entry of calcium through dihydropyridine calcium channels. References 1. Chaitman BR.: Circulation 2006, 113:2462-2472. 2. Stone PH, et al.: J Am Coll Cardiol 2010, 56:934-942. 3. Deshmukh SH, et al.: Coron Artery Dis 2009, 20:343-347. 1. Chaitman BR.: Circulation 2006, 113:2462-2472. 2. Stone PH, et al.: J Am Coll Cardiol 2010, 56:934-942. 3. Deshmukh SH, et al.: Coron Artery Dis 2009, 20:343-347. Conclusion Preoperative and postoperative WBC were not associated with development of PNAF. Reference Eff ects of perfusion pressure on the splanchnic circulation after cardiopulmonary bypass: a randomized double cross-over study Conclusion Isofl urane prevents experimental TCM and preserves LV function, an eff ect not mediated via opening of K-ATP channels. The eff ect cannot be explained entirely by attenuation of myocardial stress. Isofl urane sedation in the ICU might be an interesting approach for patients suff ering from hyperadrenergic conditions at risk of developing TCM. Introduction No randomized trial has assessed the eff ects of diff erent mean arterial pressure (MAP) targets in postcardiac surgery intensive care. We investigated the short-term eff ects of MAP of 65 or 85 mmHg on splanchnic oxygen fl ux, metabolic function, mucosal perfusion and cytokine regulation. P185 Patient demographics aff ecting the apex causing apical ballooning. TCM is frequent in patients with adrenergic overstimulation and is probably common in ICU patients [1]. In a TCM rat model we evaluated whether diff erent anesthetic agents could attenuate LV akinesia in TCM. g Methods Isoprenaline was intraperitoneal (i.p.) injected, which induces LV akinesia and apical ballooning within 90 minutes [2]. We performed the study in two diff erent settings. In the fi rst setting, spontaneously breathing rats (n = 12 in each group) were sedated with either pentobarbital, ketamine, isofl urane or no anesthetic before i.p. isoprenaline. One additional group received the K-ATP blocker glyburide before sedation with isofl urane. In the second setting, rats were anaesthetized with ketamine + midazolam, mechanically ventilated and the carotid artery was cannulated. Before i.p. isoprenaline, animals were randomized to either no isofl urane (0 MAC), isofl urane 0.5 MAC or isofl urane 1.0 MAC (n = 12 in each group). Arterial blood gas was obtained before isoprenaline and 60 minutes after isoprenaline. The heart rate (HR), systolic blood pressure (SBP) and body temperature (BT) were recorded continuously. After 90 minutes, echocardiography was performed. Extent of akinesia was expressed as the percentage of total LV endocardial length. End-diastolic and end-systolic LV volumes were measured, and stroke volume (SV) and cardiac output (CO) were calculated. Conclusion Administering morphine prior to extubation causes signifi cant delays in weaning from mechanical ventilation. We plan to introduce intraoperative and postoperative protocols to facilitate rapid weaning from mechanical ventilation for elective cardiac surgical patients. Results In spontaneously breathing rats, the degree of akinesia was signifi cantly lower with pentobarbital and isofl urane (± glyburide) but not with ketamine compared with controls. The degree of akinesia was lowest with isofl urane. In ventilated rats, the degree of apical akinesia (%) was signifi cantly lower at 0.5 MAC (8.7 ± 7.3) and 1 MAC (5.7 ± 7.4) versus 0 MAC (17.7 ± 8.0). This was accompanied by a higher CO and SV. HR was lower at 1 MAC (6%) and SBP was lower at 0.5 MAC (106 ± 7) and 1 MAC (98 ± 7) versus 0 MAC (126 ± 8). BT and pH was lower in both isofl urane groups. In a multivariate model, isofl urane was the only variable that was independently associated with the degree of LV akinesia. P188 P188 Preoperative therapy with angiotensin-converting enzyme inhibitors in cardiac surgery patients: is there any impact on postoperative renal function? F Ampatzidou, M Sileli, K Diplaris, C Koutsogiannidis, T Karaiskos, G Drossos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P188 (doi: 10.1186/cc13378) Preoperative therapy with angiotensin-converting enzyme inhibitors in cardiac surgery patients: is there any impact on postoperative renal function? P185 Reference 1. Abdelahdi et al.: Am J Cardiol 2004, 93:1176-1178. Reference 1. Abdelahdi et al.: Am J Cardiol 2004, 93:1176-1178. , , , , g Glenfi eld Hospital, University Hospitals of Leicester, UK Glenfi eld Hospital, University Hospitals of Leicester, UK Critical Care 2014, 18(Suppl 1):P185 (doi: 10.1186/cc13375) i p , y p , Critical Care 2014, 18(Suppl 1):P185 (doi: 10.1186/cc13375) Figure 1 (abstract P183). WBC at baseline (t = 0) and in the four postoperative days (t = 1 to 4), PNAF versus no PNAF. Introduction Early extubation post coronary artery bypass grafting does not increase perioperative morbidity and reduces the length of stay (LOS) in the ICU and in hospital [1]. Use of low-dose opioid- based general anaesthesia and time-directed protocols for fast- track interventions does not increase mortality or postoperative complications in low–moderate-risk patients and has been found to have a reduced time to extubation and shortened ICU stay [2]. Our mean time to extubation is 6 hours, although patients are assessed to be safe to be weaned from mechanical ventilation at 2 hours following arrival in the ICU. This study aims to identify factors that delay extubation in patients undergoing routine cardiac surgery at our institution. g g g y Methods A prospective analysis was performed on all patients post adult cardiac surgery from 14 May 2013 to 10 July 2013. Emergency surgical patients and those with intraoperative complications were excluded. Results A two-sample t test was used to analyse the data. Patient demographics are presented in Table 1. There were signifi cant delays in time of extubation in those who received morphine prior to extubation compared with those that did not (P = 0.0184) (Table 2). There were no Figure 1 (abstract P183). WBC at baseline (t = 0) and in the four postoperative days (t = 1 to 4), PNAF versus no PNAF. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S67 signifi cant diff erences in LOS in ICU or hospital. Factors such as age, EUROSCORE and type of operation did not have an infl uence on time Table 1 (abstract P185). Patient demographics Age (years) 66 (10.1) EUROSCORE (%) 2.90 (1.84) Table 2 (abstract P185). Morphine versus no morphine Morphine No morphine Patient number 51 20 Time to extubation (hours) 7:48 (4:42) 4:48 (2:19) LOS ICU (hours) 51:21 (55:16) 40:06 (28:47) LOS hospital (days) 9.83 (5.01) 10.8 (13.5) Table 1 (abstract P185). g References References 1. Park JH, et al.: Chest 2005, 128:296-302. 2. ShaoY, et al.: Int J Cardiol 2013, 168:1943-1950. 1. Park JH, et al.: Chest 2005, 128:296-302. 2. ShaoY, et al.: Int J Cardiol 2013, 168:1943-1950. Methods A single-center, randomized controlled, double cross-over trial was performed. Patients were randomized to: HLH (high–low– high) where MAP targets were 85–65–85 mmHg in sequence, with each lasting 2 hours, or LHL (low–high–low) where MAP targets were 65– 85–65 mmHg. Blood pressure was adjusted with noradrenalin infusion. Results Six + six patients were included in the study. MAP targets were achieved in all patients at all time points (64 ± 3, 84 ± 4; 65 ± 5 mmHg in the LHL group and 84 ± 3; 66 ± 2; 85 ± 5 mmHg in the HLH group at the fi rst, second and third time points), with corresponding changes in fi lling pressures. Cardiac output did not change over time. Hepatic venous saturation was 41 ± 15; 58 ± 24; 56 ± 21% in the LHL group and 50 ± 19; 43 ± 20; 41 ± 18% in the HLH group at the fi rst, second and third time points, with a signifi cant time group interaction (P <0.05). No changes were observed in global or trans-splanchnic lactate levels and cytokine levels or in gastric tonometry CO2.f Preoperative therapy with angiotensin-converting enzyme inhibitors in cardiac surgery patients: is there any impact on postoperative renal function? p p F Ampatzidou, M Sileli, K Diplaris, C Koutsogiannidis, T Karaiskos, G Drossos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P188 (doi: 10.1186/cc13378) Introduction Preoperative therapy with angiotensin-converting enzyme inhibitors (ACEI) is common in patients undergoing cardiac surgery. The aim of this study was to evaluate the still-debated impact of preoperative use of ACEI on postoperative renal function in cardiac surgery patients [1,2]. y g y 2 Conclusion Increasing MAP with norepinephrine has some eff ects splanchnic oxygenation, but has no impact on metabolic or biochemical function and key cytokine removal or release. MAP targets of 60 to 65 mmHg or 80 to 85 mmHg appear physiologically equivalent for the splanchnic circulation. y Methods A total of 624 consecutive patients, who underwent cardiac surgery from July 2012 to October 2013, were evaluated. Data were prospectively collected in our clinic’s electronic database and were retrospectively analyzed as to preoperative ACEI therapy. The chi- square test was used for correlations. Endpoints of the study were the development of postoperative acute kidney injury (AKI) and the diff erence between hospital admission and discharge glomerular fi ltration rate (GFR). The AKI defi nition was based on modifi ed RIFLE classifi cation. GFR values were estimated by the MDRD formula. References 1. Cheng DC, et al.: J Thorac Cardiovasc Surg 1996, 112:755-764. 2. Zhu F, et al.: Cochrane Database 2012, 10:CD003587. 1. Cheng DC, et al.: J Thorac Cardiovasc Surg 1996, 112:755-764. 2 Zhu F et al : Cochrane Database 2012 10:CD003587 Tissue-aggressive infl ammatory response defi nes the tissue aggressiveness of the post-infarction milieu y Methods To conduct the study we analyzed the database of a CICU of a medium-sized hospital in the city of Presidente Prudente, Brazil. Admissions that occurred during the period 1 September 2010 to 31 August 2013 were analyzed. The information was collected from the EPIMED MONITOR system and statistically analyzed using EPI INFO, version 3.5.2 software. P <0.05 two-tailed was considered signifi cant, and confi dence intervals at 95% (95% CI) were used for the logistic regression multivariate estimated in the sample. Introduction A potential measure of post-ischemic milieu in myocardium subject to acute ischemic injury is to assess the so- called myocardial salvage index (SI) by relating the fi nal infarct size to the initial myocardium at risk (MaR). Cardiac magnetic resonance (CMR) has previously been shown to enable determination of both infarct size using late gadolinium enhancement and MaR using T2- weighted imaging. This technique could thus potentially be used to identify infl ammatory responses that could be targeted to accomplish cardioprotection. The aim of this study was to relate SI, as determined by CMR, to the infl ammatory response in patients with acute myocardial infarction.i Results A total of 2,098 admissions were recorded, of which 42.1% were female and 57.9% male. The average age was 66.99 ± 13.30 years. The prognosis for SAPS 3 admission score averaged 40.8 ± 15.48 points and the mean unit length of stay was 3.5 ± 5.01 days. The main admission diagnoses were unstable angina (13.06%), non-ST-segment elevation myocardial infarction (8.29%), ST-segment elevation myocardial infarction (8.10%) and supraventricular cardiac tachyarrhythmia (6.91%). We observed a higher risk of death among patients who had, at admission, congestive heart failure NYHA 2, 3 or 4 (odds ratio (OR) = 2.81, 95% CI: 2.07 to 3.83, P = 0.001), chronic renal failure (OR = 2.48, 95% CI: 1.68 to 3.67, P = 0.001), peripheral artery disease (OR = 1.93, 95% CI: 1.04 to 3.58, P = 0.033), severe chronic obstructive pulmonary disease (OR = 3.17, 95% CI: 1.82 to 5.51, P = 0.002), and infection at admission (OR = 7.88, 95% CI: 5.27 to 11.78, P = 0.001). Methods Fifteen patients with fi rst-time ST-elevation myocardial infarction were included in the study. All patients underwent primary PCI due to an acute occlusion in one branch of the left coronary artery. Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) p J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) p J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) p J Oras1, B Redfors2, Y Shao2, H Seeman-Lodding1, SE Ricksten1, E Omerovic2 1Institute of Clinical Sciences, Gothenburg, Sweden; 2The Wallenberg Laboratory, Gothenburg, Sweden Critical Care 2014, 18(Suppl 1):P187 (doi: 10.1186/cc13377) i Results A total of 354 patients (56.7%) were treated with ACEI preoperatively. Overall, 95 patients (15.3%) developed postoperative AKI. Preoperative use of ACEI was not associated with the development Introduction Tako-tsubo cardiomyopathy (TCM) is an acute cardiac syndrome with regional hypokinesia in the left ventricle (LV), often Introduction Tako-tsubo cardiomyopathy (TCM) is an acute cardiac syndrome with regional hypokinesia in the left ventricle (LV), often Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S68 of postoperative AKI (P = 0.981). Mean GFR values on admission day were 65.23 ± 16.89 for ACEI users, and 65.06 ± 19.62 for the rest of the cohort. Mean GFR values on discharge day were 66.77 ± 21.25 and 67.35 ± 25.64 respectively. The diff erence in GFR at the time of hospital admission and on discharge day had no statistical diff erence, P = 0.511 (Table 1). Overall, 25 patients (4%) needed dialysis. Conclusion The impact of preoperative ACEI treatment on postoperative renal function after cardiac surgery is still debated. We found no association, neither protective nor harmful, between preoperative ACEI therapy and renal function impairment. References 1. Arora P, et al.: Clin J Am Soc Nephrol 2008, 3:1266-1273. 2. Ouzounian M, et al.: Ann Thorac Surg 2012, 93:559-564. Table 1 (abstract P188). 2. Polanczyk CA, et al.: Fatores de Risco Cardiovascular no Brasil: os próximos 50 Anos. Arq Bras Cardiol 2005, 84:199-201. 1. Roger VL: Heart disease and stroke statistics – 2013 update. Circulation 2013, 127:e6-e245. Hospital visit pattern and its eff ect on reperfusion time and clinical outcomes in ST-segment elevation acute myocardial infarction Introduction The reperfusion time is critical in ST-segment elevation myocardial infarction (STEMI), and it makes a diff erence to clinical outcomes. This study was designed to investigate the hospital visit pattern and its eff ect on reperfusion time and clinical outcomes of STEMI patients. Methods A total of 199 STEMI patients were registered in this study from three university hospitals in Kyungsang-do area, Korea, and were divided into two groups; group 1 (n = 69) who directly visited the hospitals capable of percutaneous coronary intervention (PCI), and group 2 (n = 130) who fi rst visited local hospitals and then transferred to PCI-capable hospitals. We analyzed the estimated distance and time to the hospitals using a driving navigation system, elapsed time from chest pain to primary PCI hospitals, chest pain to reperfusion time, and in-hospital outcomes. 1. Arora P, et al.: Clin J Am Soc Nephrol 2008, 3:1266-1273. 2. Ouzounian M, et al.: Ann Thorac Surg 2012, 93:559-564. Results There was no diff erence in fi rst medical contact time between groups 1 and 2. But the time from chest pain to PCI hospital was shorter in group 1 (206.2 ± 268.5 vs. 370.3 ± 415.2 minutes, P = 0.001). Sixty patients in group 1 and 108 patients in group 2 underwent reperfusion therapy (P = 0.473). The chest pain to reperfusion time was shorter in group 1 (294.6 ± 255 vs. 397.2 ± 341.9 minutes, P = 0.045). The diff erence in estimated time by navigator and actual hospital visit time was also shorter in group 1 (194.2 ± 269.9 vs. 321.6 ± 411.0 minutes, P = 0.009). In-hospital mortality was higher in group 2 (0 vs. 4.6%, P = 0.094). Characterization of the profi le and clinical variables associated with mortality in a Brazilian coronary ICU Characterization of the profi le and clinical variables associated with mortality in a Brazilian coronary ICU CE Bosso1, VF Paula1, OA Souza2, GE Valerio2, SV Ferreira2 1Instituto do Coração de Presidente Prudente, Brazil; 2UNOESTE – Universidade do Oeste Paulista, Presidente Prudente, Brazil Critical Care 2014, 18(Suppl 1):P189 (doi: 10.1186/cc13379) y Conclusion A primary visit to a local hospital was associated with longer reperfusion time and was associated with higher mortality. Therefore, the reperfusion time could be reduced by patient education and management of the community healthcare system. Introduction Cardiovascular disease is the leading cause of death worldwide and the outlook for 2020 data is even more alarming [1,2]. In this context, the coronary ICUs (CICUs) have become increasingly numerous. However, data concerning this are still very limited in the literature. This study aims to characterize the profi le of CICU admissions in Brazil and the main clinical variables associated with increased mortality. Isofl urane attenuates left ventricular akinesia and preserves cardiac output in the Tako-tsubo rat model AKI and GFR diff erence ACEI users (n = 354) ACEI nonusers (n = 270) P value AKI 54 (15.3%) 41 (15.2%) 0.981 GFR diff erence (mean) +1.43 +2.3 0.511 Table 1 (abstract P188). AKI and GFR diff erence ACEI users (n = 354) ACEI nonusers (n = 270) P value AKI 54 (15.3%) 41 (15.2%) 0.981 GFR diff erence (mean) +1.43 +2.3 0.511 Table 1 (abstract P188). AKI and GFR diff erence ACEI users (n = 354) ACEI nonusers (n = 270) P value P191 Tissue-aggressive infl ammatory response defi nes the tissue aggressiveness of the post-infarction milieu E Grins, L Algotsson, H Engblom, M Carlsson, H Arheden, S Jovinge Scania University Hospital, Lund University, Lund, Sweden Critical Care 2014, 18(Suppl 1):P191 (doi: 10.1186/cc13381) P190 Hospital visit pattern and its eff ect on reperfusion time and clinical outcomes in ST-segment elevation acute myocardial infarction J Park, Y Kim, D Shin, W Kim, S Lee, J Son Yeungnam University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P190 (doi: 10.1186/cc13380) P190 Hospital visit pattern and its eff ect on reperfusion time and clinical outcomes in ST-segment elevation acute myocardial infarction J Park, Y Kim, D Shin, W Kim, S Lee, J Son Yeungnam University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P190 (doi: 10.1186/cc13380) Rhabdomyolysis following cardiac surgery: from prevalence to prevention y g y Methods TTE was performed before anesthesia induction and upon ICU arrival in 15 patients with preoperative diagnosis of LV dysfunction defi ned as an EF <35%. In-ICU measurements of Ees, Ea and VAc were taken at diff erent steps of PEEP application lasting 3 minutes as follows: 0 cmH2O PEEP (ZEEP), 5 cmH2O PEEP, 10 cmH2O PEEP and 15 cmH2O PEEP. TTE and hemodynamic parameters were recorded and analyzed. Results All patients were uncoupled preoperatively (VAc >1.31, average VAc (AVAc) = 1.56) before anesthesia induction and showed worsened uncoupling postoperatively at ZEEP. PEEP application altered VAc by modifying both Ees and Ea at all steps and all patients showed further uncoupling at any level of PEEP application (AVAc at ZEEP = 1.97, at 5 cmH2O PEEP = 1.61, at 10 cmH2O PEEP = 1.87, at 15 cmH2O PEEP = 2.23) A PEEP of 5 cmH2O provided the more favorable VAc. p AS Omar1,2, HA Ewila1,3, SR Aboulnaga1,4, AK Tuli1 1Hamad Medical Corporation, Doha, Qatar; 2Beni Suef University, Egypt; 3Suez Canal University, Egypt; 4Ain Shams University, Egypt Critical Care 2014, 18(Suppl 1):P194 (doi: 10.1186/cc13384) AS Omar1,2, HA Ewila1,3, SR Aboulnaga1,4, AK Tuli1 1Hamad Medical Corporation, Doha, Qatar; 2Beni Suef University, Egypt; 3Suez Canal University, Egypt; 4Ain Shams University, Egypt Critical Care 2014, 18(Suppl 1):P194 (doi: 10.1186/cc13384) Introduction In the view of robust consequences following cardiac surgery, acute kidney injury (AKI) remains a major concern. Rhabdomyolysis (RML) following cardiac surgery and its relation to AKI need to be investigated. We aim to study the prevalence of RML development following cardiac surgery and the perioperative risk factors that may expedite the occurrence of RML. 2 p Conclusion Sb evaluation of Ea/Ees shows that following CABG the patients with depressed LV remain uncoupled, and that in such patients the application of PEEP leads to further decoupling. In this preliminary experience, 5 cmH2O PEEP seems to be the most appropriate in preventing further worsening of the VAc. A large RCT is needed to draw conclusions on the PEEP eff ect on VAc following CABG in a depressed heart. Methods All patients undergoing cardiac surgeries in our hospital were enrolled in the study during the period of 1 year in a prospective descriptive study measuring the occurrence of RML and its association with AKI, where all patients in the study underwent serial assessment of serum creatinine kinase (CK) and serum myoglobin. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P = 0.5742) response. However, the proportion of TH1 (CD4+ IFNγ+) cells was higher in the group with the lowest SI (57.47 ± 4.805 vs. 38.10 ± 6.514, P = 0.0349). P = 0.5742) response. However, the proportion of TH1 (CD4+ IFNγ+) cells was higher in the group with the lowest SI (57.47 ± 4.805 vs. 38.10 ± 6.514, P = 0.0349). predictive of mortality. The recently introduced high-sensitivity cardiac troponin T (HS cTnT) assay has resulted in an increased detection of elevated cTnT in ICU patients [1]. The aim of this study was to determine the prevalence of elevated cTnT using the HS assay and its relationship with mortality. Conclusion This is the fi rst study that identifi es infl ammatory cell patterns which infl uence the hostility of the infarction milieu in vivo. The study indicates that SI can be used as an evaluator of post- ischemic milieu and that TH1 response is associated with unfavorable post-infarction injury and could therefore be a possible target for future studies to limit infarction size. The current study is, by its size and observational character, important; an argument for rather than a substitute for future interventional studies in this area. y Methods A retrospective observational study was performed on all ICU admissions over a 12-month period. Data were obtained from the clinical information system (ICIP; Philips) and the ICU audit databases (AcuBase). Data collected included patient demographics, peak cTnT value, APACHE II score, requirement for organ support and mortality. The primary outcome measure was hospital mortality. Data were analysed using SPSS v.17.0. cTnT levels were divided into categories for analysis: normal (<14 ng/l) and elevated. The elevated category was further subdivided into quartiles. Univariate analysis was performed between potential risk factors and mortality followed by multivariate regression analysis to ascertain independent predictors of mortality. Results There were 417 admissions to the ICU during the study period, 89 of whom were excluded because of an absent cTnT value, leaving 328 patients included in the analysis. cTnT was elevated in 85% of patients. ICU mortality was 19% and hospital mortality was 28%. Hospital mortality (%) per cTnT category was: <14 ng/l = 2%; 14 to 38 ng/l = 19%; 39 to 90 ng/l = 26%; 91 to 252 ng/l = 39%; >252 ng/l = 43%. On univariate analysis, cTnT levels, age, ventilation and APACHE II score were signifi cantly associated with mortality. 1. Hajjar L, Grande S, Galas F, Roquim A, Sampaio L, Auler J: Risk factors and outcome of rhabdomyolysis after cardiac surgery. Crit Care 2008, 12(Suppl 2):P470. Rhabdomyolysis following cardiac surgery: from prevalence to prevention Serial renal function, prior statin treatment, cardiac injury, lengths of ventilation, and lengths of stay in the ICU and hospital were monitored. References Results We recruited 202 patients in our study, 185 males and 17 females with mean age 52 ± 12.4 years. According to the existence of RML (CK 2,500 U/ml or more) [1], patients were divided into group 1 where RML was identifi ed in 17 patients (8.4%), which was associated with AKI in seven patients (41%), and group 2 without RML (185 patients), where AKI occurred in 34 patients (18.4%) (P = 0.025). We observed a signifi cantly longer duration of ventilation and lengths of stay in the ICU and in hospital in the RML group (P <0.01 for all observations). 1. Guarracino F, et al.: Crit Care 2013, 17:213. 2. Chen CH, et al.: JACC 2001, 38:2028-2034. 3. Franchi F, et al.: Biomed Res Int 2013, 2013:918548. Prevalence of elevated cardiac troponin T in ICU patients using the high-sensitivity assay and the relationship with mortality p g p Conclusion Early increase in the serum CK and myoglobin in postoperative high-risk cardiac surgeries may predict the concomitance of early AKI, where proper intervention may prevent the sequelae of logistic organ dysfunction. g y y S O’Sullivan1, M Sutton2, G Fitzpatrick1 1Tallaght Hospital & Trinity College Dublin, Ireland; 2Health Research Board, Dublin, Ireland Critical Care 2014, 18(Suppl 1):P193 (doi: 10.1186/cc13383) P193 Prevalence of elevated cardiac troponin T in ICU patients using the high-sensitivity assay and the relationship with mortality S O’Sullivan1, M Sutton2, G Fitzpatrick1 1Tallaght Hospital & Trinity College Dublin, Ireland; 2Health Research Board, Dublin, Ireland Critical Care 2014, 18(Suppl 1):P193 (doi: 10.1186/cc13383) Tissue-aggressive infl ammatory response defi nes the tissue aggressiveness of the post-infarction milieu Final infarct size and MaR was determined by CMR performed 1 week after the acute event. The ischemic time was defi ned as the time from pain onset to opening of the occluded vessel. Blood samples were taken for assessment of infl ammatory response. Infl ammatory cells were analyzed by fl ow cytometry in a BD FACS Aria. Parameters were gated against control antibody and fl uorescence minus one strategy. Cytokine patterns were analyzed by BioRad BioPlex multiplex protein analysis technology. Conclusion The results reported may direct healthcare professionals to profi le the patient hospitalized in a CICU, paying attention to the most disturbing and lethal comorbidities present on admission. References Conclusion The results reported may direct healthcare professionals to profi le the patient hospitalized in a CICU, paying attention to the most disturbing and lethal comorbidities present on admission. References Results The SI did not correlate with MaR (P = 0.2720, R2 = 0.09191). The population was divided into the lowest half of the SI (representing the most hostile milieu; SI: 23 to 57%) and the upper half of the SI (representing the friendliest post-infarction milieu; SI: 71 to 95%). The patients’ profi le of adaptive infl ammatory response was characterized by fl ow cytometry. The two groups did not diff er with regard to their T-regulatory response (CD25+FoxP3+, P = 0.7203) or NK-cell (CD3–CD56+, 1. Roger VL: Heart disease and stroke statistics – 2013 update. Circulation 2013, 127:e6-e245. 2. Polanczyk CA, et al.: Fatores de Risco Cardiovascular no Brasil: os próximos 50 Anos. Arq Bras Cardiol 2005, 84:199-201. S69 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 cTnT levels were signifi cant in multivariate regression independent of age and ventilation but did not reach signifi cance (P = 0.06) in a multivariate analysis that included the APACHE II score. y Methods A retrospective observational study was performed on all ICU admissions over a 12-month period. Data were obtained from the clinical information system (ICIP; Philips) and the ICU audit databases (AcuBase). Data collected included patient demographics, peak cTnT value, APACHE II score, requirement for organ support and mortality. The primary outcome measure was hospital mortality. Data were analysed using SPSS v.17.0. cTnT levels were divided into categories for analysis: normal (<14 ng/l) and elevated. The elevated category was further subdivided into quartiles. Univariate analysis was performed between potential risk factors and mortality followed by multivariate regression analysis to ascertain independent predictors of mortality. Impact of positive end-expiratory pressure application on ventriculo-arterial coupling in decompensated left ventricles after cardiac surgery: a non-invasive echocardiographic study P Bertini, V Simone, R Baldassarri, L Doroni, F Guarracino University Hospital, Pisa, Italy Critical Care 2014, 18(Suppl 1):P192 (doi: 10.1186/cc13382) Impact of positive end-expiratory pressure application on ventriculo-arterial coupling in decompensated left ventricles after cardiac surgery: a non-invasive echocardiographic study P Bertini, V Simone, R Baldassarri, L Doroni, F Guarracino University Hospital, Pisa, Italy Critical Care 2014, 18(Suppl 1):P192 (doi: 10.1186/cc13382) y y Results There were 417 admissions to the ICU during the study period, 89 of whom were excluded because of an absent cTnT value, leaving 328 patients included in the analysis. cTnT was elevated in 85% of patients. ICU mortality was 19% and hospital mortality was 28%. Hospital mortality (%) per cTnT category was: <14 ng/l = 2%; 14 to 38 ng/l = 19%; 39 to 90 ng/l = 26%; 91 to 252 ng/l = 39%; >252 ng/l = 43%. On univariate analysis, cTnT levels, age, ventilation and APACHE II score were signifi cantly associated with mortality. cTnT levels were signifi cant in multivariate regression independent of age and ventilation but did not reach signifi cance (P = 0.06) in a multivariate analysis that included the APACHE II score. Introduction The management of ICU patients following heart surgery can be hustling when coping with severe left ventricular (LV) dysfunction. Single beat (Sb) measurements of ventriculo- arterial coupling (VAc) can be used by the intensivist when dealing with altered hemodynamic states [1]. LV elastance (Ees) and arterial elastance (Ea) can be measured by trasthoracic echocardiography (TTE) in a Sb fashion [2], so allowing quick assessment of VAc. PEEP application is common practice in the ICU but can have hemodynamic consequences and lead to instability. Speckle tracking analysis by TTE has been recently reported to help titrating PEEP in critically ill patients [3]. However, this can be demanding and require specifi c ultrasound tools. In this study we aimed to assess whether standard TTE can be useful in evaluating the eff ect of respiratory treatment with PEEP on cardiovascular effi ciency by measuring VAc after coronary artery bypass surgery (CABG). Conclusion In 85% of general ICU patients, troponin measured by HS cTnT assay was elevated. cTnT levels were signifi cantly associated with mortality and are predictive of mortality independent of age and mechanical ventilation, but not independently of APACHE II score. There was a high correlation between troponin levels and APACHE II scores. Reference 1. Abubaker et al.: Intensive Care Med 2013, 39(Suppl 2):0531. P194 95 Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review A Roberts, P Hampshire Royal Liverpool University Hospital, Liverpool, UK Critical Care 2014, 18(Suppl 1):P195 (doi: 10.1186/cc13385) Intensive care mortality increases progressively with age [1] and as the population changes we are likely to see increasing numbers of patients over the age of 80 admitted to critical care units. Methods We searched the ICNARC database from 2006 to 2013 for patients aged 80 years or over, admitted from theatre or after surgery. Data were referenced against the electronic theatre management system, and patients not undergoing abdominal cavity surgery were excluded. The data were analysed using an Excel spreadsheet (Microsoft) and Medcalc software. Results Eighty-fi ve patients were included, with ages ranging from 80 to 99 years. Fifty-one (60%) patients were male and 79 (93%) patients were categorized as having an emergency operation. ICU mortality was 34/85 (40%) and hospital mortality was 48/85 (56%). Variables assessed for association with hospital mortality can be seen in Table 1. Only invasive ventilation and time (days) from hospital admission to ICU admission were signifi cant predictors of hospital mortality. Table 1 (abstract P195). Survivors versus nonsurvivors Survivors Nonsurvivors P value Table 1 (abstract P195). Survivors versus nonsurvivors Survivors Nonsurvivors P value Age 83 83 0.82 Time 1, 1 to 8 4.5, 1 to 8 0.004 APACHE II 17, 14 to 20 17, 14 to 21 0.96 Sex (%) M57, F43 M63, F37 0.65 Ventilation (n, %) 31, 83 48, 100 0.005 RRT (n, %) 6, 16 11, 23 0.58 Data presented as median, IQR or n, %. Table 1 (abstract P195). Survivors versus nonsurvivors Table 1 (abstract P195). Survivors versus nonsurvivors Conclusion The development of ARI in SAP was associated with hemodynamic instability, whereas excessive volume expansion does not prevent ARI. Severe acute pancreatitis in ICU: a 5-year audit Introduction Severe acute pancreatitis (SAP) is associated with signifi cant mortality and morbidity. The objective of this study is to examine the profi le, outcome and resource utilization for patients with SAP admitted to the ICU in a university teaching hospital over a 5-year period. Conclusion ICU and hospital mortality rates were high at 40% and 56% respectively. Increasing age did not correlate with mortality. However, invasive ventilation and time between hospital and ICU admission were associated with a higher mortality. This may be due to a delay in diagnosis or surgical intervention. We suggest that one considers early intervention in patients aged over 80. A functional outcome measure at discharge may be a more clinically relevant endpoint. 95 Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review A Roberts, P Hampshire Royal Liverpool University Hospital, Liverpool, UK Critical Care 2014, 18(Suppl 1):P195 (doi: 10.1186/cc13385) p Results Of 145 patients, 24 patients who developed ARI at any time during hospitalization (ARI group) were contrasted with 24 patients without ARI (control group). The patients were older in the ARI group: age 57 ± 13 versus 49 ± 16, P = 0.046. Although none of the patients in the ARI group had creatinine values ≥1.5-fold from the estimated baseline, the creatinine values on admission were higher in this group: 100 ± 38 versus 68 ± 16, P = 0.001. The severity of pancreatitis was similar. On admission, the APACHE II and SOFA scores were higher in the ARI group (12.7 ± 3.7 vs. 8.6 ± 3.4, P = 0.001 and 5.6 ± 3.4 vs. 2.8 ± 1.9, P = 0.002, respectively). The patients in the ARI group had higher intra- abdominal pressure and SOFA respiratory score on admission and after 72 hours (P <0.01). Although on admission the cardiovascular SOFA score was similar in both groups, it increased signifi cantly after 72 hours in the ARI group from 1.1 ± 1.7 to 2.1 ± 1.8, P = 0.012 and became higher compared with the control group: 2.1 ± 1.8 versus 0.6 ± 1.2, P = 0.001. Lactate was higher in the ARI group on admission and after 72 hours (P <0.05). The percentage of patients requiring vasopressors over 72 hours was greater in the ARI group: 66.7% versus 29.2%, P = 0.01. Positive fl uid balance after 72 hours in ICU was higher in the ARI group: 8,594 ± 7,044 ml versus 4,192 ± 4,467 ml, P = 0.004; however, the infused volume of crystalloids did not diff er between groups. Central venous pressure was higher on admission (P = 0.036) and after 72 hours (P = 0.001) in the ARI group. Multivariable logistic regression analysis revealed that development of ARI was independently associated with cardiovascular SOFA score after 72 hours: odds ratio = 1.475, 95% CI 1.049 to 2.073, P = 0.025. A Roberts, P Hampshire Royal Liverpool University Hospital, Liverpool, UK Critical Care 2014, 18(Suppl 1):P195 (doi: 10.1186/cc13385) Introduction This review was undertaken to establish the ICU and hospital mortality rates in patients aged 80 and over admitted to our ICU following abdominal cavity surgery. g g Reference 1. Hajjar L, Grande S, Galas F, Roquim A, Sampaio L, Auler J: Risk factors and outcome of rhabdomyolysis after cardiac surgery. Crit Care 2008, 12(Suppl 2):P470. Introduction Elevated cardiac troponin levels are common in ICU patients even in the absence of acute coronary syndromes and may be Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S70 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P195 was composed of randomly selected patients with SAP who did not develop ARI. P195 Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review A Roberts, P Hampshire Royal Liverpool University Hospital, Liverpool, UK Critical Care 2014, 18(Suppl 1):P195 (doi: 10.1186/cc13385) 95 Open cavity abdominal surgery in octogenarians and nonagenarians admitted to a university teaching hospital ICU: a retrospective review A Roberts, P Hampshire Royal Liverpool University Hospital, Liverpool, UK Critical Care 2014, 18(Suppl 1):P195 (doi: 10.1186/cc13385) p Methods A retrospective observational study was carried out of all patients admitted to the ICU from 1 January 2008 to 31 December 2012 with SAP. Data were collected from the ICU database (AcuBase), the medical records and the ICU clinical information system. Data collected included patient demographics, etiology of SAP, data for APACHE II, Imrie, Ranson and Acute Kidney Injury Network (AKIN) scores, and requirement for organ support. Outcomes recorded were length of stay, ICU mortality and hospital mortality. Cost of ICU care was calculated based on previously reported methodology.i 1. Paul E, et al.: Management of the critically ill geriatric patient. Crit Care Med 2006, 34:S176-S182. p y p gy Results Thirty-eight eligible patients were identifi ed. Mean age was 51.4 years (range 24 to 86), 68% were male. The commonest etiologies were alcohol (53%) and gallstone pancreatitis (24%). The mean APACHE II score was 18.5 (IQR 14 to 23). Twenty-eight patients (74%) required mechanical ventilation, three of whom required high-frequency oscillation (all three survived). Twenty-two patients (58%) had evidence of an acute kidney injury on admission (AKIN criteria). Eighteen (47%) required renal replacement therapy and 60% required inotropes. The ICU mortality and the hospital mortality were 26%. There was no signifi cant diff erence in age, APACHE II, Imrie, or Ranson scores between survivors and nonsurvivors. The median length of stay in the ICU was 11 days (IQR 5.25 to 28.5) and the median hospital stay was 45.4 days (IQR 22.25 to 104.5). Nine patients (24%) required multiple ICU admissions and the mortality was signifi cantly higher in this group (P <0.05, chi-square test). In total, 834 ICU bed-days were taken up by 38 patients. Based on a median cost for an ICU bed-day of €2,205 [1], the total cost of ICU care for these patients is estimated at €1,838,970 or almost €50,000 per patient. Bacterial infection in severe acute pancreatitis patients admitted to the ICU Results Of the 803 APAP-ALF patients, the median age was 37 (29 to 47) years and 76% were female. A total of 238 (30%) patients were listed for and 87 (11%) received LT. A total of 531 (66%) patients recovered without LT at 21 days. Using standard logistic regression methods for all patients with complete data (n = 679), KCC (INR, creatinine, coma grade 3/4, pH) yielded an AC of 69% (Sn 90%, Sp 27%) at admission. For late-stage data (n = 341), KCC provided similar AC (70%) and Sn (97%), but suff ered poor Sp (15%). CART analysis using the KCC variables on admission off ered predictive AC (66%) and Sn (65%), with increased Sp (67%). Using day 3 to 7 data, the KCC-CART model had increased AC (82%) and Sn (86%), with improved Sp (46%) compared with logistic KCC. New CART models were developed with 18 variables based on previous literature. A new CART model on admission (MELD, lactate and MV: test AC 72%, Sn 71%, Sp 77%) performed better than KCC-CART. For days 3 to 7, a CART model (MELD, lactate, coma grade) off ered superior prediction (test AC 86%, Sn 91%, Sp 46%) compared with days 3 to 7 KCC-CART. Introduction Controversy surrounds the empirical use of antibiotics in severe acute pancreatitis (SAP). There are concerns that the widespread use of antibiotic therapy in the absence of documented infection may lead to selection of drug-resistant organisms [1]. The aim of this study was to review the profi le of pancreatic fl uid isolates in patients with SAP admitted to the ICU. Methods Data were reviewed for 38 patients admitted to the ICU over a 5-year period. We evaluated organisms cultured from pancreatic specimens, as well as the prevalence of drug-resistant organisms in this group of patients. Results Aspirate of pancreatic material for culture was obtained in 55% of patients (n = 21). The mean time to acquisition of samples for culture from admission to ICU was 15.5 days. Fluid was sterile in 67% (n = 14) of initial samples. Gram-positive organisms were cultured from 43% (n = 9) of samples, Gram-negative organisms from 5% (n = 1) and yeasts from 5% (n = 1). Antibiotic therapy was administered in 95% of patients prior to samples being obtained for culture. A multicenter retrospective cohort analysis of therapeutic hypothermia in acute liver failure y C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA y C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA y Conclusion In the majority of patients, initial aspirates of pancreatic material were sterile. This may be a result of prior antibiotic usage. Where organisms were cultured from initial aspirates, Gram-positive organisms predominated, possibly as a result of prior anti-Gram- negative antibiotic use. Therefore, in patients with ongoing sepsis who are receiving broad-spectrum antibiotic therapy, consideration needs to be given to the empiric treatment of Gram-positive infection, and in particular drug-resistant organisms such as VRE. Local epidemiology should be taken into account. Rationale use of antibiotics, in accordance with best-practice guidelines, may limit development of drug resistance; however, other risk factors for resistance may exist in this group and this would need to be further evaluated. Reference y Critical Care 2014, 18(Suppl 1):P200 (doi: 10.1186/cc13390) Introduction Cerebral edema is a severe and life-threatening complication in acute liver failure (ALF). Concerns exist that therapeutic hypothermia (TH) may increase the risk of infection, worsen coagulopathy and inhibit hepatic regeneration. We therefore reviewed the experience in use of TH in participating US Acute Liver Failure Study Group (ALFSG) centers. The aims were to determine utilization of TH in ALF patients at high risk for cerebral edema (grade III or IV hepatic encephalopathy (HE)) and to determine its eff ect on survival and complication rates. Introduction Cerebral edema is a severe and life-threatening complication in acute liver failure (ALF). Concerns exist that therapeutic hypothermia (TH) may increase the risk of infection, worsen coagulopathy and inhibit hepatic regeneration. We therefore reviewed the experience in use of TH in participating US Acute Liver Failure Study Group (ALFSG) centers. P200 P200 A multicenter retrospective cohort analysis of therapeutic hypothermia in acute liver failure C Karvellas1, R Stravitz2, H Battenhouse3, V Durkalski3, W Lee4, ML Schilsky5 1University of Alberta, Edmonton, Canada; 2Virginia Commonwealth University, Richmond, VA, USA; 3Medical University of South Carolina, Charlston, SC, USA; 4University of Texas Southwestern, Dallas, TX, USA; 5Yale University, New Haven, CT, USA Critical Care 2014, 18(Suppl 1):P200 (doi: 10.1186/cc13390) A multicenter retrospective cohort analysis of therapeutic hypothermia in acute liver failure The aims were to determine utilization of TH in ALF patients at high risk for cerebral edema (grade III or IV hepatic encephalopathy (HE)) and to determine its eff ect on survival and complication rates. 1. Tenner S, Baillie J, DeWitt J, Vege SS; American College of Gastroenterology: Am J Gastroenterol 2013, 108:1400-1415. 1. Tenner S, Baillie J, DeWitt J, Vege SS; American College of Gastroenterology: Am J Gastroenterol 2013, 108:1400-1415. Methods A retrospective cohort study of all ALF patients enrolled in the US ALFSG registry between January 1998 and September 2013 with grade III or IV HE. TH using an external cooling device was used in 97 (8%) patients while in 1,135 (92%) patients it was not (controls). Results TH ALF patients were younger (36 vs. 40, P = 0.03) and had acetaminophen etiology (63 vs. 47%, P = 0.04). Admission MELD (32 vs. 34) and lactate (5.4 vs. 5.0 mmol/l, P >0.2 for all) were similar. More TH ALF patients received renal replacement therapy (63 vs. 40%), vasopressors (61 vs. 43%) and ICP monitoring (40 vs. 22%, P <0.0002 for all). Overall (38% vs. 40%, P = 0.7) and 21-day transplant survival (45 vs. 49%, P = 0.5) were similar. There were no diff erences in bleeding (12% vs. 12%) or bloodstream infections (17 vs. 18%, P >0.7 for both). More TH ALF patients had arrhythmias (38 vs. 27%, P = 0.03). There were no diff erences in listing (43 vs. 40%, P = 0.5) or receipt of transplant (18 vs. 25%, P = 0.1). After controlling for MELD, requirement for organ support on multivariable analysis, hypothermia was not independently Methods A retrospective cohort study of all ALF patients enrolled in the US ALFSG registry between January 1998 and September 2013 with grade III or IV HE. TH using an external cooling device was used in 97 (8%) patients while in 1,135 (92%) patients it was not (controls). i References 1. Harrison et al.: Crit Care 2007, 11(Suppl 1):S1. 1. Harrison et al.: Crit Care 2007, 11(Suppl 1):S1. 2. Pavlidis et al.: Crit Care Res Pract 2013, 2013:897107. 2. Pavlidis et al.: Crit Care Res Pract 2013, 2013:897107. P196 Risk factors for acute renal impairment in patients with severe acute pancreatitis M Serpytis, N Scupakova, J Sabliauskas, A Sileikis, J Sipylaite, K Strupas Vilnius University, Vilnius, Lithuania Critical Care 2014, 18(Suppl 1):P196 (doi: 10.1186/cc13386) Introduction The aim of this study was to clarify the risk factors for acute renal impairment (ARI) in patients with severe acute pancreatitis (SAP). Methods We conducted a retrospective observational case–control study. The data of all patients admitted to the ICU at a university hospital with the diagnosis of SAP from January 2008 to December 2012 were abstracted from the hospital database. ARI was defi ned according to RIFLE criteria. Patients with any signs of renal impairment (serum creatinine value ≥1.5-fold from the estimated baseline) on admission or history of kidney disease were excluded. A control group S71 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion A hospital mortality rate of 26% is similar to that reported recently from a specialist unit in the UK [2] but less than the 42% reported in the UK in 2007 [1], suggesting some improvement in recent years. SAP is associated with prolonged ICU and hospital stay and signifi cant resource utilization. admission but not in later phases of illness. The aim was to improve determinations of prognosis on and after admission in APAP- ALF patients using the classifi cation and regression tree (CART) methodology to construct optimal binary splits on independent variables to predict outcome. Methods CART models were applied to US ALFSG registry data for prediction of 21-day spontaneous survival on admission and late stage (days 3 to 7). Analyses were carried out using R software (package rpart) for all (n = 803) APAP-ALF patients enrolled between January 1998 and September 2013 with complete outcome data. Training data were used to build CART trees and test data were used to evaluate prediction accuracy (AC), sensitivity (Sn) and specifi city (Sp). Bacterial infection in severe acute pancreatitis patients admitted to the ICU On review of all samples received from patients (including nonpancreatic specimens), vancomycin-resistant enterococci (VRE) were isolated in 13 patients. Linezolid-resistant enterococci (LRE) were isolated in six patients, fi ve of whom had VRE isolated prior to the culture of LRE. Extended- spectrum beta-lactamase organisms were isolated in two patients, and carbapenem nonsusceptible Gram-negative organisms in three patients. The mean APACHE II score was 18.5 and overall hospital mortality was 26%. Conclusion CART analysis increased predictive performance compared with traditional KCC. KCC-CART trees have higher Sp and similar predictive AC compared with traditional KCC, with newer CART trees providing marginal improvement over KCC-CART models. Acknowledgment Supported in part by a U-01 58369-014 from NIDDK to the US Acute Liver Failure Study Group. P200 P198 98 Bacterial infection in severe acute pancreatitis patients admitted to the ICU AR Prior, S Egan, R Durani, PG Murphy, J Febbell, G Fitzpatrick Tallaght Hospital, Dublin, Ireland Critical Care 2014, 18(Suppl 1):P198 (doi: 10.1186/cc13388) References 1. Choi NK, et al.: Hepatogastroenterology 2012, 59:1189-1193. 2. Monsel A, et al.: Crit Care 2011, 15:R234. 1. Choi NK, et al.: Hepatogastroenterology 2012, 59:1189-1193. 2. Monsel A, et al.: Crit Care 2011, 15:R234. pp p Critical Care 2014, 18(Suppl 1):P202 (doi: 10.1186/cc13392) g References gi References 1. Shawcross DL, Austin MJ, Abeles RD, et al.: The impact of organ dysfunction in cirrhosis: survival at a cost? J Hepatol 2012, 56:1054-1062. 1. Shawcross DL, Austin MJ, Abeles RD, et al.: The impact of organ dysfunction in cirrhosis: survival at a cost? J Hepatol 2012, 56:1054-1062. 2. Alexopoulos S, Matsuoka L, Cho Y, et al.: Outcomes after liver transplantation in patients achieving a model for end-stage liver disease score of 40 or higher. Transplantation 2013, 95:507-512. 3. Oberkofl er CE, Dutkowski P, Stocker R, et al.: Model of end stage liver disease (MELD) score greater than 23 predicts length of stay in the ICU but not mortality in liver transplant recipients. Crit Care 2010, 14:R117. Extracorporeal membrane oxygenation before and after adult liver transplantation: worth the eff ort? Results On the day of LT, 5% (28/519) of patients had a MELD score ≥40. These patients had longer fi rst ICU stay after LT (14 vs. 2 days; P <0.001). MELD score ≥40 at transplant was independently associated with fi rst ICU stay after transplant ≥10 days (OR, 3.21). These patients had longer fi rst hospital stay after LT (45 vs. 18 days; P <0.001); however, there was no signifi cant diff erence in the rate of ICU readmission (18% vs. 22%; P = 0.58) or re-transplant rate (4% vs. 4%; P = 1.00). Cumulative survival at 1 month, 3 months, 1 year, 3 years, and 5 years was 98%, 96%, 90%, 79%, and 72%, respectively. There was no signifi cant diff erence in cumulative survival stratifi ed by MELD score ≥40 versus <40 at transplant (P = 0.59). Introduction Extracorporeal membrane oxygenation (ECMO) is increasingly used for the treatment of refractory but potentially reversible respiratory and/or cardiac failure. Data on perioperative support with veno-arterial (V-A) and veno-venous (V-V) ECMO for adult liver transplant recipients are scarce [1,2]. We report our experience of ECMO support in patients with acute liver failure (ALF) as a bridge to transplant and postoperative ECMO use following complications after surgery. Methods A retrospective study in a specialist tertiary referral ICU. Patients supported with V-V or V-A ECMO before, during, or after orthotopic liver transplant (OLT) were identifi ed. Conclusion Cirrhotic patients with MELD score ≥40 at transplant utilize greater postoperative health resources; however, they derive similar long-term survival benefi t with LT. Results In total, four patients were supported during a 12-month period. Two patients required V-V and two patients V-A support. Two patients with ALF were bridged to OLT, one patient V-V ECMO for refractory respiratory failure and the other patient required emergency V-A support for treatment of intraoperative arrest. Both patients were successfully transplanted but died subsequently on ECMO: disseminated aspergillosis and haemophagocytic syndrome, and anoxic brain injury respectively. Two patients received postoperative ECMO support. The fi rst was treated with V-V ECMO for refractory persistent hypoxaemia following OLT for hepato-pulmonary syndrome, the second received emergency V-A support (eCPR) following cardiac tamponade and arrest on postoperative day 2. Both patients made a full recovery. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 associated with 21-day spontaneous survival (P = 0.93) while MELD (P <0.0001) and vasopressors (P <0.02) were. Results Twenty patients (27.1%) developed 33 episodes of infection. Among them 35% suff ered from liver dysfunction, 40% took large doses of immunosuppression and 50% were re-operated. Five out of them showed signs of infection promptly after transplantation. Medium duration of mechanical ventilation was 16.5 ± 12.75 days (vs. 3.24 ± 5.5 of those without an infection) and medium length of stay was 22.14 ± 18.35 days (vs. 6.79 ± 8.86). The medium APACHE II score was 17.8 ± 5.5 (vs. 16.4 ± 4.37, P = 0.256) and medium SOFA was 11.32 ± 2.62 (vs. 9.9 ± 2.62, P = 0.054). The majority of patients was transfused with many blood units, FFP and cryoprecipitates and was hemodynamically unstable (56.3%). MELD score and MELD-Na were higher (22.57 and 27.5 relatively vs. 19.02 and 22.5). Eight cases had VAP from Gram- negative bacteria, 15 had bacteraemia (mainly Gram-negative, but also fungaemia), 10 had intraabdominal infection (most of them with two or three re-operations) and urine infection. The majority of pathogens were multidrug resistant. Regarding Klebsiella pneumoniae Carbapenemase, 60% was sensitive to colimycine, 20% to tobramycin, 70% to gentamycin and 40% to tygecycline. In four cases the donors had an infection that was transmitted to the recipient as much as 75%. Fourteen patients died (10 in ICU and four in the transplantation clinic) soon after with multiorgan failure. associated with 21-day spontaneous survival (P = 0.93) while MELD (P <0.0001) and vasopressors (P <0.02) were. p Conclusion TH was not associated with increased bleeding/infection rates nor diff erences in survival in ALF patients with high-grade HE. ICP monitoring was not universally used in TH ALF patients (~40%). There is a need for a prospective trial to clarify the use of TH in patients with ALF. Acknowledgement Supported in part by a U-01 58369-014 from NIDDK to the US ALFSG. Determining late predictors of outcome for acetaminophen- induced acute liver failure using classifi cation and regression tree modeling analysis C Karvellas1, J Speiser2, W Lee3 1University of Alberta, Edmonton, Canada; 2Medical University of South Carolina, Charlston, SC, USA; 3University of Texas Southwestern, Dallas, TX, USA C Karvellas1, J Speiser2, W Lee3 1University of Alberta, Edmonton, Canada; 2Medical University of South Carolina, Charlston, SC, USA; 3University of Texas Southwestern, Dallas, TX, USA Critical Care 2014, 18(Suppl 1):P199 (doi: 10.1186/cc13389) Introduction Liver transplantation (LT) in acetaminophen-induced acute liver failure (APAP-ALF) patients often presents signifi cant challenges. The King’s College Criteria (KCC) have been validated on S72 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P204 Introduction Patients with liver transplantation suff er a major risk of infections immediately after the surgery due to heavy immunosuppression, severe stress and underlying pathology. The aim of this study is to fi nd their causes and consequences in our ICU. Introduction Patients with liver transplantation suff er a major risk of infections immediately after the surgery due to heavy immunosuppression, severe stress and underlying pathology. The aim of this study is to fi nd their causes and consequences in our ICU. Causes and consequences of infections in patients after liver transplantation: 2-year study in the only ICU that hospitalizes these cases in Greece Conclusion Emergency ECMO support before and after liver transplant is feasible. Despite the poor outcome in patients with ALF, we consider ECMO a valuable option to bridge selected patients to transplant. References A Karapanagiotou, C Kydona, S Papadopoulos, T Theodoridou, A Karapanagiotou, C Kydona, S Papadopoulos, T Theodoridou, E Mouloudi, C Dimitriadis, G Imbrios, N Gritsi-Gerogianni Hippokrateion Hospital of Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P202 (doi: 10.1186/cc13392) Postoperative resource utilization and survival among liver transplant recipients with Model for End-stage Liver Disease score ≥40: a retrospective cohort study Introduction Cirrhotic patients with Model for End-stage Liver Disease (MELD) score ≥40 have high risk of death without liver transplant (LT) [1]. This study aimed to evaluate these patients’ outcomes after transplant. Conclusion Patients after liver transplantation with organ dysfunction and multiple transfusions, due to surgical complications, have a major risk of developing an infection inside the ICU that aff ects their survival. Methods The retrospective cohort included 519 adult cirrhotic patients who underwent LT at one Canadian center between 2002 and 2012. Primary exposure was severity of end-stage liver disease measured by MELD score at transplant (≥40 vs. <40) [2]. The primary outcome was duration of fi rst ICU stay after LT [3]. Secondary outcomes were duration of fi rst hospital stay after LT, rate of ICU readmission, re-transplant rate, and survival rates. P203 p References p References Results In our study the criteria used to defi ne post-reperfusion syndrome relied on the hemodynamic changes that occurred at reperfusion. Preoperative echocardiography showed normal systolic and diastolic function at rest in all of the patients. For the identifi cation of patients at risk for CCM we used two of the supportive criteria from the recent defi nition of CCM: prolonged QTc interval and increased BNP levels. The study group included 28 men (53.8%) and 24 women. Mean (± SD) age was 50.5 (± 11.4). Mean MELD and MELD Na scores were respectively 15.51 (± 5.43) and 18.9(± 6.22). The value of BNP correlated well with the length of the QTc interval (P = 0.005), and with MELD and MELD Na scores (P = 0.025 and P = 0.001). In our study, post-reperfusion syndrome occurred in 63.4% of the patients. We could not fi nd a correlation between post-reperfusion syndrome and the BNP levels (P = 0.85) or the prolonged QTc interval (P = 0.38). The post-reperfusion syndrome did not correlate with the severity of the liver disease as assessed by MELD and MELD Na scores. The severity of post-reperfusion syndrome did not correlate with QTc prolongation or BNP levels. 1. Cannon RM, et al.: Transpl Int 2012, 25:1223-1228. 2. Daly RC, et al.: Transplantation 2013, 95:e2-e4. Impaired balance between coagulation and fi brinolysis plays a prominent role in patients with sepsis Introduction The balance between coagulation and fi brinolysis was a prominent factor in the pathophysiology of sepsis, but this mechanism has been poorly understood. We aimed to determine whether collapsing this balance during the fi rst day of sepsis correlates with progression of organ dysfunction and subsequent death. Methods This study included all patients with sepsis admitted to a tertiary referral hospital in Japan. Global coagulation tests and hemostatic molecular markers such as fi brin/fi brinogen degradation products (FDP), D-dimer, thrombin–antithrombin complex (TAT) and plasmin-alpha 2 plasmin inhibitor complex (PIC) were measured within 12 hours after admission, and then SOFA and APACHE II scores and in-hospital mortality were evaluated. Patients were divided into three groups based on the levels of TAT/PIC and FDP/D-dimer and diff erences of clinical outcome between groups were assessed by chi-square analysis and ANOVA. Conclusion Reperfusion is a critical time during liver transplantation. The clinical predictors of post-reperfusion syndrome are still under debate [1]. Our study showed that the post-reperfusion syndrome is not correlated with the severity of the liver disease or with the presence of risk factors indicating CCM. Reference 1. Paugham-Burtz C, et al.: Liver Transpl 2009, 15:522-529. 1. Paugham-Burtz C, et al.: Liver Transpl 2009, 15:522-529. y Results We enrolled 101 patients; 87 patients survived, and 14 died. Mortality was signifi cantly higher in the high TAT/PIC group (0%, 19% and 24% for low, middle and high TAT/PIC groups, respectively; P = 0.011). In addition, SOFA and APACHE II scores were signifi cantly higher in the low FDP/D-dimer group (APACHE II = 22.3, 18.9 and 15.3; P <0.01, SOFA = 8.6, 6.5 and 5.1; P <0.01, for low, middle and high FDP/ D-dimer groups, respectively). See Figure 1. P206 P206 Impaired balance between coagulation and fi brinolysis plays a prominent role in patients with sepsis Y Umemura1, K Yamakawa2, T Kiguchi1, H Ogura2, T Shimazu2, S Fujimi1 1Osaka General Medical Center, Osaka, Japan; 2 Osaka University Graduate School of Medicine, Osaka, Japan Critical Care 2014, 18(Suppl 1):P206 (doi: 10.1186/cc13396) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion CHLT is a life-saving operation that is performed with relatively low mortality and can be successfully performed in select patients with congenital heart disease. Patients undergoing CHLT at our institution had relatively preserved hepatic function but limited cardiac function often requiring inotropic support. Cardiac transplantation typically precedes liver transplantation during CHLT given the decreased ischemic tolerance of the cardiac graft [2]. However, liver transplantation prior to cardiac transplantation may serve to mitigate high-titer donor-specifi c antibodies. Various aforementioned operative approaches may be successfully utilized for the liver transplantation portion of these procedures. We attribute the favorable outcomes and perioperative courses to the multidisciplinary approach to care that CHLT patients receive at our institution. frequently observed and is termed post-reperfusion syndrome. Recent studies showed the existence of a specifi c heart disease associated with cirrhosis termed cirrhotic cardiomyopathy (CCM). The aim of this study was to investigate whether the CCM has an infl uence on the development or the severity of post-reperfusion syndrome. Methods Fifty-two consecutive liver transplant patients were included in a retrospective observational study. The variables recorded were: age, etiology of the liver disease, MELD and MELD Na scores, the associated pathologies, the length of the QT interval, and plasma levels of brain natriuretic peptide (BNP). The patients with known renal or heart disease and the recipients of organs from extended criteria donors were excluded from the study. The QT interval was corrected for the heart rate using Bazett’s formula (QTc). Statistical analysis was performed using SPSS Statistics v.19.1.i Is cirrhotic cardiomyopathy a risk factor for post-reperfusion syndrome during liver transplantation? y E Scarlatescu, G Droc, D Tomescu Fundeni Clinical Institute, Bucharest, Romania Methods We studied 72 cirrhotic patients who were transplanted in our hospital during the years 2011 and 2012. The cases that developed intrahospital infection, its source and microbiology, the surgical complications, the function of the transplant, the duration of mechanical ventilation and their outcome were fully examined. Fundeni Clinical Institute, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P204 (doi: 10 1 , , Critical Care 2014, 18(Suppl 1):P204 (doi: 10.1186/cc13394) Introduction A period of hemodynamic instability following revascularization of the liver graft during liver transplantation is S73 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Perioperative management of patients undergoing combined heart–liver transplantation Bacterial load is usually associated with bacteremia, LPS, high level of IL-6, PCT and also with aromatic microbial metabolites [1-3], and so forth. In our opinion, microbial metabolites can participate in hemodynamic disorders in critically ill patients, particularly due to their infl uence on NO production [4] and intestinal permeability. Methods In a prospective study we observed critically ill patients on the day of admission to a polyvalent ICU, severe cardiac pathology was excluded. The level of phenylpropionic, phenyllactic, p-OH- phenyllactic, p-OH-phenylacetic acids and total phenylcarboxylic acids (PhCAs) were measured in blood serum using gas chromatography (GC-FID). The level of PCT and NT-proBNP were measured using Elecsys 2010. Comparison between patients with hypotension (on vasopressor support) (group A) and without (group B) was performed.f Results The SF values in the DIC and the Subclinical DIC groups were signifi cantly higher than in the No DIC group. We created the receiver operating characteristic curve of SF value for DIC onset (JAAM-DIC score ≥4) and the SF value of 35 μg/ml was set as the cutoff SF value. The high Methods In a prospective study we observed critically ill patients on the day of admission to a polyvalent ICU, severe cardiac pathology was excluded. The level of phenylpropionic, phenyllactic, p-OH- phenyllactic, p-OH-phenylacetic acids and total phenylcarboxylic acids (PhCAs) were measured in blood serum using gas chromatography (GC-FID). The level of PCT and NT-proBNP were measured using Elecsys 2010. Comparison between patients with hypotension (on vasopressor support) (group A) and without (group B) was performed.f Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Figure 2 (abstract P207). ROC curve of the SF value for DIC. Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. pp g p g p p Results We studied 50 ICU patients with diff erent diseases: pneumonia (n = 15), severe kidney failure (n = 13), abdominal surgical pathology (n = 10), alcoholic cirrhosis (n = 5), soft-tissue infection (n = 7). In group A (24/50) the median of PhCAs was 17.8 (IR 11.4 to 30.0) μmol/l, and in group B (26/50) it was 7.2 (IR 3.7 to 13.2) μmol/l, P = 0.003 (t test). Perioperative management of patients undergoing combined heart–liver transplantation p DW Barbara, KH Rehfeldt, JK Heimbach, CB Rosen, RC Daly, JY Findlay Mayo Clinic, Rochester, MN, USA DW Barbara, KH Rehfeldt, JK Heimbach, CB Rosen, RC Daly, JY Findlay Mayo Clinic, Rochester, MN, USA Conclusion We demonstrated that the balance between coagulation and fi brinolysis, assessed with FDP/D-dimer and TAT/PIC ratios, was correlated with disease severity and clinical outcomes in sepsis, suggesting that impaired balance of the hemostatic system might play a pivotal role in progression of sepsis pathophysiology. Introduction Combined heart–liver transplantation (CHLT) is an uncommonly performed procedure for patients with coexisting cardiac and liver disease [1]. The purpose of this study was to examine and describe the perioperative management of patients undergoing CHLT. Methods A retrospective review was performed of patients undergoing CHLT at our institution from 1999 to 2013. Figure 1 (abstract P206). Comparison of the characteristics between the three groups. Results Twenty-seven CHLTs were performed, with 4/27 including simultaneous kidney transplantation. Familial amyloidosis was the indication for 21 CHLTs (78%), and 12 of these explanted livers were used for domino transplantations. Nineteen patients (70%) were receiving inotropic infusions at the time of organ availability. The median preoperative MELD score was 12, and elevations in preoperative international normalized ratio were due to warfarin in all but one patient. Liver transplantation immediately preceded cardiac transplantation in 2/27 cases to reduce high-titer donor-specifi c antibodies. Venovenous bypass was utilized in 14 operations (52%) performed with the caval interposition liver transplantation approach, cardiopulmonary bypass during liver transplantation in two cases (7%), and no bypass in 11 operations (41%) performed with a caval sparing (piggyback) surgical technique. Postoperatively, the median duration of mechanical ventilation, ICU stay, and hospital stay until discharge were 1 day, 5.5 days, and 15 days, respectively. Transfusions in the fi rst 48 hours following CHLT were not substantial in the majority of patients. One patient died within 30 days of CHLT. Figure 1 (abstract P206). Comparison of the characteristics between the three groups. S74 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P207 SF group (SF ≥35 μg/ml) had signifi cantly higher JAAM-DIC score, SOFA score and APACHE II score than the low SF group (SF <35 μg/ml). Mainly in the high SF group except DIC patients on admission, we found that SF increased before the JAAM-DIC score changed. Perioperative management of patients undergoing combined heart–liver transplantation See Figures 1 and 2. Conclusion We think measurement of the plasma SF level may be clinically useful in evaluating the severity of critically ill patients such as those with sepsis. P207 Clinical usefulness of measurement of plasma soluble fi brin levels in critically ill patients T Masuda, T Kobayashi, H Takahashi, K Yoshikawa, M Takahashi, E Isotani Tokyo Women’s Medical University Medical Center East, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P207 (doi: 10.1186/cc13397) Introduction The soluble fi brin monomer fi brinogen complex (SF) is a complex coupling fi brin monomer and fi brinogen molecules. As the level of SF refl ects the thrombin generation activity in plasma, we may estimate the early-activated state of blood coagulation by the measurement of SF. The aim of this study is to evaluate the clinical usefulness of SF for the hypercoagulated state. P208 P208 Value of microbial metabolites in blood serum as criteria for bacterial load in the pathogenesis of hemodynamic disorders in critically ill patients Y Sarshor, N Beloborodova, V Moroz, A Osipov, A Bedova, E Chernevskaya, M Getsina Negovsky Research Institute of General Reanimatology, Moscow, Russia Critical Care 2014, 18(Suppl 1):P208 (doi: 10.1186/cc13398) y g Methods We measured the plasma level of SF in 63 patients within 48 hours after admission and on the 1st, 3rd, 5th and 7th days after admission. Underlying disease mainly includes sepsis, shock, and so on. According to the disseminated intravascular coagulation diagnostic criteria established by the Japanese Association of Acute Medicine, we defi ned the DIC group as JAAM-DIC score more than 3 within 48 hours after admission, the Subclinical DIC group as score more than 3 within 7 days beyond 48 hours after admission, and the No DIC group as score less than 4 during the entire study period. The SF value of each group was compared with the Mann–Whitney U test. Introduction Hemodynamic disorders in critically ill patients are often connected with bacterial load. Bacterial load is usually associated with bacteremia, LPS, high level of IL-6, PCT and also with aromatic microbial metabolites [1-3], and so forth. In our opinion, microbial metabolites can participate in hemodynamic disorders in critically ill patients, particularly due to their infl uence on NO production [4] and intestinal permeability. Introduction Hemodynamic disorders in critically ill patients are often connected with bacterial load. Perioperative management of patients undergoing combined heart–liver transplantation In group A, all patients (with or without documented infections) had symptoms of infection manifestation [5], 20/24 (83.3%) of them died. In group B, the symptoms of infection manifestation were revealed in 12/26 (46%) cases, and the mortality was signifi cantly lower, 3/26 (11.5%) (P <0.05). General mortality was 23/50 (46%). The profi le of PhCAs diff ered in groups A versus B. f g p Conclusion The total level of PhCAs in critically ill patients with hypotension was considerably higher than in hemodynamically stable patients. The participation of microbial factor in pathogenesis of hemodynamic disorders in the presence of systemic infl ammation may be validated with the load of microbial metabolites (PhCAs). Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Figure 1 (abstract P207). SF values of the DIC, Subclinical DIC and No DIC groups. Acknowledgement This work was supported by the Russian Foundation for Basic Research (project number 13-04-01758/13). References 1. Beloborodova N, et al.: Crit Care 2009, 13:41. Figure 2 (abstract P207). ROC curve of the SF value for DIC. Figure 2 (abstract P207). ROC curve of the SF value for DIC. 2. Sarshor Yu, et al.: Shock 2013, 40(Suppl 1):31. 3. Beloborodova N, et al.: Biochemistry 2009, 74:1350-1355. 4. Ogiwara T, et al.: Anticancer Res 2003, 23:1317-1323. 5. Dellinger RP, et al.: Crit Care Med 2013, 41:580-637. Plasma platelet-derived microparticles to platelet count ratio as a marker of mortality in critically ill patients l Results Upon ICU admission, levels of sRAGE, HMGB1, S100A12 and CRP were higher as compared with healthy levels. HMGB1, S100A12 and CRP remained elevated throughout the ICU stay but sRAGE decreased to levels lower than in healthy volunteers by day 7. sRAGE and CRP showed distinct time profi les during the ICU stay in patients undergoing cardiac versus other surgery and in patients with versus without sepsis upon admission. Elevated sRAGE upon admission, unlike CRP, was associated with need for renal replacement therapy, liver dysfunction, circulatory failure and mortality. Except for mortality, these associations remained in multivariate logistic regression analysis correcting for baseline risk factors. Introduction While the crosstalk between coagulopathy and infl ammation plays a key role in the development of multiple organ dysfunction in critically ill patients, the mechanisms governing this crosstalk have yet to be established. Microparticles (MPs) are submicron vesicles shed from a variety of cells that are considered to have proinfl ammatory and prothrombotic properties. Although platelet- derived MPs (PDMPs) are the main form of MPs, the role of PDMPs in critically ill patients remains unclear [1]. The aims of this study were to investigate serum PDMP levels in critically ill patients and to assess their prognostic value. Conclusion Critical illness alters several components of the RAGE axis. Elevated sRAGE levels upon admission to the ICU were associated with adverse outcome, independent of baseline pathology. Conclusion Critical illness alters several components of the RAGE axis. Elevated sRAGE levels upon admission to the ICU were associated with adverse outcome, independent of baseline pathology. Reference p g Methods This study comprised 119 critically ill patients who were admitted to the ICU. PDMPs were measured by ELISA three times a week, and 372 samples were obtained. We calculated both the mean PDMP value and the mean PDMP/platelet (PDMP/PLT) ratio (converted to plasma PDMP levels per 104 platelets) during the course of the ICU stay. Baseline patient data, including APACHE II score, SOFA score, Japanese Association for Acute Medicine DIC score, International Society for Thrombosis and Haemostasis overt DIC score, blood coagulation parameters, C-reactive protein and lactate, were collected at the time of admission to the ICU. The primary outcome was in- hospital mortality. Potential predictors were analyzed for possible association with outcomes.if 1. Creagh-Brown BC, et al.: The RAGE axis in systemic infl ammation, acute lung injury and myocardial dysfunction: an important target? P209 P209 Fibrinogen at admission is an independent predictor of mortality in severe sepsis and septic shock M Azfar, M Khan, M Khurshid King Saud University, Riyadh, Saudi Arabia Critical Care 2014, 18(Suppl 1):P209 (doi: 10.1186/cc13399) M Azfar, M Khan, M Khurshid g y y Critical Care 2014, 18(Suppl 1):P209 (doi: 10.1186/cc13399) Introduction Coagulation abnormalities are common in severe sepsis or septic shock [1]. Introduction Coagulation abnormalities are common in severe sepsis or septic shock [1]. Methods A prospective observational cohort study of 100 patients above 18 years of age diagnosed with severe sepsis or septic shock on admission. The fi rst blood sample collected on admission was analyzed. Data were collected through a predesigned pro forma. Those with previous history of any coagulation disorders were excluded. Figure 2 (abstract P207). ROC curve of the SF value for DIC. S75 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Univariate analysis showed signifi cant correlation of APACHE II, platelet, PT, aPTT, fi brinogen and D-dimer with mortality in patients with severe sepsis or septic shock. Multivariate analysis showed APACHE II >20 (P = 0.001), fi brinogen <2 (P = 0.019) and D-dimer >1(P = 0.06) are independent predictors of mortality in severe sepsis or septic shock. See Table 1. in-hospital mortality. The AUCs were calculated as 0.768 ± 0.073 for the mean PDMP/PLT ratio (P = 0.00) and 0.811 ± 0.048 for the APACHE II score (P = 0.00). in-hospital mortality. The AUCs were calculated as 0.768 ± 0.073 for the mean PDMP/PLT ratio (P = 0.00) and 0.811 ± 0.048 for the APACHE II score (P = 0.00). Conclusion The PDMP/PLT ratio is a good predictor of in-hospital mortality in critically ill patients, especially in patients with DIC. Reference 1. Delabranche X, et al.: Intensive Care Med 2013, 39:1695-1703 Table 1 (abstract P209). Receptor for advanced glycation end products axis in critically ill patients Introduction Systemic infl ammation caused by infection or trauma often leads to adverse outcome in critically ill patients. Binding of ligands to the receptor for advanced glycation end products (RAGE) activates several pathways, including the nuclear factor-kappa B pathway, which generates infl ammatory cytokines, proteases and oxidative stress. RAGE activation has been suggested to link amplifi cation and perpetuation of infl ammation to subsequent organ damage and adverse outcome in sepsis, acute lung injury and myocardial dysfunction [1]. The soluble receptor, sRAGE, is thought to act as a decoy, thus protecting against further RAGE activation. High mobility group box 1 (HMGB1) is a nuclear protein that is released during cellular stress and damage. S100A12 is a neutrophil-derived protein that acts as a proinfl ammatory danger signal. Both are ligands for RAGE. We hypothesized that excessive RAGE activation is linked to adverse outcome in critically ill patients and that a diff erent pattern of RAGE activation and infl ammation may be present in patients according to underlying pathology. Conclusion The fi brinogen level at admission is an independent predictor of mortality in patients with sepsis or septic shock. It also confi rms correlation of other coagulation abnormalities with the outcome. Reference 1. Levi M, et al.: Coagulation abnormalities in critically ill patients. Crit Care 2006, 10:222. P212 Usefulness of the endotoxin activity assay as a biomarker to assess severity in ICU patients Plasma platelet-derived microparticles to platelet count ratio as a marker of mortality in critically ill patients Intensive Care Med 2010, 36:1644-1656. P210 y y Methods We measured sRAGE, HMGB1 and S100A12 serum levels upon admission, day 7 and the last day in the ICU in 405 critically ill surgical patients who needed intensive care for at least 7 days and in 69 matched healthy controls. We assessed the relation of these levels with clinical complications and outcome, in comparison with C-reactive protein (CRP) as a routinely measured clinical parameter of infl ammation. P210 Plasma platelet-derived microparticles to platelet count ratio as a marker of mortality in critically ill patients M Ohuchi, K Hashimoto, A Ushiba, T Kishimoto, T Yamane, T Hamamoto, T Tabata, Y Tsujita, M Matsushiga, K Takahashi, K Matsumura, K Fujino, Y Eguchi Shiga University of Medical Science, Otsu-shi, Japan Critical Care 2014, 18(Suppl 1):P210 (doi: 10.1186/cc13400) P209 Died Survived 95% CI Variable (n = 65) (n = 35) ARR of ARR P value Platelet <150 33 (50.8) 10 (28.6) 1.37 0.03, 1.84 0.03 PT >16 41 (63.1) 14 (40) 1.40 1.02, 1.91 0.027 aPTT >40 41 (63.1) 14 (40) 1.40 1.02, 1.91 0.027 Fibrinogen <2 40 (61.5) 15 (42.9) 1.31 0.96, 1.70 0.07 D-dimer >1 64 (98.5) 31 (88.6) 3.37 0.48, 19.5 0.03 APACHE II >20 53 (81.5) 18 (51.4) 1.80 1.15, 2.84 0.002 Age >65 27 (41.5) 8 (22.9) 1.60 1.04, 2.46 0.02 Conclusion The fi brinogen level at admission is an independent predictor of mortality in patients with sepsis or septic shock. It also confi rms correlation of other coagulation abnormalities with the outcome. Reference 1. Levi M, et al.: Coagulation abnormalities in critically ill patients. Crit Care 2006, 10:222. Usefulness of presepsin and procalcitonin levels in the diagnosis of sepsis in patients with acute kidney injury Y Nakamura, H Ishikura, R Ichiki, K Hoshino, M Mizunuma, J Tanaka, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P213 (doi: 10.1186/cc13403) Introduction The presepsin (PSEP) and procalcitonin (PCT) levels are useful biomarkers for diff erentiating between sepsis and non- infectious systemic infl ammatory response syndrome (SIRS). The PSEP and PCT levels have been reported to be abnormally high in patients with chronic renal failure. However, there have been no signifi cant investigations regarding the relationships between these biomarkers and the presence of acute kidney injury (AKI) in the diagnosis of sepsis. The purpose of this study was to clarify the diagnostic accuracy of PSEP and PCT levels in patients with AKI. Conclusion We concluded that although serum leptin may not be benefi cial in early diff erentiation between sepsis and non-infectious SIRS on admission, it may be highly specifi c on the second day. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 analysis; the area under the curve (AUC) for the PSEP and PCT levels was 0.883 and 0.870, respectively, in the non-AKI group. In addition, the AUC values for the PSEP and PCT levels in the Risk, Injury and Failure groups were 0.843 and 0.843, 0.818 and 0.922 and 0.669 and 0.804, respectively. In the Failure group, the AUC for the PSEP and PCT levels was 0.828 and 0.852, respectively, after dividing the PSEP and PCT levels by the creatinine (Cr) level. The optimal cutoff values for the PSEP and PCT levels for diagnosing sepsis were 409 pg/ml/Cr (sensitivity: 66.0%, specifi city: 91.7%) and 1.5 ng/ml/Cr (sensitivity: 63.6%, specifi city: 95.8%), respectively. the endotoxin activity assay (EAA), a newly developed rapid assay of endotoxin. Blood endotoxin levels (EA levels) of 314 patients admitted to our university hospital ICU were measured within 24 hours from admission, and their correlation with disease severity and outcome was examined. Methods The study is a single-center retrospective analysis of critically ill patients admitted to our university hospital ICU from November 2006 to March 2012. All patients whose EA and procalcitonin levels were measured and severity criteria of disease recorded were enrolled. A total of 314 patients were analyzed. i y y Conclusion The diagnostic accuracy of PSEP and PCT levels for detecting sepsis decreased in the Failure group; however, the value of these parameters for diagnosing sepsis in patients meeting the Failure criteria increased after dividing them by the Cr level. Results The mean ± SD of all ICU-admitted patients (n = 314) was 0.39 ± 0.25, and that of healthy volunteers (n = 61) was 0.10 ± 0.09. The mean APACHE II score at admission increased in parallel with increased EA levels. The mean (± SD) APACHE II score in the very low group of patients (EA <0.2) was 17.3 ± 8.9, while that in the low group (0.2 ≤EA <0.4) was 20.6 ± 9.2; it was 22.6 ± 8.1 in the intermediate group (0.4 ≤EA <0.6) and 25.3 ± 8.5 in the high group (0.6 ≤EA). The diff erence between the groups was statistically signifi cant. The diff erence between the groups was statistically signifi cant. p Reference 1. Ulevitch RJ, Tobias PS: Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol 1999, 11:19-22. 1. Ulevitch RJ, Tobias PS: Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol 1999, 11:19-22. y y Results Our patients had mean age of 52.3 ± 18.6 years, 10 males (33.3%). There were no signifi cant diff erences regarding baseline demographic and clinical data apart from blood pressures, which were lower in sepsis group. Serum leptin on day 2 only was higher in the sepsis group (44.2 ± 17.7 μg/l vs. 31.1 ± 2.1 μg/l, P = 0.008) with no diff erence on days 0 and 4 of admission. We detected a serum leptin level of 38.05 μg/l on day 2 to be 93% sensitive and 100% specifi c to diagnose sepsis. The three serum CRP levels were higher in the sepsis group compared with the SIRS group (61.2 ± 9 mg/l vs. 48.9 ± 7.1 mg/l, P <0.001 on day 0, 71.5 ± 9.6 mg/l and 196.8 ± 39.8 mg/l in the sepsis group vs. 56.9 ± 8 mg/l and 73.7 ± 32.5 mg/l in the SIRS group for days 2 and 4 respectively, P <0.001 for both). We found a CRP of 67.5 mg/l on day 2 having 87% sensitivity and 93% specifi city for the diagnosis of sepsis. Usefulness of the endotoxin activity assay as a biomarker to assess severity in ICU patients Usefulness of the endotoxin activity assay as a biomarker to asses severity in ICU patients M Noguchi, T Ikeda, K Ikeda, S Suda, A Kodaira, T Ueno, A Ohmi Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P212 (doi: 10.1186/cc13402) y p M Noguchi, T Ikeda, K Ikeda, S Suda, A Kodaira, T Ueno, A Ohmi okyo Medical University, Tokyo, Japan M Noguchi, T Ikeda, K Ikeda, S Suda, A Kodaira, T Ueno, A Ohmi Tokyo Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P212 (doi: 10.1186/cc13402) Results The mean PDMP/PLT ratio was signifi cantly diff erent when comparing hospital survivors (n = 98; median, 1.95) and nonsurvivors (n = 21; median 8.40; P = 0.00). The mean PDMP/PLT ratio was signifi cantly higher in patients with (median, 4.28) than in those without DIC (median, 1.57; P = 0.00). In DIC patients, the mean PDMP/ PLT ratio was signifi cantly higher in nonsurvivors (median, 9.39) than in survivors (median, 3.65; P = 0.02). Multivariate logistic regression analysis revealed that both the mean PDMP/PLT ratio (P = 0.04) and APACHE II score (P = 0.00) were independently associated with Introduction Endotoxin is a component of the membrane of Gram- negative bacteria and plays a key role in the pathogenesis of sepsis [1]. Measurement of endotoxin levels in patient blood is important for the early diagnosis of and appropriate determination of the treatment strategy for sepsis. The aim of this study was to investigate the prevalence of endotoxemia in Japanese critically ill patients using Introduction Endotoxin is a component of the membrane of Gram- negative bacteria and plays a key role in the pathogenesis of sepsis [1]. Measurement of endotoxin levels in patient blood is important for the early diagnosis of and appropriate determination of the treatment strategy for sepsis. The aim of this study was to investigate the prevalence of endotoxemia in Japanese critically ill patients using S76 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Diff erentiating sepsis from non-infective systemic infl ammatory response syndrome: comparison between C-reactive protein and leptin Conclusion Our patients’ EA levels were signifi cantly correlated with disease severity criteria and 28-day mortality of the patients. When the EA level and procalcitonin level were used concomitantly, disease severity could be assessed more precisely than when either marker was used alone. These results suggest that the EA level is a useful marker for disease severity assessment and outcome prediction in critically ill patients. Introduction Diff erentiation of sepsis from non-infectious SIRS is important in improving sepsis outcome. We intended in this study to evaluate the role of serum leptin and to compare it with CRP in diff erentiating sepsis from non-infectious SIRS. Introduction Diff erentiation of sepsis from non-infectious SIRS is important in improving sepsis outcome. We intended in this study to evaluate the role of serum leptin and to compare it with CRP in diff erentiating sepsis from non-infectious SIRS. f g Methods We included 30 patients with SIRS. According to the presence or absence of infection, our patients were classifi ed into the SIRS group and the sepsis group. Leptin and CRP were evaluated in all patients on admission, day 2 and day 4. P213 Usefulness of presepsin and procalcitonin levels in the diagnosis of sepsis in patients with acute kidney injury Y Nakamura, H Ishikura, R Ichiki, K Hoshino, M Mizunuma, J Tanaka, A Murai Fukuoka University, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P213 (doi: 10.1186/cc13403) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 The percentages of patients diagnosed with severe sepsis or septic shock were 19.3%, 34.5%, 50.0% and 81.3% in the very low, low, intermediate and high groups, respectively.i P214 Diff erentiating sepsis from non-infective systemic infl ammatory response syndrome: comparison between C-reactive protein and leptin N Farag1, K Taema2, E Abdel-Latif2, G Hamed2 1Alsahel Teaching Hospital, Cairo, Egypt; 2Cairo University, Cairo, Egypt Critical Care 2014, 18(Suppl 1):P214 (doi: 10.1186/cc13404) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods The observational study in ICU ventilated septic patients with peritonitis (65%), pancreonecrosis (20%) and mediastinitis (15%) was performed in 2010 and 2013. ARDS was diagnosed and staged according to the V.A. Negovsky Research Institute criteria [1,2] and the Berlin defi nition. Plasma SPA was measured on ARDS diagnosis (day 0) and days 3 and 5 by the immunoenzyme essay (BioVendor, USA). Patients were treated according to the international guidelines. Data were statistically analyzed by STATISTICA 7.0, ANOVA method, and presented as median and 25th to 75th percentiles, ng/ml; P <0.05 was considered statistically signifi cant. Areas under the receiver operating curves were calculated. Results The WBC and PCT median values were signifi cantly (P <0.05) higher in infected than control patients, during 10 postoperative days and on the third and fourth postoperative day, respectively, both variables showing a peak at 3 days in infected patients. The number of times that the WBC count surpassed its second postoperative median value (13,000 cells/mm3) during the fi rst 3 postoperative days plus the number of times that PCT surpassed its own (1.7 ng/ ml) was a parameter (ranging from 1 to 6) with three categories (R1= surpassed zero to one time; R2 = two to three times; R3 = four to six times) signifi cantly associated with risk of infection, which increased with increasing number of times (R2: OR = 2.48 (1.32 to 4.65) and R3: OR = 7.06 (3.17 to 15.71)).i Results Eighty-fi ve patients (out of 300 screened) were enrolled in the study according to the inclusion/exclusion criteria. Patients were assigned into groups: ARDS (n = 50, 48 ± 5.7 years old, M/F 35/15, mortality 26%) and noARDS (n = 35, 46 ± 8.7 years old, M/F 30/5, mortality 23%). Groups were comparable in APACHE II scores. In the ARDS group SPA was higher at all points than in the noARDS group (day 0: 32.6, 25 to 75 IQR 18.2 to 60.7 vs. 23.4, 25 to 75 IQR 17.8 to 28.6; day 3: 31.5, 25 to 75 IQR 20.9 to 31.5 vs. 24.6, 25 to 75 IQR 19.7 to 24.6; day 5: 32.5, 25 to 75 IQR 17.3 to 66.4 vs. 22.5, 25 to 75 IQR 13.4 to 29.5, P <0.05). The plasma SPA was signifi cantly lower in surviving versus dead patients with ARDS (day 0: 20.9, 25 to 75 IQR 13.0 to 35.7 vs. Reference 1. Sablotzki A, Friedrich I, Muhling J, Dehne MG, Spillner J, Silber RE, et al.: The systemic infl ammatory response syndrome following cardiac surgery: diff erent expression of proinfl ammatory cytokines and procalcitonin in patients with and without multiorgan dysfunctions. Perfusion 2002, 17:103-109. Single pro-adrenomedullin determination in septic shock and 28-day mortality A Garcia-de la Torre1, M De La Torre-Prados2, J Parez-Vacas2, C Trujillano-Fernandez2, A Puerto-Morlan2, E Camara Sola2, A Garcia-Alcantara2 1University Hospital Puerto Real, Cadiz, Spain; 2Hospital Virgen de la Victoria, Málaga, Spain Critical Care 2014, 18(Suppl 1):P216 (doi: 10.1186/cc13406) Conclusion Plasma SPA level ≥38.8 ng/ml on the day of ARDS diagnosis is a sensitive and specifi c candidate biomarker of mortality prediction in ARDS septic patients. P218 Club Cell protein: a candidate diagnostic biomarker of Pseudomonas aeruginosa nosocomial pneumonia V Moroz1, A Kuzovlev1, S Polovnikov2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology, RAMS, Moscow, Russia; 2N.N. Burdenko Main Military Hospital, Moscow, Russia Critical Care 2014, 18(Suppl 1):P218 (doi: 10.1186/cc13408) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 45.7, 25 to 75 IQR 23.5 to 67.9; day 3: 25.5, 25 to 75 IQR 11.8 to 35.5 vs. 45.0, 25 to 75 IQR 29.6 to 68.4; day 5: 24.5, 25 to 75 IQR 11.4 to 33.6 vs. 49.6, 25 to 75 IQR 31.3 to 79.0, P <0.05). Plasma SPA on day 0 had a good capacity for prediction of mortality in ARDS patients: SPA on day 0 ≥38.8 ng/ml yielded a sensitivity of 65% and specifi city of 80% (AUC 0.74; 95% CI 0.577 to 0.866; P = 0.0026). Conclusion Daily assessment of WBC and PCT during the fi rst 3 postoperative days may be of value to diagnose and predict infection in cardiac surgery patients. f Club Cell protein: a candidate diagnostic biomarker of Pseudomonas aeruginosa nosocomial pneumonia V Moroz1, A Kuzovlev1, S Polovnikov2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology, RAMS, Moscow, Russia; 2N.N. Burdenko Main Military Hospital, Moscow, Russia Critical Care 2014, 18(Suppl 1):P218 (doi: 10.1186/cc13408) V Moroz1, A Kuzovlev1, S Polovnikov2 1V.A. Negovsky Scientifi c Research Insitute of General Reanimatology, RAMS, Moscow, Russia; 2N.N. Burdenko Main Military Hospital, Moscow, Russia Critical Care 2014, 18(Suppl 1):P218 (doi: 10.1186/cc13408) Introduction Early etiological diagnosis of nosocomial pneumonia (NP) determines prompt targeted treatment. The aim of this study was to investigate the role of Club Cell protein (CCP) as a candidate diagnostic biomarker of Pseudomonas aeruginosa (PA) NP. Introduction Early etiological diagnosis of nosocomial pneumonia (NP) determines prompt targeted treatment. The aim of this study was to investigate the role of Club Cell protein (CCP) as a candidate diagnostic biomarker of Pseudomonas aeruginosa (PA) NP. g Methods The observational study in ICU ventilated septic patients with peritonitis (65%), pancreonecrosis (20%) and mediastinitis (15%) was performed in 2010 and 2013. Diagnosis of NP was made according to the standard clinical criteria. Associations of multiresistant Gram- negative bacteria were detected in sputum of all patients. PA was detected in 75% of patients. Plasma CCP was measured on the day of NP diagnosis (day 0) and days 3, 5 and 7 by the immunoenzyme essay (BioVendor, USA). Data were statistically analyzed by STATISTICA 7.0, ANOVA method, and presented as Me and 25 to 75 percentiles, ng/ml; P <0.05 was considered signifi cant. Areas under the receiver operating curves (ROC) were calculated. Conclusion Pro-adrenomedullin is an important prognostic biomarker of survival when measured on admission of septic shock patients to the ICU. Use of procalcitonin and white blood cells as combined predictors of infection in cardiac surgery patients p Methods This study was conducted as a single-center retrospective study. Blood samples were collected from patients immediately after admission to the Department of Emergency and Critical Care Medicine, Fukuoka University Hospital between June 2010 and August 2013. We enrolled 629 patients in whom both the PSEP and PCT levels were measured on admission. We classifi ed the patients into two groups according to the RIFLE criteria (Risk, Injury, Failure, Loss of kidney function and End-stage kidney disease: Loss and ESKD): the AKI group and the non-AKI group. The patients in the AKI group were further classifi ed into the sepsis group and the nonsepsis group according to each stage of AKI. We subsequently investigated the diagnostic accuracy of the PSEP and PCT levels for detecting sepsis in these groups. Results We evaluated 254 patients with AKI and 375 patients without AKI. The AKI group included 103 patients who met the Risk criteria, 65 who met the Injury criteria, 66 who met the Failure criteria and 18 who met the Loss and ESKD criteria. The mean PSEP and PCT levels were signifi cantly higher in the sepsis group than in the nonsepsis group among the non-AKI patients and those meeting the Risk, Injury and Failure criteria (P <0.01). The diagnostic accuracy of the PSEP and PCT levels for detecting sepsis was determined according to a ROC Critical Care 2014, 18(Suppl 1):P215 (doi: 10.1186/cc13405 Introduction The aim of this study is to identify a marker able to predict sepsis in cardiac surgery patients and to diff erentiate SIRS from infectious and non-infectious origin. The occurrence of sepsis after cardiac surgery increases the mortality risk. Sepsis may be mistaken for the cardiac surgery-associated systemic infl ammatory response syndrome (SIRS) [1]. Methods A prospective, observational study was carried out to compare procalcitonin (PCT), C-reactive protein (CRP) and white blood cells (WBCs) during the fi rst 10 postoperative days after cardiac surgery with cardiopulmonary bypass (CPB) between 122 patients with infection (Infection group) and 301 without (Control group) to identify predictors of infection. S77 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References Critical Care 2014, 18(Suppl 1):P216 (doi: 10.1186/cc13406) 1. Kuzovlev A, et al.: Semin Cardiothorac Vasc Anesth 2010, 14:231-241. 2. Kuzovlev A, et al.: Crit Care 2013, 17(Suppl 2):P21800. Introduction The early phase of septic shock is dominated by severe alterations of the cardiovascular system. The prognostic value of pro- adrenomedullin (pADM), a vasoactive pro-hormone, measured within 24 hours from septic shock onset was assessed. Introduction The early phase of septic shock is dominated by severe alterations of the cardiovascular system. The prognostic value of pro- adrenomedullin (pADM), a vasoactive pro-hormone, measured within 24 hours from septic shock onset was assessed. p Methods A prospective, observational study in 100 patients >18 years with septic shock in a polyvalent ICU of a university hospital. Demographic, clinical parameters and pADM, C-reactive protein (CRP) and procalcitonin (PCT) were studied during the fi rst 24 hours after admission in 2011. Descriptive and comparative statistical analysis was performed using the statistical software packages StatSoft STATISTICA 7.1 and MedCalc 9.2.1.0. Results We analyzed 100 consecutive episodes of septic shock in the ICU. The median age of the study sample was 64 years, interquartile range 16.8 years, 59% were men; the main sources of infection were respiratory tract (48%) and intra-abdominal (24%). The 28-day mortality was 36%. The profi le of dead patients showed signifi cantly higher clinical severity scores, APACHE II (27 vs. 25; P <0.001) and SOFA (12 vs. 10; P <0.001). The area under the curve was 0.72 for pADM, signifi cantly higher than those for CRP (0.62) and PCT (0.65), but similar to those for APACHE II score (0.69) and SOFA score (0.78). Kaplan–Meier survival analysis was signifi cant (P = 0.0012) for patients with pADM <1.2 nmol/l. Cox regression analysis also showed statistical signifi cance (P = 0.0004) and a likelihood ratio =1.26 per each 1 nmol/l increase in pADM. P217 f Results Ninety patients (out of 350 screened) were enrolled in the study according to the inclusion/exclusion criteria. Patients were assigned to groups: NP (n = 50, 45 ± 4.3 years old, M/F 36/14) and noNP (n = 40, 48 ± 7.2 years old, M/F 30/10). Groups were comparable in APACHE II and CPIS scores. In patients with PA NP (n = 30), plasma CCP was signifi cantly lower at all points than in the patients with no PA detected (n = 20; Figure 1). Plasma CCP on day 0 had a good capacity for the diagnosis of PA NP: CCP on day 0 ≤17.5 ng/ml yielded a sensitivity of 86.5% and specifi city of 66.7% (AUC 0.74; 95% CI 0.630 to 0.829; P = 0.0001; Figure 2).i P217 Surfactant protein A: a candidate predictive biomarker of mortality in acute respiratory distress syndrome in sepsis V Moroz, A Kuzovlev, A Goloubev V.A. Negovsky Scientifi c Research Insitute of General Reanimatology, RAMS, Moscow, Russia Critical Care 2014, 18(Suppl 1):P217 (doi: 10.1186/cc13407) Introduction Prediction of mortality in septic acute respiratory distress syndrome (ARDS) is a problem of great signifi cance. The aim of this study was to investigate the role of surfactant protein A (SPA) as a candidate predictive biomarker of mortality in ARDS. Conclusion Plasma CCP level ≤17.5 ng/ml is a sensitive and specifi c candidate diagnostic biomarker of PA NP. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S78 P219 Plasma cholinesterase activity as diagnostic marker for systemic infl ammation AR Zivkovic, K Schmidt, T Brenner, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2014, 18(Suppl 1):P219 (doi: 10.1186/cc13409) Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure Therefore early diagnosis of systemic inflammation Figure 1 (abstract P218). Club Cell protein concentration in patients with Pseudomonas aeruginosa NP. Figure 2 (abstract P218). ROC curve for Club Cell protein (day 0) for the diagnosis of Pseudomonas aeruginosa NP. Figure 1 (abstract P218). Club Cell protein concentration in patients with Pseudomonas aeruginosa NP. practice, but a defi nitive diagnostic tool for an early detection of systemic infl ammation remains to be identifi ed. The neurotransmitter acetylcholine (Ach) has been shown to play an important role in the infl ammatory response. Serum cholinesterase (butyrylcholinesterase (BChE)) is synthesized in the liver and acts as the major Ach hydrolyzing enzyme in plasma. P217 f Hence, BChE activity has been widely used as a biomarker for liver function. However, the role of this enzyme in the infl ammatory pathomechanism has not yet been fully understood. Here, we describe a strong correlation between the BChE activity and the systemic infl ammatory response. yl y p Methods In this study we measured BChE activity in healthy subjects and in critically ill patients, clinically diagnosed with systemic infl ammation or sepsis. Furthermore, we measured the levels of routine infl ammation biomarkers and liver function parameters in blood of critically ill patients. Data were statistically analyzed using unpaired Student t test, Pearson correlation or ANOVA followed by Dunnett’s post hoc analysis. P <0.05 was considered statistically signifi cant. y yi Results We found that the activity of BChE in patients with systemic infl ammation was dramatically reduced in comparison with healthy subjects and negatively correlated with the serum levels of conventional infl ammatory biomarkers. BChE synthesis depends on hepatic function. Surprisingly, the observed reduction in the BChE activity during systemic infl ammation does not correlate with liver failure. Conclusion Our results suggest that BchE activity might play an important role in the diagnosis of systemic infl ammation independent of overall hepatic function. Figure 1 (abstract P218). Club Cell protein concentration in patients with Pseudomonas aeruginosa NP. P220 Figure 1 (abstract P218). Club Cell protein concentration in patients with Pseudomonas aeruginosa NP. Lymphopenia as a predictor of bacteremia in the emergency department p R Lowsby1, C Gomes1, I Jarman2, P Nee1 1St Helens and Knowsley Teaching Hospital NHS Trust, Liverpool, UK; 2Liverpool John Moores University, Liverpool, UK Critical Care 2014, 18(Suppl 1):P220 (doi: 10.1186/cc13410) Figure 2 (abstract P218) ROC curve for Club Cell protein (day 0) for the Introduction Bloodstream infection (BSI) is associated with a reduction in circulating lymphocytes. Lymphopenia has been proposed as an early marker of BSI in pyrexial adults in the emergency department (ED) setting [1]. The aim of this study was to compare lymphocyte Figure 1 (abstract P220). ROC curve comparing blood test parameters in predicting bacteremia. Figure 2 (abstract P218). ROC curve for Club Cell protein (day 0) for the diagnosis of Pseudomonas aeruginosa NP. Figure 2 (abstract P218). ROC curve for Club Cell protein (day 0) for the diagnosis of Pseudomonas aeruginosa NP. P219 Plasma cholinesterase activity as diagnostic marker for systemic infl ammation AR Zivkovic, K Schmidt, T Brenner, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2014, 18(Suppl 1):P219 (doi: 10.1186/cc13409) Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure. Therefore, early diagnosis of systemic infl ammation ld b f h i d i i V i P219 Plasma cholinesterase activity as diagnostic marker for systemic infl ammation AR Zivkovic, K Schmidt, T Brenner, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2014, 18(Suppl 1):P219 (doi: 10.1186/cc13409) Plasma cholinesterase activity as diagnostic marker for systemic infl ammation l AR Zivkovic, K Schmidt, T Brenner, S Hofer Universitätsklinikum Heidelberg, Germany Critical Care 2014, 18(Suppl 1):P219 (doi: 10.1186/cc13409) Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure. Therefore, early diagnosis of systemic infl ammation would be of great therapeutic and prognostic importance. Various infl ammation biomarkers have been used in clinical and experimental Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure. Therefore, early diagnosis of systemic infl ammation would be of great therapeutic and prognostic importance. Various infl ammation biomarkers have been used in clinical and experimental Introduction Systemic infl ammation is a generalized response to internal or external infl ammatory stimuli often resulting in multiple organ failure. Therefore, early diagnosis of systemic infl ammation would be of great therapeutic and prognostic importance. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods In 183 patients, in a prospective observational study, serum CRP and PCT values were collected every day starting on postoperative day 1 through day 5. The defi nition of SIRS includes two or more of the following: temperature >38 or <36°C; heart rate >90 beats/minute; respiratory rate >20/minute; arterial carbon dioxide pressure <32 mmHg; white blood cell count >12,000/mm3 and <4.00/mm3. The ability of PCT to predict sepsis and other postoperative complications were determined by performing receiver operative characteristic curve analysis. Methods In 183 patients, in a prospective observational study, serum CRP and PCT values were collected every day starting on postoperative day 1 through day 5. The defi nition of SIRS includes two or more of the following: temperature >38 or <36°C; heart rate >90 beats/minute; respiratory rate >20/minute; arterial carbon dioxide pressure <32 mmHg; white blood cell count >12,000/mm3 and <4.00/mm3. The ability of PCT to predict sepsis and other postoperative complications were determined by performing receiver operative characteristic curve analysis. count with conventional markers in patients presenting to the ED of a UK hospital with suspected sepsis. count with conventional markers in patients presenting to the ED of a UK hospital with suspected sepsis. Methods A retrospective review was undertaken of adult patients presenting to the ED (annual census 90,000) with pyrexial illness over a 12-month period, 2011 to 2012. Data included white cell count (WCC), neutrophil count (NC), lymphocyte count (LC) and C-reactive protein (CRP). The results were compared. Sensitivity and specifi city were calculated for each parameter and receiver operating characteristic (ROC) curves were constructed. y y Results All patients were divided post hoc into patients with SIRS (Group 1, n = 83) and patients without SIRS (Group 2, n = 100). A PCT threshold value of 2.79 ng/ml on postoperative day 1 was able to discriminate postoperative complications in patients with or without SIRS with a sensitivity of 82.5% and a specifi city of 70% (area under curve: 0.76, P <0.01).i Results All patients were divided post hoc into patients with SIRS (Group 1, n = 83) and patients without SIRS (Group 2, n = 100). A PCT threshold value of 2.79 ng/ml on postoperative day 1 was able to discriminate postoperative complications in patients with or without SIRS with a sensitivity of 82.5% and a specifi city of 70% (area under curve: 0.76, P <0.01). P222 1. Venet F, Filipe-Santos O, et al.: Decreased T-cell repertoire diversity in sepsis: a preliminary study. Crit Care Med 2013; 41:111-119. Altered T-cell repertoire diversity in septic shock patients 2 2 2 2 Altered T-cell repertoire diversity in septic shock patients A Kutz, E Grolimund, B Mueller, P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P221 (doi: 10.1186/cc13411) A Kutz, E Grolimund, B Mueller, P Schuetz Kantonsspital Aarau, Switzerland Critical Care 2014, 18(Suppl 1):P221 (doi: 10.1186/cc13411) N Takeyama1, A Tomino2, M Hashiba2, A Hirakawa2, T Hattori2, H Miyabe2 1Fujita Health University, Aichi, Japan; 2Fuita Health University, Aichi, Japan Critical Care 2014, 18(Suppl 1):P223 (doi: 10.1186/cc13413) N Takeyama1, A Tomino2, M Hashiba2, A Hirakawa2, T Hattori2, H Miyabe2 1Fujita Health University, Aichi, Japan; 2Fuita Health University, Aichi, Japan Critical Care 2014, 18(Suppl 1):P223 (doi: 10.1186/cc13413) Introduction The occurrence of sepsis-induced immune suppression is associated with multiple organ dysfunctions, although the exact role of T-cell malfunction is obscure. We investigated the impact of sepsis on the adaptive immune system and to monitor T-cell receptor (TCR) diversity. Methods TCR diversity was analyzed in peripheral blood mononuclear cells (PBMCs) isolated from septic shock patients at three time points (days 1, 3 and 7 after diagnosis of septic shock). TCR diversity was measured in genomic DNA isolated from PBMCs using the Human Immun TraCkeRb test (ImmunID Technologies, Grenoble, France) [1]. Multi-N-plex PCR was performed using a primer specifi c to a V gene family and several primers specifi c to J segments. The signal is measured as a function of the fl uorescence intensity of the reference marker. Rearrangement validation and map generation were detected and analyzed using the Constel’ID software (ImmunID Technologies). HLA-DR expression on CD14 cells was measured by fl ow cytometry. Results TCR diversity was markedly decreased in septic patients at day 1 compared with healthy volunteers. A recovery of TCR diversity was observed at days 3 and 7 except for dead patients. HLA-DR expression was signifi cantly decreased in septic patients at day 1. The total lymphocyte count reduced in septic patients at day 1, but the lymphocyte count recovered at days 3 and 7 except for dead patients. Conclusion We observed a lower TCR diversity and lymphopenia in the early stages of septic shock. A quick recovery of TCR diversity and lymphocyte count was observed in live patients. On the other hand, dead patients stayed on the lower level over the course of the study. These results suggest that altered TCR diversity and lymphopenia might participate in increased mortality and susceptibility to secondary nosocomial infections. Altered T-cell repertoire diversity in septic shock patients 2 2 2 2 The total lymphocyte count reduced in septic patients at day 1, but the lymphocyte count recovered at days 3 and 7 except for dead patients. Results TCR diversity was markedly decreased in septic patients at day 1 compared with healthy volunteers. A recovery of TCR diversity was observed at days 3 and 7 except for dead patients. HLA-DR expression was signifi cantly decreased in septic patients at day 1. The total lymphocyte count reduced in septic patients at day 1, but the lymphocyte count recovered at days 3 and 7 except for dead patients. Conclusion We observed a lower TCR diversity and lymphopenia in the early stages of septic shock. A quick recovery of TCR diversity and lymphocyte count was observed in live patients. On the other hand, dead patients stayed on the lower level over the course of the study. These results suggest that altered TCR diversity and lymphopenia might participate in increased mortality and susceptibility to secondary nosocomial infections. Conclusion The infl uence of pre-analytic factors on the examined biomarkers is marginal and not clinically relevant. Our observations reinforce the concept of using biomarkers in algorithms with widely- separated cutoff values and overruling criteria considering the entire clinical picture, without adjustment for pre-analytic factors. Reference 1. Schuetz et al.: JAMA 2009, 302:1059-1066. Altered T-cell repertoire diversity in septic shock patients 2 2 2 2 Introduction The occurrence of sepsis-induced immune suppression is associated with multiple organ dysfunctions, although the exact role of T-cell malfunction is obscure. We investigated the impact of sepsis on the adaptive immune system and to monitor T-cell receptor (TCR) diversity. Methods TCR diversity was analyzed in peripheral blood mononuclear cells (PBMCs) isolated from septic shock patients at three time points (days 1, 3 and 7 after diagnosis of septic shock). TCR diversity was measured in genomic DNA isolated from PBMCs using the Human Immun TraCkeRb test (ImmunID Technologies, Grenoble, France) [1]. Multi-N-plex PCR was performed using a primer specifi c to a V gene family and several primers specifi c to J segments. The signal is measured as a function of the fl uorescence intensity of the reference marker. Rearrangement validation and map generation were detected and analyzed using the Constel’ID software (ImmunID Technologies). HLA-DR expression on CD14 cells was measured by fl ow cytometry. Introduction Blood biomarkers are increasingly used to diagnose, guide therapy in, and risk-stratify community-acquired pneumonia (CAP) patients in emergency departments (EDs). How pre-analytic factors aff ect these markers’ initial levels in this population is unknown. Methods In this secondary analysis of consecutive ED patients with CAP from a large multicentre antibiotic stewardship trial [1], we used adjusted multivariate regression models to determine the magnitude and statistical signifi cance of diff erences in mean baseline concentrations of fi ve biomarkers (procalcitonin (PCT), C-reactive protein (CRP), white blood cell count, proadrenomedullin (ProADM), copeptin) associated with six pre-analytic factors (antibiotic or corticosteroid pretreatment, age, gender, chronic renal failure or liver insuffi ciency). fi y Results Of 925 CAP patients (median age 73 years, 58.8% male), 25.5% had antibiotic pretreatment, 2.4%, corticosteroid pretreatment, 22.3% chronic renal failure, and 2.4% chronic liver insuffi ciency. Diff erences associated with pre-analytic factors averaged 6.1 ± 4.6%; the three largest statistically signifi cant changes (95% CI) were: PCT, +14.2% (+2.1 to +26.4%, P = 0.02) in patients with liver insuffi ciency; ProADM, +13.2% (+10.2 to +16.1%, P <0.01) in patients with age above median; and CRP, –12.8% (–25.4 to –0.2%, P = 0.05) with steroid pretreatment.l l Results TCR diversity was markedly decreased in septic patients at day 1 compared with healthy volunteers. A recovery of TCR diversity was observed at days 3 and 7 except for dead patients. HLA-DR expression was signifi cantly decreased in septic patients at day 1. Pre-analytic factors and initial biomarker levels in community- acquired pneumonia patients P223 Altered T-cell repertoire diversity in septic shock patients N Takeyama1, A Tomino2, M Hashiba2, A Hirakawa2, T Hattori2, H Miyabe2 1Fujita Health University, Aichi, Japan; 2Fuita Health University, Aichi, Japan Critical Care 2014, 18(Suppl 1):P223 (doi: 10.1186/cc13413) Lymphopenia as a predictor of bacteremia in the emergency department Various infl ammation biomarkers have been used in clinical and experimental Figure 1 (abstract P220). ROC curve comparing blood test parameters in predicting bacteremia. S79 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results A total of 2,515 patient records were screened. Patients on chemotherapy were excluded, as were those under 18 years old. In total, 1,954 patients (53% female, median age 66 years) were included in the analysis. Blood cultures were positive in 13.7% of cases. There were signifi cant diff erences between all variables measured, with the exception of WCC, between bacteremic and nonbacteremic patients. The area under the curve of 70.8 was best for lymphocyte count (Figure 1). Conclusion (1) Serum PCT values increased signifi cantly after cardiopulmonary bypass in the SIRS group in comparison with patients without SIRS on postoperative day 1 and remain elevated until postoperative day 5. (2) Serum CRP values also follow a similar pattern, but a CRP threshold value was not obtained to diff erentiate between postoperative complications in patients with or without SIRS. A PCT threshold value of 2.79 ng/ml on postoperative day 1 is a valuable marker to discriminate between patients with or without SIRS. Conclusion In adult patients presenting to the ED with pyrexial illness, lymphopenia predicts bacteremia better than the usual markers of infection. Reference Reference 1. deJager et al.: Crit Care 2010, 14:R192. P224 P224 Association between DNA haplogroups and severe sepsis in patients who underwent major surgery E Gomez1, M Heredia1, S Resino2, M Jimenez-Sousa2, J Gomez-Herreras1, E Tamayo1, P Jorge1, M Lorenzo3, P Ruiz1 1Hospital Clínico Universitario de Valladolid, Spain; 2Instituto de Salud Carlos III, Madrid, Spain; 3Hospital Río Hortega, Valladolid, Spain Critical Care 2014, 18(Suppl 1):P224 (doi: 10.1186/cc13414) Association between DNA haplogroups and severe sepsis in patients who underwent major surgery E Gomez1, M Heredia1, S Resino2, M Jimenez-Sousa2, J Gomez-Herreras1, E Tamayo1, P Jorge1, M Lorenzo3, P Ruiz1 1Hospital Clínico Universitario de Valladolid, Spain; 2Instituto de Salud Carlos III, Madrid, Spain; 3Hospital Río Hortega, Valladolid, Spain Critical Care 2014, 18(Suppl 1):P224 (doi: 10.1186/cc13414) p y yp A Baysal1, M Doğukan2, H Toman3 Association between DNA haplogroups and severe sepsis in patients who underwent major surgery E Gomez1, M Heredia1, S Resino2, M Jimenez-Sousa2, J Gomez-Herreras1, E Tamayo1, P Jorge1, M Lorenzo3, P Ruiz1 1Hospital Clínico Universitario de Valladolid, Spain; 2Instituto de Salud Carlos III, Madrid, Spain; 3Hospital Río Hortega, Valladolid, Spain Critical Care 2014, 18(Suppl 1):P224 (doi: 10.1186/cc13414) A Baysal1, M Doğukan2, H Toman3 1Kartal Kosuyolu High Speciality Research and Training Hospital, Istanbul, Turkey; 2Adıyaman University Training and Research Hospital, Adıyaman, Turkey; 3Çanakkale 18 Mart Research Hospital, Çanakkale, Turkey Critical Care 2014, 18(Suppl 1):P222 (doi: 10.1186/cc13412) A Baysal , M Doğukan , H Toman 1Kartal Kosuyolu High Speciality Research and Training Hospital, Istanbul, Turkey; 2Adıyaman University Training and Research Hospital, Adıyaman, Turkey; 3Çanakkale 18 Mart Research Hospital, Çanakkale, Turkey Critical Care 2014, 18(Suppl 1):P222 (doi: 10.1186/cc13412) Introduction The values of C-reactive protein (CRP) and procalcitonin (PCT) were investigated to determine their eff ects on postoperative complications in patients with or without systemic infl ammatory response syndrome (SIRS). Introduction The aim of this study was to analyze whether mitochondrial DNA haplogroups are associated with severe sepsis and S80 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 were measured in supernatant from peripheral blood mononuclear cells stimulated with anti-CD3 and anti-CD3+anti-CD28, incubated for 4 days. Cytokine concentrations of IL-1β, IL-5, IL-6, IL-8, IL-10, IL-13, IL-17A, IFNγ and TNFα were determined by multiplex cytometric bead array. mortality after major surgery in European populations. Mitochondrial DNA (mtDNA) variants may play an important role for predicting clinical outcome in sepsis [1]. P224 Methods We carried out a case–control study on 240 septic patients (severe sepsis or septic shock; Case group) and 267 patients with systemic infl ammatory response syndrome (SIRS; Control group). Furthermore, a longitudinal substudy for analyzing survival was performed in septic patients. mtDNA genotyping was performed by Sequenom’s MassARRAY platform. y Results Plasma levels of IFNγ and IL-13 were lower in septic patients compared with healthy participants. In contrast, plasma levels of IL-6 (see Figure 1) and IL-8 were increased in septic patients compared with both surgical patients and healthy participants. Plasma levels of IL-10 were signifi cantly higher only in comparison with surgical patients. Following incubation with anti-CD3 and anti-CD3+anti-CD28, concentrations of IL-1β, IL-5, IL-6 (see Figure 1), IL-13, IL-17A, IFNγ and TNFα were markedly decreased in samples from septic patients. In addition, stimulation with anti-CD3+anti-CD28 resulted in lower production of IL-10 in septic patients. Lower concentrations of IL-8 were detected in septic patient samples stimulated with only anti-CD3. We found cytokine levels of IL-12p70 remained unaff ected across all groups and stimuli. Results Regarding cardiac surgery, patients with clusters JT and haplogroup J had higher likelihoods of sepsis than patients with clusters HV (OR = 2.76 (95% CI = 1.27; 6.02); P = 0.010) and haplogroup H (OR = 3.68 (95% CI = 1.17; 11.54); P = 0.026), respectively. No signifi cant association was found for abdominal surgery. When analyzing survival, 45.4% patients died with a survival median of 39 (95% CI = 31.4; 46.62) days. When the clusters were analyzed for all patients, 41% (55/134) of patients within cluster HV died versus 71.4% (10/14) patients within cluster IWX (P = 0.018). The adjusted Cox regression showed that patients within cluster IWX had a higher risk of dying than patients within cluster HV (hazard ratio (HR) = 2.24 (95% CI = 1.10; 4.56); P = 0.027). No signifi cant association between haplogroups and mortality was found when patients were stratifi ed by the type of surgery. Conclusion We demonstrated a proinfl ammatory cytokine profi le in blood from septic patients, preceding a pan downregulation of all assessed cytokines following in vitro T-cell stimulation. To our knowledge, this study is the fi rst to perform an immune functional assay across these three groups. Conclusion European mitochondrial haplogroups are associated with sepsis development in patients who underwent major cardiac surgery, but not in patients who underwent major abdominal surgery. P225 T-cell receptor activation-associated cytokine release is impaired in septic patients with faecal peritonitis DG Gore1, L Preciado-Llanes2, GH Mills1, AW Heath2, RC Read3 1Sheffi eld Teaching Hospitals NHS Foundation Trust, Sheffi eld, UK; 2The University of Sheffi eld, UK; 3University of Southampton, UK Critical Care 2014, 18(Suppl 1):P225 (doi: 10.1186/cc13415) g Reference 1. Yang Y, Shou Z, Zhang P, He Q, Xiao H, Xu Y, Li C, Chen J: Mitochondrial DNA haplogroup R predicts survival advantage in severe sepsis in the Han population. Genet Med 2008, 10:187-192. Introduction Activated protein C (APC) defi ciency is prevalent in severe sepsis and septic shock patients. The aim of the study was to relate the anticoagulation activity evaluated by APC with other coagulation parameters adjusted to 28-day mortality. P224 Besides, mtDNA haplogroups also infl uence mortality. Haplogroups HV and H were related to low risk of sepsis and death, while JT and J were related to high risk of sepsis, and IWX was associated with death. P226 Activated protein C consumption and coagulation parameters in severe sepsis and septic shock M De La Torre-Prados1, A Garcia-de la Torre2, C Trujillano-Fernandez1, J Perez-Vacas1, A Puerto-Morlan1, E Camara-Sola1 1Hospital Virgen de la Victoria, Málaga, Spain; 2University Hospital Puerto Real, Cadiz, Spain Critical Care 2014, 18(Suppl 1):P226 (doi: 10.1186/cc13416) Activated protein C consumption and coagulation parameters in severe sepsis and septic shock M De La Torre-Prados1, A Garcia-de la Torre2, C Trujillano-Fernandez1, J Perez-Vacas1, A Puerto-Morlan1, E Camara-Sola1 1Hospital Virgen de la Victoria, Málaga, Spain; 2University Hospital Puerto Real, Cadiz, Spain Critical Care 2014, 18(Suppl 1):P226 (doi: 10.1186/cc13416) T-cell receptor activation-associated cytokine release is impaired in septic patients with faecal peritonitis Methods A cohort study of 150 critically ill adults. Age, sex, sources of infection and coagulation markers within 24 hours from severe sepsis or septic shock onset, defi ned according to Surviving Sepsis Campaign (SSC) criteria, were studied. We analyzed APC activity using a hemostasis laboratory analyzer (BCS® XP; Siemens). A descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Introduction Sepsis is associated with immune hyporesponsiveness but the immunological processes behind this are ill defi ned.if Introduction Sepsis is associated with immune hyporesponsiveness but the immunological processes behind this are ill defi ned.if g Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) admitted to the UCI. The median age of the study sample was 64 (interquartile range (IQR): 22.3 years; male: 60%). The main sources of infection were: respiratory tract 38%, intra-abdomen i Methods This study quantifi ed diff erences in plasma concentrations of cytokines between septic patients with faecal peritonitis, age and gender-matched surgical patients (without sepsis) and age and gender-matched healthy participants. In addition, cytokine levels Figure 1 (abstract P225). Concentration of IL-6 in plasma on day 0 (a) and supernatant on day 4 (b). igure 1 (abstract P225). Concentration of IL-6 in plasma on day 0 (a) and supernatant on day 4 (b). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 S81 Figure 1 (abstract P226). Relationship between activated protein C and antithrombin III and INR. Figure 1 (abstract P226). Relationship between activated protein C and antithrombin III and INR. Table 2 (abstract P227). Flow-cytometric data Normal value WBC (×109/l) 10.67 ± 4.82 4 to 10 Total lymphocytes (×106/l) 1,123.15 ± 547.02 1,200 to 3,000 CD3+ lymphocytes (×106/l) 67.11 ± 11.86 1,100 to 1,700 CD3+CD4+ lymphocytes (×106/l) 493.07 ± 313.92 600 to 1,400 CD3+CD8+ lymphocytes (×106/l) 250.65 ± 171.94 300 to 900 CD4+DR+ lymphocytes (×106/l) 23.19 ± 15.09 CD8+DR+ lymphocytes (×106/l) 16.26 ± 20.83 Table 2 (abstract P227). Flow-cytometric data Table 2 (abstract P227). Flow-cytometric data 45%, and 70.7% had medical pathology. The 28-day mortality was 22.7%. Nonsurvivors had a signifi cantly higher consumption of APC than survivors, 56% (IQR: 38.5) versus 68.6 (IQR: 41.4); P = 0.023. The profi le of lower levels of APC was a surgery patient with septic shock, neurological focus or catheter-related infection and Gram-negative pathogens from blood cultures. P228 Polymorphonuclear cell surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, Z Gueldue, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany Critical Care 2014, 18(Suppl 1):P228 (doi: 10.1186/cc13418) Table 1 (abstract P227). Collected data Age (years), mean ± SD 55.43 ± 23.12 Sex (M/F) 44/10 Body mass index, mean ± SD 25.51 ± 2.69 ISS, mean ± SD 29.04 ± 26.57 RTS, mean ± SD 5.76 ± 1.53 TRISS, mean ± SD 68.73 ± 28.77 Admission SAPS II, mean ± SD 44.96 ± 14.87 Admission GCS, mean ± SD 7.63 ± 3.88 Flow-cytometric analysis day (days), mean ± SD 1.69 ± 3.34 Discharge GCS, mean ± SD 10.48 ± 3.19 ICU LOS, mean ± SD 10.67 ± 8.08 Table 1 (abstract P227). Collected data T-cell receptor activation-associated cytokine release is impaired in septic patients with faecal peritonitis Spearman’s showed relationship with antithrombin III, r = 0.674 (P <0.001) and International Normalized Ratio (INR), r = –0.611 (P >0.001). See Figure 1. Conclusion Low levels of PC are associated with poor outcome and severity in severe sepsis, and it is well correlated with antithrombin III and INR. Flow-cytometric analysis in traumatic brain injury to evaluate immunosuppression Methods We retrospectively analyzed data from 54 patients, including 10 patients with isolated brain injury and 44 patients with brain and extracranial injuries. The fl ow-cytometric analysis was performed within 48 hours of trauma. Collected data are shown in Table 1. Flow-cytometric analysis in traumatic brain injury to evaluate immunosuppression S Di Valvasone, L Perretta, M Bonizzoli, F Liotta, F Annunziato, F Socci, P Ruggiano, A Peris Careggi Hospital, Firenze, Italy Critical Care 2014, 18(Suppl 1):P227 (doi: 10.1186/cc13417) pp S Di Valvasone, L Perretta, M Bonizzoli, F Liotta, F Annunziato, F Socci, P Ruggiano, A Peris Careggi Hospital, Firenze, Italy Critical Care 2014, 18(Suppl 1):P227 (doi: 10.1186/cc13417) Results Preliminary analysis is limited to descriptive statistics that show an immediate immunosuppression condition after TBI, as established by reduction of CD4+ T lymphocytes and CD8+ T lymphocytes. Signifi cant data are collected in Table 2. Introduction Flow-cytometric analysis is still restricted to cancer and immunocompromised patients. There are no clinical studies that evaluate the immunological changes in traumatic brain injury (TBI) patients. The objective of this study is to determine whether patients with severe TBI (GCS <9) manifest early (<48 hours after injury) signs of immunosuppression and whether this condition increases the incidence of infection during the ICU stay. gi Conclusion In severe TBI patients, an immunosuppression state is early developed. It is relevant to establish whether this condition could aff ect the course and prognosis of ICU patients. P228 Lymphocyte surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, L Brach, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany Critical Care 2014, 18(Suppl 1):P229 (doi: 10.1186/cc13419) Stewart’s strong ion theory can be used in the interpretations of acid– base abnormalities [1]. The Cl:Na ratio is a simple alternative test that obviates the need to solve the complex strong ion gap (SIG) equations [2]. y p y Critical Care 2014, 18(Suppl 1):P229 (doi: 10.1186/cc13419) Methods A total of 434 sepsis and severe sepsis patients hospitalized in the ICU of two centers between 2006 and 2012 were included in the study. Three groups were formed as patients having low (<0.75), normal (>0.75 to <0.80) and high (>0.80) Cl:Na ratio within the fi rst 24 hours in the ICU. Patients’ age, gender, APACHE II score, SOFA score, pH, PaCO2, HCO3, base excess, Na, K, Cl, Ca, lactate, strong ion diff erence, anion gap, length of ICU stay and mortality were recorded. Logistic regression analysis was used to calculate odd ratios and 95% CIs for the association of Cl:Na with mortality. In the fully adjusted model, pH, BE, AG, lactate and Cl:Na ratio were entered into the model. P <0.05 was considered statistically signifi cant. Introduction Lymphocytes show diff erent cell surface molecules depending on the degree of their activation or suppression. We used this surface expression to look at infl ammatory or infectious states in patients with polytrauma and patients with sepsis. The present study was performed to clarify: what is the expression profi le of lymphocyte surface markers in polytraumatized patients over time; and are there diff erences in the expression patterns of polytraumatized patients compared with healthy controls and with patients with severe sepsis and septic shock? Methods In a prospective observational study in a surgical ICU, surface expression of CD3, CD4, CD8, IL7R, CD4/25, CD8/25, CD2/86, CD28, CD3/56, CD2, CD39, CD244, CD11b, and CD56 on lymphocytes was measured by fl ow cytometry. We collected data from six healthy controls, from eight patients with severe sepsis or septic shock, and, longitudinally from eight polytraumatized patients on admission (0 hours), and at 4, 12, 24, 48, 120, and 240 hours. ISS, SAPS 3 and SOFA score refl ect severity of injury, of disease and of organ dysfunctions of the polytraumatized patients. yi Results The distribution of the patients was as follows: low Cl:Na (75, 17%), normal Cl:Na (243, 56%), high Cl:Na (116, 27%). References 1. Stewart PA: Can J Physiol Pharmacol 1983, 61:1441-1461. 2. Durward A, et al.: Intensive Care Med.2001, 5:828-835. Dysfunction of peroxisomes as one of the possible causes of multiple organ dysfunction syndrome development AN Osipenko1, AV Marochkov2 1A. Kuleshov Mogilev State University, Mogilev, Belarus; 2Mogilev Regional Hospital, Mogilev, Belarus Critical Care 2014, 18(Suppl 1):P231 (doi: 10.1186/cc13421) p g p Conclusion Surface expression molecules of polytraumatized patients diff er from those of the healthy controls and within the group. The more heavily injured polytraumatized patients are more likely to develop severe sepsis and septic shock with surface expression profi les on lymphocytes comparable with those of septic patients. Dysfunction of peroxisomes as one of the possible causes of multiple organ dysfunction syndrome development AN Osipenko1, AV Marochkov2 1A. Kuleshov Mogilev State University, Mogilev, Belarus; 2Mogilev Regional Hospital, Mogilev, Belarus Critical Care 2014, 18(Suppl 1):P231 (doi: 10.1186/cc13421) Table 1 (abstract P227). Collected data Introduction Each cell of the immune system shows a characteristic expression of cell surface molecules depending on the grade of activation or suppression. We examined the cell surface profi le on polymorphonuclear cells (PMN) of polytraumatized patients and compared them with those of healthy controls and furthermore with patients with severe sepsis or septic shock. Moreover, we wanted to fi nd out in the polytraumatized patients whether there are associations of PMN surface marker patterns with severity of injury, of disease and of organ dysfunctions. Methods In a prospective observational study, cell surface expression of CD16, CD88, CD64, CD66b, CD11b, CD95, CD33, CD39, IL-17RA, TLR- 2, TLR-4 and HLA-DR on PMN was determined using fl ow cytometry. Thirteen healthy volunteers, eight patients with severe sepsis and S82 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P229 Lymphocyte surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, L Brach, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany Critical Care 2014, 18(Suppl 1):P229 (doi: 10.1186/cc13419) Lymphocyte surface expression patterns diff er in infl ammatory and infectious stages in polytraumatized and septic shock patients M Weiss, L Brach, M Georgieff , F Gebhard, M Huber-Lang, M Schneider University Hospital Medical School, Ulm, Germany Critical Care 2014, 18(Suppl 1):P229 (doi: 10.1186/cc13419) Univariate analysis revealed that in low and high Cl:Na ratio patients, mortality was higher by 1.56-fold (0.87 to 2.81) and 2.22-fold (1.36 to 3.61) (P = 0.135 and P <0.01). In multivariate analysis, increased mortality by 1.95-fold (0.92 to 4.12) and 2.02-fold (1.01 to 4.03) was found in low and high Cl:Na ratios (P = 0.081 and P = 0.046) (Figure 1). Conclusion Stewart’s strong ion theory provides assessment for the etiology of acid–base disturbances. This evaluation can be performed easier and faster with the Cl:Na ratio. This study demonstrates that the disturbed Cl:Na ratio is associated with increased mortality in sepsis and severe sepsis patient groups. Results In septic and polytraumatized patients, the expression of CD3, CD28 and CD2 was signifi cantly lower than in controls. In polytraumatized patients, surface expression of CD244, CD39 and CD8/25 was signifi cantly higher than in controls. Within the polytraumatized patients, we found two diff erent subgroups, group A with septic progression and Group B without septic progression of disease. Especially, the septic polytraumatized patients revealed very low levels of CD3, CD2 and CD28. Moreover, their levels of CD39 and CD8/25 were less increased and their ISS higher than those of the nonseptic subgroup. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 septic shock, and longitudinally, on admission (0 hours), and 4, 12, 24, 48, 120 and 240 hours thereafter, eight polytraumatized patients were monitored. ISS, SAPS3 and SOFA score refl ect severity of injury, of disease and of organ dysfunctions. Figure 1 (abstract P230). In the multivariate model, eff ect of Cl:Na ratio group was adjusted for pH, lactate, anion gap and base excess. septic shock, and longitudinally, on admission (0 hours), and 4, 12, 24, 48, 120 and 240 hours thereafter, eight polytraumatized patients were monitored. ISS, SAPS3 and SOFA score refl ect severity of injury, of disease and of organ dysfunctions. g y Results In polytraumatized patients (24 hours measurement), but also in septic patients, CD88+ expression and CD33+/CD66b– expression were signifi cantly lower and CD33–/CD66b+ higher than in healthy controls. Results In polytraumatized patients (24 hours measurement), but also in septic patients, CD88+ expression and CD33+/CD66b– expression were signifi cantly lower and CD33–/CD66b+ higher than in healthy controls. Especially, the lowest values of CD88+ occurred in patients with polytrauma developing septic complications. signifi cantly lower and CD33–/CD66b+ higher than in healthy controls. Especially, the lowest values of CD88+ occurred in patients with polytrauma developing septic complications. Furthermore, the polytraumatized patients revealed higher levels of CD66b, CD11b and CD16, whereas CD16+ was lower. Considering the post-traumatic course, polytraumatized patients with better prognostic evaluation in SOFA and SAPS3 initially expressed higher levels of TLR-2. Polytrauma patients could be divided into two subgroups, one with and the other without septic complications. The septic subgroup presented predominantly higher values in SOFA, SAPS3 and ISS scoring. Conclusion Cell surface molecules on PMN of polytraumatized patients diff er from those of healthy volunteers. Besides, distinct expression patterns resemble those of patients with sepsis. Patents with higher ISS values go along with higher scores in SOFA and SAPS3, and are at risk to develop septic complications. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods Whole blood was taken from healthy volunteers and was placed in tubes containing EDTA and immediately transferred to the laboratory. Heparinized blood samples were diluted 1:10 in RPMI 1640 culture medium (100 μl whole blood added in 900 μl RPMI 1640). Samples were preincubated with 0‚ 5‚ 12.5‚ 25‚ 50‚ 100 and 200 mM alcohol (EtOH) for 10 minutes at room temperature. After incubation, 500 pg LPS was added to each sample for 4 hours at 37°C. At the end of the process, samples were centrifuged (1,800 rpm, 5 minutes, r.t.). Culture supernatants were collected and stored at –70°C until measurements. TNFα and TNFR levels were determined in culture supernatant using the ELISA method [2]. volunteers aged 37.7 ± 3.2 years served as control. The preanalytical phase was to prepare a solution of ethyl esters of fatty acids (FAc) and diethylacetals of fatty aldehydes (FAl) of blood plasma samples by acidic ethanolysis. Analysis of FAl and FAc in plasma was carried out using capillary gas–liquid chromatography. Quantitative evaluation of the analytes was performed as a mass percentage of the FA and FAl amount. Statistical analysis was performed using the Mann–Whitney U test (P <0.05). ( ) Results In contradistinction to triglycerides, diacylphospholipids and Pls participate in exchange of polyunsaturated fatty acids (PUFA) with a large number of double bonds, such as primarily arachidonic and docosahexaenoic PUFA, acting as an intermediate station, through which these fatty acids are transported to cell membranes [1]. Thus, the ratio of level of FAl to the level of aforementioned blood plasma PUFA may refl ect a change in the share of Pls relative to diacylphospholipids. According to our data, at MODS the ratio of fatty aldehydes to amount of arachidonic and docosahexaenoic PUFA is 0.16, while in the control group the fi gure is 0.27 (P <0.001). Thus, we can conclude that the level of Pls towards diacylphospholipid plasma levels of the experimental group decreased by approximately 40%. Currently, there is evidence that there is a reduction of cholesterol levels in blood plasma of patients with the diseases associated with peroxisome biogenesis disorders [2]. In our study, patients had low levels of plasma total cholesterol (124.6 ± 28.7 mg/dl), which may also indicate peroxisome dysfunction in MODS patients. p g [ ] Results We studied 24 healthy males volunteers aged 36.5 ± 1.4 (X ± SEM). P232f P232 Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo A Gavala1, K Venetsanou1, P Myrianthefs1, E Manolis2, C Kittas3, G Baltopoulos1 1ICU ‘Agioi Anargyroi’ General Hosrital, School of Nursing, Athens University, Athens, Greece; 2Dentistry School, Athens University, Athens, Greece; 3Medical School, Athens University, Athens, Greece Critical Care 2014, 18(Suppl 1):P232 (doi: 10.1186/cc13422) Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo y References References 1. Braverman N, Moser A: Bioch Biophys Acta 2012, 1822:1442-1452. 2. Kovacs W, et al.: J Mol Cell Biol 2004, 24:1-13. P233 Neutrophil phenotype model for extracorporeal treatment of sepsis A Malkin1, R Sheehan1, S Mathew1, H Redl2, G Clermont1 1University of Pittsburgh, PA, USA; 2Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria Critical Care 2014, 18(Suppl 1):P233 (doi: 10.1186/cc13423) References 1. Cook RT‚ et al.: Alcohol Clin Exp Res 1998, 22:1927. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. 1. Cook RT‚ et al.: Alcohol Clin Exp Res 1998, 22:1927. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 TNFα was not detected in samples treated without alcohol in the absence of LPS stimulation (control) or in the presence of alcohol alone (data not shown). TNFα production was signifi cantly decreased at a dose of 25 mM alcohol after LPS stimulation (P <0.0001) compared with LPS-challenged samples (Figure 1A). Alcohol had no eff ect on TNFR I production when incubated with or without LPS (data not shown). Alcohol at lower doses (<50 mM) seemed to decrease TNFR II levels, but an increase in TNFR II levels was observed at higher doses (>50 mM) of alcohol, which was statistically signifi cant at doses of 100 and 200 mM alcohol after LPS stimulation ex vivo (P <0.001) (Figure 1B). Conclusion Our observations indicate a suppression of pro infl am- matory response, but also a diff erential eff ect of alcohol on TNFR II production of whole blood in the presence of LPS challenge depending on the degree of alcohol intoxication. Conclusion On the basis of determined defi ciency of analyzed compound levels, we concluded the possibility of peroxysomal dysfunction in MODS. Cl:Na ratio on ICU admission as a prognostic indicator of mortality in sepsis patients Cl:Na ratio on ICU admission as a prognostic indicator of mortality in sepsis patients HK Atalan1, B Gucyetmez2, N Cakar3 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Istanbul Medical Faculty, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P230 (doi: 10.1186/cc13420) Introduction This study demonstrates that low level of plasmalogens (Pls) is an important marker of peroxisomal dysfunction. The primary- OH group in glycerol Pls was not substituted by the acyl group (fatty acid) as in diacylphospholipids but by the aldehydogenic alkenyl group (fatty aldehyde) found in the form of vinyl ether [1]. It is known that there is disruption of several organ systems in diseases connected with peroxisomal dysfunction, as in the case of multiple organ failure. Cl:Na ratio on ICU admission as a prognostic indicator of mortality in sepsis patients HK Atalan1, B Gucyetmez2, N Cakar3 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Istanbul Medical Faculty, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P230 (doi: 10.1186/cc13420) HK Atalan1, B Gucyetmez2, N Cakar3 1Atasehir Memorial Hospital, Istanbul, Turkey; 2International Hospital, Istanbul, Turkey; 3Istanbul Medical Faculty, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P230 (doi: 10.1186/cc13420) Introduction The aim of the study is to investigate the relationship between Cl:Na ratio disturbances and mortality in sepsis patients. Introduction The aim of the study is to investigate the relationship between Cl:Na ratio disturbances and mortality in sepsis patients. p y p g Methods The objects of study were 18 people with multiple organ failure (35.6 ± 8.7 years) of various etiologies. The blood of 16 healthy S83 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data K Vardas1, K Apostolou1, K Psarra2, E Botoula2, S Tsagarakis2, E Magira1, C Routsi1, E Briassouli1, D Goukos1, S Nanas1, G Briassoulis3 1Medical School, NKUA, Athens, Greece; 2Evangelismos Hospital, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2014, 18(Suppl 1):P234 (doi: 10.1186/cc13424) Results AI prevented Cr, BUN and CysC from increasing after CLP (0.43 ± 0.18, 0.2 ± 0.02, P = 0.0003; 62.2 ± 10.8, 43.7 ± 17.7, P = 0.01; 103.9 ± 54.8, 49.3 ± 30.8, P = 0.01, respectively). AICAR decreased cytokine release after CLP (IL-6: 1,818 ± 344 vs. 1,374 ± 268, P = 0.04; IL- 10: 7,592 ± 5,038 vs. 2,245 ± 2,668, P = 0.05; TNFα: 213 ± 172 vs. 39 ± 16, P = 0.03) and LPS in macrophage culture (IL-6: 241.7 ± 1.5 vs. 7.2 ± 1.5, P = 0.055; TNF: 1,882 ± 583 vs. 82 ± 28, P = 0.04). However, AI lost its protective signal when administered 4 hours pre or 2 hours after CLP (Cr CLP vs. CLP+AI: 0.2 vs. 0.2, P = 0.1 and 0.45 ± 0.3 vs. 0.56 ± 0.3, P = 0.5, respectively). AI also decreased ICAM-1 expression in vivo and in vitro, vascular leak (0.09 ± 0.01 vs. 0.06 ± 0.008, P = 0.003) and PMN adhesion (1,055 ± 179 vs. 751 ± 242, P = 0.04). Introduction Besides cortisol, prolactin might also be important in maintaining normal immune response in stress states. Its role in the hypothalamus–pituitary–adrenal axis hormonal and immune stress response in sepsis has hardly been investigated in a critically ill setting. The aims of the study were to evaluate the early (fi rst 48 hours) ACTH, cortisol and prolactin plasma levels in ICU patients with severe sepsis/ septic shock (SS) or systemic infl ammatory response syndrome (SIRS) compared with healthy control subjects (C) and to correlate their expression with heat shock proteins (HSPs), interleukins (ILs) and outcome. Conclusion AMPK activation by AICAR prevented sepsis-induced AKI. AICAR decreased cytokine release, endothelial activation and vascular leak, suggesting that organ protection may be mediated by modulation of the infl ammatory response and protection of the microvasculature. However, AICAR’s protective eff ect may be conditional to timing of administration. Methods Thirty consecutively admitted patients with SS, 22 with SIRS, and 15 C were enrolled in the study. P235 AMP-protein kinase may protect against sepsis-induced acute kidney injury through modulation of immune response and endothelial activation AMP-protein kinase may protect against sepsis-induced acute kidney injury through modulation of immune response and endothelial activation H Gomez, D Escobar, A Botero, BS Zuckerbraun University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P235 (doi: 10.1186/cc13425) p p y Results Of 16 baboons, 11 (69%) died, six (38%) within 1 day of bacterial infusion. The model was well calibrated to the data from survivors and nonsurvivors. Sensitivity analysis identifi ed six model parameters, out of 21, as key determinants of mortality. Predictions indicate best eff ect with introduction of the proposed extracorporeal intervention within 1 hour of infection for a 72-hour duration, results in the survivor population increasing from 31% to 61%. The treatment can result in harm if initiated <10 hours from infection and continued <24 hours. A delay of 10 hours increases survival by 7% on average. Treatment effi cacy quickly diminishes if not introduced within 15 hours of infection. H Gomez, D Escobar, A Botero, BS Zuckerbraun University of Pittsburgh, PA, USA Introduction Organ injury is a hallmark of sepsis, in particular acute kidney injury (AKI). Yet the mechanisms involved in sepsis-induced AKI are not well understood. Energy prioritization is an important cell defense mechanism, and thus we hypothesized that exogenous activation of AMP-protein kinase (AMPK), a master regulator of cellular energy metabolism, protects against sepsis-induced AKI. Conclusion These fi ndings support the continued development of an extracorporeal treatment that modulates CXCR-1/2 levels. Further development of the mathematical model will help guide device development and determine which patient populations should be targeted by treatment. gy p g p Methods Sixty C57BL/6 male mice, 6 to 8 weeks of age, weighing 20 to 25 g were divided into six groups: 1, cecal ligation and puncture (CLP); 2, CLP+AICAR (AI, AMPK activator, 100 mg/kg 24 hours before CLP); 3, CLP+compound C (AMPK inhibitor, 30 mg/kg; CoC); 4, sham; 5, sham+AI; 6, sham+CoC. Blood/tissue samples were collected 8 hours after CLP. Renal function (creatinine (Cr, mg/dl), BUN (mg/dl) and cystatin C (CysC, ng/ml)), cytokine expression (ELISA), endothelial activation (ICAM- 1 expression), neutrophil adhesion (PMN, fl uorescence, anti-CD11b mAb-tagged PMNs) and vascular leak (Evan’s blue) were assessed. The eff ect of AI given 4 hours (n = 12) before and 2 hours after (n = 11) CLP was also evaluated. Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo A Gavala1, K Venetsanou1, P Myrianthefs1, E Manolis2, C Kittas3, G Baltopoulos1 p 1ICU ‘Agioi Anargyroi’ General Hosrital, School of Nursing, Athens University, Athens, Greece; 2Dentistry School, Athens University, Athens, Greece; 3Medical School, Athens University, Athens, Greece Critical Care 2014, 18(Suppl 1):P232 (doi: 10.1186/cc13422) Introduction Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage. Interleukin-8 (IL-8), a key modulator of neutrophil function, signals through neutrophil specifi c surface receptors CXCR-1 and CXCR-2. Expression of these surface receptors can be altered by perfusion through an extracorporeal device. Extracorporeal methods of immune modulation have shown promise for treatment of sepsis; however, achieving an appropriate response is a major challenge [1]. In this study a mechanistic mathematical model was used to evaluate and deploy an extracorporeal sepsis treatment that modulates CXCR-1/2 levels. Introduction Acute alcohol exposure suppresses proinfl ammatory response, which may be related to increased susceptibility to infections [1].The purpose of the study was to investigate the eff ect of acute exposure to alcohol on TNFα production capacity and TNFα receptors (TNFRs) in an ex vivo model of whole-blood stimulation with lipopolysaccharide (LPS). Figure 1 (abstract P232). Figure 1 (abstract P232). S84 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A simplifi ed mechanistic mathematical model of IL-8- mediated activation of CXCR-1/2 receptors was developed. Receptor-level dynamics and systemic parameters are coupled with multiple neutrophil phenotypes to generate dynamic populations of activated neutrophils that reduce pathogen load, and/or primed neutrophils that cause adverse tissue damage when misdirected. The mathematical model was calibrated using experimental data from baboons administered a 2-hour infusion of E. coli and followed for a maximum of 28 days. An extracorporeal intervention was implemented by introducing a trapped receptor state that limits IL-8 signaling through CXCR-1/2. Time of onset, duration, and capture effi cacy of the extracorporeal device were explored to provide probabilistic predictions of the impact on mortality. Conclusion Prolactin seems to be a stress hormone, probably related to the severity of illness, increased along with IL-6 and eHSP90 levels in SS. Also in these patients, cortisol correlates to lactate and eHSP90, but in contrast to SIRS the iHSP72 and iHSP90 immune response is depressed. P234 Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data K Vardas1, K Apostolou1, K Psarra2, E Botoula2, S Tsagarakis2, E Magira1, C Routsi1, E Briassouli1, D Goukos1, S Nanas1, G Briassoulis3 1Medical School, NKUA, Athens, Greece; 2Evangelismos Hospital, Athens, Greece; 3University Hospital, University of Crete, Heraklion, Greece Critical Care 2014, 18(Suppl 1):P234 (doi: 10.1186/cc13424) Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data Prolactin, cortisol and heat shock proteins in early sepsis: preliminary data Patients’ demographics, laboratory examinations, and APACHE II, SOFA and SAPS III scores were recorded on ICU admission. Blood sampling was performed between 9:00 and 10:00 a.m. Prolactin and cortisol were measured using the ADVIA centaur system, ACTH using the Immulite 2000. Intracellular monocyte HSP (iHSP) was determined after staining with surface antigens CD33– PE/Cy5 and CD45 PE/Cy7 followed by either HSP72-FITC or HSP90a-PE intracellular staining (four-color fl ow cytometry). Mean fl uorescence intensity values for each HSP were noted and analyzed. ELISA was used to determine extracellular (e) HSP, IL-6 and IL-10 levels. Diff erential eff ect of alcohol on TNFα receptor II production in the presence of LPS challenge ex vivo Acknowledgements This research has been co-fi nanced by the European Union (European Social Fund) and Greek national funds through the Operational Program ‘Education and Lifelong Learning’ of the National Strategic Reference Framework Research Funding Program: THALES. Reference Reference Reference 1. Peng Z-Y, et al.: Kidney Int 2012, 81:363-369. 1. Peng Z-Y, et al.: Kidney Int 2012, 81:363-369. P235 AMP-protein kinase may protect against sepsis-induced acute kidney injury through modulation of immune response and endothelial activation The experiment was reproduced in vitro using cell culture (renal epithelial, endothelial cells and macrophages), with similar groups: 1, control; 2, control+AI (1 mM/1 hour pre LPS); 3, LPS (100 ng/ml for 4 hours); 4, LPS+AI; 5, control+CoC (10 μM/1 hour pre LPS); 6, LPS+CoC. Cytokines were measured with ELISA. Data are presented as mean ± SD and as LPS/CLP and LPS/CLP+AI. P236 The same procedures were done for sham-operated mice and for mice subject only to femur crush and to pneumothorax. Figure 2 (abstract P236). Ex vivo proinfl ammatory cytokine release after whole-blood LPS stimulation in older patients. with a group with arterial disease, expressed by proinfl ammatory cytokine release after ex vivo whole-blood LPS stimulation [2]. with a group with arterial disease, expressed by proinfl ammatory cytokine release after ex vivo whole-blood LPS stimulation [2]. Methods The study comprised 16 polytrauma patients admitted to the ICU, aged 78 ± 8 (Group I) and 16 with arterial disease, aged 74 ± 5 (Group II). Ten milliliters of peripheral blood were collected from each patient, divided into two tubes with/without anticoagulant. Diluted 1:10 whole-blood samples were stimulated with 500 pg/ml LPS, at 37°C, for 4 hours. Serum and cell culture supernatants (CCSP) were removed and stored at –70°C. TNFα and IL-6 were measured in serum and CCSP by ELISA. j y p Results Initial experiments with 21 mice showed that the overall death rate for this model of multitrauma was 66.7% with most deaths occurring in the fi rst 48 hours. In the second set of experiments, 12 mice remaining alive 72 hours post injury were challenged with P. aeruginosa; mortality was 37.5% compared with 75% of 12 non- injured and infected mice (log-rank: 5.77, P = 0.016). Respective mean production of TNFα from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 651 and 523 pg/ml (P = NS); post femur crush 217 and 916 pg/ml (P = 0.010); post pneumothorax 286 and 1,056 pg/ml (P = 0.018); and post both femur crush and pneumothorax 207 and 1,011 pg/ml (P = 0.019). Respective mean production of IFNγ from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 324 and 230 pg/ml (P = NS); post femur crush 278 and 840 pg/ml (P = 0.021); post pneumothorax 377 and 2,356 pg/ml (P = 0.025); and post both femur crush and pneumothorax 252 and 908 pg/ml (P = 0.019). Mean production of TNFα from splenocytes of injured mice sacrifi ced 24 hours after bacterial challenge was 1,184 pg/ ml compared with 484 pg/ml non-injured and infected mice (P = 0.024). Similar diff erences were not found for IFNγ or for quantitative tissue cultures. See Figure 1. p References References 1. Bruno L: Proc Nutr Soc 1999, 58:85-98. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. 1. Bruno L: Proc Nutr Soc 1999, 58:85-98. 2. Myrianthefs P, et al.: Cytokine 2003, 24:286-292. P236 Methods The study comprised 16 polytrauma patients admitted to the ICU, aged 78 ± 8 (Group I) and 16 with arterial disease, aged 74 ± 5 (Group II). Ten milliliters of peripheral blood were collected from each patient, divided into two tubes with/without anticoagulant. Diluted 1:10 whole-blood samples were stimulated with 500 pg/ml LPS, at 37°C, for 4 hours. Serum and cell culture supernatants (CCSP) were removed and stored at –70°C. TNFα and IL-6 were measured in serum and CCSP by ELISA. y Results Serum proinfl ammatory cytokines were signifi cantly elevated after severe trauma against control group (TNFα, P <0.001 and IL-6, P <0.001). Ex vivo cytokine release showed the opposite direction. There was a signifi cantly lower TNFα and IL-6 release for Group I (TNFα, P <0.05 and IL-6, P <0.01) compared with Group II. TNFα ex vivo release from the samples of Group II was >300 pg/ml. See Figures 1 and 2. Conclusion Older patients showed adequate immunological response, considering the limit of 300 pg/ml. The incidence of severe trauma was involved in the downregulation of immune activity and should be considered. Group I patients do not have the opportunity to precondition their immune status. Group II patients can better compensate operative therapies. Conclusion Mice survivors from multiple trauma become resistant to subsequent infection. This is probably linked with the induction of tolerance of the innate immunity to substances released after sterile tissue injury. P236 Study of the ex vivo immune response of polytrauma older patients in the ICU on admission: preliminary results L Filippou, K Venetsanou, G Voulalas, D Markopoulou, D Chroni, C Maltezos, I Alamanos KAT Hospital Athens, Kifi sia, Athens, Greece Critical Care 2014, 18(Suppl 1):P236 (doi: 10.1186/cc13426) Results Prolactin (P <0.034), cortisol (P <0.0001), and ACTH (P <0.02) levels diff ered among groups. Prolactin, cortisol, IL-6, and eHSP90 were higher in SS (P <0.03) and SIRS (P <0.04) compared with C. Cortisol and eHSP90 were higher in SS compared with SIRS (P <0.005; P <0.03); iHSP72 and iHSP90 were higher in SIRS compared with SS (P <0.05; P <0.05) or C (iHSP72, P <0.02). Cortisol related with eHSP90 (r = 0.45, P <0.02), lactate (r = 0.33, P <0.02) and HCO3 (r = –0.45), and prolactin with SAPS III (r = 0.34, P <0.02), but not with mortality or LOS. Introduction Immunological status is diff erentiated with age, infl uencing treatment and outcome [1]. The aim is to determine the immune response of severely traumatized older patients compared Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S85 Figure 1 (abstract P236). Serum proinfl ammatory cytokines in older patients. Figure 2 (abstract P236). Ex vivo proinfl ammatory cytokine release after whole-blood LPS stimulation in older patients. Figure 1 (abstract P236). Serum proinfl ammatory cytokines in older patients. Figure 1 (abstract P237). Cytokine stimulations after sterile injury. Figure 1 (abstract P236). Serum proinfl ammatory cytokines in older patients. Figure 2 (abstract P236). Ex vivo proinfl ammatory cytokine release after whole-blood LPS stimulation in older patients. Methods Ninety-seven C56Bl6 male mice were subject to multitrauma after crush of the femur and chemical pneumothorax by turpentine. Mice surviving 72 hours after the injuries were challenged intravenously with one 7log10 log-phase inoculum of P. aeruginosa and survival was recorded. In separate experiments, mice were sacrifi ced post injury; splenocytes were isolated and stimulated with 10 ng/ ml LPS and cytokines were measured in supernatants by an enzyme immunoassay. Quantitative cultures of the right lung, kidney and liver were performed. The same procedures were done for sham-operated mice and for mice subject only to femur crush and to pneumothorax. Results Initial experiments with 21 mice showed that the overall death rate for this model of multitrauma was 66.7% with most deaths occurring in the fi rst 48 hours. P236 In the second set of experiments, 12 mice remaining alive 72 hours post injury were challenged with P. aeruginosa; mortality was 37.5% compared with 75% of 12 non- injured and infected mice (log-rank: 5.77, P = 0.016). Respective mean production of TNFα from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 651 and 523 pg/ml (P = NS); post femur crush 217 and 916 pg/ml (P = 0.010); post pneumothorax 286 and 1,056 pg/ml (P = 0.018); and post both femur crush and pneumothorax 207 and 1,011 pg/ml (P = 0.019). Respective mean production of IFNγ from splenocytes isolated at sacrifi ce 24 hours and 96 hours post sham injury was 324 and 230 pg/ml (P = NS); post femur crush 278 and 840 pg/ml (P = 0.021); post pneumothorax 377 and 2,356 pg/ml (P = 0.025); and post both femur crush and pneumothorax 252 and 908 pg/ml (P = 0.019). Mean production of TNFα from splenocytes of injured mice sacrifi ced 24 hours after bacterial challenge was 1,184 pg/ ml compared with 484 pg/ml non-injured and infected mice (P = 0.024). Similar diff erences were not found for IFNγ or for quantitative tissue cultures. See Figure 1. Methods Ninety-seven C56Bl6 male mice were subject to multitrauma after crush of the femur and chemical pneumothorax by turpentine. Mice surviving 72 hours after the injuries were challenged intravenously with one 7log10 log-phase inoculum of P. aeruginosa and survival was recorded. In separate experiments, mice were sacrifi ced post injury; splenocytes were isolated and stimulated with 10 ng/ ml LPS and cytokines were measured in supernatants by an enzyme immunoassay. Quantitative cultures of the right lung, kidney and liver were performed. The same procedures were done for sham-operated mice and for mice subject only to femur crush and to pneumothorax. Methods Ninety-seven C56Bl6 male mice were subject to multitrauma after crush of the femur and chemical pneumothorax by turpentine. Mice surviving 72 hours after the injuries were challenged intravenously with one 7log10 log-phase inoculum of P. aeruginosa and survival was recorded. In separate experiments, mice were sacrifi ced post injury; splenocytes were isolated and stimulated with 10 ng/ ml LPS and cytokines were measured in supernatants by an enzyme immunoassay. Quantitative cultures of the right lung, kidney and liver were performed. Tissue oxygenation in patients with severe sepsis A Oei1, M Kok2, A Karakus3, H Endeman4 Tissue oxygenation in patients with severe sepsis A Oei1 M Kok2 A Karakus3 H Endeman4 1Academic Medical Center, Amsterdam, the Netherlands; 2University Medical Center, Utrecht, the Netherlands 3Diakonessenhuis, Utrecht, the Netherlands; 4Onze Lieve Vrouwen Gasthuis, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P240 (doi: 10.1186/cc13430) Introduction The aim of this study was to determine the correlation between regional tissue oxygenation (SrO2) measured by near-infrared spectroscopy (NIRS), central venous oxygen saturation (SvO2) and levels of serum lactate (SL) in patients with severe sepsis. Methods An observational pilot study was performed in an ICU of a medium-sized teaching hospital. Adult patients admitted with severe sepsis were included and three NIRS electrodes were attached: on the left side of the forehead (LF), the right side of the forehead (RF) and the right forearm (RA). SL and SvO2 measured in a jugular or subclavian central venous line sample were determined every 4 hours. Descriptive statistics were calculated. Pearson correlation coeffi cients (PCC) were calculated between SrO2, SvO2 and SL. Normal values for SrO2 were defi ned as the median ± 1 interquartile range in patients with SL <1.7 mmol/l. Diff erences between groups were calculated by Pearson chi-square tests. P <0.05 was considered statistically signifi cant.i f Conclusion While clopidogrel may not increase the risk of bleeding in these patients, these data suggest that clopidogrel does not reduce adverse outcomes in patients with sepsis. A potential benefi t in patients with APACHE II scores ≥25 may need further study. Future research into the mechanism of ticagrelor’s benefi t reported in the PLATO trial may be directed at the non-P2Y12 receptor mechanism. q y gi Results Twenty-fi ve patients (12 men) were included. Mean age was 68 years, mean APACHE score 31. The most frequent reasons for ICU admission were abdominal sepsis (n = 11) and pneumosepsis (n = 7). Statistically signifi cant correlations were found between SvO2 and SrO2: PCC SrO2 LF, 0.46; PCC SrO2 RF, 0.50; PCC SrO2 RA, 0.21. Low, although statistically signifi cant, correlations were found between SrO2 and SL: PCC SrO2 LF, –0.16; PCC SrO2 RF, –0.15; and PCC SrO2 RA, –0.20. Calculated normal values for SrO2 LF were 60 to 80%, for SrO2 RF were 60 to 76% and for SrO2 RA were 64 to 84%. An out-of-range SrO2 had a high positive predictive value (PPV) for an increased SL. P239 P239 Continuous administration of corticosteroids in septic shock can reduce risk of hypernatremia L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, I Grintescu1, D Mirea3 1Clinical Emergency Hospital of Bucharest, Romania; 2CI Parhon National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P239 (doi: 10.1186/cc13429) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P = 0.0041) and slightly higher when HHS was delivered as a bolus (RR 3.08, 1.32 <OR <7.25, P = 0.0071). P = 0.0041) and slightly higher when HHS was delivered as a bolus (RR 3.08, 1.32 <OR <7.25, P = 0.0071). reduction in sepsis-related mortality and pulmonary adverse events compared with clopidogrel. It is unknown whether clopidogrel also provides benefi t in patients with sepsis compared with no therapy. Knowing this information may help focus future research on determining the mechanism of ticagrelor’s benefi t. reduction in sepsis-related mortality and pulmonary adverse events compared with clopidogrel. It is unknown whether clopidogrel also provides benefi t in patients with sepsis compared with no therapy. Knowing this information may help focus future research on determining the mechanism of ticagrelor’s benefi t. Conclusion Continuous administration of hydrocortisone hemi- succinate in septic shock is associated with a lower risk of hypernatremia than bolus administration. References Methods This retrospective cohort study included all patients over the age of 40 with a confi rmed diagnosis of severe sepsis and an ICU stay at our academic medical center from 1 January 2005 to 31 March 2011. Clopidogrel use, patient demographics, and APACHE II score at the time of sepsis were collected from patient charts. Clinical outcomes included in hospital mortality, development of acute respiratory distress syndrome (ARDS), days on mechanical ventilation, in-hospital cardiac events, hospital cost, and length of stay.i 1. Sprung CL, Annane D, Keh D, et al.: Hydrocortisone therapy f ith ti h k N E l J M d 2008 358 111 124 1. Sprung CL, Annane D, Keh D, et al.: Hydrocortisone therapy for patients with septic shock. N Engl J Med 2008, 358:111-124. 1. Sprung CL, Annane D, Keh D, et al.: Hydrocortisone therapy for patients with septic shock. N Engl J Med 2008, 358:111-124. 2. Annane D, Bellissant E, Bollaert PE, et al.: Corticosteroids in the treatment of severe sepsis and septic shock in adults: a systematic review. JAMA 2009, 301:2362. 2. Annane D, Bellissant E, Bollaert PE, et al.: Corticosteroids in the treatment of severe sepsis and septic shock in adults: a systematic review. JAMA 2009, 301:2362. Continuous administration of corticosteroids in septic shock can reduce risk of hypernatremia L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, I Grintescu1, D Mirea3 L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, I Grintescu1, D Mirea3 1Clinical Emergency Hospital of Bucharest, Romania; 2CI Parhon National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania 1Clinical Emergency Hospital of Bucharest, Romania; 2CI Parhon National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P239 (doi: 10.1186/cc13429) Introduction Although the administration of hydrocortisone in septic shock generates adverse eff ects, the risk of corticosteroid-induced hypernatremia may be reduced by continuous administration of the drug [1,2]. g Methods A total of 171 patients with septic shock were randomized into three study groups: group A (n = 58), 200 mg/day hydrocortisone hemisuccinate in four doses; group B (n = 59), same dose of hydrocortisone hemisuccinate in continuous administration; group C (n = 54), no hydrocortisone hemisuccinate. Mean serum sodium values, the number of hypernatremia episodes and variations in serum sodium (Na var) were investigated for 7 days. The local ethics committee approved the study. Conclusion This pilot study shows a statistically signifi cant correlation between SvO2 and SrO2 in patients with severe sepsis. An out-of-range SrO2 LF or RF had a high predictive value for increased serum lactate. We therefore conclude that NIRS could have a role in goal-directed therapy of patients with severe sepsis. Tissue oxygenation in patients with severe sepsis A Oei1, M Kok2, A Karakus3, H Endeman4 The PPV for out-of-range SrO2 LF was 85% versus 58% for normal SrO2 LF (OR 4.27; 95% CI 2.09 to 8.72), the PPV for out-of-range SrO2 RF was 77% versus 60% for normal SrO2 RF (OR 2.32; 95% CI 1.25 to 4.31) and the PPV for out-of-range SrO2 RA 75% versus 60% for normal SrO2 RA (OR 1.94; 95% CI 0.95 to 4.00).i P240 Results We identifi ed 824 patients who met the inclusion criteria for this study. Of these patients, 76 (9.2%) had been exposed to clopidogrel. The mean APACHE II score was similar for patients receiving clopidogrel and those who did not (23.4 vs. 22.6; P = 0.426). Patients exposed to clopidogrel had a similar rate of in-hospital mortality (26.3% vs. 33.2%; P = 0.223) and ARDS (43.4% vs. 38.6%; P = 0.415). While mortality was also similar between the groups for patients with a low APACHE II score (<25), mortality was lower in clopidogrel-exposed patients with an APACHE II score ≥25 (27.3% vs. 45.6%; P = 0.045). Patients exposed to clopidogrel did not have a higher use of blood products (65.8% vs. 65.9%; P = 0.983). Patients exposed to clopidogrel did not have signifi cantly more days on mechanical ventilation, or ventilation-free days. Cardiac events during the hospital stay were not lower in patients on clopidogrel (4.0 vs. 2.4; P = 0.432). Hospital costs and length of stay did not diff er between the groups. P241 y Results There were no diff erences between the three groups at the beginning of the study regarding demographic data and the clinical characteristics. Mean values of natremia were normal in group C (140.35 ± 7.390 mEq/l to 144.79 ± 8.338 mEq/l) during the study period. High mean values appeared on day 4 in group A (147.21 ± 8.470 mEq/l to 149.37 ± 8.973 mEq/l on day 7) and on day 5 in group B (146.36 ± 8.272 mEq/l to 147.70 ± 8.865 mEq/l). Na var was 8.59 ± 5.960 mEq/l (–8 and 21 mEq/l) in group A, 6.63 ± 7.609 mEq/l (–17 and 23 mEq/l) in group B and 4.54 ± 7.455 mEq/l (–12 and 22 mEq/l) in group C. This variation is statistically signifi cant when groups A and B are compared with group C (P = 0.012) and when only group A is compared with group C (P = 0.0019). The risk of hypernatremia after hydrocortisone hemisuccinate was almost three times higher than that of patients who did not receive this drug (RR 2.82, 1.35 <OR <5.90, Delayed admission to the ICU is associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock A Schnegelsberg, J Mackenhauer, M Pedersen, H Nibro, H Kirkegaard Aarhus University Hospital, Aarhus C, Denmark Critical Care 2014, 18(Suppl 1):P241 (doi: 10.1186/cc13431) P237 Multiple trauma is linked with reversal of immunoparalysis and provides survival benefi t from Pseudomonas aeruginosa E Mandragos1 A Pistiki2 DI Droggiti2 M Georgitsi2 Does clopidogrel change morbidity and mortality in ICU sepsis patients? DG Klepser, PP Dobesh, TR McGuire, DA Roberts, AL Himmelberg, KM Olsen University of Nebraska Medical Center College of Pharmacy, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13428) Multiple trauma is linked with reversal of immunoparalysis and provides survival benefi t from Pseudomonas aeruginosa E Mandragos1, A Pistiki2, DI Droggiti2, M Georgitsi2, E Giamarellos-Bourboulis2 1Attikon Hospital, Chaidari, Attiki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2014, 18(Suppl 1):P237 (doi: 10.1186/cc13427) E Mandragos , A Pistiki , DI Droggiti , M Georgitsi , E Giamarellos-Bourboulis2 1Attikon Hospital, Chaidari, Attiki, Greece; 2University of Athens, Medical School, Athens, Greece Critical Care 2014, 18(Suppl 1):P237 (doi: 10.1186/cc13427) Introduction The purpose of this study was to evaluate whether patients with sepsis exposed to clopidogrel have lower mortality and fewer days on mechanical ventilation compared with patients not exposed to clopidogrel. A recent post-hoc analysis of the PLATO trial suggests that the antiplatelet agent ticagrelor provided a signifi cant Introduction Patients with multiple injuries are prone to hospital- acquired infections. We hypothesized that exposure to multiple injuries may modulate the innate immune response and the subsequent outcome of infections. S86 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 24 hours has been found to be signifi cantly higher compared with patients admitted directly to the ICU from the ED [2]. 24 hours has been found to be signifi cantly higher compared with patients admitted directly to the ICU from the ED [2]. pyelonephritis or intraabdominal infection were blindly assigned to placebo or clarithromycin for four consecutive days as adjunctive treatment to standard of care. Clarithromycin was administered at a dose of 1 g once daily in 1 hour of continuous infusion. Organ failures before allocation to blind treatment were defi ned according to the Surviving Sepsis Campaign 2003 defi nitions. Cox regression analysis was done to verify the eff ect of clarithromycin as a moderator. Hazard ratios (HRs) and 95% confi dence intervals (CIs) were calculated. pyelonephritis or intraabdominal infection were blindly assigned to placebo or clarithromycin for four consecutive days as adjunctive treatment to standard of care. Clarithromycin was administered at a dose of 1 g once daily in 1 hour of continuous infusion. Organ failures before allocation to blind treatment were defi ned according to the Surviving Sepsis Campaign 2003 defi nitions. Cox regression analysis was done to verify the eff ect of clarithromycin as a moderator. Hazard ratios (HRs) and 95% confi dence intervals (CIs) were calculated. Methods A retrospective cohort study of patients admitted with community-acquired sepsis to a 12-bed, tertiary ICU at a university- affi liated teaching hospital, November 2008 to October 2010. Patients were divided into two groups based on their ICU admission pattern: direct transfer from the ED (direct group); and one or more ward transfers between the ED and ICU within 48 hours (delayed group). Our primary outcome measure was mortality.i i Results Forty-nine patients of the placebo arm and 55 patients of the clarithromycin arm had acute lung injury (ALI); mortality was 51.0% and 30.9% respectively (P = 0.046). Forty-seven patients of the placebo arm and 54 patients of the clarithromycin arm had acute coagulopathy; mortality was 44.7% and 44.4% respectively (P = 1.000). Twenty patients of the placebo arm and 19 patients of the clarithromycin arm had metabolic acidosis; mortality was 55.0% and 52.6% respectively (P = 1.000). Twenty-nine patients of the placebo arm and 39 patients of the clarithromycin arm had acute oliguria; mortality was 55.2% and 48.7% respectively (P = 0.631). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ALI (HR = 2.42; 95% CI = 1.45 to 4.03, P = 0.001), acute coagulopathy (HR = 2.65; CI = 1.69 to 4.16) and cardiovascular failure (HR = 3.36, CI = 2.09 to 5.39) were independently associated with unfavorable outcome. Adding treatment with clarithromycin in the equation reduced the risk for death by ALI by 1.86-fold (HR = 0.54; CI = 0.29 to 0.99, P = 0.049). Results We identifi ed 277 patients admitted to the ICU within 48 hours from arrival in the ED. Of these, 186 were admitted directly from the ED, and 91 patients had more than one ward transfer between the ED and the ICU. In-hospital mortality in the delayed group was 32% compared with 21% in the direct group (P = 0.0477). Patients with delayed admission had signifi cantly lower APACHE II scores: 21 (16; 26) and 24 (18.75; 31) respectively (P = 0.0016). The in-hospital LOS was similar in the two groups. For patients in the delayed group, we found a trend toward increased 30-day mortality (P = 0.0723), 90-day mortality (P = 0.0838) and higher Charlson Comorbidity Index (P = 0.0609). g y Conclusion We found that patients admitted with community- acquired severe sepsis or shock are more likely to die in-hospital if they experience redundant ward transfers within 48 hours of admission. However, the delayed group was signifi cantly lower risk stratifi ed in the ICU based on their APACHE II score. Identifi cation of severe sepsis and risk assessment in the ED is crucial for patient outcome. Conclusion Clarithromycin is a major moderator of the physical course of Gram-negative sepsis complicated with ALI. Reference References 1. Vallés J, et al.: Chest 2003, 123:1615-1624. 2. Parkhe M, et al.: Emergency Med 2002, 14:50-57. 1. Vallés J, et al.: Chest 2003, 123:1615-1624. 2. Parkhe M, et al.: Emergency Med 2002, 14:50-57. 1. Giamarellos-Bourboulis EJ, et al.: J Antimicrob Chemother 2013. [Epub ahead of print] , , 2. Parkhe M, et al.: Emergency Med 2002, 14:50-57. print] P243i Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial E Giamarellos-Bourboulis1, K Lymberopoulou2, I Tsangaris3, A Antonopoulou3, A Marioli2, L Leonidou4, E Douzinas3, I Koutelidakis5, A Armaganidis3 1University of Athens, Medical School, Athens, Greece; 2Sismanogleion General Hospital, Athens, Greece; 3University of Athens, Athens, Greece; 4University of Patras, Rion, Greece; 5Aristotle University, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P242 (doi: 10.1186/cc13432) Benefi t profi le of recombinant human soluble thrombomodulin in sepsis-induced DIC K Yamakawa1, H Ogura1, S Fujimi2, M Morikawa3, Y Ogawa1, Y Umemura2, Y Inoue3, H Tanaka3, T Hamasaki1, T Shimazu1 1Osaka University Graduate School of Medicine, Osaka, Japan; 2Osaka General Medical Center, Osaka, Japan; 3Juntendo University Urayasu Hospital, Chiba, Japan Critical Care 2014, 18(Suppl 1):P243 (doi: 10.1186/cc13433) Delayed admission to the ICU is associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock Introduction The aim of this study is to determine whether ward transfers causing delayed ICU admission are associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock. Community-acquired infections are among the leading causes of ICU admission [1]. The 30-day mortality for adult emergency department (ED) patients with a delay in ICU admission of up to Introduction The aim of this study is to determine whether ward transfers causing delayed ICU admission are associated with increased in-hospital mortality in patients with community-acquired severe sepsis or shock. Community-acquired infections are among the leading causes of ICU admission [1]. The 30-day mortality for adult emergency department (ED) patients with a delay in ICU admission of up to S87 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Comprehensive assessment of the true sepsis burden using electronic health record screening augmented by natural language processing R Arnold, J Isserman, S Smola, E Jackson Christiana Care Health System, Newark, DE, USA Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) R Arnold, J Isserman, S Smola, E Jackson Table 1 (abstract P245). Patient characteristics ICU survivors ICU nonsurvivors n 47 (73%) 17 (27%) Age (years) 53 (38 to 65) 49 (33 to 62) BM transplant 24 (51%) 7 (41%) Table 2 (abstract P245). Characteristics on admission to the ICU ICU survivors ICU nonsurvivors Neutrophil count 0.5 (0.3) 0.2 (0.3)* Days of neutropenia 3 (0 to 8) 6 (0 to 19) P <0.05. Conclusion In a group of oncology patients admitted to the ICU with neutropenia and severe sepsis/septic shock, we found an in-hospital mortality of less than 50%. This is similar to the general population. Reference 1. Quenot et al.: Crit Care 2013, 17:R65. P246 Vitamin D and ICU outcome in septic patients: a diffi cult connection? FI Socci, A Cecchi, S Di Valvasone, M Ciapetti, L Perretta, ML Migliaccio, G Zagli, S Batacchi, G Cianchi, M Bonizzoli, A Terreni, A Peris AOU Careggi, Florence, Italy Critical Care 2014, 18(Suppl 1):P246 (doi: 10.1186/cc13436) Introduction Vitamin D a secosteroid hormone has roles in the Table 1 (abstract P245). Patient characteristics ICU survivors ICU nonsurvivors n 47 (73%) 17 (27%) Age (years) 53 (38 to 65) 49 (33 to 62) BM transplant 24 (51%) 7 (41%) Table 2 (abstract P245). Characteristics on admission to the ICU ICU survivors ICU nonsurvivors Neutrophil count 0.5 (0.3) 0.2 (0.3) Days of neutropenia 3 (0 to 8) 6 (0 to 19) P <0.05. Table 1 (abstract P245). Patient characteristics ICU survivors ICU nonsurvivors Table 1 (abstract P245). Patient characteristics Table 1 (abstract P245). Patient characteristics y Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) Introduction Interventional trials for sepsis have not shown an improvement in patient outcomes, often due to the lack of a diagnostic gold standard resulting in large heterogeneity of the patients enrolled. Electronic health record (EHR) screening tools have been applied to the sepsis population but limited to vital sign and laboratory data to identify target patients. Our objective was to describe an investigational database created through the application of a new EHR screening tool that applies natural language processing (NLP) analysis to clinical documentation to augment the identifi cation of infection. i Methods We acquired data from the Clinical Vigilance for Sepsis EHR screen on consecutive patients from two hospital systems over 12 months at a 300-bed community hospital and 24 months from a 500- bed academic tertiary care center. Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial E Giamarellos-Bourboulis1, K Lymberopoulou2, I Tsangaris3, A Antonopoulou3, A Marioli2, L Leonidou4, E Douzinas3, I Koutelidakis5, A Armaganidis3 1University of Athens, Medical School, Athens, Greece; 2Sismanogleion General Hospital, Athens, Greece; 3University of Athens, Athens, Greece; 4University of Patras, Rion, Greece; 5Aristotle University, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P242 (doi: 10.1186/cc13432) Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial E Giamarellos-Bourboulis1, K Lymberopoulou2, I Tsangaris3, A Antonopoulou3, A Marioli2, L Leonidou4, E Douzinas3, I Koutelidakis5, A Armaganidis3 1University of Athens, Medical School, Athens, Greece; 2Sismanogleion General Hospital, Athens, Greece; 3University of Athens, Athens, Greece; 4University of Patras, Rion, Greece; 5Aristotle University, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P242 (doi: 10.1186/cc13432) E Giamarellos-Bourboulis1, K Lymberopoulou2, I Tsangaris3, Introduction Recombinant human soluble thrombomodulin (rhTM) demonstrated promising evidence suggestive of effi cacy in a phase IIb, randomized, controlled trial [1] and is currently under evaluation in a phase III trial. However, the benefi t profi les of rhTM have not been elucidated. The purpose of this study was to explore whether patients with a high disease severity (according to Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores) might have a treatment benefi t from rhTM administration. Introduction A recent randomized trial of our group showed that blind treatment with clarithromycin decreased mortality from septic shock and multiple organ dysfunction, shortened time until resolution of infection in patients with severe sepsis/shock and decreased hospitalization costs [1]. The effi cacy of clarithromycin in relation with the type of failing organs is analyzed.l Methods Six hundred patients with systemic infl ammatory response syndrome due to primary Gram-negative bacteremia or acute Figure 1 (abstract P243). Estimated survival curves for each of three disease severities. Figure 1 (abstract P243). Estimated survival curves for each of three disease severities. S88 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods This was a post-hoc, subgroup analysis of a multicenter retrospective cohort study [2] conducted in three tertiary referral hospitals in Japan. All patients with sepsis-induced DIC who required ventilator management were included. We stratifi ed all patients with diff erent disease severity, as defi ned by APACHE II and SOFA scores to three strata. Outcomes of neutropenic patients with severe sepsis on a specialist cancer ICU S Jhanji, S Hallam, T Wigmore Royal Marsden NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P245 (doi: 10.1186/cc13435) Royal Marsden NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P245 (doi: 10.1186/cc13435) Introduction The hospital survival rate for patients with septic shock remains at approximately 50% [1]. Critically ill cancer patients have often been considered poor candidates for ICU management due to the perception of poor outcomes for this group, in particular in the presence of neutropenia. There is a paucity of data supporting this. The objective of this study was to describe clinical outcomes for a group of septic neutropenic patients admitted to a cancer ICU. y Results Eligible were 162 patients with sepsis-induced DIC; 68 patients received rhTM and 94 did not. After adjusting for imbalances, rhTM administration was signifi cantly associated with reduced mortality only in patents in the stratum II group (APACHE II, 22 to 27) (adjusted hazard ratio, 0.20; 95% confi dence interval, 0.05 to 0.74; P = 0.016), while not signifi cant in stratum I and stratum III (Figure 1). A similar tendency was observed in analysis for SOFA score (stratum I (SOFA, –10), P = 0.368; stratum II (SOFA, 11 to 12), P = 0.012; stratum III (SOFA, 13–), P = 0.673). Conclusion A survival benefi t with rhTM treatment was observed in sepsis-induced DIC and a high risk of death according to baseline APACHE II and SOFA scores. Methods All neutropenic patients (neutrophils <1,000/mm3) admitted to the ICU at the Royal Marsden hospital (London, UK) between October 2010 and December 2012 with a diagnosis of severe sepsis/septic shock were included retrospectively. Data were collated from patients’ electronic records after approval by the hospital audit committee. Data are presented as the absolute value (%) or median (IQR). Fisher’s exact test or the Mann–Whitney U test was used as appropriate. Conclusion A survival benefi t with rhTM treatment was observed in sepsis-induced DIC and a high risk of death according to baseline APACHE II and SOFA scores. P246 Results The EHR screen identifi ed 216,550 patients over a total of 36 months from the two hospitals. A total of 37,160 (17%) patients were treated with i.v. antibiotics. Sepsis patients experienced a 3% (1,186/37,160) mortality rate relative to 0.5% (448/216,550) in patients without infection at any time. Sepsis at any time represented 73% (1,186/1,634) of all in-hospital deaths. ICU transfer occurred in 18% (6,865/37,160) of patients, with septic shock (vasopressor requirement) occurring in 10% (3,837), and 13% (5,072) requiring mechanical ventilation. Comprehensive assessment of the true sepsis burden using electronic health record screening augmented by natural language processing R Arnold, J Isserman, S Smola, E Jackson Christiana Care Health System, Newark, DE, USA Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) A physician order for intravenous antibiotics was used as a surrogate for suspected infection, removing patients receiving a single dose of antibiotics without subsequent administration. Each patient’s in-hospital course was tracked from arrival to fi nal disposition, identifying vital signs, laboratory values, radiological results, and interventions as they occurred. Patients were followed for the primary outcome of mortality, with secondary outcomes of transfer to the ICU, vasopressor initiation and mechanical ventilation. Conclusion In a group of oncology patients admitted to the ICU with neutropenia and severe sepsis/septic shock, we found an in-hospital mortality of less than 50%. This is similar to the general population. Reference 1. Quenot et al.: Crit Care 2013, 17:R65. References y pp p Results Sixty-four neutropenic patients were admitted to the ICU during this period. Forty-seven (73%) patients survived to ICU discharge and 34 (53%) patients to hospital discharge. Twenty-two (34%) patients were alive at 6 months and 18 (28%) patients were alive at 12 months. Seventy-seven percent of patients had microbiologically documented infections. There was no signifi cant diff erence between ICU survivors and nonsurvivors in duration of neutropenia, the presence of, or removal of, indwelling catheters, or the source of sepsis. Mechanical ventilation and need for vasopressors were associated with worse outcomes. Patient characteristics are presented in Tables 1 and 2. 1. Vincent JL, et al.: Crit Care Med 2013, 41:2069-2079. 2. Yamakawa K, et al.: Intensive Care Med 2013, 39:644-652. P244 Eff ect of clarithromycin in patients with Gram-negative sepsis: subgroup analysis of a randomized trial Intervention eff ects estimated as hazard ratios were analyzed by Cox regression analysis adjusted for propensity model to detect subgroup heterogeneity of the eff ects of rhTM on in-hospital mortality. Comprehensive assessment of the true sepsis burden using electronic health record screening augmented by natural language processing Comprehensive assessment of the true sepsis burden using electronic health record screening augmented by natural language processing R Arnold, J Isserman, S Smola, E Jackson Christiana Care Health System, Newark, DE, USA Critical Care 2014, 18(Suppl 1):P244 (doi: 10.1186/cc13434) P248 Effi cacy of early administration of thrombomodulin alfa in patients with sepsis-induced disseminated intravascular coagulation: subanalysis from post-marketing surveillance data Y Eguchi1, S Gando2, H Ishikura3, D Saitoh4, J Mimuro5, H Takahashi6, I Kitajima7, H Tsuji8, T Matsushita9, Y Sakata5 1Shiga University of Medical Science, Shiga, Japan; 2Hokkaido University Graduate School of Medicine, Hokkaido, Japan; 3Fukuoka University, Fukuoka, Japan; 4National Defense Medical College, Saitama, Japan; 5Jichi Medical University, School of Medicine, Tochigi, Japan; 6Niigata Prefectural Kamo Hospital, Niigata, Japan; 7Graduate School of Medical and Pharmaceutical Science, University of Toyama, Japan; 8Kyoto Prefectural University of Medicine, Kyoto, Japan; 9Nagoya University Hospital, Nagoya, Japan Critical Care 2014, 18(Suppl 1):P248 (doi: 10.1186/cc13438) Results Most patients showed vitamin D levels below 20 ng/ml, and we have not demonstrated a statistical signifi cance correlation in the univariate regression between vitamin D level and both SAPS (P = 0.300) or length of stay in hospital (P = 0.154). Also our data do not demonstrate a statistical signifi cance diff erence at t test between the value of vitamin D in the dead group and the alive group. Introduction We hypothesize that the early administration of thrombo- modulin alfa (TM-alfa) (Recomodulin® injection; Asahi Kasei Pharma Corporation, Tokyo, Japan) could improve mortality in patients with sepsis-induced disseminated intravascular coagulation (DIC). TM-alfa has been approved for use as a curative medicine for DIC in Japan from 2008 that was examined in a multicenter, randomized, clinical trial [1]. Introduction We hypothesize that the early administration of thrombo- modulin alfa (TM-alfa) (Recomodulin® injection; Asahi Kasei Pharma Corporation, Tokyo, Japan) could improve mortality in patients with sepsis-induced disseminated intravascular coagulation (DIC). TM-alfa has been approved for use as a curative medicine for DIC in Japan from 2008 that was examined in a multicenter, randomized, clinical trial [1]. Methods DIC was diagnosed based on the Japanese Association for Acute Medicine (JAAM) criteria. From May 2008 to April 2010, a total of 1,787 patients with sepsis-induced DIC from post-marketing surveillance data [2] were analyzed. The survival rates on day 28 after the last TM-alfa administration were evaluated. Methods DIC was diagnosed based on the Japanese Association for Acute Medicine (JAAM) criteria. From May 2008 to April 2010, a total of 1,787 patients with sepsis-induced DIC from post-marketing surveillance data [2] were analyzed. The survival rates on day 28 after the last TM-alfa administration were evaluated. Conclusion Several groups have reported an inverse association between vitamin D levels in critically ill patients, severity of disease, outcome length of ICU stay and mortality [3,4]. Dynamic myocardial depression in septic cardiomyopathy A Darawshe, D Levingston, Y Haviv The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) Introduction Benefi cial eff ects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on vascular diseases have been demonstrated in several clinical trials. Recently discovered anti-infl ammatory eff ects of statins seem to have an important role in counteracting the harmful eff ects of sepsis on the coagulation system. Many epidemiologic studies evidence that statin treatment may be associated with a better prognosis in severe bacterial infections, and a recent randomized trial found a reduced mortality in the statin group. We decided to perform a meta-analysis of all randomized controlled trials ever performed on statin therapy in septic patients to evaluate their eff ect on survival. g The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel g The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) Introduction Depression of left ventricular contractility appears in many diseases resulting from various etiology factors. One of the most interesting features of septic cardiomyopathy is the signifi cant dynamic myocardial depression that is commonly observed. In this context, the objective of the present work is to characterize clinically, by laboratory, by echocardiography, and by invasive measures the patients with septic cardiomyopathy. f Methods Four trained investigators searched and assessed pertinent studies in BioMed Central, PubMed, Embase and the Cochrane Central Register (divergences resolved by consensus). Inclusion criteria were: random allocation to treatment; comparison of statins versus any comparator in septic patients. Exclusion criteria were: duplicate publications; nonadult studies; no data on main outcomes.i Methods A single-center database investigates all patients who were admitted and treated for severe sepsis or septic shock in the ICU over a period of 2 years (November 2011 to October 2013), and who were discharged with a diagnosis of septic cardiomyopathy or new left ventricular dysfunction. The clinical, laboratory, echocardiography, and invasive measures, and clinical outcome were recorded. Results From the 105 patients that were investigated, 15 patients were found with septic cardiomyopathy. Septic cardiomyopathy is more prevalent among men (60%). Patients with septic cardiomyopathy have an increased prevalence of immune compromised disease (46%), and hypertension (40%). There was a need for mechanical respiratory support for 86% of patients. The improvement in cardiac function Results Data from 650 patients in fi ve randomized controlled studies were analyzed. P248 Effi cacy of early administration of thrombomodulin alfa in patients with sepsis-induced disseminated intravascular coagulation: subanalysis from post-marketing surveillance data In our experience we have not found an evident correlation between low vitamin levels and ICU outcomes. However, considering the prevalence of low vitamin D levels among the medical patients admitted to the ICU, further studies should be performed. Results The 28-day survival rate was 64.5%. Use of other anticoagulants before and after TM-alfa administration did not aff ect the survival rate (present vs. absent: 63.8% vs. 65.2% (P = 0.782) and 62.5% vs. 65.3% (P = 0.606) respectively). The survival rate decreased signifi cantly in proportion to the duration of DIC before TM-alfa administration (P <0.001). More precisely, the 28-day survival rate was 66.4% when TM- alfa was injected on the same day that DIC was diagnosed, whereas it was 48.5% and 31.1% when TM-alfa was started 4 days later and 7 days or more after DIC was diagnosed, respectively. These diff erences were signifi cant (P = 0.033 and P <0.001, respectively).f p References 1. Holick MF: Vitamin D defi ciency. N Engl J Med 2007, 357:266-281.i 2. Lucidarme O, et al.: Incidence and risk factors of vitamin D defi ciency in critically ill patients: results from a prospective observational study. Intensive Care Med 2010, 36:1609-1611. 3. McKinney JD, et al.: Relationship between vitamin D status and ICU outcomes in veterans. J Am Med Dir Assoc 2011, 12:208-211.i 3. McKinney JD, et al.: Relationship between vitamin D status and ICU outcomes in veterans. J Am Med Dir Assoc 2011, 12:208-211.i 4. Higgins DM, et al.: Relationship of vitamin D defi ciency to clinical outcomes in critically ill patients. JPEN J Parenter Enteral Nutr 2012, 36:713–720. 4. Higgins DM, et al.: Relationship of vitamin D defi ciency to clinical outcomes in critically ill patients. JPEN J Parenter Enteral Nutr 2012, 36:713–720. i Conclusion The early administration of TM-alfa may be eff ective for patients with sepsis-induced DIC diagnosed based on the JAAM criteria. References Vitamin D and ICU outcome in septic patients: a diffi cult connection? Introduction Vitamin D, a secosteroid hormone, has roles in the optimal functioning of many organ systems and illnesses [1]. Recent reports show that most patients admitted to the ICU are vitamin D insuffi cient [2]. Introduction Vitamin D, a secosteroid hormone, has roles in the optimal functioning of many organ systems and illnesses [1]. Recent reports show that most patients admitted to the ICU are vitamin D insuffi cient [2]. Conclusion Application of this novel EHR screening tool to identify sepsis patients utilizes NLP applied to clinical documentation, providing greater clinical context than laboratory and vital sign screening alone. This database represents the entire sepsis acuity spectrum, allowing for a more granular description of the infectious process as well as subgroups with adequate sample size. The dataset collected as a patient-centered time series will enable future studies to focus on the trajectory of clinical deterioration and shock before its occurrence. Methods In a 10-bed general ICU at the emergency department of a tertiary teaching hospital in Florence (Italy), 71 medical patients with severe sepsis or septic shock (51% men, 40% women) admitted to the ICU between January 2013 and September 2013 were studied. Vitamin D levels were measured by radioimmunoassay at admission, as well Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S89 Table 1 (abstract P246). topic. Published data, summarized by this meta-analysis of randomized trials, show that statin therapy has no detrimental eff ect on survival in the overall population of adult septic patients. P248fi A meta-analysis of randomized controlled trials on the use of statins in septic patients 1. Saito H, et al.: J Thromb Haemost 2007, 5:31-41. 1. Saito H, et al.: J Thromb Haemost 2007, 5:31-41. 2. Mimuro J, et al.: Thromb Res 2013, 131:436-443. 1. Saito H, et al.: J Thromb Haemost 2007, 5:31-41. 2. Mimuro J, et al.: Thromb Res 2013, 131:436-443. 1. Saito H, et al.: J Thromb Haemost 2007, 5:31-41. 2. Mimuro J, et al.: Thromb Res 2013, 131:436-443. G Landoni, L Nobile, D Febres, E Frati, N Villari, AL Di Prima, R Dossi, L Pasin Vita-Salute San Raff aele University, Milan, Italy G Landoni, L Nobile, D Febres, E Frati, N Villari, AL Di Prima, R Dossi, L Pasin Vita-Salute San Raff aele University, Milan, Italy G Landoni, L Nobile, D Febres, E Frati, N Villari, AL Di Prima, R Dossi, L Pasin Vita-Salute San Raff aele University, Milan, Italy Dynamic myocardial depression in septic cardiomyopathy f y y Critical Care 2014, 18(Suppl 1):P247 (doi: 10.1186/cc13437) f y y Critical Care 2014, 18(Suppl 1):P247 (doi: 10.1186/cc13437) Dynamic myocardial depression in septic cardiomyopathy A Darawshe, D Levingston, Y Haviv The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) Overall analysis showed no diff erence in mortality between patients receiving statins (44/322 (14%)) versus control (50/328 (15%)), RR = 0.90 (95% CI 0.65 to 1.26), P = 0.6.i Conclusion Scientifi c evidence for the role of statins in septic patients is still limited and larger randomized trials should be performed on this S90 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 diagnostic and therapeutic effi cacy of these CXRs is now known to be low [1]. Routine CXRs may only be benefi cial for certain indications and the discussion regarding these indications and the safety of abandoning routine CXRs is still continuing [2]. occurred at an average of 6.9 days. E. coli is the commonest bacterial pathogen (33%). Laboratory fi ndings show elevated liver enzyme and kidney function impairment in all patients. Thirty-three percent of patients were treated with N-acetyl cysteine, and 46% were treated with renal replacement therapy. High CRP was observed in all patients. Paroxysmal atrial fi brillation was diagnosed in 46%. Invasive measures in 50% of the patients have demonstrated high cardiac index (CI) and low systemic vascular resistant index (SVRI) on their admission, and 93% demonstrate low CI and high SVRI a few hours later. Hospitalization stay was between 3 and 42 days with an average of 14.6 days. occurred at an average of 6.9 days. E. coli is the commonest bacterial pathogen (33%). Laboratory fi ndings show elevated liver enzyme and kidney function impairment in all patients. Thirty-three percent of patients were treated with N-acetyl cysteine, and 46% were treated with renal replacement therapy. High CRP was observed in all patients. Paroxysmal atrial fi brillation was diagnosed in 46%. Invasive measures in 50% of the patients have demonstrated high cardiac index (CI) and low systemic vascular resistant index (SVRI) on their admission, and 93% demonstrate low CI and high SVRI a few hours later. Hospitalization stay was between 3 and 42 days with an average of 14.6 days. Methods We prospectively included all patients who underwent major cardiac surgery in the year 2012. A direct postoperative CXR was performed only for certain specifi ed indications. An on-demand CXR could be obtained during the postoperative period according to other specifi ed indications. Evaluation of early graft function in a case series of lung-transplanted patients J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, L Rosso, D Chiumello, F Valenza, L Gattinoni Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, L Rosso, D Chiumello, F Valenza, L Gattinoni Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, J Fumagalli, I Algieri, M Brioni, AM Villa, GM Ruggeri, F Rapido, A Colombo, S Luoni, G Babini, B Safaee Fakhr, L Spada, S Froio, S Coppola, A Palleschi, Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) Critical Care 2014, 18(Suppl 1):P252 (doi: 10.1186/cc13442) Introduction The aim of the study was to investigate early graft function in terms of biological and radiological variables in a case series of lung transplant (Ltx). Conclusion The strategy of daily routine CXRs for critically ill patients has developed from a common practice (63%) in 2006 [2] to a rare practice (7%) nowadays. Intensivists still assume the value of routine CXRs to be higher than the effi cacy that is reported in the literature. This might be due to the clinical value of negative fi ndings, which has not been studied before. Methods We performed a prospective analysis of patients that underwent Ltx at Fondazione IRCCS Cà Granda Policlinico of Milan from 1 December 2012 to 10 December 2013. Donors’ lung parameters were recorded. Recipients’ clinical data were collected daily and lung computed tomography (CT) at end expiration was performed within 7 days after Ltx. On the same day, bronchoalveolar lavage was collected [1]. Quantitative CT analysis was run on separate lungs for each patient. Results Out of 25 LTx, 12 paired data for donors and recipients were analyzed. Lung donors’ PaO2/FiO2 was 392 ± 118, Oto score was 5 ± 3, and ICU length of stay was 2 ± 1 days. P252 Results Of the 83 ICUs that were contacted, 69 (83%) responded to the survey. Only 7% of ICUs still perform daily routine CXRs for all patients while 65% of ICUs say never to perform CXRs on a routine basis. A daily meeting with a radiologist is established in the majority of ICUs and is judged to be important or even essential. The therapeutic effi cacy of routine CXRs was assumed by intensivists to be lower than 10% or to be between 10 and 20%. The effi cacy of on-demand CXRs was assumed to be between 10 and 60%. There is consensus between intensivists to perform a routine CXR after endotrachial intubation, chest tube placement or central venous catheterization. P251 Table 1 (abstract P252). Quantitative CT scan analysis on transplanted lungs Table 1 (abstract P252). Quantitative CT scan analysis on transplanted lungs Lung CT Mean ± SD (n = 16) Volume (ml) 1,639 ± 437 Weight (g) 761 ± 175 Hyper infl ated (%) 0.0 ± 0.0 Normal (%) 38.9 ± 17.1 Poorly (%) 29.4 ± 8.4 Not infl ated (%) 31.7 ± 14.9 P250i Signifi cant change in the practice of chest radiography in Dutch ICUs M Tolsma1, TA Rijpstra2, MJ Schultz3, NJ Van der Meer2 1University Medical Center Utrecht, the Netherlands; 2Amphia Hospital, Breda, the Netherlands; 3Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P250 (doi: 10.1186/cc13440) Conclusion Stating clear indications for CXRs following cardiac surgery increases the effi cacy of these CXRs and safely reduces the total number of CXRs signifi cantly. Introduction We performed a survey under Dutch intensivists on the current practice of chest radiography in their departments. Chest radiographs (CXRs) are obtained frequently in ICU patients, despite the diagnostic and therapeutic effi cacy of routine CXRs being known to be low. The discussion regarding specifi c indications for CXRs in critically ill patients and the safety of abandoning routine CXRs is still continuing [1]. Methods A web-based questionnaire was sent to the medical director of all adult ICUs in the Netherlands. This survey contained questions regarding ICU characteristics, ICU patients, daily CXR strategies, indications for routine CXRs and the practice of radiologic evaluation. Introduction We performed a survey under Dutch intensivists on the current practice of chest radiography in their departments. Chest radiographs (CXRs) are obtained frequently in ICU patients, despite the diagnostic and therapeutic effi cacy of routine CXRs being known to be low. The discussion regarding specifi c indications for CXRs in critically ill patients and the safety of abandoning routine CXRs is still continuing [1]. Methods A web-based questionnaire was sent to the medical director of all adult ICUs in the Netherlands. This survey contained questions regarding ICU characteristics, ICU patients, daily CXR strategies, indications for routine CXRs and the practice of radiologic evaluation. Results Of the 83 ICUs that were contacted, 69 (83%) responded to the survey. Only 7% of ICUs still perform daily routine CXRs for all patients while 65% of ICUs say never to perform CXRs on a routine basis. A daily meeting with a radiologist is established in the majority of ICUs and is judged to be important or even essential. The therapeutic effi cacy of routine CXRs was assumed by intensivists to be lower than 10% or to be between 10 and 20%. The effi cacy of on-demand CXRs was assumed to be between 10 and 60%. There is consensus between intensivists to perform a routine CXR after endotrachial intubation, chest tube placement or central venous catheterization. References 1. Tolsma M, Kröner A, van den Hombergh CL, et al.: The clinical value of routine chest radiographs in the fi rst 24 hours after cardiac surgery. Anesth Analg 2011, 112:139-142. g 2. Ganapathy A, Adhikari NK, Spiegelman J, Scales DC: Routine chest X-rays in intensive care units: a systematic review and meta-analysis. Crit Care 2012, 16:R68. Evaluation of early graft function in a case series of lung-transplanted patients Recipients’ age was 56 ± 11 years, and body mass index was 24 ± 4 kg/m2. Four patients received double Ltx, and the warm ischemia time was 85 ± 16 and 94 ± 14 respectively for right lung and left lung. When the CT scan was performed (on References 1. Ganapathy A, Adhikari NK, Spiegelman J, Scales DC: Routine chest X-rays in intensive care units: a systematic review and meta-analysis. Crit Care 2012, 16:R68. doi: 10.1186/cc11321. 2. Graat ME, Hendrikse KA, Spronk PE, Korevaar JC, Stoker J, Schultz MJ: Chest radiography practice in critically ill patients: a postal survey in the Netherlands. BMC Med Imaging 2006, 18:6:8. 2. Graat ME, Hendrikse KA, Spronk PE, Korevaar JC, Stoker J, Schultz MJ: Chest radiography practice in critically ill patients: a postal survey in the Netherlands. BMC Med Imaging 2006, 18:6:8. Dynamic myocardial depression in septic cardiomyopathy A Darawshe, D Levingston, Y Haviv The Chaim Sheba Medical Center affi liated to the Tel-Aviv University, Ramatgan, Israel Critical Care 2014, 18(Suppl 1):P249 (doi: 10.1186/cc13439) For all patients who did not have a CXR taken before the morning of the fi rst postoperative day, a control CXR was then performed. All CXR fi ndings were noted and classifi ed, including whether or not they led to an intervention. Diagnostic and therapeutic effi cacy values were calculated. Conclusion Septic cardiomyopathy is more common among immune compromised patients. It is characterized by dynamic changes in the cardiac function based on echocardiography and invasive measures. A persistent hyperkinetic state was associated with high mortality rate. In addition to echocardiography follow-up, invasive monitoring even in their admission is of great importance for more eff ective and adequate treatment. fi Results A total of 1,351 patients were included who mainly underwent coronary artery bypass grafting, valve surgery or a combination of both. Eighteen percent of patients underwent a minimally invasive cardiac surgery. The diagnostic effi cacy for major abnormalities was clearly higher for the postoperative and on-demand CXRs, performed on indication, when compared with the next-morning routine CXR (6.7% and 6.9% vs. 2.9%) (P = 0.002). The therapeutic effi cacy was also clearly higher for the postoperative and on-demand CXRs (2.9% and 4.1%), while the need for intervention after the morning control CXR was now reduced to be minimal (0.6%) (P = 0.002). Stating clear indications for chest radiographs after cardiac surgery increases their effi cacy and safely reduces costs y p g Lung CT Mean ± SD (n = 16) fi M Tolsma1, TA Rijpstra2, PM Rosseel2, M Bentala2, HA Dijkstra2, NJ Van der Meer2 1University Medical Center Utrecht, the Netherlands; 2Amphia Hospital, Breda, the Netherlands 1University Medical Center Utrecht, the Netherlands; 2Amphia Hospital, Breda, the Netherlands Critical Care 2014, 18(Suppl 1):P251 (doi: 10.1186/cc13441) Critical Care 2014, 18(Suppl 1):P251 (doi: 10.1186/cc13441) Introduction We investigated the effi cacy and safety of chest radiographs (CXRs) performed on specifi ed indications only, directly after cardiac surgery. CXRs in the ICU are frequently obtained routinely for postoperative cardiosurgical patients, despite the fact that the S91 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 day 4 ± 1), four patients were mechanically ventilated, PaO2/FiO2 was 270 ± 93 and PaCO2 was 44.6 ± 10.0 mmHg. Primary graft dysfunction grade 2 or 3 at 72 hours post Ltx was diagnosed in six patients. All patients were discharged from ICU. Table 1 presents quantitative analysis of transplanted lungs. The alveolar protein concentration and not aerated tissue weight are signifi cantly related (R2 =0.69; P = 0.001). Conclusion Alveolar protein concentration is related to morphological features of lungs early after Ltx. However, the early postoperative period when the rate of complications is highest has been scarcely examined. Our study aimed to compare the residual pulmonary function of pulmonary lobectomy patients after VATS and the standard thoracotomy approach in the immediate postoperative period. day 4 ± 1), four patients were mechanically ventilated, PaO2/FiO2 was 270 ± 93 and PaCO2 was 44.6 ± 10.0 mmHg. Primary graft dysfunction grade 2 or 3 at 72 hours post Ltx was diagnosed in six patients. All patients were discharged from ICU. Table 1 presents quantitative analysis of transplanted lungs. The alveolar protein concentration and not aerated tissue weight are signifi cantly related (R2 =0.69; P = 0.001). Conclusion Alveolar protein concentration is related to morphological features of lungs early after Ltx. R f p p p Methods This prospective study included 14 VATS and 13 thoracotomy lobectomy (THOR) patients (age 58.8 ± 10.9 vs. 59 ± 8.9 years, P = 0.96; preoperative FVC 3.4 ± 1 vs. 3.4 ± 0.95 l, P = 0.98 and preoperative FEV1 2.7 ± 0.96 vs. 2.7 ± 0.6 l, P = 0.9, respectively). All patients received standard surgical and postoperative care with standardized pain management including i.v. p References 1. Fang WF, et al.: Application and comparison of scoring indices to predict outcomes in patients with healthcare-associated pneumonia. Crit Care 2011, 15:R32. 1. Fang WF, et al.: Application and comparison of scoring indices to predict outcomes in patients with healthcare-associated pneumonia. Crit Care 2011, 15:R32. fi y Methods We present preliminary data of a prospective observational ongoing study. Sixteen patients undergoing NIV treatment outside the ICU for ARF of any origin were evaluated with LUS at three times: before NIV application (T0), and at 5 minutes (T5) and 60 minutes (T60) of NIV treatment. US scan was performed in six regions for each emithorax. LUS patterns were defi ned as: consolidation (C); multiple coalescent B-lines (B+); multiple irregularly spaced B-lines (B) and normal aeration (A). A LUS-ReS [2] was calculated detecting changes in the US pattern when comparing T0 to T5 and T0 to T60 assessments. Outcome was defi ned as NIV failure in the case of tracheal intubation or death within 1 week from ARF outset, otherwise NIV success.i 2. Jarvis S, et al.: Combining the National Early Warning Score with an early warning score based on common laboratory test results better discriminates patients at risk of hospital mortality. In Rapid Response Systems and Medical Emergency Teams; May 13–14 2013; London. P253 Can laboratory blood tests be used to risk stratify patients admitted with pneumonia? L Apps, S Hutchinson, T Leary, L Perry Norfolk & Norwich University Hospitals NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P253 (doi: 10.1186/cc13443) P253 Can laboratory blood tests be used to risk stratify patients admitted with pneumonia? L Apps, S Hutchinson, T Leary, L Perry Norfolk & Norwich University Hospitals NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P253 (doi: 10.1186/cc13443) Lung ultrasound reaeration score: a useful tool to predict non-invasive ventilation eff ectiveness L Nobile, P Beccaria, M Zambon, L Cabrini, G Landoni, A Zangrillo Ospedale San Raff aele, Milan, Italy Critical Care 2014, 18(Suppl 1):P255 (doi: 10.1186/cc13445) Conclusion Observational results suggest an EWS based on laboratory tests can be used to risk stratify patients with pneumonia and could be used to treat those with higher scores more aggressively earlier in their illness. Further analysis is required to determine whether age is a contributing factor and how this may modify the EWS. We need to determine whether laboratory-based EWS risk stratifi cation can be used in isolation, or whether it contributes suffi ciently to an existing physiological EWS that a combined system would improve outcome in our patients. Introduction The aim of our study is to evaluate the lung ultrasound (LUS) reaeration score (ReS) as a predictive tool for non-invasive ventilation (NIV) effi cacy in general wards for acute respiratory failure (ARF) treatment. Even if ICUs are considered the safest place to perform NIV, a shortage of intensive beds has lead to NIV application outside the ICU. With appropriate patient selection, treatment-timing choice, adequate monitoring and staff training, NIV application in general wards can allow one to safely treat patients at an early stage with better cost-eff ectiveness [1]. Few data assessing the right tool to evaluate NIV treatment effi cacy are available. References 1. Cajipe MD, et al.: Am J Surg 2012, 204:607-612. 2. Ceppa DP, et al.: Ann Surg 2012, 256:487-493. 2. Ceppa DP, et al.: Ann Surg 2012, 256:487 493. 3. Varela G, et al.: Eur J Cardiothor Surg 2006, 30:644-648. 3. Varela G, et al.: Eur J Cardiothor Surg 2006, 30:644-648. Results Of 1,598 patients, 538 died during this admission. It is uncertain whether death was due to pneumonia as only admission diagnosis is recorded but overall mortality was 35%. Analysis of data showed a strongly positive relationship between increasing EWS and increased risk of mortality with a correlation coeffi cient of 0.97. Can laboratory blood tests be used to risk stratify patients admitted with pneumonia? L Apps, S Hutchinson, T Leary, L Perry L Apps, S Hutchinson, T Leary, L Perry Norfolk & Norwich University Hospitals NHS Foundation Trust, Norwich, UK Critical Care 2014, 18(Suppl 1):P253 (doi: 10.1186/cc13443) Results The nFVC and nFEV1 values were signifi cantly higher in the VATS group in the fourth and eighth postoperative hours compared with the THOR group (84.3 ± 14.4 vs. 64.3 ± 23.4, P = 0.013 and 84 ± 18.8 vs. 64 ± 22, P = 0.017, respectively). There was no statistically signifi cant diff erence between the groups in the 24th, 48th and 72nd hours, although VATS patients showed higher values at each time points. The length of ICU stay was similarly 1 day, but the length of hospital stay was signifi cantly longer in the THOR group (5 (3 to 6) vs. 7 (3 to 42) days (median and range) (P = 0.011)). Introduction Our objective was to determine whether an Early Warning Score (EWS) based on laboratory tests could risk stratify patients admitted to the Norfolk & Norwich University Hospitals NHS Foundation Trust with pneumonia. Physiological EWS systems producing an escalated response to an increasing score are used eff ectively within the Trust, yet mortality is high in patients admitted with a diagnosis of pneumonia. The CURB-65 score may not be an eff ective risk stratifi cation tool for predicting mortality in the older patient nor to guide intensive care admission [1]. Jarvis and colleagues developed an EWS based on laboratory blood tests and used it in conjunction with physiological EWS to eff ectively risk stratify all medical patients [2]. g Conclusion Preoperative lung function prediction severely over- estimates the real lung capacity of lobectomy patients in the immediate postoperative period. VATS lobectomy seems more benefi cial from the point of early postoperative lung function. VATS lobectomy should be considered in patients with poor preoperative spirometry results to ensure better postoperative outcome. Methods Of 2,158 patients admitted with pneumonia during 12 months from August 2012, data were collected for 1,598 who had received the required blood tests. Data included dates of admission, discharge and death if appropriate, gender, haemoglobin (g/dl), white cell count (109/l), sodium (mmol/l), potassium (mmol/l), urea (mmol/l), creatinine (mmol/l) and albumin (g/l) on admission. A composite EWS was calculated and measured against outcome. P255 55 Lung ultrasound reaeration score: a useful tool to predict non-invasive ventilation eff ectiveness L Nobile, P Beccaria, M Zambon, L Cabrini, G Landoni, A Zangrillo Ospedale San Raff aele, Milan, Italy Critical Care 2014, 18(Suppl 1):P255 (doi: 10.1186/cc13445) Stating clear indications for chest radiographs after cardiac surgery increases their effi cacy and safely reduces costs diclofenac combined with epidural administration of bupivacaine and fentanyl. Spirometry was performed with a bedside MIR Spirolab II spirometer (Rome, Italy) preoperatively and 4, 8, 24, 48 and 72 hours after the surgery. FVC, FEV1, PaO2, PaCO2, complication rate, and length of ICU and hospital stay were recorded. Postoperatively measured FVC and FEV1 values were divided by preoperatively predicted values and multiplied by 100 to give the normalized FVC (nFVC) and FEV1 (nFEV1) [3].i Reference 1. Matute-Bello G, Liles WC, Radella F, Steinberg KP, Ruzinski JT, Jonas M, Chi EY, Hudson LD, Martin TR: Neutrophil apoptosis in the acute respiratory distress syndrome. Am J Respir Crit Care Med 1997, 156:1969-1977. Lung function in the immediate postoperative period after video- assisted thoracoscopic and thoracotomy pulmonary resection T Végh, R Nemes, B Fülesdi p T Végh, R Nemes, B Fülesdi University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Critical Care 2014, 18(Suppl 1):P254 (doi: 10.1186/cc13444) University of Debrecen, Medical and Health Science Center, Debrecen, Hungary Critical Care 2014, 18(Suppl 1):P254 (doi: 10.1186/cc13444) Results NIV treatment failed in fi ve patients. Eleven patients have been successfully treated with NIV. Mean LUS-ReS (SD) at T0 to T5 was 0 (± 3.1) in group 0 and 2.5 (± 2.5) in group 1 (P = 0.15). Mean LUS-ReS (SD) at T0 to T60 was –1.2 (± 3.9) in group 0 and 4.2 (± 3.4) in group 1 (P = 0.03). ROC curves were obtained for the two LUS-ReS at T0 to Introduction Previous studies reported that video-assisted thoraco- scopic surgery (VATS) is more benefi cial for pulmonary lobectomy concerning the late postoperative period than open thoracotomy [1,2]. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S92 Figure 1 (abstract P255). Figure 1 (abstract P255). Methods Physician sonographers accredited by the German Medical Ultrasound Society (DEGUM) for ultrasonography in surgery, anaesthesia or medicine were invited to participate in an online survey. Frequency of exposure to patients with suspected pneumothorax, frequency of LUS use, assessment of diagnostic accuracy of LUS for ruling-out or ruling-in pneumothorax and preferences regarding technical aspects were enquired about. Results Eighty-nine physicians responded. Average exposure to pneumothorax cases was 1/week. Fifty-fi ve per cent of respondents used LUS ‘always’ or ‘frequently’. Thirty-four per cent of physicians rated LUS as ‘always accurate’, and a further 54% as ‘accurate in a majority of cases’ in ruling out pneumothorax. Twenty-one per cent rated LUS as ‘always accurate’ and 69% as ‘accurate in a majority of cases’ in ruling in pneumothorax. Physicians reporting frequent exposure to pneumothorax patients used LUS in a higher proportion of cases (‘high caseload sonographers’) and were more confi dent to rule out pneumothorax (Figure 1). In total, 16 diff erent combinations of transducers, probe orientations and scanning modes were reported. Linear transducers, sagittal probe-orientation, and B-mode and M-mode scanning were most often selected (38%). g Conclusion Physicians’ use of LUS in the diagnosis of pneumothorax was modest. Assessment of diagnostic accuracy gave markedly lower scores than reported in clinical trials [1]. Correlation between frequency of exposure, likelihood of LUS usage and confi dence in diagnostic accuracy warrants further research into the nature of the learning curve. Lung function in the immediate postoperative period after video- assisted thoracoscopic and thoracotomy pulmonary resection T Végh, R Nemes, B Fülesdi Considerable variations regarding technical aspects of LUS refl ect the ambiguity of current recommendations [2]. More research is required to establish the most effi cient way of performing LUS for suspected pneumothorax and eff orts need to be made to promote its use in these scenarios. Figure 1 (abstract P255). Figure 1 (abstract P255). T5 (AUC 0.72) and T0 to T60 (AUC 0.83) (Figure 1). A LUS-ReS cutoff value of 0 can predict NIV eff ectiveness, with a sensibility of 91% and a specifi city of 80%.i pi y Conclusion If confi rmed, our preliminary results suggest that LUS-ReS could be a useful tool in predicting NIV eff ectiveness for ARF treatment. Caution has to be applied when interpreting our results considered the small amount of patients enrolled. R f References 2. Volpicelli G, et al.: International Liaison Committee on Lung Ultrasound. International evidence-based recommendations for point-of-care lung ultrasound. Intensive Care Med 2012, 38:577-591. 1. Cabrini L, et al.: Medical emergency team and non-invasive ventilation 1. Cabrini L, et al.: Medical emergency team and non-invasive ventilation outside ICU for acute respiratory failure. Intensive Care Med 2009, 35:339-343. 1. Cabrini L, et al.: Medical emergency team and non invasive ventilation outside ICU for acute respiratory failure. Intensive Care Med 2009, 35:339-343. 2. Bouhemad B, et al.: Bedside ultrasound assessment of positive end- expiratory pressure-induced lung recruitment. Am J Respir Crit Care Med 2011, 183:341-347. References 1. Alrajhi K, et al.: Test characteristics of ultrasonography for the detection of pneumothorax. A systematic review and meta-analysis. Chest 2012, 141:703-708. 1. Alrajhi K, et al.: Test characteristics of ultrasonography for the detection of pneumothorax. A systematic review and meta-analysis. Chest 2012, 141:703-708. 2. Volpicelli G, et al.: International Liaison Committee on Lung Ultrasound. International evidence-based recommendations for point-of-care lung ultrasound. Intensive Care Med 2012, 38:577-591. Eff ects of sitting on the respiratory pattern, mechanics and work of breathing in mechanically ventilated patients F Ruiz-Ferron1, J Serrano-Simon2 1Complejo Hospitalario de Jaen, Spain; 2Hospital Reina Sofi a, Cordoba, Spain Critical Care 2014, 18(Suppl 1):P259 (doi: 10.1186/cc13449) Results The mean age of the patients was 59 years, 32 of the patients were men. The mean (SD) diameter of the bronchi in SII patients measured was 3.9 (1.5) mm (range 0.9 to 9.0 mm). There were statistically signifi cant positive associations between wall thickening (expressed as T/D ratio) and luminal narrow (expressed as LA%) and the developed pneumonia (T/D ratio: R2 = 0.56, P <0.01 and LA%: R2 = 0.19, P = 0.005) and mechanical ventilation days (T/D ratio: R2 = 0.37, P <0.0001 and LA%: R2 = 0.32, P <0.001, respectively). No statistically signifi cant associations were identifi ed between T/D ratio or LA% and initial P/F ratio, infusion volume initial 24 hours, ICU stay days, and outcome. The mean T/D ratio and LA% were 0.25 (0.04) and 25.9% (7.6) for patients with SII and 0.35 (0.04) and 44.7% (5.6) for controls. g y F Ruiz-Ferron1, J Serrano-Simon2 1Complejo Hospitalario de Jaen, Spain; 2Hospital Reina Sofi a, Cordoba, Spain Critical Care 2014, 18(Suppl 1):P259 (doi: 10.1186/cc13449) Introduction The eff ect of sitting in an armchair on mechanically ventilated patients has not been studied enough. We study a group of patients ready for weaning for the respiratory pattern, mechanics and work of breathing during reclining in bed and after sitting in an armchair. Methods Thirteen patients who needed mechanical ventilation after 18 days (1 to 60) were studied during volume assist-control mechanical ventilation and spontaneous breathing (O2T, CPAP or PSV) in both positions. Airways, esophageal pressures and fl ow were registered for posterior analysis. Passive respiratory mechanics were measured by multiple linear regression methods, respiratory drive in esophageal pressure (P01) and respiratory eff ort using the pressure time product (PTP). Conclusion We have shown with the use of HRCT scanning on admission that patients with SII have airway wall thickening compared with normal controls. Furthermore, airfl ow obstruction due to bronchial wall edema related with developed pneumonia and mechanical ventilation days in SII patients. Results On controlled mechanical ventilation the respiratory system and chest wall elastance were signifi cantly higher in sitting compared with reclining positions (Ers 39 ± 24 vs. 33 ± 25 cmH2O/l and 10 ± 3 vs. 7 ± 3 cmH2O/l), respiratory resistances were similar (15 ± 3 vs. 14 ± 5 cmH2O/l/second) in both positions. Breathing pattern did not change signifi cantly: tidal volume (0.406 ± 0.108 vs. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods We prospectively studied 42 patients with a diagnosis of SII, according to visualized bronchoscopic fi ndings at admission, and 15 control subjects. The thoracic high-resolution computed tomography (HRCT) scan was obtained within a few hours of admission to our hospital. Airway wall dimensions were calculated using a validated method. The images were viewed on a workstation using a magnifi cation of ×5, and measurements of overall (D) and internal (L) diameter of the bronchi were made using electronic calipers, with wall thickness (T) being derived from these measurements (T = (D – L) / 2). Luminal area (Ai, mm2) and total airway wall area (Ao) were calculated from L and D, respectively, using the formula: A = r2. We used both the ratio of airway wall thickness to total diameter (T/D ratio) and the percentage luminal area (LA% = (Ai / Ao + Ai) × 100). Eff ects of sitting on the respiratory pattern, mechanics and work of breathing in mechanically ventilated patients F Ruiz-Ferron1, J Serrano-Simon2 1Complejo Hospitalario de Jaen, Spain; 2Hospital Reina Sofi a, Cordoba, Spain Critical Care 2014, 18(Suppl 1):P259 (doi: 10.1186/cc13449) 0.394 ± 0.118 l), inspiratory fl ow (0.74 ± 0.27 vs. 0.69 ± 0.23 l/second), inspiratory (0.97 ± 0.23 vs. 0.97 ± 0.28 seconds) and expiratory (1.43 ± 0.55 vs. 1.39 ± 0.44 seconds) times and respiratory frequency (27 ± 7 vs. 26 ± 7 bpm). Respiratory drive and eff ort tend to be higher in the sitting position, but not signifi cantly. P01: 3.7 ± 1.9 versus 3.0 ± 1.4 cmH2O, Δpes: 19 ± 10 versus 15 ± 8 cmH2O, PTPmin: 373 ± 192 versus 284 ± 162 cmH2O/secondminute. References Speelberg B, van Beers F: Artifi cial ventilation in the semi-recumbent position improves oxygenation and gas exchange [abstract]. Chest 2003 124:203S. 2. van Beers F, Speelberg B: Eff ect of body positioning in ventilated obese patients [abstract]. In 18th Annual Congress of the European Society of Intensive Care Medicine; Amsterdam; September 25-28 2005. 3. van Beers F, Speelberg B: The eff ect of semi-recumbency in ventilated morbid obese patients [abstract]. In 19th Annual Congress of the European Society of Intensive Care Medicine, Barcelona; September 24-27 2006. P256 j y H Yamamura, T Yamamoto, S Kaga, K Kaneda, Y Mizobata H Yamamura, T Yamamoto, S Kaga, K Kaneda, Y Mizobata Osaka City University, Osaka, Japan Critical Care 2014, 18(Suppl 1):P257 (doi: 10.1186/cc13447) P256 Ultrasound in the diagnosis of pneumothorax: a survey of current practice T Berlet, T Fehr, T Merz Inselspital/University Hospital Bern, Switzerland Critical Care 2014, 18(Suppl 1):P256 (doi: 10.1186/cc13446) y y, , p Critical Care 2014, 18(Suppl 1):P257 (doi: 10.1186/cc13447 Introduction Smoke inhalation injury (SII) is progress to pulmonary edema, pneumonia, and acute respiratory distress syndrome. SII may cause bronchial mucosal edema; we hypothesized that narrowing of luminal air bronchus due to bronchial wall edema correlated with respiratory deterioration of SII patients. Introduction The purpose of this study was to survey current practice for the use of lung ultrasound (LUS) in the diagnosis of pneumothorax. Figure 1 (abstract P256). Accuracy of LUS in pneumothorax. S93 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 The win ratio method: a novel hierarchical endpoint for pneumonia trials in patients on mechanical ventilation A Montgomery1, T Abuan1, M Kollef2 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) The win ratio method: a novel hierarchical endpoint for pneumonia trials in patients on mechanical ventilation A Montgomery1, T Abuan1, M Kollef2 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) Semi-upright position improves ventilation and oxygenation in mechanically ventilated intensive care patients F Van Beers, P Vos St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P258 (doi: 10.1186/cc13448) Semi-upright position improves ventilation and oxygenation in mechanically ventilated intensive care patients F Van Beers, P Vos St Elisabeth Hospital, Tilburg, the Netherlands Critical Care 2014, 18(Suppl 1):P258 (doi: 10.1186/cc13448) Introduction A semi-upright position (45° position) in ventilated patients is recommended to prevent ventilator-associated pneumonia (VAP) and is one of the fi rst steps in progressive early mobility. We studied ventilated intensive care patients in a semi-upright position compared with a supine position to explore whether there was an improvement of oxygenation and ventilation. 2 Conclusion A sitting position for mechanically ventilated patients increased the rigidity of chest wall and the respiratory system. The eff ects of this mobilization must be evaluated because some patients show higher respiratory drive and eff ort in this position. yg Methods We retrospectively studied 60 patients in a mixed medical, surgical, neurological ICU during 2003 and 2007 [1-3]. In this study the eff ects of 45° position on the peripheral oxygen saturation (SpO2) and the end-tidal carbon dioxide (ETCO2) were measured. Body position was changed with a Total Care® bed (Hill-Rom) after results for the supine position (10°) were obtained. Half an hour after the body position was changed, measurements were taken, which included SpO2 and ETCO2. The results of body position change in the individual patients were analysed with paired-samples t test. A signifi cance level <0.05 was considered signifi cant. P260 Physiologic comparison between NAVA, PAV+ and PSV in critically ill patients E Ak i ki G P i i ki E K dili D G l E Akoumianaki, G Prinianakis, E Kondili, D Georgopoulos University Hospital of Heraklion, Greece Critical Care 2014, 18(Suppl 1):P264 (doi: 10.1186/cc13454) y Methods We retrospectively reviewed charts of all patients admitted through the ED with a diagnosis of pneumonia requiring intubation in the fi rst 24 hours between January 2011 and November 2012. Patients were classifi ed as SC collected or not collected. We recorded demographic data, SC results, antibiotic choice and de-escalation, ventilator-free days (up to day 14), and mortality in ICU and hospital. Inferential statistics were performed using SPSS version 20.0, with P <0.05 considered signifi cant. Introduction The aim of the present study was to compare, in a group of diffi cult to wean critically ill patients, the short-term eff ects of PSV, PAV+ and NAVA on breathing pattern, patient eff ort and patient– ventilator interaction. Introduction The aim of the present study was to compare, in a group of diffi cult to wean critically ill patients, the short-term eff ects of PSV, PAV+ and NAVA on breathing pattern, patient eff ort and patient– ventilator interaction. Methods Seventeen patients were studied during NAVA, PAV+ and PSV with and without artifi cial increase in ventilator demands (challenge) using either dead space (DS, n = 10) or chest elastic load (CL, n = 7) application. Airway and transdiaphragmatic (Pdi) pressures, electrical activity of the diaphragm (EAdi), volume and fl ow were measured breath by breath, while inspiratory integral of Pdi (PTPPdi) and EAdi (EAdi) were calculated. i Results Of 50 patients we reviewed, 43 (86%) were intubated in the ED, 45 had SC ordered (only eight (18%) by ED physicians), and 37 (74%) had SC collected. There was no diff erence in age, gender or severity of illness as measured by APACHE score between the two groups. ICU mortality was lower in the SC collected group (24% vs. 69%, P = 0.007), as was hospital mortality (30% vs. 77%, P = 0.007) and antibiotics were de-escalated more often (89% vs. 8%, P <0.001). Patient with SC collected showed a trend toward signifi cantly more ventilator-free days (6.5 vs. 0, P = 0.053). Results At resting conditions all modes provided equal support as indicated by a similar PTPPdi per breath, per minute and per liter of ventilation. Failure to obtain admission sputum culture is associated with higher mortality and fewer ventilator-free days for intubated pneumonia patients: a quality improvement project f Conclusion The results of this study revealed that among the NIMV nonresponsive hypercapnic patients, nonventilatory factors such as thyroid dysfunction and increased dead space ventilation should be considered. Y Mahal, K Smith, R Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P261 (doi: 10.1186/cc13451) Introduction The primary objective was to assess the impact of failure to obtain sputum culture (SC) among patients requiring intubation for pneumonia. For patients admitted to an ICU with severe pneumonia, guidelines recommend obtaining a lower respiratory tract sample for culture. Our experience suggested this is rarely ordered from the emergency department (ED). Physiologic comparison between NAVA, PAV+ and PSV in critically ill patients E Ak i ki G P i i ki E K dili D G l Apart from triggering delay, which with and without the challenge was signifi cantly higher with PAV+ than that with NAVA and PSV, patient–ventilatory synchrony did not diff er among modes. Independent of challenging conditions, inspiratory eff ort to trigger the ventilator was signifi cantly higher with PAV+ than with NAVA and PSV. Compared with PSV, PAV+ and NAVA favored a more variable breathing pattern as indicated by the signifi cantly higher coeffi cient of variation of tidal volume (VT). CL increased PTPPdi signifi cantly less with PAV+ than with PSV and NAVA, while the increase of PTPPdi after DS did not diff er among modes. The relationship between VT and PTPPdi was weaker with NAVA (median (IQR) r2 = 25.6% (2.7 to 58.1%)) than with PAV+ (55.6% (34.4 to 61.6%)) and PSV (53.9% (23.2 to 77.4%)) on account of a poor EAdi–PTPPdi relationship (r2 = 16.2% (1.4 to 30.9%)) during NAVA. Conclusion Sputum cultures were rarely ordered by ED physicians, and when not obtained in intubated patients with pneumonia, ICU and hospital mortality was higher, there was less antibiotic de-escalation, and a trend toward fewer ventilator-free days. Eff orts to improve collection of sputum cultures in these patients are warranted. P264 Physiologic comparison between NAVA, PAV+ and PSV in critically ill patients Physiologic comparison between NAVA, PAV+ and PSV in criticall ill patients E Akoumianaki, G Prinianakis, E Kondili, D Georgopoulos University Hospital of Heraklion, Greece Critical Care 2014, 18(Suppl 1):P264 (doi: 10.1186/cc13454) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 These simulation results assumed results within pairs were uncorrelated. If a positive correlation for each endpoint within pairs is assumed, power for the win ratio endpoint increases. g y Results Among the 41 patients, 28 (68%) were classifi ed as Group 1 and 13 (32%) as Group 2. No diff erences were identifi ed among the ventilatory parameters of the two groups (P >0.05). Patients in Group 1 were younger, had higher admission PaCO2 levels, higher free T3 levels and ejection fraction in echocardiography (P <0.05). VD/VT values of Group 1 measured at admission and on the second and third days of NIMV were lower than Group 2 (P <0.05). Similarly, they had lower FEV1 percent predicted values in the stable period and on the fi rst and second days of NIMV (P <0.05). FEV1/FVC was lower in Group 1 when measured in the stable period and on the third and fourth days of NIMV. In Group 2, CRP values measured during the fi rst day (P = 0.014) and third day (P = 0.031) of NIMV were identifi ed as higher. In multivariate regression analysis, admission PaCO2 (OR: 1.59, 95% CI: 1.1 to 2.3, P = 0.014), free T3 (OR: 12, 95% CI: 1.51 to 101, P = 0.019), and VD/VT (OR: 1.23, 95% CI: 1.01 to 1.52, P = 0.048) were identifi ed as independent risk factors aff ecting NIMV success. Conclusion The win ratio method is both clinically meaningful and straightforward to explain. This method could provide a new approach to powering both superiority and non-inferiority trials of novel antibiotics. In particular, this method allows for well-powered phase 2 trials, and potentially decreases the required size of phase 3 trials. Reference 1. Pocock et al.: Eur Heart J 2012, 22:176-182. 1. Pocock et al.: Eur Heart J 2012, 22:176-182. P261 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A total of 41 patients were included in this prospective cohort study, who were followed for at least 96 hours in the ICU between January 2010 and November 2012 with the diagnosis of hypercapnic respiratory failure. Patients with ≥10 mmHg decrease in PaCO2 in the fi rst 72 hours were accepted as successful (Group 1) and those without this decrease were accepted as unsuccessful (Group 2). Among the patients with similar NIMV application characteristics, the eff ect of age, APACHE II score, infection, bronchospasm (daily respiratory function tests were performed with portable spirometry), heart failure, thyroid dysfunction and physiologic dead space ventilation (VD/VT) on success were evaluated. In statistical analysis, t test, Mann–Whitney U test and regression analysis were used. loser, or is a draw. The pair is fi rst compared on mortality; if no diff erence, then ventilator-free days (VFD) are compared. If the outcomes are the same for both endpoints, a draw results. Active versus placebo groups will then be compared using the win ratio, defi ned as the number of pairs in which the active group was the winner divided by the number of pairs that did not result in a draw. We examined sample size and power characteristics of the win ratio endpoint using trial simulations. Results Assuming a 15% 28-day mortality rate in the active arm and 20% in the control arm, to have 80% power with a two-tailed 0.05-level test for mortality would require 906 subjects per arm. Under the same assumptions with a diff erence in mean VFDs of 3 favoring the active arm (with common SD of 6), 130 subjects per arm provides 80% power. In approximately 32% of simulations, the win ratio result for each pair was determined by mortality. These simulation results assumed results within pairs were uncorrelated. If a positive correlation for each endpoint within pairs is assumed, power for the win ratio endpoint increases. p p g Results Assuming a 15% 28-day mortality rate in the active arm and 20% in the control arm, to have 80% power with a two-tailed 0.05-level test for mortality would require 906 subjects per arm. Under the same assumptions with a diff erence in mean VFDs of 3 favoring the active arm (with common SD of 6), 130 subjects per arm provides 80% power. In approximately 32% of simulations, the win ratio result for each pair was determined by mortality. The win ratio method: a novel hierarchical endpoint for pneumonia trials in patients on mechanical ventilation g y 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) 1Cardeas Pharma, Seattle, WA, USA; 2Washington University, St Louis, MO, USA Critical Care 2014, 18(Suppl 1):P260 (doi: 10.1186/cc13450) i Results Mean SpO2 supine was 96.55% ± SD 2.404 versus mean SpO2 semi-upright 97.4% ± SD 2.423, and mean ET-CO2 supine was 4.62% ± SD 1.988 versus mean ET-CO2 semi-upright 4.31% ± SD 1.060. The SpO2 (P <0.0001) and the ETCO2 (P <0.0001) improved signifi cantly for the 45° position compared with <10° position.i Introduction Development of novel antibiotics for VAP is hampered by the need for large phase 3 trials with mortality endpoints. With all- cause mortality rates decreasing, the large sample size for these trials, in particular for non-inferiority trials, has become a barrier to rapid drug development. Recently, a hierarchical approach to defi ning a composite endpoint for CV trials (win ratio method) has been described [1]. Conclusion We demonstrated a signifi cant increase in oxygen saturation and a signifi cant decrease in end-tidal CO2 when the head of the bed was elevated during mechanical ventilation. We believe positional therapy in intensive care patients is very important. The semi-upright position is an easy, eff ective and safe treatment in ICU patients. This position is eff ective in the bundle prevention of VAP, is the fi rst step in progressive early mobility and is also eff ective in oxygenation and ventilation in mechanically ventilated patients. This clinical benefi t of head-of-bed elevation >30° must lead to a standard of care in mechanically ventilated patients. Since 2009 the semi-upright position is a standard of care in our hospital. Methods We have adapted this approach in an ongoing superiority trial (Clinicaltrials.gov NCT01969799) comparing adjuvant use of a combination of fosfomycin and amikacin aerosol delivered by the PARI eFlow® Inline nebulizer in patients with Gram-negative pneumonia who are on mechanical ventilation. Both groups are receiving standard- of-care i.v. antibiotics. Patients from the active and placebo groups are matched in pairs based on presence of MDR Gram-negative bacteria, and disease severity by APACHE II score. Each pair has a winner and a S94 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Determining the mechanical ventilation mode and pressure support combination that is best compatible with the rapid shallow breathing index calculated in spontaneous ventilation Determining the mechanical ventilation mode and pressure support combination that is best compatible with the rapid shallow breathing index calculated in spontaneous ventilation S Demirtas Yilmaz, G Gürsel, M Aydogdu , , y g Gazi University Medical Faculty, Ankara, Turkey y y y Critical Care 2014, 18(Suppl 1):P267 (doi: 10.1186/cc13457) Introduction Weaning from the mechanical ventilation (MV) composes about 40 to 50% of the total length of MV. Besides, there is no single reliable parameter indicating that patient will tolerate extubation safely. The rapid shallow breathing index (RSBI) is relatively the best predictive parameter for the initial assessment of readiness for discontinuation of MV support. But evaluation of the RSBI is valuable during T-tube ventilation; and in clinical practice it is not always possible to perform this assessment. In this study, we aimed to determine the best MV mode and pressure combinations that can predict successful RSBI closest to values calculated in spontaneous ventilation and estimate the patients’ readiness for weaning. Results When we analyzed patients according to their body mass indexes (BMI), pH and pCO2 values of the patients with BMI ≤30 showed a greater improvement at all three measurements in the AVAPS compared with BIPAP (P <0.001). When patient compliance was examined, the number of patients regarded as comfortable in the BIPAP period was 20 (66.7%), but this fi gure was 25 (83.3%) for the AVAPS. Conclusion Patient comfort was higher and need for sedation was lower in AVAPS. According to the results obtained from this study, the AVAPS had positive eff ects on pH, gas variation and patient comfort; therefore, it can be confi dently used in clinical practice. Results When we analyzed patients according to their body mass indexes (BMI), pH and pCO2 values of the patients with BMI ≤30 showed a greater improvement at all three measurements in the AVAPS compared with BIPAP (P <0.001). When patient compliance was examined, the number of patients regarded as comfortable in the BIPAP period was 20 (66.7%), but this fi gure was 25 (83.3%) for the AVAPS. Methods In this prospective cohort study, 25 mechanically ventilated patients were included. After 24 hours of MV, if the patients can successfully pass the daily screening test a spontaneous breathing trial (SBT) was initiated. RSBI and other weaning parameters were calculated in diff erent combinations (PS:5 PEEP:5, PS:0 PEEP:5, PS:5 PEEP:0, PS:0 PEEP:0) before T-tube trial in all patients. Oxygenation index outperforms the P/F ratio for mortality prediction Oxygenation index outperforms the P/F ratio for mortality prediction K Davies1, C Bourdeaux1, T Peiris2, T Gould1 1Bristol Royal Infi rmary, Bristol, UK; 2University of Southampton, UK Critical Care 2014, 18(Suppl 1):P266 (doi: 10.1186/cc13456) p K Davies1, C Bourdeaux1, T Peiris2, T Gould1 1Bristol Royal Infi rmary, Bristol, UK; 2University of Southampton, UK Critical Care 2014, 18(Suppl 1):P266 (doi: 10.1186/cc13456) p K Davies1, C Bourdeaux1, T Peiris2, T Gould1 1Bristol Royal Infi rmary, Bristol, UK; 2University of Southampton, UK Critical Care 2014, 18(Suppl 1):P266 (doi: 10.1186/cc13456) Introduction The P/F ratio is widely used clinically and as part of research to categorise severity of respiratory failure [1]. However, no account is made of an important determinant of oxygenation; mean airway pressure (MAP). The oxygenation index (OI) incorporates the MAP and has been suggested as a more accurate means of determining severity of respiratory failure [2]. In addition, the optimal time for this assessment is unclear. We sought to answer these questions by analysing a large database of patient and ventilator data.i Conclusion Although there was a correlation between RSBI measured in the T-tube and RSBI measured in diff erent mode and pressure combinations, especially with the combination of PS:5 PEEP:0, a threshold value for RSBI cannot be detected during MV to predict SBT success. Does average volume-assured pressure support make any diff erence compared with BIPAP? f p G Canpolat, A Ozgultekin, G Turan, A Iskender, E Adıyeke, O Ekinci Haydarpasa Numune Teaching and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P265 (doi: 10.1186/cc13455) p g Conclusion Our analysis suggests that the OI is a more sensitive descriptor of the severity of respiratory failure than the P/F ratio and that this calculation should be performed using data from the fi rst 12 hours of ventilation. Introduction Average volume-assured pressure support (AVAPS) has been developed to ensure a more fi xed tidal volume along with the convenience and advantages of pressure support ventilation. In this study we compared the AVAPS with the BIPAP. References y p Methods Approval was obtained from the hospital’s ethics committee for the study. Thirty-three patients over 18 years of age with acute respiratory failure as a result of either internal or surgical reasons were included in the study. This study was conducted with the Philips V 60 ventilator, which includes both BIPAP and AVAPS mode. The implementation protocol for non-invasive ventilation (NIV) included, fi rstly, a 2-hour BIPAP application (Period Bi) and then, without inter- ruption, a 2-hour AVAPS application (Period AV). After measuring the basal blood gas analysis, patients were informed of how NIV would be practiced and what function it would have. In BIPAP mode, the ventilator parameters were adjusted as follows; EPAP: 5 to 7 cmH2O, IPAP: 15 to 20 cmH2O. Patient comfort was analyzed with a scale ranging from 0 to 2 (0: compatible, 1: medium-compatible, 2: noncompatible). During BIPAP ventilation, levels of arterial blood gases (pH, pO2, pCO2 and SPO2), comfort scale and hemodynamic data were recorded at the 30th minute, fi rst hour and second hour. After the patient was monitored for 2 hours in BIPAP mode, the mode was changed to the AVAPS by setting the required rates without removing the mask. EPAP settings were adjusted as follows for AVAPS: 5 to 7 cmH2O, Pmin to max: 10 to 25 cmH2O, tidal volume: 6 to 8 ml/kg. As in the BIPAP mode, we analyzed and recorded the rates of arterial blood gases, comfort scale and hemodynamic parameters at the 30th minute, fi rst hour and second hour. In case of agitation that prevents NIV, patients were sedated by dexmedetomidine. 1. Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, et al.: Acute respiratory distress syndrome: the Berlin Defi nition. JAMA 2012, 307:2526-2533. 1. Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, et al.: Acute respiratory distress syndrome: the Berlin Defi nition. JAMA 2012, 307:2526-2533. 2. Seeley E, McAuley DF, Eisner M, Miletin M, Matthay MA, Kallet RH: Predictors of mortality in acute lung injury during the era of lung protective ventilation. Thorax 2008, 63:994-998. 2. Seeley E, McAuley DF, Eisner M, Miletin M, Matthay MA, Kallet RH: Predictors of mortality in acute lung injury during the era of lung protective ventilation. Thorax 2008, 63:994-998. Nonventilatory factors aff ecting noninvasive mechanical ventilation success in hypercapnic critical care patients yp p p S Demir, Y Aldağ, M Aydogdu, G Gürsel yp p p S Demir, Y Aldağ, M Aydogdu, G Gürsel ğ y g Gazi University Medical Faculty, Ankara, Turkey Gazi University Medical Faculty, Ankara, Turkey y y y Critical Care 2014, 18(Suppl 1):P263 (doi: 10.1186/cc13453) Conclusion Compared with PSV proportional modes favored breathing variability, while in the face of changing respiratory system mechanics PAV+ might be superior. However, signifi cant drawbacks of both NAVA and PAV+ limit the eff ectiveness of these modes to proportionally assist the inspiratory eff ort. Critical Care 2014, 18(Suppl 1):P263 (doi: 10.1186/cc13453) Introduction The aim was to determine the nonventilatory factors that aff ect the noninvasive mechanical ventilation (NIMV) success in patients with hypercapnic respiratory failure. S95 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P265 In comparison with survivors, nonsurvivors were older, with higher OI and 24-hour SOFA scores and lower P/F ratios. The optimal time for calculation of both OI and P/F ratio for mortality prediction is the fi rst 12 hours of ventilation. The models using worst OI are better predictors of ICU and hospital mortality than those using worst P/F ratio (area under receiver operating curve 0.840 vs. 0.822). In comparison with survivors, nonsurvivors were older, with higher OI and 24-hour SOFA scores and lower P/F ratios. The optimal time for calculation of both OI and P/F ratio for mortality prediction is the fi rst 12 hours of ventilation. The models using worst OI are better predictors of ICU and hospital mortality than those using worst P/F ratio (area under receiver operating curve 0.840 vs. 0.822). P265 Does average volume-assured pressure support make any diff erence compared with BIPAP? G Canpolat, A Ozgultekin, G Turan, A Iskender, E Adıyeke, O Ekinci Haydarpasa Numune Teaching and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P265 (doi: 10.1186/cc13455) Does average volume-assured pressure support make any diff erence compared with BIPAP? Determining the mechanical ventilation mode and pressure support combination that is best compatible with the rapid shallow breathing index calculated in spontaneous ventilation Measurements in the spontaneous ventilation was performed with the COSMOPLUS Novometrix device that has both capnography and respiratory monitorisation function; other measurements were performed with ventilators. Conclusion Patient comfort was higher and need for sedation was lower in AVAPS. According to the results obtained from this study, the AVAPS had positive eff ects on pH, gas variation and patient comfort; therefore, it can be confi dently used in clinical practice. Results The mean age of the study group was 73 ± 10 years; 11 of them were female and mean APACHE II score was 19 ± 6. RSBI did not diff er signifi cantly between spontaneous mode and other combinations, but the best correlation with spontaneous mode was found with PS:5 PEEP:0 (P = 0.0001, r = 0.719), and the worst with PS:0 PEEP:5 combination. RSBI calculated in each combination showed no predictive value for weaning success. Respiration rate (f) was higher in the SBT failure group than the SBT success group. When measured at PS:0 PEEP:5 and PS:5 PEEP:0 combinations, the threshold value of f was found to be 27/minute (P = 0.03). P268 P268 New setting of neurally adjusted ventilatory assist during mask noninvasive ventilation F Longhini1, C Pan2, G Cammarota1, R Vaschetto1, J Xie2, L Liu2, Y Yian2, F Della Corte1, P Navalesi1, H Qiu2 1Università del Piemonte Orientale, Novara, Italy; 2Southeast University, Nanjing, China Critical Care 2014, 18(Suppl 1):P268 (doi: 10.1186/cc13458) P268 New setting of neurally adjusted ventilatory assist during mask noninvasive ventilation F Longhini1, C Pan2, G Cammarota1, R Vaschetto1, J Xie2, L Liu2, Y Yian2, F Della Corte1, P Navalesi1, H Qiu2 1Università del Piemonte Orientale, Novara, Italy; 2Southeast University, Nanjing, China Critical Care 2014, 18(Suppl 1):P268 (doi: 10.1186/cc13458) y g g Methods The ICU of the Bristol Royal Infi rmary has used an electronic clinical information system (CIS) since 2008, with every hour of care available for analysis as a result. Ventilated patients were identifi ed, the P/F ratio and OI were calculated and the worst values for these determined for four time periods (fi rst 12, 24, 36 and 48 hours of ventilation). Logistic regression analysis was used to create models to predict unit and hospital mortality. Results Data for over 150,000 hours of care in 4,886 patients was available for analysis. Excluding nonventilated patients and those transferred ventilated from another ICU, 2,156 patients provided data. Introduction Noninvasive ventilation through a mask is commonly applied in pressure support ventilation (nPSV). Recent studies S96 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 showed that noninvasive neurally adjusted ventilatory assist (nNAVA) improves patient–ventilator interaction and synchrony. More recently we described a new setting for nNAVA (nNAVA15) able to reduce the peak of electrical activity of the diaphragm (EAdipeak) and dyspnea (assessed by a visual analogue scale, VASd), compared with both nPSV and nNAVA, in patients undergoing NIV through a helmet, by improving the rate pressurization. We therefore designed a physiological study to evaluate and compare the eff ects of nNAVA15 with nPSV and nNAVA on VASd, EAdipeak, pressurization rate and arterial blood gases (ABGs). Methods Fourteen patients undergoing noninvasive ventilation because of acute respiratory failure underwent three randomized 30-minute trials: nPSV (inspiratory support above positive end- expiratory pressure (PEEP) ≥8 cmH2O, fastest rate of pressurization); nNAVA (NAVA level to achieve a comparable EAdipeak as during nPSV); nNAVA15 (NAVA level at 15 cmH2O/μV and the maximum inspiratory airway pressure (Paw) set at the value corresponding to PEEP + inspiratory support during nPSV). P268 Oxygen inspiratory fraction and PEEP remained unmodifi ed throughout the study period. The last minute of each trial was analyzed. Paw-time products of the initial 200 ms from the onset of ventilator pressurization (PTP200), of the initial 300 and 500 ms from the onset of the EAdi swing indexed to the ideal PTP (PTP300i and PTP500i, respectively), and of the triggering area (PTPt) were computed. ABGs and VASd were assessed at the end of each trial. Results nNAVA15 reduced VASd (3.0 (2.0; 3.0)), compared with both nPSV (5.0 (4.0; 5.2)) and nNAVA (4.0 (3.0; 5.0)) (P <0.001), without aff ecting ABGs and EAdipeak. nNAVA15 improved PTP300i and PTP500i (42% (32.5; 46.5) and 63% (54; 68)%, respectively) compared with nPSV (25 (4; 33)% and 44 (23; 52)%, P <0.001) and nNAVA (25 (20; 34)% and 46 (33; 57)%, P <0.001). PTP200 was lower in nNAVA (62 (46; 82) cmH2Osecond) with respect to both nPSV (87 (77; 112) cmH2Osecond) and nNAVA15 (85 (70; 127) cmH2Osecond) (P = 0.001). PTPt was signifi cantly improved by both nNAVA (–00.9 (–3.2; –0.2) cmH2Osecond) and nNAVA15 (–0.6 (–2.3; –0.2) cmH2Osecond) as opposed to nPSV (–9.4 (–12.3; –5.9) cmH2Osecond, P <0.001). Results hNAVA15 reduced the EAdipeak (10.2 (7.1; 16.2) μV) with respect to both hPSV (16.9 (12.7; 19.8) μV, P <0.001) and hNAVA (15.3 (10.7; 18.8) μV, P <0.001), while decreasing VASd (3.0 (3.0; 4.0) in hPSV, 3.0 (2.0; 4.0) in hNAVA and 1.0 (1.0; 2.0) in hNAVA15; P <0.01). PTP200 and PTP500i were improved by hNAVA15 (36 (28; 57) cmH2Osecond and 40 (30; 47)%, respectively) compared with hPSV (31 (24; 45) cmH2Osecond and 17 (9; 26)%, respectively) and hNAVA (23 (16; 30) cmH2Osecond and 23 (17; 37)%, respectively) (P <0.01). PTPt was lower in hNAVA15 (2.9 (1.6; 4.4) cmH2Osecond, P <0.01) compared with both hPSV and hNAVA, and lower in hNAVA (6.0 (2.7; 11.6) cmH2Osecond), compared with hPSV (18.5 (11.2; 22.5) cmH2Osecond, P <0.01). ABGs were no diff erent between trials. f Conclusion In comparison with hPSV and hNAVA, hNAVA15 signifi cantly reduced EAdipeak and VASd, improving the pressurization and triggering performance, without aff ecting ABGs. Reference 1. Cammarota G, et al.: Intensive Care Med 2011, 37:1943-1950. 1. Cammarota G, et al.: Intensive Care Med 2011, 37:1943-1950. P270 Is neurally adjusted ventilatory assist feasible during anesthesia? P268 F Campoccia Jalde, P Sackey, P Radell, M Wallin Karolinska University Hospital Solna, Sweden Critical Care 2014, 18(Suppl 1):P270 (doi: 10.1186/cc13460) 69 A new setting to improve noninvasive neurally adjusted ventilatory assist by helmet A new setting to improve noninvasive neurally adjusted ventilatory assist by helmet y F Longhini, G Cammarota, C Olivieri, R Perucca, R Vaschetto, D Colombo, A Messina, F Della Corte, P Navalesi Università del Piemonte Orientale, Novara, Italy Critical Care 2014, 18(Suppl 1):P269 (doi: 10.1186/cc13459) l Conclusion NAVA can be applied during propofol and sevofl urane anesthesia in pigs, with well-preserved Edi and spontaneous breathing. The natural variability is maintained with NAVA even during anesthesia. In contrast to sevofl urane, propofol sedation and anesthesia is associated with a high frequency of sighs and post-sigh apneas, probably due to a centrally induced mechanism. Our data warrant studies of NAVA in humans undergoing anesthesia and surgery when neuromuscular blockade is not required. Introduction Noninvasive neurally adjusted ventilatory assist by helmet (hNAVA) was shown to improve, compared with pressure support ventilation by helmet (hPSV), patient–ventilator interaction and synchrony in patients with acute respiratory failure without aff ecting peak electrical activity of the diaphragm (EAdipeak) [1]. Recently, a new helmet is available, which improves pressurization during hPSV. We propose a new setting of hNAVA (hNAVA15) to achieve further improvement. We compare hPSV, hNAVA and hNAVA15, all delivered using the new helmet, with respect to patient’s dyspnea, assessed by a visual analogue scale (VASd), arterial blood gases (ABGs), EAdipeak, rate of ventilator pressurization and triggering performance. Is neurally adjusted ventilatory assist feasible during anesthesia? F Campoccia Jalde, P Sackey, P Radell, M Wallin Karolinska University Hospital Solna, Sweden Critical Care 2014, 18(Suppl 1):P270 (doi: 10.1186/cc13460) Introduction Neurally adjusted ventilatory assist (NAVA) has so far been used in minimally sedated intensive care patients. NAVA has not been applied in patients in the operation room. The eff ect of diff erent sedatives/anesthetics on the electrical activity of the diaphragm (Edi) has not so far been studied. The aim of our study was to compare the eff ect of sevofl urane and propofol on the Edi signal and breathing pattern during sedation and anesthesia and also in combination with remifentanil. Methods A randomized cross-over study comparing sevofl urane and propofol sedation and anesthesia in 10 juvenile pigs. Remifentanil 0.1 μg/kg/minute was added after a period of anesthetic agent administration. The animals were ventilated with NAVA with fi xed level throughout the study. Respiratory variables were measured for the last 5 minutes of each 15-minute exposure. 2 Conclusion Compared with nPSV and nNAVA, nNAVA15 through a mask reduces VASd, assuring an optimal pressurization rate and triggering performance, without aff ecting the breathing pattern, neural drive and ABGs. Results The Edi signal and spontaneous breathing were preserved with both anesthetics. The breathing variability, expressed as the coeffi cient of variation (SD/mean) of the tidal volume (CVvt), was high with both drugs. CVvt was greater during with propofol than with sevofl urane (CVvt 32 vs. 18% during sedation and CVvt 23 vs. 14% during anesthesia). The frequency of sighs was higher with propofol both during sedation (29 vs. 12 sighs/hour) and anesthesia (21 vs. 1 sighs/hour) than with sevofl urane. P271 P271 Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial A Leme1, L Hajjar1, M Amato1, J Fukushima1, C Hashizume1, E Nozawa1, E Osawa1, R Nakamura1, J Almeida2, R Ianotti1, J Auler Jr1, F Galas1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P271 (doi: 10.1186/cc13461) P271 Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial A Leme1, L Hajjar1, M Amato1, J Fukushima1, C Hashizume1, E Nozawa1, E Osawa1, R Nakamura1, J Almeida2, R Ianotti1, J Auler Jr1, F Galas1 1Heart Institute, São Paulo, Brazil; 2Instituto do Cancer do Estado de São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P271 (doi: 10.1186/cc13461) Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial Using respiratory data from an animal study we suggest a classifi cation into resistance shape categories based on the slope of the R(V) profi les. g Results A total of 320 patients were enrolled in the study, 163 patients in the standard protocol group and 157 in the intensive alveolar recruitment group. Patients of the interventional group presented a higher incidence of pneumonia than patients for the control group (5 (3.3%) vs. 19 (22%), P = 0.004). The length of the hospital stay was shorter among patients receiving intensive alveolar recruitment than among those receiving standard care (10.9 (9.9 to 11.9) vs. 12.4 days (11.3 to 13.6); P = 0.045). There was no diff erence between groups according to extrapulmonary complications and mortality. i Methods Fifteen pigs with lavage-induced lung damage were ventilated at two PEEP levels (0 and 12 mbar) and three tidal volumes (8, 12 and 16 ml/kg bodyweight). Compliance (C(V)) and resistance (R(V)) profi les for each individual animal and ventilation setting were calculated from respiratory data using the gliding-SLICE method [3]. C(V) profi les were associated with one of the six suggested shape categories. The dependency of the mean R(V) slope of all animals on the ventilation setting was used as a basis for classifi cation into resistance shape categories. Resistance shape categories were compared with compliance shape categories for each individual animal to test whether similar PEEP suggestions result from both methods. y y Conclusion In this trial, an intensive alveolar recruitment protocol associated with a protective mechanical ventilation strategy reduced pulmonary complication and length of hospital stay in patients undergoing cardiac surgery (NCT01502332). 1. Futier E, Constantin JM, Paugam-Burtz C, et al.: A trial of intraoperative low- tidal-volume ventilation in abdominal surgery. N Engl J Med 2013, 369:428-437. 1. Futier E, Constantin JM, Paugam-Burtz C, et al.: A trial of intraoperative low- tidal-volume ventilation in abdominal surgery. N Engl J Med 2013, 369:428-437. 1. Futier E, Constantin JM, Paugam-Burtz C, et al.: A trial of intraoperative low- tidal-volume ventilation in abdominal surgery. N Engl J Med 2013, 369:428-437. Results Small PEEP and VT were typically associated with increasing C(V), and decreasing C(V) corresponds to large PEEP and VT. A classifi cation of each C(V) profi le into one of six compliance shape categories was possible. The shapes of the R(V) profi les of individual animals were remarkably similar. Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial Methods Fifteen patients underwent three randomized 30-minute trials: hPSV, set with an inspiratory support above positive end- expiratory pressure (PEEP) ≥10 cmH2O and the fastest rate of pressurization; hNAVA, setting the NAVA level to achieve the same EAdipeak as during hPSV; hNAVA15 setting the NAVA level at 15 cmH2O/ μV and the maximum inspiratory airway pressure (Paw) at the value corresponding to PEEP + inspiratory support during nPSV. Oxygen inspiratory fraction and PEEP remained unmodifi ed throughout the study period. Paw-time products of the initial 200 ms from the onset of ventilator pressurization (PTP200), of the initial 500 ms from the onset of the EAdi swing indexed to the ideal PTPaw (PTP500i), and of the triggering area (PTPt) were computed. ABGs and VASd were assessed at the end of each trial. Introduction Protective mechanical ventilation has been associated with lower incidence of pulmonary and extrapulmonary complications in major surgery. The aim of the present study is evaluate whether adding an intensive alveolar recruitment protocol improves clinical outcomes and reduces healthcare utilization in patients undergoing cardiac surgery. S97 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods In this single-center, parallel-group trial, we randomly assigned adult patients presenting signals of defi cient gas exchange (PaO2/FIO2 <250 at a PEEP of 5 cmH2O) in the immediate postoperative period to either intensive alveolar recruitment or a standard protocol, both using low-tidal volume ventilation (6 ml/kg/ibw). Our hypothesis was that an aggressive alveolar recruitment protocol will be translated to better lung compliance, better gas exchange, fewer pulmonary complications and reduced length of hospital stay when compared with the control group. PEEP titration on the basis of intratidal resistance–volume profi les S Buehler1, S Schumann1, M Lichtwarck-Aschoff 2, J Guttmann1 1University Medical Center Freiburg, Germany; 2Uppsala University, Uppsala, Sweden Critical Care 2014, 18(Suppl 1):P273 (doi: 10.1186/cc13463) Critical Care 2014, 18(Suppl 1):P273 (doi: 10.1186/cc13463) Introduction Lung-protective mechanical ventilation requires positive end-expiratory pressure (PEEP) and tidal volume (VT) to be chosen with regard to the individual state of the lung. The shape of the intratidal compliance–volume profi les might refl ect the state of the lung (atelectatic, open, overdistended) and could therefore be classifi ed into shape categories that translate into PEEP suggestions [1]. Intratidal resistance–volume profi les might refl ect intratidal opening and collapse of the lung [2]. References 1. Mols et al.: Intensive Care Med 1999, 25:1084-1091. 2. Mols et al.: Br J Anaesth 2001, 86:176-182. 3. Schumann et al.: Physiol Meas 2009, 30:13415. f Methods We investigated two groups of 13 rats each. Animals were ventilated with ZEEP or PEEP of 5 mbar, FiO2 of 1.0 and tidal volume of 10 ml/kg. A double low-fl ow manoeuvre was designed, consisting of two consecutive low-fl ow manoeuvres with a peak pressure of 30 mbar, interrupted by a 5-second plateau phase at diff erent pressures (2, 4, 8 and 12 mbar). Alveolar size at the peak pressures and during the plateau levels was analyzed from the recorded videos frame by frame. Therefore the alveolar outline of 10 alveoli was marked manually and the outlined area was calculated [2]. Compliance of tidal breaths before and after the manoeuvres was calculated using two-point compliance. Results In both groups, analysis of the alveolar area revealed that alveolar size at the second peak of the manoeuvre did not diff er signifi cantly compared with the fi rst peak (100.97% in ZEEP group, 102.37% in the PEEP group). During the plateau phases there was a slight increase in alveolar size at higher plateau pressures (slope of linear regression at plateau 4 mbar: 0.1 %/500 ms for ZEEP group, 0.18%/500 ms for PEEP group; at plateau 8 mbar: 0.42%/500 ms for ZEEP group, 0.565%/500 ms for PEEP group). After the manoeuvres, compliance increased to 137.73% in the ZEEP group and 119.91% in the PEEP group. 1. Mols et al.: Intensive Care Med 1999, 25:1084-1091. 2. Mols et al.: Br J Anaesth 2001, 86:176-182. 3. Schumann et al.: Physiol Meas 2009, 30:13415. 2. Schwenninger D, et al.: IEEE Trans Biomed Eng 2010, 57:415-421. 1. Schwenninger D, et al.: J Biomed Opt 2011, 16:046002. 1. Futier E, Constantin JM, Paugam-Burtz C, et al.: A trial of intraoperative low- tidal-volume ventilation in abdominal surgery. N Engl J Med 2013, 369:428-437. P274 P274 US study of gliding in nondependent lung regions: the dark side of the moon E De Blasio1, M Venditto1, A Federico1, G Azan1, C Pellegrini1, C Di Maria1, P De Luca1, G Rossi2 1Hospital G. Rummo, Benevento, Italy; 2Hospital ‘San Giovanni Battista’, Turin, Italy Critical Care 2014, 18(Suppl 1):P274 (doi: 10.1186/cc13464) Intensive alveolar recruitment after cardiac surgery: a randomized controlled clinical trial The R(V) slope was typically largest for a PEEP and VT setting at which derecruitment was likely and smallest where overdistension was likely. Based on this a classifi cation scheme was defi ned: 10 <slope <21 mbar s/L2 (category 1, ‘increase PEEP’), slope ≥21 mbar s/L2 (category 2, ‘keep PEEP’) and slope ≤10 mbar s/L2 (category 3, ‘decrease PEEP’). Comparison of resistance and compliance shape categories for single animals shows a good correlation. US study of gliding in nondependent lung regions: the dark side of the moon y Critical Care 2014, 18(Suppl 1):P274 (doi: 10.1186/cc13464) Introduction A protective ventilatory strategy should prevent VILI, but in patients with larger nonaerated areas hyperinfl ation may occur during tidal ventilation even during a protective ventilatory strategy [1]. The gliding sign is used as a marker of pneumothorax and, in a study [2], to quantify preoperatively the degree of pleural adhesion in thoracic surgery patients. In our study we measured the variations of gliding (G) and static compliance (Cstat) according to incremental/ decremental variations of PEEP in patients with hypoxic respiratory failure. Conclusion In the healthy lung, once recruited, alveoli stay stable in size over wide pressure ranges. Further recruitment manoeuvres do not lead to further increase of alveolar size, but increase of compliance. During plateau phases, alveolar size increases dependent on pressure. This leads to the assumption that recruitment is not only pressure dependent but also time dependent. Methods Ten patients with hypoxic respiratory failure (P/F <300) were ventilated in VCV (Vt of 7 ml/kg, FiO2 100%, RR 10/minute); keeping Vt constant, PEEP was gradually increased from ZEEP to 22 cmH2O, unless there was occurrence of hypotension or SpO2 <90% or Pplat >45 cmH2O or G no more visible, and then similarly reduced from 22 cmH2O to ZEEP. The gliding was assessed at six points of intercostal P272 P272 Endomicroscopic analysis of time-dependent and pressure- dependent recruitment of subpleural alveoli H Runck, D Schwenninger, S Schumann, J Haberstroh, J Guttmann University Medical Center, Freiburg, Germany Critical Care 2014, 18(Suppl 1):P272 (doi: 10.1186/cc13462) Conclusion Resistance shape categories might provide additional guidance for PEEP setting. Combining compliance and resistance shape categories could improve lung-protective ventilation. References Introduction We used transthoracic endoscopy [1] to continuously record images of subpleural alveoli during recruitment manoeuvres and diff erent steady plateau pressures between the manoeuvres. References 1. Chiumello D, et al.: Crit Care Med 2013. [Epub ahead of print] 2. Karbing DS, et al.: Med Eng Phys 2011, 33:240-248. 1. Chiumello D, et al.: Crit Care Med 2013. [Epub ahead of print] 2. Karbing DS, et al.: Med Eng Phys 2011, 33:240-248. 1. Chiumello D, et al.: Crit Care Med 2013. [Epub ahead of print] 2. Karbing DS, et al.: Med Eng Phys 2011, 33:240-248. References 1. Terragni PP, et al.: Am J Respir Crit Care Med 2007, 175:160-166. 2. Masato S, et al.: Ann Thorac Surg 2005, 80:439-442. 1. Terragni PP, et al.: Am J Respir Crit Care Med 2007, 175:160-166. 2. Masato S, et al.: Ann Thorac Surg 2005, 80:439-442. p ρ Results PEEP improved V/Q in four patients, shunt reducing 7 to 42% with no/small increase in ΔAcPCO2. Two deteriorated, with large ΔAcPCO2 or shunt increase. No systematic changes in ΔAcPO2 were seen. Figure 1 shows response to PEEP in two patients. Changes in nonaerated regions and shunt were correlated (ρ = 0.94, P = 0.002). No correlations were found between poorly aerated regions and ΔAcPO2 (ρ = –0.09, P = 0.84) or hyperinfl ated regions and ΔAcPCO2 (ρ = 0.07, P = 0.88). P276 P276 Changes in computed tomography and ventilation/perfusion mismatch with positive end-expiratory pressure DS Karbing1, M Panigada2, N Bottino2, E Spinelli2, A Protti2, SE Rees1, L Gattinoni2 1Aalborg University, Aalborg, Denmark; 2Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P276 (doi: 10.1186/cc13466) spaces bilaterally and the movement of a hyperechoic point of pleura or a b-line was observed during tidal ventilation. For each step, the excursion of G during the inspiratory phase was measured and compared with the Cstat values. Statistical analysis was performed with the Pearson correlation coeffi cient (PCC). Introduction The purpose was to compare eff ects of PEEP on computed tomography (CT) and estimated ventilation/perfusion (V/Q) mismatch. Previously, an oxygenation-based method was shown more related to the CT-measured eff ect of PEEP than lung mechanics [1], indicating lung aeration is better quantifi ed using V/Q mismatch. Pulmonary shunt and low and high V/Q mismatch can be estimated from varying FIO2 and measuring ventilation and blood gas contents [2]. Results All patients completed the study without adverse events. In all patients we observed a reduction of G and Cstat at the increase of PEEP and specularly an increase of G and Cstat during the reduction of PEEP (Figure 1). In fi ve patients at the lower levels of PEEP (from 0 to 10) an increase of Cstat and G was observed. For all patients the PCC of Cstat and G and was >0.5 (P <0.03), ranging from 0.537 (P = 0.017) to 0.964 (P <0.0001). 2 Methods Preliminary results in six ARDS patients. CT scans were taken in static conditions at PEEP 5, 45 and 15 to 20 cmH2O. V/Q was estimated at 5 and 15 to 20 cmH2O as: shunt, low V/Q as alveolar to lung capillary PO2 diff erence (ΔAcPO2), high V/Q as alveolar to lung capillary PCO2 diff erence (ΔAcPCO2) [2]. Nonaeration, poor aeration, and normal aeration plus hyperinfl ation were calculated from Hounsfi eld units. Aeration and V/Q were compared (Pearson, ρ). Conclusion The variations of G at diff erent levels of PEEP are consensual with those of Cstat. The study of G during tidal ventilation could help to identify hyperinfl ation in nondependent lung regions and to optimize lung-protective ventilatory strategies. Protective ventilation reduces bacterial growth and lung injury in a porcine pneumonia model J Sperber1, A Nyberg1, M Lipcsey2, A Larsson2, J Sjölin2, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Uppsala, Sweden; 2Uppsala University, Uppsala, Sweden Critical Care 2014, 18(Suppl 1):P275 (doi: 10.1186/cc13465) 2 Conclusion In these preliminary cases, changes in shunt and nonaerated tissue correlated well. However, results indicate poor agreement between changes in low and high V/Q and lung morphology. References Introduction Bacterial pneumonia is a common indication for mechanical ventilation in the ICU. Ventilation with high positive end- expiratory pressure (PEEP) and low tidal volume (VT) is recommended in patients with adult respiratory distress syndrome. This improves clinical outcome compared with ventilation with low PEEP and medium-high VT [1]. However, the eff ect of VT and PEEP on bacterial growth in lung tissue is not known. This study contrasted the eff ect of a protective ventilator protocol with a standard medium-high VT and lower PEEP protocol on lung bacterial growth, lung edema formation and lung injury. It was performed in a porcine model of intensive care with the frequently found pathogen Pseudomonas aeruginosa. p References 1. Schwenninger D, et al.: J Biomed Opt 2011, 16:046002. 2. Schwenninger D, et al.: IEEE Trans Biomed Eng 2010, 57:415-421. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S98 Reference 1. N Engl J Med 2000, 342:1301-1308. P276 Changes in computed tomography and ventilation/perfusion mismatch with positive end-expiratory pressure DS Karbing1, M Panigada2, N Bottino2, E Spinelli2, A Protti2, SE Rees1, L Gattinoni2 1Aalborg University, Aalborg, Denmark; 2Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P276 (doi: 10.1186/cc13466) Table 1 (abstract P275). P. aeruginosa log mean, wet to dry ratio, and PaO2/ FiO2 at 5 hours P. aeruginosa (cfu/g) W/D PaO2/FiO2 at 5 hours (mmHg) Prot-V 3.5 ± 0.7 1.7 ± 0.2 434 ± 62 Control 4.2 ± 0.7 2.7 ± 1.3 343 ± 61 Data presented as mean ± SD. Figure 1 (abstract P274). PCC between mean G (cm) and mean Cstat at diff erent PEEP, in axis Pplat. Table 1 (abstract P275). P. aeruginosa log mean, wet to dry ratio, and PaO2/ FiO2 at 5 hours Reference 1. N Engl J Med 2000, 342:1301-1308. Table 1 (abstract P275). P. aeruginosa log mean, wet to dry ratio, and PaO2/ FiO2 at 5 hours P. aeruginosa (cfu/g) W/D PaO2/FiO2 at 5 hours (mmHg) Prot-V 3.5 ± 0.7 1.7 ± 0.2 434 ± 62 Control 4.2 ± 0.7 2.7 ± 1.3 343 ± 61 Data presented as mean ± SD. Table 1 (abstract P275). P. aeruginosa log mean, wet to dry ratio, and PaO2/ FiO2 at 5 hours Figure 1 (abstract P274). PCC between mean G (cm) and mean Cstat at diff erent PEEP, in axis Pplat. P. aeruginosa (cfu/g) W/D PaO2/FiO2 at 5 hours (mmHg) Prot-V 3.5 ± 0.7 1.7 ± 0.2 434 ± 62 Control 4.2 ± 0.7 2.7 ± 1.3 343 ± 61 Data presented as mean ± SD. P277 Gas exchange and CT for patient improving (A) or worsening (B) with PEEP change. 494 ICUs around the world [1]. The Dutch cohort covered 196 patients and the global cohort 7,952. compliance. We present a new graphical user interface (GUI) that supports the clinician to titrate the PEEP level by means of a shape category analysis of the CV curve. Results Vt was 7.6 ml/kg predicted bodyweight in the Dutch cohort versus 8.0 ml/kg and the median applied PEEP was 8 cmH2O versus 5.8 cmH2O (both P <0.01). In the subgroup of patients with ARDS, Vt ml/kg was 7.6 and applied PEEP 8.8 cmH2O in the Netherlands versus 8.6 ml/kg (P = 0.41) and 8.3 cmH2O worldwide. In the Netherlands 7.1% of admitted patients received NIV as fi rst mode of mechanical ventilation versus 15% in the global cohort. In both cohorts 18% of patients were hypercapnic at ICU admission. Fewer patients in the Dutch cohort showed an ICU-acquired pneumonia (4.1 vs. 9.4%, P = 0.007) and sepsis (5.1 vs. 9.0%, P = 0.042), but more patients evolved delirium (16 vs. 5%, P <0.01). Methods A decision support system in the form of a computer-based GUI was developed and tested using respiratory data obtained from patients of the University Medical Center Freiburg. The new clinician- oriented approach provides a breath-by-breath visualization of the patient’s individual intratidal CV curve. The dynamic compliance was analyzed using the gliding-SLICE method [1]. The resulting intratidal CV curve was classifi ed into one of six shape categories according to the defi nition from Mols and colleagues [2]. The actual shape category of the CV curve indicates whether PEEP setting should be changed or whether the volume range of maximal compliance is reached. g p Results The GUI provided a breath-by-breath visualization of the intratidal CV curve and the intuitive individual compliance shape category. Based on the compliance shape category, diff erent guidelines of PEEP titration were applied with the objective of ventilating the patient mechanically within the range of maximal compliance. Conclusion According to our hypothesis, Vt was smaller and applied PEEP was higher in the Dutch cohort. Patients in both cohorts received larger Vt than recommended in prevention of ARDS [2]. Hypercapnia is a main criterion for the use of NIV [3], which suggests that NIV could be used more often in the Netherlands. References 1. Esteban et al.: Am J Respir Crit Care Med 2013, 188:220-230. 1. Esteban et al.: Am J Respir Crit Care Med 2013, 188:220 230. 2. The Acute Respiratory Distress Syndrome Network: N Engl J Med 2000, 342:1301-1308. 3. Brochard: Eur Respir J 2003, 22:31s-37s. p , 2. The Acute Respiratory Distress Syndrome Network: N Engl J Med 2000, 342:1301-1308. P277 The lower incidence of delirium worldwide could be caused by diff erences in sedation or may be due to the used methods of screening. Conclusion The newly developed GUI allows a breath-by-breath visualization of the intratidal CV curve with high reliability. Automated classifi cation of the intratidal CV curve into compliance shape categories provides the rationale basis for patient-individual PEEP titration. References 1. Schumann S, et al.: Physiol Meas 2009, 30:1341-1356. 2. Mols G, et al.: Intensive Care Med 1999, 25:1084-1091. 1. Schumann S, et al.: Physiol Meas 2009, 30:1341-1356. 2. Mols G, et al.: Intensive Care Med 1999, 25:1084-1091. 1. Schumann S, et al.: Physiol Meas 2009, 30:1341-1356. 2 M l G t l I t i C M d 1999 25 1084 1091 P278 Graphical user interface for visualization of a decision support system for PEEP titration S Lozano-Zahonero, S Buehler, S Schumann, J Guttmann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P278 (doi: 10.1186/cc13468) P277 Ventilator settings in ICUs: comparing a Dutch with a global cohort M Van IJzendoorn1, M Koopmans1, U Strauch2, S Heines2, S Den Boer3, B Kors4, P Van der Voort5, P Dennesen6, I Van den Hul7, E Alberts7, P Egbers1, A Esteban8, F Frutos-Vivar8, M Kuiper1 1Medisch Centrum Leeuwarden, the Netherlands; 2Universitair Medisch Centrum Maastricht, the Netherlands; 3Spaarneziekenhuis, Hoofdorp, the Netherlands; 4Kennemer Gasthuis, Haarlem, the Netherlands; 5Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands; 6Medisch Centrum Haaglanden, Den Haag, the Netherlands; 7Vrije Universiteit Medisch Centrum, Amsterdam, the Netherlands; 8Hospital Universitario de Getafe, Madrid, Spain Critical Care 2014, 18(Suppl 1):P277 (doi: 10.1186/cc13467) Methods Sixteen pigs were anesthetized and randomized to mechanical ventilation with two diff erent ventilator settings for 6 hours; Prot-V (PEEP 10 cmH2O, VT 6 ml/kg, n = 8) and Control (PEEP 5 cmH2O, VT 10 ml/kg, n = 8). At 0 hours, 1×1011 colony-forming units (cfu) of P. aeruginosa were instilled intratracheally. At the end of the experiment, six postmortem lung biopsies from predefi ned declivial locations were taken from each animal for cultures and weight measurements. Introduction In data collected during the Third International Study on Mechanical Ventilation, we compared data from the Netherlands with a global cohort. We hypothesized that tidal volumes (Vt) were smaller and applied positive end-expiratory pressure (PEEP) was higher in the Netherlands, compared with the global cohort. We also compared use of non-invasive ventilation (NIV) and outcomes in both cohorts. Methods A post-hoc analysis of a prospective observational study of patients receiving invasive mechanical ventilation was conducted in Results P. aeruginosa growth in lung tissue and wet to dry ratio were lower in the Prot-V group than in the Control group (P <0.05 and P <0.05). PaO2/FiO2 was higher in the Prot-V group than in the Control group (P <0.05) (Table 1). Conclusion Protective ventilation with low VT and higher PEEP reduces P. aeruginosa growth in lung tissue, lung edema formation and lung injury in contrast with medium-high VT and lower PEEP ventilation in this porcine pneumonia model. Methods A post-hoc analysis of a prospective observational study of patients receiving invasive mechanical ventilation was conducted in Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S99 Figure 1 (abstract P276). Gas exchange and CT for patient improving (A) or worsening (B) with PEEP change. Figure 1 (abstract P276). Gas exchange and CT for patient improving (A) or worsening (B) with PEEP change. 6). P279 The aim of the study was to show time- dependent diff erences in the induction of apoptosis due to mechanical overload, by fl uorescence microscopic observations. in SF6 retention at the end of inspiration and a return to baseline during expiration similar to the changes observed in pigs with ALI at PEEP0. In ALI at PEEP15, shunt decreased throughout inspiration and returned to baseline levels during expiration. Conclusion Serial dynamic pulmonary shunt measurements during mechanical ventilation showed distinct variations over the respiratory cycle in both healthy and injured lungs. Increasing PEEP from 0 to 15 cmH2O altered the patterns of dynamic pulmonary shunt before and after ALI. Thus, serial assessment of dynamic pulmonary shunt fraction by SF6 retention with MMIMS-MIGET could prove useful for optimization of mechanical ventilation in healthy and injured lungs. l Methods Alveolar epithelial cells (A549) were grown on RGD-coated, highly fl exible polydimethyl siloxane membranes. After becoming approximately 100% confl uent, cells were stained with Hoechst 33342/TMRE/caspase-3 and caspase-7/PI and continuously observed by fl uorescence microscopy. The cells were stimulated either with ventilation-analogue or spontaneous-breathing analogue profi le [1]. For both settings the frequency was 0.25 Hz, the surface increase 20% and the cell monolayers were stimulated over a time period of 2 hours in the bioreactor [2].l P281 P281 Eff ect of positive end-expiratory pressure on right ventricle function assessed by speckle tracking echocardiography SR Orde, A Behfar, PG Stalboerger, JK Oh, GC Kane Mayo Clinic, Rochester, MN, USA Critical Care 2014, 18(Suppl 1):P281 (doi: 10.1186/cc13471) Results The analysis of fl uorescence microscopic images showed the fi rst evidence of apoptosis induction after 40 minutes of ventilation-analogue stimulation. In contrast, apoptosis induction after spontaneous-breathing analogue stimulation occurred after 90 minutes. Introduction We sought to determine in a swine model whether a novel echocardiography method, speckle tracking echocardiography (STE), could determine deterioration in right ventricle (RV) function induced by escalating levels of positive end-expiratory pressure (PEEP), and to compare STE with RV fractional area change (FAC). Acute cor pulmonale and hypotension can be induced by high levels of PEEP used in the management of acute respiratory distress syndrome [1]. Quantifying RV function by echocardiography can be challenging due to its shape and position. References 1. Luecke T, et al.: Crit Care 2005, 9:607-621. 1. Luecke T, et al.: Crit Care 2005, 9:607 621. 2. Huang SJ, et al.: Curr Opin Crit Care 2013, 19:250-257. 2. Huang SJ, et al.: Curr Opin Crit Care 2013, 19:250-2 g p 3. Fine NM, et al.: Circ Cardiovasc Imaging 2013, 6:711-721. 3. Fine NM, et al.: Circ Cardiovasc Imaging 2013, 6:711-721. Methods Pigs (n = 10) were anesthetized and ventilated at a tidal volume of 8 ml/kg and two levels of PEEP (0 and 15 cmH2O – conditions: PEEP0; PEEP15). Hemodynamic, respiratory and multiple inert gas analysis by micropore membrane inlet mass spectrometery (MMIMS- MIGET; Oscillogy, USA) measurements were taken at PEEP0 and PEEP15 before and after saline washout. Retention of SF6, measured fi ve times during one breathing cycle, was taken as a refl ection of shunt fraction. MIGET sample acquisition was synchronized by electrical impedance tomography. P279 STE is a relatively new, feasible, sensitive, angle-independent method for describing cardiac deformation (strain) [2] and is particularly useful in analyzing RV function (RV free wall strain, RVfwS), as has been shown in pulmonary hypertension cohorts [3]. Conclusion The observation of cells during stimulation with continuous fl uorescence microscopic imaging allowed us to analyse the time- dependent induction of apoptosis under the aspects of diff erent stimuli. We could prove our hypothesis that ventilation-analogue stimulation was worse for the cellular viability then spontaneous- breathing analogue stimulation. In future, the direct optical tracking of cell damage should allow one to analyse other stimulation profi les as well, and thereby to improve the stimulation profi les and ultimately the ventilation profi le as well. P279 Time-dependent apoptosis induction after spontaneous-breathing or ventilation-analogue experimental mechanostimulation of monolayer lung cell cultures S Meyer, S Schumann, J Guttmann, K Gamerdinger University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P279 (doi: 10.1186/cc13469) y S Lozano-Zahonero, S Buehler, S Schumann, J Guttmann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P278 (doi: 10.1186/cc13468) y S Lozano-Zahonero, S Buehler, S Schumann, J Guttmann University Medical Center Freiburg, Germany Critical Care 2014, 18(Suppl 1):P278 (doi: 10.1186/cc13468) Introduction The analysis of dynamic volume-dependent compliance provides the rationale basis for PEEP titration during mechanical ventilation. Due to its volume dependence, the compliance of the respiratory system is nonlinear within each single breath (intratidal), which is refl ected in the compliance–volume curve (CV curve). The shape of the CV curve is determined by the PEEP level. The mechanical stress for the mechanically ventilated lung is expected to be minimal when the lung is ventilated within the volume range of maximal Introduction The cyclic strain of lung tissue during mechanical ventilation compared with spontaneous breathing is associated with a largely increased mechanical load due to larger pressure amplitudes and higher acceleration forces. This additional load may change tissue mechanics and may lead to tissue damage. Although experimental mechanostimulation in vitro is a widely used method Introduction The cyclic strain of lung tissue during mechanical ventilation compared with spontaneous breathing is associated with a largely increased mechanical load due to larger pressure amplitudes and higher acceleration forces. This additional load may change tissue mechanics and may lead to tissue damage. Although experimental mechanostimulation in vitro is a widely used method S100 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 to analyse mechanical load on cells or tissue, in vitro experiments with ventilation-analogue stimulation are missing. In this work we compare for the fi rst time the changes of monolayer lung cells after ventilation-analogue stimulation with spontaneous-breathing analogue mechanostimulation. The aim of the study was to show time- dependent diff erences in the induction of apoptosis due to mechanical overload, by fl uorescence microscopic observations. to analyse mechanical load on cells or tissue, in vitro experiments with ventilation-analogue stimulation are missing. In this work we compare for the fi rst time the changes of monolayer lung cells after ventilation-analogue stimulation with spontaneous-breathing analogue mechanostimulation. p References e e e ces 1. Gamerdinger et al.: Acta Bioeng Biomech 2012, 14:53-62. 2. Schumann et al.: J Biomed Mater Res B Appl Biomater 2013, 101:1164-1171. 1. Gamerdinger et al.: Acta Bioeng Biomech 2012, 14:53-62. 2. Schumann et al.: J Biomed Mater Res B Appl Biomater 2013, 101:1164-1171. 1. Gamerdinger et al.: Acta Bioeng Biomech 2012, 14:53-62. 2. Schumann et al.: J Biomed Mater Res B Appl Biomater 2013, 101:1164-1171. Methods Ten pigs, 40 to 90 kg, anaesthetized, fully mechanically ventilated at 6 to 8 mg/kg were subject to stepwise escalating levels of PEEP at 2-minute intervals (0, 5, 10, 15, 20, 25 and 30 cmH2O). RV images were obtained using intracardiac echocardiography (for optimal frame- rate and endocardial defi nition) and were analyzed offl ine for FAC and RVfwS (using Velocity Vector Imaging; Siemens). Infl uence of positive end-expiratory pressure on cyclic recruitment and derecruitment during one breathing cycle in porcine acute lung injury g y g g Results Escalating levels of PEEP were strongly associated with signifi cant reductions in mean blood pressure (R2 = 0.8, P <0.0001), FAC (R2 = 0.8, P <0.0001) and RVfwS (R2 = 0.9, P <0.001). Paired t tests indicated signifi cant reductions in RVfwS with each step increase in PEEP. FAC only showed signifi cant deterioration at 15 cmH2O PEEP. Signifi cant hypotension (a decrease of more than 20 mmHg) occurred after 10 cmH2O PEEP. RVfwS decreased by a larger extent and earlier than FAC and mean blood pressure with increasing levels of PEEP. j y IS Sulyok1, A Johannes1, S Böhme1, KU Klein1, J Baumgardner2, O Kimberger1, K Markstaller1, R Ullrich1 1Medical University of Vienna, Austria; 2Oscillogy LLC, Folsom, PA, USA Critical Care 2014, 18(Suppl 1):P280 (doi: 10.1186/cc13470) Introduction Cyclic recruitment and derecruitment (R/D) of lung parenchyma during mechanical ventilation are responsible for atelectrauma. Theoretically, cyclic R/D can lead to varying degrees of pulmonary shunt fraction throughout the respiratory cycle. Measurements of dynamic pulmonary shunt fraction could help in assessing the degree of cylic R/D. However, common methods of measuring pulmonary shunt do not allow for dynamic serial measurements during one respiratory cycle. In this study, we measured serial dynamic pulmonary shunt fractions during one breathing cycle and investigated the eff ect of positive end-expiratory pressure (PEEP) on cyclic R/D in artifi cially ventilated pigs before and after saline washout induced acute lung injury. p g Conclusion RVfwS measured by STE is a sensitive method for determining RV dysfunction induced by PEEP. RVfwS displays a stronger association, greater deterioration and earlier reduction than FAC and mean blood pressure with escalating levels of PEEP. This potentially has interesting implications for the role of STE in managing PEEP levels in critically ill patients with acute lung injury. P283 Experimental VILI begins with subpleural lung lesions h We studied the development i p gy p Methods In fi ve piglets (weight 21 ± 2 kg) under general anesthesia, we recorded PV curves to quantify dynamic dissipated energy (DE) at increasing tidal volume (TV) (150, 300, 450, 600, 750, 900) and at increasing respiratory rate (RR) (3, 6, 9, 12, 15). We then recorded PV curves for the same TV infl ated with a super-syringe (100 ml), to quantify static DE. We also quantifi ed airway DE connecting the postmortem isolated tracheobronchial tree to the ventilator and recording PV curves at the respiratory setting previously described. of VILI with CT scan to determine where the fi rst lung lesion developed. Methods Piglets were sedated, orotracheally intubated and instrumented with arterial and central venous catheter and urinary catheter. The whole study was performed in the animal CT scan facility, which was equipped as an ICU, and CT scan was performed every 3 hours or if respiratory parameters (plateau/peak pressure) changed. We defi ned as new lesion lung regions the appearance of poorly infl ated/not infl ated lung regions not present in the previous CT scan image. We select the fi rst CT scan in which a relevant number of new lesions appeared (>15) and manually delineated the lesions; the lesions were classifi ed as close/not close to the pleural surface. Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). i Results Six piglets were studied (22 ± 8 kg) that were ventilated with a TV of 750 ± 71 ml (41 ± 1 ml/kg) up to development of VILI, defi ned radiologically as infi ltrates present in all pulmonary fi elds at CT scan plus development of lung edema. In the fi rst CT scan where lesions appeared, a median of 28 lesions (IQ range 22 to 30) were present. Of these lesions, 18 (17 to 22) (72%) were located near the pleura and nine (6 to 11) (28%) near vessels/bronchi. See Figure 1. Conclusion In an experimental model of VILI the fi rst lung lesions appear below the pleural surface. P283 Experimental VILI begins with subpleural lung lesions h Introduction During mechanical ventilation some of the energy delivered to the respiratory system (RS) is dissipated within it, while some is recovered during expiration. The amount of unrecovered energy represents mechanical work done on the RS by the ventilator and may be related to the development of ventilatory-induced lung injury (VILI). The unrecovered energy is measured as the hysteresis area of the pressure–volume (PV) curve in static and dynamic conditions. We explored how and where the energy is dissipated inside the RS. Introduction During mechanical ventilation some of the energy delivered to the respiratory system (RS) is dissipated within it, while some is recovered during expiration. The amount of unrecovered energy represents mechanical work done on the RS by the ventilator and may be related to the development of ventilatory-induced lung injury (VILI). The unrecovered energy is measured as the hysteresis area of the pressure–volume (PV) curve in static and dynamic conditions. We explored how and where the energy is dissipated inside the RS. Methods In fi ve piglets (weight 21 ± 2 kg) under general anesthesia, we recorded PV curves to quantify dynamic dissipated energy (DE) at increasing tidal volume (TV) (150, 300, 450, 600, 750, 900) and at increasing respiratory rate (RR) (3, 6, 9, 12, 15). We then recorded PV curves for the same TV infl ated with a super-syringe (100 ml), to quantify static DE. We also quantifi ed airway DE connecting the postmortem isolated tracheobronchial tree to the ventilator and recording PV curves at the respiratory setting previously described. Introduction We developed an experimental model of VILI, ventilating piglets at a strain (TV/FRC) >2.5. It is possible that lung inhomogeneities act as stress raisers within the lung parenchyma, locally multiplying pressures. In a healthy lung the pleural surface, vessels and bronchi are detected as natural lung inhomogeneities. We studied the development i Introduction We developed an experimental model of VILI, ventilating piglets at a strain (TV/FRC) >2.5. It is possible that lung inhomogeneities act as stress raisers within the lung parenchyma, locally multiplying pressures. In a healthy lung the pleural surface, vessels and bronchi are detected as natural lung inhomogeneities. P284 1. Maung AA, Luckianow G, Kaplan LJ: Lessons learned from airway pressure release ventilation. J Trauma 2012, 72:624-628. 1. Maung AA, Luckianow G, Kaplan LJ: Lessons learned from airway pressure release ventilation. J Trauma 2012, 72:624-628. P284 Dissipated energy inside the respiratory system during mechanical ventilation M Gotti, C Chiurazzi, M Amini, C Rovati, M Brioni, G Rossignoli, A Cammaroto, C Bacile di Castiglione, S Luoni, K Nikolla, C Montaruli, T Langer, G Conte, M Cressoni, L Gattinoni Università degli studi di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P274 (doi: 10.1186/cc13474) 2. Kaplan LJ, Bailey H, Formosa V: Airway pressure release ventilation increases cardiac performance in patients with acute lung injury/adult respiratory distress syndrome. Crit Care 2001, 5:221-226. 2. Kaplan LJ, Bailey H, Formosa V: Airway pressure release ventilation increases cardiac performance in patients with acute lung injury/adult respiratory distress syndrome. Crit Care 2001, 5:221-226. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P284 Dissipated energy inside the respiratory system during mechanical ventilation M Gotti, C Chiurazzi, M Amini, C Rovati, M Brioni, G Rossignoli, A Cammaroto, C Bacile di Castiglione, S Luoni, K Nikolla, C Montaruli, T Langer, G Conte, M Cressoni, L Gattinoni Università degli studi di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P274 (doi: 10.1186/cc13474) Figure 1 (abstract P283). improving oxygenation in ventilated patients with cardiogenic shock and severe progressive hypoxemia. improving oxygenation in ventilated patients with cardiogenic shock and severe progressive hypoxemia. Figure 1 (abstract P283). Methods All cardiac and cardiac surgical patients with cardiogenic shock and ALI/ARDS admitted to our ICU were enrolled between January 2012 and September 2013. Data were collected on admission while the patients were on the conventional mode of ventilation and after 48 hours from switching to APRV. All enrolled patients were hemodynamically monitored with a pulmonary artery catheter and frequent echocardiography assessment. A retrospective analysis of these data was performed. Results Completed datasets were obtained from 29 patients. The cardiac index was increased by 30% (P <0.013), serum lactate decreased by 37% (P <0.001), central venous saturation increased by 42% (P <0.001) and peak airway pressure decreased 19% (P <0.001), with 50% increase of mean airway pressure, hypoxemia improved within the fi rst few hours of alveolar recruitment with PaO2/FIO2 increased by 23% (P <0.018), and there was less use of vasopressors, sedation and neuromuscular blockage over the course of APRV application.i g pp Conclusion In our patient series, APRV signifi cantly improved oxygenation and allowed for spontaneous breathing and a reduction in peak airway pressures. Furthermore, this strategy improved hemodynamics and facilitated weaning from MV. Therefore, our data suggest that this ventilation modality has favorable results and appears to be an eff ective alternative for lung recruitment in patients with cardiogenic shock and acute lung injury during their course of stay in cardiac ICU [2]. Figure 1 (abstract P283). Airway pressure release ventilation restores hemodynamic stability in patients with cardiogenic shock: initial experience in cardiac intensive carei Airway pressure release ventilation restores hemodynamic stability in patients with cardiogenic shock: initial experience in cardiac intensive care A Taha, A Shafi e, M Mostafa, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2014, 18(Suppl 1):P282 (doi: 10.1186/cc13472) A Taha, A Shafi e, M Mostafa, H Hon, R Marktanner Sheikh Khalifa Medical City, Abu Dhabi, United Arab Emirates Critical Care 2014, 18(Suppl 1):P282 (doi: 10.1186/cc13472) Results We observed dynamic changes in pulmonary shunt fraction, expressed as changes in SF6 retention, within all breathing cycles recorded, before and after induction of ALI. In healthy lungs at PEEP0 there was a decrease in SF retention at the end of inspiration and a return to baseline levels during expiration. In contrast, SF6 retention increased at PEEP0 in ALI during inspiration and decreased during expiration. In healthy pigs ventilated with PEEP15 there was an increase Introduction Airway pressure release ventilation (APRV) is a pressure- limited, time-cycled mode of mechanical ventilation. It increases the cardiac index, resulting in improved organ perfusion, which is crucial in cardiogenic shock patients preventing organ failure secondary to inadequate perfusion [1]. The purpose of this study was to evaluate the eff ectiveness of APRV in restoring hemodynamic stability and S101 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P283 E i P283 Experimental VILI begins with subpleural lung lesions C Chiurazzi, M Gotti, M Amini, C Rovati, M Brioni, G Rossignoli, A Cammaroto, C Bacile, S Luoni, K Nikolla, C Montaruli, T Langer, D Dondossola, M Cressoni, L Gattinoni Fondazione IRCCS Cà Granda, Milan, Italy Critical Care 2014, 18(Suppl 1):P283 (doi: 10.1186/cc13473) Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets Results We enrolled seven patients (six males, age 54.1 ± 22.2 years, BMI 24.2 ± 4.9 kg/m2, PaO2/FiO2 186 ± 78, tidal volume 445 ± 140 ml, RR 14.5 ± 3.4 breaths/minute, PEEP 12.5 ± 3.3 cmH2O). In the 14 conditions evaluated, median LUSS was 19 (IQR 14 to 23); LUSS ≤19 (n = 8) corresponded to 34 ± 13% of nonaerated tissue at qCT; LUSS >20 (n = 6) to 48 ± 18% (P <0.05). A good linear correlation was found between reduction at LUS of consolidated areas (area score 3) versus reduction of qCT nonaerated volume (R2 = 0.66), and between reduction at LUS of g g Methods LUS and whole-lung CT scan were performed on ARDS sedated, paralyzed, mechanically ventilated patients at PEEP 5 and 15 cmH2O. LUS was performed considering six areas for each lung, with a comprehensive scan of the intercostal spaces in each area. We assigned to each area a score of aeration [1]: 0 (normal lung), 1 (≥3 noncoalescent B-lines), 2 (≥3 coalescent B-lines), 3 (consolidation). A cumulative LUS score (LUSS, ranging from 0 to 36 for the two lungs) was obtained as sum of all areas’ individual scores, each area’s score being the average of all pertaining LUS fi ndings. LUS recruiters upon PEEP increase from 5 to 15 cmH2O were defi ned by the switch of at least three areas to well aerated (area score 0). LUS-based assessment of lung aeration and lung recruitability was compared with qCT fi ndings. Methods In eight healthy piglets we measured total hysteresis and the peak inspiratory pressure (Ppeak) while randomly increasing VT (with no PEEP) or PEEP (with fi xed VT). P1 was extrapolated from the drop in airway pressure during an end-inspiratory pause [3]. Hysteresis attributable to lung parenchyma was computed as: total hysteresis – ((Ppeak – P1) × VT).i Results The main fi ndings are shown in Figure 1. P values refer to one- way repeated-measures analysis of variance. Conclusion Lung hysteresis increases with VT, but not with PEEP. Further studies are needed to prospectively evaluate the role of lung hysteresis in the pathogenesis of ventilator-induced lung injury. References Conclusion Lung hysteresis increases with VT, but not with PEEP. Further studies are needed to prospectively evaluate the role of lung hysteresis in the pathogenesis of ventilator-induced lung injury. P286f CT scan and ultrasound comparative assessment of PEEP-induced lung aeration changes in ARDS I Algieri1, S Mongodi2, D Chiumello3, F Mojoli2, M Cressoni1, G Via2, S Luoni1, A Colombo1, G Babini1, A Braschi2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Policlinico S. Matteo Hospital – University of Pavia, Italy; 3Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P285 (doi: 10.1186/cc13475) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Static DE(J) had a nonlinear relationship with the TV (ml/kg) applied: static DE = 0.0031TV1.5198, R2 = 0.96. Subtracting from the PV curve hysteresis area of a single breath (dynamic DE) the airway DE, the resulting curve overlapped static DE at every RR considered, suggesting that the amount of energy spent on the RS is equal to the static DE. Static DE can be estimated knowing fl ow (l/minute) and dynamic DE(J) since:ll poorly aerated areas (area score 1 to 2) versus reduction of qCT poorly aerated volume (R2 = 0.74). Change at LUS of at least three areas to well aerated (LUS recruiters, n = 4) corresponded to a qCT increase in well-aerated lung volume of 788 ± 262 g versus 431 ± 35 g in the LUS nonrecruiter group (n = 3) (P <0.05). Conclusion These preliminary data suggest that LUS could be an accurate tool to assess lung aeration and recruitment at the bedside, avoiding the risks and workload related to the use of CT scan. Reference y (dynamic–static)DE = 0.0014[fl ow]2 – 0.0173fl ow + 0.1387, R2 = 0.90 (Figure 1). 1. Bouhemad B, et al.: Bedside ultrasound assessment of positive end- expiratory pressure–induced lung recruitment. Am J Respir Crit Care Med 2011, 183:341-347. Conclusion According to our data, the amount of energy that may be related to the development of VILI is static DE; it is a nonlinear function of TV and can be estimated knowing fl ow and dynamic DE. 1. Bouhemad B, et al.: Bedside ultrasound assessment of positive end- expiratory pressure–induced lung recruitment. Am J Respir Crit Care Med 2011, 183:341-347. P283 Experimental VILI begins with subpleural lung lesions h Mutiple nonmutually exclusive possible explanations are possible: the pleural surface acts as a stress raiser; the mechanical friction of the lung with the ribs at very high tidal volume leads to parenchimal injury; and the lung skeleton is a fan-like structure starting from the hilum and going to the pleural surface, leading to increased stress/strain of the subpleural regions. Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). Figure 1 (abstract P284). Relationship between fl ow (l/minute) and (dynamic–static) dissipated energy (J). S102 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 CT scan and ultrasound comparative assessment of PEEP-induced lung aeration changes in ARDS Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets DT Andreis1, M Milesi1, P Pugni1, F Nicosia1, GE Iapichino1, M Monti2, B Comini2, E Votta3, A Protti2, L Gattinoni2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy; 3Politecnico di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P286 (doi: 10.1186/cc13476) Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets I Algieri1, S Mongodi2, D Chiumello3, F Mojoli2, M Cressoni1, G Via2, S Luoni1, A Colombo1, G Babini1, A Braschi2 Introduction Growing evidence suggests that, as long as the total lung capacity is not overcome, dynamic (that is, tidal volume, VT) is more injurious than static (that is, positive end-expiratory pressure, PEEP) lung deformation [1]. Because the lung behaves like a viscoelastic body [2], hysteresis may play a role in the development of ventilator- induced lung injury. The aim of the study was to investigate the eff ects of increasing VT or PEEP on lung hysteresis. Introduction Growing evidence suggests that, as long as the total lung capacity is not overcome, dynamic (that is, tidal volume, VT) is more injurious than static (that is, positive end-expiratory pressure, PEEP) lung deformation [1]. Because the lung behaves like a viscoelastic body [2], hysteresis may play a role in the development of ventilator- induced lung injury. The aim of the study was to investigate the eff ects of increasing VT or PEEP on lung hysteresis. Introduction CT-scan quantitative analysis (qCT) represents the gold standard to assess lung aeration and recruitment in ARDS patients. Lung ultrasound (LUS) has been proposed as a bedside nonirradiating alternative to assess lung recruitability, identifying patients who may benefi t from higher PEEP levels. We compared the two methods in the assessment of PEEP-induced lung aeration changes. g g Methods LUS and whole-lung CT scan were performed on ARDS sedated, paralyzed, mechanically ventilated patients at PEEP 5 and 15 cmH2O. LUS was performed considering six areas for each lung, with a comprehensive scan of the intercostal spaces in each area. We assigned to each area a score of aeration [1]: 0 (normal lung), 1 (≥3 noncoalescent B-lines), 2 (≥3 coalescent B-lines), 3 (consolidation). A cumulative LUS score (LUSS, ranging from 0 to 36 for the two lungs) was obtained as sum of all areas’ individual scores, each area’s score being the average of all pertaining LUS fi ndings. LUS recruiters upon PEEP increase from 5 to 15 cmH2O were defi ned by the switch of at least three areas to well aerated (area score 0). LUS-based assessment of lung aeration and lung recruitability was compared with qCT fi ndings. Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets P287 Evaluation and quantifi cation of pulmonary hyperinfl ation in three gravitational zones of domestic felines by computed tomography A Rodrigues de Carvalho Martins1, D Tabacchi Fantoni2, D Aya Otsuki1, A Magalhães Ambrósio2, A Brandão de Campos Fonseca Pinto2, J França dos Santos2, C Rodrigues de Carvalho Martins3, L Sá Malbouisson1 1Faculdade de Medicina da Universidade de São Paulo, Brazil; 2Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, Brazil; 3UFAPE, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P287 (doi: 10.1186/cc13477) Results In Group I relative to group II at 3 to 5 days we registered an increase in mature monocytes (CD14) – 23.1 ± 0.8% (P <0.05); reduction in the relative content of CD69 – 3.8 ± 0.21%, lymphocyte of apoptosis: (Annexin V–FITC+PI–) – 7.12 ± 0.46% and (Annexin V–FITC+PI+) – 0.79 ± 0.07% (P <0.001). The duration of mechanical ventilation was 4.1 ± 1.4 days (P <0.05). All patients survived. None of the patients showed clinical or laboratory evidence of adverse eff ects of inhaled nitric oxide. In Group II seven newborns died, and the duration of mechanical ventilation in survivors was 18 ± 3.4 days. Critical Care 2014, 18(Suppl 1):P287 (doi: 10.1186/cc13477) Introduction Mechanical ventilation (MV) aims to enhance blood oxygenation and to remove carbon dioxide. However, excessive hyperinfl ation by MV may cause lung injury. Introduction Mechanical ventilation (MV) aims to enhance blood oxygenation and to remove carbon dioxide. However, excessive hyperinfl ation by MV may cause lung injury. y Conclusion Inhalable of nitric oxide activates monocyte–macrophage immunity, stabilizes the apoptosis of T-lymphocytes, and reduces mortality and duration of mechanical ventilation in newborns with respiratory diseases on mechanical ventilation. References Methods Eighteen cats (4 ± 1 kg) were anesthetized with propofol (loading dose of 6 mg/kg and constant rate infusion of 0.5 mg/kg/ minute) and neuromuscular blockade was achieved with rocuronium at 1 mg/kg/minute. Their lungs were initially mechanically ventilated in FiO2 of 40%, with peak inspiratory pressure (Ppeak) of 5 cmH2O for 20 minutes, and then the Ppeak was increased by 5 cmH2O increments until 15 cmH2O every 5 minutes. Following that, Ppeak was decreased by 2 cmH2O every 5 minutes until reaching Ppeak of 5 cmH2O. The ventilator maintained the respiratory rate and inspiratory time at 15 breaths/minute and 1 second, respectively. Between the Ppeak increments, we applied a 4-second pause for a 5-mm computed tomography (CT) scan of the thorax area. Diaphragm microcirculatory dysfunction and lipid accumulation in endotoxemic rabbits during mechanical ventilation Diaphragm microcirculatory dysfunction and lipid accumulation in endotoxemic rabbits during mechanical ventilation Y Yang1, T Yu1, J Liu1, C Pan1, F Longhini2, L Liu1, Y Huang1, F Guo1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P289 (doi: 10.1186/cc13479) Results The three zones presented increased over-insuffl ated areas and decreased areas with normal insuffl ation with increasing Ppeak from 5 to 15 cmH2O. At 5 cmH2O, the areas of over-insuffl ation and normal insuffl ation in zones I, II and III were 13% and 36%; 4% and 22%; and 0.7% and 15%, respectively. At 15 cmH2O, the areas of over-insuffl ation and normal insuffl ation in zones I, II and III were 74% and 55%; 81% and 57%; and 82% and 71%, respectively. Introduction Sepsis-induced diaphragm dysfunction (SIDD) has been widely described in the literature as a condition aff ecting the diaphragm muscle characterized by contractility loss of function and associated with a high mortality, assessed at around 54% [1]. Previous studies have investigated the pathogenesis of ventilator-induced diaphragmatic dysfunction (VIDD), its lipid metabolic alterations [2] and microcirculatory function processes. This study was designed to investigate on diaphragm muscle the eff ects of LPS-induced endotoxemia in rabbits undergoing two diff erent modes of mechanical ventilation. Conclusion The higher proportion of overly distended pulmonary areas in high Ppeak may increase the risk of lung injury. The lowest Ppeak (5 cmH2O) showed less potential to lung injury as it has higher areas of normal insuffl ation and less areas of over-insuffl ation in all gravitational zones. Methods A prospective randomized animal study in 25 invasively monitored and mechanically ventilated New Zealand White rabbits. The rabbits were randomized to control (n = 5), controlled mechanical ventilation (CMV) (n = 5), pressure support ventilation (PSV) (n = 5), or CMV or PSV with LPS-induced endotoxemia (CMV-LPS and PSV- LPS respectively) (n = 5 for each). The endotoxemia was induced by LPS injection in the CMV-LPS and PSV-LPS groups. Rabbits were anesthetized and ventilated for 24 hours, except for the control (30 minutes). A catheter able to detect the electrical activity of the diaphragm was placed to evaluate the diaphragm contractility at baseline and after 6, 12 and 24 hours. Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets References i Results We enrolled seven patients (six males, age 54.1 ± 22.2 years, BMI 24.2 ± 4.9 kg/m2, PaO2/FiO2 186 ± 78, tidal volume 445 ± 140 ml, RR 14.5 ± 3.4 breaths/minute, PEEP 12.5 ± 3.3 cmH2O). In the 14 conditions evaluated, median LUSS was 19 (IQR 14 to 23); LUSS ≤19 (n = 8) corresponded to 34 ± 13% of nonaerated tissue at qCT; LUSS >20 (n = 6) to 48 ± 18% (P <0.05). A good linear correlation was found between reduction at LUS of consolidated areas (area score 3) versus reduction of qCT nonaerated volume (R2 = 0.66), and between reduction at LUS of 1. Protti A, et al.: Which is the most important strain in the pathogenesis of ventilator-induced lung injury: dynamic or static? Curr Opin Crit Care 2014, 20:33-38. 2. Bayliss LE, et al.: The visco-elastic properties of the lung. Q J Exp Physiol 1939, 29:27-47. 3. Bates JHT, et al.: General method for describing and extrapolating monotonic transients and its application to respiratory mechanics. Med Biol Eng Comput 1987, 25:131-135. Figure 1 (abstract P286). Lung hysteresis as a function of VT (A) and PEEP (B). P <0.05 versus VT 250 ml or PEEP 0 cmH2O (Holm–Sidak method). Figure 1 (abstract P286). Lung hysteresis as a function of VT (A) and PEEP (B). P <0.05 versus VT 250 ml or PEEP 0 cmH2O (Holm–Sidak method). S103 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P287 on the Beckman Coulter Epics XL cytometer (USA). The statistical power of the study was 80% (α ≤0.05). Eff ect of tidal volume and positive end-expiratory pressure on lung hysteresis of healthy piglets The radiographic attenuation (in Hounsfi eld units, HU) was classifi ed as over-insuffl ation (1,000 to 900 HU), normal insuffl ation (900 to 500 HU) and atelectasic (500 to 100 HU). We split the lungs into three proportional gravitational zones (I, II and III) from apex to base. 1. Dudareva MV, Estrin VV: The state of immunity in newborn infants with respiratory disease. Allergy Immunol 2008, 9:349-350. p y gy 2. Puhtinskaya MG, Estrin VV, Dudareva MV, Gulova ES: Method of treatment of RDS in neonates receiving mechanical ventilation. RU Patent No. 2434653; 27 November 2011. 2. Puhtinskaya MG, Estrin VV, Dudareva MV, Gulova ES: Method of treatment of RDS in neonates receiving mechanical ventilation. RU Patent No. 2434653; 27 November 2011. Diaphragm microcirculatory dysfunction and lipid accumulation in endotoxemic rabbits during mechanical ventilation After 24 hours, we evaluated: the diaphragm microcirculation assessed by a sidestream dark-fi eld videomicroscopy; the mitochondria membrane potential; the lipid accumulation; and the diaphragm muscular fi ber structure. Eff ect of inhaled nitric oxide on apoptosis of lymphocytes in newborns in a critical state M Puhtinskaya Research Institute of Obstetrics and Pediatrics, Rostov-on-Djn, Russia Critical Care 2014, 18(Suppl 1):P288 (doi: 10.1186/cc13478) y Research Institute of Obstetrics and Pediatrics, Rostov-on-Djn, Russia Critical Care 2014, 18(Suppl 1):P288 (doi: 10.1186/cc13478) Introduction Activation of lymphocyte apoptosis while reducing the endogenous nitric oxide is a predictor of adverse outcome in newborns on mechanical ventilation [1]. With the aim to improve the results of treatment we studied the eff ect of inhalable nitric oxide on the immune system in newborns with respiratory diseases on mechanical ventilation [2]. i Results In endotoxemic animals, after 24 hours, the diaphragm contractility and fi ber structure, the microcirculation, mitochondrial membrane potential and lipid accumulation were severely compromised, but not in the CMV and PSV groups. Moreover, a slight but signifi cant increase of lipid accumulation was observed in the CMV and PSV groups in comparison with control (P <0.05). Methods With the permission of the ethics committee in a controlled, randomized, blind clinical trial we included 27 newborns with respiratory diseases on mechanical ventilation. Randomization was performed by the method of envelopes. Group I (n = 17), patients receiving inhalation of nitric oxide at a concentration of 10 ppm for 24 hours controlling the level of methemoglobin (Pulmonox mini; Messer II NO Therapeutics, Austria). Group II (n = 10) did not receive inhaled NO. At admission and at 3 to 5 days we studied subpopulations of lymphocytes by one-parameter immunophenotyping using reagents (Immunotech Beckman Coulter, USA): fi tz-labeled CD3, CD4, CD8, CD14, CD19, CD34, CD56, CD69, CD71, CD95 monoclonal antibody, the relative content of lymphocytes in early and late apoptosis using Annexin V+-labeled FITK and propidium iodide (PL+), labeled with PE (Saltag, USA), with results Conclusion In endotoxemic rabbits, the impaired microcirculation resulted in an increased lipid accumulation and in a disturbance of the mitochondria membrane potential and contractility of the diaphragm. No microvascular alterations have been observed in ventilated non-endotoxemic animals. Moreover, the diaphragm contractility dysfunction was more pronounced in endotoxemic animals. References EIT comparison of airway pressure release ventilation and conventional ventilation S Jog, S Sable, D Patel, P Tambur g, , , Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P291 (doi: 10.1186/cc13481) Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P291 (doi: 10.1186/cc13481) g Conclusion EIT may help to identify patients with severe ARDS on VCV with a potential of increasing recruitment by tidal redistribution of ventilation with APRV. Introduction The aim was to study EIT as a monitoring tool for tidal ventilation (TV) redistribution following switching patients from volume-controlled ventilation (VCV) to airway pressure release ventilation (APRV) in patients with severe ARDS. High-frequency oscillatory ventilation use in patients with H1N1: a single-centre review Introduction Limited evidence exists as to the value of high-frequency oscillatory ventilation (HFOV) for patients with H1N1 [1,2]. We describe a subgroup of patients, from a single UK centre, who received HFOV and had H1N1-positive virology. gy Methods Local permissions for research were obtained. Patients with confi rmed H1N1 who underwent HFOV between 2008 and 2012 were included. Data collected retrospectively included demographics, diagnosis, illness severity and outcome measures.i g y Results We identifi ed 10 patients (Table 1) who received both HFOV and had confi rmed H1N1. Two patients were transferred to a tertiary centre to receive ECMO and both critical care and 6-month mortality were 40%. Table 1 (abstract P290). Patient characteristics Table 1 (abstract P290). Patient characteristics Male (%) 60 Age (years) 39.4 ± 9.75 Mean APACHE II 19.5 ± 4.88 Pre-HFOV neuromuscular blockade infusion (%) 40 Pre-HFOV vasoactive drug infusion (%) 50 Mean pre-HFOV VT exp (ml/kg) 8.54 ± 2.68 Median P/F ratio (mmHg) Pre HFOV 67.5 0 hours 94.35 4 hours 110.4 Table 1 (abstract P290). Patient characteristics Table 1 (abstract P290). Patient characteristics Conclusion This study adds to the literature on the use of HFOV in H1N1 patients. We managed to replicate some of the existing evidence in respect to the population age [1,2] and the incidence of mortality. Whilst P/F ratios improved on initiation of HFOV, these patients subsequently had long critical care and hospital stays. There is uncertainty regarding the use of HFOV in ARDS, but it may be a valuable treatment for H1N1 patients. The ventilation strategies employed and the subsequent consequences for H1N1 patients require further evaluation. References Figure 1 (abstract P291). EIT comparison of VCV and APRV. and percentage of tidal ventilation distribution in the four ROIs were recorded at steps 1 and 2. Analyses were performed by paired t test. Results Patients on VCV had P/F ratio of 79.5 ± 12.5 with PEEP of 14.16 ± 1.32 There was a signifi cant improvement in P/F ratios on switching to APRV (126.16 ± 23.69, P = 0.002) at 30 minutes of ventilation on APRV. There was a trend to decrease in FiO2 (0.82 ± 0.15 vs. 0.68 ± 0.10, P = 0.068) and PCO2 (52.5 ± 6.15 vs. 45.00 ± 8.67, P = 0.071) and increase in PaO2 (65.83 ± 14.53 vs. 84.83 ± 12.22, P = 0.056) at step 2. P292 Methods Six patients with severe ARDS having Pplat ≥30 cm were included in the study. Patients ventilated with the ARDSnet strategy were subjected to EIT analysis. Regional TV distribution was monitored by an EIT system (PulmoVista 500®; Dräger Medical GmbH, Lübeck, Germany), dividing the lung fi eld into four same-size regions of interest (ROIs): ventral right (ROI 1) and left (ROI 2) and dorsal right (ROI 3) and left (ROI 4). In step 1, patients ventilated with VCV as per the ARDSnet protocol were subjected to EIT analysis. In step 2, patients were switched to APRV. Ventilation parameters, arterial blood gas analysis Lung-protective ventilation suppresses plasma levels of cell-free DNA in porcine experimental postoperative sepsis A Nyberg1, J Sperber1, M Lipcsey2, J Jylhävä3, M Hurme3, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Eskilstuna, Sweden; 2Uppsala University, Uppsala, Sweden; 3University of Tampere, Finland Critical Care 2014, 18(Suppl 1):P292 (doi: 10.1186/cc13482) High-frequency oscillatory ventilation use in patients with H1N1: a single-centre review The proportional distribution of ventilation in the dorsal ROI 3 and ROI 4 also improved on switching to APRV. TV in ROI 3 during VCV, 12.76 ± 6.76%, improved to 24.58 ± 6.61% (P = 0.067). Similarly TV in ROI 4 during VCV, 24.58 ± 6.61%, improved to 26.6 ± 6.09% (P = 0.068). Due to small sample size, improvement in TV in dorsal ROIs was not statistically signifi cant. Upper panel of Figure 1 shows end-inspiratory and end-expiratory images of EIT on VCV with poor TV in dorsal ROI 3 and ROI 4. Lower panel of fi gure shows two EIT images at Phigh = 30 cm showing improved TV in dorsal ROIs. and percentage of tidal ventilation distribution in the four ROIs were recorded at steps 1 and 2. Analyses were performed by paired t test. Results Patients on VCV had P/F ratio of 79.5 ± 12.5 with PEEP of 14.16 ± 1.32 There was a signifi cant improvement in P/F ratios on switching to APRV (126.16 ± 23.69, P = 0.002) at 30 minutes of ventilation on APRV. There was a trend to decrease in FiO2 (0.82 ± 0.15 vs. 0.68 ± 0.10, P = 0.068) and PCO2 (52.5 ± 6.15 vs. 45.00 ± 8.67, P = 0.071) and increase in PaO2 (65.83 ± 14.53 vs. 84.83 ± 12.22, P = 0.056) at step 2. The proportional distribution of ventilation in the dorsal ROI 3 and ROI 4 also improved on switching to APRV. TV in ROI 3 during VCV, 12.76 ± 6.76%, improved to 24.58 ± 6.61% (P = 0.067). Similarly TV in ROI 4 during VCV, 24.58 ± 6.61%, improved to 26.6 ± 6.09% (P = 0.068). Due to small sample size, improvement in TV in dorsal ROIs was not statistically signifi cant. Upper panel of Figure 1 shows end-inspiratory and end-expiratory images of EIT on VCV with poor TV in dorsal ROI 3 and ROI 4. Lower panel of fi gure shows two EIT images at Phigh = 30 cm showing improved TV in dorsal ROIs. 1. Riscilli BP, et al.: PLOS One 2011. doi: 10.1371/journal.pone.0018166. 2. Boots RJ, et al.: Anaesth Intensive Care 2011, 39:837-846. 1. Riscilli BP, et al.: PLOS One 2011. doi: 10.1371/journal.pone.0018166. 2. Boots RJ, et al.: Anaesth Intensive Care 2011, 39:837-846. dysfunction References Demoule A, et al.: Am J Respir Crit Care Med 2013, 188:213-219. Picard M, et al.: Am J Respir Crit Care Med 2012, 186:1140-1149. Demoule A, et al.: Am J Respir Crit Care Med 2013, 188:213-219. Picard M, et al.: Am J Respir Crit Care Med 2012, 186:1140-1149. Demoule A, et al.: Am J Respir Crit Care Med 2013, 188:213-219. Picard M, et al.: Am J Respir Crit Care Med 2012, 186:1140-1149. S104 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P290 High-frequency oscillatory ventilation use in patients with H1N1: a single-centre review N Smith, N Pathmanathan, V Martinson, J Glazebrook, R Rao, L Sleight, A Gratrix Hull and East Yorkshire NHS Trust, Hull, UK Critical Care 2014, 18(Suppl 1):P290 (doi: 10.1186/cc13480) Figure 1 (abstract P291). EIT comparison of VCV and APRV. Comparison of HFOV and conventional ventilation in H1N1 infl uenza ARDS l S Jog, S Sable, D Patel, N Kapadnis, P Rajhans, P Akole, B Pawar, B Bhurke, M Kothari, S Gururaj D th M hk H it l d R h C t P I di j Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P293 (doi: 10.1186/cc13483) Deenanath Mangeshkar Hospital and Research Center, Pune, India Critical Care 2014, 18(Suppl 1):P293 (doi: 10.1186/cc13483) g y ( g p y ) Results Eighteen patients (male 12, age 56.0 ± 18.6 years, BMI 25.6 ± 5.2 kg/m2, PaO2/FiO2 166 ± 70, tidal volume 496 ± 84 ml, RR 19 ± 7.4 breaths/minute, PEEP 11.4 ± 3.7 cmH2O) were enrolled. The fraction of lung parenchyma that could be recruited at plateau pressure above 30 cmH2O was highly variable with a median of 5% (IQ range 1 to 17%) corresponding to a median 16% (IQ range 10 to 47%) of total lung recruitability. Indeed we observed a statistically relevant diff erence in lung recruitment between airway pressures of 30 and 45 cmH2O (P = 0.016). With PEEP 5 cmH2O, median opening/closing was 129 g (IQ range 124 to 145 g) in the HR group and 50 g (IQ range 24 to 76 g) in LR (P = 0.006), corresponding to 8% of lung parenchyma (IQ range 6 to 8%) in HR and 4% (2 to 7%) in LR (P = 0.053). Increasing the PEEP level to 15 cmH2O, median opening/closing was 67 g (IQ range 24 to 95 g) in the HR group and 45 g (0 to 80 g) in the LR group (P = 0.512), corresponding to 3% (1 to 5%) in HR and 3% (0 to 5%) in LR (P = 0.93). We observed a statistical diff erence between recruitments with PEEP 5 and PEEP 15 in the HR group (P = 0.013) but not in the LR group (P = 0.781).i Introduction HFOV is a promising rescue therapy for refractory hypoxia in severe ARDS. Methods This is a retrospective comparative study. We retrieved data for all patients with H1N1 infl uenza-related severe ARDS treated in the ICU during October 2009 to April 2013. Our ICU had only one HFOV machine during the pandemic. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Introduction Intratidal opening/closing is believed to be one of the main triggers of ventilator-induced lung injury; the application of higher PEEP united with the limitation of plateau pressure at 30 cmH2O may limit this phenomenon. We aim to evaluate the intratidal opening/ closing at diff erent PEEP levels and the amount of lung parenchyma that regains infl ation going from 30 (pressure limit of tidal ventilation) to 45 cmH2O. Conclusion In experimental postoperative sepsis, protective ventila tion suppresses arterial levels of cfDNA. The liver seems to be a signifi cant contributor to systemic cfDNA levels, an eff ect that is suppressed during protective ventilation. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 levels of cell-free DNA (cfDNA) increase and have prognostic value in sepsis [2]. In the present study, the eff ect of tidal volume and PEEP on arterial and transorgan levels of cfDNA was investigated in a porcine postoperative sepsis model. (P = 0.02, odds ratio (CI) 71.60 (1.85 to 2,766.59)) compared with the HFOV group. levels of cell-free DNA (cfDNA) increase and have prognostic value in sepsis [2]. In the present study, the eff ect of tidal volume and PEEP on arterial and transorgan levels of cfDNA was investigated in a porcine postoperative sepsis model. Conclusion HFOV when applied as rescue therapy for refractory hypoxia due to severe ARDS caused by H1N1 infl uenza pneumonia is associated with better outcome compared with CLPV. Methods Two groups of anaesthetised pigs were ventilated with either protective ventilation (VT 6 ml/kg, PEEP 10 cmH2O; n = 20) or controls (VT 10 ml/kg, PEEP 5 cmH2O; n = 10) for 7 hours. An artery, the hepatic vein, the portal vein and the jugular bulb were catheterized. Continuous endotoxin infusion at 0.25 μg/kg/hour for 5 hours was started after 2 hours of laparotomy that simulated a surgical procedure. Results The group receiving protective ventilation showed lower levels of cfDNA in arterial blood compared with controls (P = 0.02). Trans- hepatic levels of cfDNA were higher compared with trans-splanchnic levels during the experiment (P = 0.02), but this eff ect was attenuated in the group receiving protective ventilation. No diff erence between the groups was detected in blood samples from the jugular bulb. P294 P294 Opening pressures and intratidal opening and closing in ARDS lung I Algieri1, D Chiumello2, M Cressoni1, A Colombo1, G Babini1, S Luoni1, M Brioni1, F Crimella1, C Chiurazzi1, B Comini2, M Monti2 1Università degli Studi di Milano, Milan, Italy; 2Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P294 (doi: 10.1186/cc13484) Introduction Intratidal opening/closing is believed to be one of the main triggers of ventilator-induced lung injury; the application of higher PEEP united with the limitation of plateau pressure at 30 cmH2O may limit this phenomenon. We aim to evaluate the intratidal opening/ closing at diff erent PEEP levels and the amount of lung parenchyma that regains infl ation going from 30 (pressure limit of tidal ventilation) to 45 cmH2O. References 1. Sperber J, et al.: PLOS One 2013. doi: 10.1371/journal.pone.0083182. 2. Saukkonen K, et al.: Clin Chem 2008, 54:1000-1007. 2 Methods Patients with ARDS underwent whole-lung low-dose (60 mAs, 120 kV) computed tomography at PEEP 5 cmH2O end expiration and end inspiration, PEEP 15 cmH2O end expiration and end inspiration, and airway pressure 30 and 45 cmH2O end inspiration. Quantitative analysis of CT data was performed and recruitability was defi ned as the fraction of lung parenchyma that regains infl ation going from 5 cmH2O end expiration to 45 cmH2O end inspiration. Patients were classifi ed as high recruiters (HR) and low recruiters (LR) according to the median lung recruitability (14% of lung parenchyma). Comparison of HFOV and conventional ventilation in H1N1 infl uenza ARDS Patients were divided into two groups: HFOV group (received HFOV at fi rst eligibility) and conventional lung protective ventilation (CLPV) group (did not receive HFOV at fi rst eligibility due to nonavailability of HFOV). Eligibility criteria for rescue therapy by HFOV were: P/F ratio ≤100; PEEP needed above 12 cm; Pplat ≥30 cm on CLPV. There was no selection or omission bias for HFOV application and HFOV was applied to the fi rst eligible patient. Patient demographic data, laboratory parameters, hemodynamic variables, and oxygenation and ventilator settings were recorded while on CLPV at fi rst HFOV eligibility in all patients. i g y Results The total of 43 patients who met the rescue therapy criteria were further grouped into the HFOV group (24 patients) and the CLPV group (19 patients) depending upon modality of ventilation received after satisfying fi rst-time HFOV eligibility criteria. Both groups were comparable for diff erences with Fisher’s t test for qualitative variables and ANOVA for quantitative variables (Table 1), except for higher mortality in the CLPV group (16/19 (84.4%) vs. 12/12 (50%), P = 0.026). On logistic regression analysis to fi nd independent variables diff erentiating the two groups, mortality was higher in the CLPV group Conclusion A highly variable and signifi cant fraction of lung parenchyma is always closed with tidal ventilation at 30 cmH2O plateau pressure, regardless of the PEEP level applied; this implies that sigh or periodic recruitment maneuvers may lead to opening and closing. Intratidal opening/closing was reduced but not abolished in all patients while ventilating at higher PEEP level (15 cmH2O). P295 P295 Lung-protective ventilation suppresses plasma levels of cell-free DNA in porcine experimental postoperative sepsis p p p p p A Nyberg1, J Sperber1, M Lipcsey2, J Jylhävä3, M Hurme3, M Castegren1 1Centre for Clinical Research Sörmland, Uppsala University, Eskilstuna, Sweden; 2Uppsala University, Uppsala, Sweden; 3University of Tampere, Finland Critical Care 2014, 18(Suppl 1):P292 (doi: 10.1186/cc13482) Introduction Mechanical ventilation aff ects systemic infl ammation where protective ventilation attenuates the response [1]. Plasma S105 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1. Boles JM, et al.: Weaning from mechanical ventilation. Eur Respir J 2007, 29:1033-1056. References References 1. ARDsnet: N Engl J Med 2002, 342:1301-1308. 2. Young D, et al.: N Engl J Med 2013, 368:806-813. 3. The ARDS Defi nition Task Force: JAMA 2012, 307:2526-2533. 1. ARDsnet: N Engl J Med 2002, 342:1301-1308. 2. Young D, et al.: N Engl J Med 2013, 368:806-813. 3. The ARDS Defi nition Task Force: JAMA 2012, 307:2526-2533. Results The study was completed with 36 patients overall, being 11 patients in failure and 25 patients in the success group. In both groups, areas under the curve (AUCs) were calculated for each minute via ROC analysis using minute SPIF and SPEF values measured during SBT. The maximum AUC was calculated at minute 23 for SPIF (0.564; 95% CI: 0.363 to 0.764) and at minute 9 for SPEF (0.542; 95% CI: 0.316 to 0.376). Cutoff values were determined for the minutes in which maximum AUC values for SPIF and SPEF were detected; and sensitivity and specifi city values were calculated. When the cutoff value for SPIF was accepted as >26.7 l/minute at minute 23, sensitivity and specifi city was calculated as 72.0% and 28.0%, respectively. When the cutoff value for SPEF was accepted as >24.7 l/minute at minute 9, sensitivity and specifi city were calculated as 63.6% and 48.8%, respectively. P298 P298 Lung ultrasound fi ndings predict weaning failure from mechanical ventilation f References 1. Schumann S. et al.: Determination of respiratory system mechanics during inspiration and expiration by FLow-controlled EXpiration (FLEX): a pilot study in anesthetized pigs. Minerva Anestesiol 2014, in press. 2. Goebel U. et al.: Flow-controlled expiration (FLEX): a novel ventilation mode to attenuate experimental porcine lung injury. Br J Anaesth 2014, in press. 1. Schumann S. et al.: Determination of respiratory system mechanics during inspiration and expiration by FLow-controlled EXpiration (FLEX): a pilot study in anesthetized pigs. Minerva Anestesiol 2014, in press. y p g p 2. Goebel U. et al.: Flow-controlled expiration (FLEX): a novel ventilation mode to attenuate experimental porcine lung injury. Br J Anaesth 2014, in press. Mechanisms underlying the lung-protective eff ects of FLow- controlled EXpiration p S Schumann, U Goebel, J Haberstroh, J Guttmann p S Schumann, U Goebel, J Haberstroh, J Guttmann University Medical Center Freiburg, Germany y g, y Critical Care 2014, 18(Suppl 1):P296 (doi: 10.1186/cc13486) Introduction During mechanical ventilation the expiration occurs passively and is determined by the recoil forces of the respiratory system. In an experimental study in pigs we could fi nd that linearization of the expiratory fl ow via FLow-controlled EXpiration (FLEX) [1] is lung protective [2]. Utilizing electrical impedance tomography (EIT) we aimed at investigating the mechanisms underlying the lung-protective eff ects of FLEX. p y Conclusion We think that minute SPIF measurement which has better sensitivity and minute SPEF measurement which has better specifi city compared with available traditional predictors [1] may be used as a potential bedside weaning predictor when evaluated in comprehensive studies. f Methods All experiments were approved by the local animal welfare committee. Twelve pigs with oleic acid-induced lung injury were ventilated in the volume-controlled mode (VCV). In six animals, expiratory fl ow was linearized via FLEX. PEEP was set to achieve similar mean airway pressure in the control group (n = 6) and in the FLEX group (n = 6). Using EIT, the local distribution of ventilation was measured and alveolar derecruitment during the no-fl ow phase in late expiration was quantifi ed. Reference P297 l Value of peak fl ow rates measured during a spontaneous breathing trial to predict success of weaning from mechanical ventilation K Gundogan, S Baldane, R Coskun, I Bahar, G Altunyurt, H Mumcuoglu, M Guven, M Sungur Erciyes University, Kayseri, Turkey Critical Care 2014, 18(Suppl 1):P297 (doi: 10.1186/cc13487) Value of peak fl ow rates measured during a spontaneous breathing trial to predict success of weaning from mechanical ventilation K Gundogan, S Baldane, R Coskun, I Bahar, G Altunyurt, H Mumcuoglu, M Guven, M Sungur Erciyes University, Kayseri, Turkey Critical Care 2014, 18(Suppl 1):P297 (doi: 10.1186/cc13487) Table 1 (abstract P295). Tidal volume With ARDS (%) At risk of ARDS (%) <6 ml/kg 25.3 15.7 6 to 8 ml/kg 31.7 29.3 >8 ml/kg 43 55 Introduction Numerous parameters have been suggested for the prediction of weaning from mechanical ventilation; however, these parameters have limited success in the prediction of weaning outcome. The aim of this study is to assess the success of minute peak fl ow rates (spontaneous peak inspiratory fl ow rate (SPIF) and spontaneous peak expiratory fl ow rate (SPEF)) measured during a spontaneous breathing trial (SBT) in the prediction of weaning outcome. Conclusion Compliance with protective lung ventilation in our ICU is suboptimal. This may be due to the lack of education and guidelines in the unit regarding protective lung ventilation. Moreover, accurate recording of patient height and determination of predicted body weight should be documented to facilitate accurate tidal volume calculation and protective lung ventilation. Methods Patients managed and receiving mechanical ventilation support for at least 24 hours in the medical and surgical ICUs of Erciyes University between March 2011 and May 2012 were included in the present study. Over 30 minutes, SPIF and SPEF values were measured during a SBT in patients spontaneously breathing by T tube. Patients who tolerated 30 minutes of SBT were extubated. Patients who did not need reintubation for 48 hours after extubation were considered successful weaning, while those needing reintubation were considered weaning failure. Reference 1. Boles JM, et al.: Weaning from mechanical ventilation. Eur Respir J 2007, 29:1033-1056. P298 P295 Compliance with protective lung ventilation in an Irish teaching hospital Table 1 (abstract P293). Variable CLPV (n = 19) HFOV (n = 24) P value Age 41.15 32.83 0.07 SOFA 4.95 4.62 0.41 Pplat 29.57 30.46 0.26 O.I. 38.88 35.70 0.54 P/F 70.63 76.79 0.50 PCO2 59.89 59.92 0.99 Death 16 12 0.02 g p Critical Care 2014, 18(Suppl 1):P295 (doi: 10.1186/cc13485 Introduction The importance of protective lung ventilation in reducing mortality in adult respiratory distress syndrome (ARDS) patients is well described [1]. However, suboptimal compliance with the recommended tidal volumes has been reported [2]. Therefore, we wished to assess our compliance in adhering to protective lung ventilation in patients with, or at risk of developing, ARDS. p g Methods A retrospective review was done on all mechanically ventilated patients in the ICU of Tallaght Hospital over a 6-month period (February to July 2013). Hourly tidal volumes were recorded S106 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 automatically in electronic charts. Compliance was assessed by calculating the total time patients with, or at risk of developing, ARDS were ventilated with tidal volumes <6 ml/kg, 6 to 8 ml/kg, and >8 ml/ kg during the fi rst 72 hours on mechanical ventilation. ARDS is defi ned as per the Berlin criteria [3]. Exclusion criteria were patients who did not receive invasive ventilation or who were ventilated for less than 72 hours. We also assessed whether patients’ height was documented. Results A total of 72 patients were ventilated for >72 hours. Of these patients (44 males, 28 females, mean age 65.5 years), ARDS criteria were met in 17 patients and 22 patients were determined to be at risk of developing ARDS. For patients with ARDS, the ventilated time with tidal volumes <6 ml/kg, 6 to 8 ml/kg, and >8 ml/kg was 25.3%, 31.7% and 43% respectively. For patients at risk of developing ARDS, the ventilated time with tidal volumes <6 ml/kg, 6 to 8 ml/kg, and >8 ml/ kg was 15.7%, 29.3%, 55%, respectively (Table 1). A total of 16 patients (nine who had ARDS) had no documentation of their height. distribution of ventilation is more homogeneous. These mechanisms of alveolar recruitment maintenance can explain the lung-protective eff ects of FLEX. Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients AC Pecanha Antonio1, M Basso Gazzana2, P Souza Castro1, M Knorst1 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P299 (doi: 10.1186/cc13489) Introduction The rapid shallow breathing index (RSBI) is considered a good parameter to predict mechanical ventilator liberation. We hypothesized that the RSBI provides no benefi t when clinical readiness criteria are met. AC Pecanha Antonio1, M Basso Gazzana2, P Souza Castro1, M Knorst1 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P299 (doi: 10.1186/cc13489) AC Pecanha Antonio1, M Basso Gazzana2, P Souza Castro1, M Knorst1 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P299 (doi: 10.1186/cc13489) Introduction Fluid balance (FB) has been associated with weaning and extubation failure, particularly of cardiac origin. Also, the increased right ventricular afterload, a feature common in chronic obstructive pulmonary disease (COPD) patients especially during a weaning test, may hinder diastolic fi lling of the left ventricle through a biventricular interdependence mechanism. We aimed to investigate the relationship of the FB in the 48 hours prior to a spontaneous breathing trial (SBT) and weaning outcomes in a subgroup of COPD patients admitted to a medical–surgical ICU. Methods Adults with acute respiratory who required MV for more than 24 hours, excluding COPD, were assessed daily as a liberation protocol (Figure 1). During the RSBI step, RSBI was recorded and blinded to the Figure 1 (abstract P300). Standard mechanical ventilator weaning protocol. Methods We conducted a 2-year prospective, multicenter, obser- vational study in two adult medical–surgical ICUs. All enrolled patients met eligibility criteria for ventilation liberation. Patients with tracheostomy were excluded. We collected demographic, physiologic, 48-hour FB (measured inputs minus outputs) and lung ultrasound fi ndings immediately before a SBT in 29 COPD patients. Our main outcome of interest was weaning failure (WF), defi ned as the inability to tolerate a T-tube trial during 30 to 120 minutes, in which case patients were not extubated. p Results Weaning success (WS) (n = 19) and WF (n = 10) patients were similar in relation to age, sex, APACHE II score, reason for mechanical ventilation (MV) and comorbidities. Mean duration of MV was 11 days. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Therefore, the presence of cardiovascular dysfunction can contribute to weaning failure (WF). A randomized trial concluded that bedside lung ultrasound could predict post-extubation distress through changes in aeration during a T-tube test; however, it could not screen patients before submission to a SBT [1]. We aim to assess the reliability of lung ultrasound as a predictor of weaning outcomes. Figure 1 (abstract P299). Association between positive fl uid balance WF in COPD patients. Figure 1 (abstract P299). Association between positive fl uid balance WF in COPD patients. Methods We conducted a prospective, multicenter, observational study in two adult medical–surgical ICUs. Lung ultrasound was performed immediately before SBT. Three or more B-lines in a single view were called a B-pattern. B-predominance was defi ned as a B-pattern on at least one of the four anterior chest wall zones. All enrolled patients met eligibility criteria for ventilation liberation. Patients with tracheostomy were excluded. Results During 2 years, 250 SBTs were analyzed. WF, defi ned as an inability to tolerate a T-tube trial during 30 to 120 minutes, occurred in 51 (20.4%). There was a higher prevalence of chronic obstructive pulmonary disease in the WF group as well as higher duration of mechanical ventilation. WF patients were also younger. Patients succeed at SBT and were extubated at fi rst time in 75.9% of cases. We observed a signifi cant association between B-predominance prior to submission to SBT and WF (OR = 1.99 (1.04 to 3.84)). For diagnosing WF, B-predominance showed 69% sensitivity, 48% specifi city, 25% positive predictive value, and 86% negative predictive value.i Figure 1 (abstract P299). Association between positive fl uid balance WF in COPD patients. Reference Conclusion The fi nding of B-predominance at bedside lung ultrasound performed before SBT predicts WF, although it shows low accuracy. Reference . Perren A, Brochard L: Managing the apparent and hidden diffi culties of weaning from mechanical ventilation. Intensive Care Med 2013, 39:1885-1895. 1. Soummer A, Perbet S, Brisson H, Arbelot C, Constantin JM, Lu Q, Rouby JJ; Lung Ultrasound Study Group: Ultrasound assessment of lung aeration loss during a successful weaning trial predicts postextubation distress. Crit Care Med 2012, 40:2064-2072. P300 P300 Role of the rapid shallow breathing index to predict the success of mechanical ventilator liberation in acute respiratory failure C Chanpharm, R Bhurayanontachai Prince of Songkla University, Hat Yai, Songkhla, Thailand Critical Care 2014, 18(Suppl 1):P300 (doi: 10.1186/cc13490) Lung ultrasound fi ndings predict weaning failure from mechanical ventilation i Results During ventilation with FLEX the no-fl ow phase in late expiration was reduced by 50% compared with passive expiration. Derecruitment during the no-fl ow phase was clearly reduced by FLEX compared with VCV. Furthermore, intratidal ventilation was more homogeneously distributed during ventilation with FLEX compared with conventional passive expiration. Lung ultrasound fi ndings predict weaning failure from mechanical ventilation AC Pecanha Antonio1, P Souza Castro1, LF Schulz1, J Maccari1, R Oliveira1, C Teixeira1, M Knorst2 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P298 (doi: 10.1186/cc13488) AC Pecanha Antonio1, P Souza Castro1, LF Schulz1, J Maccari1, R Oliveira1, C Teixeira1, M Knorst2 1Hospital Moinhos de Vento, Porto Alegre, Brazil; 2UFRGS, Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P298 (doi: 10.1186/cc13488) Conclusion In comparison with conventional VCV with passive expiration, the no-fl ow phase in late expiration is reduced and so is the time the lung persists on the lowest pressure level (PEEP) during the breath. The reduced low-pressure time is associated with reduced end- tidal derecruitment. In a lung mechanically stabilized and recruited by sustained airway pressure throughout the expiration phase, the Introduction Lung ultrasound is increasingly becoming a diagnostic tool in the critical care setting. The B-line is an artifact that correlates with interstitial edema. Decreases in intrathoracic pressure during a spontaneous breathing trial (SBT) will augment venous return and impede left ventricular ejection, increasing intrathoracic blood volume. Introduction Lung ultrasound is increasingly becoming a diagnostic tool in the critical care setting. The B-line is an artifact that correlates with interstitial edema. Decreases in intrathoracic pressure during a spontaneous breathing trial (SBT) will augment venous return and impede left ventricular ejection, increasing intrathoracic blood volume. S107 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P302 Microbiology and outcomes of severe pneumonia in critically ill cancer patients L Rabello1, J Salluh1, M Soares1, M Rosolem1, V Bogado1, I Souza1, J Lapa e Silva2, L Azevedo3 1DOr Institute of Research, Rio de Janeiro, Brazil; 2Postgraduate Program of Internal Medicine – Universidade Federal do Rio de Janeiro, Brazil; 3Hospital Sirio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P302 (doi: 10.1186/cc13492) researcher. The liberation process was continued regardless of the RSBI result. The primary outcome was the success rate of mechanical ventilator liberation with or without RSBI. Introduction Pneumonia is the most frequent types of infection in cancer patients. The presence of multiresistant pathogens (MR) is often associated with inadequate antimicrobial therapy. The aims of this study were to describe the microbiology and outcomes of cancer patients with severe pneumonia requiring ICU admission. Results Analysis of 120 cases with clinical characteristics as presented in Table 1. There was no statistically signifi cant diff erence between using only clinical readiness and using clinical readiness and the RSBI (92% vs. 89%, P = 0.43). Methods A secondary analysis of a prospective cohort study was performed from 2002 to 2011 at Instituto Nacional de Cancer and Hospital Sirio-Libanes, Brazil. Adult patients with a diagnosis of cancer with pneumonia (not acquired in the hospital setting) were evaluated at ICU admission. Demographic, clinical and laboratory data were collected during the fi rst day in the ICU, severity scores, comorbidities, performance status, cancer-related data, microbiologic identifi cation, empiric antibiotics and the adherence to treatment guidelines. Conclusion The inclusion of RSBI in our standard mechanical ventilator liberation protocol for patients who met the clinical readiness criteria did not signifi cantly increase the success rate of mechanical ventilator liberation. p g Results A total of 268 patients were enrolled: 187 (69.8%) patients with solid tumors and 81 (30.2%) patients with hematological malignancies. In total, 167 (62.3%) patients had septic shock, and ICU and hospital mortality rates were 45.5% and 67.9%. Microbiological confi rmation was present in 140 (52%) patients with 56% Gram-negative. The most frequent pathogens were methicillin-sensitive S. aureus (36 (26%)), P. aeruginosa (35 (25%)) and S. pneumoniae (16 (12%)). Low incidence of MR (16 (11.4%)) was observed. Adequate antibiotic therapy based on microbiological identifi cation was prescribed in 120 (85.72%) patients. Adherence to ATS/IDSA guidelines was observed in 41 (15.3%) patients. There were no diff erences regarding ATS/IDSA guideline adherence (MR 3 (18.8%) vs. no MR 38 (15.1%), P = 0.719). We observed a trend towards higher hospital mortality in the MR patients (MR 14 (87.5%) vs. no MR 168 (66.7%), P = 0.101). P301 Determinants of ventilator weaning outcome in a medical–surgical ICU G Georgiev1, S Milanov1, L Todorova2, M Matveev2 1University Emergency Hospital ‘Pirogov’, Sofi a, Bulgaria; 2Bulgarian Academy of Sciences, Sofi a, Bulgaria i Critical Care 2014, 18(Suppl 1):P301 (doi: 10.1186/cc13491) Introduction The purpose of this study is to prospectively evaluate the determinants of weaning outcome in a selected sample of ventilator- dependent patients.i Introduction The purpose of this study is to prospectively evaluate the determinants of weaning outcome in a selected sample of ventilator- dependent patients.i p p Methods After fulfi lling a set of inclusion/exclusion criteria, 46 patients treated in a single medical–surgical ICU were prospectively evaluated between October 2011 and January 2013. The study protocol followed the generic course of mechanical ventilatory support and its discontinuation. A period of CMV was followed by stepwise reduction in pressure support. The outcomes were recorded at the second and 24th hour after the beginning of the PSV (8 cmH2O PS) spontaneous breathing trial (SBT). A short period of ZEEP breathing was introduced after the CMV as an additional respiratory stressor. A large number of parameters (including those derived via indirect calorimetry and esophageal balloon catheter) were analyzed. The following statistical methods were used: Student’s t test or its nonparametric alternatives for intergroup comparisons, repeated-measurements ANOVA for intragroup comparisons, cross-tabulation and Fisher’s exact test to compare categorical variables. Logistic regression analysis was conducted with regard to success/failure classifi cation. i Results Twenty-two (63.04% of all) patients successfully passed the 2-hour SBT. Of them, fi ve failed the subsequent 24-hour SBT. Almost all of the latter (four out of fi ve) were ventilated due to chronic respiratory or cardiac diseases. The following parameters were signifi cantly diff erent between the groups of patients who successfully and unsuccessfully completed 2-hour SBT: pre-inclusion body weight; PEEPi (end-expiratory occlusion method), peak and mean airway pressures during CMV; PaO2, PaO2/FiO2 and oxygenation index during the SBT; respiratory rate, tidal volume, f/Vt, patient work of breathing (modifi ed Campbell method), total body oxygen consumption, respiratory quotient and energy expenditure during ZEEP and the SBT. The logistic regression model devised to predict the outcome of the 2-hour SBT included the following parameters: type of artifi cial airway (tracheotomy vs. translaryngeal), oxygen consumption, f/Vt and energy expenditure during the SBT. Fluid balance predicts weaning failure in chronic obstructive pulmonary disease patients FB in the 48 hours prior to the SBT did not diff er between the WS and WF groups (1,091.11 ± 2,195.89 ml and 2,398.80 ± 1,533.15 ml, respectively). Nevertheless, comparing individuals with 48-hour FB above and under the cutoff value of 0 ml according to weaning outcomes resulted in signifi cant association between positive FB and WF in COPD patients (odds ratio = 1.77 (1.24 to 2.53)). That cutoff point was obtained on the ROC curve. See Figure 1. g Conclusion Positive FB in the 48 hours preceding the SBT predicted WF in COPD individuals. We recognized that no intervention was performed in order to accelerate the weaning process. Brain natriuretic peptide levels were not available. Figure 1 (abstract P300). Standard mechanical ventilator weaning protocol. Figure 1 (abstract P300). Standard mechanical ventilator weaning protocol. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S108 Table 1 (abstract P300). Character CR CR + RSBI P value Age 55.79 ± 19.6 56.62 ± 19.4 0.74 Male sex (%) 58.3 59.1 0.90 Success rate (%) 89.2 92.2 0.43 P302 Microbiology and outcomes of severe pneumonia in critically ill cancer patients L Rabello1, J Salluh1, M Soares1, M Rosolem1, V Bogado1, I Souza1, J Lapa e Silva2, L Azevedo3 1DOr Institute of Research, Rio de Janeiro, Brazil; 2Postgraduate Program of Internal Medicine – Universidade Federal do Rio de Janeiro, Brazil; 3Hospital Sirio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P302 (doi: 10.1186/cc13492) P302 Microbiology and outcomes of severe pneumonia in critically ill cancer patients L Rabello1, J Salluh1, M Soares1, M Rosolem1, V Bogado1, I Souza1, J Lapa e Silva2, L Azevedo3 1DOr Institute of Research, Rio de Janeiro, Brazil; 2Postgraduate Program of Internal Medicine – Universidade Federal do Rio de Janeiro, Brazil; 3Hospital Sirio-Libanês, São Paulo, Brazil Critical Care 2014, 18(Suppl 1):P302 (doi: 10.1186/cc13492) In multivariate analysis, mechanical ventilation (OR 2.52 (1.19 to 5.32)), dialysis (3.86 (1.23 to 12.10)) and higher SAPS2 (OR per point 1.03 (1.01 to 1.05)) were associated with increased hospital mortality whereas successful noninvasive ventilation was associated with lower mortality (0.32 (0.13 to 0.77)). MR were forced into MV analysis but were not associated with outcomes. Conclusion Severe pneumonia in cancer patients presents high hospital mortality, with particular clinical and microbiology features. Despite low adherence to ATS/IDSA guidelines, antibiotic therapy was adequate in most of patients following local guidelines based on local bacterial profi les. Further investigation is needed to clarify the impact of MRs in clinical outcomes of cancer patients with severe pneumonia. P304 g Results Median age and SOFA scores on ICU admission were 66 (interquartile range: 50 to 77) years and 6 (4 to 8) years, respectively. Primary diagnosis on ICU admission was coma including post-cardiac arrest syndrome (31%), trauma (29.4%), and sepsis (9.8%). Prediction of MDRPs was signifi cantly higher in the risk group than in the nonrisk group (36.8% vs. 15.2%, P = 0.004). Guideline adherence was lower in the risk group (45.6% vs. 65.2%, P = 0.016). Treatment adequacy was greater with guideline adherence than with nonadherence (75.7% vs. 55.9%, P <0.01). Hospital mortality was not aff ected by guideline adherence (P = 0.70). Multivariate analysis revealed that the independent factors related to hospital mortality were adequate antimicrobial treatment (odds ratio: 0.53, 95% CI: 0.29 to 0.95; P = 0.034), age (1.02, 1.00 to 1.04; P = 0.004), history of malignancy (8.59, 1.48 to 49.6; P = 0.016), trauma (0.41, 0.2 to 0.87; P = 0.02), acute kidney injury (5.54, 2.69 to 11.4: P <0.001), and severe sepsis at VAP onset (2.7, 1.4 to 5.1; P = 0.002). Conclusion The prediction of MDRPs using the 2005 ATS/IDSA guidelines was acceptable. To ensure adequate antimicrobial treatment, strict guideline adherence is required in the Japanese setting. P304 Clinical pulmonary infection score calculator in the early diagnosis and treatment of ventilator-associated pneumonia in the ICU O Celik, N Koltka, S Devrim, B Sen, M Gura Celik Istanbul Medeniyet University, Goztepe Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P304 (doi: 10.1186/cc13494) Introduction Ventilator-associated pneumonia (VAP) is a frequently occurring nosocomial infection in ICU patients and has been associated with increased morbidity, prolonged duration of ventilation and ICU stay and increased costs for healthcare. It was shown that early diagnosis of VAP and immediate initiation of appropriate antibiotics is associated with reduced morbidity and mortality. The aim of this study is to evaluate the potential ability of a screening test based on the clinical pulmonary infection score (CPIS) to identify and treat patients with VAP.i y Methods All fi les belonging to patients between 18 and 80 years old admitted to the ICU and supported by mechanical ventilation for longer than 48 hours were evaluated retrospectively. Demographic data of the patients, the time of mechanical ventilation, duration of the ICU stay and results (survival or death) were recorded. The CPIS was calculated after 48 hours for the diagnosis of VAP. P305 non-VAP groups and receiver operating characteristics (ROC) curves were constructed for individual biomarkers and combinations of markers before optimum cutoff points were determined. non-VAP groups and receiver operating characteristics (ROC) curves were constructed for individual biomarkers and combinations of markers before optimum cutoff points were determined. Validation of the 2005 American Thoracic Society/Infectious Diseases Society of America guidelines for ventilator-associated pneumonia: a Japanese multicenter observational study N Saito1, N Shime2, K Sugiyama3, H Yasuda4, K Ishii5, Y Masuda6, T Kanemura7, W Sakamoto8, T Hirayu9 1Nippon Medical School Chiba Hokusoh Hospital, Inzai, Chiba, Japan; 2Kyoto Medical Center, Kyoto, Japan; 3Tokyo Metropolitan Hospital Bokuto, Tokyo, Japan; 4Musashino Red Cross Hospital, Tokyo, Japan; 5Fukuyama City Hospital, Fukuyama, Japan; 6Nagasaki Medical Center, Omura, Japan; 7National Disaster Medical Center, Tokyo, Japan; 8Nippon Medical School Hospital, Tokyo, Japan; 9Kurume University Hospital, Kurume, Japan Critical Care 2014, 18(Suppl 1):P305 (doi: 10.1186/cc13495) f Results A total of 150 patients had paired semi-quantitative culture and biomarker results. Fifty-three (35%) patients had VAP and 97 (65%) patients formed the non-VAP group. All BALF biomarkers were signifi cantly higher in the VAP group (P <0.001). The area under the ROC curve for IL-1β was 0.81, for IL-8 was 0.736, for MMP-8 was 0.758, for MMP-9 was 0.785 and for HNE was 0.777. Using a cutoff value of 17 pg/ ml, IL-1β had a sensitivity of 96.2%, a specifi city of 43.3%, a negative predictive value (NPV) of 95.5% and a post-test probability of 4.5% (95 CI: 1 to 16%). A combination of IL-1β and IL-8 excluded VAP with a sensitivity of 100%, a specifi city of 44.3% and a NPV of 1. There was no signifi cant diff erence in serum biomarkers between the VAP and non- VAP groups. Introduction To select the empirical antimicrobial treatment for patients with ventilator-associated pneumonia (VAP), the 2005 American Thoracic Society/Infectious Disease of America (ATS/IDSA) guidelines classify patients according to time of onset and risk factors for potential multidrug-resistant pathogens (MDRPs). This study aimed to evaluate the eff ectiveness of microbial prediction and validate the adequacy of these guidelines for antibiotic strategy in VAP patients. g p Conclusion This study demonstrates that IL-1β eff ectively excludes VAP when validated in a multicentre study. The performance is further improved by the addition of IL-8, and the combination could form a rapid diagnostic assay to exclude VAP. P305 Biomarker analysis appears to have the potential to improve antibiotic stewardship, and this concept should be formally tested in the setting of a randomised controlled trial. y y Methods We retrospectively analyzed 296 patients who received empiric antimicrobial treatment for VAP between January 2006 and December 2010 in 10 ICUs of Japanese tertiary hospitals. VAP was diagnosed according to CDC criteria. After assigning patients to the ATS/IDSA risk group (n = 250; late onset or risk factors for MDRPs) or non-risk group (n = 46; early onset without risk factors for MDRPs), we determined the accuracy of the guidelines for predicting pathogens and the impact of guideline adherence on patient outcome. References References 1. Vincent JL, et al.: JAMA 2009, 302:2323-2329. 2. Chastre J, et al.: Am J Respir Crit Care Med 2002, 165:867-903. 3. Conway Morris A, et al.: Thorax 2010, 65:201-207. 4. Wilkinson TS, et al.: Chest 2012, 142:1425-1432. P304 The patients with CPIS >5 intubated were evaluated VAP(+) and the others with CPIS ≤5 were thought VAP(–). The diagnosis of VAP was bacteriologically confi rmed with the culture of endotracheal aspirate. Statistical evaluations were done according to the results on the day of intubation and the results on days 2, 3, 5, 8 and 10 after intubation. Scores of APACHE II and CRP levels were also recorded on the same days. Biomarker-based exclusion of ventilator-associated pneumonia: a multicentre validation study T Hellyer, J Simpson T Hellyer, J Simpson Newcastle University, Newcastle, UK Critical Care 2014, 18(Suppl 1):P303 (doi: 10.1186/cc13493) Introduction Ventilator-associated pneumonia (VAP) remains a leading cause of nosocomial infection in the ICU [1]. VAP is confi rmed by positive microbiology in approximately one-third of patients with suspected VAP [2], implying that there is scope to improve antibiotic stewardship. In a single-centre study, bronchoalveolar lavage fl uid (BALF) infl ammatory mediators (in particular interleukin-1 beta (IL-1β)) [3] and neutrophil proteases [4] demonstrated potential as biomarkers to exclude VAP. We aimed to validate these fi ndings in a multicentre study. Methods We conducted a prospective, multicentre observational study of 167 patients with clinically suspected VAP from 12 ICUs across the UK. VAP was confi rmed by growth of a potential pathogen in BALF at >104 colony-forming units/ml. IL-1β, IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were measured in BALF by cytometric bead array. IL-6, IL-8, MMP-8, MMP-9 and HNE were measured in serum. Patients were dichotomised into VAP and Conclusion The presence of serious respiratory and/or cardiac comorbidities might require longer duration of the SBT. In our study, respiratory load–muscle capacity balance and metabolic activity appear to play a major role in determining the weaning outcome. A short period of ZEEP breathing is safe and might have prognostic utility regarding the outcome of mechanical ventilatory support discontinuation. S109 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P306 P306 Surveillance and evaluation of ventilator-associated events as per Centers for Disease Control and Prevention guidelines M Brown, B Rose Lewisham and Greenwich NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P306 (doi: 10.1186/cc13496) P307 Extracorporeal carbon dioxide removal as a bridge to lung transplantation in life-threatening hypercapnia K Riss, G Lang, T Staudinger, R Ullrich, CG Krenn, C Sitzwohl, A Bojic, P Wohlfarth, WR Sperr, W Rabitsch, C Aigner, S Taghavi, P, W Klepetko, P Schellongowski Medical University of Vienna, Austria Critical Care 2014, 18(Suppl 1):P307 (doi: 10.1186/cc13497) Extracorporeal carbon dioxide removal as a bridge to lung transplantation in life-threatening hypercapnia K Riss, G Lang, T Staudinger, R Ullrich, CG Krenn, C Sitzwohl, A Bojic, P Wohlfarth, WR Sperr, W Rabitsch, C Aigner, S Taghavi, P, W Klepetko, P Schellongowski Medical University of Vienna, Austria Critical Care 2014, 18(Suppl 1):P307 (doi: 10.1186/cc13497) Results Sixteen subjects were recruited for this study. There was a signifi cant increase in paO2 (13.6 ± 2.35 kPa, P = 0.01) and decrease in paCO2 (4.82 ± 1.27 kPa, P = 0.02) in the chair group at 1 hour after sitting out in the chair when compared with baseline (10.9 ± 2.44 kPa; 5.41 ± 1.32 kPa). Also there was a signifi cant increase in tidal volume in the chair group after 1 minute of sitting out (403 ± 118 ml) compared with baseline (314 ± 105 ml). There was no diff erence in the electric bed group for all physiological parameters. The chair group had a better CPAX score on discharge from intensive care (chair group 24; electric bed group 13) and on discharge from the hospital (chair group 39; electric bed group 16). There were no adverse cardiovascular responses to either position. Introduction The introduction of the lung allocation score has resulted in a growing number of patients who are considered for lung transplantation (LTX) while being acutely decompensated. In the sickest of these patients, mechanical ventilation (MV) alone may not be suffi cient to establish adequate gas exchange. Thus, diff erent modes of extracorporeal life support have come to the focus of interest in this setting. Conclusion Sitting suitable critically ill patients out into a chair is safe and can signifi cantly improve the arterial blood gas measurement and the tidal volume when compared with sitting a patient into the sitting position in an electric bed. P307 g Methods A retrospective analysis of 17 patients (male/female ratio: 6/11; median age: 35 (range 16 to 63)) who underwent arteriovenous or venovenous interventional lung assist (iLA; Novalung, Germany) support as bridging to primary LTX (n = 11) or re-LTX (n = 6) between 2005 and 2013. P309 Quantifying sputum production in intensive therapy C Pope, R Spacie, S Reynolds, H Jones Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P309 (doi: 10.1186/cc13499) p Reference p Reference 1. Zhiqiang L, et al.: Arch Phys Med Rehab 2013, 94:551-561. 1. Zhiqiang L, et al.: Arch Phys Med Rehab 2013, 94:551-561. 1. Zhiqiang L, et al.: Arch Phys Med Rehab 2013, 94:551-561. Results The underlying diagnosis was bronchiolitis obliterans syndrome III in re-LTX patients (n = 6), cystic fi brosis (n = 5), idiopathic pulmonary fi brosis (n = 2), emphysema (n = 1), adult respiratory distress syndrome (n = 1), hemosiderosis (n = 1), and chronic obstructive lung disease (n = 1), respectively. The type of iLA was arteriovenous in 10 and venovenous (iLA active) in seven patients. The median bridging time was 14 (1 to 58) days. The type of transplantation was bilateral LTX (n = 6), size-reduced bilateral LTX (n = 5), lobar bilateral LTX (n = 4), and right single LTX with contralateral pneumonectomy (n = 1), respectively. Hypercapnia was eff ectively corrected in all patients within the fi rst 12 hours of iLA therapy: PaCO2 levels declined from 145 (70 to 198) to 60 (36 to 99) mmHg, P <0.0001. iLA was initiated during non-invasive ventilation in three patients, of whom one was intubated prior to LTX. All other patients (n = 14) were placed on iLA while on invasive MV. Of those, three patients were extubated and remained on iLA until LTX, one patient was weaned from iLA and remained on MV until LTX, and one patient was weaned from iLA and MV prior to LTX. Five patients were switched to extracorporeal membrane oxygenation (venovenous n = 2, venoarterial n = 3) after 5 (1 to 30) days on iLA support. One patient died prior to LTX due to septic multiorgan failure (SMOF). All others (n = 16; 94%) were successfully transplanted. Of these, two patients died in the ICU due to SMOF. The remaining 14 patients (82%) survived to hospital discharge and were alive at a median follow-up of 20 (1 to 63) months. Reference 1. Magill SS, et al.: Developing a new national approach to surveillance for ventilator-associated events: executive summary. Crit Care Med 2013, 41:2467-2475. 1. Magill SS, et al.: Developing a new national approach to surveillance for ventilator-associated events: executive summary. Crit Care Med 2013, 41:2467-2475. Surveillance and evaluation of ventilator-associated events as per Centers for Disease Control and Prevention guidelines M Brown, B Rose Introduction While ventilator-associated pneumonia (VAP) can aff ect all patients ventilated for >2 days in an ICU, an issue during the last two decades has been identifi cation of a universally accepted defi nition. The Centers for Disease Control and Prevention (CDC) in Atlanta, GA, USA, have designed a new surveillance paradigm aiming to increase the objectivity of VAE diagnosis ranging from ventilator-associated condition (VAC), infective VAC to possible and probable VAP [1]. We assessed the feasibility and accessibility of the audit tool developed from the CDC guidelines as a marker of quality assurance. y Results The duration of mechanical ventilation and ratio of death were signifi cantly higher in the patients with VAP(+). CPIS levels in the patients with VAP(+) were signifi cantly higher than the patients with VAP(–) in the days after the diagnosis. CPIS levels were also higher in the patients with VAP(+) on the day of diagnosis. At the same day the parameters, which included the CPIS, body temperature, leukocyte number, tracheal secretions, PaO2/FiO2 levels and the presence of infi ltrates on the chest radiograph, were signifi cantly higher in VAP(+) patients (P <0.05). ROC curves were formed for CPIS scores to be used in diagnosis VAP and the cutoff point had a sensitivity of 97.44% and a specifi city of 100% for diagnosing VAP. Methods A prospective audit of patients admitted to University Hospital Lewisham ICU for intubation and ventilation was performed for September and October 2013. Recording of minimum PEEP and FiO2, minimum and maximum temperatures, WBC count, specimens sent, antibiotics administered and date of organisms found was performed on a daily basis. VAEs were recorded as per modifi ed CDC criteria (absence of quantitative microbiological testing) and compared Conclusion At the end of the study, it was concluded that using the CPIS for early diagnosis and treatment of VAP and thinking that the patients with CPIS >5 were VAP(+) are guiding factors to resolve the problems associated with VAP in ICU patients. S110 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Diff erence in physiological parameters between sitting out of bed into a chair or sitting up on an electric bed in the adult ICU A Fitzgerald, D Breen Diff erence in physiological parameters between sitting out of bed into a chair or sitting up on an electric bed in the adult ICU A Fitzgerald, D Breen Cork University Hospital, Cork, Ireland Critical Care 2014, 18(Suppl 1):P308 (doi: 10.1186/cc13498) Introduction The primary aim of this study was to identify in adult intensive care patients whether there was a diff erence in the acute physiological response when a patient is sat out into a chair compared with when a patient is placed in a chair position using an electric bed. The secondary aim of this study was to observe the functional outcome of these patients [1]. Conclusion While similar numbers of VAEs are triggered using CDC and clinical criteria, we noted a disparity in the VAP incidence. Four clinical VAPs were not triggered due to the hierarchical nature of the CDC criteria because the initial criterion of sustained minimum PEEP or FiO2 rise was not met despite clear clinical criteria based on increased infl ammatory markers, temperature spikes and new microbiology. We are therefore doubtful of the robustness of this tool in its current format as an accurate measure of quality assurance and agree with recent statements from the CDC [1] that modifi cations may be necessary. R f Methods The study was conducted in an adult tertiary referral ICU over a 3-month period. Patients that met predetermined inclusion/exclusion criteria were allocated to either sitting in a chair or sitting up in an electric bed. Heart rate, respiratory rate, tidal volume and mean arterial pressure were obtained for all patients when they were supine in bed, at 1 minute and at 1 hour in the new position. Arterial blood gases were obtained at 1 hour in their new position. A functional outcome measure known as the Chelsea Critical Care Physical Assessment Tool (CPAX) was also taken on the day of admission, on the day of sitting out, on the day of discharge from intensive care and at ward level. All data were analysed using Student’s t test. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion In patients with life-threatening hypercapnia, bridging to LTX with iLA is feasible, and results in favorable short-term and long- term outcome. Conclusion In patients with life-threatening hypercapnia, bridging to LTX with iLA is feasible, and results in favorable short-term and long- term outcome. against a surrogate marker of clinically diagnosed VAEs based on information available to the clinician.i Results Forty patients were admitted with 29 fulfi lling inclusion criteria. Of n = 29, mean age was 58 (SD 21) with a median number of 5 (2 to 29) ventilator-days. We recorded eight VAEs based on CDC criteria, one VAC, four IVAC and three VAPs. Clinical decision-making indicated seven VAEs, of which fi ve were VAPs. CDC and clinical criteria correlated in only fi ve of eight VAPs and clinical criteria alone would have indicated 40% more VAPs than CDC criteria. Median time to VAE trigger was 8 days (4 to 20), median number of ventilator-days if a CDC VAE triggered was 14 compared with 3 if no CDC VAE was triggered (P <0.01), and 11 compared with 3 (P <0.01) for clinical VAEs. Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit p MC Faulds, S Lobaz, AJ Glossopfi Introduction Bilevel non-invasive ventilation (NIV) is an established therapy in chronic obstructive pulmonary disease (COPD) and cardiogenic pulmonary oedema but evidence for its use in other acute respiratory conditions is less robust. Reported ICU mortality after NIV treatment of pneumonia ranges between 18 and 33% [1,2], compared with 10% in exacerbations of COPD [3]. We aimed to study the outcomes of patients with acute respiratory failure of mixed aetiology treated with NIV in our critical care unit and compare fi ndings with those already published. Conclusion In this study, NHF gradually improved oxygenation. Additionally, NHF reduces the respiratory rate and the value of PaCO2. This result might suggest that NHF decreased dead-space ventilation. There was no diff erence between the success group and the failure group. So we can use NHF as the fi rst choice for postoperative respiratory failure, but it is diffi cult to predict success or failure. y p Methods Data were collected retrospectively on patients admitted to our critical care unit with acute respiratory failure requiring NIV over a 3-year period using the Metavision electronic patient record system. Patients with a primary surgical problem and those who received continuous positive airway pressure as a primary intervention were excluded. We recorded: primary respiratory diagnosis causing respiratory failure; patient demographics; serial arterial blood gas results; success of NIV as defi ned by the British Thoracic Society (BTS) [4]; and mortality statistics. Inhalation injury and clinical course in major burned patients S Henrich, TH Rech, IC Warwzeniak, RB Moraes, E Parolo, K Prado, SR Vieira Hospital de Clínicas de Porto Alegre, Brazil Critical Care 2014, 18(Suppl 1):P312 (doi: 10.1186/cc13502) Results In total, 113 consecutive patients were identifi ed. Mean age was 64 and 50% (56/113) were male. The primary diagnosis was pneumonia in 55 patients and exacerbation of COPD in 40 patients. The overall mortality on critical care, in hospital and at 1 year was 19% (22/113), 34% (38/113) and 41% (46/113) respectively. Success of NIV as defi ned by BTS criteria (pH >7.3 or reduction in PaCO2 by 0.5 kPa) in the fi rst 6 hours was seen in 72% (80/111) of patients. In NIV responders, 1-year mortality was 31% (25/80) compared with 65% (20/31) in nonresponders. Introduction Inhalation injury is the primary determinant of mortality in major burned patients, particularly when associated with pneumonia. Eff ect of nasal high fl ow for postoperative respiratory failure: a prospective observational study Eff ect of nasal high fl ow for postoperative respiratory failure: a prospective observational study p p y S Okahara, K Shimizu, M Hayashi, H Morimatsu Okayama University Hospital, Okayama City, Japan Critical Care 2014, 18(Suppl 1):P311 (doi: 10.1186/cc13501) Introduction We studied the eff ect of nasal high fl ow (NHF) for postoperative respiratory failure after extubation in our general surgical ICU. Recently some studies have reported that NHF improves oxygenation and reduces respiratory rate. However, the usefulness of NHF in the general surgical ICU has not been fully determined. NHF in the general surgical ICU has not been fully determined. Methods A prospective observational study was conducted in our general surgical ICU to investigate the eff ect of NHF on respiratory parameters in patients with postoperative respiratory failure. Patients who were admitted to the ICU for postoperative respiratory failure (defi ned as oxygen saturation as measured by pulse oximetry <96% and/or respiratory rate>24 beats/minute while receiving more than 6 l/ minute oxygen through a facemask) were eligible in this study. Pre and 1 and 6 hours after NHF treatment, we collected PaO2, PaCO2, respiratory rate, heart rate, and blood pressure. Data were presented with means and standard deviations. P <0.05 was considered statistically signifi cant. Results Forty-two patients were treated using NHF in our ICU from February 2013 to November 2013. The mean age of the patients was 62.8 ± 15.8 years, and the male:female ratio was 22:19. PaO2 values after 1 and 6 hours of NHF (104 ± 34 mmHg, 107 ± 26 mmHg, respectively) were signifi cantly higher than that before NHF (89 ± 38 mmHg; P <0.02). The PaO2/FiO2 ratio was increased from 1 hour to 6 hours of NHF (from 218 ± 90 mmHg to 236 ± 86 mmHg, P <0.05). Respiratory rate after NHF (19.6 ± 4.7/minute) was signifi cantly lower than that at baseline (22.3 ± 4.8/minute; P = 0.0006), whereas PaCO2 after NHF was reduced compared with baseline (from 41 ± 7 mmHg to 39 ± 5 mmHg, P <0.02). Thirty-two (76%, success group) patients did not need other positive ventilation. On the other hand, 10 (24%, failure group) patients required non-invasive positive pressure ventilation or intubation. We compared the failure group with the success group. However, there were no signifi cant diff erences in vital signs, total bleeding, operative duration and preoperative respiratory function between the groups. P311 decisions to extubate or decanulate. The quantifi cation of sputum was inconsistent: 39% of respondents counted the frequency of suctioning, 24% measured the quantity of sputum in the suction tubing, whereas 25% used another method. An eff ective cough, consistency and colour were felt to be more important features of sputum than blood staining. Conclusion Our results showed a very high level of agreement on the importance of knowing sputum load for decisions to extubate, decanulate or discharge from the ITU. In contrast, there was little consensus on how we should quantify sputum load in ventilated patients. This lack of standard approach may contribute to uncertainty in the clinical decision-making process. We have developed an objective sputum scoring system. Components identifi ed as important by our survey such as suction frequency, sputum consistency and colour are included. We have recognised the benefi ts of the standardised Bristol stool chart to facilitate communication and believe this can be achieved with sputum load in ventilated patients. R f Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit This study sought to describe the association between severity of inhalation injury on bronchoscopic examination and clinical course among fi re victims of Santa Maria, RS, Brazil. Two hundred and forty-two people were killed in the disaster. Methods Eighteen patients with inhalation injury secondary to smoke and fi re exposure in an enclosed space admitted to Hospital de Clínicas de Porto Alegre were divided into groups according to the severity of injury as determined by bronchoscopic criteria: grade 1 (moderate edema and hyperemia), grade 2 (marked edema and hyperemia, with or without carbonaceous debris), or grade 3 (mucosal ulceration or necrosis). Duration of mechanical ventilation (MV), length of ICU stay, overall length of hospital stay, and PaO2/FiO2 ratio on days 1 and 3 were compared among these groups by means of ANOVA with Tukey’s post- hoc correction. Conclusion NIV is used to treat acute respiratory failure due to a wide range of aetiologies in our unit with comparable mortality rates to large published series [1,2,5]. A successful response to NIV in the fi rst 6 hours is associated with a reduction in 1-year mortality when compared with nonresponders. P310 P310 Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P310 (doi: 10.1186/cc13500) P310 Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P310 (doi: 10.1186/cc13500) Outcomes of patients with acute respiratory failure of mixed aetiology treated with non-invasive ventilation in a large teaching hospital critical care unit MC Faulds, S Lobaz, AJ Glossop Sheffi eld Teaching Hospitals NHS FT, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P310 (doi: 10.1186/cc13500) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Eff ect of nasal high fl ow for postoperative respiratory failure: a prospective observational study Conclusion In this study, NHF gradually improved oxygenation. Additionally, NHF reduces the respiratory rate and the value of PaCO2. This result might suggest that NHF decreased dead-space ventilation. There was no diff erence between the success group and the failure group. So we can use NHF as the fi rst choice for postoperative respiratory failure, but it is diffi cult to predict success or failure. 1. Fahy et al.: Airway mucus function and dysfunction. N Engl J Med 2010, 363:2233-2247 1. Fahy et al.: Airway mucus function and dysfunction. N Engl J Med 2010, 363:2233-2247 2. Kulkarni et al.: Extubation failure in intensive care: predictors and management. Indian J Crit Care Med 2008, 12:1-9. P310 Quantifying sputum production in intensive therapy C Pope, R Spacie, S Reynolds, H Jones Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P309 (doi: 10.1186/cc13499) Introduction Sputum is essential for the protection of the respiratory tract but also plays a signifi cant role in the pathophysiology of lung disease [1]. This is evident in critical care where high sputum loads contribute to respiratory failure [2]. The quantity of sputum produced by a patient can impact on key decisions such as weaning, extubation and discharge. We undertook a survey to establish whether there was a consensus on how we quantify sputum on our intensive therapy unit (ITU). Methods We conducted a multidisciplinary team questionnaire of our 28-bed tertiary ITU. Staff were asked how they quantifi ed sputum load in intubated patients. They were also asked to rate statements on a fi ve-point scale pertaining to sputum characteristics. The results were analysed in Excel 2010.f Results One hundred members of staff completed the sputum production in intensive therapy (SPIT) questionnaire (21% doctors, 71% nurses, 8% physiotherapists). Sputum load was deemed to be important or essential by more than 95% of respondents when making S111 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References 1. Carillo et al.: Intensive Care Med 2012, 38:458-466. 2. Jolliet et al.: Intensive Care Med 2001, 27:812-821. 3. Lightowler et al.: BMJ 2003, 326:185-189. 4. 2013 BTS NIV Audit [http://www.brit-thoracic.org.uk/Portals/0/Audit%20 Tools/SummaryReports/NIV%20Audit%202013-FINAL.pdf] 5. Confalonieri et al.: Am J Respir Crit Care Med 1999, 160:1585-1591. P314 Advanced respiratory care techniques in a severe adult respiratory failure unit D Rafi q, A Needham, R Porter, G Lau Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P314 (doi: 10.1186/cc13504) yg References References 1. Cancio L, et al.: Clin Plastic Surg 2009, 36:555-567. 2. Mlcak RP, et al.: Burns 2007, 33:2-13. Figure 1 (abstract P314). Frequency of interventions in the base hospital (BH) and SARF centre. Reference 1. Peek GJ, Mugford M, Tiruvoipati R, et al.: Effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet 2009, 374:1351–1363. doi: 10.1016/ S0140-6736(09)61069-2. g Results Thirteen casualties had an average Injury Severity Score of 50.3 ± 10.5 (age 27.7 ± 8.6 years, 69.2% male) and were supported 9.9 ± 4.8 days on ECMO (n = 7) and 7.16 ± 5.9 days on iLA (n = 6). All suff ered severe chest injuries, including one cardiac perforation. Most were coagulopathic prior to initiation of ECMO/iLA support. Among the seven patients with TBI, four had active intracranial hemorrhage. Only 30% of the patients received continuous anticoagulation during the fi rst 24 hours of support without clotting of the system or diagnosis of a thromboembolic event. Complications directly related to support therapy were not lethal; these included hemorrhage from a cannulation site (n = 1), accidental removal of a cannula (n = 1) and pressure sores (n = 3). Deaths occurred due to septic (n = 3) and cardiogenic shock (n = 1). Survival rates were 57 and 83% on ECMO and iLA, respectively. Follow-up of survivors detected no neurological deterioration. p References 1. Carillo et al.: Intensive Care Med 2012, 38:458-466. 2. Jolliet et al.: Intensive Care Med 2001, 27:812-821. 3. Lightowler et al.: BMJ 2003, 326:185-189. 4. 2013 BTS NIV Audit [http://www.brit-thoracic.org.uk/Portals/0/Audit%20 Tools/SummaryReports/NIV%20Audit%202013-FINAL.pdf] 5. Confalonieri et al.: Am J Respir Crit Care Med 1999, 160:1585-1591. 1. Carillo et al.: Intensive Care Med 2012, 38:458-466. 2. Jolliet et al.: Intensive Care Med 2001, 27:812-8 Results Three patients had grade 1 injury, four had grade 2 injury, and 11 had grade 3 injury. Seven patients developed ventilator-associated pneumonia. Mean duration of MV increased progressively in relation S112 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P314). Frequency of interventions in the base hospital (BH) and SARF centre. to injury severity (grade 1: 2.7 ± 0.6 days vs. grade 2: 5.7 ± 2.1 days vs. grade 3: 13.0 ± 5.4 days; P = 0.004), as did length of ICU stay (grade 1: 4 days vs. grade 2: 7.2 ± 2.2 days vs. grade 3: 19.9 ± 7.7 days; P = 0.001) and overall length of hospital stay (grade 1: 5 days vs. grade 2: 14.5 ± 5.0 days vs. grade 3: 63.4 ± 43.6 days; P = 0.025). There were no signifi cant diff erences in PaO2/FiO2 ratio between groups at day 1. However, as expected, diff erences in PaO2/FiO2 ratio were found on day 3 (grade 1: 501 vs. grade 2: 424 ± 115 vs. grade 3: 318 ± 120; P = 0.049). Conclusion In this cohort of patients with major burns, the severity of inhalation injury was associated with prolonged MV, length of ICU stay, and overall length of hospital stay, as well as with deterioration in oxygenation on day 3. Severe respiratory failure in multiple trauma patients: extracorporeal support as a salvage therapy – a single-center experience PB Bid Methods All patients transferred to the Severe Adult Respiratory Failure (SARF) unit between April 2012 and April 2013 for advanced respiratory care who did not require ECMO, identifi ed from the ECMO database, underwent retrospective notes’ review to identify the advanced respiratory techniques performed at the SARF unit and the base hospital. Methods All patients transferred to the Severe Adult Respiratory Failure (SARF) unit between April 2012 and April 2013 for advanced respiratory care who did not require ECMO, identifi ed from the ECMO database, underwent retrospective notes’ review to identify the advanced respiratory techniques performed at the SARF unit and the base hospital. Rabin Medical Center, Petah Tikva, Israel Critical Care 2014, 18(Suppl 1):P313 (doi: 10.1186/cc13503) Introduction Use of extracorporeal life support (ECLS) in trauma casualties is limited by concerns regarding hemorrhage, particularly in the presence of traumatic brain injury (TBI). We report usage of ECMO/ interventional lung assist (iLA) as salvage therapy in 13 trauma patients. A high-fl ow technique without anticoagulation was used in cases with coagulopathy or severe TBI. p Results Ten patients were admitted for advanced respiratory care who did not require ECMO. Eight patients had community-acquired pneumonia, one had an inhalational injury and one had a previously undiagnosed cardiac sarcoma. The techniques utilised in the SARF unit and base hospital are shown in Figure 1. Nine patients were discharged from the SARF unit alive (90%). Conclusion A negative fl uid balance and targeting higher haemoglobin are more frequently achieved in a SARF unit. Further studies are required to fully elucidate the advanced respiratory care techniques undertaken at specialist SARF units. g p y Methods Data were collected from all adult trauma cases referred to one center for ECMO/iLA treatment due to severe hypoxemic respiratory failure. Thirteen consecutives cases are reported. The type of assistance was chosen based on a fl owchart. Type of study: therapeutic, level of evidence IV. We analyzed patient data, injury data, blood gases before connection, methods of assistance, coagulation study, complications, survival and neurological outcome. Nebulized C1-esterase inhibitor treatment does not attenuate pulmonary complement activation in a rat model of severe Streptococcus pneumoniae pneumonia Introduction While complement protein defi ciencies are associated with severe and recurrent pulmonary infections, excessive complement activation plays a role in the pathogenesis of lung injury. We hypo- thesized that inhibition of the complement system by repetitive treatment with nebulized plasma-derived human C1-esterase inhibitor (C1-INH) reduces pulmonary complement activation and subsequently attenuates lung injury and lung infl ammation in a model of severe Streptococcus pneumoniae pneumonia. Conclusion ECMO/iLA therapy can be used as rescue therapy in adult trauma cases with severe hypoxemic respiratory failure, even in the presence of coagulopathy, bleeding and/or brain injury. The benefi ts of oxygenation and circulatory support must be weighed individually against the risk of hemorrhage. Further research should determine whether ECMO therapy also confers survival benefi t. Methods Thirty-two male rats were intratracheally challenged with S. pneumoniae to induce pneumonia. Rats were repeatedly exposed to nebulized C1-INH or saline, 30 minutes before induction of pneumonia and every 6 hours thereafter. Rats were sacrifi ced 20 or 40 hours after inoculation to investigate early and late eff ects. BALF and lung tissue were obtained for measuring levels of complement activation (C4b/c in BALF), lung injury (total protein levels in BALF), and infl ammation (IL-6 levels in lung tissue). Advanced respiratory care techniques in a severe adult respiratory failure uniti D Rafi q, A Needham, R Porter, G Lau Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P314 (doi: 10.1186/cc13504) Introduction Outcome is improved when patients with severe adult respiratory distress syndrome are transferred to an extracorporeal membrane oxygenation (ECMO)-capable unit [1]. Not all patients transferred require ECMO, and this service evaluation examines which techniques are utilised in patients who ultimately do not require ECMO. Introduction Outcome is improved when patients with severe adult respiratory distress syndrome are transferred to an extracorporeal membrane oxygenation (ECMO)-capable unit [1]. Not all patients transferred require ECMO, and this service evaluation examines which techniques are utilised in patients who ultimately do not require ECMO. Results Pneumonia was characterized by bilateral macroscopic infi ltrates, bacterial outgrowth in the lung and clinical signs of illness. Pneumonia was associated with pulmonary complement activation. In rats treated with nebulized C1-INH, a functional fraction of C1-INH was detectable in BALF. However, C1-INH treatment did not aff ect S113 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). Conclusion Severe S. pneumoniae pneumonia is associated with pulmonary complement activation in rats. Nebulized C1-INH treat- ment, in an attempt to reduce pulmonary complement activation, neither aff ects pulmonary complement activation or lung injury and infl ammation. P316 Novel carbon dioxide removal device driven by a renal-replacement system without hemofi lter: an experimental approach and validation T Godet1, A Combes2, E Zogheib3, M Jabaudon1, E Futier1, J Constantin1 1CHU Clermont-Ferrand, France; 2CHU Pitié-Salpêtrière, Paris, France; 3CHU Amiens, France Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Introduction Management of acute respiratory diseases must avoid ventilator-induced lung injuries along with the rise of PaCO2 and respiratory acidosis [1]. Eff orts are made to fi nd devices assisting protective ventilation and able to remove arterial CO2 and correct acidosis [2]. An extracorporeal CO2 removal device driven by the widely used Prismafl ex® platform called PrismaLung® was tested in vivo. Methods Five hypercapnic ventilated pigs were equipped with the PrismaLung® system designed to remove CO2 from the bloodstream through a decarboxylation membrane mounted on a renal replacement device without any hemofi lter. Experiments examined the potential for blood decarboxylation by gas-exchanger membrane with diff erent sets of parameters (blood fl ow rate: 200, 300 and 400 ml/minute, sweep gas fl ow: 2, 5, 10 and 50 l/minute, FiO2: 21 and 100%). P316 P316 Novel carbon dioxide removal device driven by a renal-replacement system without hemofi lter: an experimental approach and validation T Godet1, A Combes2, E Zogheib3, M Jabaudon1, E Futier1, J Constantin1 1CHU Clermont-Ferrand, France; 2CHU Pitié-Salpêtrière, Paris, France; 3CHU Amiens, France Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Advanced respiratory care techniques in a severe adult respiratory failure uniti Statistical analysis was performed with the Student t test. Results The extracorporeal device allowed effi cient CO2 removal rates at FiO2 1 (Figure 1) and 0.21 (Figure 2), ranging from 40 to 60 ml/ minute. Effi ciency was increased with blood and sweep gas fl ows. Carbia and pH of animals were signifi cantly modifi ed after 10 minutes Figure 1 (abstract P315). Figure 1 (abstract P316). CO2 removal rates at FiO2 1. pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). Conclusion Severe S. pneumoniae pneumonia is associated with pulmonary complement activation in rats. Nebulized C1-INH treat- ment, in an attempt to reduce pulmonary complement activation, f Figure 1 (abstract P315). pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). Figure 1 (abstract P315). Figure 1 (abstract P315). Figure 1 (abstract P315). pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). pulmonary complement activation (Figure 1A), lung injury (Figure 1B) or infl ammation (Figure 1C). Figure 1 (abstract P316). CO2 removal rates at FiO2 1. l g Conclusion Severe S. pneumoniae pneumonia is associated with pulmonary complement activation in rats. Nebulized C1-INH treat- ment, in an attempt to reduce pulmonary complement activation, neither aff ects pulmonary complement activation or lung injury and infl ammation. 3 6 Novel carbon dioxide removal device driven by a renal-replacement system without hemofi lter: an experimental approach and validation T Godet1, A Combes2, E Zogheib3, M Jabaudon1, E Futier1, J Constantin1 1CHU Clermont-Ferrand, France; 2CHU Pitié-Salpêtrière, Paris, France; 3CHU Amiens, France Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Critical Care 2014, 18(Suppl 1):P316 (doi: 10.1186/cc13506) Introduction Management of acute respiratory diseases must avoid ventilator-induced lung injuries along with the rise of PaCO2 and respiratory acidosis [1]. Eff orts are made to fi nd devices assisting protective ventilation and able to remove arterial CO2 and correct acidosis [2]. An extracorporeal CO2 removal device driven by the widely used Prismafl ex® platform called PrismaLung® was tested in vivo. Figure 1 (abstract P316). CO2 removal rates at FiO2 1. of parameters (blood fl ow rate: 200, 300 and 400 ml/minute, sweep gas fl ow: 2, 5, 10 and 50 l/minute, FiO2: 21 and 100%). Statistical analysis was performed with the Student t test.fi Methods Five hypercapnic ventilated pigs were equipped with the PrismaLung® system designed to remove CO2 from the bloodstream through a decarboxylation membrane mounted on a renal replacement device without any hemofi lter. Experiments examined the potential for blood decarboxylation by gas-exchanger membrane with diff erent sets Results The extracorporeal device allowed effi cient CO2 removal rates at FiO2 1 (Figure 1) and 0.21 (Figure 2), ranging from 40 to 60 ml/ minute. Effi ciency was increased with blood and sweep gas fl ows. Carbia and pH of animals were signifi cantly modifi ed after 10 minutes Results The extracorporeal device allowed effi cient CO2 removal rates at FiO2 1 (Figure 1) and 0.21 (Figure 2), ranging from 40 to 60 ml/ minute. Effi ciency was increased with blood and sweep gas fl ows. Carbia and pH of animals were signifi cantly modifi ed after 10 minutes S114 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 2 (abstract P316). CO2 removal rates at FiO2 0.21. centre and mortality among patients with severe 2009 infl uenza A(H1N1). JAMA 2011, 306:1659-1668. 3. Davies A, et al.: Australia and New Zealand Extracorporeal Membrane Oxygenation (ANZ ECMO) Infl uenza investigators. Extracorporeal membrane oxygenation for 2009 infl uenza A(H1N1) acute respiratory distress syndrome. JAMA 2009, 302:1888-1895. 4. NI Stewart, et al.: Regionalisation of intensive care and extra-corporeal membrane oxygenation services in the UK: beliefs about the evidence, benefi t and harm. J Intensive Care Med 2012, 13:244-250. Reference 1. Berra et al.: Crit Care Med 2001, 1:119-124. p p Results See Figure 1. ECMO services have expanded rapidly in the last 5 years in England following the publication of evidence for its effi cacy and concerns regarding an infl uenza pandemic. The referral rates for ECMO for severe hypoxic respiratory failure vary greatly around the country from 88 per 1,000,000 population in Leicester City to no referrals in 32 PCTs. Possible explanations could include: the distributions of swine fl u around the country, referring doctors’ beliefs about the effi cacy of ECMO, local access to high-frequency oscillation ventilation and possible reluctance of teaching hospitals to refer to specialist centres. Further investigation to account for this variation appears indicated. P318 Assessment of an endotracheal tube cleaning closed-suctioning system by micro-computed tomography: preliminary clinical data A Coppadoro1, G Bellani1, A Bronco1, N Eronia1, A Barletta1, M Teggia Droghi1, R Borsa1, M Battistini1, S Bramati2, L Berra3, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy;3Massachusetts General Hospital, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P318 (doi: 10.1186/cc13508) Assessment of an endotracheal tube cleaning closed-suctioning system by micro-computed tomography: preliminary clinical data A Coppadoro1, G Bellani1, A Bronco1, N Eronia1, A Barletta1, M Teggia Droghi1, R Borsa1, M Battistini1, S Bramati2, L Berra3, A Pesenti1 1University of Milan-Bicocca, Monza, Italy; 2San Gerardo Hospital, Monza, Italy;3Massachusetts General Hospital, Boston, MA, USA Critical Care 2014, 18(Suppl 1):P318 (doi: 10.1186/cc13508) Introduction Using micro-computed tomography (MicroCT), we assessed the eff ectiveness of a cleaning closed-suctioning system (CSS) to remove secretions from the endotracheal tube (ETT) lumen. Biofi lm growing within the ETT, soon after intubation, increases the patient’s risk to develop ventilator-associated pneumonia, and new cleaning devices have been designed to keep the ETT clean from secretions [1]. Methods In a bench test, we injected a water-based gel into unused ETTs to evaluate MicroCT scan (SkyScan 1172; Bruker, Belgium) eff ectiveness to measure secretions. In six critically ill patients, a cleaning CSS (Airway Medix Closed Suction system; Biovo, Tel Aviv) was used three times a day to keep the ETT clean. After extubation, we measured ETT secretions volume by MicroCT scanning over a length of 20 cm from the ETT tip. We also collected ETTs from 11 patients treated with a standard CSS as controls, and evaluated ETT microbial colonization. of treatment. No signifi cant modifi cation of blood oxygenation was obtained with pure oxygen, allowing the use of ambient air as the sweep gas through the membrane. No side eff ects or fi lter clotting occurred during experiments.lfi Conclusion The device based on the Prismafl ex® platform effi ciently removed CO2 from blood and decreased PaCO2 and acidosis of hypercapnic pigs. Benefi ts for patients with acute to chronic respiratory diseases need evaluation. P317 Results The volume of gel measured by MicroCT strongly correlated with the volume of injected gel (P <0.001, R2 = 0.99). At extubation, a lower amount of secretions was measured in the ETTs treated with the cleaning CSS as compared with controls (0.031 ± 0.029 vs. 0.350 ± 0.417 mm3, P = 0.028), corresponding to a smaller occupation of the cross-sectional area (average 0.3 ± 0.4 vs. 3.8 ± 4.5% respectively, P = 0.030). Microbial colonization tended to be reduced in the ETTs treated with the cleaning CSS (total bacterial charge 1.3 ± 1.7 vs. 3.6 ± 2.7 log(CFU/ml), P = 0.08). References 1. Slutsky AS, et al.: N Engl J Med 2013, 369:2126-2136. 1. Slutsky AS, et al.: N Engl J Med 2013, 369:2126-2136. 2. Pesenti A, et al.: Crit Care Med 2010, 38:S549-S554. Does geography aff ect referral rates for extracorporeal membr oxygenation in England? J Barnett Royal United Hospital, Bath, UK Critical Care 2014, 18(Suppl 1):P317 (doi: 10.1186/cc13507) Royal United Hospital, Bath, UK Introduction Referral for ECMO has been demonstrated to reduce mortality in severe hypoxic respiratory failure [1-3]. The numbers of patients that undergo ECMO is still small and the service depends on timely referral from regional ICUs. There is evidence that intensivists’ views on the role of ECMO are mixed [4]. The purpose of this study is to determine whether there are variations in the geographical distribution of patients that receive ECMO. Conclusion MicroCT scan showed high precision and accuracy in measuring the volume of secretions in bench tests and can thus be used to evaluate the eff ectiveness of actions or devices studied to reduce ETT biofi lm accumulation. In a small nonrandomized population of critically ill patients, the use of an ETT cleaning device appeared eff ective to reduce the volume of secretions present in the ETT at extubation. p Methods NHS England provided the home primary care trust (PCT) of all adult patients referred for ECMO for potentially reversible respiratory failure from 2008 to 2012. The referrals from each PCT were indexed to the population of each area to produce a referral rate per 1,000,000 people. Reference Does cost aff ect endotracheal tube performance? P Lichtenthal, UB Borg University of Arizona, Tucson, AZ, USA Critical Care 2014, 18(Suppl 1):P319 (doi: 10.1186/cc13509) Does cost aff ect endotracheal tube performance? P Lichtenthal, UB Borg University of Arizona, Tucson, AZ, USA Critical Care 2014, 18(Suppl 1):P319 (doi: 10.1186/cc13509) Introduction Today’s healthcare environment has forced providers to constantly evaluate the materials used, and to fi nd less expensive alternatives. Recently, low-cost endotracheal tubes (ETTs) have been introduced to the market. The aim of this study was to test these tubes (Portex AirCare and Cardinal Health ETT) compared with endotracheal tubes with known performance (Hi-Lo Covidient pre ISO standard and Taper-Guard Covidient, post ISO standard). Conclusion The referral rates for ECMO vary greatly around the country. References Conclusion The referral rates for ECMO vary greatly around the country. References 1. Peek GJ, et al.; CESAR Trial Collaboration: Effi cacy and economic assessment of Conventional Ventilatory Support Versus Extra-Corporeal Membrane Oxygenation for Severe Adult Respiratory Failure (CESAR): a multicentre randomised controlled trail. Lancet 2009, 374:1351-1363. 1. Peek GJ, et al.; CESAR Trial Collaboration: Effi cacy and economic assessment of Conventional Ventilatory Support Versus Extra-Corporeal Membrane Oxygenation for Severe Adult Respiratory Failure (CESAR): a multicentre randomised controlled trail. Lancet 2009, 374:1351-1363. Methods We used the required test setups according to the ISO standard for cuff sealing performance. The tubes were tested versus each other in size 7.0, 7.5 and 8.0 mm. Kink performance was done in the routine manner. 2. Noah MA, et al.: Referral to an extracorporeal membrane oxygenation S115 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P317). Figure 1 (abstract P317). Figure 1 (abstract P317). Results The leak test showed that the Portex AirCare ETT leaked signifi cantly more compared with both the Hi-Lo (P <0.05) or Taper- Guard (P <0.001) ETTs. The standard deviation leak rate for AirCare was large, suggesting varying seal performance between tubes from various lots. The kink test showed no diff erence among tubes.f Results The leak test showed that the Portex AirCare ETT leaked signifi cantly more compared with both the Hi-Lo (P <0.05) or Taper- Guard (P <0.001) ETTs. The standard deviation leak rate for AirCare was large, suggesting varying seal performance between tubes from various lots. The kink test showed no diff erence among tubes.f price alone but should take into account documented performance in standardized tests. price alone but should take into account documented performance in standardized tests. P320 P320 Tracheostomy in obese patients: the best tube choice issue L Marullo, G Izzo, A Torino, A D’Elia, L Vessicchio, F Ferraro Second University of Naples, Italy Critical Care 2014, 18(Suppl 1):P320 (doi: 10.1186/cc13510) Conclusion Although tubes look the same there may be diff erences in performance. This study demonstrated that the new cheaper ETTs had greater cuff leak compared with the two tubes used for comparison. We can only speculate whether diff erences found in this study is a result of cost cutting. However, the great variability in sealing performance between ETTs of the same size from diff erent lots would indicate that manufacturing controls may be less stringent. Although we cannot demonstrate that the higher incidence of leak will result in adverse patient outcomes, one can surmise that the possibility exists. Thus, selection of endotracheal tubes should not be based on purchase Introduction Obesity is not an absolute contraindication for percutaneous tracheostomy (PDT). Video-endoscopy (video-FBS) and ultrasound (US) facilitate PDT techniques and reduce complications in obese patients (OPs) [1,2]. OPs may have a higher trachea–skin distance that makes it diffi cult to place or to manage a tracheostomy tube (TT). S116 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A retrospective review was performed using data in the last 5 years. All OPs were from the ICU of our university hospital. Only OPs who underwent a PDT were selected with BMI >30 kg/m2. All OPs needed prolonged mechanical ventilation. A total of 67 OPs were identifi ed, with 60 PDTs placed using the Ciaglia Blue Rhino (CBR) Introducer Kit and seven PDTs with the UniPerc PDT Kit. All PDTs were performed by dedicated staff including residents. Valuation of clinical anatomical and physio-pathological features of the OPs and US scan of the neck came before the procedure. At the beginning of the procedure we placed a 5 mm ID orotracheal tube by tube exchange with video-FBS assistance, as already described [3]. An 8 to 9 mm ID wire-reinforced silicone tracheostomy tube (rTT) with adjustable fl ange was chosen instead of a standard PVC or silastic TT (sTT) in all OPs treated with CBR because of the anatomical particularities of OPs and because of external traction by the weight of the tubing attached to the TT. P322 3 Development of the novel Tracoe Twist Plus tracheostomy tube L Barrass, M Healy, D Kennedy, H Drewery Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P322 (doi: 10.1186/cc13512) Introduction The 4th National Audit project, conducted by the Royal College of Anaesthetists in 2011, reviewed major airway complications in the UK [1]. It highlighted tracheostomy tube displacement as a signifi cant risk in critical care patients. Prior to 2011, at the Royal London Hospital ICU, the Tracoe Twist (marketed by Kapitex) was primarily used as the default choice of tracheostomy tube. However, over a 5-year period, there were a signifi cant number of critical incidents related to tracheostomy tube displacement or blockage. On investigation of these incidents, root-cause analysis identifi ed inadequate length of the Tracoe Twist as a major contributing factor. As a consequence of this, there was also an increasing requirement for the use of adjustable fl ange tubes. This was particularly apparent in obese patients. Our objective was to develop a new tracheostomy tube that would reduce the number of incidents related to inadequate length. An additional aim was to maximise the inner diameter for a given external diameter, thus reducing airway resistance and potentially aiding weaning. Results No major complications (aborting procedure, >50 ml bleed- ing, TT misplacement, death) were observed. We had only minor complications (<50 ml bleeding: 3%; ring fracture: 2%; diffi cult insertion: 21% only with CBR rTT because of the step between the tip of the rTT and its introducer). UniPerc eTT placement has always been easy. Conclusion In our experience, the data do not support what previous studies have shown suggesting increased risk of complication in OPs [1,2]. We know that the sTT could not be eff ective in OPs. The use of US, video-FBS assistance [3], and rTT with an adjustable fl ange allows a safe and eff ective adjustment to anatomical OP particularities, avoiding collected risks. Impact of high-fl ow oxygen therapy delivered through a tracheostomy on arterial blood gases and endotracheal pressure D Natalini, FA Idone, DL Grieco, L Spaziani, MT Santantonio, F Toni, M Antonelli, SM Maggiore A Gemelli Hospital, University of the Sacred Heart, Rome, Italy Critical Care 2014, 18(Suppl 1):P321 (doi: 10.1186/cc13511) i Results We developed a prototype tracheostomy tube that had an increased length for a given internal diameter. We also increased the mobility to fl ange, in order to reduce pressure areas and assist with fi xation. A third modifi cation was to ensure a maximal internal diameter for a given external diameter. We piloted its use in 20 patients. No adverse events were observed and the clinical impression was that the increased length was benefi cial to the patients. Introduction High-fl ow oxygen therapy (HFOT) delivered through nasal cannulas can improve oxygenation as compared with low-fl ow oxygen devices. It has been shown that nasal HFOT can generate a positive airway pressure, which increases linearly with the gas fl ow rate. Data on the use of HFOT delivered through a tracheostomy are scarce. The aim of the present randomized, controlled, cross-over trial was to assess the eff ects of HFOT delivered through a tracheostomy on arterial blood gases and endotracheal pressure in critically ill patients. i Conclusion Following the success of the pilot study, this new tracheostomy tube was developed and then marketed by Kapitex. This tube was named the Tracoe Twist Plus. We have now used this tube for 2 years. We have since performed an extensive retrospective audit of tracheostomy usage and the eff ect of the introduction of the novel Tracoe Twist Plus, and the complication rate was reduced in terms of displacement and obstruction. y Methods Tracheostomized patients underwent HFOT with three gas fl ow rates (10 l/minute, 30 l/minute and 50 l/minute), randomly applied for 20-minute periods. At the end of each period, arterial blood gases, respiratory rate, and endotracheal pressure (Ptrach) were measured. Ptrach was recorded over the last 3 minutes of each study period: the maximum expiratory pressure (MEPtrach) and mean expiratory pressure were measured and averaged for all respiratory cycles during 1-minute recording with stable breathing. FiO2 was kept constant during the whole study. P320 An extralong TT (eTT) was chosen because the pretracheal tissue was too thick for a regular-sized TT in 16 OPs with BMI >40 kg/m2 treated with CBR. For three OPs treated with CBR we needed to change rTT to eTT because of tube dislodgement and subocclusion X-ray and video-FBS diagnosis. The UniPerc technique was chosen for OPs with BMI >40 kg/m2. 1.32 ± 0.4 cmH2O, and 1.89 ± 0.5 cmH2O at 10 l/minute, 30 l/minute and 50 l/minute, respectively, P <0.01) and mean expiratory pressure (0.54 ± 0.27 cmH2O, 0.91 ± 0.29 cmH2O, and 1.36 ± 0.35 cmH2O, at 10 l/ minute, 30 l/minute and 50 l/minute, respectively, P <0.01) increased with fl ow. Changes in PaO2/FiO2 were not correlated with changes in expiratory pressures. Conclusion When HFOT is used through a tracheostomy at increasing gas fl ow rate, oxygenation increases up to 30 l/minute while CO2 clearance and the respiratory rate do not vary. Tracheal expiratory pressure increases with fl ow, but changes are small and probably of limited clinical relevance. Changes in oxygenation are not related to the variations of tracheal expiratory pressure. HFOT through a tracheostomy has diff erent eff ects from when a nasal interface is used. Reference 1. Cook TM, Woodall N, Harper J, Benger J; Fourth National Audit Project: Major complications of airway management in the UK: results of the Fourth National Audit Project of the Royal College of Anaesthetists and the Diffi cult Airway Society. Part 2: intensive care and emergency departments. Br J Anaesth 2011, 106:632-642. Results Seventeen tracheostomized patients were enrolled (SAPS II 52 ± 10, PaO2 96 ± 27 mmHg, PaCO2 33 ± 10 mmHg). Increasing the gas fl ow rate from 10 l/minute to 30 l/minute was associated with an increase in PaO2/FiO2 that did not improve further when 50 l/minute was used (259 ± 66, 317 ± 79, and 325 ± 76, respectively, P <0.001). The same trend was observed with PaO2 (89 ± 19 mmHg, 109 ± 26 mmHg, and 113 ± 29 mmHg, respectively, P <0.001) and SaO2 (96 ± 3%, 98 ± 2%, 98 ± 2%, respectively, P <0.001). PaCO2 (32 ± 8 mmHg on average) and respiratory rate (27 ± 7 breaths/minute on average) did not change with diff erent gas fl ow rates. MEPtrach (0.96 ± 0.43 cmH2O, References 1. McCague A, et al.: Laryngoscope 2012, 122:1031-1034. 2. Guinot PG, et al.: Crit Care 2012, 16:R40. 3. Ferraro et al.: Chest 2004, 126:159-164. 1. McCague A, et al.: Laryngoscope 2012, 122:1031-1034. 2. Guinot PG, et al.: Crit Care 2012, 16:R40. 3. Ferraro et al.: Chest 2004, 126:159-164. Methods We formed a consultation committee, consisting of consultants in critical care, a consultant surgeon and a consultant anaesthetist with a specialist interest in head and neck anaesthesia and tracheostomy care. We collaborated with the Kapitex design team to develop a new tracheostomy tube which addressed some of the perceived defi cits of existing devices. P321 Impact of high-fl ow oxygen therapy delivered through a tracheostomy on arterial blood gases and endotracheal pressure D Natalini, FA Idone, DL Grieco, L Spaziani, MT Santantonio, F Toni, M Antonelli, SM Maggiore A Gemelli Hospital, University of the Sacred Heart, Rome, Italy Critical Care 2014, 18(Suppl 1):P321 (doi: 10.1186/cc13511) Ability to speak in ventilator-dependent tracheostomized ICU patients Methods The nonlinear constant of the Rohrer equation (K2) was calculated as resistive properties, during a continuous fl ow of 10 to 90 l/minute, for a conventional endotracheal tube (ETT) with and without FOB (ETT size 7, 7.5, 8, 7f, 7.5f, 8f), ventilation tube of TLT (F4 and F5) and DLET. The variation of gas exchange (Δ) was measured with arterial blood gas samples obtained before and after the PT. During PT, all patients received sedation, analgesia, neuromuscular blocking and volume-controlled ventilation set with FiO2 100%, TV 500 ml, RR 15 breaths/minute, PEEP 5 cmH2O. Methods The aim of this study was to compare weaning from MV by gradually decreasing the level of support in cuff -defl ated ventilation with use of a BiPAP Vision® and a Passy Muir® speaking valve, or by trials of spontaneous breathing with use of a speaking valve, both for progressively longer periods of time. We examined the diff erences in the ability to speak, the duration of the weaning period, the occurrence of delirium and the frequency of tracheal suctioning. We performed a single-centre retrospective and prospective observational study in a 22-bed mixed ICU during 1 year. Data were collected using the patient data management system. Baseline criteria were age, gender, APACHE IV score, ICU length of stay and duration of MV before placement of the tracheostomy.ii 2 Results In vitro evaluation showed that the DLET had the lowest K2 (7 = 11.33; 7.5 = 8.74; 8 = 7.57; 7f = 6.13; 7.5f = 10.52; 8f = 12.28; F4 = 130.0; F5 = 11.12; DLET = 5.25 cmH2O/l/minute). During in vivo evaluation, PT was performed with the conventional ETT with FOB and DLET for fi ve patients in each group (age 69 ± 13 vs. 71 ± 16; SAPS II: 56 ± 14 vs. 52 ± 20; GCS 3 vs. 4). Gas exchange before and after the procedure did not diff er between the groups, but the Δ values of pH, PaO2 and PaCO2 measured before and after the procedure were, ETT+FOB versus DLET: ΔpH: –0.05 ± 0.05 versus 0.01 ± 0.02, P = 0.04; ΔPaO2: –112.6 ± 112.6 versus 41.6 ± 25.3, P = 0.01; ΔPaCO2: 14.5 ± 10.8 versus 0.6 ± 1.1, P = 0.02; ΔHCO3: 0.5 ± 2 versus –0.04 ± 0.3, P = 0.6. Ability to speak in ventilator-dependent tracheostomized ICU patients p R Bultsma, M Koopmans, M Kuiper, P Egbers Medisch Centrum Leeuwarden, the Netherlands Critical Care 2014, 18(Suppl 1):P323 (doi: 10.1186/cc13513) p R Bultsma, M Koopmans, M Kuiper, P Egbers Medisch Centrum Leeuwarden, the Netherlands Critical Care 2014, 18(Suppl 1):P323 (doi: 10.1186/cc13513) Introduction Mechanical ventilation (MV) and inability to speak increases psycho-emotional distress [1]. Although not commonly used S117 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P323). Number of patients able to speak. Figure 1 (abstract P324). Point 1 just before vocal cord, point 2 asymmetric elliptical cuff just before the carena. Figure 1 (abstract P324). Point 1 just before vocal cord, point 2 asymmetric elliptical cuff just before the carena. Figure 1 (abstract P323). Number of patients able to speak. Figure 1 (abstract P323). Number of patients able to speak. in ICU patients, MV with a defl ated cuff in patients with a tracheostomy can be provided safely and comfortably, by use of a BiPAP Vision®. Air leakage to the upper airway enables speech [2]. By adding a Passy- Muir® speaking valve as second step, the quality of speech and cough will improve. The ability to speak provides an important improvement in communication. (FOB), and a lower channel exclusively dedicated to the patient’s ventilation. The aim of this study is to achieve an in vitro and an in vivo evaluation of PT performed with the DLET. (FOB), and a lower channel exclusively dedicated to the patient’s ventilation. The aim of this study is to achieve an in vitro and an in vivo evaluation of PT performed with the DLET. Methods The nonlinear constant of the Rohrer equation (K2) was calculated as resistive properties, during a continuous fl ow of 10 to 90 l/minute, for a conventional endotracheal tube (ETT) with and without FOB (ETT size 7, 7.5, 8, 7f, 7.5f, 8f), ventilation tube of TLT (F4 and F5) and DLET. The variation of gas exchange (Δ) was measured with arterial blood gas samples obtained before and after the PT. During PT, all patients received sedation, analgesia, neuromuscular blocking and volume-controlled ventilation set with FiO2 100%, TV 500 ml, RR 15 breaths/minute, PEEP 5 cmH2O. Ability to speak in ventilator-dependent tracheostomized ICU patients Results Ten patients were included, fi ve in the BiPAP group and fi ve in the spontaneous group. There were no signifi cant diff erences in the baseline criteria. On the second day after tracheostomy, three out of fi ve patients in the BiPAP group were able to speak compared with one in the spontaneous method group. A diff erence in speaking ability remained until day 9 (see Figure 1). At fi rst time of speaking, the BiPAP group had higher PEEP level (10 vs. 7.5 cmH2O) and higher SOFA score (6.2 vs. 4.6) compared with the spontaneous group. There was no signifi cant diff erence in delirium, duration of weaning and tracheal suctioning between both groups.fl 3 Conclusion The DLET resulted in adequate airway patency and minimal obstruction due to the lower channel exclusively dedicated to patient’s ventilation. Gas exchange in PT with the DLET remained stable without any variation in oxygenation and carbon dioxide levels, although the same settings of mechanical ventilation. Conclusion Cuff -defl ated MV in ICU patients enables speaking during ventilator dependence. With this technique the ability to speak started in an earlier phase of weaning compared with weaning with spontaneous breathing trials and a speaking valve. National survey of ICUs in the UK: discharging patients with tracheostomies 1. Engström A, et al.: People’s experiences of being mechanically ventilated in an ICU: a qualitative study. Intensive Crit Care Nurs 2013, 29:88-95. 1. Engström A, et al.: People’s experiences of being mechanically ventilated in an ICU: a qualitative study. Intensive Crit Care Nurs 2013, 29:88-95. A Garrett, C Strauss, S Saha Barking, Havering and Redbridge University Hospitals NHS Trust, Romford, UK Critical Care 2014, 18(Suppl 1):P325 (doi: 10.1186/cc13515) A Garrett, C Strauss, S Saha Barking, Havering and Redbridge University Hospitals NHS Trust, Romford, UK Critical Care 2014, 18(Suppl 1):P325 (doi: 10.1186/cc13515) 2. Hess DR: Facilitating speech in the patient with a tracheostomy. Respir Care 2005, 50:519-525. 2. Hess DR: Facilitating speech in the patient with a tracheostomy. Respir Care 2005, 50:519-525. Introduction Respiratory weaning in ICUs can be a lengthy and expensive process [1], but may be facilitated by the use of tracheostomies. Discharging patients with tracheostomies to general wards improves ICU bed availability but raises potential patient safety issues. This is demonstrated by the increased mortality compared with patients decannulated before discharge from the ICU [2]. We investigated how often ICUs in the UK discharge patients with tracheostomies to wards, which wards these are and whether systems are in place to ensure adequate safety on discharge.f Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A retrospective review of all repeat bedside percutaneous dilatational tracheostomies (Ciaglia technique with direct broncho- scopic guidance) performed on our cardiothoracic critical care unit from January 2004 to February 2013 was conducted. Methods A retrospective review of all repeat bedside percutaneous dilatational tracheostomies (Ciaglia technique with direct broncho- scopic guidance) performed on our cardiothoracic critical care unit from January 2004 to February 2013 was conducted.i 39 did occasionally. Five discharged to the high dependency unit only, 60 to respiratory wards only, 70 to specialist wards and 15 to any or most wards. Eighty-fi ve out of 190 units discharged patients with tracheostomy cuff s both up and down, 72 discharged with the cuff down or cuffl ess and 16 with the cuff ‘usually down’. A total of 141 hospitals had routine follow-up for tracheostomy patients from critical care outreach or other services. Critical care outreach was available 24 hours a day in 65 hospitals. y y Results From a total of 1,001 patients undergoing PDT, we identifi ed 36 patients with repeat PDT. Patients’ previous tracheostomies dated back between 5 days and 2.7 years (mean 122 days). Mean age was 60.3 ± 14.3 years, 42% of patients were female. The mean time from intubation to PDT was 3.7 ± 3.9 days. There were no deaths associated with PDT but one major procedure-related complication: one patient suff ered from a periprocedural laceration of the brachiocephalic trunk. After emergency surgery and surgical tracheotomy (ST), the patient recovered completely. In all other patients, no conversion to ST, no loss of airway, no paratracheal insertion, and no accidental tracheal extubation was observed. No pneumothorax, pneumomediastinum, hypotension, hypoxemia, or arrhythmias were recorded. A mild bleeding was observed in 13 patients (36%). A moderate but not signifi cant bleeding was observed in only one patient (2.8%). A tracheal ring fracture occurred in six patients (16.7%). Fourteen patients (38.9%) could be weaned successfully from the respirator and the tracheostomy could be removed. Thirteen patients (36.1%) were transferred to another ICU with the tracheostomy in place. The functional and cosmetic outcomes of PDT were excellent. y p Conclusion The vast majority of ICUs in the UK perform tracheostomies for respiratory weaning and many routinely discharge patients to the wards prior to decannulation. Routine follow-up is usually available, but cover may only be available during the day. P328 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) 3 8 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) y p itical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) Introduction Percutaneous dilatational tracheostomy (PDT) is the standard airway access in critically ill patients who require prolonged mechanical ventilation. However, patients with severe coagulopathy or thrombocytopenia might have an increased risk of periprocedural bleeding. Introduction The tracheostomy is an ancient technique that more recently has developed a percutaneous technique. Percutaneous tracheostomies (PCT) have been shown to be safer and reduce infection, cost and other complications over surgical techniques [1-3]. Ventilator- associated pneumonia (VAP) is a serious complication resulting from the use of endotracheal tubes (ETT) and tracheostomies. Changes in design of these tubes by the addition of a subglottic suction port have been shown to improve VAP rates in mechanically ventilated patients [4,5]. A large meta-analysis review showed that subglottic drainage reduced the number of days of mechanical ventilation required and reduced the number of days stayed on the ICU [4]. Methods We retrospectively reviewed the records of all patients who underwent PDT (using the Ciaglia technique with bronchoscopic guidance) on our cardiothoracic ICU between January 2004 and February 2013. Patients were stratifi ed into two groups: no coagulopathy (group 1), and coagulopathy/thrombocytopenia defi ned as international normalized ratio >1.5, partial thromboplastin time >50 seconds and/or platelet count <50 × 109/l (group 2). p g p Results From a total of 1,001 patients (46% male, mean age 68.1 years) that underwent PDT, we identifi ed 441 patients (44.1%) with a severe coagulopathy (group 2). There were no procedure-related deaths. Major procedure-related complications included a severe bleeding (requiring transfusion and/or surgery) in two patients in each group (one laceration of the brachiocephalic trunk, one venous bleeding, two bleedings from a thyroid vessel), injury of the membranous wall of the trachea in two patients in group 2 as well as a pneumothorax and a device failure in group 1. P328 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) The incidence of moderate periprocedural bleeding was comparable between the two groups (n = 43 (9.75%) vs. n = 41 (7.3%), P = NS). y y Methods We contacted all ICUs in the UK by telephone and spoke to the nurse-in-charge to ascertain their normal practice with regards to PCT and subglottic suction use. g Results We contacted a total of 246 general ICUs, 72% of which we received a response. The average number of beds per ICU from all units who responded was 11. Ninety-eight per cent of ICUs that we questioned did use PCT. For three units, the average bed number per unit was 11 and the other 2% of ICUs who did not use PCT had fi ve beds per unit on average. The proportion of ICUs that employed subglottic suction ports on their ETTs was 43% having on average 11 beds per unit, whilst the proportion of ICUs that did not employ subglottic suction ports was 57%, also with 11 beds per unit on average. Regarding PCT subglottic suction ports, 38% of ICUs did utilise these tubes whilst 62% did not. Of the group of ICUs that did use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 12. Of the group of ICUs that did not use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 10. Conclusion Signifi cant diff erences in practise exist with PCT and subglottic suction ports on tubes. The size of the ICUs in these groups is variable. The larger units are more likely to use PCT over the smaller units. Regarding subglottic suction ports on ETT and tracheostomy tubes, the size of the ICU does not necessarily dictate their use. We propose that all ICUs review their policy on the use of PCT and subglottic suction-assisted tubes to help improve surgical complications, cost, VAP rates and ICU stays. g Results We contacted a total of 246 general ICUs, 72% of which we received a response. The average number of beds per ICU from all units who responded was 11. Ninety-eight per cent of ICUs that we questioned did use PCT. For three units, the average bed number per unit was 11 and the other 2% of ICUs who did not use PCT had fi ve beds per unit on average. P326 P Percutaneous dilatational tracheostomy in patients with severe coagulopathy or thrombocytopenia K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P326 (doi: 10.1186/cc13516) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Patients may go to a specialist ward with trained nurses but this is not always the case. Patients are often discharged to wards with their tracheostomy cuff up, raising major safety issues if their tracheostomy tubes block and nurses are not trained for such emergencies. Twenty-four hours a day critical care outreach cover may improve patient safety, but further research and the production of guidelines is needed to facilitate the safe discharge of patients with tracheostomies from ICU to the wards. R f 1. Vitacca M, et al.: [Experience of an intermediate respiratory intensive therapy in the treatment of prolonged weaning from mechanical ventilation] [Review in Italian]. Anestesiology 1996, 62:57-64. 1. Vitacca M, et al.: [Experience of an intermediate respiratory intensive therapy in the treatment of prolonged weaning from mechanical ventilation] [Review in Italian]. Anestesiology 1996, 62:57-64. Conclusion Repeat PDT is a safe procedure in experienced hands and should not be generally considered a contraindication. However, special attention should be paid to the anatomical situation with an increased risk of vascular complications. 2. Martinez GH, et al.: Tracheostomy tube in place at intensive care unit discharge is associated with increased ward mortality. Respir Care 2009, 54:1644-1652. P328 National UK survey: a review of percutaneous tracheostomy and auxiliary subglottic suction port use J Rees, R McCartney, S Saha, T Wickrama Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P238 (doi: 10.1186/cc13518) The proportion of ICUs that employed subglottic suction ports on their ETTs was 43% having on average 11 beds per unit, whilst the proportion of ICUs that did not employ subglottic suction ports was 57%, also with 11 beds per unit on average. Regarding PCT subglottic suction ports, 38% of ICUs did utilise these tubes whilst 62% did not. Of the group of ICUs that did use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 12. Of the group of ICUs that did not use subglottic suction ports on their tracheostomy tubes, the average beds per unit was 10.if Conclusion Periprocedural bleeding complications during and after PDT are rare, even in patients with a severe coagulopathy, and thus PDT can be safely performed in these patients. Double-lumen endotracheal tube for percutaneous tracheostomy: in vitro and in vivo preliminary data Double-lumen endotracheal tube for percutaneous tracheostomy: in vitro and in vivo preliminary data M Vargas1, G Servillo1, A Marra1, D Salami2, P Pelosi3 1University of Naples ‘Federico II’, Naples, Italy; 2AOU San Martino IST, Genoa, Italy; 3University of Genoa, Italy Critical Care 2014, 18(Suppl 1):P324 (doi: 10.1186/cc13514) p y M Vargas1, G Servillo1, A Marra1, D Salami2, P Pelosi3 1University of Naples ‘Federico II’, Naples, Italy; 2AOU San Martino IST, Genoa, Italy; 3University of Genoa, Italy Critical Care 2014, 18(Suppl 1):P324 (doi: 10.1186/cc13514) Methods We telephoned 217 ICUs in the UK. Nursing staff answered a series of questions regarding the discharge of patients with tracheostomies to the wards and their follow-up. Introduction A double-lumen endotracheal tube (DLET; bilumen ventilation tube, PCT/IT2012/000154; Deas S.r.l., Italy) (Figure 1) has been developed to improve the safety of patients and procedural comfort during percutaneous tracheostomy (PT). The DLET is divided into an upper channel, for placement of a fi beroptic bronchoscope Introduction A double-lumen endotracheal tube (DLET; bilumen ventilation tube, PCT/IT2012/000154; Deas S.r.l., Italy) (Figure 1) has been developed to improve the safety of patients and procedural comfort during percutaneous tracheostomy (PT). The DLET is divided into an upper channel, for placement of a fi beroptic bronchoscope Results We obtained information from 203 ICUs. A total of 201 units used tracheostomies for respiratory weaning. In total, 151 routinely discharged patients to wards with tracheostomies, 11 never did and S118 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P327 P327 Repeat bedside percutaneous tracheostomy: still a contraindication? K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P327 (doi: 10.1186/cc13517) Eff ect of subglottic secretion drainage for preventing ventilator-associated pneumonia Eff ect of subglottic secretion drainage for preventing ventilator-associated pneumonia Conclusion There is a wide variation in post-ICU/HDU management of tracheostomy patients throughout the UK. Although there are well established UK national guidelines for the management of tracheostomy patients, outside the ICU/HDU environment there is a lack of full implementation of the NTSP recommendations, increasing the risk of tracheostomy-related morbidity and mortality. References A Koker1, F Gok2, I Erayman2, A Yosunkaya2 1Konya Education and Research Hospital, Konya, Turkey; 2Necmettin Erbakan University, Meram Faculty of Medicine, Konya, Turkey Critical Care 2014, 18(Suppl 1):P331 (doi: 10.1186/cc13521) A Koker1, F Gok2, I Erayman2, A Yosunkaya2 1Konya Education and Research Hospital, Konya, Turkey; 2Necmettin Erbakan University, Meram Faculty of Medicine, Konya, Turkey Critical Care 2014, 18(Suppl 1):P331 (doi: 10.1186/cc13521) y y y y Critical Care 2014, 18(Suppl 1):P331 (doi: 10.1186/cc13521) Introduction We aimed to assess the eff ect of continuous drainage of subglottic secretion in the prevention of ventilator-associated pneumonia (VAP) in patients requiring prolonged mechanical ventilation for more than 48 hours in the ICU as a prospective, randomized, controlled study. 1. Cook TM, Woodall N, Frerk C (Eds): The Fourth National Audit Project of the Royal College of Anaesthetists and the Diffi cult Airway Society: Major Complications of Airway Management in the UK. Report and Findings. London: Royal College of Anaesthetists; 2011. 2. National Tracheostomy Safety Project Manual 2013 [http://www. tracheostomy.org.uk] Methods Our study was performed with a document from the ethics committee and written informed consent from the relatives of patients between April 2011 and February 2012 in our 14-bed ICU. Fifty-four patients whose mechanical ventilation requirements were expected to be longer than 72 hours were included in our study. Patients were randomly divided into two groups. These were formed as the group using a conventional intubation tube (Group C) and the group using an intubation tube allowing aspiration of subglottic secretions (Group S). In Group S, continuous subglottic aspiration occurred under constant pressure with a special device. In both groups, the cuff pressure was maintained at a constant pressure of 20 to 30 using a digital cuff pressure device [1]. References 1. Rello J, et al.: Chest J 2002, 122:2115-2121. 2. Shi Z, et al.: Cochrane Database Syst Rev 2013, 8:CD008367, 3. Barkvoll et al.: J Clin Periodontol 1989, 16:593-595. 4. Kolahi J, et al.: Quintessence Int 2006, 37:605-612. 1. Rello J, et al.: Chest J 2002, 122:2115-2121. 1. Rello J, et al.: Chest J 2002, 122:2115-2121. 2. Shi Z, et al.: Cochrane Database Syst Rev 2013, 8:CD008367, 3. Barkvoll et al.: J Clin Periodontol 1989, 16:593-595. 4. Kolahi J, et al.: Quintessence Int 2006, 37:605-612. P327 Repeat bedside percutaneous tracheostomy: still a contraindication? K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P327 (doi: 10.1186/cc13517) P327 Repeat bedside percutaneous tracheostomy: still a contraindication? K Pilarczyk, F Dusse, G Marggraf, B Schönfelder, H Jakob University Hospital Essen, West German Heart Center, Essen, Germany Critical Care 2014, 18(Suppl 1):P327 (doi: 10.1186/cc13517) Conclusion Signifi cant diff erences in practise exist with PCT and subglottic suction ports on tubes. The size of the ICUs in these groups is variable. The larger units are more likely to use PCT over the smaller units. Regarding subglottic suction ports on ETT and tracheostomy tubes, the size of the ICU does not necessarily dictate their use. We propose that all ICUs review their policy on the use of PCT and subglottic suction-assisted tubes to help improve surgical complications, cost, VAP rates and ICU stays. Introduction Percutaneous tracheostomy has become an established procedure in airway management of critically ill patients. Repeat PDT is considered a (relative) contraindication as a result of distorted anatomy. S119 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P329 The frequency of chlorhexidine application varied between ICUs; 15 (9.9%) applied 4-hourly, 91 (59.9%) 6-hourly, 20 (13.2%) 8-hourly, 19 (12.5%) 12-hourly and seven (4.6%) applied at variable times. Ninety-seven per cent (n = 152) brushed patient’s teeth; 86% (n = 130) used toothpaste, 3% (n = 5) used chlorhexidine gel and 11% (n = 17) used both. Ninety-seven per cent (n = 147) of ICUs using chlorhexidine also brushed patient’s teeth with toothpaste. Forty-eight per cent (n = 70) administered chlorhexidine within 30 minutes of toothpaste application (Table 1). Introduction Over 5,000 tracheostomies are performed in the UK per year [1]. The 4th National Audit Project identifi ed signifi cant morbidity and mortality associated with tracheostomy care [1]. The National Tracheostomy Safety Project (NTSP) 2013 manual highlighted the need for: local policy; an appropriate care environment; immediate availability of emergency equipment; trained staff and local training programmes; and bed-head sign and emergency algorithms for tracheostomy patients [2]. Following these guidelines, we asked: how are adult tracheostomy patients managed post discharge from the intensive care/high dependence unit (ICU/HDU) throughout the UK? Introduction Over 5,000 tracheostomies are performed in the UK per year [1]. The 4th National Audit Project identifi ed signifi cant morbidity and mortality associated with tracheostomy care [1]. The National Tracheostomy Safety Project (NTSP) 2013 manual highlighted the need for: local policy; an appropriate care environment; immediate availability of emergency equipment; trained staff and local training programmes; and bed-head sign and emergency algorithms for tracheostomy patients [2]. Following these guidelines, we asked: how are adult tracheostomy patients managed post discharge from the intensive care/high dependence unit (ICU/HDU) throughout the UK? Methods In November 2013, 200 adult ICU/HDUs throughout the UK were contacted to take part in a telephone survey. Data were collected on tracheostomy weaning, post-ICU/HDU care, safety guidelines, emergency protocols and training for clinicians and nurses. g g p Results Sixty-fi ve per cent of ICUs in the UK responded to our survey (n = 157). Ninety-seven per cent (n = 152) used chlorhexidine and 96% (n = 150) used it as part of a ventilator care bundle. Forty-six per cent (n = 70) used a gel, 32% (n = 48) used a mouthwash and 23% (n = 34) used both preparations. References References 1. Baumann HJ, et al.: Pneumologie 2010, 64:769-776. 2. Engels PT, et al.: Can J Surg 2009, 52:427-433. 3. Muscedere J, et al.: Crit Care Med 2011, 39:1985-1991. 4. Bouza E, et al.: Chest 2008, 134:938-946. 5. Smulders K, et al.: Chest 2002, 121:858-862 Table 1 (abstract P330). Timing diff erence between toothpaste and chlorhexidine (minutes) Minutes Number of ICUs (%) >30 74 (50.3%) <30 32 (21.8%) Nil 38 (25.9%) Variable 3 (2.0%) Table 1 (abstract P330). Timing diff erence between toothpaste and chlorhexidine (minutes) P329 Is the post-critical care environment safe for tracheostomy patients? J Siah, S Begum, S Wijayatilake, G De La Cerda, T Jovaisa Queen’s Hospital, Romford, UK Critical Care 2014, 18(Suppl 1):P329 (doi: 10.1186/cc13519) ill patients, as studies suggest a risk reduction in VAP [2]. Chlorhexidine reacts with soaps in toothpaste to form inactive insoluble salts [3]. A minimum delay of 30 minutes between tooth brushing and the sub- sequent application of chlorhexidine is therefore recommended [4]. Methods A telephone questionnaire was conducted on all ICUs in the UK to assess current oral decontamination procedures with regards to chlorhexidine use and the timing of tooth brushing with toothpaste. Results Sixty-fi ve per cent of ICUs in the UK responded to our survey (n = 157). Ninety-seven per cent (n = 152) used chlorhexidine and 96% (n = 150) used it as part of a ventilator care bundle. Forty-six per cent (n = 70) used a gel, 32% (n = 48) used a mouthwash and 23% (n = 34) used both preparations. The frequency of chlorhexidine application varied between ICUs; 15 (9.9%) applied 4-hourly, 91 (59.9%) 6-hourly, 20 (13.2%) 8-hourly, 19 (12.5%) 12-hourly and seven (4.6%) applied at variable times. Ninety-seven per cent (n = 152) brushed patient’s teeth; 86% (n = 130) used toothpaste, 3% (n = 5) used chlorhexidine gel and 11% (n = 17) used both. Ninety-seven per cent (n = 147) of ICUs using chlorhexidine also brushed patient’s teeth with toothpaste. Forty-eight per cent (n = 70) administered chlorhexidine within 30 minutes of toothpaste application (Table 1). ill patients, as studies suggest a risk reduction in VAP [2]. Chlorhexidine reacts with soaps in toothpaste to form inactive insoluble salts [3]. A minimum delay of 30 minutes between tooth brushing and the sub- sequent application of chlorhexidine is therefore recommended [4]. Methods A telephone questionnaire was conducted on all ICUs in the UK to assess current oral decontamination procedures with regards to chlorhexidine use and the timing of tooth brushing with toothpaste. Results Sixty-fi ve per cent of ICUs in the UK responded to our survey (n = 157). Ninety-seven per cent (n = 152) used chlorhexidine and 96% (n = 150) used it as part of a ventilator care bundle. Forty-six per cent (n = 70) used a gel, 32% (n = 48) used a mouthwash and 23% (n = 34) used both preparations. P329 The frequency of chlorhexidine application varied between ICUs; 15 (9.9%) applied 4-hourly, 91 (59.9%) 6-hourly, 20 (13.2%) 8-hourly, 19 (12.5%) 12-hourly and seven (4.6%) applied at variable times. Ninety-seven per cent (n = 152) brushed patient’s teeth; 86% (n = 130) used toothpaste, 3% (n = 5) used chlorhexidine gel and 11% (n = 17) used both. Ninety-seven per cent (n = 147) of ICUs using chlorhexidine also brushed patient’s teeth with toothpaste. Forty-eight per cent (n = 70) administered chlorhexidine within 30 minutes of toothpaste application (Table 1). g y p g Results Out of the 200 adult ICU/HDUs contacted, 134 took part in the survey. Out of these, 44% have a tracheostomy weaning protocol, 69% initiate weaning whilst the patient is mechanically ventilated, and 92% use a speaking valve in their weaning process. Also, 87% allow tracheostomy patients to have oral nutritional intake and in 59% of these the decision involves speech and language therapy. Post ICU/ HDU, 67% of units discharge to specialised wards, 22% to nonspecialised wards, 4% to dedicated step-down units and 6% do not step down their tracheostomy patients. A critical care outreach team reviews the patients in 73% of the hospitals surveyed. Furthermore, only 11% of the hospitals have a consultant lead tracheostomy ward round and 17% have a tracheostomy multidisciplinary team (MDT). Also within these hospitals, 57% have their own tracheostomy safety guidelines and 70% have emergency tracheostomy management protocols. On the wards: 34% have tracheostomy bed-head information signs, 93% have emergency bed-side tracheostomy equipment, 89% have a tracheostomy training programme, and 50% have a MDT approach to decannulation. Conclusion Chlorhexidine is being used too soon after the application of toothpaste in 48% of ICUs in the UK. This results in attenuation of its eff ect and may remove its benefi cial risk reduction in VAP. Awareness of this interaction should be emphasised. References Survey of the use and practicalities of subglottic suction drainage in the UK Methods Anesthetized Yorkshire swine were randomized (n = 6/ group) to receive a bolus of the PFC Oxycyte™ either 45 minutes before (PFC-B) or after (PFC-A) induction of ARDS or nothing as a control (NON). ARDS was induced via intravenous oleic acid infusion (time 0 (T0)) over 30 minutes. Animals were monitored for physiological and hematological parameters. They were euthanized at T180 minutes and a full necropsy and histopathological analysis was performed. F Baldwin, R Gray, A Chequers y y p g Critical Care 2014, 18(Suppl 1):P332 (doi: 10.1186/cc13522) Introduction Subglottic secretion drainage (SSD) has been shown to reduce the incidence of ventilator-associated pneumonia (VAP) [1]. We reviewed current UK practice and practicalities surrounding implementation of SSD using a survey. p y p g y p Results Survival was 100% in the NON group, 80% in the PFC-A group and 20% in the PFC-B group. Mean arterial pressure (MAP) and mean pulmonary artery pressure (MPAP) were signifi cantly increased during infusion of PFC and during ARDS in the PFC-B group, while cardiac output (CO) was signifi cantly reduced. In the PFC-A group it was observed that MAP and MPAP increased and CO decreased during ARDS induction, but not during PFC infusion. Those changes were signifi cant in comparison with the NON group. Oxygen delivery and consumption in the PFC-A group were signifi cantly increased. Histopathological analysis is currently being performed. Interim analysis showed a trend to reduced alveolar damage in PFC-A animals. y Methods We constructed a survey of 10 questions using SurveyMonkey and circulated it via an email link to the Linkmen of the UK Intensive Care Society. Responses were received between August and November 2013. Results We had 77 responses. The majority were from doctors (88%) and the rest from nurses. Of respondents, 63% worked in district general hospitals, 28% in teaching hospitals and the rest in specialist units. Overall, 54% of respondents worked in units using SSD. From these responses, the types of patients receiving SSD are summarised in Table 1. One hundred per cent of units used intermittent SSD. Seventy- one per cent of respondents reported that SSD tubes were stored only on their ICU, with 26% reporting availability in acute areas and the rest hospital wide. Twenty-eight per cent of respondents indicated it was unit policy to reintubate patients to facilitate SSD. P333 statistically signifi cant diff erences were detected (P = 0.276). However, in the fi rst 5 days the development of VAP was signifi cantly higher in Group C (respectively, 4.3% vs. 25%, P = 0.046). The growth rate of VAP (VAP number/ventilator-day × 1,000) in Group C was 17.48, while in Group S it was 11.62. Between the groups there were no statistically signifi cant diff erence according to ICU mortality, length of ICU stay and length of hospital stay (P ≥0.05). Intravenous perfl uorocarbons increased oxygen delivery/ consumption in ARDS in swine p A Scultetus1, A Haque1, F Arnaud1, G McNamee2, L Dickson1, C Auker1, R McCarron1, R Mahon1 1Naval Medical Research Center, Silver Spring, MD, USA; 2Uniformed Services University of the Health Sciences, Bethesda, MD, USA Critical Care 2014, 18(Suppl 1):P333 (doi: 10.1186/cc13523) Conclusion This prospective randomized controlled study demonstrated that the incidence of VAP (especially early development of the VAP) with continuous aspiration of subglottic secretions without creating any undesirable clinical damage in the airways was signifi cantly reduced. Introduction Emulsifi ed perfl uorocarbons (PFC) are synthetic hydrocarbons that can carry 50 times more oxygen than human plasma. Their properties may be advantageous in applications requiring preservation of tissue viability in oxygen-deprived states, which makes them a potential candidate for combat and civilian pre- hospital resuscitation. Our hypothesis was that an intravenous dose of PFC increases vital organ tissue oxygenation, improves survival and reduces or prevents the development of ventilator-associated ARDS. Here we report data from the second part (ARDS only) of a multiphase swine study to investigate the benefi ts of PFC in treating hemorrhagic shock and preventing ARDS. Reference 1. Bouza E, Perez MJ, Munoz P, Rincon C, et al.: Continuous aspiration of subglottic secretions in the prevention of ventilator-associated pneumonia in the postoperative period of major heart surgery. Chest 2008, 134:938-946. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1. Muscedere J, et al.: Crit Care Med 2011, 39:1985-1991. Survey of the use and practicalities of subglottic suction drainage in the UK More than 90% of units had a ventilator care bundle and regularly measured cuff pressures. Overall, 83% of those surveyed thought SSD was benefi cial in the prevention of VAP. g Conclusion Administration of PFC before induction of ARDS was detrimental, while giving PFC after ARDS improved oxygen delivery and increased oxygen consumption. Although survival in this group was lower than in the NON control group (80% vs. 100%, not signifi cant), a reduction in alveolar damage was observed. This might improve long- term outcome after ARDS. Based on these data we will continue to the fi nal phase of this project and evaluate the capacity of PFC to prevent ARDS in combination with HS. Table 1 (abstract P332). SSD in specifi c patient groups (more than one per responder) Patients Number All 23 Tube >72 hours 6 Tracheostomy 13 Selected 7 Table 1 (abstract P332). SSD in specifi c patient groups (more than one per responder) Prevention of pneumothorax using venovenous ECMO in acute respiratory distress syndrome with emphysematous/cystic changes in the lung T Otani, S Ohshimo, K Ota, Y Kida, T Inagawa, J Itai, S Yamaga, K Une, Y Iwasaki, N Hirohashi, N Kohno, K Tanigawa Hiroshima University, Hiroshima, Japan Critical Care 2014, 18(Suppl 1):P334 (doi: 10.1186/cc13524) 2. High Impact Intervention [http://nhschoicestraining.spinningclock.com/ Documents/HII_-_Ventilator_associated_pneumonia.pdf] Survey on the use of chlorhexidine and toothpaste as part of oral care in UK ICUs I Kangesan1, F Millinchamp1, JV Williams1, LA Burrows2 1Royal United Hospital, Bath, UK; 2Frenchay Hospital, Bristol, UK Critical Care 2014, 18(Suppl 1):P330 (doi: 10.1186/cc13520) Introduction Ventilator-associated pneumonia (VAP) is the most common nosocomial infection among ventilated patients and is associated with increased mortality and morbidity [1]. Oral chlorhexidine has been used to decontaminate the airway in critically Results In Group C, VAP was developed in 10 (35.7%) of 28 patients. In group S, VAP was developed in fi ve (21.7%) of 23 patients. In both groups when compared according to the development of VAP, no S120 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Forty-one patients were included in this study (ECMO and non- ECMO group, 21 and 20 patients, respectively). There were no signifi cant diff erences between ECMO and non-ECMO groups as regards age, sex, underlying disease, PaO2/FIO2 ratio, duration of ICU stay, and survival. In the ECMO group, the mean duration of ECMO use was 17 ± 13 days, and bleeding due to anticoagulation was observed in fi ve patients. The mean airway pressure in the ECMO group was signifi cantly lower than in the non-ECMO group (12 ± 6 cmH2O, 22 ± 6 cmH2O, respectively; P <0.0001). The incidence of pneumothorax was also signifi cantly lower in the ECMO group than the non-ECMO group (10%, 45%, respectively; P = 0.015). In Kaplan–Meier analysis, the proportion of pneumothorax- free patients was signifi cantly higher in the ECMO group (P = 0.014). In multivariate analysis, conventional ventilator management, presence of interstitial pneumonia and the duration of intubation were the independent risk factors of pneumothorax (hazard ratio (HR), 18.0, P = 0.010; HR 33.3, P = 0.025; HR 1.05, P = 0.041, respectively).f secretion of Th17 (IL-6, IL-8, IL-9, IL-17) and Th1 (TNFα, IL-15, IL-12p70) cytokines [1,2]. However, the exact role of T-helper cells (Th) in the ALI model remains unknown. We hypothesized that there might be Th imbalance within the lung in the early phase of LPS-induced ALI. This study was to assess the role of lung Th polarization response in ALI mice. Methods C57BL/6 mice were randomly divided into two groups: control group and ALI group. ALI animals received 2 mg/kg LPS. Lung wet weight/body weight (LW/BW) was recorded to assess lung injury. The pathological changes were examined under an optical microscope. The mRNA expression levels of T-bet, GATA-3 and RORγt were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. Meanwhile, levels of IL-6, IFNγ, IL-4 and IL-17 in lung homogenates were assessed by enzyme-linked immunosorbent assay. Results The increase in LW/BW was induced in ALI mice. Histologically, widespread alveolar wall thickening caused by edema, severe hemorrhage in the interstitium and alveolus, and marked and diff use interstitial infi ltration with infl ammatory cells were observed in the ALI group. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. Comparison of CD80 level on dendritic cells in acute lung injury mice 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Results LPS-challenged and ventilated older rats showed higher mortality, larger change in wet-to-dry ratio and larger decline in P/F ratio, compared with other age groups. Increases in neutrophil infl ux and infl ammatory mediators were age dependent, with higher levels with increasing age. Compared with controls, ventilated LPS- challenged rats showed a decrease in mRNA expression of ACE, ACE2, AT-1 and Mas receptor in all age groups, except for infants. The relative decrease in the mRNA expression of the Mas receptor was most extensive in older rats, thereby shifting the balance towards the lung injurious axis in this age group.f Critical Care 2014, 18(Suppl 1):P337 (doi: 10.1186/cc13527) Critical Care 2014, 18(Suppl 1):P337 (doi: 10.1186/cc13527) Introduction Dendritic cells (DC) may play an important role in acute lung injury (ALI) [1]. CD80 is the crucial co-stimulatory molecule that is expressed on the surface of DCs. However, little is known about the expression of CD80. The purpose of this study was to observe the expression of CD80 on circulating, lung and splenic dendritic cells (DC) in ALI mice. Methods Twelve C57BL/6 mice were randomly divided into two groups: control group and ALI group. Blood, lungs and spleens were harvested at 6 hours after LPS or PBS administration. The level of CD80 on DC was assessed by fl ow cytometry (FCM). The IL-6 level in the lung was measured by enzyme-linked immunosorbent assay. Lung wet weight/ body weight (LW/BW) was recorded to assess lung injury. Meanwhile, pathological changes were examined under an optical microscope. Results LPS-ALI resulted in a signifi cant increase in lung W/D ratio. Histologically, widespread alveolar wall thickening caused by edema, severe hemorrhage in the interstitium and alveolus, and marked and diff use interstitial infi ltration with infl ammatory cells were observed in the ALI group. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. FCM analysis showed that the level of CD80 on circulating DC in control group was (3.3 ± 1.5)%, CD80 expression on lung DC was (3.6 ± 1.2)%, and expression of CD80 on splenic DC was (9.0 ± 3.6)%, which was Methods Twelve C57BL/6 mice were randomly divided into two groups: control group and ALI group. Blood, lungs and spleens were harvested at 6 hours after LPS or PBS administration. References 1. Bermejo-Martin JF, et al.: Crit Care 2009, 13:R201. 2. Hagau N, et al.: Crit Care 2010, 14:R203. 1. Bermejo-Martin JF, et al.: Crit Care 2009, 13:R201. 2. Hagau N, et al.: Crit Care 2010, 14:R203. g j y Methods Infant (~1/2 month), juvenile (~1 month), adult (~4 months) and older (~19 months) Wistar rats were challenged with intratracheal LPS and injurious ventilation using tidal volumes of 15 ml/kg for 4 hours. Lung injury was assessed by wet-to-dry ratio and changes in P/F ratio. Levels of infl ammatory mediators were measured in bronchoalveolar lavage fl uid; mRNA expression of key genes of the pulmonary RAS was determined in lung homogenates. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 The mRNA expression of T-bet and RORγt was upregulated in ALI mice at 24 hours and 48 hours relative to normal mice (P <0.05 vs. Con). There was no signifi cant diff erence in the expression of GATA-3 among groups at 24 hours and 48 hours. Meanwhile, the levels of IFNγ, IL-17 and IL-6 in lung tissue were signifi cantly enhanced at 24 hours and 48 hours in the LPS-induced ALI mice. In addition, the levels of IL-4 in lung tissue were signifi cantly enhanced at 48 hours in the LPS-induced ALI mice. The expression of T-bet mRNA and RORγt mRNA had a strong correlation with the IL-6 concentration. However, there was no signifi cant correlation of GATA- 3 with the IL-6 concentration. In addition, there was a signifi cant correlation of IFNγ, IL-4 and IL-17 with the IL-6 level in LPS-induced ALI at 24 hours and 48 hours. y Conclusion Although the survival rate was not statistically diff erent, the use of ECMO for ARDS patients complicated with emphysematous/ cystic changes in the lung markedly reduced the incidence of pneumothorax. Injurious ventilation has an age-dependent aff ect on the pulmonary renin–angiotensin system in LPS-challenged rats LR Schouten1, HJ Helmerhorst1, GT Wagenaar2, AP Bos1, MJ Schultz1, RM Wösten-van-Asperen1 1Academic Medical Center, Amsterdam, the Netherlands; 2Leiden University Medical Center, Leiden, the Netherlands Critical Care 2014, 18(Suppl 1):P335 (doi: 10.1186/cc13525) Introduction The underlying molecular mechanisms for the association between aging and a higher susceptibility to develop ARDS are poorly understood. The pulmonary renin–angiotensin system (RAS), with a lung-protective (angiotensin-converting enzyme (ACE)2–angiotensin (Ang)-1,7–Mas receptor) axis and a lung-injurious (ACE–Ang II–Ang II receptor (AT1)) axis, has been implicated in the pathogenesis of ARDS and changes with age. We hypothesized that injurious ventilation has an age-dependent eff ect on the pulmonary RAS in LPS-challenged rats that is associated with increased lung injury. Conclusion ALI provokes Th1 and Th17 polarization response. Th1 and Th17 may participate in the early infl ammatory response to ALI. Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. References Conclusion ALI provokes Th1 and Th17 polarization response. Th1 and Th17 may participate in the early infl ammatory response to ALI. y p p yl y p Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. References Comparison of CD80 level on dendritic cells in acute lung injury mice The level of CD80 on DC was assessed by fl ow cytometry (FCM). The IL-6 level in the lung was measured by enzyme-linked immunosorbent assay. Lung wet weight/ body weight (LW/BW) was recorded to assess lung injury. Meanwhile, pathological changes were examined under an optical microscope.i Conclusion The eff ects of injurious ventilation on lung injury are age dependent in a model of LPS-challenged rats, which is associated with a more pronounced imbalance of the pulmonary RAS at the expense of the lung-protective axis with increasing age. Prevention of pneumothorax using venovenous ECMO in acute respiratory distress syndrome with emphysematous/cystic changes in the lung Introduction Venovenous extracorporeal membrane oxygenation (VV ECMO) is a treatment option for acute respiratory distress syndrome (ARDS) to minimize ventilator-induced lung injury including life- threatening pneumothorax. The purpose of our study was to investigate the safety and effi cacy of VV ECMO for preventing pneumothorax in ARDS patients who were complicated with emphysematous/cystic changes in the lung. Conclusion Despite specifi c recommendations from the UK Department of Health [2], only about one-half of respondents work in ICUs where SSD has been adopted. Most studies show benefi t in patients expected to be ventilated for greater than 72 hours [1], but most units used SSD in all intubated patients. The reintubation rate to facilitate SSD was also reasonably high, despite a lack of evidence to support this practice. In the vast majority of hospitals, SSD endotracheal tubes are stored only on the ICU and so the need for reintubation may result from a lack of available appropriate tubes at the point of fi rst intubation. References Methods We have retrospectively analyzed data of ARDS patients complicated with emphysematous/cystic changes in the lung who were admitted to our ICU from 2006 through 2012. We divided the subjects into two groups, patients treated with VV ECMO (ECMO group), and those treated only by conventional ventilator management (non- ECMO group). Correlations between age, sex, underlying disease, PaO2/ FIO2 ratio on admission, duration of ICU stay, survival and incidence of pneumothorax were evaluated. S121 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P338 Five-year single-centre review of ARDS patients receiving high-frequency oscillatory ventilation N Pathmanathan1, N Smith1, V Martinson1, V Allgar2, R Rao1, J Glazebrook1, L Gray-Nicholson1, A Gratrix1 1Hull and East Yorkshire Hospitals, Hull, UK; 2Hull and York Medical School, York, UK Critical Care 2014, 18(Suppl 1):P338 (doi: 10.1186/cc13528) Results The increase in LW/BW induced by LPS was partly prevented by pretreated with losartan. Histologically, losartan eff ectively attenuated the LPS-induced lung hemorrhage, and leukocyte cell infi ltration in the interstitium and alveolus. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. With pretreatment of ALI mice with losartan, the level of IL-6 in lungs markedly decreased. The mRNA expression of T-bet and RORγt was upregulated in ALI mice at 24 hours and 48 hours relative to normal mice (P <0.05 vs. Con). There was no signifi cant diff erence in the expression of GATA-3 among groups at 24 hours and 48 hours. Of note, pretreatment of ALI mice with losartan resulted in signifi cantly reduced mRNA expression of T-bet at 24 hours and 48 hours and RORγt mRNA expression at 48 hours (P <0.05 vs. ALI). Meanwhile, the levels of IFNγ, IL-4, IL-17 and IL-6 in lung tissue were signifi cantly enhanced at 24 hours and 48 hours in the LPS-induced ALI mice. In addition, both IFNγ and IL-17 in lung tissue at 24 hours and 48 hours decreased signifi cantly in losartan-pretreated mice compared with the ALI mice. With pretreatment of ALI mice with losartan, the level of IL-4 in lungs was not changed. Introduction Two recent RCTs (OSCAR and OSCILLATE [1,2]) showed that high-frequency oscillatory ventilation (HFOV) had no positive impact on mortality. We present our experience over 5 years. p y p p y Methods Adult ARDS patients who received HFOV from 2008 to 2012 were included. Demographics, illness severity and outcomes were collected retrospectively. Results A total of 118 patients were included; 56.8% were male, mean age was 54.8 years. RRT use was 45% during admission. Vasoactive agent use and neuromuscular blockade infusion rate was 81.9 and 29.7% pre HFOV respectively. The 28-day and 6-month mortality was 61.9 and 70.3%. A total of 60.1% had less than 48 hours conventional ventilation (CV) pre HFOV. The 6-month mortality was 64.8% for this group. Patients who had over 48 hours CV pre HFOV had a 6-month mortality of 76.6%. See Table 1. y Conclusion Mortality rates were higher than in recent trials [1,2]. Our patients represent a more critically unwell group with lower PF ratios pre HFOV and high vasoactive and RRT use. P339 signifi cantly higher than that on circulating DC and lung DC (P <0.05). In the ALI mouse, the level of CD80 on peripheral blood DC was (5.1 ± 2.1)%; the CD80 level on lung DC was (9.6 ± 2.50)%, which was signifi cantly higher than that on peripheral blood DC (P <0.05); and the level of CD80 on splenic DC was (25.2 ± 4.7)%, which was signifi cantly higher than CD80 levels on the peripheral blood and lung DC (P <0.05). The CD80 level on lung and splenic DC in ALI mice was signifi cantly higher than that on lung and splenic DC in control mice (P <0.05 vs. Con). Blocking angiotensin type 1 receptor modulates Th1-mediated and Th17-mediated responses in lipopolysaccharide-induced acute lung injury mice J Liu1 W Li2 J Liu , W Li 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Research Institute of General Surgery, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Critical Care 2014, 18(Suppl 1):P339 (doi: 10.1186/cc13529) Conclusion There is a dynamic characteristic in the expression of CD80 on DC populations in normal and ALI mice. Elevated expression of CD80 on DC seems to play an important role in the pathogenesis of ALI. Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. Reference Introduction Losartan, an antagonist of angiotensin II (Ang II) type 1 receptor, is a potential therapeutic drug for acute lung injury (ALI). Recent reports suggest that losartan inhibits T-helper (Th)-1 immune response and ultimately attenuates infl ammation in several angiotensin II-mediated infl ammatory diseases [1,2]. However, the possible protective mechanisms of losartan in ALI remain poorly understood. This study was to assess the eff ect of losartan on the lung Th polarization response in ALI. 1. Venet F, et al.: Am J Pathol 2010, 176:764-773. Methods C57BL/6 mice were randomly divided into three groups: control group, ALI group and ALI + losartan group. ALI animals received 2 mg/kg LPS; ALI + losartan animals received 2 mg/kg LPS and 15 mg/kg losartan 30 minute before intratracheal injection of LPS. The pathological changes were examined under an optical microscope. The mRNA expression levels of T-bet, GATA-3 and RORγt were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. P338 Five-year single-centre review of ARDS patients receiving high-frequency oscillatory ventilation N Pathmanathan1, N Smith1, V Martinson1, V Allgar2, R Rao1, J Glazebrook1, L Gray-Nicholson1, A Gratrix1 1Hull and East Yorkshire Hospitals, Hull, UK; 2Hull and York Medical School, York, UK Critical Care 2014, 18(Suppl 1):P338 (doi: 10.1186/cc13528) HFOV may still have a role in the treatment of these very sick patients with treatment refractory to conventional ventilation. g Conclusion Ang II-induced Th1 and Th17 polarization response could upregulate infl ammatory response and induce lung injury, and losartan may be a promising substance for clinical use in LPS-induced ALI. Acknowledgements Supported by the Research Project CPSFG 2013M542578, JSPSFG 1301005A, SYS201251 and 2013NJZS50. References 1. Hoch NE, et al.: Am J Physiol Regul Integr Comp Physiol 2009, 296:R208-R216. 2. Platten M, et al.: Proc Natl Acad Sci U S A 2009, 106:14948-14953. P336 Role of Th1 and Th17 imbalance in acute lung injury mice J Liu1 W Li2 p g g p p Results LPS-ALI resulted in a signifi cant increase in lung W/D ratio. Histologically, widespread alveolar wall thickening caused by edema, severe hemorrhage in the interstitium and alveolus, and marked and diff use interstitial infi ltration with infl ammatory cells were observed in the ALI group. Meanwhile, the levels of IL-6 in lung tissue were signifi cantly enhanced in the LPS-induced ALI mice. FCM analysis showed that the level of CD80 on circulating DC in control group was (3.3 ± 1.5)%, CD80 expression on lung DC was (3.6 ± 1.2)%, and expression of CD80 on splenic DC was (9.0 ± 3.6)%, which was , 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China , 1Suzhou Municipal Hospital (Eastern) Nanjing Medical University, Suzhou, China; 2Jinling Hospital, Nanjing University School of Medicine, Nanjing, China Critical Care 2014, 18(Suppl 1):P336 (doi: 10.1186/cc13526) Critical Care 2014, 18(Suppl 1):P336 (doi: 10.1186/cc13526) Introduction Acute lung injury (ALI) is characterized by an excessive infl ammatory response. Several recent clinical observations have shown that severe H1N1 infl uenza with ARDS is characterized by early S122 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Risk factors for multi-resistant organisms in sepsis L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P342 (doi: 10.1186/cc13532) Introduction The increasing prevalence of infections by multi-resistant organisms (MDR) has increased over the last decades, with implications not only in the overall level of therapeutic success, but also in the selective pressure exerted by the use of broad-spectrum antibiotics to defeat increasingly resistant agents, thereby creating a vicious cycle [1,2]. The aim of this study is to describe risk factors associated with infection by MDR organisms among septic patients. g p Methods We report a retrospective analysis of out-of-center ECMO implantation in patients with severe ARDS. Our center provides a 24/7 service for out-of-hospital ECMO implantation with a team consisting of two intensive care physicians and a perfusionist. In all patients, ECMO therapy was initiated in a non-ECMO center prior to transportation to our hospital. Implantation of a dual-lumen catheter was fi rst choice in all patients and was guided echocardiographically. y g g p p Methods A retrospective cohort study including all adult patients with microbiological documented sepsis, admitted to the emergency room of a tertiary care, university hospital between 1 July 2011 and 30 June 2012. g g y Results Between January 2011 and November 2013, a total of 56 patients (average age 53.3 years) underwent out-of-center venovenous ECMO implantation. In 52 cases (94.6%), a dual-lumen catheter could be implanted successfully using echocardiographic guidance. Either 31 Fr (65.0%) or 27 Fr (35.0%) Avalon Elite catheters were employed. No patient developed any major or even fatal complications related to ECMO implantation. In three out of 56 patients, poor imaging quality or technical issues hampered the implantation of a dual-lumen catheter and a femoral approach had to be made. Concerning short-term survival, 81.0% of all patients could be dismissed from the ICU. Results During the study period, 162 patients were admitted to the emergency room with severe sepsis; 79 (49%) had microbiological documentation, and were included in this study. The mean (SD) age was 71 (15) years; 62% were men. Forty patients (51%) had an infection by a MDR organism. References 1. Young D, et al.: High-frequency oscillation for ARDS. N Engl J Med 2013, 368:806-813. 1. Hoch NE, et al.: Am J Physiol Regul Integr Comp Physiol 2009, 296:R208-R216. 2. Platten M, et al.: Proc Natl Acad Sci U S A 2009, 106:14948-14953. 2. Ferguson N, et al.: High-frequency oscillation in early ARDS. N Engl J Med 2013, 368:795-805. 1. Hoch NE, et al.: Am J Physiol Regul Integr Comp Physiol 2009, 296:R208-R216. 2. Platten M, et al.: Proc Natl Acad Sci U S A 2009, 106:14948-14953. Table 1 (abstract P338). OSCAR OSCILLATE Hull HFOV Control HFOV Control Mean APACHE II 22.0 21.8 21.7 29 29 Mean Vt (ml/kg predicted) 8.6 8.7 8.3 7.2 7.1 PaO2:FiO2 (mmHg) 81.5 113 113 121 114 Vasoactive agents (%) 81.9 43.5 (day 1) 44.6 (day 1) 67 63 Hospital mortality (%) 66.1 50.1 48.4 46.9 35.2 Table 1 (abstract P338). Table 1 (abstract P338). S123 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 loads were higher in TA compared with NP, up to 2 log viral copies. Duration of positivity of infl uenza in daily samples was up to two times longer in TA samples than in NP samples. Of all 35 virus-positive patients, 10 had received viral diagnostics via routine care. loads were higher in TA compared with NP, up to 2 log viral copies. Duration of positivity of infl uenza in daily samples was up to two times longer in TA samples than in NP samples. Of all 35 virus-positive patients, 10 had received viral diagnostics via routine care. P340 Echocardiographic guidance for Avalon Elite dual-lumen catheter implantation D Staudacher1, A Schmutz2, P Biever1, J Kalbhenn2, C Bode1, T Wengenmayer1 1University Heart Center Freiburg, Germany; 2University Clinic Freiburg, Germany Critical Care 2014, 18(Suppl 1):P340 (doi: 10.1186/cc13530) Conclusion The prevalence of respiratory viruses in this unselected patient population is high. Forty percent would have been missed if only NP was sampled. Thereby, TA sampling adds to the detection of respiratory viruses in critically ill patients. Accuracy and implications of these results needs to be investigated further. Introduction Venovenous ECMO is a therapeutic option in patients with severe ARDS. The dual-lumen catheter (Avalon Elite; Maquet Medical) off ers excellent oxygenation and decarboxylation through a single insertion site and facilitates patient mobilization as it avoids femoral access. Lower airway sampling greatly increases detection of respiratory viruses in critically ill patients: the COURSE studyf Lower airway sampling greatly increases detection of respiratory viruses in critically ill patients: the COURSE studyf j Conclusion The presence of diabetes and decreased functional capacity should be considered risk factors for infection by a MDR organism and should be taken into consideration in the empirical therapy prescription. g 1Academic Medical Center, Amsterdam, the Netherlands; 2VU Medical Center, Amsterdam, the Netherlands; 3Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2014, 18(Suppl 1):P341 (doi: 10.1186/cc13531) P341 Lower airway sampling greatly increases detection of respiratory viruses in critically ill patients: the COURSE study F Van Someren Gréve1, KF Van der Sluijs1, NP Juff ermans1, T Winters1, SP Rebers1, KD Verheul1, R Molenkamp1, AM Spoelstra-de Man2, P Spronk3, MD De Jong1, MJ Schultz1 1Academic Medical Center, Amsterdam, the Netherlands; 2VU Medical Center, Amsterdam, the Netherlands; 3Gelre Hospitals, Apeldoorn, the Netherlands Critical Care 2014, 18(Suppl 1):P341 (doi: 10.1186/cc13531) P342 Risk factors for multi-resistant organisms in sepsis L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P342 (doi: 10.1186/cc13532) Risk factors for multi-resistant organisms in sepsis L Serpa-Pinto, T Cardoso Oporto Hospital Center, Oporto, Portugal Critical Care 2014, 18(Suppl 1):P342 (doi: 10.1186/cc13532) Risk factors associated with infection by a MDR organism were the presence of any comorbidity (OR = 3.542, P = 0.022), diabetes mellitus (OR = 4.500, P = 0.006), Karnofsky performance status (KPS) <70% (OR = 3.882, P = 0.005), the presence of modifi ers of etiology (OR = 5,040, P = 0.010), chronic wounds (OR = 5.371, P = 0.040), healthcare-associated infections (OR = 3.325, P = 0.026) and hospital- acquired infections (OR = 5.225, P = 0.016). The multivariate model retained as independent variables associated with infection by a MDR organism: the presence of diabetes mellitus (adjusted OR = 4.1,95% CI: 1.4 to 12.6) and the need for assistance in daily activities (KPS <70%, adjusted OR = 3.6, 95% CI: 1.3 to 9.7). Conclusion Echocardiographic guidance for out-of-center Avalon Elite catheter implantation is a safe and effi cient option when performed by an experienced team. References As incorrect placement of the catheter might result in perforation and cardiac tamponade, fl uoroscopy guidance is advocated in the literature. Transporting an unstable patient for fl uoroscopy, however, can endanger the patient. y References 1. Siegel JD, et al.: Am J Infect Control 2007, 35(10 Suppl 2):S165-S193. 2 Wright SW et al : Am J Emerg Med 2000 18:143 146 1. Siegel JD, et al.: Am J Infect Control 2007, 35(10 Suppl 2):S165-S193. 1. Siegel JD, et al.: Am J Infect Control 2007, 35(10 Suppl 2):S165-S193. 2. Wright SW, et al.: Am J Emerg Med 2000, 18:143-146. Introduction The prevalence of viral respiratory infections in critically ill patients on the ICU and the diagnostic potential of tracheal aspirate sampling are unknown. For this study, the prevalence of respiratory viruses was investigated in intubated patients by simultaneous sampling of nasopharynx and tracheal aspirate. 2. Wright SW, et al.: Am J Emerg Med 2000, 18:143-146. Retrospective observational analysis of the infective risk of arteria lines in a general ICU M Wylie, M Clark Victoria Hospital, Kirkcaldy, UK Critical Care 2014, 18(Suppl 1):P345 (doi: 10.1186/cc13535) g M Wylie, M Clark y Victoria Hospital, Kirkcaldy, UK P344 Clostridium diffi cile infection in ICU patients E Belesiotou, C Routsi, M Nepka, E Magira, P Kaltsas, Z Psaroudaki, E Kraniotaki, A Argyropoulou, T Pittaras, S Zakynthinos Evangelismos Hospital, Athens, Greece Critical Care 2014, 18(Suppl 1):P344 (doi: 10.1186/cc13534) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 between potential risk factors and development of MDR infection. Categorical variables were compared using the Fisher test. Odds ratios (ORs) and 95% confi dence intervals (CIs) were calculated to evaluate the strength of any association. Continuous variables were compared using the Student t test or the Wilcoxon test where appropriate. Multivariable analysis was performed using multiple logistic regression. Variables with P <0.20 in bivariable analyses were considered for inclusion in a multivariable model. Methods During the last year, all stool specimens received in the microbiology department from patients hospitalized in the 30-bed, multidisciplinary ICU of a tertiary-care hospital were evaluated. Specimens were ordered by physicians in the presence of clinical features compatible with C. diffi cile-associated infection. Each specimen was subjected to diagnostic tests for C. diffi cile infection including toxin enzyme immunoassays for C. diffi cile toxins A and B detection (DUO Toxin A&B; VEDA.LAB, France), and glutamate dehydrogenase (GDH) for cell wall antigen detection (C. DIFF Quik Chek Complete®; USA). Results Out of 882 patients, 135 (15.3%) developed MDR infection. The following factors showed statistical signifi cant diff erence (P <<0.01): length of stay in the ICU, origin of patient, APACHE II score, previous colonization, previous use of carbapenems, duration of mechanical ventilation, immunosuppression, diabetes mellitus, hypoproteinemia and the presence of CVC. The multivariant analysis showed statistically signifi cant correlations for the following independent risk factors: previous use of carbapenems (OR = 632.64, P <<0.01), origin of patient (OR = 19.60, P = 0.014), presence of CVC (OR = 14.19, P = 0.015), previous colonization (OR = 4.71, P = 0.037) and duration of ventilator use (OR = 1.10, P = 0.029). g p Results During the study period, 335 stool specimens were evaluated. Results obtained with the two-stage immunoassay tests are shown in Figure 1. All infected patients were treated with metronidazole or vancomycin. Following a course of therapy, 2% of the infected patients had recurrence or relapse. The crude mortality rate was 17%. Conclusion GDH antigen was positive in 12% of the stool specimens received from ICU patients with suspected C. diffi cile-associated infections. The majority of these specimens (51.4%) produce both C. diffi cile toxins A and B, whereas toxin B is produced in 31.4% and toxin A in the remaining 17.2%. References P343 Infections from MDR strains (K. pneumoniae, P. aeruginosa, A. Baumannii complex) in a multivalent ICU K Kontopoulou, E Antypa, I Sgouropoulos, G Voloudakis, P Tasioudis, E Antoniadou G. Genimatas General Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P343 (doi: 10.1186/cc13533) Methods During March and April 2013, consecutive acutely admitted, intubated patients were included in three ICUs in the Netherlands, regardless of diagnosis at admission. Daily sampling of the nasopharynx (NP) with fl ocked swab and tracheal aspirate (TA) was performed until successful weaning from mechanical ventilation or death. Admission samples were tested via multiplex RT-PCR for infl uenza A and B, parainfl uenza, RSV, human metapneumovirus, bocavirus, coronavirus, rhinovirus, enterovirus, parechovirus and adenoviruses. Of the infl uenza-positive patients, subsequent daily samples were tested for infl uenza. Results of viral diagnostics performed by routine care were collected, and compared with results found in this study. p Critical Care 2014, 18(Suppl 1):P343 (doi: 10.1186/cc13533) Introduction The infection incidences from MDR strains in our ICU are in accordance with the results of respective studies in other ICUs of our country. Our aim is to identify potential risk factors for the development of MDR infection in ICU patients. Methods The sample consisted of 882 patients, admitted to our ICU from 1 January 2010 until 30 June 2013. The factors studied were: age, gender, length of stay in the ICU, origin of patient (for example, home or transferred from another institution), APACHE II score, adjusted mortality score, previous colonization with MDR, prior use of antimicrobial agents, duration of ventilator use, immunosuppression, diabetes, hypoproteinemia and presence of central venous catheter (CVC). Bivariable analyses were conducted to determine the association y Results As part of an ongoing observational study (COURSE study), 128 patients were included, of which 35 were virus-positive (27%). Of these, 13 patients were positive in both NP and TA, eight only in NP, and 14 only in TA. Thereby, 40% of the viruses would have been missed if only NP was performed in this study group. In eight out of 12 coronavirus- positive patients, only the TA sample was virus-positive, with negative NP. In subsequent daily samples of infl uenza-positive patients, viral S124 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References Conclusion The analysis showed a very strong correlation between prior use of carbapenems and development of MDR infection. Other signifi cant factors are presence of CVC, origin of patient, previous colonization and duration of ventilator use. The identifi cation of the above factors and the eff ort for their restriction redound to the reduction of the MDR infections and, consequently, to the reduction of the morbidity and the mortality of the patients hospitalized in ICUs. 1. Bobo LB, et al.: Chest 2011, 140:1643. 1. Bobo LB, et al.: Chest 2011, 140:1643. 2. Cohen SH, et al.: Infect Control Clin Epidemiol 2010, 315:431. 2. Cohen SH, et al.: Infect Control Clin Epidemiol 2010, 315:431. P345 Reducing CR-BSI in a general ICU Results Out of 588 patients, 30 (5.1%) developed candidemia (frequency: 12 per 1,000 days of hospitalization). The mortality of patients with candidemia was 66.7% (20/30). The monovariant analysis showed statistical signifi cant diff erence in the following factors: immunosuppression (P = 0.0005), diabetes mellitus (P = 0.0005), hypoproteinemia (P = 0.0005), the presence of central venous catheter (P = 0.0005), the coexistence of bacteremia and the prior use of antimicrobial agents (P = 0.0005), length of stay in the ICU >5 days (P = 0.004) and the type of intervention (P = 0.001). As independent risk factors from the multivariant analysis were found the following: immunosuppression (exp(B) = 218.37, 95% CI = 11.76 to 4,053.72, P = 0.0005), hypoproteinemia (exp(B) = 25.69, 95% CI = 2.82 to 296.8, P = 0.009), presence of central venous catheter (exp(B) = 13.79, 95% CI = 1.86 to 102.51, P = 0.01) and the coexistence of bacteremia in combination with prior use of antimicrobial agents (exp(B) = 404.94, 95% CI = 11.61 to 14190.41, P = 0.001). D Dawson, P Riley, A Rhodes D Dawson, P Riley, A Rhodes , y, St George’s Healthcare NHS Trust, London, UK St Georges Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P346 (doi: 10.1186/cc13536) Introduction Central venous catheters (CVCs) are essential for the delivery of medications and fl uids in the ICU patient; however, they carry a substantial infection risk. Evidence from a collaborative, cohort study suggests bundled interventions can provide a sustained decrease in infection [1]. Methods Since December 2009, data have been collected daily on the number of patients with one or more CVC. All positive blood cultures are reviewed monthly against predefi ned criteria to judge whether these are genuine bacteraemic episodes and thus classifi ed as laboratory-confi rmed bloodstream infections. A monthly rate for CR-BSI per 1,000 dwell-days is calculated from these data. Since July 2010, a number of interventions aimed at reducing CR-BSI have been introduced to the ICU. Conclusion Early candidemia identifi cation, prompt collaboration of intensivists with biopathologists and immediate initiation of the proper antifungal treatment is of great signifi cance. The concurrent understanding of the predisposing risk factors constitutes a signifi cant supportive tool for the prediction of such infections. Results Data are presented on 15,644 CVC dwell-days over 47 months. Table 1 presents data for three full years and one part year (January to November 2013). K Kontopoulou, E Antypa, I Sgouropoulos, N Voloudakis, E Chassou, E Antoniadou G G G l H l Th l k G K Kontopoulou, E Antypa, I Sgouropoulos, N Voloudakis, E Chassou, E Antoniadou G. Genimatas General Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P347 (doi: 10.1186/cc13537) Introduction The prediction for and the mortality of patients with candidemia are highly adverse. The aim of this study was to identify the risk factors for the development of candidemia in postoperative patients. Introduction The prediction for and the mortality of patients with candidemia are highly adverse. The aim of this study was to identify the risk factors for the development of candidemia in postoperative patients. Conclusion These results suggest that CRLI rates for arterial lines are low at 1.003 per 1,000 arterial line-days. However, there is a signifi cant bacterial colonisation rate of the arterial lines sampled. Three arterial lines (6.25%) grew organisms that could represent an important potential source of ongoing bacteraemia. There is evidence to suggest that the risk of infection and colonisation of arterial lines may be similar to that of central lines [1]. Further prospective work is needed to assess the impact of catheter care bundles on colonisation and infection rates of arterial lines in the ICU. Methods From 1 July 2010 to 30 June 2013 all postoperative patients (n = 588) admitted to the multivalent ICU of our hospital were enrolled in this study. We recorded the age, sex, length of stay in the ICU, APACHE II score upon admission to the ICU, adjusted mortality score, underlying conditions, recent operations, invasive therapeutic procedures and prior usage of antimicrobial agents. Initial bivariable statistical comparisons were conducted using the χ2 test for categorical data and the Student t test or Wilcoxon test for continuous data. Relative risks (RRs) and their 95% confi dence intervals (CIs) were calculated. Statistical signifi cance was set at P <0.05. To identify patient characteristics associated with candidemia we used multivariable logistic regression. In the multivariant analysis we also included the independent risk factors reported in recent medical literature. Results of the logistic regression analysis are reported as adjusted odds ratio (OR) with 95% CI. P347 Thirty-seven arterial lines returned no growth (77.08%). Seven cultures grew organisms likely to be contaminants (14.58%). Four cultures grew signifi cant organisms (yield of 8.33%). There were two cases with documented clinical signs of catheter-related local infection (CRLI) at the arterial line puncture site. In one case of CRLI the primary source of infection was felt to be remote from the arterial line. The second represented a local infection with organisms that are typically skin commensals. Of the four cultures likely to represent invasive pathogens, three had clinical suspicion that the primary source was a site remote from the arterial line. In two of these cases this was confi rmed by growing the same organism at an alternative site more likely to be the source of infection. Risk factors of candidemia in postoperative ICU patients: a prospective study K Kontopoulou, E Antypa, I Sgouropoulos, N Voloudakis, E Chassou, E Antoniadou G. Genimatas General Hospital, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P347 (doi: 10.1186/cc13537) Reference 1. Lucet JC, et al.: Infectious risk associated with arterial catheters compared with central venous catheters. Crit Care Med 2010, 38:1030-1035. Reducing CR-BSI in a general ICU Reducing CR-BSI in a general ICU Despite an increase in bed-days per year, there has been a sustained reduction in infection rates and a reduction in dwell-days. Table 1 (abstract P346). Catheter infection rates, APACHE score and bed-days 2010 to 2014 1. Pronovost et al.: N Engl J Med 2006, 355:26. 2. Dixon-Woods et al.: Implement Sci 2013, 8:702. P348 P348 Escherichia coli infection in Polish neonatology ICUs in 2009 to 2012 J Wójkowska-Mach1, A Chmielarczyk1, D Romaniszyn1, E Helwich2, R Lauterbach2, M Pobiega1, M Borszewska-Kornacka2, E Gulczyńska2, A Kordek2 1Jagiellonian University Medical School, Krakow, Poland; 2Polish Neonatal Surveillance Network, Warsaw, Poland Critical Care 2014, 18(Suppl 1):P348 (doi: 10.1186/cc13538) y Total CR-BSI/ CVC CR-BSI 1,000 APACHE II Bed- Dwell-days/ Year dwell-days annually dwell-days (mean) days bed-days 2010 4,037 6 1.5 15.8 5,870 0.69 2011 3,942 2 0.5 16.3 5,967 0.66 2012 3,822 3 0.8 15.9 6,117 0.62 2013* 3,863 1 0.3 16.0 6,059 0.64 Introduction The aim of our study was the analysis of the epidemiology of infections caused by ECO in Polish NICUs and their resistance to antibiotics, with particular reference to their impact on the safety of infants with very low birth weight, and their relationship to the care of women who are pregnant and in labour. The routes of transmission of ECO strains were also evaluated. Conclusion CR-BSI rates of 1.5/1,000 dwell-days in the fi rst year were similar to the post-intervention rate of 1.4 in the Pronovost study [1]. Subsequent rates have reduced, suggesting we are outperforming the secular trend [2]. The proportion of dwell-days to bed-days was reduced, which may suggest a reduction in duration and/or quantity of CVC placements. Methods Continuous prospective infection surveillance was conducted in 2009 to 2012 in fi ve NICUs and included 1,768 newborns whose birth weight was lower than 1,500 g. Results The incidence of ECO infections was 5.4% and 2.0/1,000 patient- days. The occurrence of ECO infections depended signifi cantly on the NICU and ranged from 3.9 to 17.9%. Multivariate analysis that took into account the combined eff ect of demographic data (gender, gestational 3 Clostridium diffi cile infection in ICU patients p , y, Critical Care 2014, 18(Suppl 1):P345 (doi: 10.1186/cc13535) Introduction This study aims to describe the infective morbidity of arterial line catheters in a general ICU. Introduction This study aims to describe the infective morbidity of arterial line catheters in a general ICU. Methods All ICU admissions for the year 2012 were listed on the WardWatcher database. All cases in which arterial line tip samples returned any growth were reviewed in conjunction with the medical, nursing and daily chart recordings. Data were collected on the organisms grown and antimicrobial sensitivities. A qualitative decision of clinical relevance was established based on necessitated change in patient management, signs of local site infection or evidence of bacteraemia with the same organism. Introduction Clostridium diffi cile infection is becoming more common worldwide. Critically ill patients are at particularly high risk for this disease due to multiple risk factors in this population. Accurate diagnosis is essential for patient management, infection control and epidemiology. There are a variety of methods to detect the presence of toxigenic C. diffi cile in stools [1,2]. The aim of this study was to evaluate the incidence of C. diffi cile infection in our ICU. Laboratory results of C. diffi cile toxin detection performed by the methods available in our institution are presented. Results During 2012 there were 416 ICU admissions, representing a total of 2,440 ITU-days and 1,994 arterial line-days. A total of 48 arterial line tips suspected of being infected were sent for culture. Figure 1 (abstract P344). Illustration of the tests performed for detection of C. diffi cile. Figure 1 (abstract P344). Illustration of the tests performed for detection of C. diffi cile. S125 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Surveillance for nosocomial pathogens in our ICU Y Kawano, H Ishikura, T Nishida, N Matusmoto, H Ohya, M Mizunuma, Y Nakamura, T Umemura Fukuoka University Hospital, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P350 (doi: 10.1186/cc13540) Surveillance for nosocomial pathogens in our ICU Y Kawano, H Ishikura, T Nishida, N Matusmoto, H Ohya, M Mizunuma, Y Nakamura, T Umemura Fukuoka University Hospital, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P350 (doi: 10.1186/cc13540) Introduction The use of contact precautions is recommended to reduce the transmission of these pathogens. However, there is little research regarding the relationship between the rate of patients with culture- positive fi ndings for Acinetobacter baumannii and the consumption of hand disinfectant. The objective of this study was therefore to evaluate trends in nosocomial bacterial detection, including A. baumannii, and the use of hand disinfectant in our ICU. y Conclusion Unfortunately, the presented data indicate that antibiotic prophylaxis in the presence of symptoms such as chorioamnionitis and PROM did not help to reduce the risk of ECO infection in the group of examined infants. In addition, multivariate analysis demonstrated only one signifi cant risk factor for ECO infection among infants with a birth weight <1,500 g; that is, the impact of the NICU. Epidemiology of ECO infections clearly indicated that observed cases of infections have no connection with the horizontal transmission (thus no proof of a link between the observed ECO infections with possible negligence in hand hygiene or excessive congestion on NICUs). This is confi rmed by the fact that no epidemic clones were observed (DEC-2011/01/D/ N27/00104). Methods A single-center, retrospective, observational study was carried out. The results of all cultures (sputum, urine, blood, and so forth) were used to examine trends in the detection of microbiology in our ICU over a 4-year period from April 2009 through March 2013. The rate of culture-positive patients, frequency of use of hand disinfectant and the antimicrobial use density (AUD) values were investigated for the described periods and compared between the early period group (April 2009 to March 2011) and the late period group (April 2011 to March 2013). Results We encountered 3,302 patients in our ICU during this period. No patients with culture-positive fi ndings for multidrug-resistant A. baumannii were identifi ed in this study. The rates of patients with culture-positive fi ndings for multidrug-resistant Pseudomonas aeruginosa (0‰ vs. 0.05‰ per 1,000 patient-days, P = 0.99) and P. aeruginosa (6.96‰ vs. 7.21‰ per 1,000 patient-days, P = 0.7) were not signifi cantly diff erent between the early period group and the late period group. The rates of patients with culture-positive fi ndings for MRSA (6.96‰ vs. 1. Nalapko Y, Peicheva O, Iegorov O, Nekrylov A: The dependence of infectious complications from the trauma severity. Intensive Care Med 2013, 39(Suppl. 2):P.0791. P350 age and birth weight) and place of birth (NICUs, where the baby was born) showed that only the place of hospitalisation had a signifi cant eff ect on the ECO infection risk. The highest levels of resistance among all ECO isolates were observed against ampicillin (88.8%) and amoxicillin/clavulanic acid (62.2%). ECO isolates showed very diff erent pulsotypes and dominant epidemic clones were not detected. Cluster analysis based on PFGE of the 90 isolates showed 71 unique types, some of which were less than 70% similar, suggesting a genotypically variable population. Isolates that have identical pulsotypes usually were derived from the same patient (as in the case of 11 isolates) or were isolated from diff erent patients of the same NICU in the same period of time (in the case of seven isolates). The location of the NICU and the site of the isolation did not appear to have a correlation in the cluster analysis P351 35 Comparison of multidrug-resistant Acinetobacter and non-Acinetobacter infections in terms of outcome in critically ill patients A Madaan1, V Singh1, P Shastri2, C Sharma3 1Patel Cancer and Superspeciality Hospital, Jalandhar, India; 2Sir Gangaram Hospital, New Delhi, India; 4Lovely Professional University, Jalandhar, India Critical Care 2014, 18(Suppl 1):P351 (doi: 10.1186/cc13541) Surveillance for nosocomial pathogens in our ICU Y Kawano, H Ishikura, T Nishida, N Matusmoto, H Ohya, M Mizunuma, Y Nakamura, T Umemura Fukuoka University Hospital, Fukuoka, Japan Critical Care 2014, 18(Suppl 1):P350 (doi: 10.1186/cc13540) 8.94‰ per 1,000 patient-days, P <0.05) and A. baumannii (4.98‰ vs. 6.51‰ per 1,000 patient-days, P <0.05) were signifi cantly higher in the late period group than in the early period group. The frequency of use of hand disinfectant (99.5 ml/patient- day vs. 85 ml/patient-day, P <0.05) was signifi cantly lower in the late period group. The AUD values for fl uoroquinolones (34 vs. 31.1 defi ned daily doses/1,000 bed-days, P = 0.69), third-generation cephalosporins (23.5 vs. 38.4 defi ned daily doses/1,000 bed-days, P = 0.55) and glycopeptides (34.9 vs. 35.9 defi ned daily doses/1,000 bed-days, P = 0.66) were not signifi cantly diff erent between the early period group and the late period group. However, the AUD values for carbapenems were signifi cantly higher in the early period group (40.6 vs. 71.8 defi ned daily doses/1,000 bed-days, P < 0.05). P349 Infection control as a nonpharmacologic strategy for the prevention of healthcare-associated infections in a Ukrainian hospital Y Nalapko Lugansk State Medical University, Lugansk, Ukraine Critical Care 2014, 18(Suppl 1):P349 (doi: 10.1186/cc13539) p References S126 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P349 Infection control as a nonpharmacologic strategy for the prevention of healthcare-associated infections in a Ukrainian hospital Y Nalapko Lugansk State Medical University, Lugansk, Ukraine Critical Care 2014, 18(Suppl 1):P349 (doi: 10.1186/cc13539) p Lugansk State Medical University, Lugansk, Ukraine Introduction Healthcare-associated infections (HAI) in ICU patients are related to intubation, mechanical ventilation, and central venous and/ or urinary catheters. The incidence of HAI is too high, and antibiotic strategies suff er from resistance to the common classes of antibacterial agents [1]. Methods A complex program including staff teaching on the basic approaches of hand hygiene, microbiological passport of the ICU departments, and detection of the sources of the pathogens polluting the treatment area of the ICU was implemented at the Lugansk Regional Clinical Hospital. The incidence of the HAI was studied in polytrauma patients in 1999 to 2003 (before the implementation of the program) and in 2008 to 2012 (after its implementation). Conclusion Improving compliance with hand hygiene and appropriate use of carbapenems is important for decreasing the rate of patients with culture-positive fi ndings for MRSA and A. baumannii. ( p ) Results Before the implementation of the infection control program, the incidence of respiratory tract infections in polytrauma patients staying in ICUs was 57.4% of patients. Urinary infections occurred in 51.9% of patients. Surgical site infections were found in 32.8% of the patients. Combination of the infections was detected in 33.8% of patients. The incidence of the multiple-drug-resistant bacterial colonies was 5.8%. In contrast, recent data obtained after the implementation of the program of the infection control showed that the respiratory infections occurred in 29.3% of the patients, catheter-related urinary tract infections were detected in 32.8%, and surgical site infections were detected in 8.6% of the polytrauma patients. On the other hand, the incidence of the multiple-drug-resistant bacterial colonies increased signifi cantly up to 14.2% (P <0.001). Other types of HAI changed nonsignifi cantly. Results Before the implementation of the infection control program, the incidence of respiratory tract infections in polytrauma patients staying in ICUs was 57.4% of patients. Urinary infections occurred in 51.9% of patients. Surgical site infections were found in 32.8% of the patients. Combination of the infections was detected in 33.8% of patients. The incidence of the multiple-drug-resistant bacterial colonies was 5.8%. References 1. Mahgoub S, et al.: Infection Control Hosp Epidemiol 2002, 23:477-479. 2. Hsueh PR, et al.: Emerg Infect Dis 2002, 8:827-832. P352 Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) M Albert1, DR Willamson1, J Muscedere2, F Lauzier3, C Rostein4, S Kanji5, X Jiang2, M Hall6, DK Heyland2 1Hopital du Sacré-Coeur de Montréal, Canada; 2Kingston General Hospital, Kingston, Canada; 3Centre hospitalier affi lié universitaire de Québec, Canada; 4Toronto General Hospital, Toronto, Canada; 5The Ottawa Hospital Research Institute, Ottawa, Canada; 6Nationwide Children’s Hospital, Colombus, OH, USA Critical Care 2014, 18(Suppl 1):P352 (doi: 10.1186/cc13542) Retrospective analysis of respiratory isolates post out-of-hospital cardiac arrest to establish choices in empirical antibiotic cover Introduction We analysed positive respiratory isolates for antibiotic resistance in our out-of-hospital cardiac arrest (OOHCA) population to establish adequacy of current empirical regimes. Pneumonia commonly complicates OOHCA, with studies suggesting a prevalence up to 48% [1]. Results MDR AB-infected patients (n = 45, 43%) had greater number of organ dysfunctions, and higher mean lengths of hospital stay (8.4 days vs. 6.1 days) and ICU stay (5.3 days vs. 0.8 days) after the index day than MDR non-AB infection (n = 59, 57%) (Table 1). In-hospital mortality rates for patients with MDR AB infections (42%) were higher than for MDR non-AB infection (10%). F critical = 3.934253. Methods Patients admitted to the ICU between May 2007 and September 2013 who underwent therapeutic hypothermia post OOHCA were included in this study. Demographics and antibiotic resistance were collected from an electronic database. Conclusion We demonstrated in patients infected with MDR strains of any organism that patients infected with Acinetobacter have greater number of organ dysfunction, longer lengths of stay in both the hospital and the ICU as well as increased mortality than patients infected with non-Acinetobacter infection when we controlled for severity of illness. Results A total of 160 patients were admitted to the ICU post OOHCA. In total, 37.5% (60/160) had no respiratory sample sent within 72 hours of admission and were excluded. Forty per cent (40/100) grew a clinically important isolate. Gram-negative bacteria (GNB) were most frequently isolated (42.5%, 17/40) followed by Gram-positive bacteria (32.5%, 13/40) and mixed bacterial growth (25%, 10/40). S. aureus (n = 14) isolates were often resistant to penicillin (92.8%, 13/14 isolates tested) but maintained macrolide (erythromycin 92.8%, 13/14), clindamycin (92.8%, 13/14) and vancomycin (100%, 13/13) sensitivity, if tested. S. pneumoniae isolates (n = 8) maintained penicillin (87.5%, 7/8), levofl oxacin (100%, 6/6), erythromycin (87.5%, 7/8) and vancomycin (100%, 6/6) sensitivity. The most commonly isolated GNB, H. infl uenzae, maintained high-level sensitivity to amoxicillin (81.8%, 9/11) and co- amoxiclav (90.9%, 10/11). Other isolated GNB, however, demonstrated variable resistance to co-amoxiclav (69.2%, 9/13). Isolates were 100% sensitive to pipericillin–tazobactam (17/17), amikacin (12/12) and meropenem (16/16). The British Thoracic Society suggests co-amoxiclav and clarithromycin for fi rst-line treatment of severe community- acquired pneumonia [2]. Based on this, 77.5% (31/40) of our patients would have received adequate cover. If pipericillin–tazobactam replaced co-amoxiclav, 95% (38/40) of our patients would have been treated with appropriate antibiotics. 1. Delisle MS, Williamson DR, Perreault MM, Albert M, Jiang X, Heyland DK: The clinical signifi cance of candida colonization of respiratory tract secretions in critically ill patients. J Crit Care 2008, 23:11-17. References 1. Nielsen N, et al.: Adverse events and their relation to mortality in out-of- hospital cardiac arrest patients treated with therapeutic hypothermia. Crit Care Med 2011, 39:57-64. 1. Nielsen N, et al.: Adverse events and their relation to mortality in out-of- hospital cardiac arrest patients treated with therapeutic hypothermia. Crit Care Med 2011, 39:57-64. hospital cardiac arrest patients treated with therapeutic hypothermia. Crit Care Med 2011, 39:57-64. p Methods We conducted a double-blind, placebo-controlled, multi- center, pilot randomized trial of antifungal therapy in critically ill patients with a clinical suspicion of ventilator-associated pneumonia with positive airway secretion specimens for Candida spp. We also included an observational group without Candida spp. in their airway secretions. We measured the recruitment rate, infl ammatory profi les over time and clinical outcomes. 2. Lim WS, et al.: BTS guidelines for the management of community acquired pneumonia in adults: update 2009. Thorax 2009, 64:1-55. 2. Lim WS, et al.: BTS guidelines for the management of community acquired pneumonia in adults: update 2009. Thorax 2009, 64:1-55. P354 Ph Pharmacokinetics of antituberculosis drugs in critically ill patients with tuberculosis and acute respiratory failure CH Hernandez-Cardenas, GL Lugo-Goytia Instituo Nacional de Enfermedades Respiratorias, Mexico City, DF, Mexico Critical Care 2014, 18(Suppl 1):P354 (doi: 10.1186/cc13544) Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) Candida in the respiratory tract secretions of critically ill patients and the impact of antifungal treatment: a randomized placebo- controlled pilot trial (CANTREAT study) M Albert1, DR Willamson1, J Muscedere2, F Lauzier3, C Rostein4, S Kanji5, X Jiang2, M Hall6, DK Heyland2 1Hopital du Sacré-Coeur de Montréal, Canada; 2Kingston General Hospital, Kingston, Canada; 3Centre hospitalier affi lié universitaire de Québec, Canada; 4Toronto General Hospital, Toronto, Canada; 5The Ottawa Hospital Research Institute, Ottawa, Canada; 6Nationwide Children’s Hospital, Colombus, OH, USA Critical Care 2014, 18(Suppl 1):P352 (doi: 10.1186/cc13542) M Albert , DR Willamson , J Muscedere , F Lauzier , C Rostein , S Kanji , X Jiang2, M Hall6, DK Heyland2 1Hopital du Sacré-Coeur de Montréal, Canada; 2Kingston General Hospital, Kingston, Canada; 3Centre hospitalier affi lié universitaire de Québec, Canada; 4Toronto General Hospital, Toronto, Canada; 5The Ottawa Hospital Research Institute, Ottawa, Canada; 6Nationwide Children’s Hospital, Colombus, OH, USA Critical Care 2014, 18(Suppl 1):P352 (doi: 10.1186/cc13542) Introduction Candida spp. are frequently recovered from endotracheal secretions in critically ill patients suspected of having ventilator- associated pneumonia. Observational studies reported an association with worse clinical outcomes [1] but the eff ect of antifungal therapy in these patients remains unclear. We designed this pilot study to assess the feasibility of a larger trial and to evaluate infl ammatory profi les and clinical outcomes in these patients. Introduction Candida spp. are frequently recovered from endotracheal secretions in critically ill patients suspected of having ventilator- associated pneumonia. Observational studies reported an association with worse clinical outcomes [1] but the eff ect of antifungal therapy in these patients remains unclear. We designed this pilot study to assess the feasibility of a larger trial and to evaluate infl ammatory profi les and clinical outcomes in these patients. Conclusion Respiratory samples post OOHCA frequently grow potentially pathogenic bacteria but current antibiotic guidelines fail to provide adequate cover in this population. Pharmacokinetics of antituberculosis drugs in critically ill patients with tuberculosis and acute respiratory failure CH H d C d GL L G i Results We recruited 60 patients into the randomized trial; 29 patients into the observational study. Recruitment was halted before the end of the study because of diffi culty in recruiting patients. Markers of infl ammation and all clinical outcomes were comparable between placebo and antifungal treatment groups at baseline and overtime. At baseline, TNFα levels were higher in the VAP with Candida compared with the observational group (mean ± SD) (21.8 ± 23.1 vs. 12.4 ± 9.3 pg/ ml, P = 0.02) and these patients had lower response to the LPS stimulation test (854.8 ± 855.2 vs. 1,559.4 ± 1,290.6 pg/ml, P = 0.01).fi Instituo Nacional de Enfermedades Respiratorias, Mexico City, DF, Mexico Critical Care 2014, 18(Suppl 1):P354 (doi: 10.1186/cc13544) Introduction The purpose of this study was to describe the pharmacokinetics of antituberculosis drugs, isoniazid (INH), rifampicin (RIF) and pyrazinamide (PZA), in eight critically ill patients with tuberculosis and acute respiratory failure. Methods We analyzed plasma concentrations of RIF, INH and PZA. Blood samples were obtained at steady state. Plasma concentrations were determined with HPLC. A population pharmacokinetic approach using a nonlinear mixed-eff ect model was implemented [1]. Interindividual variability in PK parameters was ascribed to an exponential model according to the equation: θj = θp × exp(nj), where θj is the estimate for a pharmacokinetic parameter in the jth patient, θp is the typical population PK parameter value (ka, CL/F, V/F), and n is a random variable from a normal distribution with zero mean and variance ω2. Residual variability was estimated using additive and additive-proportional error models; Cij = Cj + εadd and Cij = Cj(1 + εp) + εadd, where Cij and Cj are observed–predicted concentrations for the g Conclusion Given the diffi culty recruiting patients and the lack of signal in clinical or infl ammatory outcomes, this study does not provide evidence to support a larger trial examining the effi cacy of empiric antifungal treatment in patients with a clinical suspicion of ventilator- associated pneumonia and Candida in the endotracheal secretions. The presence of Candida in the lung may be associated with persistent infl ammation and immunosuppression. Comparison of multidrug-resistant Acinetobacter and non-Acinetobacter infections in terms of outcome in critically ill patients Outcome evaluated MDR AB MDR non-AB P value F value Mean length of stay 8.488889 6.101695 0.029299 4.886684 after index day Mean ICU stay after 5.311111 0.864407 1.73×10–11 57.30083 index day P353 Retrospective analysis of respiratory isolates post out-of-hospital cardiac arrest to establish choices in empirical antibiotic cover JL Wong, H Harb, K Bamford, A Ramesh, P Patel Imperial College Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P353 (doi: 10.1186/cc13543) Comparison of multidrug-resistant Acinetobacter and non-Acinetobacter infections in terms of outcome in critically ill patients Comparison of multidrug-resistant Acinetobacter and non-Acinetobacter infections in terms of outcome in critically ill patients p g In contrast, recent data obtained after the implementation of the program of the infection control showed that the respiratory infections occurred in 29.3% of the patients, catheter-related urinary tract infections were detected in 32.8%, and surgical site infections were detected in 8.6% of the polytrauma patients. On the other hand, the incidence of the multiple-drug-resistant bacterial colonies increased signifi cantly up to 14.2% (P <0.001). Other types of HAI changed nonsignifi cantly. p A Madaan1, V Singh1, P Shastri2, C Sharma3 1Patel Cancer and Superspeciality Hospital, Jalandhar, India; 2Sir Gangaram Hospital, New Delhi, India; 4Lovely Professional University, Jalandhar, India Critical Care 2014, 18(Suppl 1):P351 (doi: 10.1186/cc13541) Introduction Acinetobacter infections have increased and gained attention because of the organism’s prolonged environmental survival and propensity to develop antimicrobial drug resistance [1,2]. We performed a retrospective, matched cohort investigation at a tertiary care hospital in a tier II city hospital of India to examine morbidity and mortality outcome of patients with multidrug-resistant (MDR) Acinetobacter (AB) infection compared with patients with MDR non- Acinetobacter (non-AB) infections. gi y Conclusion In the era of the total antibacterial resistance, the education strategies and organization approaches (for example, infection control, antibiotic susceptibility, hand hygiene, and so forth) become more potent and eff ective than pharmacology innovations. Polytrauma patients, as one of the most severe categories suff ering from the HAI, demand a high level of compliance of the infection control approaches from the side of doctors as well as staff of and visitors to ICUs. Methods Patient records from the last 3 years from 2011 through 2013 were studied (n = 104) to examine outcomes of hospitalized patients in terms of number of organ dysfunctions, ICU length of stay, hospital length of stay and mortality in patients infected with MDR AB infection compared with the patients with MDR non-AB infection. 1. Nalapko Y, Peicheva O, Iegorov O, Nekrylov A: The dependence of infectious complications from the trauma severity. Intensive Care Med 2013, 39(Suppl. 2):P.0791. S127 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P351). P355 Eight-year study of the Staphylococcus epidermidis resistance profi le against glycopeptides, oxazolidinones and glycylcyclines in an ICU of a Greek tertiary hospital A Stylianakis, V Kaldis, D Argyris, D Chatzilia, V Kasouris, F Kalogeropoylos, S Kamariotis, K Daskalakis, S Stratoyli, I Alamanos 1General Hospital Kat-Eka, Kifi sia Athens, Greece Critical Care 2014, 18(Suppl 1):P355 (doi: 10.1186/cc13545) Methods The faecal vancomycin concentration was measured in eight patients who suff ered a Clostridium diffi cile infection and were treated with 4×500 mg vancomycin orally per day. In vitro, a (1:2) dilution series of vancomycin (range 6,250 to 0.4 μg/ml) was created and 1 ml vancomycin solution was then added to 1 ml standardized inoculum. One vancomycin-susceptible enterococcus isolate (VSE, MIC = 3 μg/ ml) and two VRE isolates (MIC = 16 μg/ml) were studied. After 1, 7 and 14 days incubation at 35°C, growth was defi ned as macroscopic visible turbidity. To test for surviving bacteria, all inocula were cultured to sheep blood agar plates, which were read after 24-hour incubation at 35°C. E-tests to measure MIC were performed on relevant samples. The in vitro experiment was performed twice. Introduction Staphylococcus epidermidis (SE) is the most often isolated species of coagulase-negative staphylococci, which are recognized as one of the main causes of ICU infections [1]. In this study we aimed to study the resistance profi le of SE clinical isolates against last-line antibiotics (vancomycin (VA), teicoplanin (TEC), linezolid (LZ) and daptomycin (DA)) for treating CNS infections during an 8-year period p p Results The faecal vancomycin concentration in patients treated orally with vancomycin was 8,000 μg/ml on average. VSE growth at day 14 was detected at up to 1.5 μg/ml vancomycin, whereas VRE growth was detected at up to 98 μg/ml. The MIC of these VRE species growing at 98 μg/ml vancomycin was increased (≥256 μg/ml). For both VSE and VRE, surviving bacteria were detected at very high concentrations of vancomycin (>98 μg/ml): the MIC of these survivors was not increased. Conclusion Oral treatment with vancomycin results in extremely high faecal concentrations. At these high concentrations, VRE bacteria are killed in vitro; however, a minority of the VRE is able to survive. Vancomycin thus seems unsuitable for eradication. However, high concentrations of vancomycin dramatically reduce the bacterial VRE load. l Reference 1. Delisle MS, Williamson DR, Perreault MM, Albert M, Jiang X, Heyland DK: The clinical signifi cance of candida colonization of respiratory tract secretions in critically ill patients. J Crit Care 2008, 23:11-17. S128 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P354). Mean population parameters of antituberculosis drugs Parameter RIF INH PZA ka 0.459 3.58 1.41 CL/F 6.69 2.74 1.56 V/F 105 42.3 33.4 ωka 0.645 38.7 0.98 ωCL/F 0.391 65.6 0.23 ωV/F 0.469 46.8 0.38 ε 0.441 0.235 0.089 Table 1 (abstract P354). Mean population parameters of antituberculosis drugs Table 1 (abstract P354). Mean population parameters of antituberculosis dr gs Reference 1. Zieburhr W, et al.: Nosocomial infections by Staphylococcus epidermidis. Int J Antimicrob Agents 2006, 28(Suppl 1):S14-S20. Table 1 (abstract P355). 2005 2006 2007 2008 2009 2010 2011 2012 (n = 77) (n = 76) (n = 79) (n = 89) (n = 92) (n = 93) (n = 46) (n = 59) VA 0 0 0 0 0 0 0 0 TEC 1.3 2.6 0 0 0 1.1 0 0 LZ 0 0 1.4 1.2 6.8 12.6 23.1 27.1 DA Not Not Not 0 0 0 0 0 tested tested tested Table 1 (abstract P355). jth patient at time i, respectively, and ε is the error, a random variable with a normal distribution with zero mean and variance σ2. Bayesian estimates were obtained and the pharmacokinetic parameters Cmax, Tmax and AUC0–24 hours were calculated. P356 Vancomycin-resistant enterococci: eradication using vancomycin? J Van Prehn1, AM Stemerding2, HK Van Saene3, PE Spronk2 1VU University Medical Center, Amsterdam, the Netherlands; 2Gelre Hospitals, Apeldoorn, the Netherlands; 3University of Liverpool, UK Critical Care 2014, 18(Suppl 1):P356 (doi: 10.1186/cc13546) P356 Vancomycin-resistant enterococci: eradication using vancomycin? J Van Prehn1, AM Stemerding2, HK Van Saene3, PE Spronk2 1VU University Medical Center, Amsterdam, the Netherlands; 2Gelre Hospitals, Apeldoorn, the Netherlands; 3University of Liverpool, UK Critical Care 2014, 18(Suppl 1):P356 (doi: 10.1186/cc13546) Results Cmax of PZA was above the recommended concentration (>20 mg/l). For RIF the Cmax was below the recommended level (>8 mg/l), and the Cmax of INH was below the recommended levels (>3 mg/l). See Table 1. g Conclusion Large interindividual pharmacokinetic variability and concentrations below the recommended levels for RIF and ISO. We need to monitor drugs and to re-evaluate the doses. Reference Introduction Patient-to-patient transmission enables vancomycin- resistant enterococci (VRE) outbreaks. l Reference Outbreak management is expensive and time consuming, and therefore the possibility of VRE eradication is desirable. Since vancomycin is scarcely absorbed in the gastrointestinal tract, treatment with vancomycin per os may result in very high gastrointestinal concentrations (many times the minimum inhibiting concentration (MIC)). The purpose of this study is to measure in vivo gastrointestinal concentrations of vancomycin in patients that are treated with a standard dose orally, and to investigate in vitro whether vancomycin is able to kill VRE at concentrations up to 2,000 times the MIC. Introduction Patient-to-patient transmission enables vancomycin- resistant enterococci (VRE) outbreaks. Outbreak management is expensive and time consuming, and therefore the possibility of VRE eradication is desirable. Since vancomycin is scarcely absorbed in the gastrointestinal tract, treatment with vancomycin per os may result in very high gastrointestinal concentrations (many times the minimum inhibiting concentration (MIC)). The purpose of this study is to measure in vivo gastrointestinal concentrations of vancomycin in patients that are treated with a standard dose orally, and to investigate in vitro whether vancomycin is able to kill VRE at concentrations up to 2,000 times the MIC. 1. Immanuel CP: Indian J Med Res 2003, 118:109-114. P355 Therefore oral treatment with vancomycin may help to terminate VRE outbreaks: a dramatic reduction in bacterial load of the colonised patient will minimise the risk of patient-to-patient transmission. daptomycin (DA)) for treating CNS infections, during an 8-year period. Methods From January 2005 until December 2012 we examined 518 nonduplicated SE isolates recovered from blood cultures of 421 patients hospitalized in a surgical ICU of our hospital. Species identifi cation and susceptibility testing were performed using the automated VITEK II system (Biomerieux). Additionally we used the E-test method (Biomerieux, ABI-Biodisk) in order to confi rm some isolation resistances against TEC and LZ found by the VITEK II system and to estimate the MIC levels of DA and VA. Mueller–Hinton agar adjusted to contain physiologic levels of free calcium ions (50 μg/ml) was used when testing DA susceptibility. Isolates with MIC >4 mg/l were considered resistant to TEC and LZ and those with MIC <1 mg/l and MIC <4 mg/l susceptible to DA and VA, respectively. Results The percentage resistance rate of the examined SE isolates is shown in Table 1. Methicillin resistance was observed with an overall prevalence of approximately 84.6%. All of the resistant isolates to TEC and LZ were also resistant to methicillin. The MIC values of VA were lower than 2 mg/l (Table 1). P357 Demographics and incidence of bacteraemia a strategy to treat bacteraemia, unless specifi cally indicated otherwise (for example, endocarditis, osteomyelitis). We aimed to determine the impact of this strategy on antibiotic resistance patterns and patient outcomes compared with a similar exercise we conducted in 2000 [1]. Methods We conducted a retrospective study of all patients with bacteraemia or fungaemia (community-acquired, hospital-acquired, and ICU-acquired) treated in our university hospital ICU over a 6-month period (December 2012 to May 2013). We compared this against data from blood culture-positive patients admitted between February and July 2000. Information was collected on bacteraemia episodes, causative pathogens, antimicrobial resistance patterns, antibiotic use and duration, and patient outcomes. Notably, our ICU admits many immunosuppressed patients (for example, haemoncology). a strategy to treat bacteraemia, unless specifi cally indicated otherwise (for example, endocarditis, osteomyelitis). We aimed to determine the impact of this strategy on antibiotic resistance patterns and patient t d ith i il i d t d i 2000 [1] outcomes compared with a similar exercise we conducted in 2000 [1]. Methods We conducted a retrospective study of all patients with bacteraemia or fungaemia (community-acquired, hospital-acquired, and ICU-acquired) treated in our university hospital ICU over a 6-month period (December 2012 to May 2013). We compared this against data from blood culture-positive patients admitted between February and July 2000. Information was collected on bacteraemia episodes, causative pathogens, antimicrobial resistance patterns, antibiotic use and duration, and patient outcomes. Notably, our ICU admits many immunosuppressed patients (for example, haemoncology). Results Table 1 presents demographics and incidence of bacteraemia. Antimicrobial resistance remained low in the 2013 cohort with few multi-resistant Gram-negative organisms, few fungaemia episodes and a marked decrease in methicillin-resistant Staphylococcus aureus (MRSA) (Figure 1). The number of relapses and breakthrough bacteraemias remained low. Conclusion A strategy of short-course antibiotic monotherapy is associated with low breakthrough and relapse rates and a low rate of antibiotic resistance. P357 35 Audit of bacteraemia management in a university hospital ICU V De Santis, MG Gresoiu, A Peter, R Wilson, M Singer University College London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P357 (doi: 10.1186/cc13547) Audit of bacteraemia management in a university hospital ICU V De Santis, MG Gresoiu, A Peter, R Wilson, M Singer University College London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P357 (doi: 10.1186/cc13547) Conclusion The examined SE isolates present a scattered resistance to TEC and they show a remarkable continuing increase of resistance to LZ. These fi ndings enforced the necessity to take the appropriate measures in the ICU environment and during the clinical practice to limit the dissemination and the amplifi cation of these resistances. DA and VA possess an excellent in vitro activity against SE isolates and they could be very good alternative solutions for treating ICU infections caused by this species. Introduction The optimal duration of antibiotic treatment in critically ill patients remains a subject of debate. In our multidisciplinary ICU, a short course of antibiotic monotherapy (5 to 7 days) is generally used as S129 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P357). Microorganisms isolated in each bacteraemia group. Left panel, 2000; right panel, 2013. Top panel, community-acquired bacteraemia; Middle panel, hospital-acquired bacteraemia; lower panel, ICU-acquired bacteraemia. Shaded areas represent numbers of methicillin-resistant strains for S. aureus and coagulase-negative staphylococci; vancomycin-resistant strains for Enterococcus spp.; multidrug-resistant strains for Gram-negative pathogens; and fl uconazole-resistant strains for Candida spp. Figure 1 (abstract P357). Microorganisms isolated in each bacteraemia group. Left panel, 2000; right panel, 2013. Top panel, community-acquired bacteraemia; Middle panel, hospital-acquired bacteraemia; lower panel, ICU-acquired bacteraemia. Shaded areas represent numbers of methicillin-resistant strains for S. aureus and coagulase-negative staphylococci; vancomycin-resistant strains for Enterococcus spp.; multidrug-resistant strains for Gram-negative pathogens; and fl uconazole-resistant strains for Candida spp. Table 1 (abstract P357). Demographics and incidence of bacteraemia Variable 2000 cohort 2013 cohort Total ICU population 713 1,318 Patients with bacteraemia 91 87 Episodes of bacteraemia 102 113 Community-acquired bacteraemia 13 37 Hospital-acquired bacteraemia 28 39 ICU-acquired bacteraemia 60 37 Hospital mortality 45% 32% Monotherapy treatment (%); duration median (IQR) Community-acquired bacteraemia 57%; 6 (5 to 6) 65% 5 (3 to 5) Hospital-acquired bacteraemia 78%; 6 (5 to 8) 63%; 5 (4 to 7) ICU-acquired bacteraemia 80%; 5 (5 to 7) 62%; 4 (3 to 6) Table 1 (abstract P357). P359 Safety and effi cacy of amphotericin B inhalation for Candida spp. in the respiratory tract of critically ill patients P359 Safety and effi cacy of amphotericin B inhalation for Candida spp. in the respiratory tract of critically ill patients P Van der Geest, E Dieters, B Rijnders, J Groeneveld Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P359 (doi: 10.1186/cc13549) Introduction Nosocomial pneumonia (NP) is frequently caused by multiresistant Gram-negative bacteria. The inhaled antibiotics provide us with a new treatment modality for NP in sepsis. The aim of this study was to estimate the effi cacy of inhaled tobramycin (IT) as an adjunct to systemic antibiotics in the treatment of NP in sepsis. P Van der Geest, E Dieters, B Rijnders, J Groeneveld , , j , Erasmus Medical Center, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P359 (doi: 10.1186/cc13549) y p Methods Fifty ICU ventilated septic patients with NP were enrolled in the study (all male, 55.3 ± 7.3 years old; primary reason for ICU stay – intraabdominal infections (78%), mediastinitis (13%), other (9%)). Diagnosis of NP was made according to the standard clinical and CPIS criteria. Associations of multiresistant Gram-negative bacteria were detected in bronchoalveolar lavage (BAL) of all patients. Seventy- two percent of bacteria were sensitive to tobramycin. Patients were randomized into two groups: IT (group 1, n = 25), addition of IT to systemic antibiotics (carbapenems, aminoglycosides, protected penicillins); and no IT (group 2, n = 25), shift of systemic antibiotics according to sensitivity. Groups were comparable in APACHE II and CPIS scores. IT (Bramitob) was administered 300 mg twice daily via nebulizer. The data were statistically analyzed by STATISTICA 7.0 (M, σ, Newman– Keuls test; P <0.05). Introduction Candida spp. are increasingly isolated in the critically ill, but the clinical signifi cance hereof is hard to establish [1]. Candida spp. colonization has been suggested as a risk factor for ventilator-associated pneumonia (VAP) [2]. The effi cacy and safety of inhalational amphotericin B (AB) is unknown [3]. The hypothesis was that inhalational AB deoxycholate is a safe and eff ective treatment for Candida spp. colonization of the respiratory tract and thereby prevents VAP and prolonged need for mechanical ventilation. p g Methods All patients admitted to the ICU from December 2010 to 2011 with positive Candida spp. cultures of the respiratory tract and requiring mechanical ventilation >48 hours were included. AB treatment was decided by attending intensivists. The colonization index was calculated to determine the eff ect of AB. Reference S130 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P359). Duration of mechanical ventilation grouped by amphotericin B. P358 P360 Inhaled tobramycin for the treatment of nosocomial pneumonia in sepsis References 1. Dellinger RP, et al.: Crit Care Med 2008, 36:296-327. 2. Levy MM, et al.: Intensive Care Med 2010, 36:222-231. 3. Kumar A, et al.: Crit Care Med 2006, 34:1589-1596. 4. Kumar A, et al.: Chest 2009, 136:1237-1248. 1. Dellinger RP, et al.: Crit Care Med 2008, 36:296-327. 2. Levy MM, et al.: Intensive Care Med 2010, 36:222-231. 3. Kumar A, et al.: Crit Care Med 2006, 34:1589-1596. 4. Kumar A, et al.: Chest 2009, 136:1237-1248. p A Kuzovlev, V Moroz, A Goloubev V.A. Negovsky Scientifi c Research Insitute of General Reanimatology RAMS, Moscow, Russia Critical Care 2014, 18(Suppl 1):P360 (doi: 10.1186/cc13550) Critical Care 2014, 18(Suppl 1):P360 (doi: 10.1186/cc13550 P358 Sepsis: impact of timely and appropriate empirical antibiotic therapy on mortality Introduction The administration of timely and appropriate antibiotic therapy is a well-known prognostic factor among severe sepsis patients [1-4]. The purpose of this study is to describe the magnitude of the impact of early and appropriate empirical antibiotic therapy on hospital mortality. y Methods A retrospective cohort study including all adult patients with sepsis admitted to the emergency room of a tertiary care, university hospital between 1 July 2011 and 30 June 2012. p y Results A total of 1,219 patients were admitted to the emergency room during the study period, of which 162 (13%) had severe sepsis. Forty (25%) patients had withheld therapeutic decisions and were excluded from the current analysis; 20 additional patients transferred from other acute healthcare facilities were excluded due to missing or inaccurate data, leaving 102 patients to be included with a hospital mortality rate of 45%. The median time to antibiotics administration was 36 minutes (IQR 0 to 174), 59 (58%) patients had antibiotic administered within the fi rst hour after sepsis recognition; 60 (59%) had positive microbiology, 74% with appropriate empiric antibiotic therapy. An association was found for hospital mortality with: heart failure (OR = 4.297; P = 0.037), decreased functional status (Karnofsky performance status <70%) (OR = 2.368; P = 0.034) and SOFA score (OR per point = 1.415; P <0.001). Two multivariate models with hospital mortality as the dependent variable were built using alternative severity scores: one with SAPS II that was retained in the fi nal model (adjusted OR = 1.068, 95% CI = 1.020 to 1.119) along with heart failure (adjusted OR = 5.859, 95% CI = 0.996 to 34.474); and another with SOFA score that was also retained in the fi nal model (adjusted OR = 1.659, 95% CI = 1.227 to 2.242) along with heart failure (adjusted OR = 12.636, 95% CI = 1.423 to 112.229). Figure 1 (abstract P359). Duration of mechanical ventilation grouped by amphotericin B. associated with a higher CPIS and LIS, even in those with similar degree and duration of colonization and thus probably Candida spp. load at baseline (P <0.001) (Figure 1). There was no diff erence in occurrence of VAP or mortality.f y Conclusion AB deoxycholate treatment is eff ective for the treatment of Candida spp. colonization of the respiratory tract in critically ill patients. However, patients are mechanically ventilated for longer. References 1. P358 Sepsis: impact of timely and appropriate empirical antibiotic therapy on mortality El-Ebiary et al.: Am J Respir Crit Care Med 1997, 156:583-590. 2. Azoulay E, et al.: Chest 2006, 129:110-117. 3. Knechtel SA, et al.: Expert Opin Drug Saf 2007, 6:523-532. Conclusion Contrarily to what has been described previously, early and appropriate empirical antibiotic therapy was not associated with better prognosis. The most probable explanation is the higher compliance found with the current recommendations, reinforcing the need for period audits and feedback to the team. 3. Knechtel SA, et al.: Expert Opin Drug Saf 2007, 6:523-532. 3. Knechtel SA, et al.: Expert Opin Drug Saf 2007, 6:523-532. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 of resources and false results is well established and unnecessary cultures can cause patient harm. was reliable in 80% of the patients of group 1 (P <0.02). It is noteworthy that 21% of group 1 patients were in vitro resistant to tobramycin, but it was clinically eff ective, probably due to a local superconcentration. Treatment with IT was associated with an increase of sensitivity of microbes to antibiotics they were prior resistant to (32% of patients). This is probably due to IT eff ects on biolayers. De-escalation of antibiotic therapy was possible in group 1 by day 5 in 42% of patients. The treatment with IT made it possible to wean 40% of patients by day 5.3 ± 1.8 after treatment cessation (vs. 35% and 11.2 ± 1.3 days in group 2, P = 0.02). Hearing loss and tinnitus was detected only in three patients of group 1. There were no cases of bronchospasm. The mortality was 12% (n = 3) in group 1 and 16% (n = 4) in group 2 (P >0.05), and was not related to a progression of NP [1]. Methods All ICU admissions in 2011 were listed retrospectively. Notes were reviewed for those with blood cultures taken during, or within 24 hours prior to or following, ICU admission. Data collected for positive blood cultures included organism, antimicrobial sensitivity, culture timing with respect to ICU admission and antibiotic therapy before and after positive blood culture. Qualitative decisions were made regarding clinical utility of each positive blood culture. Results were deemed useful if management changed – starting, changing or stopping antibiotics or altering antibiotic duration. Confi rmatory results or those not altering treatment were not deemed useful. Statistical analysis was performed using the chi-squared test. p g q Results During 2011 and 2012, there were 450 ICU admissions. In total, 698 blood cultures were taken during, or within 24 hours prior to or following, ICU admission. A total of 135 blood cultures were taken in the 24 hours prior to ICU admission. Of these, 26 grew signifi cant organisms (19.3%). Nine of these cultures were deemed clinically useful (6.7%). A total of 542 blood cultures were taken during ICU admission. Thirty-three of these yielded signifi cant results (6.1%) but only nine (1.7%) were deemed clinically useful. Sternal wound infections in cardiac surgery: eff ects of vancomycin prophylaxis F Ampatzidou, M Sileli, C Koutsogiannidis, A Vlachou, A Baddour, G Drossos F Ampatzidou, M Sileli, C Koutsogiannidis, A Vlachou, A Baddour, G Drossos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P361 (doi: 10.1186/cc13551) Introduction Appropriate antibiotic prophylaxis plays a crucial role in preventing sternal wound infection after cardiac surgery [1]. In institutions with high prevalence of methicillin-resistant Staphylococci species, vancomycin prophylaxis is recommended either as a monotherapy or as an adjuvant agent [2]. In our study, we assessed sternal wound infection rates before and after the introduction of a vancomycin prophylaxis protocol. y y Methods Twenty-six of a total 227 consecutive cardiac surgical patients, between July and December 2012, developed sternal wound infection (Group A). All of the patients received a standard empirical antibiotic prophylaxis. From January to July 2013, 308 patients underwent cardiac surgery (Group B). In this group, we applied a more restricted antibiotic protocol, considering the resistance patterns and the results of microbiological tests of group A. We also evaluated the results of MIC susceptibility testing of fi ve antibiotics: oxacillin, linezolide, daptomycin, teicoplanin and vancomycin. In the new protocol the fi rst vancomycin dose was given 1 hour before sternal incision followed by three additional doses (48 hours duration). 1. Coburn B, et al.: Does this adult patient with suspected bacteraemia require blood cultures? JAMA 2012, 308:502-510. P363 P363 Employing quality improvement methodology in sepsis: an electronic sepsis order set further improves compliance with the Surviving Sepsis Campaign 3-hour bundle S Rossi, A Shields, N Gauge, M Kinirons, A Hopper, G Glover, R Beale Guys and St Thomas NHS Foundation Trust, London, UK Critical Care 2014, 18(Suppl 1):P363 (doi: 10.1186/cc13553) Results In group A, 26 patients (11.45%) developed sternal wound infection. Twenty out of 26 patients had staphylococcal infections characterized by high prevalence of oxacillin resistance while six patients had Gram-negative infections. In the vancomycin prophylaxis group (group B) we observed a signifi cant reduction in the incidence of sternal wound infection rate (P <0.01). Among 308 patients, six patients (1.94%) developed sternal wound infections, two of them caused by coagulase-negative oxacillin-resistant staphylococci while in four patients Gram-negative microorganisms were cultured. Mortality in infected patients was 0% in both groups. Introduction The Surviving Sepsis Campaign (SSC) has developed guidelines to promote evidence-based management for patients with severe sepsis [1]. Improvements in bundle compliance have been demonstrated over time, but compliance remains below 40%. Sepsis has been studied in acute and critical care environments, but little research has focused on the management in level 1 wards. An initial audit of patients with sepsis who were referred to the GSTT critical care outreach team revealed very low overall compliance to the SSC 3-hour bundle. A novel quality improvement campaign was instituted with the aim of improving bundle compliance. g Conclusion Appropriate antibiotic prophylaxis in cardiac surgery patients is of paramount importance in preventing sternal wound infections, resulting in dramatic reduction of postoperative morbidity and in signifi cant economic benefi ts. i References References 1. Kappeler R, et al.: Antimicrob Chemother 2012, 67:521-522. 2. Garey KW, et al.: Antimicrob Agents Chemother 2008, 52:446-451. References 1. Kappeler R, et al.: Antimicrob Chemother 2012, 67:521-522. 2. Garey KW, et al.: Antimicrob Agents Chemother 2008, 52:446-451. References 1. Kappeler R, et al.: Antimicrob Chemother 2012, 67:521-522. 2. Garey KW, et al.: Antimicrob Agents Chemother 2008, 52:446-451. Methods A retrospective cohort study in a university hospital was performed. Patients on level 1 wards with severe sepsis registered in the adult critical care response team (CCRT) database in November 2012 were identifi ed (Cohort A). Physiological observation, track and trigger scores, compliance with the 3-hour bundle elements (measured lactate, blood cultures before antibiotics, fl uid challenge, early antibiotics), antimicrobial stewardship and 28-day mortality were recorded. Following this, a quality improvement project was initiated: central to this was an electronic ‘SEPSIS’ order set, containing appropriate investigations and a step-by-step management guide for use on level 1 wards. A ‘viral’ print and social media campaign were also undertaken. Compliance to the SSC early care bundle was re-examined Reference 1. Moroz VV, et al.: Gen Reanimatol 2012, VIII:5-10. P359 Safety and effi cacy of amphotericin B inhalation for Candida spp. in the respiratory tract of critically ill patients The clinical pulmonary infection score (CPIS) and the lung injury score (LIS) were calculated to determine pulmonary eff ects of AB. Results Administration of IT as an adjunct to systemic antibiotics was associated with a decrease of systemic infl ammation and acute respiratory insuffi ciency signs 2.3 ± 1.2 days after the treatment onset (vs. 6.3 ± 1.5 days in group 2, P = 0.03). The decrease of microbial titer to 103 to 104 CFU/ml was detected in both groups by days 5 to 7, but it yf Results Fifty-fi ve of 181 patients had been treated with AB. The AB decreased indicators of Candida spp. load but increased the duration of mechanical ventilation as compared with nontreated patients, S131 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 A total of 102 cultures were taken in the fi rst 24 hours of the ICU, of which 17 were positive (16.7%) and six were useful (5.9%). Fifty-three were taken over the next 24 hours, of which two were positive and two were useful (3.8%). Forty-four cultures were taken over the following 24 hours, of which two were positive (4.5%) and one was useful (2.3%). A total of 343 cultures were taken subsequent to this in the ICU, of which 12 were positive (3.5%) and three were deemed useful (0.9%). Of 22 blood cultures taken in the 24 hours post ICU, one was positive but deemed useful (4.5% yield). Conclusion These results demonstrate overall low clinical utility of blood cultures but specifi cally that utility of ICU cultures is signifi cantly lower than pre ICU (1.7% vs. 6.7%; P = 0.0001). The yield of positive blood cultures and their clinical utility also decrease during ICU stay. This may refl ect appropriate empirical antibiotics and lower bacteraemia burden in later illness. Given the low clinical yield and a lack of established sensitive or specifi c triggers [1], we suggest further work in an ICU setting to maximise utility whilst minimising harm. Reference Conclusion Administration of IT as an adjunct to systemic antibiotics is effi cient in treatment of NP caused by multiresistant Gram-negative bacteria in sepsis. Reference Early detection of postoperative acute kidney injury by Doppler renal resistive index in major lung and cardiac operations M Arslantas, I Cinel, A Kararmaz Marmara University Pendik Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P365 (doi: 10.1186/cc13555) Early detection of postoperative acute kidney injury by Doppler renal resistive index in major lung and cardiac operations M Arslantas, I Cinel, A Kararmaz Marmara University Pendik Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P365 (doi: 10.1186/cc13555) Results The mean age of all patients studied (n = 79) was 66.5 years. Fifty-three per cent of the patients were male. Thirty-one per cent were in septic shock at the time of sepsis identifi cation. Overall SSC bundle compliance was 6.60% (Cohort A), 24% (Cohort B) and 45.5% (Cohort C). Improvements in other bundle parameters were also seen, including blood culture (54%, 72%, 91%), antibiotic administration (50%, 69%,76%) and fl uid administration in septic shock (50%, 42%, 75%) in Cohort A, Cohort B and Cohort C respectively. Introduction Lung and cardiac operations cause signifi cant changes in the fl uid balance, and thus have a high incidence of development of postoperative acute kidney injury (AKI). The renal resistive index (RRI) calculated by the pattern of the renal artery fl ow is an indicator of renal artery fl ow. In this research work, we aimed to evaluate the effi ciency of the RRI on the early prediction of postoperative kidney injury in major lung and cardiac operations. p y Conclusion Baseline compliance with the SSC 3-hour bundle on level 1 wards was very low. An electronic sepsis order set was associated with marked improvement. Novel quality improvement methodology may be important to achieve optimal compliance with evidence-based guidelines and an electronic sepsis order set is recommended. Reference g p Methods Twenty-two patients who have undergone lung or cardiac surgery were included in the study. After the kidneys were localized by ultrasonography, the best regions of blood fl ow were detected using color Doppler and then the arterial waveforms of these regions were obtained and optimized by Doppler. The measurements taken from three diff erent regions were averaged. The RRI was calculated at the preoperative and postoperative fi rst and 24th hours respectively.i 1. Dellinger RP, et al.: Crit Care Med 2013, 41:580-637. Results A signifi cant correlation was established between RRI and postoperative creatinine levels (P <0.01). RRI values reached their highest point at the postoperative fi rst day whereas the creatinine levels reached their highest level at the postoperative third day. P365 in two cohorts of patients in July 2013; patients that were referred to the CCRT as before (Cohort B) and also patients who had the electronic order set activated (Cohort C). Early detection of postoperative acute kidney injury by Doppler renal resistive index in major lung and cardiac operations M Arslantas, I Cinel, A Kararmaz Marmara University Pendik Education and Research Hospital, Istanbul, Turkey Critical Care 2014, 18(Suppl 1):P365 (doi: 10.1186/cc13555) Although there was no correlation between postoperative creatinine level and duration of staying in hospital, a signifi cant relationship was detected between duration of staying in ICU and the creatinine levels (P <0.01). When the cases were divided into two groups as RRI is less (n = 13) and larger (n = 9) than 0.7, signifi cant diff erences were present with regard to age and creatinine levels. 1. Dellinger RP, et al.: Crit Care Med 2013, 41:580-637. References Results Our literature search yielded 316 potential papers, of which 57 were included (20 papers on AHF, 15 ACS and 22 CS). A risk of bias analysis showed a low risk for selection bias in 55% of the studies and prospective data collection in 45%. AKIRIFLE was used in 33 studies (RIFLE in 22, AKIN in 14, KDIGO in four), AKIWRF, with six variants, in 24 studies and use of RRT (AKIRRT) in 20 studies. The incidence of AKI in CRS-1 patients defi ned by AKIRIFLE and AKIWRF was similar (22.5%, respectively 22.4%, P = 0.401), and greater than AKIRRT (2.6%, both P <0.001). AKIRIFLE occurred more frequently in AHF patients compared with ACS and CS patients (55.0% vs. 14.9% vs. 19.3%; P = 0.009 respectively P = 0.001, P = NS for ACS vs. CS). This was similar when defi ned by AKIWRF. AKIRRT was evenly distributed among CRS- 1 subtypes (AHF 4.3%, ACS, 1.7%, and CS 3.1%, P = 0.611). Despite predominant low severity of AKIRIFLE (stage 1: 16.9%, stage 2: 3.7%, and stage 3: 3.6%), AKIRIFLE was associated with increased mortality (RR = 5.4), LOSICU (MD 1.7 days), and LOShosp (MD 4.4 days), and increasing AKIRIFLE severity was associated with increase in these three outcomes in all CRS-1 patients as well as in the three subgroups. The impact of AKIRIFLE on mortality was greatest in CS patients (AHF RR = 2.8, ACS RR = 3.5, and CS RR = 9.1). Not surprisingly, AKIWRF had similar impact on outcomes, but AKIRRT had greater impact compared with AKIRIFLE (mortality RR = 9.16, LOSICU MD = 10.6 days, and LOShosp, MD = 20.2 days). Retrospective analysis of the clinical utility of blood cultures taken surrounding intensive care admission V Humphrey, M Clark V Humphrey, M Clark p y Victoria Hospital, Kirkcaldy, UK Victoria Hospital, Kirkcaldy, UK p y Critical Care 2014, 18(Suppl 1):P362 (doi: 10.1186/cc13552) Introduction This study aimed to establish clinical utility of blood cultures taken surrounding ICU admission. Blood culture cost in terms S132 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P364 P364 Acute kidney injury in cardiorenal syndrome type 1: a meta-analysis W Vandenberghe, S Gevaert, H Peperstraete, I Herck, J Decruyenaere, E Hoste University Hospital Ghent, Belgium Critical Care 2014, 18(Suppl 1):P364 (doi: 10.1186/cc13554) Introduction Cardiorenal syndrome type 1 (CRS-1) refl ects an abrupt worsening in cardiac function leading to acute kidney injury (AKI). Acute cardiac conditions contributing to CRS-1 include acute heart failure (AHF), acute coronary syndrome (ACS) and cardiac surgery (CS). The objective of this study was to evaluate the epidemiology of AKI in CRS-1. Conclusion The RRI, which is used to evaluate renal arterial fl ow, is directly related with increasing renal vascular resistance in the case of AKI. The usefulness of RRI for prediction of AKI was shown both clinically following a renal allograft and experimentally in the acute tubular necrosis modeling [1,2]. Also in septic patients this was asserted, as RRI is a better marker than cystatin C, which is one of the popular markers of recent times for prediction of development of AKI [3]. Our results show that RRI could be a simple, non-invasive and useful technique for early diagnosis of AKI in patients undergoing major operations such as lung and cardiac surgeries. Methods This is a systematic review and meta-analysis. AKI defi ned by the RIFLE defi nition and its modifi cations AKIN and KDIGO is grouped as AKIRIFLE. Similarly, AKI defi ned by variations of worsening renal failure is grouped as AKIWRF. Incidence of AKI is reported by the diff erent defi nitions of AKI. In addition, we report on mortality and length of intensive care and hospital stay (LOSICU and LOShosp) for AKIRIFLE. Data are reported as percentage, risk ratio (RR), and mean diff erence (MD). References References 1. Tranquart F, et al.: Transpl Int 1993, 6:14-17. 2. Yoon DY, et al.: Invest Radiol 1995, 30:168-172. 3. Schnell D, et al.: Shock 2012, 38:592-597. P367 P367 Acute kidney injury and cardiac surgery: impact of fl uid balance on AKI classifi cation and prognosis EM Moore1, A Tobin2, D Reid2, J Santamaria2, R Bellomo1 1Monash University, Melbourne, Australia; 2St Vincent’s Hospital, Melbourne, Australia Critical Care 2014, 18(Suppl 1):P367 (doi: 10.1186/cc13557) Figure 1 (abstract P368). Cox-hazard prediction of AKI class on hospitalization (ICU LOS fi xed at median). Critical Care 2014, 18(Suppl 1):P367 (doi: 10.1186/cc13557) Introduction We assessed the eff ect of fl uid balance (FB) on acute kidney injury (AKI) classifi cation/prognosis in cardiac surgical patients by comparing patients classifi ed with AKI, before and after adjusting the creatinine (used to classify AKI) for FB. Fluid accumulation is associated with negative outcomes including development of AKI in critically ill patients [1]. Cardiac surgical patients commonly receive large volumes of fl uid postoperatively and could be at risk for the harmful eff ects of fl uid accumulation. Furthermore, fl uid accumulation may infl uence serum creatinine concentration and mask AKI [2]. Methods We performed a retrospective analysis of prospectively collected data on all cardiac surgical patients admitted to St Vincent’s Hospital ICU, Melbourne, Australia from 1 July 2004 to 30 June 2012. AKI Network creatinine criteria were used to classify AKI in the usual method and then using FB-adjusted creatinine (FB at 18 hours and an assumption that total body water is 60% of weight involved). FB (total i.v. input minus (total urine output + chest drain losses)) was calculated for 18 hours post surgery as most patients were in the ICU for this period. p Results Patients classifi ed with AKI increased from 27.7% to 37.2% (n = 2,171) after adjusting creatinine for FB. Patients were categorised into four groups based on presence or absence of AKI before and after adjustment for FB: group A, no AKI before or after adjustment for FB; group B, no AKI before/AKI after; group C, AKI before/no AKI after; and group D, AKI before and after. Group B (n = 209) had an in-hospital mortality rate similar to patients in group D (n = 599) (3.4% vs. 4.3%, P = 0.53) and greater than those in group A (n = 1,333) (3.4% vs. 1.6%, P = 0.07). Group B also had an ICU mortality rate similar to patients in group D (2.9% vs. 2.7%, P = 0.88) and signifi cantly greater than those in group A (2.9% vs. P368 on day 3. Within the fi rst 5 days a total of 17 patients had AKI stage 2 or 3. The RI at admission was associated with APACHE II score, hypotension at admission, baseline serum lactate and history of type II diabetes treated with insulin. Patients who developed AKI stage 2 or 3 had signifi cantly higher RI on admission (0.79 vs. 0.67, P = 0.021). A cutoff value of 0.72 best diff erentiated patients with AKI 2 or 3. Raise of RI by ≥0.05 during the fi rst 24 hours was associated with the development of AKI stage 2 or 3 (P = 0.034). Patients with persistent AKI on day 3 had higher RI values on admission, and those who recovered lowered their RIs by >0.05. In multivariate analysis, admission RI and raise of RI by ≥0.05 were independently associated with development of AKI stage 2 or 3 and ICU mortality. Best sensitivity for AKI was achieved when both criteria were used: elevated RI at admission (>0.72) and raise of RI by ≥0.05 at 24 hours post admission. 1. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. 2. Liu KD, et al.: Crit Care Med 2011, 39:2665-2671. 1. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. Acute kidney injury of all severity is associated with extended hospitalization after critical illness Acute kidney injury of all severity is associated with extended hospitalization after critical illness Introduction Acute kidney injury (AKI) complicates over 50% of ICU admissions and is associated with signifi cantly increased mortality, length of stay, and costs across a broad spectrum of conditions [1]. Methods We performed a single-centre, retrospective analysis of AKI diagnosis in patients with ICU admissions of 5 days or more who survived to hospital discharge between 2009 and 2011. We examined the relationship between hospital length of stay, AKI diagnosis, demographics and clinical characteristics in a multivariable Cox-hazard analysis. p Conclusion The renal RI measured at admission and its dynamics after the fi rst 24 ICU hours are predictive of the development of AKI and ICU mortality. Further studies are needed to confi rm these results. y Results We identifi ed 700 cases, with a 66% incidence of AKI. The AKI was associated with older age, greater initial illness severity and longer ICU and hospital length of stay in univariate analysis (Table 1). In Cox-hazard analysis, only AKI category and ICU length of stay were signifi cantly associated with lower probability of discharge over time (Figure 1). AKI-1 was associated with a hazard ratio for hospital discharge of 0.66 (0.55 to 0.79), AKI-2 with 0.55 (0.42 to 0.71) and AKI-3 with 0.54 (0.44 to 0.66). P366 Renal resistive index at ICU admission and its change after 24 hours predict acute kidney injury in sepsis I Gornik, A Godan, V Gašparović University Hospital Centre Zagreb, Croatia Critical Care 2014, 18(Suppl 1):P366 (doi: 10.1186/cc13556) Introduction The renal resistive index (RI) measured by Doppler ultrasound refl ects the changes in renal microvascular resistance. It was recently shown that the RI measured at ICU admission was associated with development of AKI and with persistent AKI on day 3 after admission. The aim of this study was to investigate whether change of RI after the fi rst 24 ICU hours has an additional predictive value for the development of AKI. Methods This non-interventional study included adult patients with sepsis admitted to a medical ICU. Patients with renal transplant and those in terminal renal failure were excluded. The RI was measured within 2 hours from admission and 24 ± 2 hours after ICU admission. Occurrence of AKI within the fi rst 5 days was classifi ed according to the AKI Network criteria. There was no intervention guide by RI measurements, nor were the results known to the attending physicians. Results There were 52 patients included in the study. At admission, eight patients had AKI stage 2 or 3 and it was persistent in fi ve patients Conclusion Almost one-quarter of patients with an acute cardiac condition had AKI, and RRT was used in approximately 3%. AKI was associated with signifi cant worse outcomes. AHF patients experienced the highest incidence of AKI, but the impact on mortality was greatest in CS patients. S133 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966 973. 2. Liu KD, et al.: Crit Care Med 2011, 39:2665-2671. P367 0.7%, P = 0.003). The need for renal replacement therapy (RRT) in group B was also high as for patients in group D (7.7% vs. 12.4%, P = 0.06) and was signifi cantly greater than those in group A (7.7% vs. 1.6%, P <0.001). Thus, hospital and ICU mortality and use of RRT in patients classifi ed with AKI only after adjustment for FB were similar to patients with AKI before and after adjustment for FB and were notably higher than those of patients without AKI. Table 1 (abstract P368). Patient characteristics by AKI No AKI AKI P value Age 46 (32 to 60) 51 (37 to 64) <0.001 SAPS-2 35 (27 to 42) 41 (32 to 49) <0.001 ICU LOS 8 (6 to 12) 12 (7 to 18) <0.001 Hospital LOS 27 (17 to 42) 41 (28 to 72) <0.001 Data presented as median (IQR). Table 1 (abstract P368). Patient characteristics by AKI No AKI AKI P value Table 1 (abstract P368). Patient characteristics by AKI Conclusion AKI was a signifi cant predictor of remaining in hospital at all levels of AKI severity even after allowing for longer ICU stay. Even mild AKI is associated with extended recovery from critical illness and healthcare costs even after ICU discharge. Reference Conclusion Lack of adjustment for FB post cardiac surgery may mask the presence of AKI that is associated with increased risk for death and RRT, which could hinder optimal treatment. References Conclusion Lack of adjustment for FB post cardiac surgery may mask the presence of AKI that is associated with increased risk for death and RRT, which could hinder optimal treatment. Reference S134 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P369 using CG, MDRD and MCQE equations. The predictive capacity of these formulae was tested and compared in relation to AKI and RD incidence by constructing receiver operating characteristic curves for each of the models. Reference Reference 1. Macedo E, et al.: Crit Care 2010, 14:R82. Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery M Sileli, F Ampatzidou, S Tsagkaropoulos, K Diplaris, C Koutsogiannidis, G Drosos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P370 (doi: 10.1186/cc13560) Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery M Sileli, F Ampatzidou, S Tsagkaropoulos, K Diplaris, C Koutsogiannidis, G Drosos General Hospital ‘G. Papanikolaou’, Thessaloniki, Greece Critical Care 2014, 18(Suppl 1):P370 (doi: 10.1186/cc13560) Fluid accumulation increases the risk of AKI progression and death in critically ill patients with early AKI MR Raimundo1, S Crichton2, Y Syed2, J Martin2, M Ostermann2 1Hospital Beatriz Ângelo, Loures, Portugal; 2Guy’s & St Thomas Hospital, London, UK Critical Care 2014, 18(Suppl 1):P371 (doi: 10.1186/cc13561 Introduction Fluid therapy is a cornerstone in the management of patients with evolving acute kidney injury (AKI). Despite the wide availability of advanced hemodynamic monitoring to guide therapy, there are few data on how to manage hemodynamics optimally once early AKI has occurred. Our aim was to investigate the association between cumulative fl uid balance (FB)/fl uid administration and outcome (progression to AKI III and hospital mortality) in critically ill patients with early AKI, and its interaction with other hemodynamic parameters. y Conclusion The prevalence of early AKI in nonsepsis, noncardiac patients admitted to the general surgical ICU was 41.7%. Surgical patients with risk factors should be carefully cared for to prevent the development of AKI. Receiving 6% 130/0.4 HES >20 ml/kg/day and crystalloid >30 ml/kg/day increased the risk of early AKI in nonsepsis, noncardiac surgical patients. p Methods A retrospective analysis (2-year period) of all patients admitted to an adult ICU with AKI (defi ned by KDIGO criteria) who had hemodynamic monitoring within 12 hours of AKI I. We recorded FB, urinary output (UO), fl uid administered and hemodynamic parameters including mean arterial pressure (MAP) on the day of AKI I and in the following 72 hours. Logistic regression was employed to determine independent predictors of outcome. Reference P369 Early acute kidney injury in nonsepsis, noncardiac surgical patients admitted to a general surgical ICU KS Kongsayreepong, N Rojanapithayakorn Siriraj Hospital, Mahidol University, Bangkok, Thailand Critical Care 2014, 18(Suppl 1):P369 (doi: 10.1186/cc13559) Results The mean age of the cohort was 64.7 ± 0.45 years and the mean value of estimated kidney function from MCQE, MRDR and CG formulae was 83.7 ± 1.03 ml/minute/1.73 m2, 69.03 ± 0.82 ml/minute/1.73 m2 and 75.3 ± 1.18 ml/minute/1.73 m2 respectively. AKI was identifi ed in 75 (14.2%) patients, whereas early RD was necessary in 16 (3%) patients. All of the three variables showed a good predictive value for estimating AKI and RD after cardiac surgery. The area under the curve values for the early RD group was 0.887, 0.867 and 0.804, respectively and for the AKI group was 0.701, 0.651 and 0.691, respectively. Introduction Perioperative AKI is a signifi cant factor determining morbidity/mortality in surgical patients. We sought to determine the prevalence and risk factors of early AKI (within 72 hours of ICU admission) in nonsepsis, noncardiac surgical patients admitted to the general surgical ICU. Methods This prospective observational study was done in 600 nonsepsis, noncardiac surgical patients admitted to the 14-bed general surgical ICU of Siriraj Hospital. The following data were collected: patient demographic data, ASA, comorbidity, type and urgency of surgery, type of anesthesia, preoperative and the fi rst 72 hours laboratory data, amount of bleeding, type/amount of fl uid and blood replacement, average intraoperative and the fi rst 72 hours MAP, and severity score. Outcome as ventilator-hours, ICU length of stay and ICU mortality were also determined. Risk factors were identifi ed by multiple logistic regression. AKI was defi ned and classifi ed according to the AKIN criteria using adjusted serum creatinine (Cr) [1]. g p p y Conclusion On the basis of our fi ndings, all of the above algorithms seem to be accurate in predicting AKI and RD incidence in the early ICU postoperative period after elective cardiac surgery. Nevertheless, the MCQE equation more accurately classifi ed individuals compared with MDRD and CG formulae. Our results extend knowledge from previous studies [1,2]. Further investigations should be performed to determine whether these costless formulae could be used as an additional validated predictor in this group of patients and hence whether they could be incorporated into clinical practice. References Results In total, 41.7% of the study patients developed AKI (AKIN-I 31.0%, AKIN-II 10%, AKIN-III 4.8%) and 4.8% received RRT. The following factors were diff erent between AKI and non-AKI patients: baseline expected GFR <60 ml/minute/1.73 m2, baseline serum Cr and baseline serum albumin, major abdominal surgery, vascular surgery, combined regional and general anesthesia, APACHE II score, receiving 6% 130/0.4 hydroxyethyl starch (HES) >20 ml/kg/day, 4% gelatin >20 ml/kg/day, crystalloid >30 ml/kg/day and positive fl uid balance in the fi rst 48 hours. Multiple logistic regression showed that independent risk factors of AKI included: baseline eGFR <60 ml/minute/1.73 m2 (OR = 1.53; 95% CI, 1.08 to 1.27, P = 0.02), baseline serum albumin <2 mg/dl (OR = 1.75; 95% CI, 1.01 to 3.06, P = 0.049), admitting hemoglobin <8 g/dl (OR = 2.41; 95% CI, 1.14 to 5.11, P = 0.02), receiving 6% 130/0.4 HES >20 ml/kg/day (OR = 2.02; 95% CI, 1.09 to 3.76, P = 0.03), receiving crystalloid >30 ml/ kg/day (OR = 3.15; 95% CI, 1.53 to 6.48, P = 0.01), vascular surgery (OR = 1.67; 95% CI, 1.05 to 2.64, P = 0.03), and major abdominal surgery (OR = 1.68; 95% CI, 1.11 to 2.55, P = 0.01). AKI patients had higher ventilator- hours (P = 0.02) and ICU length of stay (P = 0.04). 1. Tomaszuk-Kazberuk A, et al.: Ren Fail 2011, 33:983-899. 2. Ekmekci A, et al.: Angiology 2013. [Epub ahead of print] 1. Tomaszuk-Kazberuk A, et al.: Ren Fail 2011, 33:983-899. 2. Ekmekci A, et al.: Angiology 2013. [Epub ahead of print] P371 Fluid accumulation increases the risk of AKI progression and death in critically ill patients with early AKI MR Raimundo1, S Crichton2, Y Syed2, J Martin2, M Ostermann2 1Hospital Beatriz Ângelo, Loures, Portugal; 2Guy’s & St Thomas Hospital, London, UK Critical Care 2014, 18(Suppl 1):P371 (doi: 10.1186/cc13561) Incidence and outcomes of contrast-induced nephropathy in adult ICU patients S Alderson1, S Pillai2, A Manoras1, J Papanikitas1, D Lewis1, S McKechnie1 1John Radcliff e Hospital, Oxford, UK; 2Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P374 (doi: 10.1186/cc13564) S Alderson1, S Pillai2, A Manoras1, J Papanikitas1, D Lewis1, S McKechnie1 1John Radcliff e Hospital, Oxford, UK; 2Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P374 (doi: 10.1186/cc13564) Introduction Critically ill patients cared for in ICUs often require radiological investigation using iodinated contrast agents. Contrast- induced nephropathy (CIN) – a form of acute kidney injury (AKI) – is a complication following the use of such contrast. Although CIN has been thoroughly studied in some populations (for example, those undergoing coronary angiography), it has not been investigated in large numbers of ICU patients [1]. p Conclusion Our results demonstrate that AKI occurs in 8.8% of the patients following surgery for gynecologic malignancies. Patients who had AKI were older, had higher body mass index with higher preoperative CRP levels, more frequent distant organ metastasis and longer operation times and higher amounts of blood transfused intraoperatively. Methods We conducted a single-centre retrospective review of the electronic patient records of general adult ICU patients who received iodinated contrast over a 3-year period (2009 to 2011). Our review identifi ed evidence of CIN or AKI post scan (assessed using CIN and KDIGO criteria); and clinical outcomes post scan (renal replacement therapy (RRT), ICU length of stay, mortality). Patients were excluded if: they had received pre-scan RRT; they did not have pre/post-scan creatinine measurements. Univariate analyses investigated the relationship between CIN or AKI and ICU outcomes (use of RRT post scan, ICU mortality and ICU length of stay), using chi-squared tests for categorical outcomes and nonparametric tests for continuous outcomes. Results A total of 479 scans involving 331 patients were included. In total, 303 (63%) scans involved males, median age 61 (IQR 46 to 71) years, 119 (25%) diabetic, with median pre-scan eGFR of 85 (38 to 113) ml/minute/1.73 m2. Scans occurred a median of 2.9 (0.8 to 8.8) days from admission. A total of 266 (56%) scans were associated with CIN (grade 0, n = 167; grade 1, n = 39; grade 2, n = 60). Clinical outcomes were signifi cantly worse in patients developing higher grades of CIN (increased use of RRT post scan, P = 0.02; increased ICU length of stay, P = 0.04; increased ICU mortality, P = 0.01). P374 Incidence and outcomes of contrast-induced nephropathy in adult ICU patients S Alderson1, S Pillai2, A Manoras1, J Papanikitas1, D Lewis1, S McKechnie1 1John Radcliff e Hospital, Oxford, UK; 2Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P374 (doi: 10.1186/cc13564) Reference Methods A total of 1,000 patients were enrolled retrospectively from January 2007 through March 2013. Patients under 18 years of age, those with chronic kidney disease and patients who died within the fi rst week after surgery were excluded. AKI was defi ned according to the KDIGO 2012 Clinical Practice Guideline for Acute Kidney Injury. Perioperative variables of patients were collected from medical charts. Results The mean age was 55.4 ± 12.4 years. The incidence of postoperative AKI was 8.8%, stage 1 occurred in 5.9%, stage 2 in 2.4% and stage 3 in 0.5% of the patients. Patients who had AKI were signifi cantly older (57.9 ± 12.8 vs. 55.1 ± 12.3 years, P = 0.046), had higher body mass index (30.1 ± 7.1 vs. 28.6 ± 6.4, P = 0.031), higher preoperative C-reactive protein (CRP) levels (157.8 ± 121.5 vs. 76.6 ± 81.1 mg/dl, P = 0.037) and more frequently had history of distant organ metastasis (13.3 vs. 7.8%, P = 0.022) when compared with those who did not have AKI. When compared with patients who did not develop AKI postoperatively, longer operation times (149.1 ± 62.5 vs. 123.8 ± 56.6 minutes, P = 0.001), intraoperative usage of higher amounts of erythrocyte suspension (279.5 ± 616.7 vs. 128.3 ± 296.1 ml, P = 0.001) and fresh frozen plasma (165.9 ± 284.8 vs. 94.1 ± 185.9 ml, P = 0.001) were seen in those who developed AKI. 1. Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group: KDIGO Clinical Practice Guideline for Acute Kidney Injury. Kidney Int 2012, 2(Suppl):1-138. P373 Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) Postoperative acute kidney injury in patients with gynecologic malignancies haematocrit and low preoperative glomerular fi ltration rate (GFR) were signifi cant risk factors (P = 0.03, P = 0.04 and P <0.01 respectively) for developing postoperative AKI following cardiac surgery. See Table 1. Introduction Postoperative acute kidney injury (AKI) is an important cause of mortality and morbidity among surgical patients. Less is known about the occurrence of AKI after operations for gynecologic malignancies. The aim of this study was to determine the incidence of AKI in patients who underwent surgery for gynecologic malignancies and to compare patients with and without postoperative AKI. p g p p g g y Conclusion Our results suggest that we should focus on LV function and GFR as predictors for developing AKI following cardiac surgery. Strategies to increase preoperative haematocrit should be investigated to reduce in the incidence and severity of postoperative AKI. R f P372 Postoperative acute kidney injury in patients with gynecologic malignancies M Didik, P Zeyneloglu, A Pirat, A Ayhan, Z Kayhan Baskent University Faculty of Medicine, Ankara, Turkey Critical Care 2014, 18(Suppl 1):P372 (doi: 10.1186/cc13562) Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery Results A total of 210 patients (median age 70 years; 138 male) had hemodynamic monitoring within 12 hours of AKI I. In total, 41.5% progressed to AKI III and 43.3% died. Patients with fl uid overload after the diagnosis of AKI I (FB >1 l/day; n = 85) had a higher rate of progression to AKI III (63.5 vs. 23.3%, respectively; P <0.001) and in- hospital mortality (43.5 vs. 24.8%, respectively; P = 0.004) compared with patients with FB <1 l/day. There was no diff erence in the other parameters (demographics, comorbidities, severity scores, hemodynamic parameters on day of AKI I, vasopressor use), with the exception of MAP on the day of AKI I (71 vs. 74 mmHg, respectively; P = 0.01). In multivariate analysis after adjustment for demographics, severity scores, comorbidities and hemodynamic parameters on the day of AKI I, a higher FB was associated with an increased risk of progression to AKI III and death (odds ratio (OR) per each 1 l/day increase 2.8; P <0.001) and death (OR 1.6; P = 0.001). Similarly, a higher amount of fl uid administered was associated with an increased risk of AKI progression (OR per each 1 l/day increase 1.8; P = 0.011), even with adjustment for UO. There were no signifi cant interactions between the risk of progression/death and other hemodynamic parameters. With the exception of MAP, improvements in other hemodynamic Introduction The Cockcroft–Gault (CG) equation and the four-variable Modifi cation of Diet in Renal Disease (MDRD) formula are the most commonly used methods to provide estimation of kidney function. The newly developed Mayo Clinic quadratic equation (MCQE) is another alternative. The scope of this study is to investigate the prognostic value of the above algorithms as prediction models for development of acute renal injury (AKI) and early renal dialysis (RD) in cardiac surgery patients during the postoperative ICU stay. Methods A retrospective single-centre study of 528 consecutive patients admitted to the ICU, who underwent elective cardiac surgery under extracorporeal circulation from July 2012 to November 2013. Patients undergoing urgent or emergent surgery were excluded prior to the study. Preoperative estimation of renal function was obtained S135 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P373). Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) g p Results A total of 479 scans involving 331 patients were included. In total, 303 (63%) scans involved males, median age 61 (IQR 46 to 71) years, 119 (25%) diabetic, with median pre-scan eGFR of 85 (38 to 113) ml/minute/1.73 m2. Scans occurred a median of 2.9 (0.8 to 8.8) days from admission. A total of 266 (56%) scans were associated with CIN (grade 0, n = 167; grade 1, n = 39; grade 2, n = 60). Clinical outcomes were signifi cantly worse in patients developing higher grades of CIN (increased use of RRT post scan, P = 0.02; increased ICU length of stay, P = 0.04; increased ICU mortality, P = 0.01). Ninety-fi ve (20%) scans were associated with AKI (stage 1, n = 45; stage 2, n = 13; stage 3, n = 37). Clinical outcomes were again signifi cantly worse in patients developing AKI (increased use of RRT post scan, P <0.001; increased ICU mortality P = 0.01). Introduction Acute kidney injury (AKI) is a signifi cant complication following cardiac surgery associated with an increase in morbidity, hospital stay and mortality. Although the estimated incidence of AKI following cardiac surgery is 30%, few studies have identifi ed the incidence of AKI following cardiac surgery as defi ned by the Kidney Disease: Improving Global Outcomes (KDIGO) group [1]. We conducted a prospective observational study at our institution to identify the incidence and staging of AKI according to the KDIGO defi nition. We also aim to identify the factors that predispose adult patients to developing AKI post cardiac surgery. Methods A prospective analysis was performed on 103 adult patients admitted to ICU post-cardiac surgery from September to October 2013. Data for perioperative risk factors and renal biochemical markers were collected up to the sixth postoperative day and are expressed as mean (SD). Conclusion Renal impairment/injury is common in adult ICU patients undergoing investigations using iodinated contrast and the incidence varies depending upon the classifi cation used. Whichever classifi cation is used, patients developing CIN/AKI following contrast administration have poorer clinical outcomes than patients who do not. Reference Results Ordered logistic regression was used to analyse the data. Thirty- three per cent of cardiac surgery patients at our institution developed AKI. Factors such as poor left ventricular (LV) function, low preoperative . Incidence and outcomes of contrast-induced nephropathy in adult ICU patients Ninety-fi ve (20%) scans were associated with AKI (stage 1, n = 45; stage 2, n = 13; stage 3, n = 37). Clinical outcomes were again signifi cantly worse in patients developing AKI (increased use of RRT post scan, P <0.001; increased ICU mortality P = 0.01). Impact of kidney function calculation formulae on predicting early adverse renal events in cardiac surgery Results parameters in the days after AKI I diagnosis did not have a signifi cant impact on outcome.l Age (years) 66 (13.8) Male 69 Female 34 No AKI 69 KDIGO 1 22 KDIGO 2 6 KDIGO 3 6 Conclusion In critically ill patients with early AKI, a positive fl uid balance, induced by excessive fl uid administration, is associated with an increased risk of AKI progression and death. Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) Lameire et al.: Contrast-induced acute kidney injury and renal support for acute kidney injury: a KDIGO summary (Part 2). Crit Care 2013, 17:205. Lameire et al.: Contrast-induced acute kidney injury and renal support for acute kidney injury: a KDIGO summary (Part 2). Crit Care 2013, 17:205. S136 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P375 of AKI. Signifi cant risk factors for AKI again included male gender (P = 0.009), reduced pre-scan eGFR (P <0.001), and decreasing time from admission to scan (P = 0.003), but also included emergency admission to ICU (P = 0.03), pre-scan shock (P <0.001), and pre-scan oliguria (P <0.001). P375 Human acute kidney injury is associated with a proinfl ammatory phenotype N Gauge, M Varrier, D Boardman, M Hernandez-Fuentes, M Ostermann Guy’s and St Thomas’ Hospital, London, UK Critical Care 2014, 18(Suppl 1):P375 (doi: 10.1186/cc13565) Conclusion Male gender, reduced pre-scan eGFR and decreasing time from admission to scan are risk factors for the development of both CIN and AKI. The association with time from admission to scan may refl ect inadequate patient optimisation prior to contrast administration; this hypothesis is supported by the risk factors signifi cantly associated with AKI alone (emergency admission to ICU and pre-scan shock). Given the adverse clinical outcomes associated with the development of CIN/AKI, these fi ndings necessitate a review of current practice. Reference Introduction Animal research suggests that acute kidney injury (AKI) is an infl ammatory condition [1]. Our aim was to describe the immune phenotype in human AKI. Introduction Animal research suggests that acute kidney injury (AKI) is an infl ammatory condition [1]. Our aim was to describe the immune phenotype in human AKI. y Methods We enrolled patients with: AKI grade II/III (defi ned by KDIGO criteria) and systemic infl ammatory response syndrome (SIRS) without sepsis; SIRS without AKI; and AKI II/III without SIRS. A healthy control population was used for baseline comparison. Serial blood samples were taken on days 0, 2 and 7. Cells were separated using Percoll gradients and phenotyped using fl ow cytometry.i 1. Lameire et al.: Contrast-induced acute kidney injury and renal support for acute kidney injury: a KDIGO summary (Part 2). Crit Care 2013, 17:205. References 1. Martensson J, et al.: Intensive Care Med 2010, 36:1333-1340. 2. Nejat et al.: Crit Care 2010, 14:R85. 1. Martensson J, et al.: Intensive Care Med 2010, 36:1333-1340. 2. Nejat et al.: Crit Care 2010, 14:R85. 2. Nejat et al.: Crit Care 2010, 14:R85. Acute kidney injury after elective adult cardiac surgery A Darbar, G Lau, R Porter Glenfi eld Hospital, Leicester, UK Critical Care 2014, 18(Suppl 1):P373 (doi: 10.1186/cc13563) l Results The results from 24 day 0 samples identifi ed statistically signifi cant diff erences between SIRS, AKI, AKI + SIRS and healthy controls amongst: CD8+ cytotoxic T cells, CD45–CD25+++ regulatory T cells, and CD45–CD25++ cytokine secreting non-T-regulatory cells (Table 1). The percentage of CD69-positive neutrophils was signifi cantly increased across all three groups relative to controls, with little variation between AKI, SIRS and AKI + SIRS patients. P376 Risk factors for the development of contrast-induced nephropathy in ICU patients f Results The changes of plasma NGAL, plasma cystatin C, urine NGAL and urine cystatin C with time were similar to the changes of plasma IL-6. However, only plasma NGAL levels were closely correlated with levels of plasma IL-6 (R2 = 0.36, P <0.05). The analysis for plasma cystatin C, urine NGAL and urine cystatin C at 22 hours for severe AKI showed AUCs of 0.78, 0.71 and 0.75 respectively (all P <0.05), and the AUC for plasma NGAL was 0.62 (P = 0.11). There were no signifi cant diff erences in plasma NGAL at 22 hours between severe AKI and no AKI (2,143.32 vs. 2,077.02 U/ml, P = 0.21). Introduction Critically ill patients cared for in ICUs often require radiological investigation using iodinated contrast agents. Contrast- induced nephropathy (CIN) – a form of acute kidney injury (AKI) – is a complication following the use of such contrast. Although CIN has been thoroughly studied in some populations (for example, those undergoing coronary angiography), it has not been investigated in large numbers of ICU patients [1]. Conclusion In this animal model of CLP sepsis, plasma NGAL levels were aff ected by the systemic infl ammatory response, and did not discriminate for AKI. Urine NGAL, plasma cystatin C and urine cystatin C were able to diff erentiate severe AKI from no AKI in CLP sepsis. References Methods We conducted a single-centre retrospective review of the electronic patient records of general adult ICU patients who received iodinated contrast over a 3-year period (2009 to 2011). Our review identifi ed: patient demographics; ICU admission details (specialty, time of admission, elective/emergency admission); physiological status pre scan (evidence of shock, eGFR, urine output); volume of iodinated contrast; and evidence of CIN or AKI post scan (assessed using CIN and KDIGO criteria). Patients were excluded if they had pre-scan renal replacement therapy. Analyses investigated the risk factors for CIN or AKI using chi-squared tests for categorical variables and nonparametric tests for continuous variables (as no continuous variable was normally distributed, even with log transformation). Test characteristics of acute kidney injury biomarkers in animal models of sepsis p Z Peng, J Zhang, F Zhou, J Kellum Z Peng, J Zhang, F Zhou, J Kellum University of Pittsburgh Medical Center, Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P377 (doi: 10.1186/cc13567) Table 1 (abstract P375). AKI + SIRS AKI no SIRS SIRS alone Control % CD8+ cytotoxic T cells 23.3 16.7 11.6 31.1 % Fr. II T-regulatory cells 4.1 2.7 1.6 1.2 % Fr. III cytokine T cell 11.5 21.2 13.9 8.0 % CD69+ neutrophils 83.9 69.7 65.5 7.35 Conclusion Human AKI is associated with a proinfl ammatory phenotype. Reference 1. Akcay A, et al.: Mediators of infl ammation in acute kidney injury. Mediators Infl amm 2009, 2009:137072. Table 1 (abstract P375). Introduction Approximately 50% of acute kidney injury (AKI) is associated with sepsis. Neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C are the two most widely used biomarkers for AKI. However, these two markers are also aff ected by the systemic infl ammatory response, and their diagnostic value in sepsis-induced AKI is disputed [1,2]. Unlike clinical AKI, animal models can be used to explore single etiology. The purpose of this study is to examine the relationship between infl ammatory mediators and biomarkers for AKI in a sepsis model in rats. p Methods Sepsis was induced by cecal ligation and puncture (CLP) in 60 adult SD rats and then observed for AKI and survival. Blood and urine samples were collected at baseline, and 18, 22, and 48 hours after CLP. AKI severity was assessed by RIFLE criteria (creatinine only). The associations between plasma IL-6 and plasma NGAL, plasma cystatin C, urine NGAL and urine cystatin C were analyzed. The area under the receiver-operator characteristic curves (AUC) was used to evaluate the diagnostic capability between severe AKI (RIFLE-I or RIFLE-F) and no AKI (includes RIFLE-R) for diff erent biomarkers. P378 Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? L Desteghe1, W Boer2, Q Swennen3, V Pennemans3, M Vander Laenen2, K Engelen2, C De Deyne2, F Jans2 1Hasselt University, Diepenbeek, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium; 3Biomedical Research Institute, Diepenbeek, Belgium Critical Care 2014, 18(Suppl 1):P378 (doi: 10.1186/cc13568) Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? L Desteghe1, W Boer2, Q Swennen3, V Pennemans3, M Vander Laenen2, K Engelen2, C De Deyne2, F Jans2 1Hasselt University, Diepenbeek, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium; 3Biomedical Research Institute, Diepenbeek, Belgium Critical Care 2014, 18(Suppl 1):P378 (doi: 10.1186/cc13568) Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? L Desteghe1, W Boer2, Q Swennen3, V Pennemans3, M Vander Laenen2, K Engelen2, C De Deyne2, F Jans2 1Hasselt University, Diepenbeek, Belgium; 2Ziekenhuis Oost-Limburg, Genk, Belgium; 3Biomedical Research Institute, Diepenbeek, Belgium Critical Care 2014, 18(Suppl 1):P378 (doi: 10.1186/cc13568) P376 P376 Risk factors for the development of contrast-induced nephropathy in ICU patients S Pillai1, S Alderson2, A Manoras2, J Papanikitas2, D Lewis2, S McKechnie2 1Morriston Hospital, Swansea, UK; 2John Radcliff e Hospital, Oxford, UK Critical Care 2014, 18(Suppl 1):P376 (doi: 10.1186/cc13566) Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? Results In total, 479 scans involving 331 patients were included. A total of 266 (56%) scans were associated with CIN. Signifi cant risk factors for the development of CIN included male gender (P = 0.02), reduced pre- scan GFR (P <0.001), and decreasing time from admission to scan (P = 0.03). Ninety-fi ve (20%) scans were associated with the development Introduction Acute kidney injury (AKI) remains a common complication after cardiopulmonary bypass (CPB) and can be diagnosed by S137 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 serum creatinine [1]. However, serum creatinine is an insensitive and nonspecifi c biomarker [2]. This study was designed to investigate whether a correlation exists between urinary oxygen tension (UOT) and early markers of AKI. The aim was to evaluate whether UOT could provide warning signs of an insuffi cient renal oxygen supply, which can lead to postoperative AKI. serum creatinine [1]. However, serum creatinine is an insensitive and nonspecifi c biomarker [2]. This study was designed to investigate whether a correlation exists between urinary oxygen tension (UOT) and early markers of AKI. The aim was to evaluate whether UOT could provide warning signs of an insuffi cient renal oxygen supply, which can lead to postoperative AKI. dose, mean arterial pressure, fl uid balance and urine output in the fi rst 6 hours after admission were signifi cantly diff erent for patients with low and high risk for AKI. The negative predictive value (NPV) of 0.41 for urine output <1 ml/kg/hour over the fi rst 6 hours after admission was signifi cantly lower if compared with NPV of 0.86 for GFBP7/TIMP-2 ≤0.3 for exclusion of AKI. Similarly NPV of urine output <1 ml/kg/hour of 0.89 for moderate and severe AKI (stage 2 and 3) was lower than the NPV of 1.00 for GFBP7/TIMP-2 ≤0.3. Methods Fourteen subjects undergoing cardiac surgery with CPB were included in this prospective clinical pilot study. UOT was measured perioperatively in all patients, both in the operating room (before, during and after CPB) and in the ICU. Biomarkers of AKI in blood and urine were measured preoperatively and postoperatively at 3, 6, 12 and 24 hours after the initiation of CPB. These included serum creatinine and the early urinary biomarkers kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C. Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? Student’s t tests and Mann–Whitney tests were used to compare continuous variables.i Conclusion The novel urine biomarkers IGFBP7 and TIMP-2 enable early risk stratifi cation of surgical ICU patients at risk for development of AKI. The predictive values of biomarkers were better for early prediction than clinical parameters such as urine output within the fi rst 6 hours after admission to ICU. P380 Urine TIMP2 × IGFBP7 increases 24 hours before severe AKI M Ostermann1, L Chawla2, L Forni3, J Kellum, on behalf of Sapphire Investigators4 1Guys & St Thomas Foundation Hospital, London, UK; 2George Washington University, Washington, DC, USA; 3Western Sussex Hospital, Worthing, UK; 4University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P380 (doi: 10.1186/cc13570) Urine TIMP2 × IGFBP7 increases 24 hours before severe AKI M Ostermann1, L Chawla2, L Forni3, J Kellum, on behalf of Sapphire Investigators4 Results There was a signifi cant decrease in UOT between the start of CPB (138.44 ± 22.19 mmHg) and the lowest UOT during CPB (107.70 ± 23.28 mmHg) (P = 0.001). Dividing the subjects into two groups according to the Acute Kidney Injury Network (AKIN) classifi cation, no signifi cant diff erences were found in mean UOTs between the group of patients with a normal kidney function (n = 7) and the group with AKIN stage 1 or 2 (n = 7). For KIM-1, a signifi cant diff erence between the two groups was found at 3 hours (P = 0.041) after the initiation of CPB. Further, for NGAL a signifi cant increase in biomarker concentrations compared with the preoperative value was observed in the group with an AKIN stage 1 or 2 at all diff erent postoperative time points (3 hours (P = 0.013), 6 hours (P = 0.003), 12 hours (P = 0.009) and 24 hours (P = 0.003)). On the contrary, there were no signifi cant increased urinary NGAL levels measured in the group with the normal kidney function. P379 Postoperative acute kidney injury can be predicted by the novel biomarkers insulin-like growth factor-binding protein 7/tissue inhibitor of metalloproteinases-2 as early as 6 hours after surgery I Göcze, R Herzog, M Koch, P Renner, F Zeman, BM Graf, HJ Schlitt, T Bein University Medical Center Regensburg, Germany Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) Postoperative acute kidney injury can be predicted by the novel biomarkers insulin-like growth factor-binding protein 7/tissue inhibitor of metalloproteinases-2 as early as 6 hours after surgery I Göcze, R Herzog, M Koch, P Renner, F Zeman, BM Graf, HJ Schlitt, T Bein University Medical Center Regensburg, Germany Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) Postoperative acute kidney injury can be predicted by the novel biomarkers insulin-like growth factor-binding protein 7/tissue inhibitor of metalloproteinases-2 as early as 6 hours after surgery I Göcze, R Herzog, M Koch, P Renner, F Zeman, BM Graf, HJ Schlitt, T Bein University Medical Center Regensburg, Germany Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) Conclusion The TIMP2 × IGFBP7 biomarker combination identifi es patients who ultimately develop moderate or severe AKI 24 hours earlier than serum creatinine. Perioperative measurement of urinary oxygen tension as a tool in the prevention of acute kidney injury? g 1Guys & St Thomas Foundation Hospital, London, UK; 2George Washington University, Washington, DC, USA; 3Western Sussex Hospital, Worthing, UK; 4University of Pittsburgh, PA, USA Introduction We recently reported a 728-patient multicenter study (Sapphire) where a biomarker combination of tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) were validated for risk stratifi cation for moderate or severe acute kidney injury (AKI) KDIGO stage 2 and 3 [1].f Methods We subsequently selected two clinical cutoff values for the TIMP2 × IGFBP7 combination from the Sapphire dataset, one (0.3) with high sensitivity (89%) (specifi city = 50%) and one (2.0) with high specifi city (95%) (sensitivity = 42%) for the development of AKI KDIGO stage 2 and 3 within 12 hours of study enrolment. We examined the timing of change in TIMP2 × IGFBP7 relative to change in creatinine using the sign test. g y Conclusion This pilot study was not able to demonstrate any association between perioperatively measured UOT and markers of postoperative AKI. Additional laboratory and clinical studies will be necessary to further defi ne the relationship between the UOT and new biomarkers of AKI. References References 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. g g Results TIMP2 × IGFBP7 results were available for 178 patients who developed AKI stage 2 or 3. The median TIMP2 × IGFBP7 result was signifi cantly greater than the cutoff value of 0.3 from 24 hours before to 24 hours after AKI 2 or 3 (P <0.01) (Figure 1). Conversely, median serum creatinine was not diff erent from baseline prior to development of AKI 2 or 3. 1. Mariscalco G, et al.: Ann Thorac Surg 2011, 92:1539-1547. 2. Vaidya VS, et al.: Annu Rev Pharmacol Toxicol 2008, 48:463-493. P382 P382 Urine microscopy score combined with albumin creatinine ratio score improves prediction of future acute kidney injury (AKI) and worsening AKI JJ Dixon1, K Lane1, W Fleming-Nouri2, H Cheema2, P Walker2, I MacPhee3, B Philips1 1St George’s Hospital and University of London, UK; 2St George’s, University of London, UK; 3St George’s Hospital, London, UK Critical Care 2014, 18(Suppl 1):P382 (doi: 10.1186/cc13572) Reference Reference y g g, y Critical Care 2014, 18(Suppl 1):P379 (doi: 10.1186/cc13569) 1. Kashani K, et al.: Discovery and validation of cell cycle arrest biomarkers in human acute kidney injury. Crit Care 2013, 17:R25. Introduction Acute kidney injury (AKI) in surgical critically ill patients is an independent risk factor for early mortality. Two novel urine biomarkers, insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), may help to detect clinically silent episodes of AKI in the golden hours prior to irreversible damage of the kidney. We evaluated the early predictive value of these biomarkers for AKI, moderate and severe AKI, early requirement of renal replacement therapy (RRT), and ICU mortality, with a cutoff value of IGFBP7/TIMP-2 >0.3. Figure 1 (abstract P380). NC, Nephrocheck (TIMP2 × IGFBP7); Cr, creatinine. Methods Four to six hours after admission to the surgical ICU, urine biomarkers were prospectively evaluated in all patients with present exposures and susceptibilities for AKI according to the KDIGO recommendation. The incidence and severity of AKI (KDIGO 2012) and requirement of RRT were assessed over 48 hours after admission. In addition, ICU mortality and variables such a norepinephrine dose, mean arterial pressure, hemoglobin level, cumulative fl uid balance and urine production were noted at the time of biomarker evaluation (4 to 6 hours) and for the fi rst 24 hours after admission. i Results A total of 120 patients were included in the study. The area under the curve (AUC) for IGFBP7/TIMP2 >0.3 was 0.83 for early detection of AKI, 0.86 for moderate and severe AKI, 0.86 for RRT in the fi rst 48 hours after admission and 0.86 for ICU mortality. Patients with IGFBP7/ TIMP-2 >0.3 had a higher risk (odds ratio 11.7) for development of AKI compared with patients with IGFBP7/TIMP-2 ≤0.3. The norepinephrine Figure 1 (abstract P380). NC, Nephrocheck (TIMP2 × IGFBP7); Cr, creatinine. S138 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P381 who developed AKI or worsening AKI (n = 58) had a mean score of 2.79 ± 1.21 versus 2.30 ± 1.14 in those who never developed AKI or improved (n = 150), P = 0.006. UMS-ACRS score >2 on admission had a sensitivity of 0.89 for identifying progressive AKI. Reference UMS-ACRS score of 5 to 8 on admission had a positive predictive value for worsening AKI of 60%, a negative predictive value of 69% and a likelihood ratio 3.1 for developing AKI or worsening AKI. who developed AKI or worsening AKI (n = 58) had a mean score of 2.79 ± 1.21 versus 2.30 ± 1.14 in those who never developed AKI or improved (n = 150), P = 0.006. UMS-ACRS score >2 on admission had a sensitivity of 0.89 for identifying progressive AKI. UMS-ACRS score of 5 to 8 on admission had a positive predictive value for worsening AKI of 60%, a negative predictive value of 69% and a likelihood ratio 3.1 for developing AKI or worsening AKI. P381 Urinary tissue inhibitor of metalloproteinases-2 and insulin-like growth factor-binding protein 7 as early biomarkers of acute kidney injury and renal recovery following cardiac surgery A Zarbock1, M Meersch1, C Schmidt1, S Martens1, J Rossaint1, K Singbartl2, D Görlich1, J Kellum3, H Van Aken1 1University of Münster, Germany; 2Penn State College of Medicine Hershey, PA, USA; 3University of Pittsburgh, PA, USA Critical Care 2014, 18(Suppl 1):P381 (doi: 10.1186/cc13571) Conclusion The mean UMS-ACRS is higher in patients with AKI. Combining UMS with ACRS improves prediction and stratifi cation of which patients will develop AKI, or progressive AKI, after ICU admission. Clinical implications are that urine microscopy and ACR calculation are safe, inexpensive, non-invasive and may improve prediction of AKI in critically ill patients, potentially leading to earlier intervention and improved outcomes. Introduction Diffi culties in prediction and early identifi cation of acute kidney injury (AKI) have hindered the ability to develop preventive and therapeutic measures for this syndrome. We tested the hypothesis that a urine test measuring insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor of metalloproteinases-2 (TIMP-2), both inducers of G1 cell cycle arrest, a key mechanism implicated in AKI, could predict AKI in cardiac surgery patients. P383 Resveratrol ameliorates apoptosis induced by contrast medium ioxitalamate in HK-2 human renal proximal tubule cells in vitro YY Chen, CC Cheng, TC Lin, Huang Buddhist Tzu Chi General Hospital, Hualien, Taiwan Critical Care 2014, 18(Suppl 1):P383 (doi: 10.1186/cc13573) y Methods We studied 50 patients at high risk for AKI undergoing cardiac surgery with cardiopulmonary bypass. Serial urine samples were analyzed for [TIMP-2] × [IGFBP7] concentrations. The primary outcome measure was AKI as defi ned by international consensus criteria following surgery. Furthermore, we investigated whether urine [TIMP- 2] × [IGFBP7] could predict renal recovery from AKI prior to hospital discharge. Introduction Computed tomography with contrast medium is a common diagnostic tool in emergency and critical medicine. Contrast- induced nephropathy (CIN) is one of the leading causes of hospital- acquired acute kidney injury. Focus on renal tubule protection may be a hope to improve the eff ectiveness of current strategies. Results Twenty-six patients (52%) developed AKI. Diagnosis based on serum creatinine and/or oliguria did not occur until 1 to 3 days after cardiopulmonary bypass. In contrast, urine concentration of [TIMP-2] × [IGFBP7] rose from a mean of 0.49 (0.24) at baseline to 1.51 (0.57) 4 hours after cardiopulmonary bypass in patients who developed AKI. The maximum urinary [TIMP-2] × [IGFBP7] concentration achieved in the fi rst 24 hours following surgery (composite time point) demonstrated an area under the receiver-operating characteristic curve of 0.84. Sensitivity was 0.92, and specifi city was 0.81 for a cutoff value of 0.50. The decline in urinary [TIMP-2] × [IGFBP7] values was the strongest predictor for renal recovery. f Methods We used HK-2 human renal proximal tubule cells to evaluate the therapeutic potential of resveratrol, a polyphenol phytoalexin Figure 1 (abstract P383). Immunostaining of cytoplasmic DNA fragmentation in HK-2 cells. Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. Figure 1 (abstract P383). Immunostaining of cytoplasmic DNA fragmentation in HK-2 cells. Figure 1 (abstract P383). Immunostaining of cytoplasmic DNA fragmentation in HK-2 cells. p y Conclusion Urinary [TIMP-2] × [IGFBP7] serves as a sensitive and specifi c biomarker to predict AKI early after cardiac surgery and to predict renal recovery. Recovery from AKI by KDIGO criteria Recovery from AKI by KDIGO criteria D Schrijvers, J Gunst, G van den Berghe, M Schetz KU Leuven University Hospital, Leuven, Belgium Critical Care 2014, 18(Suppl 1):P386 (doi: 10.1186/cc13576) p p p Methods This single-centre, randomized, double-blind, placebo- controlled study included 75 patients scheduled for CABG with preexisting renal impairment (estimated glomerular fi ltration rate based on p-cystatin C <60 ml/minute and >15 ml/minute). The patients either received a single high dose of EPO (400 IU/kg) or placebo preoperatively. The primary endpoint was renal protection evaluated by p-cystatin C at the third postoperative day compared with the preoperative values. Incidence of acute kidney injury and other renal biomarker changes were among secondary endpoints.if y p g Critical Care 2014, 18(Suppl 1):P386 (doi: 10.1186/cc13576) Introduction Data on recovery of AKI are mainly limited to persistent dialysis dependency in patients with dialysis-requiring AKI. The aim of this analysis is to evaluate recovery from diff erent stages of AKI. y yf g Methods In a large database (n = 4,640) of a previous RCT [1] we estimated renal recovery from AKI defi ned by KDIGO criteria (without urine output criteria). Patients with end-stage renal disease (n = 56), kidney transplantation (n = 15) or incomplete data (n = 9) were excluded. Patients were classifi ed according to their maximal AKI stage (AKImax) during the ICU stay. Recovery was evaluated by AKI stage at hospital discharge. Complete recovery was defi ned as the absence of AKI, partial recovery as persistent AKI with a decrease in AKI stage compared with AKImax and no recovery as persistence of AKImax or worsening of AKI after ICU discharge. A persistent 0.3 mg/dl increase of Screat was also considered as no or partial recovery. Results There was no signifi cant diff erence on the third postoperative day for p-cystatin C levels (2.1 ± 0.8 mg/l for the study group and 1.9 ± 0.5 mg/l for the control group, P = 0.51). There were no signifi cant diff erences in other renal biomarkers or measures between the groups (p-NGAL, p-creatinine, p-urea, and estimated glomerular fi ltration rate). There were no other diff erences in outcome variables between the groups. g p Conclusion Intravenous administration of a single high dose (400 IU/ kg) of EPO did not have a renal protective eff ect in patients with reduced kidney function undergoing coronary artery bypass surgery. Recovery from AKI by KDIGO criteria References y Results A total of 1,296 patients (28%) developed AKI. AKImax was stage 1 in 580 (45%) (416 with >50% increase of Screat), stage 2 in 207 (16%) and stage 3 in 509 (39%) (348 needing RRT). Mortality increased from 12 to 42% (P <0.0001), hospital stay increased from 21 (14 to 37) to 34 (17 to 63) days (P <0.0001) and complete recovery in survivors decreased from 82 to 53% (P <0.0001) with increasing severity of AKI. In patients requiring RRT, 51% of survivors left the hospital without AKI whereas 16% remained dialysis dependent. Within the AKI 3 group, the need for RRT signifi cantly increased mortality (P = 0.0002), but did not aff ect complete recovery in survivors (51% vs. 58%, P = 0.16). Patients with a ‘0.3 mg/dl increase of serum creatinine only’ had a signifi cantly higher mortality than patients without AKI in the ICU (P = 0.0004). They also had a worse kidney outcome at hospital discharge (P = 0.006). 1. Bahlmann FH, Fliser D: Erythropoietin and renoprotection. Curr Opin Nephrol Hypertens 2009, 18:15-20. 2. Moore E, Bellomo R: Erythropoietin (EPO) in acute kidney injury. Ann Intensive Care 2011, 1:3. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ICU nonsurvivors (n = 229), patients on dialysis at ICU discharge (n = 77) and patients for whom Clcr on the last day of ICU was not available (n = 206), 784 patients were included in this analysis. We compared eGFR (MDRD equation) with measured Clcr (based on 24-hour urine collection and corrected for BSA) at ICU discharge for patient groups with diff erent ICU stays. We also evaluated the impact of using the two GFR measurements on the estimation of complete recovery relative to baseline eGFR. Parameters were compared with the paired t test and McNemar’s test. produced naturally by several plants, for contrast ioxitalamate-induced toxicity in vitro. Cytotoxicity was determined by MTT assay. Patterns of cell death were observed by fl ow cytometry. Cytoplasmic DNA fragmentation was examined by ELISA. Western blots were used to analyze the expression of related proteins. y p p Results In a 48-hour administration, ioxitalamate elicited cytotoxicity on HK-2 cells. Annexin V+ cells were signifi cantly increased after 30 mg/ ml ioxitalamate exposure. A decrease in bcl-2 expression explained the ioxitalamate-induced apoptosis. Co-treatment with resveratrol ameliorated the cytotoxicity induced by ioxitalamate. Resveratrol at 12.5 μM decreased ioxitalamate-induced DNA fragmentation through upregulated expression of survivin. See Figures 1 and 2. Results Amongst the 784 patients with AKI, 456 (58%) reached stage 1, 143 (18%) stage 2 and 185 (24%) stage 3. Mean ± SD Clcr and eGFR at ICU discharge were respectively 54.5 ± 28 and 76 ± 55 ml/minute/1.73 m2 (P <0.0001). eGFR was not signifi cantly diff erent from Clcr in patients with ICU stay <7 days. In patients with ICU stay between 8 and 14 days, eGFR was signifi cantly higher than Clcr (79 ± 51 vs. 48.5 ± 20, P <0.0001) and the diff erence increased even further in patients with ICU stay over 14 days (102 ± 70 vs. 42.6 ± 20, P <0.0001). The percentage of patients with complete recovery diff ered signifi cantly when evaluated by eGFR (35.3%) or Clcr (28.7%) (P = 0.007). In patients with ICU stay >14 days, this diff erence increased to 56.4% by eGFR versus 14.1% by Clcr (P <0.0001). Conclusion Resveratrol ameliorated apoptosis induced by contrast medium ioxitalamate in human renal proximal tubule cells. Investigations using animal models will be conducted in the future. Estimated GFR versus creatinine clearance for evaluation of recovery from acute kidney injury y y j y P Timmermans, J Gunst, G Van den Berghe, M Schetz y y j y P Timmermans, J Gunst, G Van den Berghe, M Schetz KU Leuven University Hospital Leuven Belgium g KU Leuven University Hospital, Leuven, Belgium y p g Critical Care 2014, 18(Suppl 1):P385 (doi: 10.1186/cc13575) Conclusion Increasing severity of AKI according to the KDIGO criteria is associated with increased mortality and decreased recovery of kidney function. The need for RRT signifi cantly increases mortality but complete recovery in survivors of AKI 3 is not diff erent with or without RRT. The 0.3 mg/dl criterion proves valid with regard to mortality and kidney outcome. Introduction The aim of this study is to quantify the impact of using eGFR instead of measured creatinine clearance (Clcr) on the evaluation of recovery from acute kidney injury (AKI). Introduction The aim of this study is to quantify the impact of using eGFR instead of measured creatinine clearance (Clcr) on the evaluation of recovery from acute kidney injury (AKI). Methods From a large RCT’s database [1] we excluded patients with end-stage renal disease and kidney transplants. In the remaining 4,560 patients, 1,296 (28%) developed AKI (KDIGO criteria). After exclusion of Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P384 P384 Erythropoietin and Protection of Renal function in Cardiac Surgery (EPRICS) trial A Dardashti Lund University and Skane University Hospital, Lund, Sweden Critical Care 2014, 18(Suppl 1):P384 (doi: 10.1186/cc13574) Conclusion Estimated GFR at ICU discharge is signifi cantly higher than the measured Clcr in patients with prolonged ICU stay. This diff erence can be explained by loss of muscle mass with decreased creatinine production and results in an important overestimation of recovery. Reference Introduction To date, there are no known methods for preventing acute kidney injury after cardiac surgery. Increasing evidence suggests that erythropoietin (EPO) has renal anti-apoptotic and tissue protective eff ects [1], and in animal models a single high dose of EPO has been shown to ameliorate reperfusion injury after ischemia [2]. However, recent human studies have shown confl icting results. We aimed to study the eff ect of a single high dose of EPO preoperatively on renal function after coronary artery bypass grafting (CABG) in patients with preoperative impaired renal function. 1. Casaer et al.: N Engl J Med 2011, 365:506-517. 1. Casaer et al.: N Engl J Med 2011, 365:506-517. Casaer et al.: N Engl J Med 2011, 365:506-517. Urine microscopy score combined with albumin creatinine ratio score improves prediction of future acute kidney injury (AKI) and worsening AKI g JJ Dixon1, K Lane1, W Fleming-Nouri2, H Cheema2, P Walker2, I MacPhee3, B Philips1 1St George’s Hospital and University of London, UK; 2St George’s, University of London, UK; 3St George’s Hospital, London, UK Critical Care 2014, 18(Suppl 1):P382 (doi: 10.1186/cc13572) B Philips 1St George’s Hospital and University of London, UK; 2St George’s, University of London, UK; 3St George’s Hospital, London, UK Critical Care 2014, 18(Suppl 1):P382 (doi: 10.1186/cc13572) Introduction Patients with AKI have a high morbidity and mortality. Diagnosis of AKI may be improved by examination of the urinary sediment with microscopy and by measurement of urine albumin. A urine microscopy score (UMS; 0 to 4 points) of renal tubular epithelial cells and granular casts has previously been developed to aid diagnosis. We have devised a urine albumin:creatinine ratio score (ACRS; 0 to 4 points) and have combined this with the UMS with the aim of stratifying the risk of developing future AKI or worsening AKI (UMS- ACRS; 0 to 8 points). The aims were to compare UMS-ACRS in critically ill patients with and without AKI at ICU admission, and to determine whether a high UMS-ACRS can predict if critically ill patients develop AKI or worsening AKI. Figure 1 (abstract P383). Immunostaining of cytoplasmic DNA fragmentation in HK-2 cells. Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. g Methods Investigators were blinded to diagnosis prior to urine collection. Microscopy was performed on centrifuged urine obtained from 227 consecutive critically ill patients in a general ICU on day 1 of admission. Five photographs were taken and the mean UMS was calculated. An independent reviewer scored the photographs. The urine albumin:creatinine ratio was calculated and the ACRS determined. The UMS was then combined with the ACRS. Results Mean UMS-ACRS ± SD was higher (2.66 ± 1.57) in patients with AKI on ICU admission (n = 106) compared with those without AKI (mean UMS-ACRS = 2.40 ± 1.03; n = 120), unpaired t test P = 0.14. Patients Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. Figure 2 (abstract P383). Upregulated survivin expression by resveratrol. S139 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P390 P390 Relation between preoperative use of diuretics and renal replacement therapy after cardiac surgery: a propensity score analysis E Curiel-Balsera1, M Delange van der Kroft2, I Macias-Guarasa1, R Hinojosa Perez1, J Ravina-Sanz1, M Garcia-Delgado1, R Rivera-Fernandez1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain Critical Care 2014, 18(Suppl 1):P390 (doi: 10.1186/cc13580) Recovery of renal function after acute kid continuous renal replacement therapy p py HY Jung, KH Kim, SC Park, JY Choi, SH Park, CD Kim, YL Kim, JH Cho Kyungpook National University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P389 (doi: 10.1186/cc13579) y HY Jung, KH Kim, SC Park, JY Choi, SH Park, CD Kim, YL Kim, JH Cho Kyungpook National University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P389 (doi: 10.1186/cc13579) g, , , , , , , Kyungpook National University Hospital, Daegu, South Korea Critical Care 2014, 18(Suppl 1):P389 (doi: 10.1186/cc13579) Introduction Acute kidney injury (AKI) is increasingly common in critically ill patients and many patients with severe kidney injury require continuous renal replacement therapy (CRRT). However, little is known regarding the incidence rate and associated factors for developing chronic kidney disease after CRRT in AKI patients. This study aimed to investigate renal outcome and the factors associated with incomplete renal recovery in AKI patients who received CRRT. Conclusion By the KDIGO defi nition, AKI occurred in two-thirds of patients following LTx. AKI portended greater risk of death and loss of kidney function. y Methods Between January 2011 and August 2013, 397 patients received CRRT in our ICU. Among them, patients who had normal renal function before AKI and were discharged without maintenance renal replacement therapy (RRT) were included in this study. We examined the incidence of incomplete renal recovery with estimated glomerular fi ltration rate (eGFR) <60 ml/minute/1.73 m2 during follow-up. Factors that increased risk of incomplete renal recovery after AKI were investigated with multiple logistic regression. y Reference Casaer et al.: N Engl J Med 2011, 365:506-517. S140 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P387 Incidence and outcomes of acute kidney injury following orthotopic lung transplant: a population-based cohort study P Fidalgo1, M Ahmed1, SR Meyer1, D Lien1, J Weinkauf1, FS Cardoso1, K Jackson2, SM Bagshaw1 1University of Alberta, Edmonton, Canada; 2Alberta Health Services, Edmonton, Canada Critical Care 2014, 18(Suppl 1):P387 (doi: 10.1186/cc13577) P387 criteria were used to classify AKI using creatinine adjusted for fl uid balance. Results Renal replacement therapy was performed on 136 patients in this group (4.2%). The fl uid accumulation percentage was associated with an 8% increase in odds for AKI (OR (CI), 1.08 (1.04 to 1.12)), and a 13% increase in odds for requiring renal replacement therapy (1.13 (1.05 to 1.21)) for each percent increase in fl uid accumulation (l/kg%) after cardiac surgery, after adjusting for variables including APACHE score, cardiac failure, type of surgery, and inotrope use in multivariate analysis. Introduction Acute kidney injury (AKI) is a serious complication following lung transplantation (LTx) [1-3]. We aimed to describe the incidence and outcomes associated with AKI following LTx. Introduction Acute kidney injury (AKI) is a serious complication following lung transplantation (LTx) [1-3]. We aimed to describe the incidence and outcomes associated with AKI following LTx. y Conclusion In this relatively homogeneous patient group undergoing cardiac surgery, postoperative percent fl uid accumulation at 18 hours was associated with AKI and need for renal replacement therapy. Whether there is residual confounding due to indication for fl uid use or unmeasured risk factors requires further investigation in controlled trials. g Methods A retrospective population-based cohort study of all adult recipients of LTx at the University of Alberta between 1990 and 2011 was performed. The primary outcome was AKI, defi ned and classifi ed according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria, in the fi rst seven postoperative days. Secondary outcomes included risk factors, utilization of renal replacement therapy (RRT), occurrence of postoperative complications, mortality and kidney recovery. y References 1. Arnaoutakis GJ, George TJ, Robinson CW, et al.: Severe acute kidney injury according to the RIFLE (risk, injury, failure, loss, end stage) criteria aff ects mortality in lung transplantation. J Heart Lung Transplant 2011, 30:1161-1168. 2. Wehbe E, Brock R, Budev M, et al.: Short-term and long-term outcomes of acute kidney injury after lung transplantation. J Heart Lung Transplant 2012, 31:244-251. Results Forty-one AKI patients were discharged without further RRT and followed up for a mean of 7 months. Sixteen (39.0%) of 41 patients incompletely recovered their renal function. Patients with incomplete renal recovery showed older age and longer duration of anuria compared with complete renal recovery patients (69.7 ± 7.0 vs. 54.8 ± 16.9 years, P = 0.002; 128.6 ± 192.1 vs. 26.9 ± 66.6 hours, P = 0.019). Multivariate analysis adjusting for sex, initial eGFR, hemoglobin level, diabetes mellitus and hypertension showed that old age and long duration of anuria were independent risk factors for incomplete renal recovery (OR = 1.143, 95% CI = 1.020 to 1.281, P = 0.021 and OR = 1.011, 95% CI = 1.001 to 1.032, P = 0.038, respectively). 3. Paradela de la Morena M, De La Torre Bravos M, Prado RF, et al.: Chronic kidney disease after lung transplantation: incidence, risk factors, and treatment. Transplant Proc 2010, 42:3217-3219. 3. Paradela de la Morena M, De La Torre Bravos M, Prado RF, et al.: Chronic kidney disease after lung transplantation: incidence, risk factors, and treatment. Transplant Proc 2010, 42:3217-3219. Fluid accumulation post cardiac surgery and risk for renal replacement therapy p py EM Moore1, A Tobin2, D Reid2, J Santamaria2, R Bellomo1 EM Moore , A Tobin , D Reid , J Santamaria , R Bellomo 1Monash University, Melbourne, Australia; 2St Vincent’s Hospital, Melbourne, Australia Conclusion The renal outcome of severe AKI requiring CRRT was poor even in patients with previous normal renal function. Long-term monitoring of renal function is needed especially in severe AKI patients with old age and long duration of anuria. itical Care 2014, 18(Suppl 1):P388 (doi: 10.1186/cc13578) Critical Care 2014, 18(Suppl 1):P388 (doi: 10.1186/cc13578) Introduction We assessed the impact of fl uid accumulation on the development of acute kidney injury (AKI) and need for continuous renal replacement therapy in cardiac surgical patients. Fluid accumulation has been associated with negative outcomes including development of AKI in critically ill patients [1-3]. As cardiac surgical patients commonly receive large volumes of i.v. fl uid within 24 hours of surgery, they could be at risk of the harmful eff ects of fl uid accumulation. References 1. Bouchard J, et al.: Kidney Int 2009, 76:422-427. 1. Bouchard J, et al.: Kidney Int 2009, 76:422-427. 2 Payen D, et al.: Crit Care (London) 2008, 12:R74. 3. Grams ME, et al.: Clin J Am Soc Nephrol 2011, 6:966-973. y Results Of 445 LTx recipients included, AKI occurred in 306 (68.8%), with severity classifi ed as stage I in 38.9% (n = 173), stage II in 17.5% (n = 78) and stage III in 12.4% (n = 55). RRT was received by 36 (8.1%). Independent risk factors associated with AKI included longer duration of cardiopulmonary bypass (per minute, odds ratio (OR) 1.003; 95% CI, 1.001 to 1.006; P = 0.02), and mechanical ventilation (per hour (log-transformed), OR 5.30; 95% CI, 3.04 to 9.24; P <0.001), and use of cyclosporine (OR 2.03; 95% CI, 1.13 to 3.64; P = 0.02). In-hospital and 1-year mortality were signifi cantly higher in those with AKI compared with no AKI (7.2% vs. 0%, adjusted P = 0.001; 14.4% vs. 5.0%, adjusted P = 0.02, respectively). At 3 months, those with AKI had greater sustained loss of kidney function compared with no AKI (estimated glomerular fi ltration rate (mean (SD)) 68.9 (25.7) vs. 75.3 (22.1) ml/minute/1.73 m2, P = 0.01).i Renal replacement therapy in very elderly critical care patients K Brown, HG Jones Renal replacement therapy in very elderly critical care patients K Brown, HG Jones Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P392 (doi: 10.1186/cc13582) Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P392 (doi: 10.1186/cc13582) Introduction The very elderly (>80 years) UK population is increasing and along with it there is an increased utilisation of critical care resources [1]. The use of chronological age as a bar to treatment is unethical, yet a discrepancy of opinion remains between the disinclined and the elderly advocate intensivist [2]. Acute kidney injury requiring dialysis is a frequent complication of critical illness and is associated with high morbidity and mortality. An increased risk is seen with age due to the high prevalence of risk factors and chronic kidney disease in the elderly [3]. g p Results The total cohort was composed of 7,276 patients with 63.91 ± 12.45 years and 61.1% male gender. Elective scheduled surgery was done in 85.9% of patients. Surgical risk assessed by the additive EuroSCORE was 5.86 ± 3.14 points and predicted mortality by the Logistic EuroSCORE was 8.10%. Mortality in the ICU was 7.6% and in- hospital mortality was 10.1% (8.1% missing data). Prior to surgery, 10.4% of patients had a creatinine level between 1.2 and 2 mg/dl, 1.1% between 2 and 2.3 mg/dl, 0.8% between 2.3 and 3.5 mg/dl and 0.3% above 3.5 mg/dl. In the nonmatched cohort, 180 patients (2.5%) needed RRT. RRT was needed in 3.5% of 3,771 patients with diuretics and in 1.4% of 3,505 patients not treated with them (P <0.001); 2.61 (1.87 to 3.65). After adjusting with logistic regression by additive EuroSCORE, SAPS 3, bypass time exceeding 120 minutes and prior renal dysfunction, the OR was 1.67 (1.15 to 2.45). When we analysed 3,426 matched patients according to the propensity score, RRT was needed in 3% of 1,713 patients with diuretics and in 1.6% of 1,713 not treated with them (P <0.009), 1.85 (1.16 to 2.94). Methods Very elderly patients admitted to our tertiary referral ITU were included in a 2-year (December 2011 to November 2013) retrospective cohort analysis. The outcome of those receiving RRT was reviewed. Results There were 2,297 admissions to the ITU, of which 323 (14%) were classifi ed as very elderly with a mean APACHE II score of 17.3. Continuous renal replacement therapy (CVVHD) for acute kidney injury in critical care: incidence and outcome across South West Wales K Brown, M Challis, A Mikhail K Brown, M Challis, A Mikhail Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P391 (doi: 10.1186/cc13581) Critical Care 2014, 18(Suppl 1):P391 (doi: 10.1186/cc13581) Introduction Renal replacement therapy in critical care is associated with increased mortality. It is not known for which patients RRT confers the most benefi t, or who will recover function and remain dialysis independent on a long-term basis.f Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods A prospective cohort of adult patients who underwent cardiac surgery in 11 institutions all over the Andalusia community from March 2008 to July 2012 was included in the ARIAM adult cardiac surgery project. Diuretic users prior to the intervention were pair-matched to nonusers on the basis of a propensity score based on demographics, comorbidities, medication and surgical data. We analysed diff erences in RRT in both groups. P393 P393 Preventing continuous renal replacement therapies (CRRT)-induced hypophosphatemia using a phosphate-containing CRRT solution in the setting of regional citrate anticoagulation V Pistolesi1, S Morabito1, L Tritapepe1, E Vitaliano2, L Zeppilli1, F Polistena1, MI Sacco1, E Fiaccadori3, A Pierucci1 1Umberto I, Policlinico di Roma, Rome, Italy; 2Pertini Hospital, Rome, Italy; 3University of Parma Medical School, Parma, Italy Critical Care 2014, 18(Suppl 1):P393 (doi: 10.1186/cc13583) Preventing continuous renal replacement therapies (CRRT)-induced hypophosphatemia using a phosphate-containing CRRT solution in the setting of regional citrate anticoagulation V Pistolesi1, S Morabito1, L Tritapepe1, E Vitaliano2, L Zeppilli1, F Polistena1, MI Sacco1, E Fiaccadori3, A Pierucci1 1Umberto I, Policlinico di Roma, Rome, Italy; 2Pertini Hospital, Rome, Italy; 3University of Parma Medical School, Parma, Italy Critical Care 2014, 18(Suppl 1):P393 (doi: 10.1186/cc13583) Preventing continuous renal replacement therapies (CRRT)-induced hypophosphatemia using a phosphate-containing CRRT solution in the setting of regional citrate anticoagulation Conclusion The reported incidence of AKI in critical care ranges from 20 to 50%, with the highest rates seen in sepsis [1]. Utilisation of CRRT for AKI is higher at our centre than the described 5% [2], potentially due to close collaboration between critical care and nephrology [3] or relatively lenient ICU admission criteria. Increasing mortality was seen with age, APACHE II score and delay in initiation of RRT. Prospective analysis is required to look at determining biomarkers for AKI and risk factors for mortality; dynamic monitoring of haemodynamic responders (MAP, vasopressor requirement <24 hours), percentage creatinine decrease [4], severity-of-illness scores and urine output. Results of current RCTs are awaited, which may provide more information on mode of clearance, fl ow rates and early versus standard initiation of RRT to more accurately prognose patients’ outcomes. Introduction Phosphate depletion is a known issue during continuous renal replacement therapies (CRRT) with an incidence of hypophosphatemia up to 80% when standard CRRT solutions are used. The aim was to evaluate the eff ects on serum phosphate and phosphorus supplementation needs of a regional citrate anticoagulation (RCA) protocol for CRRT combining the use of citrate with a phosphate-containing CRRT solution. References 1. Bagshaw SM, et al.: Crit Care 2009, 13:R45. 2. Nguyen YL, Et al.: Ann of Intensive Care 2011, 1:29. 3. Stevens LA, et al.: Adv Chronic Kidney Dis 2010, 17:293-301. 4. Cracknell R: The Ageing Population. London: House of Commons Library Research; 2010. 5. Ansell D, et al., eds: UK Renal Registry Report: 2002. 6. Wight JP, et al.: Qual Health Care. 1998, 7:209-221. 7. McDermid RC, et al.: Crit Care 2011, 15:125. Methods All ICU patients receiving CVVHD (diff usive haemodialysis) for AKI were analysed in a retrospective cohort study of mixed non/surgical patients at a tertiary renal centre over a 2-year period (December 2011 to November 2013). Children <18 years, patients on intermittent haemodialysis or patients requiring plasma exchange were excluded from analysis. y Results A total of 2,297 patients were admitted to the ICU, of which 14% (319) required CRRT. Thirteen patients were excluded from analysis; n = 306, of which 58% were male. Causes of AKI included sepsis (37%), surgery (13%, of which 82% were emergency procedures) and vascular events (9%). Mean values for patients requiring RRT versus all ICU patients: age (65 vs. 62.5 years), APACHE II score (21.2 vs. 14.8), length of stay (13 vs. 8.2 days). Forty-seven per cent of patients received incident dialysis <24 hours of admission, with mean fl ow rates of 31 ml/kg/hour (2 l) in 39%. Mortality at hospital discharge was 56% for RRT versus 20% in all ICU patients admitted over the same time period. Renal replacement therapy in very elderly critical care patients K Brown, HG Jones RRT was utilised in 12.4% (n = 40) of the very elderly patients with a mean APACHE II score of 25. Forty-fi ve per cent of AKI was due to sepsis and 25% due to emergency surgery. ITU very elderly patient mortality was 30.1%, and in those who received RRT was 42% and 67% at ITU and hospital discharge respectively. Recovery of renal function was seen in 19 patients at ITU discharge, of these 11 survived to hospital discharge. Two patients required ongoing IHD in the community. Conclusion Preoperative use of diuretics is associated with an increased risk of need for RRT. P390 Relation between preoperative use of diuretics and renal replacement therapy after cardiac surgery: a propensity score analysis E Curiel-Balsera1, M Delange van der Kroft2, I Macias-Guarasa1, R Hinojosa Perez1, J Ravina-Sanz1, M Garcia-Delgado1, R Rivera-Fernandez1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain E Curiel-Balsera1, M Delange van der Kroft2, I Macias-Guarasa1, R Hinojosa Perez1, J Ravina-Sanz1, M Garcia-Delgado1, R Rivera-Fernandez1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain fl Methods We performed a retrospective analysis of prospectively collected data on all patients admitted after cardiac surgery to St Vincent’s Hospital ICU, Melbourne, Australia from 1 July 2004 to 30 June 2012 (n = 3,207). The fl uid accumulation percentage (total urine and chest drain losses subtracted from total i.v. intake (l) /weight (kg) × 100) was calculated for 18 hours post surgery as most patients were in the ICU for this period. Acute Kidney Injury Network (AKIN) creatinine q g p Critical Care 2014, 18(Suppl 1):P390 (doi: 10.1186/cc13580) Introduction The aim was to evaluate the relation between preoperative use of diuretics and renal replacement therapy (RRT) in postoperative patients of cardiac surgery. Introduction The aim was to evaluate the relation between preoperative use of diuretics and renal replacement therapy (RRT) in postoperative patients of cardiac surgery. S141 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P391 g g y Conclusion Current predictions estimate that the very elderly UK population will almost double by 2030 [4]. The biggest uptake in acceptance of chronic RRT has been in the elderly [5], where no diff erence has been shown in health-related quality of life from the general dialysis population [6]. Our experience suggests that RRT has a role in the critically ill elderly patient, with 33% surviving to hospital discharge. The challenge is identifying those most likely to benefi t from a cohort with multiple comorbidities, against the available resources, rising demand for critical care in an aging population and increasing expectations [7]. Continuous renal replacement therapy (CVVHD) for acute kidney injury in critical care: incidence and outcome across South West Wales K Brown, M Challis, A Mikhail Morriston Hospital, Swansea, UK Critical Care 2014, 18(Suppl 1):P391 (doi: 10.1186/cc13581) Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients Although we noticed a greater loss of dose and absolute creatinine clearance in the citrate group, this had no statistical signifi cance (P = 0.18 and P = 0.13). The mortality found for citrate and heparin groups was 60.4% and 39.4% respectively. The diff erences with statistical signifi cance related to dialitrauma emerged in K+ (P = 0.03), Ca2+ (P = 0.02), Na+ (P = 0.004), platelets (P = 0.002), pH (P = 0.02) and bicarbonate (P = 0.0001). Logistic regression for mortality in the citrate versus heparin groups showed the following values: eff ective dose (ROC 0.435 vs. ROC 0.663), clearance (ROC 0.606 vs. ROC 0.663), SAPS II (ROC 0.482 vs. ROC 0.696), SOFA (ROC 0.713 vs. ROC 0.696) and RIFLE (ROC 0.695 vs. ROC 0.636). Conclusion We may say that there are functional differences that must Methods We performed a retrospective and analytical study including patients exclusively submitted to citrate or heparin through years 2011 and 2012. Included were demographic data with SAPS II, SOFA, RIFLE scores and mortality rate. For functional analysis we consider the timing of the beginning, duration, loss of dose and loss of creatinine clearance. We analyzed dialitrauma, considering the variation of: potassium, total and ionized calcium, magnesium, sodium, phosphorus, pH, lactates, bicarbonate, platelets, albumin, creatinine and urea. Data are presented as the average and standard deviations. To access the infl uence of dose and clearance losses on mortality, we used logistic regression test. yp p p Conclusion The use of a phosphate-containing CRRT solution, accounting for about 50 to 60% of the CRRT dose in the setting of RCA-CVVH or RCA-CVVHDF, allowed one to prevent CRRT-induced phosphate depletion in most of the patients, minimizing the need for phosphate supplementation and maintaining phosphorus levels in a near-normal range throughout CRRT days. Results The study included 44 patients in the citrate group versus 61 in the heparin group. We found no statistical signifi cant diff erences for: age (P = 0.06); SAPS II (P = 0.28); SOFA (P = 0.19); the timing of beginning of the technique (P = 0.61), with 34.8% versus 47.5% of patients in R (RIFLE), 27.8% versus 18% in I (RIFLE) and 16% versus 21% in F (RIFLE); duration of the technique (P = 0.74) and length of stay. P395 corrected for predilution, was at least 25 ml/kg/hour with about 50 to 60% of dialysis dose given as phosphate-containing solution. By convention, hypophosphatemia was defi ned as follows: mild (<0.81 mmol/l), moderate (<0.61 mmol/l) and severe (<0.32 mmol/l). Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients V Goulão, E Lafuente, J Silva, M Fernandes, F Santos, A Ferreira CHTS, Penafi el, Portugal Critical Care 2014, 18(Suppl 1):P395 (doi: 10.1186/cc13585) Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients Elimination rates of electrolytes, vitamins and trace elements during continuous renal replacement therapy with citrate CVVHD: infl uence of treatment dose Conclusion We may say that there are functional diff erences that must be taken into account. Despite not having statistical signifi cance on this sample, losses of dose and creatinine clearance showed a direct relation with mortality. Introduction During continuous renal replacement therapy (CRRT), relevant losses of nutritional substrates, vitamins and trace elements due to diff usive transport and elimination via the fi lter may occur. We investigated the amount of these losses with regard to treatment dose during a 72-hour treatment period. Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients Although we noticed a greater loss of dose and absolute creatinine clearance in the citrate group, this had no statistical signifi cance (P = 0.18 and P = 0.13). The mortality found for citrate and heparin groups was 60.4% and 39.4% respectively. The diff erences with statistical signifi cance related to dialitrauma emerged in K+ (P = 0.03), Ca2+ (P = 0.02), Na+ (P = 0.004), platelets (P = 0.002), pH (P = 0.02) and bicarbonate (P = 0.0001). Logistic regression for mortality in the citrate versus heparin groups showed the following values: eff ective dose (ROC 0.435 vs. ROC 0.663), clearance (ROC 0.606 vs. ROC 0.663), SAPS II (ROC 0.482 vs. ROC 0.696), SOFA (ROC 0.713 vs. ROC 0.696) and RIFLE (ROC 0.695 vs. ROC 0.636).f y p References Methods In critically ill heart surgery patients undergoing CRRT for acute kidney injury, we adopted RCA in a CVVH or CVVHDF modality combining a commercially available citrate solution (18 mmol/l) with a phosphate-containing CRRT solution as dialysate and/or replacement fl uid (HCO3 – 30 mmol/l, phosphate 1.2). The prescribed CRRT dose, 1. Case J, et al.: Crit Care Res Pract 2013, 2013:479730 4. Herrera-Gutiérrez ME, et al.: ASAIO J 2006, 52:670-676. S142 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Reference Hetzel GR, Schmitz, Wissing H, et al.: Regional citrate versus systemic heparin for anticoagulation in critically ill patients on continuous venovenous haemofi ltration: a prospective randomized multicentre trial. Nephrol Dial Transplant 2011, 26:232-239. Methods Forty-one patients with CRRT were investigated. Patients were treated with a citrate CVVHD with diff erent standard doses resulting in a treatment dose range from 14 to 53 (median 27) ml/ kg/hour. Losses were calculated from substrate concentrations in the dialysate × (dialysate + ultrafi ltration) fl ow. During a 72-hour treatment period, each 24 hours were determined separately. Regression analysis was performed for the respective per-day losses related to treatment doses. P394 Elimination rates of electrolytes, vitamins and trace elements during continuous renal replacement therapy with citrate CVVHD: infl uence of treatment dose P Von Freyberg1, W Stahl2, H Reinelt2, K Träger2 1Klinikum Heidenheim, Germany; 2University Hospital Ulm, Germany Critical Care 2014, 18(Suppl 1):P394 (doi: 10.1186/cc13584) P396 Results Regression analyses of ER for Ca2+, Mg2+, PO4 2–, zinc, folic acid and vitamin B12 are shown in Table 1 as the median and interquartile range. 396 Development of key performance indicators for renal replacement therapy in adult intensive care to guide safe and cost-eff ective therapy Evaluation of functional diff erences between two anticoagulation methods used in continuous renal replacement therapy in critical patients V Goulão, E Lafuente, J Silva, M Fernandes, F Santos, A Ferreira CHTS, Penafi el, Portugal Critical Care 2014, 18(Suppl 1):P395 (doi: 10.1186/cc13585) Results Forty-eight patients were treated with RCA-CRRT for at least 72 hours (total running time 12,502 hours). Two-hundred and nineteen RCA-CVVH circuits were used with a mean fi lter life of 57.1 ± 41.7 hours (median 47, IQR 24 to 83). Acid–base status was adequately maintained without the need for additional interventions on RCA-CRRT parameters (pH 7.43 (7.40 to 7.47), bicarbonate 25.3 mmol/l (23.8 to 26.6), BE 0.9 (–0.7 to 2.4); median (IQR)). Serum phosphate was steadily maintained in a narrow range throughout RCA-CRRT days (1.2 mmol/l (0.97 to 1.45); median (IQR)). At some times during CRRT, only 10 out of 48 patients (20.8%) received a low amount of phosphate supplementation (D-fructose-1,6-diphosphate 1.05 ± 2.04 g/day) for mild (n = 7) to moderate (n = 3) hypophosphatemia. In particular, considering all patients, only 33 out of 513 serum phosphorus determinations met the criteria for mild (n = 24) to moderate (n = 9) hypophosphatemia. Severe hypophosphatemia was never observed. Introduction The aim of this study is to analyze the functional alterations that may infl uence the fi nal result when using citrate versus heparin anticoagulation in critically ill patients [1]. p g y p Methods We performed a retrospective and analytical study including patients exclusively submitted to citrate or heparin through years 2011 and 2012. Included were demographic data with SAPS II, SOFA, RIFLE scores and mortality rate. For functional analysis we consider the timing of the beginning, duration, loss of dose and loss of creatinine clearance. We analyzed dialitrauma, considering the variation of: potassium, total and ionized calcium, magnesium, sodium, phosphorus, pH, lactates, bicarbonate, platelets, albumin, creatinine and urea. Data are presented as the average and standard deviations. To access the infl uence of dose and clearance losses on mortality, we used logistic regression test. Results The study included 44 patients in the citrate group versus 61 in the heparin group. We found no statistical signifi cant diff erences for: age (P = 0.06); SAPS II (P = 0.28); SOFA (P = 0.19); the timing of beginning of the technique (P = 0.61), with 34.8% versus 47.5% of patients in R (RIFLE), 27.8% versus 18% in I (RIFLE) and 16% versus 21% in F (RIFLE); duration of the technique (P = 0.74) and length of stay. Development of key performance indicators for renal replacement therapy in adult intensive care to guide safe and cost-eff ective therapy Development of key performance indicators for renal replacement therapy in adult intensive care to guide safe and cost-eff ective therapy Table 1 (abstract P394). ER elimination rates Table 1 (abstract P394). ER elimination rates Table 1 (abstract P394). ER elimination rates Table 1 (abstract P394). ER elimination rates Parameter Average daily ER Day 1 R2 Day 2 R2 Day 3 R2 Ca2+ (mmol/day) 97 (83 to 107) 0.2125 0.2448 0.2315 Mg2+ (mmol/day) 6 (3 to 9) 0.0247 0.0708 0.0733 PO4 2– (mmol/day) 53 (40 to 69) 0.003 0.0556 0.0012 Zinc (μmol/day) 46 (27 to 73) 0.005 0.0527 0.0502 Folic acid (nmol/day) 268 (180 to 388) 0.0935 0.0031 0.0353 Vitamin B12 (pmol/day) 2,060 (1,690 to 2,558) 0.0009 0.0092 0.0152 Conclusion Only Ca2+ showed a correlation of ER and treatment dose. Mg2+, PO4 2– zinc, folic acid and vitamin B12 were eliminated without a correlation to treatment dose. y A Fischer, S Finney Table 1 (abstract P394). ER elimination rates Parameter Average daily ER Day 1 R2 Day 2 R2 Day 3 R2 ab e (abst act 3 ). e at o ates Parameter Average daily ER Day 1 R2 Day 2 R2 Day 3 R2 Ca2+ (mmol/day) 97 (83 to 107) 0.2125 0.2448 0.2315 Mg2+ (mmol/day) 6 (3 to 9) 0.0247 0.0708 0.0733 PO4 2– (mmol/day) 53 (40 to 69) 0.003 0.0556 0.0012 Zinc (μmol/day) 46 (27 to 73) 0.005 0.0527 0.0502 Folic acid (nmol/day) 268 (180 to 388) 0.0935 0.0031 0.0353 Vitamin B12 (pmol/day) 2,060 (1,690 to 2,558) 0.0009 0.0092 0.0152 Royal Brompton Hospital, London, UK y p p Critical Care 2014, 18(Suppl 1):P396 (doi: 10.1186/cc13586) Introduction Renal replacement therapy (RRT) is common. We undertook to develop and report some key performance indicators (KPIs) to monitor our provision of this costly therapy. We utilised data already collected in an electronic clinical information system that records the care received by our patients. We reduced our prescribed RRT dose to 25 ml/kg/hour in December 2011 following an appraisal of the literature [1]. We assessed whether the KPIs informed us if our practice changed and such changes were sustained.fl Methods We calculate the hourly effl uent rate corrected for a patient’s predicted body weight, and the lifespan of haemofi lters. This takes less than 30 minutes each month. Statistical process control charts (SPCs) are used to assimilate the indicators over time. S143 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P396). KPI for renal replacement therapy 2013 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 AICU-RBH no. of patients on CVVHD 8 8 17 15 6 7 13 12 9 15 21 AICU-RBH no. y A Fischer, S Finney Whilst higher than our guideline dose, they are signifi cantly less than the doses prior to the change in practice (both P <0.001). The SPC indicates that the change in practice has been sustained. See Figure 1 and Table 1. P397 Eff ectiveness of sub-albumin protein leakage membrane EMIC2 in post-cardiac surgery rhabdomyolysis G Paternoster, A Covino, R Mercorella, C Di Leo, M Calabrese, G Pittella San Carlo Hospital, Potenza, Italy Critical Care 2014, 18(Suppl 1):P397 (doi: 10.1186/cc13587) P397 Eff ectiveness of sub-albumin protein leakage membrane EMIC2 in post-cardiac surgery rhabdomyolysis G Paternoster, A Covino, R Mercorella, C Di Leo, M Calabrese, G Pittella San Carlo Hospital, Potenza, Italy Critical Care 2014, 18(Suppl 1):P397 (doi: 10.1186/cc13587) Introduction A high postoperative serum myoglobin (MyG) concentration predicts the incidence of acute kidney injury (AKI) and need for renal replacement therapy (RRT), as reported in several surgical settings. The incidence of such events in the ICU is reported to be 2 to 5% of all causes of AKI [1], but can even worsen in the case of cardiac surgery (40.3%) [1]. MyG is a small protein (17.8 kDa) that can be removed with RRT, typically in convection cases. New-generation membranes, removing sub-albumin protein molecular weight solutes, can be used in diff usive treatments (CVVHD) with the advantage of limiting albumin loss and easily combining with citrate anticoagulation, pivotal for cardio-surgical settings. We assessed the eff ectiveness of EMIC2 with citrate anticoagulation in AKI prevention of post-cardiac surgical patients.i g Conclusion The KPIs could be produced quickly and allowed monitoring of the reduction in RRT dosing, assuring us that it is in excess of 20 ml/ kg/hour. The KPIs did not require additional data collection processes. We are developing similar indicators for other organ systems, therapies and processes. y A Fischer, S Finney Methods This is a c 1,154 1,463 1,769 1,532 1,317 1,565 6 544 602 763 689 636 695 2 4,994 5,526 7,004 6,325 5,838 6,380 2 39.90 28.17 29.10 30.53 33.93 31.52 3 32.46 23.44 22.23 23.60 29.32 24.43 2 0.79 0.84 0.75 0.67 0.84 0.77 0 36.06 48.77 34.02 32.60 38.74 31.30 2 2,477 2,322 4,025 3,638 2,632 3,870 2 680 753 954 861 795 869 3 3,157 3,075 4,979 4,499 3,427 4,739 2 8,151 8,601 11,983 10,824 9,265 11,119 5, 1,019 1,075 705 722 1,544 1,588 3 ) 134 118 122 126 143 131 1 during the study. Prior to doses were 39 and 31 ml/ and delivered doses were gher than our guideline es prior to the change in at the change in practice y and allowed monitoring at it is in excess of 20 ml/ ata collection processes. organ systems, therapies nal-replacement therapy in 7. P397 Eff ectiveness of sub-albumin prote post-cardiac surgery rhabdomyoly G Paternoster, A Covino, R Mercorella, San Carlo Hospital, Potenza, Italy Critical Care 2014, 18(Suppl 1):P397 (d Introduction A high postopera concentration predicts the inciden and need for renal replacement the surgical settings. The incidence of su be 2 to 5% of all causes of AKI [1], b cardiac surgery (40.3%) [1]. MyG is a be removed with RRT, typically in c membranes, removing sub-albumin can be used in diff usive treatment limiting albumin loss and easily com pivotal for cardio-surgical settings. EMIC2 with citrate anticoagulation surgical patients. M h d Thi i i f i h 602 763 689 636 695 294 629 5,526 7,004 6,325 5,838 6,380 2,699 5,774 28.17 29.10 30.53 33.93 31.52 31.25 29.90 23.44 22.23 23.60 29.32 24.43 21.34 23.93 0.84 0.75 0.67 0.84 0.77 0.66 0.77 48.77 34.02 32.60 38.74 31.30 26.65 23.70 2,322 4,025 3,638 2,632 3,870 2,012 4,644 753 954 861 795 869 368 786 3,075 4,979 4,499 3,427 4,739 2,380 5,430 8,601 11,983 10,824 9,265 11,119 5,079 11,204 1 1,075 705 722 1,544 1,588 391 934 118 122 126 143 131 115 146 Results A total of 736 patients received RRT during the study. Prior to the dose change, the mean set and delivered doses were 39 and 31 ml/ kg/hour respectively. Thereafter the mean set and delivered doses were 33 and 26 ml/kg/hour respectively. y A Fischer, S Finney of patient-days on CVVHD 61 73 98 86 65 85 44 77 112 119 137 AICU-RBH fi lter started 32 30 52 47 34 50 26 60 71 62 74 AICU-RBH hours of CVVHD 1,154 1,463 1,769 1,532 1,317 1,565 693 1,422 2,215 2,157 2,393 AICU-RBH estimated no bags 544 602 763 689 636 695 294 629 995 952 1,054 AICU-RBH fl uid cost (£) 4,994 5,526 7,004 6,325 5,838 6,380 2,699 5,774 9,134 8,739 9,676 AICU-RBH ml/kg/hour actual 39.90 28.17 29.10 30.53 33.93 31.52 31.25 29.90 30.40 32.55 32.36 AICU-RBH ml/kg/hour 24 hours 32.46 23.44 22.23 23.60 29.32 24.43 21.34 23.93 25.25 25.38 24.01 AICU-RBH percent on CVVHDF 0.79 0.84 0.75 0.67 0.84 0.77 0.66 0.77 0.82 0.76 0.73 AICU-RBH average fi lter life (hours) 36.06 48.77 34.02 32.60 38.74 31.30 26.65 23.70 31.20 34.79 32.34 AICU-RBH fi lter cost (£) 2,477 2,322 4,025 3,638 2,632 3,870 2,012 4,644 5,495 4,799 5,728 AICU-RBH effl uent bags cost (£) 680 753 954 861 795 869 368 786 1,244 1,190 1,318 AICU-RBH total consumable (£) 3,157 3,075 4,979 4,499 3,427 4,739 2,380 5,430 6,739 5,989 7,045 AICU-RBH total cost CVVHD (£) 8,151 8,601 11,983 10,824 9,265 11,119 5,079 11,204 15,873 14,728 16,721 AICU-RBH average cost per patient (£) 1,019 1,075 705 722 1,544 1,588 391 934 1,764 982 796 AICU-RBH average cost per patient day (£) 134 118 122 126 143 131 115 146 142 124 122 Table 1 (abstract P396). KPI for renal replacement therapy 2013 ived RRT during the study. Prior to delivered doses were 39 and 31 ml/ mean set and delivered doses were . Whilst higher than our guideline n the doses prior to the change in dicates that the change in practice d Table 1. ced quickly and allowed monitoring uring us that it is in excess of 20 ml/ dditional data collection processes. s for other organ systems, therapies ntinuous renal-replacement therapy in 09, 361:1627. P397 Eff ectiveness of su post-cardiac surge G Paternoster, A Cov San Carlo Hospital, P Critical Care 2014, 18 Introduction A concentration pre and need for renal surgical settings. Th be 2 to 5% of all ca cardiac surgery (40 be removed with R membranes, remov can be used in dif limiting albumin lo pivotal for cardio-s EMIC2 with citrate surgical patients. Plasma fi ltration with dialysis (plasma diafi ltration) in critically ill patients with acute liver failure p Y Eguchi1, H Nakae2, T Furuya3, M Isono4, Y Kishi5, T Yoshioka6 1Shiga University of Medical Science, Shiga, Japan; 2Akita University School of Medicine, Akita, Japan; 3Akita Red Cross Hospital, Akita, Japan; 4Otsu Municipal Hospital, Shiga, Japan; 5Hikone Municipal Hospital, Hikone, Japan; 6Nishi-Kyoto Hospital, Kyoto, Japan Critical Care 2014, 18(Suppl 1):P399 (doi: 10.1186/cc13589) Y Eguchi1, H Nakae2, T Furuya3, M Isono4, Y Kishi5, T Yoshioka6 1Shiga University of Medical Science, Shiga, Japan; 2Akita University School of Medicine, Akita, Japan; 3Akita Red Cross Hospital, Akita, Japan; 4Otsu Municipal Hospital, Shiga, Japan; 5Hikone Municipal Hospital, Hikone, Japan; 6Nishi-Kyoto Hospital, Kyoto, Japan Critical Care 2014, 18(Suppl 1):P399 (doi: 10.1186/cc13589) y g Results The pretreatment MyG median value was 10,789 ng/ml; it signifi cantly reduced on average 92.8% during CVVHD (see Figure 1) and it remained low at ICU discharge (median value 114 ng/ml). sCr remained stable (average time value equal to 0.94 mg/dl) during CVVHD; PCT also decreased over time with a reduction rate equal to 78% (from 5.35 ± 4.39 mg/dl to 1.23 ± 1.09 mg/dl at the end of CVVHD). Finally, six patients survived at 90 days. Introduction Removing the middle molecular weight substances including cytokines and albumin-bound toxin could be eff ective for patients with acute liver failure (ALF). We have developed a new system, plasma fi ltration with dialysis (plasma diafi ltration (PDF)) [1,2], and assessed its effi cacy in multicenter analysis. y y Conclusion This small experience confi rms that serum MyG is likely to increase in post-cardiac surgical high-risk patients and suggests a benefi cial eff ect of CRRT treatments with EMIC2 membranes and citrate on serum MyG, potentially preventing AKI. Further larger assessment can be advised for confi rmation. R f fi y y Methods A subgroup analysis of an observational study conducted in the ICUs of six hospitals. In PDF, simple plasma exchange is performed using a selective membrane plasma separator (Evaxclio EC-2A; Kawasumi Chemical Inc., Tokyo Japan), which has a sieving coeffi cient of 0.3 for albumin, while the dialysate fl ows outside the hollow fi bers. The fl ow rate of the blood, dialysate, substitute and additional substitute was 80 to 100 ml/minute, 600 ml/hour, and 0 to 450 ml/hour according to the rate of water elimination and 150 ml/ hour, respectively. 1. Keil R, et al.: Comp Met Progr Biomed 2013, S0169:2607. References Methods We report results of EMIC2-based treatments in seven patients (four male/three female) with diff erent causes of rhabdomyolysis (trauma, sepsis, limb ischemia). Five patients had classic dialysis indications (persistent anuria) while in two patients treatment was prophylactically started. CRRT were delivered in CVVHD mode with the EMIC2 dialyzer and with loco-regional trisodium–citrate anticoagulation. Mb plasma levels were assessed each 12 hours while the removal rate, total body and dialyzer clearances were estimated by kinetic modeling as previously described [1]. Clinical data were also collected and both global and renal patient survival was reported. 1. Mori T, et al.: Ther Apher 2002, 6:463-466. 2. Nakae H, et al, Ther Apher Dial 2010, 14:444-450. 1. Mori T, et al.: Ther Apher 2002, 6:463-466. 2. Nakae H, et al, Ther Apher Dial 2010, 14:444-450. P399 and mean aortic cross-clamping of 98 minutes (range 25 to 190). We measured MyG, procalcitonin (PCT), and creatinin (sCr) at ICU admission and, if serum MyG was higher than 600 mg/dl, the patient was treated with CVVHD-EMIC2–citrate anticoagulant within 12 hours of ICU admission for 72 hours and a dose of 2,000 ml/hour. Biochemical assays were obtained at 12, 24, and 72 hours and at ICU discharge. P400fi Effi cacy of continuous plasma diafi ltration therapy T Komura, T Taniguchi, T Noda, M Okajima, S Kaneko Kanazawa University, Kanazawa, Japan Critical Care 2014, 18(Suppl 1):P400 (doi: 10.1186/cc13590) g p p Results The median Mb value at CRRT start was 6,971 ng/ml (range 4,679 to 48,011 ng/ml). CRRT were delivered with an average blood fl ow rate of 143 ± 45 ml/minute and a dialysate fl ow rate of 2,134 ± 1,334 ml/hour. These operating conditions allowed one to stop treatment on average after 75 ± 47 hours (median 54 hours) with a Mb reduction of 82.2% (range 99.4 to 44.4%). Overall median Mb removal per treatment was 59 mg (range 33 to 279 mg) mainly due to the fi rst 24 hours of treatment (54 mg, range 20 to 187 mg). Only two patients had residual renal function that was in one case measured to account for only 7.45 mg Mb removal during the entire treatment. Six patients survived and recovered renal function with no dialysis need at present follow-up. One patient died during the ICU stay. Introduction Acute liver failure (ALF) is a critical illness with high mortality. Plasma diafi ltration (PDF) is a blood purifi cation therapy in which simple plasma exchange is performed using a selective membrane plasma separator while the dialysate fl ows outside the hollow fi bers. While several studies demonstrated that PDF therapy is a useful blood purifi cation therapy for patients with ALF, PDF therapy is often diffi cult to employ in ALF patients complicated with multiple organ failure, especially in those with unstable hemodynamics. Furthermore, it is likely to re-occur immediately after PDF therapy. We developed continuous PDF (CPDF) as a new concept in PDF therapy, and assessed its effi cacy and safety in ALF patients compared with conventional plasma exchange (PE) plus continuous hemodiafi ltration (CHDF) therapy in this study. Conclusion Our data measured high performance of the EMIC2 membrane in Mb removal and confi rm theoretical models indicating that CRRT with a high cutoff membrane can achieve major Mb removal within 24 hours with great superiority in comparison with all other available techniques. Methods Ten ALF patients (gender: male/female = 6/4, age: 47 ± 14) employed CPDF therapy. The primary outcomes were altered liver function, measured by the model for end-stage liver disease (MELD) score, and total bilirubin and prothrombin time International Myoglobin removal of small-protein leakage membrane (EMIC2) in patients in the ICU: a case series p P Fabbrini1, R Rona1, M Migliari1, M Viganò1, A Pesenti2, A Stella2 1AO San Gerardo, Monza, Italy; 2Università degli Studi Milano-Bicocca, Milan, Italy Critical Care 2014, 18(Suppl 1):P398 (doi: 10.1186/cc13588) y p g Results A multicenter study was underway from October 2005 to August 2011. We performed PDF on 65 patients with ALF (severe sepsis, 22; post operation, 15; fulminant hepatitis, 11; alcohol hepatitis, 3; graft versus host disease, 4; and others, 10). The serum total bilirubin, plasma PT-INR and the model for end-stage liver disease (MELD) score before the PDF procedure were 15.0 ± 8.15 mg/dl (average ± SD), 2.3 ± 1.5 and 35.8 ± 9.3, respectively. PDF was performed as 9.2 ± 13.2 sessions per patient and the overall 28-day survival rate was 68.5%. According to the severity of the MELD score, we stratifi ed patients into three categories defi ned by the MELD score. The numbers of patients were 15 (23%) in score 20 to 29, 30 (46%) in score 30 to 39 and 19 (29%) in score over 40. The 28-day survival rates were 73.3%, 40% and 16%, respectively. Introduction Rhabdomyolysis is characterized by breakdown of striated muscle due to a great number of causes. Acute kidney injury (AKI) is a common complication as a consequence of high concentrations of circulating myoglobin (Mb). The AKI degree can vary but often requires dialysis, a condition which drastically worsens the ICU stay and prognosis. Since Mb overconcentration represents the cause of AKI, one of the therapy’s aims should be its removal to prevent further kidney damage and to allow faster renal recovery. Both intermittent hemodialysis and high-volume CVVHF are poorly eff ective in removing Mb, while small- protein leakage membranes seem to be promising in this setting. The aim of our study was therefore to measure effi cacy of Mb removal of a new high cutoff membrane (EMIC2; Fresenius, cutoff value 40 kDa) for continuous renal replacement therapies (CRRT) in the ICU setting. Conclusion PDF may be a useful blood purifi cation therapy for ALF, but PDF should be performed below a MELD score of 30. Reference 1. RENAL Investigators: Intensity of continuous renal-replacement therapy in critically ill patients. N Engl J Med 2009, 361:1627. EMIC2 with citrate anticoagulation in AKI prevention of post cardiac surgical patients. Methods This is a case series of eight patients (mean age 62.7 years, fi ve male, EuroSCORE log 15.61) in cardiac surgery on CPBP for 150 minutes Figure 1 (abstract P396). Delivered dose of RRT each month. Figure 1 (abstract P397). Figure 1 (abstract P396). Delivered dose of RRT each month. Figure 1 (abstract P396). Delivered dose of RRT each month. g p Methods This is a case series of eight patients (mean age 62.7 years, fi ve male, EuroSCORE log 15.61) in cardiac surgery on CPBP for 150 minutes Figure 1 (abstract P397). Figure 1 (abstract P397). Figure 1 (abstract P397). S144 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Plasma fi ltration with dialysis (plasma diafi ltration) in critically ill patients with acute liver failure As the substitute from the additional fl uid line, we added 1,200 ml (150 ml/hour) of fresh frozen plasma followed by 50 ml of 25% albumin considering the loss of albumin by diff usion. As an anticoagulant, nafamostat mesilate (Torii Pharmaceutical Co. Ltd, Tokyo, Japan) was used at a rate of 15 to 25 mg/hour. 1. Benedetto U, et al.: J Thorac Cardiovasc Surg 2010, 140:646-670. 1. Benedetto U, et al.: J Thorac Cardiovasc Surg 2010, 140:646-670. P403 Pharmacokinetics of meropenem during continuous renal replacement therapy in critically ill patients J Solé Violán1, J Ferrer Agüero1, B Sádaba2, S Sancho3, R Zaragoza3, P Luque4, M Nieto4, M López5, F García6, C Hernández6, J Azanza2 1HUGC Dr Negrín, Las Palmas de GC, Spain; 2Universidad de Navarra, Pamplona, Spain; 3Hospital Dr Peset, Valencia, Spain; 4Hospital Clínico, Zaragoza, Spain; 5Hospital General Yagüe, Burgos, Spain; 6Hospital Morales Meseguer, Murcia, Spain Critical Care 2014, 18(Suppl 1):P403 (doi: 10.1186/cc13593) p p g Methods Patients with microbiologically confi rmed severe sepsis/ septic shock and AKI (RIFLE criteria F or more) treated with HCO- CVVHD, started within 12 hours from the diagnosis, were prospectively included in the study. The cumulative vasopressor index (CVI), C-reactive protein levels (CRP), serum lactate concentration (Lac) and central venous oxygen saturation (ScvO2) were measured before (T0h) and at 24 hours and 48 hours after HCO-CVVHD initiation. Data are expressed as the median (range). The Mann–Whitney U test was applied to detect diff erences in CVI, CRP, Lac and ScvO2 at the three time points (statistical signifi cance for P <0.05). Introduction Antibiotic dosing for patients with acute renal failure receiving continuous renal replacement therapy (CRRT) is a clinical challenge. The aim of this study was to investigate the pharmacokinetics of meropenem (M) during CRRT. Methods A prospective and multicenter study was conducted at seven hospitals. Fifteen critically ill patients undergoing either continuous venovenous hemofi ltration (CVVHF) or hemodiafi ltration (CVVHDF) were included. Serum and ultrafi ltrate (UF) levels of M were determined by liquid chromatography. Blood samples were drawn 24 hours after starting CRRT at 08:00 a.m., 09:00 a.m., 10:00 a.m., 01:00 p.m., 06:00 p.m., 20:00 p.m. and 08:00 a.m. of the following day. CRRT clearance (Cl), total amount of M in the UF (MUF), percentage of the dose extracted by CRRT (EF) and the AUC (ng/hour/ml) were calculated. gi Results In 16 ICUs, a total of 16 patients (six cardiac surgery, four abdominal surgery and six medical) met the inclusion criteria and were enrolled in the study. A signifi cant reduction in CRP levels was observed over time: 263 (216 to 358) mg/dl at T0h to 153 (56 to 186) mg/dl at T48h (P <0.05). ScvO2 signifi cantly increased from 45 (40 to 55)% at T0h to 75 (68 to 77)% at T48h (P <0.05). Pharmacodynamics and pharmacokinetics of ciprofl oxacin during sustained low-effi ciency dialysis Pharmacodynamics and pharmacokinetics of ciprofl oxacin during sustained low-effi ciency dialysis fi K Olsen, T Plumb, N Reardon, K Bogard, A Branch-Woods, G Peitz University of Nebraska Medical Center, Omaha, NE, USA Critical Care 2014, 18(Suppl 1):P402 (doi: 10.1186/cc13592) Results The MELD score (34.5 to 28.0; P = 0.005), total bilirubin (10.9 to 7.25 mg/dl; P = 0.048), PT-INR (1.89 to 1.31; P = 0.084), and SOFA score (10.0 to 7.5; P <0.039) were improved 5 days after CPDF therapy. Nine patients were alive and one patient died due to acute pancreatitis, complicated by ALF. The effi cacy of CPDF therapy for maintaining liver function and renal function was not inferior to PE plus CHDF therapy. Parameters of renal function such as the creatinine value were also improved 5 days after CPDF therapy. Circulation parameters such as mean arterial pressure and heart rate were maintained without inotropic and vasopressor support during the CPDF treatment period. The oxygenation index (PaO2/FiO2) as a measure of pulmonary function tended to increase after this treatment. We could employ this treatment without any adverse events, such as infections and unstable hemodynamics. Introduction Little information is available regarding ciprofl oxacin pharmacokinetics and pharmacodynamics in sepsis patients receiving sustained low-effi ciency dialysis (SLED). This study determined the pharmacokinetics and simulated pharmacodynamics of ciprofl oxacin in ICU patients during SLED. Methods This study was a prospective evaluation of ciprofl oxacin pharmacokinetics in patients with sepsis and >18 years of age, urine output <200 ml/day and receiving SLED for at least 8 hours. Following informed consent, plasma samples were collected at baseline and 1, 2, 4, and 8 hours after a ciprofl oxacin 400 mg dose i.v. during SLED and post-SLED therapy at the same times. Dialysate samples were collected at 4-hour intervals during SLED. Pharmacokinetic parameters were determined using WinNonlin and compared between the two periods. Simulated pharmacodynamic parameters were determined for Pseudomonas aeruginosa using MIC = 2. y Conclusion In the present study, CPDF therapy safely supported liver function and generally improved the condition of critically ill patients with ALF. Results A total of seven patients (four male, three female, age 56.9 ± 7.6, APACHE II 26.8 ± 2.4) were enrolled. Ciprofl oxacin was cleared relatively rapidly with a half-life of 6.9 hours and a Ke of 0.108/hour during SLED compared with 11.9 hours and 0.057/hour post-SLED (P <0.05). Simulated pharmacodynamics demonstrated inadequate coverage for P. 1. Morgera S: Crit Care Med 2006, 34:2099-2104. P403 Finally, serum lactate decreased from 5.1 (3.0 to 9.5) mmol/l at T0h to 1.6 (1.0 to 4.6) mmol/l at T48h (P <0.05). Conversely, CVI did not signifi cantly reduce over time (8.2 (4 to 9) at T0h vs. 4.5 (4 to 8) at T48h, P >0.05). g Results Nine patients were treated with CVVHDF and six with CVVHF. M (0.5 to 2 g) was administered every 6 to 12 h by i.v. infusion over 15 minutes. Data (mean and SD) concerning the dialysate fl ow rate (DF; ml/hour), blood fl ow rate (ml/minute) and the average UF rate (ml/ kg/hour) for CRRT techniques are shown in Table 1. Pharmacokinetic Conclusion Our preliminary data show that patients with sepsis- related AKI may benefi t from early treatment with HCO-CVVHD. The modulation of proinfl ammatory and anti-infl ammatory mediators, as previously demonstrated [1], may improve microcirculation, tissue perfusion and cellular oxygenation. Although promising, our results must be confi rmed at the end of the study with larger observations. Finally, a subgroup analysis is absolutely mandatory in order to explore diff erent behaviors of tissue perfusion indexes in diff erent populations of patients. Table 1 (abstract P403). Parameters of CRRT techniques CVVHF (n = 6) CVVHDF (n = 9) DF rate 964/94 Blood fl ow rate 190/33 187/29 UF rate 37.7/5.7 29.8/15.6 Table 1 (abstract P403). Parameters of CRRT techniques Reference S145 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 http://ccforum.com/supplements/18/S1 Normalized Ratio (PT-INR), 5 days after CPDF therapy. Secondary outcomes included Sequential Organ Failure Assessment (SOFA) scores, 5 days after CPDF therapy, and the survival rate 14 days after this therapy. Pharmacodynamics and pharmacokinetics of ciprofl oxacin during sustained low-effi ciency dialysis aeruginosa during SLED with Cmax/MIC ratio 5.7 ± 1.2 and AUC/MIC 77.5 ± 22.3.l P401 P401 Hemodialysis with high cutoff membranes improves tissue perfusion in severe sepsis: preliminary data of the Sepsis in Florence sTudy (SIFT) G Villa, C Chelazzi, S Valente, AR De Gaudio, S Romagnoli University of Florence, Italy Critical Care 2014, 18(Suppl 1):P401 (doi: 10.1186/cc13591) Conclusion Ciprofl oxacin is rapidly cleared during SLED similar to clearance during normal renal function, which may result in adequate pharmacodynamic coverage for some pathogens. Introduction It has been demonstrated that blood purifi cation therapy performed by means of venovenous hemodialysis with high cutoff membranes (HCO-CVVHD) may modulate the host infl ammatory response in septic patients with acute kidney injury (AKI), potentially limiting organ dysfunction. Improvement in hemodynamics and respiratory function has been described during HCO-CVVHD treatment [1]. The Sepsis in Florence sTudy (SIFT) has been designed to evaluate changes in infl ammatory biomarkers and tissue oxygenation/perfusion indexes in septic ICU patients with AKI during HCO-CVVHD.i P406 Long-term outcomes in acute kidney injury patients treated with renal replacement therapy who were alive at hospital discharge V Sergoyne, W De Corte, J Vanhalst, A Dhondt, S Claus, S Oeyen, J Decruyenaere, E Hoste University of Ghent, Belgium Critical Care 2014, 18(Suppl 1):P406 (doi: 10.1186/cc13596) Results Data from 33 patients receiving renal replacement therapy were analysed. Mean time (± SD) of treatment interruption was 1.4 ± 2.7 hours/day due to fi lter changes, transfers for CT scans and surgical procedures. Therefore, 94% of the prescribed dose was delivered. There was a mean of 2.3 fi ltration dose prescriptions per patient over the observation period (due to changing clinical conditions), with a total of 77 dose adaptations. Mean E/ABW was signifi cantly higher than calculated IBW (76.3 kg vs. 61.4 kg), and thus a diff erence of 14.9 kg (95% CI = 9.2 to 20.6 kg). This resulted in a signifi cant diff erence in mean fi ltration dose delivered of 33.1 ml/kg/ hour for E/ABW versus 39.7 ml/kg/hour for IBW respectively (P <0.001). As a consequence, in 29.6% (8/27) of cases where HVHDF (>35 ml/kg/ hour) was prescribed, standard volume haemodiafi ltration (SVHDF) (≤35 ml/kg/hour) was delivered. In 10% (5/50) of cases where SVHDF was prescribed, HVHDF was delivered (P <0.001). Introduction Acute kidney injury (AKI) treated with renal replacement therapy (RRT) in ICU patients is associated with high mortality, chronic kidney disease (CKD) (eGFR <60 ml/minute/1.73 m2, or CKD stage ≥3) and end-stage kidney disease (ESKD). Data on long-term outcomes vary among studies due to diff erences in age, CKD, severity of illness, RRT modality and timing of initiation of RRT. Long-term patient and kidney outcomes in AKI-RRT patients were evaluated in this study. y p y Methods A retrospective study of all consecutive treated AKI-RRT patients in a 50-bed ICU academic hospital from August 2004 to December 2012. Data were retrieved from the electronic ICU, RRT and hospital patient fi les. Long-term outcomes data were obtained by a telephone survey. p Conclusion We conclude that the delivered UF rate in our cohort of patients diff ered signifi cantly depending on measured or calculated body weight. In almost one-third of cases where HVHDF was prescribed, SVHDF was delivered. As many interventions in the ICU are based on IBW and daily weighing of patients is not uniformly practiced, this makes comparison of data diffi cult. Impact of ideal versus estimated body weight on haemofi ltration dosing in critically ill patients with AKI y p y Results During the 9-year study period, a total of 1,291 AKI-RRT patients were included. Short-term mortality at day 30 was 47.2%; mortality at 1 year was 64.3%. Compared with nonsurvivors, 1-year survivors had similar age (65 vs. 67 years, P = 0.077), worse kidney function at baseline (eGFR 46 vs. 52 ml/minute/1.73 m2, P = 0.001; CKD stage ≥3 65% vs. 58%, P = 0.019), a greater proportion was male (69.0% vs. 63.2%, P = 0.048), and more were admitted to the cardiac surgery ICU (39% vs. 46%, P = 0.012). They were less severely ill as illustrated by lower SAPS 2 score at ICU admission (52 vs. 69, P <0.001), and at the time of initiation of RRT, and a lower SOFA score (9 vs. 11; P <0.001). A smaller proportion was on mechanical ventilation (85.1% vs. 89.7%, P = 0.042) and on vasoactive drugs (45.7% vs. 63.9%, P <0.001). Survivors had earlier initiation of RRT (2 vs. 3 days, P = 0.034), and more frequently intermittent RRT was used (84.8% vs. 63.2%; P <0.001). When corrected for gender, age, severity of illness, and modality and timing of RRT, worse baseline kidney function, defi ned as CKD stage ≥3, remained associated with better 1-year survival (odds ratio 1.6, 95% CI: 1.1 to 2.2, P = 0.011). Introduction Acute kidney injury (AKI) in the critically ill is an independent risk factor for adverse outcome [1]. Previously, it was suggested that high-volume haemofi ltration (HVHF) may confer a mortality benefi t and lead to a reduction in organ failure compared with standard ultrafi ltration rates (UF) [2]. This was not confi rmed by a recent investigation [3]. It has also not been determined whether ideal (IBW) or estimated/actual body weight (E/ABW) was used to calculate UF rates. We sought to determine what impact diff erent weight calculations have on delivered UF rates. Methods A retrospective single-centre study in a tertiary referral institution. Continuous venovenous haemodiafi ltration (CVVHDF) was administered according to the patient’s IBW. The delivered UF rate was then calculated both for IBW and E/ABW. The latter was based on measurements obtained at the time of ICU admission. We compared the highest predilution, postdilution and dialysate volume administered each day according to the diff erent weight estimate measurements respectively. Student’s t tests and chi-square tests were used for statistical analysis (P <0.05). 1. Chertow GM, et al.: J Am Soc Nephrol 2005, 16:3365-3370. 2. Ronco C, et al.: Lancet 2000, 355:26-30. 3. Bellomo R, et al.: N Engl J Med 2009, 361:1627-1638. Reference Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S146 Table 2 (abstract P403). Pharmacokinetic parameters for meropenem CVVHF CVVHDF AUC 357/188 433/369 M dose (g/day) 1.7/0.4 2.2/1.4 MUF (mg) 338/104 625/499 EF (%) 28/13 30/26 M Cl (l/hour) 1.2/0.7 1.7/1.5 parameters for M are depicted in Table 2. No diff erences in either EF or M Cl between the two CRRT techniques were observed. Conclusion A signifi cant removal of M by CVVHF/CVVHDF was observed. Dose adjustment is necessary in critically ill patients receiving CRRT. Acknowledgement Supported by EC 81/00469. Table 2 (abstract P403). Pharmacokinetic parameters for meropenem CVVHF CVVHDF AUC 357/188 433/369 M dose (g/day) 1.7/0.4 2.2/1.4 MUF (mg) 338/104 625/499 EF (%) 28/13 30/26 M Cl (l/hour) 1.2/0.7 1.7/1.5 P405 ICU patients treated with RRT for AKI who have chronic kidney disease have better 1-year outcome compared with patients with better kidney function W De Corte1, V Sergoyne2, J Vanhalst3, A Dhondt4, S Claus4, S Oeyen4, J Decruyenaere4, E Hoste4 1AZ Groeninge, Kortrijk, Belgium; 2Stedelijk Ziekenhuis, Aalst, Belgium; 3Sint- Jozefziekenhuis, Izegem, Belgium; 4Ghent University Hospital, Ghent, Belgium Critical Care 2014, 18(Suppl 1):P405 (doi: 10.1186/cc13595) Introduction Chronic kidney disease (CKD) is a risk factor for developing acute kidney injury (AKI) with need for renal replacement therapy (RRT). AKI-RRT is associated with important short-term mortality, and recent data showed there is also important increased risk for 1-year mortality. The aim of this study is to evaluate variables associated with 1-year survival, and in particular the impact of baseline CKD in a cohort of AKI-RRT patients. parameters for M are depicted in Table 2. No diff erences in either EF or M Cl between the two CRRT techniques were observed.i q Conclusion A signifi cant removal of M by CVVHF/CVVHDF was observed. Dose adjustment is necessary in critically ill patients receiving CRRT. g Acknowledgement Supported by EC 81/00469. Methods A single-center observational study in a 50-bed ICU tertiary care hospital. During the study period August 2004 to December 2012, all consecutive adult AKI-RRT patients were included. Data were retrieved from the electronic ICU patient fi le, the electronic hospital patient fi le and the electronic ICU-RRT database. Long-term outcome data were collected by a telephone survey. Impact of ideal versus estimated body weight on haemofi ltration dosing in critically ill patients with AKI Conclusion In ICU patients who had AKI-RRT, 1-year survival was associated with lower severity of illness. Surprisingly, worse kidney function or CKD stage ≥3 was also associated with long-term survival. This eff ect remained present after adjudication for relevant covariates. Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility bundle in adult ICU patients: a preliminary cost analysis Results Study 1: (1) the number of CD4+ T cells was lower and the percentage of Tregs in CD4+ T cells was higher in septic shock patients compared with those without shock. A signifi cant increase in the number of CD4+ T cells, a signifi cant decrease in the percentage of Tregs in the CD4+ T-cell population, and a signifi cant decrease in serum IL-10 levels were observed 24 hours after PMX-DHP in septic shock patients who survived compared with those who did not. (2) IFNγ production by PBMCs was signifi cantly lower in patients with sepsis than in healthy volunteers. IFNγ production by IL-2-stimulated and IL-12-stimulated PBMCs signifi cantly increased after PMX-DHP therapy. Study 2: IFNγ production by PBMCs in patients with sepsis increased signifi cantly in the PMX and PLB groups compared with that in the sepsis group. y G Peitz1, K Dvoracek2, J Sankaranarayanan3, M Balas4, K Olsen1 1University of Nebraska Medical Center, Omaha, NE, USA; 2Avera McKennan Hospital and University Health Center, Sioux Falls, SD, USA; 3University of Conneticut/Hartford School of Pharmacy, Storrs, CT, USA; 4The Ohio State University, Columbus, OH, USA C l C (S l ) P (d / ) y G Peitz1, K Dvoracek2, J Sankaranarayanan3, M Balas4, K Olsen1 1University of Nebraska Medical Center, Omaha, NE, USA; 2Avera McKennan Hospital and University Health Center, Sioux Falls, SD, USA; 3University of Conneticut/Hartford School of Pharmacy, Storrs, CT, USA; 4The Ohio State University, Columbus, OH, USA y Critical Care 2014, 18(Suppl 1):P409 (doi: 10.1186/cc13599) Introduction We previously demonstrated a signifi cant increase in the number of ventilator-free days and reduced the rate of delirium in ICU patients treated with the Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility (ABCDE) bundle. This study investigated whether implementation of the ABCDE bundle was cost- eff ective at an academic medical center.f Introduction We previously demonstrated a signifi cant increase in the number of ventilator-free days and reduced the rate of delirium in ICU patients treated with the Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility (ABCDE) bundle. This study investigated whether implementation of the ABCDE bundle was cost- eff ective at an academic medical center.f Conclusion PMX-DHP directly decreased the number and percentage of Tregs in peripheral blood circulating CD4+ T cells in patients with septic shock. PMX-DHP improved IFNγ production by natural killer (NK)/NKT cells in patients with septic shock. P409 P409 Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility bundle in adult ICU patients: a preliminary cost analysis G Peitz1, K Dvoracek2, J Sankaranarayanan3, M Balas4, K Olsen1 1University of Nebraska Medical Center, Omaha, NE, USA; 2Avera McKennan Hospital and University Health Center, Sioux Falls, SD, USA; 3University of Conneticut/Hartford School of Pharmacy, Storrs, CT, USA; 4The Ohio State University, Columbus, OH, USA Critical Care 2014, 18(Suppl 1):P409 (doi: 10.1186/cc13599) Polymyxin B-immobilized fi ber hemoperfusion therapy improves sepsis-related immunosuppression Table 1 (abstract P408). Multivariate analysis for ICU mortality risk factors P value OR (95% CI) SAPS II score 0.04 1.1 (1.01 to 1.1) Septic shock 0.02 10.4 (1.5 to 71) PMX-DHP 0.04 0.2 (0.1 to 0.9) Source control 0.01 0.1 (0.03 to 0.5) Table 1 (abstract P408). Multivariate analysis for ICU mortality risk factors Introduction Sepsis-induced immunosuppression has long been considered a factor in the late mortality of sepsis patients, but little is known about the immunity of immunocompetent cells and the eff ect of polymyxin B-immobilized fi ber hemoperfusion therapy (PMX- DHP) on sepsis-induced immunosuppression. The present study was designed to evaluate the eff ect of PMX-DHP on recovery from sepsis- related immunodefi ciency. i Methods Patients with septic shock who were treated with PMX- DHP were enrolled in this study. Study 1: (1) numbers of peripheral lymphocytes and CD4+ T cells, especially regulatory T cells (Tregs), and serum cytokine levels were examined to evaluate the eff ects of PMX- DHP in septic shock patients. (2) Peripheral blood mononuclear cells (PBMCs) in these patients were examined to evaluate infl ammatory cytokine production before and after PMX-DHP. The obtained PBMCs were stimulated with interleukin (IL)-2 and IL-12, anti-CD3 antibody, or lipopolysaccharide for 24 hours, and tumor necrosis factor alpha and interferon-gamma (IFNγ) production in the culture supernatants was measured using enzyme-linked immunosorbent assay. Study 2: whole blood from patients with sepsis was incubated with a polymyxin B-immobilized fi lter (cut into small sizes) for small animals for 2 hours (PMX group), or were treated with 200 μg polymyxin B for 2 hours (PLB group), or were not treated (sepsis group). IFNγ production by PBMCs was compared among the three groups. Conclusion EAA is a rapid and reliable method to identify patients who may be treated with polymyxin B hemoperfusion. Source control and extracorporeal endotoxin removal have appeared as two eff ective interventions that should be implemented in the early management of patients with SS&SSh. 1. Cruz DN, Antonelli M, et al.: Early use of polymyxin B hemoperfusion in abdominal septic shock. The EUPHAS randomized controlled trial. JAMA 2009, 301:2445-2452. 1. Cruz DN, Antonelli M, et al.: Early use of polymyxin B hemoperfusion in abdominal septic shock. The EUPHAS randomized controlled trial. JAMA 2009, 301:2445-2452. P407 Polymyxin B-immobilized fi ber hemoperfusion therapy improves sepsis-related immunosuppression A Kimura, S Ono, S Hiraki, R Takahata, H Tsujimoto, M Kinoshita, S Aosasa, K Hatsuse, D Saitoh, K Hase, J Yamamoto National Defense Medical College, Tokorozawa, Japan Critical Care 2014, 18(Suppl 1):P407 (doi: 10.1186/cc13597) Endotoxin activity assay and polymyxin B hemoperfusion use in a cohort of critically ill patients Endotoxin activity assay and polymyxin B hemoperfusion use in a cohort of critically ill patients y SL Cutuli, G De Pascale, V Alicino, S Cicconi, V Di Gravio, D Silvestri, D Giacobelli, E Gasperin, S Marsili, MS Vallecoccia, M Antonelli Sacro Cuore Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P408 (doi: 10.1186/cc13598) Introduction Endotoxin plays a crucial role in the pathogenesis of severe sepsis and septic shock (SS&SSh) [1]. The aim of this study is to analyze the impact of extracorporeal endotoxin removal with polymyxin B hemoperfusion (PMX-DHP) (Toraymyxin®). y y y y Methods All patients admitted to our ICU between 1 April 2011 and 30 June 2013 who developed SS&SSh and underwent endotoxin activity assay (EAA) measurement were retrospectively evaluated. y Conclusion The annual mortality in AKI-RRT hospital survivors is approximately 10% per year during the fi rst 3 years of follow-up. One- third of patients had an increase of CKD stage at 1 year of follow-up and almost one-half of patients who had eGFR >60 ml/minute/1.73 m2 developed CKD. Patients who had an increase of CKD had similar severity of illness, but lower Scr at baseline and at ICU admission, and had later initiation of RRT compared with patients who had stable or decreased CKD stage. y p y Results During the study period, EAA was dosed in 100 patients. Eighty- one patients were aff ected by septic shock. The source of infection was identifi ed in 70.4% of cases (45% abdominal) and the percentage of microbiologically confi rmed episodes was 77% (81% Gram-negatives). The mortality rate was 49%. The mean EAA level was 0.66 ± 0.2, and in 66% of patients the value was higher than 0.6. No signifi cant diff erences were found in terms of SAPS II (P = 0.32) and SOFA score (P = 0.67), according to EAA level (>/≤0.6). Patients with levels >0.6 presented a higher percentage of microbiologically confi rmed infections (84% vs. 66%; P = 0.09). Thirty-two of 66 patients with EAA >0.6 were treated with Toraymyxin®. No complications leading to treatment interruption were recorded and a relevant decrease of cardiovascular SOFA score and lactate levels were observed 72 hours after treatment (P = 0.05 and P = 0.06, respectively). Source control and Toraymyxin® treatment resulted as the only modifi able factors improving the ICU survival rate (Table 1). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 1.4 mg/dl, P = 0.162, respectively 46 vs. 51 ml/minute/1.73 m2) and we observed an increase of CKD stage in 36.0% of patients, a decrease in 36.3%, and stable CKD stage in 27.7%. A total 43.1% of patients with CKD stage <3 at baseline had an increase to CKD stage ≥3 at 1 year. A total 26.6% of patients with CKD stage ≥3 at baseline decreased to CKD stage <3. A total of 8.3% of hospital survivors developed ESKD. Patients with increase of CKD stage had similar age (67 vs. 64 years, P = 0.145), SAPS 2 (57 vs. 51, P = 0.858), and SOFA score (9 vs. 9, P = 0.275) compared with patients with stable or decreased CKD stage. There were no diff erences in type of ICU, modality of RRT, or number of patients treated with vasopressors (44% vs. 47%, P = 0.617) or invasive ventilation (84% vs. 81%, P = 0.496). However, these patients were more probably male (76.9% vs. 65.6%, P = 0.042), had lower Scr at baseline (1.1 vs. 1.7 mg/dl, P <0.001) and at ICU admission (1.7 vs. 2.4 mg/dl, P <0.001), and were started on RRT later (3 vs. 2 days, P = 0.008). P408 Endotoxin activity assay and polymyxin B hemoperfusion use in a cohort of critically ill patients SL Cutuli, G De Pascale, V Alicino, S Cicconi, V Di Gravio, D Silvestri, D Giacobelli, E Gasperin, S Marsili, MS Vallecoccia, M Antonelli Sacro Cuore Catholic University, Rome, Italy Critical Care 2014, 18(Suppl 1):P408 (doi: 10.1186/cc13598) P406 References Results During the study period 1,291 ICU patients were treated with RRT for AKI. Mortality was 47.2% at day 30 and 57.2% at hospital discharge. Mortality in hospital survivors showed an important increase until 3-year follow-up, and a moderate increase later (1 year: 14.4%, 2 years: 20%, 3 years: 35.7%, and 7 years: 39%). In-hospital survivors’ Scr and eGFR at baseline were comparable to 1-year follow-up (1.4 vs. S147 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 An assessment of long-term sleep quality using actigraphy in survivors of critical illness Methods Fifty-nine patients consented to complete the RCSQ for each night of their ICU admission. Alongside this, the nurses who had cared for these patients were asked to assess their patients’ sleep using the RCSQ. These were then matched with their patients’ responses. The Bland–Altman method was applied to assess for agreement between patient and nurse SEIs in order to reveal whether nurses could accurately estimate their patients’ night sleep. Additionally, Cronbach’s alpha was derived to assess for internal consistency. Ethical approval was gained prior to the start of the study.f An assessment of long-term sleep quality using actigraphy in survivors of critical illness K Solverson, C Doig University of Calgary, Canada Critical Care 2014, 18(Suppl 1):P410 (doi: 10.1186/cc13600) University of Calgary, Canada y y Critical Care 2014, 18(Suppl 1):P410 (doi: 10.1186/cc13600) Critical Care 2014, 18(Suppl 1):P410 (doi: 10.1186/cc13600) Introduction Recent studies have suggested that critical illness survivors experience long-term sleep disruption [1,2]. The Actigraph device is a sleep watch that has been shown to have equivalent accuracy to polysomnography and previously used during critical illness to show sleep disruption [3]. Our objective was to assess patients long-term sleep quality using the Actigraph device. Results A total of 126 pairs or RCSQs were gathered. The mean diff erence between nurse and patient SEIs was –0.9, suggesting there is no signifi cant trend regarding nurses overestimating or underestimating patients’ SEIs. In total, 94.2% of nurses’ estimations fell within the limits of agreement. The variability of the diff erences was consistent across the range of averages. Cronbach’s alpha was 0.63 between nurse and patient scores, suggesting questionable reliability between the RCSQ pairs. See Figure 1. g y g g Methods Study patients were selected from a 24-bed multidisciplinary ICU. Thirteen patients who were ≥18 years old, stayed longer than 4 days in the ICU and did not have and acute brain injury were followed up at 2 months post hospital discharge. The Actigraph device was given to patients to take home and worn for 72 hours. Previously validated algorithms were used to analyze sleep and wake cycles [4]. Additionally, patient completed the Pittsburgh Sleep Quality Index (PQSI), as a measure of subjective sleep quality. p g Conclusion The data gathered here demonstrate that nurses are not able to accurately estimate their patients sleep using the RCSQ, and hence alternative methods of sleep monitoring should be developed for routine use. P411 P411 Study to assess whether staff are able to accurately assess sleep quality and quantity in intensive care patients J Patel, J Baldwin, P Bunting, S Laha Royal Preston Hospital, Manchester, UK Critical Care 2014, 18(Suppl 1):P411 (doi: 10.1186/cc13601) P411 Study to assess whether staff are able to accurately assess sleep quality and quantity in intensive care patients J Patel, J Baldwin, P Bunting, S Laha Royal Preston Hospital, Manchester, UK Critical Care 2014, 18(Suppl 1):P411 (doi: 10.1186/cc13601) yi Results Data were analyzed from 146 and 150 patients in the pre- ABCDE and post-ABCDE groups respectively. There were mean decreases of 0.59 MV days and 0.83 delirium days between the pre and post bundle implementation. The mean costs (per patient per ICU stay) of implementing the bundle components were signifi cantly higher in the post-ABCDE cohort versus the pre-ABCDE cohort ($191 vs. $92, P <0.0001), with an additional increase in total costs ($8,930 vs. $8,624, P = 0.0233). Costs to prevent 1 day of MV and to prevent 1 day of delirium were $552 and $368 respectively. In the non-MV patients, the post-ABCDE bundle demonstrated fewer days of delirium than the pre- ABCDE cohort (0.8 vs. 1.13 days, P <0.05) but had higher overall costs ($5,417 vs. $4,546, P <0.05), resulting in a cost of $2,639 to prevent 1 day of delirium in a non-MV patient. Introduction Sleep deprivation is recognised as an important cause of morbidity after ICU admission, but most centres do not routinely assess their patients’ sleep. Considering the invasive nature and costs associated with objective sleep measurements, they are unsuitable for routine use. Subjective measurements off er an easy-to-use and economical alternative, the most well validated of which is the Richards–Campbell Sleep Questionnaire (RCSQ). This can be used to derive an accurate estimation of the sleep effi ciency index (SEI), a well-validated measure of sleep. However, there are several concerns regarding patients reporting their sleep quality and quantity using these questionnaires [1]. Additionally, they cannot be used to assess sleep in sedated or delirious patients. It has been suggested that one way to bypass the drawbacks of patients assessing their own sleep would be to utilise nursing staff [1]. Previous smaller scare studies have since agreed with this suggestion [2]. This study aimed to assess whether staff were able to use the RCSQ to accurately assess their patients’ sleep. P411 Conclusion Implementing the ABCDE bundle in adult ICU patients appears to be a feasible cost-eff ective strategy when considering costs of mechanical ventilation and ICU delirium. References consisted of the following components: daily spontaneous awakening trials (SATs); daily spontaneous breathing trials coordinated with the SATs; delirium monitoring; and early mobility. The study’s primary endpoint was the cost-eff ectiveness of the ABCDE bundle in terms of the bundle’s cost to prevent 1 day of mechanical ventilation (MV) or 1 day of delirium for all patients, as well as for a subgroup of non-MV patients. All cost-eff ectiveness ratios (CERs) were constructed from the hospital’s cost perspective. The economic analyses were carried out in two steps. First, the mean cost per patient per hospital stay and total costs in the pre-ABCDE and post-ABCDE bundle periods were compared. Next, CERs of each respective primary endpoint were computed as incremental costs of implementing the bundle to prevent 1 day of MV and to prevent 1 day of delirium. P <0.05 was considered statistically signifi cant. References 1. Orwelius L, et al.: Crit Care 2008, 12:R97. 2. Lee C, et al.: Intensive Care Med 2009, 35:314-320. 3. Beecroft J, et al.: Intensive Care Med 2008, 34:2076-2083. 4. Hedner J, et al.: Sleep 2004, 27:1560-1566. 5. Buysse D, et al.: J Clin Sleep Med 2008, 4:563-571. References 1. Orwelius L, et al.: Crit Care 2008, 12:R97. 2. Lee C, et al.: Intensive Care Med 2009, 35:314-320. 3. Beecroft J, et al.: Intensive Care Med 2008, 34:2076-2083. 4. Hedner J, et al.: Sleep 2004, 27:1560-1566. 5. Buysse D, et al.: J Clin Sleep Med 2008, 4:563-571. Awakening and Breathing Coordination, Delirium Monitoring and Early Mobility bundle in adult ICU patients: a preliminary cost analysis Therefore, PMX-DHP could improve sepsis-related immunosuppression. Methods A post-hoc cost-eff ectiveness study was done following the before–after ABCDE bundle implementation study. The bundle S148 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 An assessment of long-term sleep quality using actigraphy in survivors of critical illness y Results Sixty-two percent of patients at 2 months post hospital discharge reported poor sleep quality as per the PSQI. The Actigraph results showed patients’ average total sleep time was 6.15 hours, with a sleep effi ciency of 78%. The mean time to fall asleep was 12 minutes. Patients had an average of 11 awakenings per night and were awake for an average of 7 minutes during the awakenings. There were no associations found between patients’ perceived sleep quality and total sleep, sleep effi ciency or sleep disruptions. Patients’ severity of illness, as measured by the APACHE II score, was statistically associated with lower total sleep time (β = –12.6, P = 0.019), reduced sleep effi ciency (β = –1.18, P = 0.042) and higher number of sleep disruptions (β = 0.64, P = 0.023). The number of days ventilated or ICU and hospital length of stay were not statistically associated with the Actigraph sleep parameters. P414 P414 Haemodynamic eff ects of clonidine in an ovine model of severe sepsis with septic acute kidney injury P Calzavacca1, Y Lankadeva2, L Booth2, S Bailey2, M Bailey3, R Bellomo4, CN May2 1AO Melegnano, PO Uboldo, Cernusco sul Naviglio, Italy; 2Melbourne University, Parkville, Australia; 3Monash University, Clayton, Australia; 4Austin Health, Heidelberg, Australia Critical Care 2014, 18(Suppl 1):P414 (doi: 10.1186/cc13604) References 1. Bourne RS, et al.: Crit Care 2007, 11:226. 1. Bourne RS, et al.: Crit Care 2007, 11:226. 2. Kamdar et al.: Am J Crit Care 2012, 21:261-269. Figure 1 (abstract P411). 2. Kamdar et al.: Am J Crit Care 2012, 21:261-269. Figure 1 (abstract P411). g Conclusion Survivors of critical illness have high levels of sleep dysfunction as measured by actigraphy. Patients’ severity of illness while critically ill appears to increase the level of long-term sleep dysfunction experienced. There is discordance between objective and subjective measures of sleep quality, which has been shown previously [5]. Objective measures of sleep quality are needed on a larger number of patients to confi rm these fi ndings. Figure 1 (abstract P411). S149 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P412i nursing staff reported they were signifi cantly more confi dent in titrating opioids after implementation (3 (2; 3) and 3 (3; 4) respectively; P <0.01), despite a lack of signifi cant diff erence in their reported confi dence to assess pain. Compliance was good, with a median daily documentation rate of 66% with the standard being once per 8-hour shift. Median BPS score rank was signifi cantly higher on patient movement (2 (1; 2)) compared with at rest (1 (1; 2); P <0.001) (1 = no pain, 2 = mild pain). No statistically signifi cant diff erence was seen in the length of stay, duration of mechanical ventilation, VAP rate or median sedation exposure. Pre-implementation median opioid administration when looking at morphine (mg) equivalence was 455.8 (203.1; 1,174.8), and post implementation the median increased to 620.3 (218.1; 1,502); however, this did not reach statistical signifi cance (P = 0.235). There was no statistically signifi cant change in the prescribing of analgesic adjuncts. The sample size was underpowered to detect signifi cant diff erences. nursing staff reported they were signifi cantly more confi dent in titrating opioids after implementation (3 (2; 3) and 3 (3; 4) respectively; P <0.01), despite a lack of signifi cant diff erence in their reported confi dence to assess pain. Compliance was good, with a median daily documentation rate of 66% with the standard being once per 8-hour shift. Haemodynamic eff ects of clonidine in an ovine model of severe sepsis with septic acute kidney injury y j y P Calzavacca1, Y Lankadeva2, L Booth2, S Bailey2, M Bailey3, R Bellomo4, CN May2 1AO Melegnano, PO Uboldo, Cernusco sul Naviglio, Italy; 2Melbourne University, Parkville, Australia; 3Monash University, Clayton, Australia; 4Austin Health, Heidelberg, Australia Critical Care 2014, 18(Suppl 1):P414 (doi: 10.1186/cc13604) Introduction In sepsis, sympathetic nerve activity is increased, which helps maintain arterial pressure in the face of nitric oxide-induced vasodilatation. Accordingly, we investigated the haemodynamic eff ects of the centrally acting α-adrenoceptor agonist clonidine in an ovine model of severe sepsis. Conclusion Using standard EEG in this small cohort of mechanically ventilated ICU patients, a deep sedation level was frequently observed. Our preliminary data suggest that simplifi ed EEG with the SedLine system is less accurate than the standard 19-channel EEG to assess the depth of sedation in the ICU setting. Methods A prospective interventional blinded crossover study in 12 Merino ewes with cardiac and renal fl ow probes implanted to continuously measure cardiac output and renal blood fl ow. Arterial pressure was continuously monitored and blood and urine samples were taken. After 24 hours of control, sepsis was induced by an intravenous bolus and continuous infusion of live Escherichia coli for 32 hours. After 24 hours of sepsis, animals were randomly and blindly allocated to vehicle infusion or clonidine (1 μg/ml/kg/minute) for 8 hours. The E. coli infusion was then discontinued, gentamycin 150 mg given i.m. and the animals were followed for 16 hours during recovery. The animals that survived were crossed over to the alternative treatment 2 weeks later. Implementation of the Behavioural Pain Scale in sedated mechanically ventilated patients in a UK ICU J Jennings, R Bourne Sheffi eld Teaching Hospital, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P413 (doi: 10.1186/cc13603) Implementation of the Behavioural Pain Scale in sedated mechanically ventilated patients in a UK ICU J Jennings, R Bourne Sheffi eld Teaching Hospital, Sheffi eld, UK Critical Care 2014, 18(Suppl 1):P413 (doi: 10.1186/cc13603) Results Complete data were collected on eight animals/group, three animals died/group. Hyperdynamic sepsis with hypotension and acute kidney injury of similar degree developed in the two groups. Haemodynamic and renal eff ects of clonidine are shown in Figure 1. Conclusion In ovine hyperdynamic sepsis, clonidine transiently increased urine output without aff ecting creatinine clearance. Results Complete data were collected on eight animals/group, three animals died/group. Hyperdynamic sepsis with hypotension and acute kidney injury of similar degree developed in the two groups. Haemodynamic and renal eff ects of clonidine are shown in Figure 1. Introduction Pain is a common problem in mechanically ventilated patients and assessing pain in the sedated patient is diffi cult, as patients are often unable to verbalise [1]. Behavioural pain assessment tools have been developed and validated for the assessment of pain in this patient group. A behavioural pain assessment tool was implemented as part of a wider ventilator-associated pneumonia (VAP) prevention programme within a large UK ICU. Conclusion In ovine hyperdynamic sepsis, clonidine transiently increased urine output without aff ecting creatinine clearance. P412 Simplifi ed versus standard EEG to measure the depth of sedation in mechanically ventilated ICU patients Introduction The accurate measure of sedation depth among mechanically ventilated ICU patients remains challenging. The Patient State Index (PSI) is a quantitative measure calculated using an algorithm derived from a simplifi ed four-channel EEG. The aim of this study was to examine the value of the PSI to assess the level of sedation, and to verify its accuracy in comparison with the quantitative spectral analysis derived from a standard 19-channel EEG. Methods Using the SEDLine four-channel simplifi ed EEG system (Masimo Corporation, Irvine, CA, USA), which assessed depth of sedation through two frontal leads, we prospectively studied mechanically ventilated sedated ICU patients and examined whether the PSI was accurate to quantify the individual level of sedation. Pain stimuli were applied and changes in PSI were examined and compared with changes in electrical activity (% of delta power), measured by the frontal Fp2–Fp1 electrodes using a standard 19-channel EEG system (Viasys Neurocare, Madison, WI, USA). EEG recordings were performed simultaneously during 20 minutes, and the relationship between PSI and % of delta power was analyzed with the Pearson’s correlation coeffi cient. f Conclusion The BPS was successfully implemented into routine nursing practice and signifi cantly improved nurse confi dence in opioid infusion titration. Analgesic use was not signifi cantly diff erent between evaluation periods, but the results indicate that further education on anticipating pain and treating pain pre-emptively with timely administration of opioids is needed. References References 1. Puntillo K, et al.: Chest 2009; 135:1069-1074. 2. Payen JF, et al.: Crit Care Med 2001; 29:2258-2263. fi Results Ten consecutive patients (mean age 59 ± 12 years) were included. EEG recordings were performed on average 6 ± 6 days from ICU admission. Sedation consisted of propofol (seven patients), midazolam (two patients) or both (one patient). The median PSI was 54 (range 10 to 97), indicating large individual variations in PSI values. In contrast, the median delta power was 86.3% (range 55.2 to 99.4), indicating a deep sedation level. Pain stimuli were associated with a signifi cant increase of PSI value from baseline 51 (range 16 to 96) to 68 (42 to 95) (P = 0.009). However, we found no correlation between the PSI and the % delta power, both at baseline (R = 0.32, P = 0.37) and after pain stimulation (R = 0.17, P = 0.63). The % delta power from frontal electrodes (Fp2–Fp1) was well correlated with that obtained from posterior electrodes (P4–Pz; R = 0.42, P < 0.0001), and remained unchanged after pain stimulation, indeed confi rming a deep sedation level in our patient cohort. References Median BPS score rank was signifi cantly higher on patient movement (2 (1; 2)) compared with at rest (1 (1; 2); P <0.001) (1 = no pain, 2 = mild pain). No statistically signifi cant diff erence was seen in the length of stay, duration of mechanical ventilation, VAP rate or median sedation exposure. Pre-implementation median opioid administration when looking at morphine (mg) equivalence was 455.8 (203.1; 1,174.8), and post implementation the median increased to 620.3 (218.1; 1,502); however, this did not reach statistical signifi cance (P = 0.235). There was no statistically signifi cant change in the prescribing of analgesic adjuncts. The sample size was underpowered to detect signifi cant diff erences. P412 Simplifi ed versus standard EEG to measure the depth of sedation in mechanically ventilated ICU patients T Suys, P Bouzat, A Rossetti, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2014, 18(Suppl 1):P412 (doi: 10.1186/cc13602) P412 Simplifi ed versus standard EEG to measure the depth of sedation in mechanically ventilated ICU patients T Suys, P Bouzat, A Rossetti, M Oddo Lausanne University Hospital, Lausanne, Switzerland Critical Care 2014, 18(Suppl 1):P412 (doi: 10.1186/cc13602) P415f Methods The Behavioural Pain Scale (BPS) [2] was selected and implemented into daily clinical practice supported by a tailored education programme and incorporation into the clinical information system (MetaVision). Questionnaires pre and post BPS implementation were used to assess nursing staff opinions on pain assessment and opioid infusion titration. Four months of data were compared pre and post implementation, examining aspects of patient sedation and analgesia exposure and ventilated patient outcome parameters. 5 Off -label use of clonidine for sedation in Dutch ICUs J Van der Valk, M Zeeman, M Arbouw, H Van den Oever Deventer Hospital, Deventer, the Netherlands Critical Care 2014, 18(Suppl 1):P415 (doi: 10.1186/cc13605) Off -label use of clonidine for sedation in Dutch ICUs J Van der Valk, M Zeeman, M Arbouw, H Van den Oever Deventer Hospital, Deventer, the Netherlands Critical Care 2014, 18(Suppl 1):P415 (doi: 10.1186/cc13605) Off -label use of clonidine for sedation in Dutch ICUs J Van der Valk, M Zeeman, M Arbouw, H Van den Oever Deventer Hospital, Deventer, the Netherlands Critical Care 2014, 18(Suppl 1):P415 (doi: 10.1186/cc13605) Off -label use of clonidine for sedation in Dutch ICUs Introduction Clonidine is an α2-agonist, licensed in most countries for treatment of hypertension. Other pharmacologic characteristics of α2-agonists are sedation, hypnosis, anxiolysis, sympatholysis, and analgesia [1]. Because of these central nervous eff ects, clonidine can Results In the pre-implementation and post-implementation question- naire (response rate of 38% and 37% respectively of nurses surveyed), S150 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P414). Haemodynamic and renal eff ects of clonidine in established sepsis. li h d i Figure 1 (abstract P414). Haemodynamic and renal eff ects of clonidine in established sepsis. varied 10-fold and loading doses 15-fold. Clinical studies are required to establish the safety and effi cacy of clonidine in the ICU setting. References 1. Blaudszun et al.: Anesthesiology 2012, 116:1312-1322. 2. Martin et al.: Crit Care 2005, 9:117-23. varied 10-fold and loading doses 15-fold. Clinical studies are required to establish the safety and effi cacy of clonidine in the ICU setting. References varied 10-fold and loading doses 15-fold. Clinical studies are required to establish the safety and effi cacy of clonidine in the ICU setting. References be used off -label to augment sedation and delirium treatment. To our knowledge there have been no publications evaluating the effi cacy and safety of clonidine in ventilated critically ill adults. A survey in German ICUs showed that clonidine was used for sedation in 62% of units [2]. We undertook an enquiry to investigate the off -label use of clonidine in Dutch ICUs. 1. Blaudszun et al.: Anesthesiology 2012, 116:1312-1322. 2 M ti t l C it C 2005 9 117 23 Methods An inventory was sent by email to 25 ICUs in the Netherlands, determined by the diffi culty to identify a contact person. Comparison of dexmedetomidine and propofol for sedation in patients with traumatic brain injury Results The response rate was 56%. The results are summarized in Table 1. Clonidine was used in all 14 responding ICUs; in 36% this use was reported as often. Licensed use (for hypertension) was 50%. Indications for off -label use were: substance withdrawal (50%), delirium (71%), and sedation (29%). The route of administration was intravenous in all cases. Nine ICUs reported the use of a loading dose, irrespective of indication, varying from 10 to 150 μg, median 150 μg. Ten ICUs reported the use of continuous infusion, varying from 10 to 100 μg/ hour, median 50 μg/hour. Introduction Both propofol and dexmedetomidine decrease systemic blood pressure, heart rate, and cardiac output in a dose-dependent manner. The aim of this study was to compare their safety and effi cacy for intravenous sedation during mechanical ventilation. Introduction Both propofol and dexmedetomidine decrease systemic blood pressure, heart rate, and cardiac output in a dose-dependent manner. The aim of this study was to compare their safety and effi cacy for intravenous sedation during mechanical ventilation. Conclusion Off -label use of clonidine for sedation and treatment of withdrawal symptoms and delirium was common practice in Dutch ICUs. There was a wide range of dosing regimens: infusion schedules Methods Eighty-four patients with traumatic brain injury (Glasgow scale 7 to 8) entered the study, mean age 44 ± 13.37 years. All patients S151 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P415). Indication and doses of clonidine used in Dutch ICUs Table 1 (abstract P415). Indication and doses of clonidine used in Dutch ICUs IC levela Clonidine use Indication Loading dose (μg) Continuous infusion dose (μg/hour) 3 Often Hypertension sedation ns ns 3 Often Hypertension withdrawal; hypertension delirium; sedation 40 to 120 10 to 80 3 Often Substance withdrawal; sedation 150 50 3 Often Substance withdrawal 75 to 150 50 3 Often Substance withdrawal; hypertension; delirium 10 40 to 100 3 Sometimes Delirium ns 30 to 100 3 Sometimes Hypertension; delirium 150 ns 3 Sometimes Substance withdrawal; delirium ns 40 2 Sometimes Substance withdrawal; delirium ns 20 to 50 2 Sometimes Hypertension; sedation 150 40 2 Sometimes Substance withdrawal; hypertension; delirium 150 12 to 25 1 Sometimes Hypertension; delirium 150 ns 1 Sometimes Delirium 50 50 to 100 1 Sometimes Delirium ns ns ns, not specifi ed. Comparison of dexmedetomidine and propofol for sedation in patients with traumatic brain injury aLevel of ICU care, with level 3 being the most advanced. Loading dose (μg) Continuous infusion dose (μg/hour) dexmedetomidine, by evaluation of the systemic vascular system and cardiac function. dexmedetomidine, by evaluation of the systemic vascular system and cardiac function. underwent mechanical lung ventilation. Patients were divided into two groups depending on the type of intravenous sedation. In the fi rst group (n = 42), sedation was performed by intravenous propofol infusion at a dose of 4 to 12 mg/kg/hour. In patients of the second group (n = 42), sedation was carried out by intravenous infusion of dexmedetomidine at a dose of 0.2 to 1.4 mg/kg/hour. The level of sedation was assessed with bispectral index monitoring, targeting index 70. For comparison of methods we evaluated heart rate, blood pressure, SpO2 30, 90 and 180 minutes after the start of sedation. Both groups were matched for sex, age and comorbidity. Methods After animal preparation and under PiCCO monitoring (BL), endotoxemic shock was induced by an intravenous bolus of lipopolysaccharide (LPS, 055:B5) in ketamine-anesthetized rabbits. After fl uid resuscitation and norepinephrine infusion (SD0), animals were randomized to propofol (PROP, n = 8) or dexmedetomidine (DEX, n = 8) sedation, with two incremental doses (SD1 and SD2). The mean arterial pressure (MAP) and central vein pressure (CVP) were monitored. Pulse pressure variation (PPV) and stroke volume variation (SVV) were assessed to evaluate the preload dependency. Global end-diastolic volume, vascular resistances, mean systemic fi lling pressure, cardiac functional index and vascular resistances were assessed at each time point. Normality was assessed by the Kolmogorov–Smirnov test. Two- way analysis of variance for repeated measures was applied, and the Student–Newmann–Keuls post-hoc test when indicated. P <0.05 was considered signifi cant. y Results Thirty minutes after the start of sedation in patients of the fi rst group, HR was 83 ± 11.31 beats/minute, blood pressure was 127 ± 12.87/64 ± 8.54 mmHg, SpO2 was 97 ± 3.01%. In patients of the second group 30 minutes after the beginning of sedation, HR was 87 ± 10.01 beats/minute, blood pressure was 131 ± 11.67/68 ± 8.19 mmHg, SpO2 was 97 ± 2.98%. After 90 minutes in the fi rst group of patients, we observed HR was 81 ± 6.27 beats/minute, blood pressure was 119 ± 11.46/59 ± 4.29 mmHg, SpO2 was 98 ± 2.35%. Comparison of dexmedetomidine and propofol for sedation in patients with traumatic brain injury In the second group, HR was 82 ± 7.31 beats/minute, blood pressure was 94 ± 13.62/55 ± 7.81 mmHg, SpO2 was 97 ± 2.76%. In the fi rst group 180 minutes after the start of sedation, HR was 86 ± 6.19 beats/minute, blood pressure was 105 ± 10.34/54 ± 4.28 mmHg, SpO2 was 98 ± 1.32%. In the second group, HR was 75 ± 6.27 beats/minute, blood pressure was 92 ± 12.54/51 ± 6.91 mmHg, SpO2 was 96 ± 2.91%. i Results At the increasing dose of propofol, both PPV and SVV signifi - cantly increased in SD1 versus SD0 (P <0.01) and SD2 versus SD0 (P <0.001), but only PPV in SD2 versus SD1 (P = 0.024). Dexmedetomidine infusion did not aff ect PPV and SVV. At SD1 and SD2, PPV and SVV were higher in PROP with respect to DEX (P <0.001). Moreover, propofol infusion increased the heart rate and had no eff ects on cardiac contractility and vascular resistances. On the contrary, in the DEX group we recorded a signifi cant decrease in heart contractility and increment of vascular resistances at SD2. Nonetheless, both drugs aff ected the MAP, CVP, mean systemic fi lling pressure, global end-diastolic volume and venous return. 2 Conclusion Using dexmedetomidine at a dose of 0.2 to 1.4 mg/kg/hour for intravenous sedation is safe in terms of hemodynamic stability and blood oxygenation for sedation during mechanical lung ventilation in traumatic brain injury patients. Conclusion In an endotoxemic shock model, after fl uid resuscitation and during norepinephrine infusion, propofol increased more PPV and SVV in comparison with dexmedetomidine. At high dosage the vascular resistances and heart contractility were infl uenced by dexmedetomidine, but not by propofol. P417f P417 Diff erent eff ects of propofol and dexmedetomidine on preload dependency in endotoxemic shock with norepinephrine infusion: a randomized case–control study T Yu1, C Pan1, S Liu1, F Guo1, F Longhini2, Y Yang1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P417 (doi: 10.1186/cc13607) P418 T Yu1, C Pan1, S Liu1, F Guo1, F Longhini2, Y Yang1, H Qiu1 1Zhong-Da Hospital, Southeast University, Nanjing, China; 2Eastern Piedmont University ‘A. Avogadro’, Novara, Italy Critical Care 2014, 18(Suppl 1):P417 (doi: 10.1186/cc13607) Propofol: monitoring for complications l Propofol: monitoring for complications F Yau, M Healy Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P418 (doi: 10.1186/cc13608) Infl uence of increased intracranial pressure on sevofl urane-fentanyl anesthesia in major abdominal surgery N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Critical Care 2014, 18(Suppl 1):P419 (doi: 10.1186/cc13609) Infl uence of increased intracranial pressure on sevofl urane-fentanyl anesthesia in major abdominal surgery N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Critical Care 2014, 18(Suppl 1):P419 (doi: 10.1186/cc13609) N Trembach, I Zabolotskikh Introduction Increased intracranial pressure (ICP) may adversely aff ect sevofl urane anesthesia and the recovery period due to a disturbed cerebral blood fl ow. Eff ect of sevofl urane on cerebral hemodynamics remains controversial and depends on the extent of the initial value of ICP [1]. This study was designed to evaluate the safety of sevofl urane- fentanyl anesthesia during major abdominal surgery in patients with increased ICP. Methods A total of 124 ASA 3 patients, undergoing major abdominal surgery (duration 5.6 (4.1 to 6.4) hours), were divided into two groups: with normal ICP (≤12 mmHg) (N group, 70 patients) and with ICP >12 mmHg (H group, 54 patients). Initial ICP was evaluated by venous ophthalmodynamometry [2]. ICP, mean arterial pressure (MAP) and cerebral perfusion pressure (CPP) were assessed every hour of anesthesia. Time of recovery of consciousness after anesthesia, complications and length of stay in the ICU and in the hospital were also evaluated. Conclusion Deep sedation resulted in greater physician satisfaction with procedural conditions as well as lower BIS scores but no signifi cant diff erence in patient satisfaction compared with conscious sedation. Patients with no recall of the procedure had lower BIS scores at the nadir and end of the procedure. BIS may be a novel tool to monitor procedural depth, allowing proceduralists to better monitor the narrow window between adequate sedation and dangerous oversedation. Results Initial ICP was 8 ± 3 mmHg in the N group and 15 ± 2 mmHg in the H group. During the anesthesia ICP increased in the H group with a total increase of 33% (from 15 ± 2 to 20 ± 3 mmHg (P <0.05)). In the N group ICP was stable without any signifi cant change. Decrease of MAP after induction of anesthesia was similar in the two groups and was stable during anesthesia. CPP was stable in the N group (above 70 mmHg during the anesthesia), but in the H group CPP decreased signifi cantly (from 82 mmHg to 63 mmHg (P <0.05)). Time of recovery of consciousness in the H group was higher (32 ± 6 minutes vs. 20 ± 4 minutes (P <0.05)). Quantifying sedation satisfaction during bronchoscopy using the Bispectral Index Quantifying sedation satisfaction during bronchoscopy using the Bispectral Index Quantifying sedation satisfaction during bronchoscopy using th Bispectral Index J Palminteri, J Dziodzio, D Seder, J Zuckerman, R Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P420 (doi: 10.1186/cc13610) p J Palminteri, J Dziodzio, D Seder, J Zuckerman, R Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P420 (doi: 10.1186/cc13610) Introduction Sedation monitoring with the Bispectral Index (BIS) is uncommon during bronchoscopy but allows for a more objective measure of sedation; we hypothesized that higher BIS scores would correlate with lower patient satisfaction and that lower BIS scores (deep sedation) would result in better patient and physician satisfaction. Introduction Sedation monitoring with the Bispectral Index (BIS) is uncommon during bronchoscopy but allows for a more objective measure of sedation; we hypothesized that higher BIS scores would correlate with lower patient satisfaction and that lower BIS scores (deep sedation) would result in better patient and physician satisfaction. Methods Between October 2012 and August 2013, bronchoscopy using conscious (CS) or monitored anesthetic deep sedation (DS) was monitored with BIS. Medication administration and procedures were time-stamped. Sedation was administered blinded to BIS score. DS cases used propofol infusions, CS cases used bolus midazolam and fentanyl. Providers rated sedation satisfaction, cough, and the ability to perform the intended procedure using a 100 mm visual analog scale at the end of the procedure blind to BIS (0 unsatisfi ed to 100 satisfi ed). Patients were surveyed at 1 hour and 24 hours regarding overall sedation, symptoms, and procedure recall (unpleasant recall 1, no recall 4). Group diff erences were considered statistically signifi cant at P <0.05. p p y Methods Between October 2012 and August 2013, bronchoscopy using conscious (CS) or monitored anesthetic deep sedation (DS) was monitored with BIS. Medication administration and procedures were time-stamped. Sedation was administered blinded to BIS score. DS cases used propofol infusions, CS cases used bolus midazolam and fentanyl. Providers rated sedation satisfaction, cough, and the ability to perform the intended procedure using a 100 mm visual analog scale at the end of the procedure blind to BIS (0 unsatisfi ed to 100 satisfi ed). Patients were surveyed at 1 hour and 24 hours regarding overall sedation, symptoms, and procedure recall (unpleasant recall 1, no recall 4). Group diff erences were considered statistically signifi cant at P <0.05. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 life-threatening complication [1]. An audit done in 2011 highlighted that lipid profi le and electrocardiograms (ECGs) were rarely monitored. We recommended regular monitoring of these parameters when propofol sedation is used for over 3 days and that propofol-sparing agents are considered in these patients at risk of developing PRIS. higher in the H group (6 ± 2 days vs. 4 ± 2 days (P <0.05) and 15 ± 3 days vs. 11 ± 2 days in N group (P <0.05)).l Conclusion Sevofl urane-fentanyl anesthesia in patients with increased ICP was characterized by a delayed recovery with a higher incidence of postoperative delirium and higher length of stay in the ICU and in the hospital. The main cause of this is a decrease of CPP in patients with high ICP due to low craniocerebral compliance. References g p p g Methods In patients who required propofol sedation for 4 days or more, we prospectively monitored: frequency of performing lipid profi le and 12-lead ECG; and frequency of co-administration of a propofol-sparing agent. 1. Dahyot-Fizelier C, et al.: Ann Fr Anesth Reanim 2012, 31:e229-e234. 2. Firsching R, et al.: J Neurosurg 2011, 115:371-374. g Results We collected data from 25 patients. The duration of propofol infusion was 4 to 15 days (mean 8.5 days). Mean propofol dose in the fi rst 4 days was 2.2 mg/kg/hour. Maximum daily propofol dose ranged from 2.1 to 3.8 mg/kg/hour. Lipid profi le was checked in 16/25 (64%) patients (20% in 2011) whilst on propofol sedation. However, these were checked following 3 days continuous infusion in only three patients. The triglyceride level was ≥2.2 mmol/l (very high) on fi rst testing in 75% of patients. All patients had a 12-lead ECG done on admission. Seventeen of 25 (68%) had a further ECG performed whilst on continuous propofol infusion (45% in 2011). Additional ECGs were done in 13/17 patients (zero in 2011). ECG changes that we feel were attributable to propofol occurred in two patients, one of whom developed severe PRIS. Fourteen of 25 (56%) patients (55% in 2011) were on concomitant sedative agents. This included the patient who developed PRIS. P419l Results Twenty-six procedures were monitored, 20 with CS and six endobronchial ultrasound procedures with DS. There was no diff erence with respect to age or gender. The mean doses of midazolam and fentanyl were 5 mg and 85 μg, respectively. BIS values were lower at all predefi ned points of the procedure for DS cases versus CS. Physicians were more satisfi ed with sedation and the lack of cough with DS, but there was no signifi cant diff erence in patient satisfaction between the two groups with regards to overall sedation, procedure-related symptoms or willingness to have repeat bronchoscopy. Patients with no recall had lower nadir BIS scores (46 vs. 71, P = 0.03) and scores at procedure end (76 vs. 94, P = 0.04) compared with those with any recall. There was no diff erence in the doses of midazolam or fentanyl in CS cases despite statistically signifi cant diff erences in patient recall and BIS scores. Junior fellows scored greater satisfaction with sedation, were less bothered by cough and more often felt able to perform the intended procedure compared with senior fellows. Quantifying sedation satisfaction during bronchoscopy using the Bispectral Index Conclusion Awareness of complications from prolonged propofol sedation is increasing amongst our clinicians. This is refl ected in the increased frequency of lipid profi le and ECG monitoring compared with 2011. However, we feel that more regular and routine monitoring is essential to aid early detection of this potentially fatal complication. We recommend that all patients at risk of developing PRIS should have these parameters monitored on a daily basis as standard. Reference 1. Jacobi J, et al.: Clinical practice guidelines for the sustained use of sedative and analgesics in the critically ill adult. Crit Care Med 2002, 30:119-141. 1. Dahyot-Fizelier C, et al.: Ann Fr Anesth Reanim 2012, 31:e229-e234. 2. Firsching R, et al.: J Neurosurg 2011, 115:371-374. Propofol: monitoring for complications F Yau, M Healy Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P418 (doi: 10.1186/cc13608) Propofol: monitoring for complications F Yau, M Healy Royal London Hospital, London, UK Critical Care 2014, 18(Suppl 1):P418 (doi: 10.1186/cc13608) Introduction Septic shock is a status characterized by the simultaneous use of vasopressors and sedative drugs in critical care patients. Little is known about the possible interference of these drugs during shock status. We aimed to clarify how propofol could diff erently aff ect the preload dependency in an endotoxemic model in comparison with Introduction This re-audit aims to test our strategy for monitoring patients on propofol sedation for prolonged periods. Propofol infusion syndrome (PRIS), once established, is diffi cult to treat and currently there is limited guidance on how best to monitor for this potentially Introduction This re-audit aims to test our strategy for monitoring patients on propofol sedation for prolonged periods. Propofol infusion syndrome (PRIS), once established, is diffi cult to treat and currently there is limited guidance on how best to monitor for this potentially S152 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Infl uence of increased intracranial pressure on sevofl urane-fentanyl anesthesia in major abdominal surgery N Trembach, I Zabolotskikh Kuban State Medical University, Krasnodar, Russia Critical Care 2014, 18(Suppl 1):P419 (doi: 10.1186/cc13609) The incidence of postoperative delirium was higher in the H group (22.2% vs. 12.8% in the N group (P <0.05)). There were no signifi cant diff erences between two groups in other complications. Total length of stay in the ICU and in the hospital was P421 P423 P423 Delirium screening, prevention and treatment in the ICU: a systematic review of implementation strategies Z Trogrlic1, M Van der Jagt1, J Bakker1, MC Balas2, EW Ely3, PH Van den Voort4, E Ista5 1Erasmus MC University Medical Center Rotterdam, the Netherlands 2The Ohio State University, Columbus, OH, USA; 3Vanderbilt University Medical Center, Nashville, TN, USA; 4Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands; 5Erasmus MC University Medical Center Rotterdam – Sophia Children’s Hospital, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P423 (doi: 10.1186/cc13613) P423 Delirium screening, prevention and treatment in the ICU: a systematic review of implementation strategies Z Trogrlic1, M Van der Jagt1, J Bakker1, MC Balas2, EW Ely3, PH Van den Voort4, E Ista5 1Erasmus MC University Medical Center Rotterdam, the Netherlands 2The Ohio State University, Columbus, OH, USA; 3Vanderbilt University Medical Center, Nashville, TN, USA; 4Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands; 5Erasmus MC University Medical Center Rotterdam – Sophia Children’s Hospital, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P423 (doi: 10.1186/cc13613) g p Methods A total of 932 patients were admitted to our PICU from 1 October 2011 to 31 March 2013. Of these, 127 paediatric patients were supported on mechanical ventilation on the fi rst day in the PICU and received intravenous analgesics and sedatives. A retrospective review of a prospectively collected database. The statistical method was the Mann–Whitney U test, and P <0.05 was considered statistically signifi cant. Results Twenty-eight patients were scored with the WAT-1 during weaning from the drugs. Median age was 14 months (1 to 73 months) and the most common reasons for ventilation were airway trouble and pneumonia. Eight of 28 were diagnosed withdrawal syndrome, 20 were not. There were no signifi cant diff erences in age, body weight, cumulative morphine and benzodiazepine dose before weaning. In the withdrawal group, the lactate level, catecholamine index, Paediatric Index of Mortality 2 and heart rates were greater when they were admitted to hospital. Further, there are longer length of PICU stay and shorter ventilator-free days for patients with WAT-1 score >3. But all of them are alive at 28 days, so there is no diff erence in 28-day mortality. Conclusion Our study suggest that severely ill paediatric patients tended to suff er from withdrawal syndrome and then resulted in short ventilator-free days and longer PICU stay. But there is no diff erence in mortality. P421 Introduction The occurrence of delirium heralds a circumstance of higher risk of death, longer stay, higher cost, and greater likelihood of long-term brain dysfunction, and yet the majority of ICU patients worldwide do not get routinely monitored for delirium, thus obstructing timely prevention and management strategies. Determination of eff ective implementation strategies regarding screening, treatment and prevention of delirium is critical to address needed modifi cations in the culture of patient care in the ICU. The aim of this study was to summarize the effi cacy of and the barriers to implementation of delirium management. yf y y Conclusion Our study suggest that severely ill paediatric patients tended to suff er from withdrawal syndrome and then resulted in short ventilator-free days and longer PICU stay. But there is no diff erence in mortality. g Methods We searched PubMed, Embase, PsychINFO, Cochrane and CINAHL for studies published between January 2000 and October 2012, and included them when implementation strategies and their effi cacy and/or potential barriers of implementation were described. Results In all studies (n = 34) multifaceted education strategies were used and combined (median: 5; IQR: 4 to 6). Positive results were reported for both process and clinical outcomes: adherence to delirium screening – improvement after implementation compared with the before measurement, by 15 to 57%; only measured after implementation, between 84 and 92%; delirium knowledge (on a 10-point scale; improved from 6.1 and 6.2 before to 8.2 and 7.4 respectively (P <0.001) after implementation); length of stay in the ICU (4.1 vs. 5.9 days; P = 0.21 and 6.3 to 5.35 days; P < 0.009) and hospital stay (12 days vs. 18 days; P = 0.036 and from 55 to 27 days; P <0.0001); and decreased mortality (29.4% vs. 22.9%; P = 0.009; and OR = 0.45; 95% CI = 0.22 to 0.92; P = 0.03). The major barriers found impairing implementation concerned clinicians’ attitude and healthcare professionals’ knowledge. Reference Reference Reference 1. Franck LS, et al.: Pain 2012, 153:142-148. Reference 1. Franck LS, et al.: Pain 2012, 153:142-148. P421 P421 Risk factor of withdrawal syndrome in the paediatric ICU C Tanaka1, N Shimizu2, O Staito2, M Motomura2, I Watanabe2 1Japanese Musashino Red Cross Hospital, Tokyo, Japan; 2Tokyo Metropolitan Children’s Centre, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P421 (doi: 10.1186/cc13611) Introduction Critically ill paediatric patients supported on mechanical ventilation frequently receive analgesia and sedation. Iatrogenic withdrawal syndrome occurs with the abrupt discontinuation or too rapid weaning of opioids and benzodiazepines. We induce the Introduction Critically ill paediatric patients supported on mechanical ventilation frequently receive analgesia and sedation. Iatrogenic withdrawal syndrome occurs with the abrupt discontinuation or too rapid weaning of opioids and benzodiazepines. We induce the S153 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 merits further investigation and the decision to insert the epidural catheter should be discussed with the patient considering both local experience and legal dispute in case of medical complications. merits further investigation and the decision to insert the epidural catheter should be discussed with the patient considering both local experience and legal dispute in case of medical complications. Withdrawal Assessment Tool-1 (WAT-1) [1] to evaluate children during weaning from analgesics and sedatives. The patient is diagnosed with withdrawal syndrome when the score is 3 or >3. We compared the subjects who ever had a score over 3 and those with lower scores and assessed the risk factors and outcome of withdrawal syndrome between two groups. P422 Epidural analgesia reduces perioperative myocardial infarction and all-cause mortality after cardiac surgery: but at least 25 epidural hematomas have already happened G Landoni, M Greco, I Francesca, D Taddeo, O Saleh, A Belletti, A Putzu, R Lembo, A Zangrillo Vita-Salute San Raff aele University, Milan, Italy Critical Care 2014, 18(Suppl 1):P422 (doi: 10.1186/cc13612) Introduction Epidural analgesia on top of general anesthesia in cardiac surgery might improve relevant clinical outcomes but the incidence of epidural hematoma is under-reported. Introduction Epidural analgesia on top of general anesthesia in cardiac surgery might improve relevant clinical outcomes but the incidence of epidural hematoma is under-reported. Methods An international web-based, online, viral, anonymous survey, a systematic review of the literature, and a meta-analysis of randomized and matched studies were performed.i p g Conclusion Implementation strategies used suggest a strong potential for multifaceted strategies to be able to aff ect both process and clinical outcomes. The majority of studies focused only on implementation of delirium screening. There is a knowledge defi cit regarding effi cacy of well-prepared implementation of integral delirium management considering screening, prevention, and treatment preceded by analysis of barriers. Further research on the extent and contents of implementation interventions aimed at integral delirium management such as those described in the recent ABCDE bundle and PAD guideline are necessary. Results Nine epidural hematomas were identifi ed in 198 published manuscripts. The risk of epidural hematoma (9:13,429) calculated on all published evidence might be therefore estimated to be 1:1,492 (95% confi dence interval (CI) = 1:2,857 to 1:833). Through an anonymous, web-based, viral, international survey, we identifi ed at least 16 further nonpublished epidural hematomas together with at least 72,400 epidural analgesia catheters positioned in cardiac surgery in the last 20 years. The risk of epidural hematoma (25:85,829) is therefore 1:3,436 (95% CI = 1:2,325 to 1:5,076) including both published and unpublished data. Out of the 66 randomized and case-matched studies, 57 trials reported the incidence of all-cause mortality at the longest available follow-up with a signifi cant reduction in the epidural group (59/3,137 (1.9%) in the epidural group vs. 108/3,246 (3.3%) in the control arm, RR 0.64 (95% CI 0.48 to 0.85), P = 0.002, NNT = 69). P424f A nutritional protocol and personalized support reduce the cumulative caloric defi cit of cardiac surgery patients A nutritional protocol and personalized support reduce the cumulative caloric defi cit of cardiac surgery patients E De Waele, K De Bondt, J Czapla, J Nijs, D Nguyen, PM Honoré, H Spapen Universitair Ziekenhuis Brussel, Jette, Belgium Critical Care 2014, 18(Suppl 1):P427 (doi: 10.1186/cc13617) i g y p E De Waele, K De Bondt, J Czapla, J Nijs, D Nguyen, PM Honoré, H Spapen Universitair Ziekenhuis Brussel, Jette, Belgium Critical Care 2014, 18(Suppl 1):P427 (doi: 10.1186/cc13617) Introduction Providing adequate feeding in cardiac surgery patients is gaining importance [1]. The aims were to assess the preoperative nutritional status, to compare postoperative energy needs with eff ectively delivered amounts, and to evaluate the impact of a dietitian on optimizing postoperative energy balances. Methods Following ethical approval, the practice of measuring GRVs and the subsequent management of EN was retrospectively reviewed over a 3-month period, September to December 2012, and prospectively reviewed over a 3-month period, September to December 2013, following the change in practice. Recorded parameters included: length of ICU admission; admitting diagnosis; duration of EN; GRVs; prescribed calories; delivered calories via an enteral route; requirement for PN; duration of PN; and cost of total parenteral nutrition (TPN). Methods A prospective interventional study in adult patients after elective CABG and/or heart valve surgery. The patients’ nutritional risk was determined by the NRS 2002 and the Malnutrition Universal Screening Tool (MUST) [2]. A dedicated dietitian managed and assisted the nutritional approach. For each patient, global energy intake (intentional and non-intentional) and balance (diff erence between energy target and global caloric intake) were calculated daily during and after ICU stay until hospital discharge. The Harris–Benedict equation was used to calculate daily energy requirements. When energy delivery did not reach 60% of calculated needs, a protocol- driven nutritional intervention was initiated. Results The change of practice in terms of GRVs and management of EN led to an increased percentage of prescribed calories that was delivered. There was a reduced requirement for PN and reduced cost of TPN also associated with this change in practice. Conclusion Increasing the threshold of GRV prior to reducing or stopping EN can result in increased calories delivered to ICU patients and a reduced requirement for and cost of TPN. Results Two hundred patients were enrolled during a 10-month period, representing 2,690 study-days. Mean age was 67 ± 11 years. Early enteral feeding in the septic critically ill patient: evaluation of our feeding protocol h d k l l b l k M Theodorakopoulou, I Dimopoulou, S Karambi, A Strilakou, A Diamantakis, S Orfanos, A Armaganidis University Hospital of Athens Greece ‘Attikon’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P426 (doi: 10.1186/cc13616) M Theodorakopoulou, I Dimopoulou, S Karambi, A Strilakou, A Diamantakis, S Orfanos, A Armaganidis University Hospital of Athens Greece ‘Attikon’, Attiki, Greece Critical Care 2014, 18(Suppl 1):P426 (doi: 10.1186/cc13616) Introduction It has been established that early enteral nutrition in critically ill patients improves overall outcome and mortality. In our unit, feeding protocols were established based on the ESPEN recommendations and have been implemented for the last 2 years. The purpose of this study was to evaluate the compliance of our septic patients’ nutritional approach with our feeding protocols. Conclusion Close monitoring of caloric intake shouldered by interventions based on a predefi ned algorithm under supervision of a dedicated nutrition team restricts the caloric defi cit in cardiac surgery patients. References Increased threshold for gastric residual volumes and impact on nutrition in the ICU J Brohan, M Richardson, S O Riain Limerick Regional Hospital, Limerick, Ireland Critical Care 2014, 18(Suppl 1):P425 (doi: 10.1186/cc13615) Conclusion Although the initiation of early enteral feeding seems adequate for a good number of septic patients on D0, is still far off for a signifi cant percentage of those patients on D3 and is even worse on D7. The caloric goal achievements were better on D3 but very suboptimal on D7. There was no association, however, between nutritional status and compliance with the feeding protocols. It is therefore mandatory to follow daily the nutritional therapy of the septic patient and not rely only on the feeding protocols. Introduction Gastric residual volumes (GRVs) as measured at regular intervals are considered a marker of tolerance of enteral nutrition (EN). There is controversy surrounding this practice, however. The absolute value for the designated cutoff value for GRVs varies widely in the literature. No prospective randomized control trials have suggested that their use improves patient outcomes in the ICU. It has also not been shown, therefore, that GRVs are an accurate predictor of aspiration or pneumonia. However, the use of GRVs as a guide to the continuation of EN can result in a reduction in the percentage of goal calories received by patients. Prior to February 2013, in our ICU the threshold of GRVs prior to withholding of EN was 200 ml. This tolerated volume was increased to 500 ml in February 2013, in compliance with recent guidelines. By increasing the acceptable GRVs to this level, the aim was to increase the percentage of goal calories received by patients via the enteral route. P427 A nutritional protocol and personalized support reduce the cumulative caloric defi cit of cardiac surgery patients In total, 42.5% of the patients had a NRS 2002 score >3 and were considered to be nutritionally at risk. MUST identifi ed a high, medium, or low risk in respectively 1%, 4% and 95% of subjects. Mean energy requirement was 2,046 ± 347 kcal and mean daily intake 1,452 ± 335 kcal. Sixty-two percent of the caloric need was met during the entire hospital stay. Nutritional intervention was necessary in 52% of cases: oral feeding supplementation for 546 study-days, enteral feeding: 401 days, parenteral feeding: 367 days. This reduced the mean cumulative caloric defi cit to 9,085 kcal. P424 Eff ect of enteral and/or parenteral glutamine supplementation on mortality and morbidity in the critically ill H Sungurtekin, I Ozturk, B Beder, S Serin Pamukkale University Medical Faculty, Denizli, Turkey Critical Care 2014, 18(Suppl 1):P424 (doi: 10.1186/cc13614) P424 Eff ect of enteral and/or parenteral glutamine supplementation on mortality and morbidity in the critically ill H Sungurtekin, I Ozturk, B Beder, S Serin Pamukkale University Medical Faculty, Denizli, Turkey Critical Care 2014, 18(Suppl 1):P424 (doi: 10.1186/cc13614) Conclusion Epidural analgesia on top of general anesthesia in cardiac surgery might reduce the incidence of all-cause mortality (NNT 69). The incidence of epidural hematoma in this setting is 1:3,436 (95% CI = 1:2,325 to 1:5,076) including both published and unpublished data. In fact, we identifi ed at least 25 epidural hematomas that occurred so far from the following countries: Belgium (n = 1), Brazil (n = 1), France (n = 1), Germany (n = 2), India (n = 2), Italy (n = 1), Japan (n = 2), Korea (n = 1), Malaysia (n = 1), Norway (n = 2), Russia (n = 3), Sweden (n = 1), the UK (n = 3), and the USA (n = 4). Even if from the public health point of view the benefi ts seem to encourage the use of epidural analgesia in cardiac surgery with a possible reduction in perioperative mortality, this topic Introduction In this study we aimed to compare the eff ectiveness of enteral, parenteral and combined enteral–parenteral glutamine supplementations in the nutrition of critical care patients. Methods This is a single-center, randomized controlled clinical trial. During the 5-day study period, all patients received standard enteral nutrition product and were divided into three groups, including parenteral glutamine (Group I), enteral glutamine (Group II) and enteral + parenteral glutamine (Group III) supplementations. Blood S154 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Reference 1. Estívariz CF, et al.: J Parenter Enteral Nutr 2008, 32:389-402. P425 1. Estívariz CF, et al.: J Parenter Enteral Nutr 2008, 32:389-402. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 met the CCM criteria for sepsis upon admission to the ICU. APACHE II score, SOFA score, weight, BMI and nutritional status were calculated. Patients were initiated for enteral feeding based on the established feeding protocol within 48 hours of admission. The feeding status of the patient was recorded on the start day (D0), day 3 (D3) and day 7 (D7). Factors aff ecting the feeding process and its progression were also recorded biochemistry, rates of infections, length of stay in the ICU and duration of mechanical ventilation were evaluated. biochemistry, rates of infections, length of stay in the ICU and duration of mechanical ventilation were evaluated. Results Sixty patients were included in this study. There was no statistically signifi cant diff erence for biochemical values between the diff erent feeding groups. Frequency of infections ranged as Group II >Group III >Group I and mortality as Group II = Group III >Group I. Length of stay in the ICU and duration of mechanical ventilation were signifi cantly longer in Group II than the others. Results The patient mean age was 71.4 ± 12.2. LOS in the ICU was 9 to 21 days. Based on BMI, 18% of the patients were malnourished upon admission. APACHE II was 26 ± 7.8 and SOFA was 9.2 ± 4.6. The mortality rate was 42.5%. Enteral nutrition started early in 64 (77.1%) of the patients (D0), on day 3 (D3) 29 (45.31%) patients met their caloric goals and on day 7 (D7) only 18 (28.1%) patients achieved their caloric goals. Discontinuation of enteral feeding was mainly due to procedures, whereas late start and/or decreased hourly intake were due to GI complications, GI intolerance, excessive diarrhoea and hemodynamic instability. There was no association between compliance with the feeding protocol and the LOS, nutritional status, severity or disease progression. gi y g p Conclusion Although mortality was not signifi cantly diff erent between groups, parenteral glutamine administration causes less stay of ICU and mechanical ventilation. This needs a more powerful randomized controlled study [1]. P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) Introduction The renal system compensates respiratory disorders of acid–base equilibrium by modifying the urinary electrolyte composition [1]. Such a condition mainly involves changes in urinary ammonium excretion as a reaction to prolonged periods of acid–base disequilibrium, leaving the renal response to acute derangements unexplored. We aimed to determine the acute variations of urinary ammonium [NH4+]u, sodium [Na+]u, potassium [K+]u and chloride [Cl–]u concentrations following controlled minimal respiratory acid– base imbalances, and to further investigate whether urinary anion gap ([AG]u = [Na+]u + [K+]u – [Cl–]u) and sodium and chloride diff erence ([Na+]u – [Cl–]u) might unveil an early activation of the renal response. Conclusion The level of vitamin C was markedly decreased. Replacement of vitamin C should be considered for the homeless who visit the emergency department after alcohol ingestion. u u Methods Patients admitted to the ICU after major surgery were enrolled, during intubation and sedation. Patients with chronic renal failure were excluded. A urinary catheter was connected to the quasi- continuous urinary analyzer KING®, measuring [NH4+]u, [Na+]u, [K+]u and [Cl–]u over time. Based upon the arterial pH (pHa) at entrance, patients were randomly assigned to controlled hypoventilation (30% reduction of minute ventilation if pHa ≥7.40) or hyperventilation (30% increase of minute ventilation if pHa <7.40) for 2 hours. Samples for blood gas analysis were collected every 30 minutes. P429 Acid–base disorders according to the Stewart approach in septic patients J Szrama, P Smuszkiewicz, I Trojanowska University Hospital, Poznan, Poland Critical Care 2014, 18(Suppl 1):P429 (doi: 10.1186/cc13619) Introduction ICU patients often develop acid–base disorders. In clinical practice, there are several methods (for example, Henderson– Hasselbalch) used to interpret acid–base data, but most of them provide little information and fail to identify the cause of the problem. Stewart’s approach which is based on physicochemical principles has growing popularity among clinicians in critical care. This method includes three independent variables that determine plasma pH (strong ion diff erence (SID), PCO2, and total weak acid concentration – mainly albumins and phosphate). Introduction ICU patients often develop acid–base disorders. In clinical practice, there are several methods (for example, Henderson– Hasselbalch) used to interpret acid–base data, but most of them provide little information and fail to identify the cause of the problem. y y Results Thirty patients were enrolled; 20 were hypoventilated, 10 hyperventilated. Ramadoss J, et al.: Can J Physiol Pharmacol 2011, 89:227-231. References 1. Cresci G, et al.: Nutrition intervention in the critically ill cardiothoracic patient. Nutr Clin Pract 2012, 27:323-334. p 2. Lomivorotov VV, et al.: Prognostic value of nutritional screening tools for 2. Lomivorotov VV, et al.: Prognostic value of nutritional screening tools for patients scheduled for cardiac surgery. Interact Cardiovasc Thorac Surg 2013, 16:162-168. Methods A prospective study was done on a 24-bed mixed ICU over a period of 18 months. Eighty-three patients ≥18 years were included in the study. All patients were dependent on mechanical ventilation and patients scheduled for cardiac surgery. Interact Cardiovasc Thorac Surg 2013, 16:162-168. S155 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P428 P428 Vitamin B and C levels of homeless patients who visit the emergency department with alcohol ingestion HJ Lee, JH Shin, E Kang, J Jung, DK Kim SMG-SNU Boramae Medical Center, Seoul, South Korea Critical Care 2014, 18(Suppl 1):P428 (doi: 10.1186/cc13618) ninety-three ABG results met the criteria of metabolic acidosis, 473 were categorized into the metabolic alkalosis group and 324 results were within the range value of BE according to the Henderson–Hasselbalch concept. In the metabolic acidosis group (BE <–2), 34.7% of the results had elevated lactate concentration, 100% revealed hypoalbuminemia, 96.9% had Cl/Na ratio >0.75 revealing SID acidosis, while 42.5% met the criteria of SIG acidosis. In the normal range BE group, 21.3% revealed lactate concentration >2 mmol/l, 100% had hypoalbuminemia, 98.4% had Cl/Na ratio >0.75 revealing SID acidosis and hyperchloremia, while 14.5% showed SIG acidosis. The analysis of the ABG with BE >2 group showed that 18.4% had elevated lactate concentration, 99.1% revealed hypoalbuminemia, 88.8% had Cl/Na ratio >0.75 (SID acidosis) and 4.6% showed SIG acidosis. ninety-three ABG results met the criteria of metabolic acidosis, 473 were categorized into the metabolic alkalosis group and 324 results were within the range value of BE according to the Henderson–Hasselbalch concept. In the metabolic acidosis group (BE <–2), 34.7% of the results had elevated lactate concentration, 100% revealed hypoalbuminemia, 96.9% had Cl/Na ratio >0.75 revealing SID acidosis, while 42.5% met the criteria of SIG acidosis. In the normal range BE group, 21.3% revealed lactate concentration >2 mmol/l, 100% had hypoalbuminemia, 98.4% had Cl/Na ratio >0.75 revealing SID acidosis and hyperchloremia, while 14.5% showed SIG acidosis. P430 P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) Table 1 (abstract P428). Reference Lower than Higher than Mean (SD) range reference (%) reference (%) Vitamin B1 158.1 (80.2) 59 to 213 3.1 13.8 Vitamin B12 741.5 (432.2) 200 to 950 2.7 22.6 Vitamin B6 51.9 (53.8) 20 to 202 23.1 1.9 Vitamin C 15.57 (13.4) 26.1 to 84.6 82.7 0 Conclusion The level of vitamin C was markedly decreased. Replacement of vitamin C should be considered for the homeless who visit the emergency department after alcohol ingestion. References The analysis of the ABG with BE >2 group showed that 18.4% had elevated lactate concentration, 99.1% revealed hypoalbuminemia, 88.8% had Cl/Na ratio >0.75 (SID acidosis) and 4.6% showed SIG acidosis. Introduction Vitamins are essential micronutrients and depletions are reported for chronically ill patients. It is well known that the general nutrition status of the homeless is poor, especially for heavy alcoholics. But there were few data about the actual vitamin status of homeless patients. We want to evaluate the vitamin levels of homeless patients. Methods This study was conducted at a single urban teaching hospital emergency department. We performed a retrospective chart review of blood vitamin B1, B12, B6 and C levels of homeless patients. These vitamins are a common supplement in our center and sometimes blood levels are drawn if the patient is drunk, needs i.v. hydration and has cachexic features. Conclusion In critically ill patients with the BE values <–2, with BE in the range of normal value and BE >2 we observed complex acid–base disturbances with coexistence of hyperchloremic acidosis, hyperlactatemia, SIG acidosis and alkalosis caused by hypoalbuminemia. The Stewart approach is more eff ective in detecting acid–base disturbances and quantifying individual components of acid–base abnormalities and provides a detailed insight into their pathogenesis. Results During study periods, vitamin levels were checked for 156 patients. The number of male patients was 146 (94%) and the mean age was 50 ± 10.2. Vitamin C levels were 15.8 ± 1.3 mg/l. For 84 patients, levels of vitamin C were decreased. For vitamin B1 (152 ± 7.2 nmol/l), vitamin B12 (725.5 ± 35.4 pg/ml), and vitamin B6 (50.3 ± 5.5 ng/ml), there were three, two and 23 patients below the reference ranges respectively. See Table 1. P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) At 2 hours from ventilation change, pHa was respectively decreased from 7.44 ± 0.02 to 7.34 ± 0.02 and increased from 7.37 ± 0.03 to 7.44 ± 0.02 (P <0.001) in the two groups. Mean [NH4+]u rose by 2.6 ± 3.3 mEq/l in hypoventilated patients and fell by 2.5 ± 2.4 mEq/l in the hyperventilated (P <0.001). No diff erence in mean [K+]u was observed at any time, while [Na+]u and [Cl–]u progressively decreased in both groups (P <0.05). [Na+]u was reduced by 48 ± 43 mEq/l during hypoventilation and by 32 ± 41 mEq/l during hyperventilation (P = 0.14), while [Cl–]u decreased by 29 ± 69 and by 46 ± 66 mEq/l respectively (P = 0.93). Whereas [AG]u did not diff er between groups, [Na+]u – [Cl–]u variation of hyperventilated patients was greater than that of hypoventilated (17 ± 34 vs. –18 ± 54 mEq/l, P <0.05). Stewart’s approach which is based on physicochemical principles has growing popularity among clinicians in critical care. This method includes three independent variables that determine plasma pH (strong ion diff erence (SID), PCO2, and total weak acid concentration – mainly albumins and phosphate). Methods The prospective analysis of arterial blood gases (ABG) was performed according to the Henderson–Hasselbalch approach and the Stewart method. The results were categorized into three groups according to the Henderson–Hasselbalch concept and the BE values: BE <–2, metabolic acidosis; BE between –2 and 2, normal values; BE >2, metabolic alkalosis. The aim of the study was to compare the effi cacy of the traditional Henderson–Hasselbalch approach with acid–base disturbances with the Stewart concept in the population of critically ill septic patients. Conclusion During acute respiratory modifi cations, changes in urinary ammonium can be observed within the fi rst 2 hours even while maintaining arterial pH within a physiologic range. This response seems to be better associated with changes in the diff erence between urinary sodium and chloride rather than anion gap. Reference Results The analysis included 990 arterial blood gases taken from 43 consecutive septic patients admitted to the ICU. One hundred and Ramadoss J, et al.: Can J Physiol Pharmacol 2011, 89:227-231. P431 Admission hypomagnesemia as a mortality predictor in medical critically ill patients y p A Permata Sari, C Pitoyo, D Aditianingsih, C Rumende Medical Faculty Indonesia University, Jakarta Pusat, Indonesia Critical Care 2014, 18(Suppl 1):P431 (doi: 10.1186/cc13621) y p A Permata Sari, C Pitoyo, D Aditianingsih, C Rumende Medical Faculty Indonesia University, Jakarta Pusat, Indonesia Critical Care 2014, 18(Suppl 1):P431 (doi: 10.1186/cc13621) Introduction Magnesium is the second most abundant intracellular cation and serves as a cofactor in more than 300 enzymatic reactions. Hypomagnesemia is a common electrolyte imbalance in critically ill patients; yet it is frequently overlooked. Previous studies highlighted the relationship between hypomagnesemia and mortality in these patients [1,2]. This study was carried out on patients admitted to the critical care unit and seeks to fi nd admission hypomagnesemia’s role as a 28-day mortality predictor in medical critically ill patients. References P432 Impact of reduced frequency of phosphate testing on detected phosphate levels and phosphate prescription in critical care D Hepburn, H Roberts, S Zouwail University Hospital of Wales, Cardiff , UK Critical Care 2014, 18(Suppl 1):P432 (doi: 10.1186/cc13622) Eff ect of albumin and total protein concentration on plasma sodium measurements in the ICU Results This study was conducted consecutively from April to July 2013. Most subjects were male (62%) with mean age 52.6 ± 15.93 years. The occurrence of sepsis (33.3%) and cardiac disturbances (24.7%) were the most common problems among patients. The mean MSOFA score for the hypomagnesemics was higher than the normal group (4.91 ± 4.19 vs. 3.77 ± 3.52). Subgroup analysis in the MSOFA 0 to 7 group had signifi cant P = 0.015 (chi-square) and crude RR = 2.2 (95% CI = 1.19 to 4.06). From survival analysis, the survival mean of hypomagnesemia patients was lower than the normal group (19.4 vs. 22.8 days), and so was the survival percentage (52.7% vs. 74.7%). Introduction Direct ion-selective electrode without dilution is the most eff ective method for determination of the concentration of the ionized fraction of sodium [1]. We tested the hypothesis that the diff erence between indirect and direct sodium assays would be related to the plasma albumin concentration or the total protein concentration. Methods A retrospective observational study was conducted in which plasma sodium concentrations, from 101 paired venous and arterial samples from patients admitted to the ICU, were respectively analyzed on the arterial blood gas (ABG) analyzers (direct ion-selective electrode) and the central laboratory auto-analyzers (indirect ion-selective electrode). A paired t test was performed comparing the central laboratory and ABG measurements. Correlation and regression analysis were performed between total protein concentration, albumin and the diff erences between the central laboratory and ABG assays for sodium. Conclusion There was a high prevalence of hypomagnesemia in medical critically ill patients and it was associated with a high mortality rate. Reference Reference 1. Burtis C, et al.: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Elsevier; 2006:983-1018. Reference 1. Burtis C, et al.: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Elsevier; 2006:983-1018. P434 p p p p p p Results A total of 4,253 tests were performed in phase 1, and 3,641 in phase 2 – a reduction of 612 (14.4%). The ICU workload was similar in both phases. There was no signifi cant diff erence in mean phosphate levels or detected incidence of abnormal phosphate levels in phase 1 versus phase 2. Mean level was 1.13 ± 0.40 mmol/l versus 1.14 ± 0.43 mmol/l (P = 0.42). Severe hypophosphatemia (<0.4 mmol/l) was relatively uncommon: n = 18 in phase 1 (0.42% of tests) versus 19 (0.52%), P = 0.42, in phase 2. Mild hypophosphatemia (0.4 to 0.7 mmol/l) was frequent, with 1,203 episodes (28.2% of tests) versus 1,088 (29.8%), P = 0.42. Hyperphosphatemia (>1.5 mmol/l) was also common in both phases with 608 detected episodes (14.3% of tests) versus 572 (15.7%), P = 0.49. Pharmacy data show phosphate replacement fell signifi cantly, from 687 prescriptions in phase 1 to 395 in phase 2, with drug cost- savings estimated at £1,430. 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. P430 Changes in urinary electrolytes during acute respiratory acid–base modifi cations M Ferrari Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P430 (doi: 10.1186/cc13620) S156 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P431 Conclusion Reducing testing from daily to three times weekly was not associated with a signifi cant change in mean phosphate levels nor with detection of abnormally high/low phosphate levels. Daily testing, however, is associated with higher rates of phosphate prescription. It is known that there is signifi cant diurnal variation in serum phosphate [2] and we speculate that mild hypophosphataemia self-corrects without intervention. Treating mild hypophosphatemia may therefore not be indicated. P431 Admission hypomagnesemia as a mortality predictor in medical critically ill patients A Permata Sari, C Pitoyo, D Aditianingsih, C Rumende Medical Faculty Indonesia University, Jakarta Pusat, Indonesia Critical Care 2014, 18(Suppl 1):P431 (doi: 10.1186/cc13621) Impact of reduced frequency of phosphate testing on detected phosphate levels and phosphate prescription in critical care D Hepburn, H Roberts, S Zouwail University Hospital of Wales, Cardiff , UK Critical Care 2014, 18(Suppl 1):P432 (doi: 10.1186/cc13622) p p p p D Hepburn, H Roberts, S Zouwailf University Hospital of Wales, Cardiff , UK Introduction Phosphate is essential for cell and bone function [1]. In critical illness, hypophosphatemia is common and practice is often to correct even mild derangement [2]. Its supplementation has signifi cant cost and risk including hypotension and hypocalcaemia. We investigated whether changing frequency of routine serum phosphate testing had eff ects on the detected incidence of abnormal plasma levels and prescription of phosphate. y Conclusion The diff erence between indirect and direct sodium assays was found to be statistically related to the plasma albumin concentration and the total protein concentration. Although the relationship was found to be weak, the total protein concentration should be monitored when measuring sodium by indirect ion-selective electrode. Methods This was a service improvement project using observational, anonymous data. We collected data on serum phosphate levels in a 33-bed ITU over two 6-month periods before and after introduction of a new testing regime (phases 1 and 2). In phase 1, phosphate levels were tested daily. In phase 2, phosphate levels were tested three times per week. Replacement was at clinical discretion. Pharmacy data on phosphate prescription were compared for both phases. Reference References References 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. 1. Grissom CK, et al.: Am Med Assoc 2010, 4:277-284. 2. Safavi MR, et al.: MEJ Anesth 2007, 19:645-660. y y Results The central laboratory sodium measurement was, on average, 1.46 mmol/l more than the ICU assay, limits of agreement 1.18 to 1.74 mmol/l greater, P <0.001. Bland–Altman analysis of the central laboratory result minus the ICU sodium measurement had limits of agreement of 1.3 to –4.2 mmol/l. The correlation between the assay diff erences and total protein concentration and albumin were respectively r = 0.24 (P = 0.01) and r = 0.20 (P = 0.04). The diff erence in plasma sodium concentration between the assays increased as the plasma concentration albumin or total protein concentration decreased (respectively: r2 = 0.04 and r2 = 0.06).f References 1. Geerse et al.: Treatment of hypophosphatemia in the intensive care unit. Crit Care 2010, 14:R147. 1. Geerse et al.: Treatment of hypophosphatemia in the intensive care unit. Crit Care 2010, 14:R147. 2. Pocock et al.: Diurnal variations in serum biochemical and haematological measurements. J Clin Pathol 1989, 42:172-179. 2. Pocock et al.: Diurnal variations in serum biochemical and haematological measurements. J Clin Pathol 1989, 42:172-179. y y y Methods This is a cohort prospective study with prognostic research recruiting 150 critically ill patients in a major tertiary hospital. Blood samples were collected for estimation of serum total magnesium on admission, and then the patients were followed over 28 days. Cardiac surgery alters the sensitivity of the dynamic interaction between the pituitary and adrenal glands Methods Inclusion criteria: mechanical ventilation previewed at ICU admission >8 days and mortality predicted at ICU admission over 13% (SAPS II >32 points). After the clinical run-in period of 48 hours, samplings were taken to measure baseline blood melatonin and TAC, beginning from the third ICU night and day (midnight and 02:00 p.m.). At 8:00 p.m. of the third ICU day, treatment with 3 mg + 3 mg melatonin (Group M) or placebo (Group P) began: each patient received two tablets per day, at 8:00 p.m. and midnight, until ICU discharge. Further samplings were taken during the early (fourth night and day) and the late (eighth night and day) treatment phases. Melatonin was measured through an ELISA essay; TAC was measured with a specifi c kit. B Gibbison1, J Walker1, G Russell1, K Stevenson2, Y Kershaw1, G Asimakopoulos2, GD Angelini1, SL Lightman1 1University of Bristol, UK; 2University Hospitals Bristol NHS FT, Bristol, UK Critical Care 2014, 18(Suppl 1):P435 (doi: 10.1186/cc13625) Introduction Both ACTH and cortisol are secreted in a diurnal rhythm. Underlying this is an ultradian rhythm of discrete pulses [1] as a result of the feedforward:feedback interactions between cortisol and ACTH [2]. These pulses are critical for normal function; pulsatile and constant infusions yield diff erent transcriptional responses [3] and patients on optimal (nonpulsatile) glucocorticoid replacement have twice the age-related mortality of the general population [4]. We have now characterised the ultradian rhythm and pituitary–adrenal interaction of patients undergoing coronary artery bypass grafting (CABG). i Results Sixty-four critically ill patients were enrolled. Endogenous melatonin was shown decreased in the run-in period and in the placebo group compared with healthy subjects. All patients reached satisfying pharmacological values with enteral administration: peak of blood melatonin value (pg/ml) was 2,514 (982 to 7,148) for the M group versus 20 (15 to 62) for the P group (P <0.001) during the fi rst treatment night, while maintaining signifi cant diff erences also during the daytime: 51 (23 to 180) M group versus 14 (11 to 24) P group (P = 0.001). The same trend was observed in the late treatment samples (eighth ICU day). Regarding TAC values (nmol Trolox equivalent/μl plasma), a signifi cant diff erence was highlighted during the night (107 (97 to 123) M group vs. 61 (42 to 89) P group, P <0.001), but not during the daytime (37 (30 to 69) M group vs. Cardiac surgery alters the sensitivity of the dynamic interaction between the pituitary and adrenal glands 28 (25 to 50) P group, P = 0.092). Correlation between melatonin and TAC: Spearman’s rho = 0.33 (P <0.001). Methods Twenty male patients presenting for elective CABG (on-pump and off -pump) were recruited. Blood samples were taken for 24 hours from placement of the fi rst venous access. Cortisol was sampled every 10 minutes, ACTH was sampled every hour and cortisol binding globulin (CBG) was sampled at baseline, at the end of operation and at the end of the 24-hour period. Results Cortisol and ACTH were pulsatile throughout the perioperative period and the cortisol–ACTH interaction persists (Figure 1). The sensitivity of this interaction (calculated by the ratio of cortisol to ACTH pulse amplitude) changed at about 8 hours post surgery such that the adrenal sensitivity to ACTH increased. Conclusion Enteral administration of melatonin was adequate in the early phase of critically illness, with pharmacokinetics similar to published data [2]. The administration of melatonin seems to increase the TAC, with a possible meaningful role in critically ill patients. References Figure 1 (abstract P435). Figure 1 (abstract P435). 1. Mistraletti G, et al.: Intensive Care Med 2011, 37:S181. 2. Mistraletti G, et al.: J Pineal Res 2010, 48:142-147. 1. Mistraletti G, et al.: Intensive Care Med 2011, 37:S181. 2. Mistraletti G, et al.: J Pineal Res 2010, 48:142-147. References 1. Veldhuis JD, et al.: Am J Physiol 1989, 257:E6-E14. 2. Walker JJ, et al.: Proc Biol Sci 2010, 277:1627-1633. 2. Walker JJ, et al.: Proc Biol Sci 2010, 277:1627-1633. 3. Conway-Campbell BL, et al.: J Neuroendocrinol 2010, 22:1093-1100. 4. Bergthorsdottir R, et al.: J Clin Endocrinol Metab 2006, 91:4849-4853. g Results Central diabetes insipidus (CDI) developed in 106 patients. Against the background of substitution therapy, we did not observe the development of complications, hypovolemia, hypernatremia, or hyperosmolality. Out of 48 cases of CSWS, in 12 cases the symptoms interleaved with the symptoms of CDI. For 24 patients, CSWS was complicated by depressed consciousness, for three patients by convulsions against the background of hypovolemia, hyponatremia, or hypo-osmolality. No correlation between BPN level and absence of CSWS symptoms was found. 3. Conway-Campbell BL, et al.: J Neuroendocrinol 2010, 22:1093-1100. 4. Bergthorsdottir R, et al.: J Clin Endocrinol Metab 2006, 91:4849-4853. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Russian Polenov’s Neurosurgical Institute from 2001 to 2012. Patients were between 16 and 55 years old. A total of 142 patients were operated for brain tumor, 72 of them of basal–supratentorial localization; eight severe brain trauma; 62 of the hemorrhagic type of stroke, one herpes encephalitis. We excluded from this study the patients with heart and renal failure receiving diuretics. We measured BP, HR, CVP, hourly and daily urine output, level of K and Na in plasma, brain natriuretic peptide (BNP) one to four times a day, and levels of K and Na in urine in single and daily servings. All patients were receiving dexamethasone at a dose between 8 and 32 mg/day as an anti-edema therapy, and thus levels of ACTH and cortisol were not investigated. Conclusion Both cortisol and ACTH remain pulsatile during and after cardiac surgery and the pituitary–adrenal interaction persists, although the sensitivity of the adrenal glands changes throughout the perioperative period. Our study shows that endogenous glucocorticoid levels reach very high oscillating levels following cardiac surgery, which not only invalidate the interpretation of point measures of adrenal function to diagnose adrenal insuffi ciency but also demonstrate that constant infusions of hydrocortisone are unphysiological. References Melatonin blood values and total antioxidant capacity in critically ill patients p LA D’Amato, G Mistraletti, D Longhi, IR Piva, F Marrazzo, C Villa, M Tozzi, R Paroni, E Finati, G Iapichino Università degli Studi di Milano, Milan, Italy Critical Care 2014, 18(Suppl 1):P436 (doi: 10.1186/cc13626) Conclusion In patients with acute brain trauma we observed two syndromes causing water–electrolyte disturbance – CDI and CSWS. CSWS generally is more severe than CDI. We could not determine a correlation between BPN level and the absence of presence of the CSWS symptoms. Introduction Endogenous melatonin is decreased in critically ill patients; oral supplementation could help them to cope with sleep disruption and sepsis. Among patients who took part in a trial on melatonin and need for sedation [1], some were studied with blood sampling to describe their melatonin blood values and the relationship with total antioxidant capacity (TAC) in plasma. Cardiac surgery alters the sensitivity of the dynamic interaction between the pituitary and adrenal glands B Gibbison1, J Walker1, G Russell1, K Stevenson2, Y Kershaw1, G Asimakopoulos2, GD Angelini1, SL Lightman1 1University of Bristol, UK; 2University Hospitals Bristol NHS FT, Bristol, UK Critical Care 2014, 18(Suppl 1):P435 (doi: 10.1186/cc13625) Main causes of water–electrolyte disturbances in patients with acute brain injury: central diabetes insipidus and cerebral salt wasting syndrome Main causes of water–electrolyte disturbances in patients with acute brain injury: central diabetes insipidus and cerebral salt wasting syndrome L Tsentsiper, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint Petersburg, Russia Critical Care 2014, 18(Suppl 1):P434 (doi: 10.1186/cc13624) L Tsentsiper, N Dryagina L Tsentsiper, N Dryagina Russian Polenov’s Neurosurgical Institute, Saint Petersburg, Russia Critical Care 2014, 18(Suppl 1):P434 (doi: 10.1186/cc13624) Russian Polenov’s Neurosurgical Institute, Saint Petersburg, Russia Critical Care 2014, 18(Suppl 1):P434 (doi: 10.1186/cc13624) Introduction Water–electrolyte disturbances are one of the most common complications of acute brain injury of various origins, threatening the life of the patient and requiring timely correction. In this work we studied the structure of water–electrolyte complications in patients in the neurological intensive care with acute brain injury. Methods We analyzed 259 cases of water–electrolyte disturbances that developed in patients treated in the Department of Intensive Care of S157 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Evaluation of blood glucose control in ICU patients with Space GlucoseControl: a European study Evaluation of blood glucose control in ICU patients with Space GlucoseControl: a European study J Blaha, on behalf of the SGC NIS Study Group General University Hospital, Prague, Czech Republic Critical Care 2014, 18(Suppl 1):P438 (doi: 10.1186/cc13628) p p g p Methods Adult surgical patients with planned ICU admission of ≥24 hours at four medical centers were consented. Following admission to the ICU, the skin of an upper arm was cleaned and a 6 mm diameter site was prepared with controlled micro-abrasion using the Symphony CGM system. A transdermal CGM sensor containing glucose oxidase was applied. Following a 1-hour warm-up period, a calibration was performed. Blood samples were obtained from a radial artery catheter approximately every hour, centrifuged to plasma, and glucose was measured using a YSI 2300 STAT Plus Glucose Analyzer (reference BG). A maximum of 30 reference BG samples were collected for each patient. Samples were collected as frequently as every 15 minutes for trend analysis. CGM was prospectively calibrated every 4 hours. All treatment decisions were based on BG alone. Safety was assessed by visual inspection of the site using a dermatological scale following sensor removal. A study was defi ned as evaluable for CGM sessions >16 hours. Results Thirty-two subjects completed the study. Additional subjects were not considered evaluable due to early discharge from the ICU, failure or early removal of the radial artery catheter, or administration of intravenous acetaminophen. The study cohort was 19% female, 28% diabetes, 56% cardiac surgery, with a mean age of 65 ± 13 years. Overall mean absolute relative diff erence between CGM and reference BG was 12.5%. Continuous glucose error-grid analysis, which assesses point and trend accuracy, showed 98.2% of readings in the A zone (clinically accurate) and 1.2% in the B zone (benign errors). Glucose values ranged from 49 to 324 mg/dl. No device or study-related adverse events were reported. y J Blaha, on behalf of the SGC NIS Study Group y J Blaha, on behalf of the SGC NIS Study Group y p General University Hospital, Prague, Czech Republic y p , g , p Critical Care 2014, 18(Suppl 1):P438 (doi: 10.1186/cc13628) Introduction Regardless of the ongoing debate on optimum target ranges, glycaemia control (GC) remains an important therapeutic goal in critically ill patients. Dozens of diff erent insulin protocols for ICUs have been developed with diff erent complexity, eff ectiveness, blood glucose (BG) variability and safety. P437 3 Continuous prediction of glucose-level changes using an electronic nose in critically ill patients JH Leopold, RT Van Hooijdonk, LD Bos, T Winters, PJ Sterk, A Abu-Hanna, MJ Schultz Academic Medical Center, Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P437 (doi: 10.1186/cc13627) Introduction Many if not most critically ill patients are treated with insulin during their stay in the ICU [1]. Intensive monitoring of the blood glucose level is a prerequisite for effi cient and safe insulin titrations Introduction Many if not most critically ill patients are treated with insulin during their stay in the ICU [1]. Intensive monitoring of the blood glucose level is a prerequisite for effi cient and safe insulin titrations S158 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 management (enteral, parenteral or both) was carried out at the discretion of the each centre. in these patients [2]. Current continuous glucose measurement techniques rely on subcutaneous glucose measurements [3] or measurements in blood [4]. We hypothesized that changes in volatile organic compound (VOC) concentrations in exhaled breath refl ect changes in the blood glucose level. Changes in VOC concentrations can be analyzed continuously using a so-called electronic nose (eNose) [5]. Our aim was to investigate exhaled breath analysis to predict changes in glucose levels in intubated ICU patients. Results Seventeen centres from nine European countries included a total of 508 patients. During the study a total of 29,575 BG values were entered into the SGCs and the same number of recommendations were rendered. The mean time-in-target was 77.5 ± 20.9%. The mean proposed next measurement time was 2.0 ± 0.5 hours. Only four episodes of hypoglycaemia <2.2 mmol/l occurred (0.01% of measurements).fi g p Methods Exhaled breath was analyzed in 15 intubated ICU patients who were monitored with a subcutaneous CGM device. eNose results were compared with subcutaneous glucose measurements and linear regression models were built, including subject-specifi c models, and whole-sample models. The models were validated using temporal validation by training the model on the fi rst 75% of measurements and prospectively testing on the last 25% of measurements. Performance of the models was measured using an R2 value, Clarke error grids (CEG) and rate-error grid analysis (R–EGA). Conclusion SGC is a safe and very effi cient system to control BG in ICU patients. P439 P439 Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients J Joseph1, M Torjman2, J Reich3, A Furnary4, S Nasraway3, M McNamara-Cullinane5, D Olson5, D Walton5 1Thomas Jeff erson University Hospital, Philadelphia, PA, USA; 2Cooper University Hospital, Camden, NJ, USA; 3Tufts Medical Center, Boston, MA, USA; 4Providence Heart and Vascular Institute, Portland, OR, USA; 5Echo Therapeutics, Philadelphia, PA, USA Critical Care 2014, 18(Suppl 1):P439 (doi: 10.1186/cc13629) P439 Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients J Joseph1, M Torjman2, J Reich3, A Furnary4, S Nasraway3, M McNamara-Cullinane5, D Olson5, D Walton5 1Thomas Jeff erson University Hospital, Philadelphia, PA, USA; 2Cooper University Hospital, Camden, NJ, USA; 3Tufts Medical Center, Boston, MA, USA; 4Providence Heart and Vascular Institute, Portland, OR, USA; 5Echo Therapeutics, Philadelphia, PA, USA Critical Care 2014, 18(Suppl 1):P439 (doi: 10.1186/cc13629) Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients J Joseph1, M Torjman2, J Reich3, A Furnary4, S Nasraway3, M McNamara-Cullinane5, D Olson5, D Walton5 1Thomas Jeff erson University Hospital, Philadelphia, PA, USA; 2Cooper University Hospital, Camden, NJ, USA; 3Tufts Medical Center, Boston, MA, USA; 4Providence Heart and Vascular Institute, Portland, OR, USA; 5Echo Therapeutics, Philadelphia, PA, USA Critical Care 2014, 18(Suppl 1):P439 (doi: 10.1186/cc13629) Evaluation of Symphony CGM, a non-invasive, transdermal continuous glucose monitoring system for use in critically ill patients Conclusion Exhaled breath prediction of glucose levels seems promising. However, performance of the current models is too low to be used in daily practice. References 1. Schultz MJ, et al.: Intensive Care Med 2010, 26:173-174. 2. Preiser J-C, et al.: Intensive Care Med 2009, 35:1738-1748. 3. Kosiborod M, et al.: Crit Care 2013, 17(Suppl 2):462. 4. Schierenbeck F, et al.: Diabetes Technol Ther 2013, 15:26-31. Introduction Glycemic control in the ICU has been shown to reduce morbidity, mortality and length of stay. However, current methods of blood glucose (BG) monitoring are invasive, intermittent and labor- intensive. Continuous glucose monitoring (CGM) has potential to improve safety/effi cacy of BG control. The performance of a non- invasive, transdermal CGM system (Symphony CGM; Echo Therapeutics, Philadelphia, PA, USA) was evaluated in post-surgical ICU patients. 5. Röck F, et al.: Chem Rev 2008, 108:705-725. References 1. Jacobi J, et al.: Crit Care Med 2012, 40:3251-3276. 2. Cordingley JJ, et al.: Intensive Care Med 2009, 35:123-128. 3. Blaha J, et al.: Diab Care 2009, 32:757-761. 4. Amrein K, et al.: Diabetes Technol Ther 2012, 14:690-695. g y Results Changes in VOC concentrations were associated with changes in subcutaneous glucose levels. R2 performance had a mean value of 0.67 (0.34 to 0.98) for subject-specifi c models, and a mean value of 0.70 (0.52 to 0.96) for the model for the whole sample. However, when externally validating the model, the predictive performance dropped to a mean R2 of 0.19 (0.00 to 0.70) for subject-specifi c models, and 0.04 for the model for the whole sample. Point accuracy in CEG was mostly good with >99% in zones A and B; trend accuracy, as visualized with R–EGA, was low. Time-course evaluation of blood glucose changes in response to insulin delivery in critically ill patients Very Very Variable low Low Control High high Number of patients 11,471 70,106 521,839 77,423 187,553 ICU mortality* (%) 55 31 13 18 26 ICU LOS* 7 6 4 5 6 P <0.001. y y p F Bass1, S Bird1, N Hammond1, J Myburgh2, S Finfer1 1Royal North Shore Hospital, St Leonards, NSW, Australia; 2The George Institute for Global Health, Sydney, Australia Critical Care 2014, 18(Suppl 1):P440 (doi: 10.1186/cc13630) Introduction Intravenous insulin by infusion is commonly used for blood glucose control in the ICU and blood glucose is almost exclusively monitored by intermittent sampling. The rate of change in blood glucose concentration [BG] when the insulin infusion rate is changed is not known, and as a result the optimum time to measure [BG] after changing the infusion rate is unclear. during the fi rst 24 hours from admission were recorded. BG control value was defi ned as BG (≥4.0 ≤9.9 mmol/l). Other BG levels were defi ned as: very low, ≤2.2 mmol/l (≤40 mg/dl); low, >2.2 ≤3.9 mmol/l (40 to 70 mg/dl); high, ≥10.0 <11.1 mmol/l (180 to 200 mg/dl); and very high, ≥11.1 mmol/l (200 mg/dl). g g Methods Following institutional ethics approval and patient consent, using a GluCath Continuous Glucose Monitoring system sensor deployed via a radial artery catheter we studied the change in [BG] in response to a 1 unit/hour increase in the insulin infusion rate during the fi rst 48 hours after cardiac surgery. [BG] was recorded every 10 seconds. Insulin was infused at a concentration of 1 unit/5 ml/hour via a volumetric pump. We recorded [BG] for 2 hours after changing the insulin infusion rate. Data aff ected by artifacts produced by blood draws and subsequent fl ushing of the arterial catheter were excluded and linear interpolation was used to estimate missing [BG] data.i Results There was an increased incidence of mortality in those patients with at least one BG measure below 3.9 mmol/l (70 mg/dl) compared with those without (Table 1). There was a link between BG levels and LoS for surviving patients with the longest hospital stays (critical care and total hospital) experienced by those with BG levels below 2.2 mmol/l (40 mg/dl). g Conclusion There is a strong association between BG levels during admission and mortality and LoS outcomes. Evaluation of blood glucose control in ICU patients with Space GlucoseControl: a European study Although comparison of existing protocols is diffi cult due to signifi cant diff erences in processes and outcome measures, computerized clinical decision support systems generally achieved better GC with consistently lower hypoglycaemia rates than that achieved with paper-based protocols [1]. The enhanced Model Predictive Control (eMPC) algorithm, developed by the CLINICIP group, is the eff ective clinically proven protocol, which has been successfully tested at multiple institutions on medical and surgical patients with diff erent nutritional protocols [2,3]. The eMPC models the behaviour of glucose and insulin in ICU patients with a variable sample interval based on the accuracy of the BG prediction. It has been integrated in the B.Braun Space GlucoseControl (SGC) system, which allows direct data communication between pumps and Space Control with the incorporated eMPC algorithm. Although SGC is already clinically used in dozens of ICUs worldwide, there are few only published experiences with its use [4]. Methods The primary objective of this multicentre European non- interventional study was to evaluate the performance (effi ciency) of SGC under routine conditions in adult ICU patients requiring BG control. The primary endpoint was the percentage of time within the target range, and secondary outcome measures were the frequency of hypoglycaemic episodes and BG measurement intervals. Patients in this trial were assigned to the target range 4.4 to 8.3 mmol/l. Nutritional Conclusion The Symphony CGM system demonstrated clinically relevant accuracy and excellent safety in a variety of patients and ICU environments. Future studies are needed to determine whether Symphony CGM can be used to direct therapy and improve BG control in this patient population. S159 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P441). Critical care outcomes Table 1 (abstract P441). Critical care outcomes P440 P440 Time-course evaluation of blood glucose changes in response to insulin delivery in critically ill patients F Bass1, S Bird1, N Hammond1, J Myburgh2, S Finfer1 1Royal North Shore Hospital, St Leonards, NSW, Australia; 2The George Institute for Global Health, Sydney, Australia Critical Care 2014, 18(Suppl 1):P440 (doi: 10.1186/cc13630) Time-course evaluation of blood glucose changes in response to insulin delivery in critically ill patients Although it is not possible to make the link with causation from our dataset, we present results from the largest single dataset of critical care unit patients [1,2]. Results There were fi ve episodes where the insulin infusion increased by 1 unit/hour. [BG] decreased modestly for 50 minutes, after which there was marked interpatient variability in subsequent [BG] trend (Figure 1). The median (range) change in BG (mmol/l) at 30 minutes: –0.5 (–0.7/+0.1), 60 minutes: –0.1 (–0.7/+1.0), 90 minutes: –0.3 (–0.7/+0.6) and 120 minutes: –0.5(–1.2/+2.9). Acknowledgement Funding from Edwards Lifesciences. References 1. Van den Berghe G, et al.: Intensive insulin therapy in critically ill patients. N Engl J Med 2001, 345:1359-1367. g 2. The NICE-SUGAR Study Investigators: Intensive versus conventional glucose control in critically ill patients. N Engl J Med 2009, 360:1283-1297. Conclusion Within the fi rst hour the change in [BG] was consistent but beyond this time it was highly variable. Further studies are needed to understand the dynamics of [BG] in response to changes in insulin infusion rates, but these data suggest [BG] should be checked hourly until stable. 2. The NICE-SUGAR Study Investigators: Intensive versus conventional glucose control in critically ill patients. N Engl J Med 2009, 360:1283-1297. First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICUi Introduction The aim of this study was to determine the safety and effi cacy of treating patients using a novel intravenous continuous glucose monitoring (CGM) system (GlucoClear™; Edwards Lifesciences). The practical benefi t of controlling blood glucose in the critically ill remains contentious, largely because of the lack of tools to adequately measure and therefore manage levels in real time. A system that is able to provide instant, constantly calibrated, accurate values is a major bonus to ICU care and we report here the fi rst clinical use of a novel CGM system directly used to manage postoperative glycaemia. Methods All consecutive consenting adult patients undergoing cardiac surgery involving cardiopulmonary bypass and requiring postoperative insulin (>98% of patients) were enrolled in the study with a target enrolment of 100. Blood glucose was measured via a dedicated peripheral intravenous catheter with values reported every 5 minutes. The primary outcome was the number of data points in the target glycaemic control range (4.4 to 8.0 mmol/l), using a dynamic insulin protocol. Secondary endpoints include mean glucose levels, time in range and number of hypoglycaemic and hyperglycaemic episodes.i Results For the fi rst 45 patients, mean age was 67.7 years (male 52.8%), 54% had undergone valve surgery with or without CABG and the majority (67%) had no history of diabetes. The CGM sensor was typically sighted in the forearm or hand (90.5%) and was resited on 7.5% occasions. Median monitoring time in the ICU was 28.4 hours. Mean glycaemic value was 6.8 mmol/l. From the possible 18,609 glucose values (one per 5 minutes), 16,808 values were recorded (90.3%). Overall, 13,389/16,808 values (79.6%) were within the target range. No hypoglycaemic episodes (≤2.2 mmol/l) were recorded at any point. There were 219/16,808 values (1.3%) in the hyperglycaemic range (≥10.0 mmol/l). Impact of corticosteroid administration in septic shock on glycemic variability L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, D Mirea3, I Grintescu1 1Clinical Emergency Hospital of Bucharest, Romania; 2National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P445 (doi: 10.1186/cc13635) Conclusion This new CGM system enabled signifi cantly improved glycaemic control despite the challenges of working with an entirely new system of glucose control in a unit with over 200 nurses. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 refi ned to maximise the benefi t of GGM. Overall, the evidence from the fi rst applied clinical use of this novel CGM showed that proper safe, tight glycaemic control can be achieved. Further investigations are required to demonstrate the reduction in morbidity and mortality using CGM, and we plan to embark on further studies to address this. autocalibration unit. We have now completed a 34-patient trial using this device and present the data collected from this study. autocalibration unit. We have now completed a 34-patient trial using this device and present the data collected from this study. y Methods The study used GlySure sterile, single-use sensors and a 5-lumen 9.5-Fr CVC device, allowing the fl uorescence optical-based sensor to be placed into the patient’s right internal jugular vein. The screen data were blinded to the bedside staff . Data from the monitor were later compared with sample measurement from the Yellow Springs (YSI) glucose analyzer. The data accuracy was measured using the mean absolute relative diff erence (MARD), an error calculation tool. Results The device met the primary safety and eff ectiveness endpoints of the trial. The 456 sample values recorded by the monitor based on 8-hour calibrations were correlated with samples taken from the YSI and the MARD for the study was 9.40%. The analysis showed that 89.23% of the data fell within the A zone of the Clark error grid, with the rest falling within the B zone. g Conclusion The results demonstrate a good correlation with the accepted standard of blood glucose determination in ICU practice. Early detection of glycemic excursions can provide carers with the opportunity for an early intervention and thus achieve the elusive target of TGC around the chosen target range. 1. Mackenzie I, Whitehouse T, Nightingale P: Intensive Care Med 20 37:435-443. 2. Krinsley JS: Crit Care Med 2008, 36:3008-3013. 3. Krinsley JS: Crit Care 2011, 15:R173. 4. Krinsley JS: Crit Care 2013, 17:R37. 5. Mulavisala KP, Gopal PB, Crane B: Crit Care 2012, 16: P175. 1. Mackenzie I, Whitehouse T, Nightingale P: Intensive Care Med 2011, 37:435-443. 2. Krinsley JS: Crit Care Med 2008, 36:3008-3013. 3. Krinsley JS: Crit Care 2011, 15:R173. 4. Krinsley JS: Crit Care 2013, 17:R37. 5. Mulavisala KP, Gopal PB, Crane B: Crit Care 2012, 16: P175. Impact of corticosteroid administration in septic shock on glycemic variability L Mirea1, R Ungureanu1, D Pavelescu1, IC Grintescu1, C Dumitrache2, D Mirea3, I Grintescu1 1Clinical Emergency Hospital of Bucharest, Romania; 2National Institute of Endocrinology, Bucharest, Romania; 3Elias University Emergency Hospital, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P445 (doi: 10.1186/cc13635) Glycaemia and critical care outcomes 1Care Hospital Nampally, Hyderabad, India; 2STAR Hospitals, Hyderabad, India Critical Care 2014, 18(Suppl 1):P442 (doi: 10.1186/cc13632) y M Cecconi1, C Ryan2, D Dawson2, N Di Tomasso2, G McAnulty2, B Philips2, A Rhodes2 1St George’s Hospital, London, UK; 2St George’s Healthcare NHS Trust, London, UK Critical Care 2014, 18(Suppl 1):P441 (doi: 10.1186/cc13631) M Cecconi1, C Ryan2, D Dawson2, N Di Tomasso2, G McAnulty2, B Philips2, A Rhodes2 Introduction While there is ongoing discussion of the optimal range for glycemic control in hospital intensive care, recent publications from Mackenzie and colleagues [1] and Krinsley [2-4] suggest not only that mean BG should be considered, but also that glucose variability and complexity may be equally important. This has increased the need for continuous systems which can provide early warnings of hypoglycemia and eff ectively measure variability. GlySure Ltd has developed an intravascular glucose monitoring system to simplify the application of hospital protocols for tight glycemic control (TGC) at the point of care. Experience with the original research-based instrumentation [5] has now been incorporated into a combined pre-production monitor and Critical Care 2014, 18(Suppl 1):P441 (doi: 10.1186/cc13631) Introduction The aim of this study was to determine the impact of preadmission or fi rst 24-hour blood glucose (BG) measurements in UK ICUs on mortality. Introduction The aim of this study was to determine the impact of preadmission or fi rst 24-hour blood glucose (BG) measurements in UK ICUs on mortality. Methods The Intensive Care National Audit & Research Centre case-mix programme database on adult admissions to general, neuroscience and cardiothoracic critical care units was used for analysis. Within the database, the highest and lowest blood glucose (BG) values measured Figure 1 (abstract P440). Blood glucose change after infusion change and after calibration. S160 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P443 P443 First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICU N Scawn1, N Coulson1, I Kemp1, P Candolfi 2, F Andrews1, V Mather1, C McGee1, J Goldstein2 1The Liverpool Heart and Chest Hospital, Liverpool, UK; 2Edwards Lifesciences, Nyon, Switzerland Critical Care 2014, 18(Suppl 1):P443 (doi: 10.1186/cc13633) First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICU N Scawn1, N Coulson1, I Kemp1, P Candolfi 2, F Andrews1, V Mather1, C McGee1, J Goldstein2 1The Liverpool Heart and Chest Hospital, Liverpool, UK; 2Edwards Lifesciences, Nyon, Switzerland Critical Care 2014, 18(Suppl 1):P443 (doi: 10.1186/cc13633) Anti-infl ammatory and antioxidant eff ects of ranolazine on primary cultured astrocytes Introduction Because of its ability to block late INa [1], ranolazine is used as an antianginal agent for the treatment of chronic angina pectoris when angina is not adequately controlled by other agents [2]. Besides its cardiovascular eff ects, ranolazine improves diff erent neuronal functions, and thus its use has been proposed for the treatment of pain and epileptic disorders [3,4]. Since astrocytes are involved in neuronal infl ammatory processes, and autoimmune and neurodegenerative diseases [5], we have investigated the anti- infl ammatory and antioxidant eff ects of ranolazine in primary cultured astrocytes. y Results There were no diff erences between the three groups at the beginning of the study regarding demographic data and the clinical characteristics, including BG value. BG levels were strongly correlated with severity of septic shock estimated by APACHE II score (r = +0.241; P = 0.005) or Simplifi ed Acute Physiology Score II (r = 0.280; P = 0.001) – Pearson correlation. The risk of death is signifi cantly increased if SD of BG is more than 20 mg/dl (67.7% vs. 20.8%, P = 0.000). A total 94.4% of deceased patients in group A registered a SD of BG more than 20 mg/dl versus 89.5% in group B or 40% in group C (P = 0.006). In total, 53.5% of patients in group A needed insulin therapy versus 25.5% in group B or 27.3% in group C. The dose was between 30.28 ± 6.65 UI/day in group A, 37.85 ± 11.95 UI/day in group B, and 14.28 ± 5.76 UI/day in group C (P >0.05). Methods We incubated diff erentiated rat astrocytes in primary culture (10 days of culture) [5] for 24 hours with ranolazine (10–5, 10–6, 10–7 M). We measured the protein expression levels of PPARγ and Cu/Zn-SOD by western blot technique. Protective eff ect of ranolazine on cell viability was assayed using MTT conversion assay. Finally, to evaluate the eff ect of ranolazine on the IL-1β cytokine and TNFα mediators, we used the enzyme-linked immunosorbent assay technique. Conclusion BG variability is highly associated with mortality compared with BG mean daily value or insulin dose. SD levels above 20 mg/dl were associated with a signifi cantly higher mortality rate relative to those with SD levels below 20 mg/dl. N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK C iti l C 2014 18(S l 1) P448 (d i 10 1186/ 13638) Introduction Tuberculous meningitis (TBM) is the least common extrapulmonary manifestation of tuberculosis. Although the UK incidence of TBM is relatively low, it carries a high mortality and morbidity [1]. Neurological deterioration continues to be an important reason for ICU admission [2]. Little is known about the outcomes for TBM patients requiring ICU admission. Our aim is to evaluate patient demographics, TBM clinical data, and the necessity for organ support, and whether this can be associated with outcome. i Methods Ninety-six patients were studied. The following parameters were calculated during the fi rst week after ICU admission. (1) Maximum value of SOFA score (SOFAmax). (2) Mean, standard deviation, maximum, minimum, and diff erence of BG levels (BGm, BGsd, BGmax, BGmin, BGd (BGmax – BGmin), respectively). BG levels were measured basically every 6 hours. (3) Correlation between SOFAmax and BG parameters using two-dimensional (correlation coeffi cient rt) and linear regression analysis (rl). Methods A retrospective study at a tertiary centre of patients with TBM admitted to our ICU between 2000 and 2012. Data were retrieved on demographics, microbiology, radiology and pharmacological fi ndings and type and level of organ support. APACHE II and SOFA scores were calculated. Patients were stratifi ed into two groups: CSF PCR+ve and CSF PCR–ve. l Results (1) Mortality of the patients with SOFAmax 0 to 3, 4 to 5, 6 to 7, 8 to 9, and 10 or more were 0%, 14%, 23%, 40%, and 89%, respectively. (2) rt and rl (rt/rl) between SOFAmax and BG parameters: BGsd (0.49/0.36), BGmax (0.47/0.32), BGm (0.45/0.23), BGd (0.44/0.32), and BGmin (0.25/0.06). (3) BG ranges that indicate SOFAmax less than 5 calculated from the two-dimensional correlation curves between SOFAmax and BGsd, BGmax, BGm, and BGd were 35 ± 25, 250 ± 112, 150 ± 33, and 156 ± 115 mg/dl, respectively. Results Eight patients (six males:two females) were evaluated. Total mean age was 43.9 ± 8.09 years. A reduction in GCS was the main reason for ICU admission. All patients received ≥3 anti-TBM drugs and steroids. Two CSF PCR+ve patients had primary drug resistance to isoniazid. There was also a longer mean delay in the time of onset of anti-TBM treatment in CSF PCR+ve patients (4 ± 2.88 days). Higher APACHE II and SOFA scores on admission (mean = 23, 8.5) were Conclusion BG parameters except BGmin were two-dimensionally related to the severity. P448 g j 1Shisei Hospital, Saitama, Japan; 2Keiyo Hospital, Tokyo, Japan gl References References 1. Belardinelli L, et al.: J Pharmacol Exp Ther 2013, 344:23-32. 2. Siddiqui MA, et al.: Drugs 2006, 66: 693-710. 3. Park YY, et al.: J Neurophysiol 2013, 109:1378-1390. 4. Kahlig KM, et al.: Br J Pharmacol 2010, 161:1414-1426. 5. Vallés SL, et al.: Brain Pathol 2004, 14:365-371. 2. Siddiqui MA, et al.: Drugs 2006, 66: 693-710. 2. Siddiqui MA, et al.: Drugs 2006, 66: 693-710. 3. Park YY, et al.: J Neurophysiol 2013, 109:1378-1390. Anti-infl ammatory and antioxidant eff ects of ranolazine on primary cultured astrocytes y y q Results Compared with control cells, treatment with ranolazine induced an increment of anti-infl ammatory PPARγ and reduced the proinfl ammatory mediators IL-1β and TNFα in primary cultured astrocytes. Ranolazine (10–6 M) also increased the expression of antioxidant protein Cu/Zn-SOD and caused a signifi cant increase in cell viability.l P446 q g 3. Park YY, et al.: J Neurophysiol 2013, 109:1378-1390. Blood glucose target in acute phase suggested by the analysis of the relationship between blood glucose profi le and the severity of the diseases y 4. Kahlig KM, et al.: Br J Pharmacol 2010, 161:1414-1426. 5. Vallés SL, et al.: Brain Pathol 2004, 14:365-371. M Hoshino1, Y Haraguchi2, K Oda1, S Kajiwara1 M Hoshino1, Y Haraguchi2, K Oda1, S Kajiwara1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Methods In a prospective, controlled study, 134 patients with septic shock were randomized into three study groups: group A (n = 43), 200 mg/day hydrocortisone hemisuccinate in four daily doses; group B (n = 47), same dose of hydrocortisone hemisuccinate in continuous administration; group C (n = 44), no hydrocortisone hemisuccinate. Patients with diabetes mellitus were excluded. The duration of hydrocortisone treatment was a maximum 7 days. The target blood glucose (BG) level was below 180 mg/dl. BG values were analyzed by calculating mean daily values, standard deviation (SD) of BG values as an index of glycemic variability, and insulin doses. The local ethics committee approved the study.f First clinical study data from therapeutic use of a novel continuous glucose monitoring system in the ICUi Performance notably improved with experience and also highlighted that even previously validated dynamic algorithms will need to be Introduction The purpose of this study was to assess the relation between glycemic control and the severity of sepsis in a cohort of patients with vasopressor-dependent septic shock treated with corticosteroids [1,2]. S161 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P447 P447 Anti-infl ammatory and antioxidant eff ects of ranolazine on primary cultured astrocytes FB El Amrani, S Guerra, D Aguirre-Rueda, MD Mauricio, P Marchio, JM Vila, SL Vallés, F Fernández, M Aldasoro University of Valencia, Spain Critical Care 2014, 18(Suppl 1):P447 (doi: 10.1186/cc13637) References 1. Waeschle RM, Moerer O, Hilgers R, et al.: The impact of the severity of sepsis on the risk of hypoglycemia and glycemic variability. Crit Care 2008, 12:R129.f Conclusion Ranolazine decreases infl ammatory mediators IL-1β and TNFα, and increases anti-infl ammatory PPARγ as well as the antioxidant Cu/Zn-SOD in astrocytes in culture. These results suggest that ranolazine could be useful as a neuroprotective drug in pathologies inducing infl ammatory damage and oxidant processes. 2. Loisa P, Parviainen I, Tenhunen J, et al.: Eff ect of mode of hydrocortisone administration on glycemic control in patients with septic shock. A prospective randomized trial. Crit Care 2007, 11:R21. Tuberculous meningitis: a 10-year case analysis of critical care admissions p p y p y p Critical Care 2014, 18(Suppl 1):P446 (doi: 10.1186/cc13636) Introduction Blood glucose (BG) control in acute illness improves outcome. However, how to manage BG levels, or BG target, is not clearly elucidated. In this study, the BG target was suggested by the analysis of the relationship between BG profi le and the severity of the diseases. Methods Ninety-six patients were studied. The following parameters were calculated during the fi rst week after ICU admission. (1) Maximum value of SOFA score (SOFAmax). (2) Mean, standard deviation, maximum, minimum, and diff erence of BG levels (BGm, BGsd, BGmax, BGmin, BGd (BGmax – BGmin), respectively). BG levels were measured basically every 6 hours. (3) Correlation between SOFAmax and BG parameters using two-dimensional (correlation coeffi cient rt) and linear regression analysis (rl). Introduction Blood glucose (BG) control in acute illness improves outcome. However, how to manage BG levels, or BG target, is not clearly elucidated. In this study, the BG target was suggested by the analysis of the relationship between BG profi le and the severity of the diseases. N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 to compare known clinical predictors of MV and determine predictors of prolonged MV. associated with a positive CSF PCR result. Increased requirements for mechanical ventilation (100%), tracheostomy (50%), inotropes (75%), neurosurgical intervention (predominantly CSF drainage) (100%) and enteral feeding (50%) were all signifi cant for this group. Mortality and long-term neurological morbidity were substantially higher for PCR+ve patients (75% and 25%). In contrast, the majority of culture-negative patients survived (75%), and experienced good recoveries at follow-up. Overall mortality was 50%. associated with a positive CSF PCR result. Increased requirements for mechanical ventilation (100%), tracheostomy (50%), inotropes (75%), neurosurgical intervention (predominantly CSF drainage) (100%) and enteral feeding (50%) were all signifi cant for this group. Mortality and long-term neurological morbidity were substantially higher for PCR+ve patients (75% and 25%). In contrast, the majority of culture-negative patients survived (75%), and experienced good recoveries at follow-up. Overall mortality was 50%. Methods We conducted a retrospective chart review of consecutive CSI admitted between 1 January 2005 and 1 March 2009. We recorded data related to the injury, the duration of MV, respiratory complications, ICU and hospital length of stay and patients’ outcomes. A review of the literature identifi ed known predictors (ASIA level, ISS, level of injury, and so forth). Univariate and multivariate logistic regression were used to identify predictors of MV and prolonged MV. y Conclusion This is the second documented case analysis of TBM and ICU admission in adult patients [1,2]. Findings show that poor outcomes are associated with positive CSF PCR results. Factors linked to poor outcomes include delays in initiating anti-TBM treatment, neurosurgical interventions, and an increased requirement for multiorgan support. Earlier drug susceptibility testing would be preferable particularly for patients with positive CSF PCR cultures. Given the potential severity of TBM, a high index of clinical suspicion remains critical towards optimizing outcomes. y p p g Results Of the 208 patients, 82% were male and the mean age was 51 years. Hospital mortality was 8.7%. Main causes of injury were motor vehicle accidents (39.7%) and falls (43.2%). Injuries below C4 level represented 51.5% of the population. A complete loss of motor function (ASIA level A and B) was found in 34.9% of patients. The mean and median ISS score was 20.7. P452 P452 Evaluation of the ocular microcirculation in brain-dead patients: fi rst step towards a new method of multimodal neuromonitoring? T Tamosuitis1, A Pranskunas1, N Balciuniene1, V Pilvinis1, EC Boerma2 1Lithuanian University of Health Sciences, Kaunas, Lithuania; 2Medical Centre Leeuwarden, the Netherlands Critical Care 2014 18(Suppl 1):P452 (doi: 10 1186/cc13642) g y Methods During surgery, samples of cerebrospinal fl uid and arterial blood were simultaneously withdrawn to evaluate lactate concentration. Samples were collected at fi ve fi xed times during and after surgery: T1 (beginning of the intervention), T2 (15 minutes after aortic cross-clamping), T3 (just before unclamping), T4 (end of surgery), and T5 (4 hours after the end of surgery).l ritical Care 2014, 18(Suppl 1):P452 (doi: 10.1186/cc13642) Introduction Multimodal neuromonitoring is a part of goal- directed therapy in severe neurosurgical pathology that leads to better understanding and therefore accurate and timely correction of disturbances of cerebral perfusion. The aim of our study was to evaluate and compare microcirculation in the conjunctiva of the eye and sublingual mucosa in brain-dead patients and healthy volunteers. No studies to our knowledge with this purpose were performed previously. We hypothesized that direct videomicroscopic evaluation of conjunctival microcirculation is linked to cerebral blood fl ow. Results Mean lactate levels in cerebrospinal fl uid rose consistently from the beginning of the intervention steadily until after surgery (T1 = 1.83 mmol/l, T2 = 2.10 mmol/l, T3 = 2.72 mmol/l, T4 = 3.70 mmol/l, T5 = 4.31 mmol/l). Seven patients developed spinal cord injury; two of them had delayed injury occurring 24 hours after the end of surgery; the remaining fi ve had early onset. In this group of fi ve patients, preoperative cerebrospinal fl uid lactate levels were signifi cantly (P = 0.04) higher than those of the other 40 patients preoperatively (2.12 ± 0.35 vs. 1.79 ± 0.29 mmol/l). jl Methods We evaluated microcirculation of the eye conjunctiva and sublingual mucosa of 10 brain-dead diagnosed patients and 10 healthy volunteers. Brain-death diagnoses were certifi ed by cerebral angiography. Direct in vivo observation of the microcirculation was obtained with sidestream dark-fi eld imaging. Assessment of microcirculatory parameters of convective oxygen transport (microvascular fl ow index (MFI), proportion of perfused vessels (PPV)), and diff usion distance (perfused vessel density (PVD) and total vessel density (TVD)) was performed according to international criteria. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 In total, 78 patients required MV (37.5%) and 30 patients required prolonged MV (14.4%). After multivariate analysis, four predictors of MV were identifi ed: pneumonia (OR = 52.83); ISS score >22 (OR = 4.09); age (OR = 1.02); level C1 to C4 (2.34); and two predictors of prolonged MV: ASIA score A and B (OR = 5.57) and pneumonia (OR = 8.76). References G Landoni, M Pieri, V Testa, S Silvetti, M Zambon, G Borghi, M Azzolini, AL Di Prima, L Nobile, R Lembo, A Zangrillo Vita-Salute San Raff aele University, Milan, Italy Critical Care 2014, 18(Suppl 1):P449 (doi: 10.1186/cc13639) References 1. Krueger H, et al.: Chronic Dis Inj Can 2013, 33:113-122. 2. Devivo MJ: Spinal Cord 2012, 50:365-372. 3. Como JJ, et al.: J Trauma Inj Infect Crit Care 2005, 59:912-916. 4. Durga P, et al.: Anesth Anal 2010, 110:134-140. 5. Honarmand A, et al.: Ulus Travma Acil Cerrahi Derg 2008, 14:110-117. 6. Branco BC, et al.: J Trauma 2011, 70:111-115. Introduction The aim was to evaluate the role of intrathecal lactate as an early predictor of spinal cord injury during thoracoabdominal aortic aneurysmectomy. Forty-four consecutive patients were scheduled to undergo thoracoabdominal aortic aneurysmectomy. Two patients had a type B dissecting aneurysm; all other 42 patients suff ered from degenerative aneurysm. P452 Conclusion The preoperative cerebrospinal lactate concentration is elevated in patients who will develop early-onset spinal cord injury after thoracoabdominal aortic aneurysmectomy. This may allow a better stratifi cation of these patients, suggesting a more aggressive strategy of spinal cord function preservation and possibly guaranteeing them a better outcome. N Ojukwu, C Talati, S Wijayatilake, G De la Cerda, A Bellini, R Jain Queen’s Hospital, London, UK C iti l C 2014 18(S l 1) P448 (d i 10 1186/ 13638) Therefore, a part of the severe patients seemed to have lower BG levels and lower BG variability. Targeting those BG parameters in early phase within the abovementioned levels was considered to link to better outcome. S162 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References 1. Pehlivanoglu F, et al.: Scientifi cWorldJournal 2012, 2012:169028. 2. Verdon R, et al.: Clin Infect Dis 1996, 22:982-988. 1. Pehlivanoglu F, et al.: Scientifi cWorldJournal 2012, 2012:169028. 2. Verdon R, et al.: Clin Infect Dis 1996, 22:982-988. Conclusion In our study ISS, cervical level and age were associated with MV but not with the need for prolonged MV, whereas pneumonia was an independent risk factor for both. This is a potentially preventable risk factor where specifi c strategies can be applied to improve patients’ outcome. P450 Predictors of ventilatory outcome in cervical spinal injuries HT Wang, DW Williamson, MA Albert Hopital Sacre-Coeur de Montreal, Canada Critical Care 2014, 18(Suppl 1):P450 (doi: 10.1186/cc13640) y g Results All brain-dead patients required vasopressor support to sustain perfusion of donor organs. The MFI of small vessels was signifi cantly lower in brain-dead patients in comparison with healthy controls in ocular conjunctiva (2.6 (2.4 to 2.8) vs. 3.0 (3.0 to 3.0), P = 0.03) and in sublingual mucosa (2.8 (2.6 to 2.9) vs. 3.0 (3.0 to 3.0), P = 0.04). TVD and PVD of small vessels were signifi cantly lower in brain-dead patients in comparison with healthy controls in ocular conjunctiva (10.2 (5.2 to 14.8) vs. 17.0 (15.4 to 27.2) mm/mm2, P = 0.023 and 5.2 (2.9 to 7.2) vs. 10.1 (8.5 to 16.7) l/mm, P = 0.023), but there was no diff erence in sublingual mucosa.i Introduction Spinal cord injuries aff ect 50 persons per million every year in North America [1], with over 50% occurring at the cervical level [2]. Cervical spinal cord injuries (CSI) are at particular risk for mechanical ventilation (MV), pulmonary complications and increased length of hospital stay. A few small cohort studies looked at predictors of MV [3-6], and to our knowledge there are no studies addressing factors associated with prolonged MV. The purpose of this study was Conclusion We were able to identify a signifi cant reduction of capillary density in the eye conjunctiva but not in sublingual mucosa when S163 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 medical quality between the urban and depopulated areas. To improve the problem, telemedicine using a mobile device between general physicians and stroke specialists became important with the increasing demand for rapid and correct diagnosis for treatment of acute stroke. We developed a system for rapidly exchanging diagnostic images and clinical information in depopulated areas to develop the standard comparing microcirculation of brain-dead diagnosed patients and healthy volunteers. However, the presence of conjunctival fl ow in case of general cerebral fl ow is completely absent, making it diffi cult to use conjunctival fl ow as a substitute for brain fl ow. P453 thrombolytic therapy using alteplase for acute ischemic stroke [1,2]. External validation of an early warning alert for elevated intracranial pressure in the Avert-IT database Introduction After severe traumatic brain injury (TBI), episodes of elevated intracranial pressure (ICP) are associated with poor outcome. Previously we developed a model to predict increased ICP, 30 minutes in advance, using the dynamic characteristics of routinely monitored minute-by-minute ICP and mean arterial blood pressure (MAP) signals [1]. The model was developed using data from the Brain-IT database [2]. Here we present external validation results of this model, on a more recent cohort of adult TBI patients from the AVERT-IT project [3]. y Results The k-support system was managed in 102 emergency patients, 65 patients (64%) were classifi ed as neurological disease, 41 (40%) as stroke, 11 (11%) as head injury, two (2%) as epilepsy. The detail of stroke was ischemia in 35 (34%), hemorrhage in four (4%) and SAH in two (2%). Two ischemic stroke patients were treated with intravenous thrombolysis of alteplase using the k-support system and a ‘drip- and-ship’ paradigm. One patient using alteplase showed complete recanalization of the middle cerebral artery. The consultations resulted in hospitalization in 42%, transfer in 37% and return home in 20%. ece t co o t o adu t pat e ts o t e p oject [3]. Methods A retrospective analysis of physiological data collected at the minute resolution, from 43 adult patients from the AVERT-IT project. A total of 67 episodes of ICP above 30 mmHg lasting at least 10 minutes were identifi ed in this cohort. Four-hour time series of ICP and MAP anteceding each episode by 30 minutes were analyzed. Additional time series not preceding elevated ICP episodes were used for validation. Results Table 1 summarizes the main fi ndings. Performance of the model in the original study [1] is reproduced in the fi rst column. The model retains identical performance for all criteria in the cohort of more recent TBI patients of the AVERT-IT database. Methods A retrospective analysis of physiological data collected at the minute resolution, from 43 adult patients from the AVERT-IT project. A total of 67 episodes of ICP above 30 mmHg lasting at least 10 minutes were identifi ed in this cohort. Four-hour time series of ICP and MAP anteceding each episode by 30 minutes were analyzed. References 1. Guiza et al.: Crit Care Med 2013, 41:554. 2. [http://www.brainit.org] 3. [http://www.avert-it.org] 1. Guiza et al.: Crit Care Med 2013, 41:554. 2. [http://www.brainit.org] 3. [http://www.avert-it.org] External validation of an early warning alert for elevated intracranial pressure in the Avert-IT database Additional time series not preceding elevated ICP episodes were used for validation.i p Conclusion Before introduction of the k-support system, the standard thrombolytic therapy using alteplase for acute ischemic stroke could not operate in the Kaifu area due to the absence of stroke specialists and the long distance to a neighboring stroke center. The telemedicine system using a mobile device as the k-support system can be used anytime, anywhere and by anyone. This system can communicate with the doctors, between general physicians in depopulated areas and specialists in urban areas, and may become a useful tool for acute patient management in not only stroke but also other emergency diseases g Results Table 1 summarizes the main fi ndings. Performance of the model in the original study [1] is reproduced in the fi rst column. The model retains identical performance for all criteria in the cohort of more recent TBI patients of the AVERT-IT database. Table 1 (abstract P453). Database Brain-IT Avert-IT AUROC 0.85 0.83 aBrier SS (%) 34 30 Cal large 0.00 0.01 Cal slope 0.99 1.03 Accuracy (%) 77 77 References 1. Takao H, et al.: Stroke 2012, 43:236-239. 2. Tekle WG, et al.: Stroke 2012, 43:1971-1974. References 1. Takao H, et al.: Stroke 2012, 43:236-239. 2. Tekle WG, et al.: Stroke 2012, 43:1971-1974. P450 Methods A system was consisted of communicating patient data and imaging between the hospital system and participating staff members in and out of the hospital using mobile devices. The system can transfer clinical data and large volumes of CT and MRI, and expert opinion in real time. We developed the system (k-support) in the Kaifu area, which is a typical depopulated area in Tokushima Prefecture, Japan, between the general physicians in Tokushima Prefectural Kaifu Hospital and specialists in stroke and cardiovascular disease at Tokushima University Hospital from February 2013. External validation of an early warning alert for elevated intracranial pressure in the Avert-IT database M Beckers1, F Güiza1, B Depreitere1, I Piper2, R Donald3, G Van den Berghe1, G Meyfroidt1 1University Hospitals Leuven, Belgium; 2Southern General Hospital, Glasgow, UK; 3University of Glasgow, UK Critical Care 2014, 18(Suppl 1):P453 (doi: 10.1186/cc13643) P454 New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas T Kageji1, H Oka1, F Obata2, K Tani3, H Bando2, R Tabata3, M Kohno3 1Tokushima University Hospital, Tokushima, Japan; 2Tokushima Prefectural Kaifu Hospital, Kaifu, Japan; 3The University of Tokushima Graduate School, Tokushima, Japan Critical Care 2014, 18(Suppl 1):P454 (doi: 10.1186/cc13644) References Eff ects of cardiac output-guided hemodynamic management on fl uid administration after aneurysmal subarachnoid hemorrhage B Bergmans, M Egal, J Van Bommel, J Bakker, M Van der Jagt Erasmus MC – University Medical Center, Rotterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P455 (doi: 10.1186/cc13645) Conclusion The obtained external validation results, on a previously unseen cohort of adult TBI patients, confi rm the robustness of the model to accurately predict future increased ICP events 30 minutes in advance. The general applicability of the model is probably due to its sparseness, as it only uses two routinely monitored signals as input, namely ICP and MAP. These results are a large step forward in our work toward an early warning system for elevated ICP that can be used worldwide. Introduction In patients with aneurysmal subarachnoid hemorrhage (SAH), hypervolemic therapy may result in fl uid overload that may be associated with adverse clinical outcomes [1,2]. We hypothesized that a goal-directed transpulmonary thermodilution (TPT) monitoring protocol aiming for normovolemia may result in decreased fl uid intake while sustaining adequate volume status in poor-grade SAH patients. Methods Following the introduction of the hemodynamic protocol in 2011, 26 consecutive patients with SAH were included until 2013. Using TPT (PiCCO; Pulsion), cardiac output (CO), global end- diastolic volume index (GEDVI) and extravascular lung water index (EVLWI) were determined. Fluid administration was targeted at fl uid unresponsiveness. Indications for start of the protocol were: hypotension (in spite of fl uids), pulmonary edema or cardiac stunning, daily fl uid balance ≤–1 l, cerebral ischemia (DCI) with progressive symptoms. Data were collected on fl uid intake and output up to 3 days before and 3 days after the start of TPT. We assessed the course of fl uid input and output and hemodynamic parameters before and after the start of the protocol with the generalized estimating equation. P454 New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas T Kageji1, H Oka1, F Obata2, K Tani3, H Bando2, R Tabata3, M Kohno3 1Tokushima University Hospital, Tokushima, Japan; 2Tokushima Prefectural Kaifu Hospital, Kaifu, Japan; 3The University of Tokushima Graduate School, Tokushima, Japan p g g q Results Mean age was 55 ± 16, and median Glasgow Coma Scale on admission was 8 (IQR 6 to 13). TPT was started at a median of 1 day after ICU admission (IQR 0 to 4). DCI developed in 70% and the in-hospital death rate was 45%. Compared with days preceding the protocol (day –3 to –1), TPT (day 0 to 3) was associated with decreased fl uid intake (compared with day 3 as reference; day –1: +1.14 ± 0.31 l, P <0.001; day 0: +0.68 ± 0.29 l, P = 0.019; day 1: +0.73 ± 0.31 l, P = 0.02), increased p Critical Care 2014, 18(Suppl 1):P454 (doi: 10.1186/cc13644) Introduction Stroke is the main cause of deterioration of activity of daily living in Japan. The social problem in Japan is the diff erence in S164 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 PbrO2 (Licox, GMS) ICP, CPP, MABP, CVP, local brain temperature, body core temperature, PCO2, and blood glucose among others. The cerebral monitoring probes were inserted via a Bolt (ICP, PbrO2, microdialysis) and an additional burr hole (CBF). All probes were positioned in the penumbra and location was verifi ed by brain CT. The PbrO2 arm of this study and its signifi cance is still underway and will be announced later. Thirteen of the patients were successfully discharged from the ICU while eight did not survive. fl uid output (day –2: –0.91 ± 0.46 l, P <0.05 compared with day 3), and consequently a strong decrease in fl uid balance (day –3: +2.10 ± 0.56 l, P <0.001; day –2: +2.66 ± 1.14 l, P = 0.02; day –1: +1.41 ± 0.37 l, P <0.001). The decreased fl uid intake and fl uid balances did not result in decreased CO, GEDVI or EVLWI on days 1 to 3 compared with day 0 (mean PCCI 3.5 l/minute/m2, mean GEDVI 761 ml/m2, mean EVLWI 10.8 ml/kg). P454 New support system using a mobile device for diagnostic image display and treatment of acute stroke in Japanese depopulated areas Conclusion Our data suggest that in poor-grade SAH patients goal- directed fl uid management with TPT is feasible to decrease intake and increase diuresis without adverse eff ects on cardiac output or preload parameters. Future research should assess the eff ect of such a protocol on clinical outcomes. g Results The fi nal data are currently under statistical evaluation, which will be completed at the time of presentation. However, there is indication of a link between brain glucose levels and CBF values, but it is not clear as to the CBF–PbrO2 correlation that is the second part of this study under evaluation. This may be due to the fl uctuation of brain glucose because of brain ischemia, hyperemia, hypermetabolism or hypometabolism. So far we are able to establish a correlation of CBF and lactate/pyruvate ratio only in persistently low CBF values. References 1. Mutoh T, et al.: Stroke 2009, 40:2368-2374. 2. Ibrahim GM, Macdonald RL: Neurocrit Care 2013, 19:140. 1. Mutoh T, et al.: Stroke 2009, 40:2368-2374. 1. Mutoh T, et al.: Stroke 2009, 40:2368-2374. 2. Ibrahim GM, Macdonald RL: Neurocrit Care 2013, 19:140. y y y Conclusion This will be a fi nal report of a study in human patients with severe subarachnoid hemorrhage and traumatic brain injury. The results indicate correlations of varying signifi cance between the pooled data still under statistical analysis. We hope that the outcome of our study will be able to answer questions regarding the pathophysiology of severe brain injury and guide us in the titration of therapy, as it is needed by each individual patient [1-4]. y References g y Critical Care 2014, 18(Suppl 1):P456 (doi: 10.1186/cc13646) References 1. Morgan Stuart R, et al.: Neurocrit Care 2010, 12:188-198. 2. Tisdall MM, et al.: Br J Anaesth 2007, 99:61-67. 3. Nordström CH: Crit Care 2008, 12:R9I. 4. Jaeger M, et al.: Acta Neurochir (Wien) 2005, 147:51-56. References 1. Morgan Stuart R, et al.: Neurocrit Care 2010, 12:188-198. 2. Tisdall MM, et al.: Br J Anaesth 2007, 99:61-67. 3. Nordström CH: Crit Care 2008, 12:R9I. 4. Jaeger M, et al.: Acta Neurochir (Wien) 2005, 147:51-56. Introduction The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily that has been implicated in multiple neuronal and infl ammatory stress processes. In the present study, we investigated changes in RAGE immunoreactivity and its protein levels in the gerbil hippocampus (CA1 to 3 regions) after 5 minutes of transient global cerebral ischemia. 4. Jaeger M, et al.: Acta Neurochir (Wien) 2005, 147:51-56. 58 New look at the 20 mmHg ICP threshold New look at the 20 mmHg ICP threshold New look at the 20 mmHg ICP threshold FG Guiza1, BD Depreitere1, IP Piper2, GV Van den Berghe1, GM Meyfroidt1 1University Hospitals Leuven, Belgium; 2Southern General Hospital, Glasgow UK Critical Care 2014, 18(Suppl 1):P458 (doi: 10.1186/cc13648) g g Methods The ischemic hippocampus was stained with cresyl violet (CV), NeuN (a neuron-specifi c soluble nuclear antigen) antibody and Fluro-Jade B (a marker for neuronal degeneration). g FG Guiza1, BD Depreitere1, IP Piper2, GV Van den Berghe1, GM Meyfroidt1 1University Hospitals Leuven, Belgium; 2Southern General Hospital, Glasgow, UK FG Guiza1, BD Depreitere1, IP Piper2, GV Van den Berghe1, GM Meyfroidt1 1University Hospitals Leuven, Belgium; 2Southern General Hospital, Glasgow, UK Results Five days after ischemia–reperfusion, delayed neuronal death occurred in the stratum pyramidale (SP) of the CA1 region. RAGE immunoreactivity was not detected in any regions of the CA1 to 3 regions of the sham group. RAGE immunoreactivity was detected only in the CA1 region from 3 days post ischemia, and the RAGE immunoreactivity was newly expressed in astrocytes, not in neurons. In addition, the level of RAGE protein was highest at 5 days post ischemia. In brief, both the RAGE immunoreactivity and protein level were distinctively increased in astrocytes in the ischemic CA1 region from 3 days after transient cerebral ischemia. Critical Care 2014, 18(Suppl 1):P458 (doi: 10.1186/cc13648) Critical Care 2014, 18(Suppl 1):P458 (doi: 10.1186/cc13648) Critical Care 2014, 18(Suppl 1):P458 (doi: 10.1186/cc13648 Introduction We present a method based on minute-by-minute ICP monitoring and outcome (GOS) to visualize in a clinically useful way the dynamic aspects of secondary injury. Introduction We present a method based on minute-by-minute ICP monitoring and outcome (GOS) to visualize in a clinically useful way the dynamic aspects of secondary injury. Methods A retrospective analysis of data from 165 adult patients from the Brain-IT database [1]. A color-coded contour plot was made for the association between good outcome and the number of secondary insults of ICP during the ICU stay, as defi ned by continuous values of insult duration and thresholds. Conclusion These results indicate that the increase of RAGE expression in astrocytes at post ischemia may be related to the ischemia-induced activation of astrocytes in the ischemic CA1 region. Figure 1 (abstract P458). Eff ect of transient cerebral ischemia on the expression of receptor for advanced glycation end products in the gerbil hippocampus proper p p JH Cho, CW Park, HY Lee, MH Won, JY Lee, DJ Chung Kangwon National University, Chuncheonsi, South Korea Model of intracranial hypertension of tumor etiology in laboratory ratsi Model of intracranial hypertension of tumor etiology in laboratory rats AN Kolesnikov, VI Cherniy, KA Kardash, TA Mustafi n, VN Stasyuk, IV Koktishev Donetsk National Medical University of Maxim Gorky, Donetsk, Ukraine Critical Care 2014, 18(Suppl 1):P459 (doi: 10.1186/cc13649) AN Kolesnikov, VI Cherniy, KA Kardash, TA Mustafi n, VN Stasyuk, IV Koktishev AN Kolesnikov, VI Cherniy, KA Kardash, TA Mustafi n, VN Stasyuk, IV Koktishev Donetsk National Medical University of Maxim Gorky, Donetsk, Ukraine Critical Care 2014, 18(Suppl 1):P459 (doi: 10.1186/cc13649) Introduction The objective of this research is the creation of an experimental model of intracranial hypertension (ICH) of the tumor etiology in white nonlinear rats. Figure 1 (abstract P460). Methods Work was executed in the neurophysiology laboratory on 12 white nonlinear rats, weight 280 to 320 g, preselected according to criteria of bioethical rules. For modeling ICH in animals, standard anesthesia was carried out. A longitudinal section was spent in the projections of saggital suture from the frontal to occipital bone. The off ensive surgical stage anesthesia showed loss of palpebral and lingual refl exes. Further, according to the stereotactic atlas coordinates, a burr hole was performed, through which sterile two-component anti- allergen and depyrogenized gel in the amount of 0.025 ml was applied into the fourth ventricle with an intracerebroventricular microinjector. The injector was at an angle of 12° in order to avoid injuring vessels, its distal end entering a depth 4.5 mm in the fourth ventricle, and was installed and documented in the burr hole for measuring intracranial pressure. Monitoring during operations included: respiration, heart rate, ECG, thermometry, and control of diuresis. Figure 1 (abstract P460). Results In all animals the heart rate decreased to an average of 180 beats/minute during 90 minutes after the introduction of the intracerebroventricular two-component gel. Also there was a decrease in respiration rate by an average of <70 breaths/minute. Behavioral responses of animals were evaluated. The average intensity and the duration of such actions as shaking, friction, licking and carding decreased. Decline in grooming was possibly associated with breached central regulation in the brain of studied rats. Macroscopically in the preparation we observed a pronounced hyperemia of obtained materials, which may indirectly indicate the syndrome of ICH. Survival of the animals was: 3 days – 100%, 7 days – 100%, 14 days – 92%, 21 days – 83%. Correlation of thermal Doppler fl owmetry and microdialysis values in patients with severe subarachnoid hemorrhage and traumatic brain injury DC Papadopoulos1, P Papamichalis1, As Filippidis2, D Karangelis3, TH Bekiari4, KN Fountas5, G Vretzakis5, KN Paterakis5, A Komnos1 1General Hospital of Larisa, Greece; 2Boston Medical Center, Boston, MA, USA; 3Onassis Cardiac Surgey Center, Athens, Greece; 4Prefectural Authority of Thessaly, Larisa, Greece; 5University Hospital of Larissa, School of Medicine, University of Thessaly, Larisa, Greece C l C (S l ) P (d / ) y y Critical Care 2014, 18(Suppl 1):P457 (doi: 10.1186/cc13647) Introduction The purpose of this study is to investigate the relationship between continuously monitored regional cerebral blood fl ow (CBF) and microdialysis values in severe subarachnoid hemorrhage and traumatic brain injury patients. Methods Advanced multimodal neuromonitoring including measure- ments of CBF (QFlow, Hemedex) and brain lactate, pyruvate, lactate/ pyruvate ratio, glycerol and glucose values using microdialysis (CMA600, microdialysis) were performed in 21 patients with severe subarachnoid hemorrhage (n = 17) and traumatic brain injury (n = 4). Thirteen of the patients were successfully discharged from the ICU while eight did not survive. Additional recorded parameters include Figure 1 (abstract P458). S165 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Figure 1 visualizes in blue the region associated with good outcome for continuous values of thresholds and durations of secondary insults. The thick black line indicates the transition towards the negative association region in red. A best-fi t exponential curve is superimposed. This clearly shows that insults below 20 mmHg of ICP can be detrimental if sustained for long periods. Alternatively, they can be tolerated for higher thresholds but only for shorter periods. Figure 1 (abstract P460). Conclusion Secondary injury is dynamic such that not only thresholds but also insult duration are relevant. The proposed visualization goes beyond the static 20 mmHg ICP threshold introduced in [2] and provides a more accurate representation that could help the clinician in identifying when the patient’s outcome could be making a turn for the worse. Model of intracranial hypertension of tumor etiology in laboratory ratsi y Conclusion The main result of this work can be considered creation of a successful and workable model of ICH. The developed model is the testing ground to assess especially anesthesia protection in patients with the syndrome of ICH including tumor etiology and has no analogs. lactate, serum sodium and C-reactive protein (CRP) between arterial and jugular venous bulb blood. j g Methods An observational study. Between 1 January and 31 October 2013 we included neurocritical patients (NCP) with multimodal neuromonitoring (MMN). Daily samples of arterial blood and venous jugular bulb blood were obtained for measuring pCO2, lactate, serum sodium and CRP.i j g Methods An observational study. Between 1 January and 31 October 2013 we included neurocritical patients (NCP) with multimodal neuromonitoring (MMN). Daily samples of arterial blood and venous jugular bulb blood were obtained for measuring pCO2, lactate, serum sodium and CRP.i P460 Arterial–jugular bulb diff erences in pCO2, lactate, serum sodium and C-reactive protein in neurocritical patients M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P460 (doi: 10.1186/cc13650) References References 1. [http://www.brainit.org] 2. Marmarou et al.: J Neurosurg 1991, 75:59-66. 1. [http://www.brainit.org] 2. Marmarou et al.: J Neurosurg 1991, 75:59-66. Effi cacy of terutroban in preventing delayed cerebral ischemia after subarachnoid hemorrhage: a functional isotope imaging study on a rat model D Lagier1, B Guillet2, L Velly1, N Bruder1, M Alessi2 D Lagier , B Guillet , L Velly , N Bruder , M Alessi 1CHU Timone, Marseille, France; 2Aix Marseille University, Marseille, France Critical Care 2014, 18(Suppl 1):P461 (doi: 10.1186/cc13651) 1CHU Timone, Marseille, France; 2Aix Marseille Univer p g Methods Consecutive patients admitted to our ICU for aSAH were enrolled in the study. CBF-Vm and the pulsatility index (PI) were obtained by TCCS in both mean cerebral arteries (MCAs) at two scheduled time points, <3 days (T1) and 7 to 10 days (T10), and at least once every other day after bleeding. The highest Vm between left and right and the corresponding PI were chosen for analysis. All patients underwent brain MRI plus TOF-MRI angiography at T1 and T10 to assess radiologic VSP. Symptomatic VSP was defi ned as a new neurologic defi cit associated with radiological VSP. The occurrence of DCI was evaluated on DWI sequences. The C statistic (ROC curves) and nonparametric t test were used for analysis. Introduction After a subarachnoid hemorrhage (SAH), delayed cerebral ischemia (DCI) remains the principal cause of morbid mortality. F2-isoprotanes are recognized as biomarkers of DCI. These lipid metabolites, by fi xing the thromboxane and prostaglandin (TP) receptor, induce vasoconstriction and platelet aggregation. Our objective was to evaluate the effi cacy of terutroban (TER), a TP receptor- specifi c antagonist, in preventing DCI after SAH. i g g Methods Twenty rats were assigned to one of three groups: a double 250 μl intracisternal injection (ICI) was realized with saline in the CONTROLS group (n = 6) or with autologous arterial blood in the SAH (n = 8) and SAH+TER (n = 6) groups. Treated animals received an oral administration of 30 mg/kg/day TER during 5 days following blood injection. Rats were evaluated using a functional isotope imaging technique (high-resolution microSPECT). Brain capture of three 99m technetium radiolabeled tracers was evaluated: HMPAO at day (D) 0, 2 and 5 for cerebral perfusion quantifi cation, DTPA at D3 for blood–brain barrier (BBB) integrity study and annexin V-128 at D4 for apoptotic activity study. Radioactivity was measured in a predefi ned region of interest: cerebrum, cerebellum and brainstem. Statistical analysis: one- way ANOVA followed by Student’s t test. Results Forty-three consecutive patients were recruited. Thirty-seven patients had simultaneous TCCS measures and MRI (mean age 58 years, 28% WFNS 4 to 5). Reference Figure 1 (abstract P461). HMPAO uptake at day 2 and day 5. P <0.01 versus day 0; #P <0.01 versus day 2; ‡P <0.01. 1. Carrera E, et al.: Transcranial Doppler for predicting delayed cerebral ischemia after subarachnoid hemorrage. Neurosurgery 2009, 65:316-324. References 1. Futier et al.: Crit Care 2010, 14:R19. 1. Futier et al.: Crit Care 2010, 14:R19. F Di Corte, S Piff eri, L Capuano, S Magnoni, V Conte, F Ortolano, N Stocchetti Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P462 (doi: 10.1186/cc13652) 2. Chieregato et al.: Acta Neurochir 2005, Suppl 95. 3. Stochetti et al.: Anesthesiology 2005, 103:957. 4. Chieregato et al.: J Neurosurg Anesthesiol 2007, 19:222. Introduction Cerebral vasospasm (VSP) and delayed cerebral ischemia (DCI) play a central role in worsening the neurological outcome in patients with aneurismal subarachnoid hemorrhage (aSAH) [1]. Transcranial color-coded duplex sonography (TCCS) is a non-invasive tool to detect VSP and predict DCI, but its clinical utility is limited by low accuracy. Our aim was to perform a sensitivity/specifi city analysis of TCCS in predicting clinical VSP and DCI. Arterial–jugular bulb diff erences in pCO2, lactate, serum sodium and C-reactive protein in neurocritical patients Results There were 45 NCP, six (13%) with MMN (fi ve men). Mean age was 37 ± 11 years (35 to 61). Diagnostics: two TBI, two SAH, one stroke, one lupus encephalitis. APACHE II was 27 ± 6.5 (25 to 39). Glasgow Coma Scale at admission was 14 ± 4 (4 to 14). pCO2 (mmHg): arterial 41 ± 6.3 versus jugular 45 ± 7.4 (r = 0.7, P = 0.007). Lactate (mg/dl): arterial 11 ± 5.6 versus jugular 13.5 ± 3.9 (r = 0.7, P = 0.9). Sodium (mEq/dl): Results There were 45 NCP, six (13%) with MMN (fi ve men). Mean age was 37 ± 11 years (35 to 61). Diagnostics: two TBI, two SAH, one stroke, one lupus encephalitis. APACHE II was 27 ± 6.5 (25 to 39). Glasgow Coma Scale at admission was 14 ± 4 (4 to 14). pCO2 (mmHg): arterial 41 ± 6.3 versus jugular 45 ± 7.4 (r = 0.7, P = 0.007). Lactate (mg/dl): arterial 11 ± 5.6 versus jugular 13.5 ± 3.9 (r = 0.7, P = 0.9). Sodium (mEq/dl): Introduction Several reports indicate the potential usefulness of monitoring brain metabolic parameters and their correlation with the system [1-4]. We want to establish diff erences and correlation in pCO2, S166 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 arterial 141 ± 4.5 versus 141 ± 4.4 (r = 0.8, P = 0.15). CRP (mg/dl): arterial 8 ± 7.4 versus 17 ± 11.6 (r = 0.9, P <0.001). The correlation and trend curves are shown in Figure 1. arterial 141 ± 4.5 versus 141 ± 4.4 (r = 0.8, P = 0.15). CRP (mg/dl): arterial 8 ± 7.4 versus 17 ± 11.6 (r = 0.9, P <0.001). The correlation and trend curves are shown in Figure 1. Conclusion We made the fi rst microSPECT scan description of a DCI rat model. After induction of SAH, TER improves cerebral perfusion, prevents BBB disruption in the brainstem and decreases apoptotic phenomenon in the cerebrum. g Conclusion A suitable correlation is observed for the arterial–jugular bulb in diff erent variables. There is a signifi cant diff erence in CRP and pCO2 values being persistently higher in the jugular, particularly for CRP. Studies are required to defi ne its interpretation and potential usefulness. R f P462 Accuracy of transcranial color-coded duplex sonography in predicting clinical vasospasm and delayed cerebral ischemia in patients with subarachnoid hemorrhage F Di Corte, S Piff eri, L Capuano, S Magnoni, V Conte, F Ortolano, N Stocchetti Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy Critical Care 2014, 18(Suppl 1):P462 (doi: 10.1186/cc13652) Effi cacy of terutroban in preventing delayed cerebral ischemia after subarachnoid hemorrhage: a functional isotope imaging study on a rat model Eleven patients (30%) developed symptomatic VSP and 26 (70%) did not (NVSP). Vm increased signifi cantly from T1 to T10 in both groups (VS: P = 0.03, NVSP P = 0.01). The accuracy of TCCS at T10 (Vm) for predicting vasospasm was described by an AUC of 0.86 (CI = 0.74 to 0.98) and a cutoff value >115 cm/seconds (90% sensitivity, 73% specifi city). Interestingly, a cutoff value >168 cm/second corresponded to the best specifi city (92% specifi city), but low sensitivity (45%). The accuracy of PI for predicting VSP was lower (AUC 0.75). The accuracy of TCCS for predicting DCI was also poor (AUC 0.57). Considering larger DCI only (volume >2 ml), the accuracy was slightly increased (AUC 0.75). Conclusion TCCS is useful to predict clinical VSP with good accuracy. The Vm cutoff value >115 cm/second might be considered as a warning threshold, while higher values (>168 cm/second) identify high-risk patients. The accuracy of TCCS in predicting DCI is low, which indicates that mechanisms other than VSP are likely to play a role. Reference y y Results Brain HMPAO perfusion microSPECT (Figure 1) reveals a transient hypoperfusion after ICI (D2) and a lasting hypoperfusion in the SAH group. TER curtailed the SAH-induced decrease of the HMPAO uptake. TER also signifi cantly counteracted the SAH-induced increase of DTPA in the brainstem (SAH 2.6 ± 0.7 vs. SAH+TER 1.3 ± 0.1 ppm/ mm3; P <0.05) and increase of annexin V-128 in the cerebrum (SAH 1.2 ± 0.1 vs. SAH+TER 1 ± 0.06 ppm/mm3; P <0.05). Brain death determination in Europe: one condition with too many nuances IA Crippa, A Bronco, A Vargiolu, G Citerio San Gerardo Hospital, Monza, Italy Critical Care 2014, 18(Suppl 1):P464 (doi: 10.1186/cc13654) y y gi Results Of a total 1,221 patients admitted to our ICU, 37 patients (18 men, 19 women, age 65 ± 20) were clinically diagnosed with brain death. Primary diagnoses were 18 post-cardiac arrest syndromes, 14 cerebrovascular diseases, and four traumatic subarachnoid hemorrhages. GCS on admission was 4.5 ± 2.7 and days to cardiac arrest after diagnosed brain death was 8.6 ± 13.2. Urine volume for the last 24 hours was 997 ± 1,712 and serum potassium level was 4.8 ± 1.7. PaO2 and PaCO2 were 131.9 ± 80.6 and 48.9 ± 13.8, respectively. The fi nal causes of cardiac arrest were 20 cardiac failures, 11 renal failures and six respiratory failures, although with mechanical ventilator support. The fi nal cause of cardiac arrest due to respiratory failure had signifi cantly longer stay in the ICU and days after diagnosis of brain death than cardiac failure or renal failure (27.2 ± 22.6 days vs. 9.2 ± 10.0 and 8.1 ± 4.6, P <0.05; 23.2 ± 24.0 vs. 6.0 ± 9.0 and 5.4 ± 5.2, P <0.05, respectively).ii Introduction A patient is declared brain dead (BD) when physicians determine permanent loss of brain functions. Unfortunately, criteria for defi ning BD vary across diff erent countries [1]. We therefore decide to survey BD diagnostic modalities in Europe in order to describe diff erences. Methods A multiple-choice questionnaire was developed on an online platform [2]. Direct link was sent to national representatives of the European Society of Intensive Care Medicine and NeuroIntensive Care section’s members. Thirty-three countries were contacted. Answers were reviewed. In cases of discrepancies or missing data, participants were contacted for further clarifi cation. Descriptive statistics have been applied. Results Twenty-eight participants returned the questionnaire (85%). Every country has either specifi c law (93%) or guidelines issued by the scientifi c society (89%). Clinical examination, essential to the diagnosis, is the only requirement in 50% of countries. Coma, apnea, absence of corneal and cough refl exes are always necessary. Blood pressure and electrolytes are checked in 64% as mandatory prerequisites. The apnea test is legally defi ned in 86% of countries. Eighty-two percent of countries require achievement of a target paCO2 level while the Netherlands’ law states target apnea duration. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 of the legal issues involved. Therapy is continued except in the case of donation of organs for transplantation from brain-dead patients until their cardiac arrest. Consequently, we may determine what the fi nal cause of death is in brain-dead patients. Our hypothesis is that brain- dead patients ultimately die of cardiac failure. Results Wernicke’s area activation was observed in nine patients (eight patients with TBI, one with hypoxia). During the subsequent examination, which lasted from 3 to 12 months, seven patients with activated Wernicke’s area showed further consciousness expansion up to the minimal consciousness state (further consciousness expansion was seen in two patients). Two other patients with activated Wernicke’s area did not show any signs of consciousness. Two patients revealed signifi cant activity in the Broca’s area. of the legal issues involved. Therapy is continued except in the case of donation of organs for transplantation from brain-dead patients until their cardiac arrest. Consequently, we may determine what the fi nal cause of death is in brain-dead patients. Our hypothesis is that brain- dead patients ultimately die of cardiac failure. p y Methods From January 2011 to December 2012, brain-dead adult patients in our ICU were investigated. The declaration of brain death is determined by clinical neurological examinations, electroencephalography, and the auditory brain-stem response test. Age, sex, primary diagnosis, Glasgow Coma Scale (GCS) on admission, the number of days from the diagnosis of brain death until cardiac arrest (days), the fi nal cause of cardiac arrest, urine volume for the last 24 hours (ml), serum potassium levels (mEq/l), PaO2 (mmHg) and PaCO2 (mmHg) on the last day were evaluated. Values are expressed as mean ± SD. Data were analyzed by Kruskal–Wallis test and Mann– Whitney U test. P <0.05 was considered statistically signifi cant. gi y Conclusion According to the fi rst results of the study one can conclude that behind the outwardly similar clinical symptoms in patients in VS lies a diverse (due to the organization of brain functions) group of patients. fMRI enables one to reveal the fi rst signs of cognitive activity; that is, reveals the linguistic value of speech addressed to the patient which cannot be detected during routine neurological examination. Acute and long-term outcomes of ICU-acquired weakness: a cohort study and propensity matched analysis Acute and long-term outcomes of ICU-acquired weakness: a coho study and propensity matched analysis G Hermans1, H Van Mechelen2, B Clerckx2, T Vanhullenbusch2, D Mesotten2, A Wilmer1, MP Casaer2, P Meersseman1, Y Debaveye2, PJ Wouters2, R Gosselink2, G Van den Berghe2 1UZ Leuven, Belgium; 2KU Leuven, Belgium Critical Care 2014, 18(Suppl 1):P466 (doi: 10.1186/cc13656) y p p y y G Hermans1, H Van Mechelen2, B Clerckx2, T Vanhullenbusch2, D Mesotten2, A Wilmer1, MP Casaer2, P Meersseman1, Y Debaveye2, PJ Wouters2, R Gosselink2, G Van den Berghe2 1UZ Leuven, Belgium; 2KU Leuven, Belgium Critical Care 2014, 18(Suppl 1):P466 (doi: 10.1186/cc13656) G Hermans1, H Van Mechelen2, B Clerckx2, T Vanhullenbusch2, D Mesotten2, A Wilmer1, MP Casaer2, P Meersseman1, Y Debaveye2, PJ Wouters2, R Gosselink2, G Van den Berghe2 1UZ Leuven, Belgium; 2KU Leuven, Belgium Critical Care 2014, 18(Suppl 1):P466 (doi: 10.1186/cc13656) Introduction ICU-acquired weakness is a frequent complication of critical illness. It is unclear whether it is a marker or mediator of poor outcomes. We aimed to determine acute and long-term outcomes and costs of ICU-acquired weakness among long-stay (≥8 days) ICU patients and to assess the impact of recovery of weakness at ICU discharge. Methods Data were prospectively collected during an RCT (Clinical trials.gov: NCT00512122) [1,2]. Impact of weakness (MRC sum <48) on outcomes and costs was analyzed with 1:1 propensity score-matching for baseline characteristics, illness severity and risk factor exposure prior to assessment. Among weak patients, impact of persisting weakness at ICU discharge on risk of death after 1 year was examined with multivariable Cox proportional-hazard analysis. l Conclusion There are still areas of uncertainty and disparities in brain death diagnosis in European countries. This predisposes to misdiagnosis and confusion both for clinicians and families. Measures to promote uniformity of brain death procedures and clinical practice are therefore desirable. References 1. Wijdicks EF: Brain death worldwide: accepted fact but no global consensus in diagnostic criteria. Neurology 2002, 58:20-25. 2. [www.surveyplanet.com] p p y Results A total 227 of the 405 (56%) long-stay assessable ICU patients were weak; 122 weak patients were matched to 122 not-weak patients. As compared with matched not-weak patients, weak patients had a lower likelihood at any time for live weaning from mechanical ventilation (HR: 0.709 (0.548 to 0.918), P = 0.009), live ICU (HR: 0.738 (0.570 to 0.955), P = 0.021) and hospital discharge (HR: 0.682 (0.521 to 0.893), P = 0.005). In-hospital costs/patient (+30.5%, €+5,443/patient, P = 0.04) and 1-year mortality (30.6% vs. 17.2%, P = 0.015) were also higher. The 105/227 (46%) weak patients not matchable to not-weak patients had even worse prognosis and higher costs. At any time within the fi rst year following ICU admission, compared with patients who recovered from weakness and adjusted for potential confounders, those with persistent weakness and MRC sum between 36 and 47 at P465 P465 What do brain-dead patients ultimately die of? A Kawasaki, M Hotta, K Mochiduki, N Saito, M Namiki, M Takeda, T Harada, A Yaguchi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P465 (doi: 10.1186/cc13655) Functional MRI examination results in patients with vegetative state: revealing of the Wernicke’s area EA Kondratyeva1, SV Diment2, SA Kondratyev EA Kondratyeva1, SV Diment2, SA Kondratyev1 1Russian Polenovs Neurosurgical Institute, St Petersburg, Russia; 2International Institute of Biological Systems, St Petersburg, Russia Critical Care 2014, 18(Suppl 1):P463 (doi: 10.1186/cc13653) Introduction The aim was to evaluate the prognostic importance of the MRI examination using Block Design fMRI in patients in a vegetative state (VS). Introduction The aim was to evaluate the prognostic importance of the MRI examination using Block Design fMRI in patients in a vegetative state (VS). Methods Twenty-two patients corresponding to the international standards of VS underwent high-resolution MRI (T2 and T1, isomatrix with slice thickness 1.00 mm, T2 FLAIR FS, DWI, SWI) examination and Block Design fMRI (paradigm of passive speech). The patients were aged from 4 to 42 years. Causes of VS: TBI in 19 patients, hypoxia in three patients. By the time of the examination the patients were in VS from 1 to 4 months. Figure 1 (abstract P461). HMPAO uptake at day 2 and day 5. *P <0.01 versus day 0; #P <0.01 versus day 2; ‡P <0.01. S167 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Brain death determination in Europe: one condition with too many nuances IA Crippa, A Bronco, A Vargiolu, G Citerio San Gerardo Hospital, Monza, Italy Critical Care 2014, 18(Suppl 1):P464 (doi: 10.1186/cc13654) Number of physicians (median 2, range 1 to 4), number of clinical examinations (median 2, 1 to 3), and minimum observation time (median 3 hours, 0 to 12) are variable requisites in diff erent countries. In 50% of nations, additional tests are required. Hypothermia (4%), anoxic injury (7%), inability to complete clinical examination (61%), toxic drug levels (57%), and inconclusive apnea test (54%) are legal indications to perform additional tests. Cerebral blood fl ow investigation is mandatory in 18% of countries, while it is either optional or used only in selected cases in 82%. Conventional angiography is still the preferred method (50%), followed by transcranial Doppler (43%), angioCT (39%), CT perfusion and angioMR (11%). EEG is always (21%) or optionally (14%) recorded. Russia and Croatia evaluate both EEG and cerebral blood fl ow (7%). Conclusion There were signifi cantly longer days until fi nal cardiac arrest that was caused by respiratory failure than by cardiac failure or renal failure. Many diff erent observations in each case were shown, such as urine volume or potassium level on the last day. Not only cardiac failure but also other failures might lead to fi nal cardiac arrest. P466 P467 Early electrophysiological diagnosis of ICU-acquired weakness L Wieske, C Verhamme, E Witteveen, A Bouwes, MJ Schultz, IN Van Schaik, J Horn Academic Medical Center, University of Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P467 (doi: 10.1186/cc13657) Introduction Cerebral near-infrared spectroscopy (NIRS) represents an exciting prospect for the non-invasive monitoring of cerebral tissue oxygenation in traumatic brain injury (TBI). Earlier attempts at clinical application of cerebral NIRS demonstrated that further work was needed [1]. The basic layout of the probe portion of these devices consists of a light source and a light detector, arranged at calculated distances and confi gurations in order to observe target tissue. We aim to determine which commercially available NIRS probe represents the most sensitive layout of sources/detectors for the greatest sensitivity in observing tissue oxygenation at the optimal depth for TBI. Academic Medical Center, University of Amsterdam, the Netherlands Critical Care 2014, 18(Suppl 1):P467 (doi: 10.1186/cc13657) Introduction An early diagnosis of ICU-acquired weakness (ICU-AW) is diffi cult because disorders of consciousness preclude strength assessment [1]. Electrophysiological (EMG) studies may be an alternative approach [2]. In this study we investigated feasibility and diagnostic accuracy of EMG studies to diagnose ICU-AW in unconscious patients. y Methods The optimal depth for grey matter target tissue (grey–white matter junction) was ascertained by reviewing a series of brain CT scans of patients who had sustained a TBI. Set (average) measurements were derived from these identifying the target depth of the grey matter strip from the point of probe placement. Currently there are fi ve commercially available cerebral NIRS systems. Table 1 presents the variety of confi gurations off ered by each device. Source detector layouts were modelled and simulated using the NIRFAST® computational light modelling software (developed at the University of Birmingham) [2]. The novel approach of this modelling system makes no extrapolated assumptions based on subtraction. p Methods Newly admitted unconscious ICU patients (RASS <–3), ventilated for ≥2 days, were included in this single-center prospective cohort study. EMG testing included ulnar (motor/sensory), peroneal (motor) and sural (sensory) studies. Myography was performed when coagulation was normal (dorsal interossei I/II, deltoid and tibial muscles). Reliability of results was checked by an experienced neurophysiologist, blinded for strength. Motor/sensory studies were abnormal if amplitudes were below the 2.5th percentile reference values [3]. Myography was abnormal if spontaneous abnormal activity was found in ≥1 muscles. P468 P468 Choosing a cerebral near-infrared spectroscopy system for use in traumatic brain injury: deriving the ideal source detector layout D Davies1, M Clancy2, Z Su1, H Denghani2, A Belli1 1National Institute for Health Research Surgical Reconstruction and Microbiology, Edgbaston, Birmingham, UK; 2University of Birmingham – School of Computational Science, Birmingham, UK Critical Care 2014, 18(Suppl 1):P468 (doi: 10.1186/cc13658) Acknowledgement GH and HVM contributed equally to this study. R f Acknowledgement GH and HVM contributed equally to this study. References 1. Casaer M, et al.: N Engl J Med 2011, 365:506-517. 1. Casaer M, et al.: N Engl J Med 2011, 365:506-517. 2. Hermans G, et al: Lancet Respir Med 2013, 1:621-629. 1. Casaer M, et al.: N Engl J Med 2011, 365:506-517. 2. Hermans G, et al: Lancet Respir Med 2013, 1:621-629. , g , 2. Hermans G, et al: Lancet Respir Med 2013, 1:621-629. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 ICU discharge had a higher likelihood of death (HR: 1.937, 95% CI: 1.048 to 3.581, P = 0.035). This likelihood of late death was even higher for those patients with a more severe degree of persistent weakness (MRC sum <36) (HR: 1.815, 95% CI: 3.693 to 7.514, P <0.001). P465 What do brain-dead patients ultimately die of? Introduction After a patient is declared brain dead, the cessation or withdrawal of therapy in Japan is quite a diffi cult operation because Introduction After a patient is declared brain dead, the cessation or withdrawal of therapy in Japan is quite a diffi cult operation because S168 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References References 1. Crit Care Med 2009, 37:S299-S308. 2. Crit Care Med 2009, 37:2632-2637. 3. J Neurol 2004, 251:1491-1497. Conclusion Patients with ICU-acquired weakness had worse acute morbidity outcomes, consumed more resources and revealed higher mortality after 1 year than patients without weakness. Persistence of weakness at ICU discharge further increased late mortality. P467 P469 Single-subject assessment of the distribution of white matter abnormalities measured by diff usion tensor imaging in patients with severe traumatic brain injury S Magnoni1, M Macrì2, F Stretti2, C Mac Donald3, R Biffi 2, E Zanier4, A Snyder3, J Shimony3, N Stocchetti1, D Brody3 1Fondazione IRCCS Cà Granda – Ospedale Maggiore Policlinico, Milan, Italy; 2Milan University, Milan, Italy; 3Washington University, St Louis, MO, USA; 4IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy Critical Care 2014, 18(Suppl 1):P469 (doi: 10.1186/cc13659) P469 Single-subject assessment of the distribution of white matter abnormalities measured by diff usion tensor imaging in patients with severe traumatic brain injury Introduction Traumatic axonal injury (TAI) is a major contributor to adverse outcomes following traumatic brain injury (TBI). Diff usion tensor imaging (DTI) is a magnetic resonance imaging technique, which provides a robust measure of white matter integrity in patients with TBI. Certain brain regions have been identifi ed as particularly susceptible to TAI by means of DTI. Few studies, however, have focused on describing the distribution of DTI abnormalities in individual TBI patients. The aim of this project was to conduct an exploratory analysis of the extent and distribution of axonal injury in TBI patients at a single- subject level. Methods Patients admitted to the ICU for severe TBI underwent brain MRI and DTI (32 directions, b = 1,000, voxel size 2 × 2 × 2 mm3) between 2 weeks and 3 years after injury. For each individual patient, we enrolled fi ve age-and-sex-matched healthy volunteers who underwent the same DTI protocol and were used as controls (31 total). We used a region of interest (ROI) automated analysis [1] to quantify white matter integrity. The fractional anisotropy (FA) maps were segmented using a white matter parcellation map covering the entire brain. Our primary outcome was the normalized diff erence in FA between each patient and the controls. Abnormalities were defi ned as values that were more than 2 SD below the mean of the values for the matched controls for each ROI. Figure 2 (abstract P468). CT measurements and sensitivities at depth. Results Twelve TBI patients with a median age of 30 years (range 20 to 38) and a median GCS of 5 (range 5 to 7) were included. The majority of them had diff use axonal injury according to CT and conventional MRI. Reference [www.mristudio.org] [www.mristudio.org] y Conclusion Based on the computational modelling of our work, the FORE-SIGHT NIRS device by CAS Medical source detector layout provides the greatest sensitivity at depth for the purposes of cortical monitoring in trauma. Variations in the layout have a signifi cant impact on the quality of signal detected. 1. Wolf M, et al.: J Biomed Opt 2007, 12:062104. P470 Long-term outcome after severe traumatic brain injury M Ciapetti, M Migliaccio, A Cecchi, M Bonizzoli, A Peris Intensive Care Unit, Florence, Italy Critical Care 2014, 18(Suppl 1):P470 (doi: 10.1186/cc13660) P467 Analysis of individual ROIs revealed that all subjects had numerous abnormal ROIs, varying from 14 to 64 (median 21) out of 78 regions assessed. The most frequently altered regions were the frontal lobe, orbital–frontal regions, corpus callosum, internal capsules, superior and inferior longitudinal fasciculi, cingulum, cerebellar structures and corona radiata. Thalamus, hippocampus and occipital lobes were less frequently aff ected. q yf Conclusion The extent and distribution of TAI varies on a patient basis. These fi ndings highlight the need for standardized methods to precisely assess TAI in single subjects. Such single-subject methods will probably improve prognostic accuracy and may be useful in clinical trials. Results We reviewed 32 trauma series CT brain images and the average depth to grey matter was derived as 21.37 mm (range 16.59 to 31.03 mm, SD 2.33) from the surface (Figure 1). The spectrum of sensitivity of the fi ve probes was modelled (Figure 2). As is apparent here, the FORE-SIGHT (CAS Medical, CT, USA) probe currently off ers the greatest sensitivity at our derived target depth. 2. Dehghani H, et al.: Commun Numer Methods Eng 2008, 25:711-732. P467 Upon awakening, strength was assessed (ICU- AW: average MRC <4 [1]), blinded for EMG. Feasibility was determined as the percentage of measurements that could be performed and were reliable. Diagnostic accuracy was analyzed using sensitivity and specifi city. Figure 1 (abstract P468). Light scatter predictions of available systems/ probes illustrated. i y Results We included 35 patients (ICU-AW: 17). EMG testing was done on day 4 (IQR: 3 to 6). Feasibility was 94%, 89%, 77%, 34% and 31% for ulnar motor, peroneal motor, ulnar sensory, sural sensory and myography studies, respectively. Figure 1 displays amplitude values. Sensitivity/specifi city was 100%/0%, 100%/31%, 31%/100%, 50%/25% and 67%/38%, respectively. Conclusion Feasibility of ulnar and peroneal studies was acceptable; feasibility of sural and myography studies was low. Diagnostic accuracy was low for all studies. This may be improved with new reference values. Figure 1 (abstract P467). Dotted lines represent 2.5th percentile reference values [3]. Figure 1 (abstract P468). Light scatter predictions of available systems/ probes illustrated. Figure 1 (abstract P468). Light scatter predictions of available systems/ probes illustrated. Figure 1 (abstract P467). Dotted lines represent 2.5th percentile reference values [3]. S169 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (abstract P468). Available source detector layouts Probe Wavelengths (nm) Sources Detectors Spacing (mm) Peak depth sensitivity Optiplex TX (ISS, IL, USA) 690, 830 4 1 30, 35, 40, 45 8 INVOS (Covidien, MA, USA) 730, 810 1 2 30, 40 8 EQUANOX (Nonin, MN, USA) 730, 810, 880 2 2 20, 40 7 FORE-SIGHT (CAS Medical, CT, USA) 690, 780, 805, 850 1 2 20, 50 10 NIRO-200NX (Hamamatasu, Japan) 735, 810, 850 1 2 19.2, 20 9 Table 1 (abstract P468). Available source detector layouts Figure 2 (abstract P468). CT measurements and sensitivities at depth. Vitamin D level could aff ect the recovery rate in traumatic brain injury: a retrospective study Introduction Severe traumatic brain injury (TBI) is a major cause of death in people between 19 and 45 years old. Gastrointestinal dysfunction is the most common complication due to mucosal ischemia, motility disorders, and disruption of the gut barrier, with severe consequences: malnutrition, weight loss, and high risk of infections [1]. Therefore, maintaining the intestinal barrier function is a systematic engineering project. Selected new probiotics due to the capacity to bind and neutralize toxins, and to interfere with pathogen adherence, by immunomodulatory properties, mopping up the infection, could improve recovery of critically ill patients [2]. Our aim was to assess the eff ects of a new probiotic in an early enteral regimen on clinical outcome of severe TBI patients, in terms of VAP incidence, tolerance to enteral nutrition, duration of mechanical ventilation, and mortality rate. FI Socci, S Di Valvasone, A Cecchi, M Bonizzoli, A Terreni, A Peris AOU Careggi, Florence, Italy FI Socci, S Di Valvasone, A Cecchi, M Bonizzoli, A Terreni, A Peris AOU Careggi, Florence, Italy FI Socci, S Di Valvasone, A Cecchi, M Bonizzoli, A Terreni, A Peris AOU Careggi, Florence, Italy gg y Critical Care 2014, 18(Suppl 1):P471 (doi: 10.1186/cc13661) Critical Care 2014, 18(Suppl 1):P471 (doi: 10.1186/cc13661) Introduction Recent studies have shown that 1,25-dihydroxyvitamin D3 (vitamin D) defi ciency may aff ect negatively the clinical course of traumatic brain injury (TBI) [1]. This problem becomes important with respect to the older patient considering a 50% prevalence of vitamin D defi ciency [2]. Data from the Third National Health and Nutrition Examination Survey [3] document more than 60% of Caucasians aff ected by D defi ciency [4] so that all patients with TBI of any age are theoretically at risk of unfavorable outcome [2]. The objective of this preliminary study was to determine whether low levels of vitamin D at admission to the ICU (<24 hours) could negatively aff ect neurological recovery of patients with TBI. y Methods A prospective randomized 1-year study of 64 patients 19 to 78 years old allocated to receive for 10 days either an early enteral diet plus a new probiotic (Bioent; Lactobacillus bulgaricus 10 trillion CFU/cp + activated charcoal) every 6 hours (Group A) or the same formula without probiotics (Group B). The diets were isocaloric and isonitrogenous, and there were no diff erences between groups in gender, age, and nutritional status. Vitamin D level could aff ect the recovery rate in traumatic brain injury: a retrospective study We assessed the VAP incidence, duration of mechanical ventilation, tolerance to enteral nutrition, length of ICU stay, duration of diarrhea episodes, and mortality rate. The ANOVA test and t test were carried out; P <0.05 was considered signifi cant. y p Methods We retrospectively analyzed the data of 46 patients aff ected by TBI (65% severe, 9.5% moderate, 28.5% moderate) both isolated or associated with other extracranial lesions. The sampling of vitamin D was carried out within 24 hours from ICU admission. We had registered GCS at the moment of presentation (GCS in) and at discharge (GCS out) and their diff erence (GCS diff ) compared with levels of vitamin D. Patients that died in the ICU were assigned a GCS out = 0. See Table 1. Results Our data, according to other studies [5], confi rm the presence of a defi ciency of vitamin D (Table 1); however, they do not demonstrate a statistical signifi cance correlation at the univariate regression (R = 0.04; P = 0.786) between vitamin D level and outcome from the ICU. There was no correlation stratifying patients for age, for TBI class, for Injury Severity Score and for BMI. Methods We retrospectively analyzed the data of 46 patients aff ected by TBI (65% severe, 9.5% moderate, 28.5% moderate) both isolated or associated with other extracranial lesions. The sampling of vitamin D was carried out within 24 hours from ICU admission. We had registered GCS at the moment of presentation (GCS in) and at discharge (GCS out) and their diff erence (GCS diff ) compared with levels of vitamin D. Patients that died in the ICU were assigned a GCS out = 0. See Table 1.i Results Our data, according to other studies [5], confi rm the presence of a defi ciency of vitamin D (Table 1); however, they do not demonstrate a statistical signifi cance correlation at the univariate regression (R = 0.04; P = 0.786) between vitamin D level and outcome from the ICU. There was no correlation stratifying patients for age, for TBI class, for Injury Severity Score and for BMI. Results The infection rate was higher in group B, the duration of mechanical ventilation was shorter in group A, and the patients in group A received 91.7% of total caloric needs by an enteral route versus 74.68% in group B. 1. Chatterjee M, et al.: Vitamin D and genomic stability Mutat Res 2001; 475:69-87. P471 , , Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2014 18(Suppl 1):P472 (doi: 10 1186/cc13662) , , Emergency Hospital Floreasca, Bucharest, Romania Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P472 (doi: 10.1186/cc13662) g y p Critical Care 2014, 18(Suppl 1):P472 (doi: 10.1186/cc13662) Could selected probiotics have benefi cial eff ects on clinical outcome of severe traumatic brain injury patients? D Pavelescu, L Mirea, I Grintescu Emergency Hospital Floreasca, Bucharest, Romania Critical Care 2014, 18(Suppl 1):P472 (doi: 10.1186/cc13662) D Pavelescu, L Mirea, I Grintescu Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Table 1 (P471) profound impact of TBI is not only felt by the individuals who suff er the injury but also their caregivers and society as a whole. In this study we observed the long-term outcome of patients with severe traumatic brain injury admitted to our intensive care. TBI Descriptive statistics Isolated Associated P value Dead 8.70% Percentage 22 78 Male 87% GCS in 4 ± 3 8 ± 5 0.043 Age 50 ± 21 GCS out 10 ± 4 10 ± 5 0.933 BMI 26.36 ± 4.14 GCS diff 6 ± 4 3 ± 6 0.151 ISS 29 ± 15 Vitamin D (ng/ml) 17 ± 8.5 17 ± 9.8 0.597 Vitamin D 73.90% Length of 12 ± 7 8 ± 9 0.261 defi ciency stay ICU (days) (<30 ng/ml) j y Methods This study includes all patients (n = 160) with severe head trauma (GCS <9) admitted to the ICU of the emergency department of a tertiary referral center (Careggi Teaching Hospital, Florence, Italy) from 2009 to 2012. All patients will undergo a clinical assessment after 1 year, which is routine post-intensive follow-up. As an objective index of ability to function after injury, the Glasgow Outcome Scale (GOS) will be used. The neurological evaluation to determine the outcome of patients by GOS is performed in two diff erent modes. All eligible patients 1 year after discharge from the ICU are contacted by telephone by a nurse of the intensive care staff and invited to make a visit to the surgery follow-up, where a intensivist evaluates the patient and determines the GOS. For the patients who were still hospitalized at 6 months in rehabilitation departments, GOS assessment is performed by a doctor of the structure and notifi ed by telephone. 2. Milos C, et al.: Combination treatment with progesterone and vitamin D hormone may be more eff ective than monotherapy for nervous system injury and disease. Neuroendocrinology 2009, 30:158-172. yi y Results The ICU and hospital mortality was respectively 33.7% (n = 54) and 36.9% (n = 59). The mortality at 1 year was 44.4% (n = 71). The results of the neurological follow-up at 1 year were: GOS 2: 5.6% (n = 9), GOS 3: 10% (n = 16), GOS 4: 13.1% (n = 21), GOS 5: 26.9% (n = 43).i 3. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Plan and Operation of the Third National Health and Nutrition Examination Survey, 1988–94. DHHS publication No. (PHS)94-1308 – Vital and Health Statistics, series 1, no. 32. Hyattsville, MD: US Department of Health and Human Services, Public Health Service, CDC; 1994. Conclusion According to the other studies, our data confi rm that the severe traumatic brain injury is associated with a high mortality at 1 year. One-half of the survivors have a diff erent level of disability. References 4. Khazai N, et al.: Calcium and vitamin D: skeletal and extraskeletal health. Curr Rheumatol Rep 2008, 10:110-117. 4. Khazai N, et al.: Calcium and vitamin D: skeletal and extraskeletal health. Curr Rheumatol Rep 2008, 10:110-117. 5. Cecchi A, et al.: Vitamin D defi ciency in septic patients at ICU admission is not a mortality predictor. Minerva Anest 2011, 77:1184-1189. 1. Nichol AD, et al.: Measuring functional and quality of life outcomes following major head injury: common scales and checklists. Injury 2011, 42:281-287. 2. Roozenbeek B, et al.: Prediction of outcome after moderate and severe traumatic brain injury: external validation of the(IMPACT) and (CRASH) prognostic models. Crit Care Med 2012, 40:1609-1617. References Introduction Traumatic brain injury is a major public health issue, which results in signifi cant mortality and long-term disability [1,2]. The S170 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P474 P474 Long-term outcome prediction using IMPACT and APACHE II in patients with traumatic brain injury treated in the ICU R Raj, R Kivisaari, J Siironen, M Skrifvars Helsinki University Central Hospital, Helsinki, Finland Critical Care 2014, 18(Suppl 1):P474 (doi: 10.1186/cc13664) Critical Care 2014, 18(Suppl 1):P473 (doi: 10.1186/cc13663) Introduction We investigated whether blood alcohol levels (BAL) had any impact on presentation and outcome in patients with traumatic brain injury (TBI). Introduction The International Mission for Prognosis and Analysis of Clinical Trials (IMPACT) is currently the most robust prognostic model in patients with traumatic brain injury (TBI). No studies have compared this TBI-specifi c prediction model with general ICU models, such as the Acute Physiology and Chronic Health Evaluation II (APACHE II) [1,2]. This study investigates the performance and the correlation of the IMPACT and APACHE II models for long-term outcome prediction in patients with TBI. y Methods Forty-six patients with TBI requiring intubation between January 2000 and December 2012 were included. Patients were grouped into BAL-positive (>0.5‰; n = 24) and BAL-negative (<0.5‰; n = 22). Physiological parameters and outcome (survival to hospital discharge (STHD)) and neurological outcomes (Glasgow Outcome Scale (GOS), Cerebral Performance Category (CPC) and Glasgow Coma Scale (GCS)) were analyzed. Diff erences between groups were analyzed using Student’s t test and results presented as mean ± SD. Methods The study population consisted of TBI patients admitted to a designated ICU in southern Finland during a 4-year period (2009 to 2012). The IMPACT and APACHE II performances were assessed by calculating discrimination (by area under the curve), calibration (by GiViTI calibration test and Hosmer–Lemeshow C statistic) and precision (by Brier score). Correlation between the IMPACT and APACHE II was tested by Spearman’s rho. Primary outcome was 6-month mortality. Results Of the total 897 included patients, overall 6-month mortality was 22%. The IMPACT and APACHE II showed similar AUCs (0.81, 95% CI = 0.77 to 0.84; 0.80, 95% = CI 0.76 to 0.83) and Brier scores (0.134, 0.135). Calibration tests revealed signifi cant (P <0.05) deviations between observed and predicted risk for both models. A moderately strong, positive correlation between the IMPACT and the APACHE II g Results There were no signifi cant diff erences in gender and age distributions between the BAL-negative and BAL-positive groups (73% vs. 88% male; P = 0.218; 53 ± 21 vs. 43 ± 17 years; P = 0.098). Vitamin D level could aff ect the recovery rate in traumatic brain injury: a retrospective study There is a signifi cant diff erence in the number of diarrhea episodes, and the ICU length of stay was signifi cantly lower in group A; there was no signifi cant diff erence in the mortality rate between groups. See Figure 1. y Conclusion Vitamin D defi ciency is really prevalent in our TBI cases but does not seem to aff ect neurological recovery at ICU discharge; however, these preliminary results should be exposed to several criticisms and need to be confi rmed with prospective studies. Conclusion Early administration of new probiotics to severe TBI patients could have benefi cial eff ects in terms of reduction of GI dysfunction, VAP incidence and length of ICU stay. S171 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 pp http://ccforum.com/supplements/18/S1 Figure 1 (abstract P472). Figure 1 (abstract P472). Figure 1 (abstract P472). Figure 1 (abstract P472). Conclusion BAL did not have a signifi cant eff ect on presenting physiological parameters such as systolic BP and RR. It did, however, aff ect the presenting GCS score, which was signifi cantly lower in the BAL-positive group. BAL did not seem to have an eff ect on functional outcome or mortality measured by STHD. No favorable neuroprotective or deleterious eff ect was observed, demonstrated by no signifi cant diff erences in CPC, GOS or GCS scores at discharge. Conclusion BAL did not have a signifi cant eff ect on presenting physiological parameters such as systolic BP and RR. It did, however, aff ect the presenting GCS score, which was signifi cantly lower in the BAL-positive group. BAL did not seem to have an eff ect on functional outcome or mortality measured by STHD. No favorable neuroprotective or deleterious eff ect was observed, demonstrated by no signifi cant diff erences in CPC, GOS or GCS scores at discharge. P473 Eff ect of blood alcohol level on outcome of patients with traumatic brain injury k k d l l Eff ect of blood alcohol level on outcome of patients with traumatic brain injury V Borovnik Lesjak, M Strnad, V Vujanović Popović, T Pelcl ZD dr. Adolfa Drolca Maribor, Slovenia Critical Care 2014, 18(Suppl 1):P473 (doi: 10.1186/cc13663) Functional status after 3 years in ICU patients with traumatic brain injury Figure 1 (abstract P474). Area under the curve for the new models. Introduction The aim was to study the functional status at 1 year and after 3 years in ICU patients with traumatic brain injury (TBI). Introduction The aim was to study the functional status at 1 year and after 3 years in ICU patients with traumatic brain injury (TBI). Methods A prospective cohort study of patients with TBI admitted to Carlos Haya Hospital, Malaga, between 2004 and 2008. l d d Introduction The aim was to study the functional status at 1 year and after 3 years in ICU patients with traumatic brain injury (TBI). Methods A prospective cohort study of patients with TBI admitted to Carlos Haya Hospital, Malaga, between 2004 and 2008. y p j y ( ) Methods A prospective cohort study of patients with TBI admitted to Carlos Haya Hospital, Malaga, between 2004 and 2008. was noted (Spearman’s rho = 0.566, P <0.001). The IMPACT and the APACHE II were found to identify slightly diff erent groups of patients that eventually do not survive (Figure 1). y p , g , Results We studied 531 patients, age 40.35 ± 19.75 years, APACHE II 17.94 ± 6.97, GCS admission 7.53 ± 3.83 points. Cranial tomography at admission was: diff use injury type I (10.4%), diff use injury type II (28.1%), diff use injury type III (24.5%), diff use injury type IV (8.3%), evacuated mass lesion (22.6%), non-evacuated mass lesion (6.2%). Hospital mortality was 28.6%. One-year mortality was 171 (32.2%) (missing: 6.6%). After 3 years, mortality was 181(34.1%) (16.2% missing). We followed 496 patients for 1 year after admission (35 were missing) and assessed their functional status using the GOS. Thus, 22.7% were normal and 20% presented some limitations but were self-suffi cient, representing 55.6% of those who survived hospital admission. A total of 57.3% presented bad evolution (died: 34.5%, vegetative: 3%, disabled not self-suffi cient: 19.8%). After 3 years we followed 445 patients and 86 (16.2%) were missing. Thus, 132 patients (29.7% of 445 patients) were normal and 15.3% presented some limitations but were self-suffi cient, representing 75.76% of those who survived hospital admission. Fifty-six percent of 445 patients presented bad evolution (died: 40.7%, vegetative: 2.2%, disabled not self-suffi cient: 12.1%). Functional status after 3 years in ICU patients with traumatic brain injury Of 445 patients studied after 3 years of admission 171 died, and of the 274 who survived 198 (72.2%) were in a similar functional situation after 1 year, 11 (4.01%) had worsened functional situation, of which 10 had died, and 65 (27.72%) had improved situation (P <0.001). y g Conclusion The IMPACT and the APACHE II models showed equal performance for 6-month mortality prediction. A moderately strong, positive correlation, with some major discrepancies between the models, was found. Thus, features of both the IMPACT and APACHE II models are valuable for optimal outcome prediction in patients with TBI treated in the ICU. Validating and comparing the CAM-ICU and the ICDSC in mild and moderate traumatic brain injury patients: a multicenter prospective study Conclusion This study shows that approximately 75% of the patients who survived after 3 years from admission to the ICU with traumatic brain injury presented good recovery with functional situation normal or with some limitations but self-suffi cient. Between 1 and 3 years after admission, approximately 25% of patients improved their functional situation. Introduction The Confusion Assessment Method for the ICU (CAM- ICU) and the Intensive Care Delirium Screening Checklist (ICDSC) are recommended for routine delirium screening. However, literature about delirium assessment in traumatic brain injury (TBI) remains scarce. The aim of our study was to evaluate the validity and reliability of the CAM-ICU and the ICDSC for delirium assessment in patients with mild to moderate TBI. P474 There were also no diff erences in initial systolic blood pressure (BP; 144 ± 30 vs. 132 ± 37 mmHg; P = 0.241) and respiratory rate (RR; 13 ± 6 vs. 12 ± 7/ minute; P = 0.891) but BAL did aff ect the initial GCS score (7 ± 3 vs. 5 ± 2; P = 0.022). There was no eff ect on CPC (3.5 ± 1.9 vs. 2.8 ± 1.8; P = 0.185), GOS (2.4 ± 1.8 vs. 3.0 ± 1.7; P = 0.270), and GCS at discharge from the hospital (14 ± 2 vs. 14 ± 2; P = 0.801). There were also no diff erences in length of hospital stay (LOHS; 15 ± 23 vs. 23 ± 34; P = 0.372) and STHD (1.6 ± 0.5 vs. 1.3 ± 0.5; P = 0.083). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 S172 Figure 1 (abstract P474). Area under the curve for the new models. Results During an 8-month period, 61 patients (mean age 56.4 ± 18.5 years, mean APACHE II score 11.4 ± 6.5, mean GCS 13 ± 2) were enrolled. The overall sensitivity and specifi city for CAM-ICU (62% and 74%, respectively) and ICDSC (64% and 79%, respectively) were similar. The overall kappa for inter-rater reliability for the CAM-ICU and ICDSC was 0.64 and 0.68, respectively. In subgroup analyses, the CAM- ICU and ICDSC showed increased sensitivity and decreased specifi city in moderate TBI as well as deeper levels of sedation (RASS –2 to –3). Conclusion Criterion validity and inter-rater reliability of the CAM-ICU and ICDSC were good. Severity of TBI and depth of sedation infl uence delirium assessments. Clinicians should be aware of those limitations before using these clinical tools in this population. Results During an 8-month period, 61 patients (mean age 56.4 ± 18.5 years, mean APACHE II score 11.4 ± 6.5, mean GCS 13 ± 2) were enrolled. The overall sensitivity and specifi city for CAM-ICU (62% and 74%, respectively) and ICDSC (64% and 79%, respectively) were similar. The overall kappa for inter-rater reliability for the CAM-ICU and ICDSC was 0.64 and 0.68, respectively. In subgroup analyses, the CAM- ICU and ICDSC showed increased sensitivity and decreased specifi city in moderate TBI as well as deeper levels of sedation (RASS –2 to –3). Conclusion Criterion validity and inter-rater reliability of the CAM-ICU and ICDSC were good. P475 Validating and comparing the CAM-ICU and the ICDSC in mild and moderate traumatic brain injury patients: a multicenter prospective study E Bebawi1, L Deslauriers1, AA Tessier1, AJ Frenette1, MM Perreault2, MS Delisle2, JC Bertrand1, M Desjardins2, F Bernard1, P Rico1, K Khwaja2, L Burry3, DR Williamson1 1Hôpital du Sacré-Coeur de Montréal, Canada; 2McGill University Health Center, Montreal, Canada; 3Mount Sinai Hospital, Toronto, Canada Critical Care 2014, 18(Suppl 1):P475 (doi: 10.1186/cc13665) P474 Severity of TBI and depth of sedation infl uence delirium assessments. Clinicians should be aware of those limitations before using these clinical tools in this population. References 1. Lingsma et al.: Lancet Neurol 2010, 9:543-554. 1. Lingsma et al.: Lancet Neurol 2010, 9:543-554. 2. Maas et al.: Lancet Neurol 2013, 12:1200-1210. 2. Maas et al.: Lancet Neurol 2013, 12:1200-1210. P476 P476 Functional status after 3 years in ICU patients with traumatic brain injury M Delange van der Kroft1, M Arias-Verdú2, E Banderas-Bravo2, E Curiel-Balsera2, A Muñoz-Lopez2, J Fernandez-Ortega2, R Rivera-Fernandez2, M Prieto-Palomino2 1County Hospital of Axarquia, Velez Malaga, Spain; 2Carlos Haya University Hospital, Malaga, Spain Critical Care 2014, 18(Suppl 1):P476 (doi: 10.1186/cc13666) Outcome measures in randomized controlled trials of patients with severe traumatic brain injury: a systematic review g Results A total of 1,625 patients were included. Overall 6-month mortality was 33%. The APACHE II and SAPS II-based models showed good discrimination (area under the curve (AUC) 0.79, 95% confi dence interval (CI) = 0.75 to 0.82; and 0.80, 95% CI = 0.77 to 0.83), calibration (P >0.05) and precision. The SOFA-based model showed poor discrimination (AUC 0.68, 95% CI = 0.64 to 0.72) and precision but good calibration (P >0.05). The adjusted SOFA model displayed better discrimination (AUC 0.79, 95% CI = 0.76 to 0.82). The reference model showed comparable performance with all scoring system-based models regarding discrimination (AUC 0.77, 95% CI = 0.74 to 0.80), precision and calibration. See Figures 1 and 2. j y y G Lalonde1, M Shemilt1, F Lauzier1, P Desjardins1, A Boutin1, L Moore1, D Fergusson2, R Zarychanski3, A Turgeon1 1Laval University, Quebec, Canada; 2OHRI, Ottawa, Canada; 3University of Manitoba, Winnipeg, Canada Critical Care 2014, 18(Suppl 1):P478 (doi: 10.1186/cc13668) Introduction Traumatic brain injury (TBI) is a major cause of death and long-term disability worldwide, and understanding its eff ect on health is critical. Although mortality has been a gold standard for years, functional scales and quality of life have been used as main outcome measures over the last decades due to their usefulness and aid in decision-making for medical teams, patients, and families. Despite this preference, no consensus exists for the most optimal outcome measure. We systematically reviewed outcome measures used in randomized controlled trials (RCTs) in patients with severe TBI admitted to an acute care hospital. Conclusion A simple prognostic model, based only on age and GCS, displayed a fairly good prognostic performance in predicting 6-month mortality of ICU-treated patients with moderate-to-severe TBI. The use of more complex scoring systems added little to the prognostic performance. p References p Methods We searched MEDLINE, EMBASE, Cochrane Central, BIOSIS and references of eligible trials. RCTs published over the last 7 years (2006 to October 2013) in 18 selected journals (based on impact factor) were considered for inclusion. RCTs performed in adults with severe TBI (GCS ≤8) were eligible. The primary endpoint was the outcome measures used in RCTs. The secondary outcomes were the timing of assessment and the methodological quality of trials using the Cochrane risk of bias assessment tool. Two independent reviewers selected trials and collected data using a standardized form. 1. Rothen et al.: Curr Opin Crit Care 2008, 14:513-519. 1. Rothen et al.: Curr Opin Crit Care 2008, 14:513-519. 2. Woodhouse et al.: Curr Opin Crit Care 2009, 15:450-455. 3. Ridley et al.: Anaesthesia 1998, 53:1185-1194. 2. Woodhouse et al.: Curr Opin Crit Care 2009, 15:450-455. 3. Ridley et al.: Anaesthesia 1998, 53:1185-1194. P479 Predicting 6-month mortality of patients with traumatic brain injury: usefulness of common severity scores MB Skrifvars1, R Raj1, S Bendel2, T Selander2, R Kivisaari1, J Siironen1, M Reinikainen3 1Helsinki University Central Hospital, Helsinki, Finland; 2Kuopio University Hospital, Kuopio, Finland; 3North Karelia Central Hospital, Joensuu, Finland Critical Care 2014, 18(Suppl 1):P479 (doi: 10.1186/cc13669) p Conclusion Young adults and older people involved mainly in motor vehicle accidents and falls respectively were among the high-risk groups for severe TBI. Management of these patients goes beyond the ICU and involves, but is not limited to, social support, emotional motivation and community reintegration of these patients [2]. TBI among the high-risk groups is largely preventable. Public awareness and prevention programmes will go some way in reducing the incidence of TBI amongst the high-risk groups. Introduction Severity of illness scoring systems is paramount for the evaluation of quality of care of critically ill and trauma patients [1-3]. The purpose of the present study was to evaluate the usefulness of the Acute Physiology and Chronic Health Evaluation II (APACHE II), Simplifi ed Acute Physiology Score II (SAPS II) and Sequential Organ Failure Assessment (SOFA) scores in predicting long-term outcome of patients with moderate-to-severe traumatic brain injury (TBI). Methods A Finnish multicenter ICU database was screened for TBI patients admitted in 2003 to 2012. Logistic regression was used for customization of the APACHE II, SAPS II and SOFA for 6-month mortality prediction. An adjusted SOFA model, including age, and a reference model, including only age and Glasgow Coma Scale, were built for comparison. A randomized split-sample technique was used for internal validation and prognostic performance was determined by assessing discrimination, calibration and precision. 1. Ministry of Health 2012 Health Facts Singapore [http://www.moh.gov.sg/ content/moh_web/home/statistics/Health_Facts_Singapore/Principal_ Causes_of_Death.html] 2. Chua KS, et al.: A brief review of traumatic brain injury rehabilitation. Ann Acad Med Singapore 2007, 36:31-42. 2. Chua KS, et al.: A brief review of traumatic brain injury rehabilitation. Ann Acad Med Singapore 2007, 36:31-42. 2. Chua KS, et al.: A brief review of traumatic brain injury rehabilitation. Ann Acad Med Singapore 2007, 36:31-42. P478 Outcome measures in randomized controlled trials of patients with severe traumatic brain injury: a systematic review G Lalonde1, M Shemilt1, F Lauzier1, P Desjardins1, A Boutin1, L Moore1, D Fergusson2, R Zarychanski3, A Turgeon1 1Laval University, Quebec, Canada; 2OHRI, Ottawa, Canada; 3University of Manitoba, Winnipeg, Canada Critical Care 2014, 18(Suppl 1):P478 (doi: 10.1186/cc13668) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Conclusion Outcome measures used to evaluate the eff ect of intervention in RCTs performed in patients with severe TBI in acute care are heterogeneous. A signifi cant proportion of trials did not consider evaluating the functional status or other clinically meaningful outcomes, and very few trials assessed outcomes beyond 6 months. Thus, a signifi cant proportion of RCTs in patients with severe TBI are based on outcome measures not clinically useful to guide or change practice in this population. demographic profi les of severe TBI in the local context, with implications in the management of severe TBI, particularly the utilisation of critical care resources. Results A total of 780 patients were admitted with TBI during the study period, of which 365 patients (46.8%) sustained severe TBI. The majority (75.3%) of the severe TBI patients were male. There was a bimodal preponderance of severe TBI cases in young adults (age 21 to 40) and older people (age >61). Motor vehicle accidents (48.8%) and falls from <2 m (35.1%) were the main mechanisms of injury. Invasive monitoring was frequently employed in these patients with severe TBI: arterial blood pressure monitoring in 298 patients (81.6%), central venous pressure monitoring in 219 patients (60.0%), and intracranial pressure monitoring in 173 patients (47.4%). The incidence of use of tiered therapy such as sedation, mild hyperventilation, osmotherapy with mannitol, cerebrospinal fl uid drainage, barbiturate coma and decompressive craniectomy to control ICP converged with international practices. P479 P477 P477 Demographic profi les and extent of critical care resources utilisation in patients with severe traumatic brain injury: a Tan Tock Seng Hospital National Neuroscience Institute study J Yang1, JZ Wee2, CT Chong1 1Tan Tock Seng Hospital, Singapore; 2Ministry of Health, Singapore Critical Care 2014, 18(Suppl 1):P477 (doi: 10.1186/cc13667) Methods A prospective observational study of mild to moderate TBI adult patients in two critical care trauma centers. Patients underwent delirium assessment on days 3, 5 and 7 with the CAM-ICU and the ICDSC. Psychiatrists or neurointensivists evaluated delirium using the DSM-IV criteria for delirium. Assessments results were blinded and performed independently. Criterion validity of the CAM-ICU and the ICDSC were calculated from 2 × 2 frequency tables using standard defi nitions of sensitivity, specifi city, positive and negative predictive value, and overall accuracy. Because delirium was assessed repeatedly, estimates of 95% CIs for binary repeated data using generalized estimating equation in conjunction with the Huber–White estimator were performed. Inter-rater reliability for the CAM-ICU and ICDSC was assessed with the kappa coeffi cient. Introduction Trauma is the fi fth principal cause of death in Singapore, with traumatic brain injury (TBI) being the leading specifi c subordinate cause [1]. Introduction Trauma is the fi fth principal cause of death in Singapore, with traumatic brain injury (TBI) being the leading specifi c subordinate cause [1]. Methods This 8-year retrospective review of patients with severe TBI admitted to the neuroICU (NICU) of the National Neuroscience Institute, Tan Tock Seng Hospital between 2004 and 2011 reports the S173 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P480 Work activities after 3-year follow-up in ICU patients with traumatic brain injury j y M Arias-Verdu1, M Delange van der Kroft2, E Curiel-Balsera1, A Muñoz-Lopez1, J Fernandez-Ortega1, R Rivera-Fernandez1, M Prieto-Palomino1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain Critical Care 2014, 18(Suppl 1):P480 (doi: 10.1186/cc13670) j y M Arias-Verdu1, M Delange van der Kroft2, E Curiel-Balsera1, A Muñoz-Lopez1, J Fernandez-Ortega1, R Rivera-Fernandez1, M Prieto-Palomino1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain Critical Care 2014, 18(Suppl 1):P480 (doi: 10.1186/cc13670) j y M Arias-Verdu1, M Delange van der Kroft2, E Curiel-Balsera1, A Muñoz-Lopez1, J Fernandez-Ortega1, R Rivera-Fernandez1, M Prieto-Palomino1 1Carlos Haya University Hospital, Malaga, Spain; 2County Hospital of Axarquia, Velez Malaga, Spain Critical Care 2014, 18(Suppl 1):P480 (doi: 10.1186/cc13670) Results From 5,602 citations retrieved after removal of duplicates, 36 RCTs met eligibility criteria. The outcome measures most frequently used were neurophysiologic indices (n = 18, 50%), the Glasgow Outcome Scale (n = 17, 47%), mainly at 6 months, nonspecifi c complications (n = 15, 42%), mortality (n = 12, 33%), and biomarkers (n = 10, 28%). Nine trials reported only physiologic indices and did not present any clinical or functional outcome measures. The methodological quality of included RCTs was heterogeneous. We observed a low risk of bias for sequence generation (n = 29, 80%), allocation concealment (n = 20, 55%), complete data reporting (n = 30, 83%), selective reporting (n = 36, 100%), and sample size (n = 21, 58%) but a high risk of bias for blinding (n = 20, 55%). Introduction The aim was to study work activities after 3 years in patients admitted to the ICU with traumatic brain injury (TBI). Introduction The aim was to study work activities after 3 years in patients admitted to the ICU with traumatic brain injury (TBI). Introduction The aim was to study work activities after 3 years in patients admitted to the ICU with traumatic brain injury (TBI). Methods A prospective cohort study in an ICU of a reference hospital. We studied ICU-admitted patients consecutively with TBI between 2004 and 2008. We used validated scales including the Glasgow Outcome Scale (GOS) and the Project for the Epidemiological Analysis of Critical Care Patients Quality of Life questionnaire. Methods A prospective cohort study in an ICU of a reference hospital. We studied ICU-admitted patients consecutively with TBI between 2004 and 2008. P481 P481 Enteral administration of antiepileptic agents could have effi cacy for prevention of post-traumatic seizures in severe traumatic brain injury K Mocjiduki, H Suzuki, A Kawasaki, M Hotta, M Namiki, T Harada, M Takeda, A Yaguchi Tokyo Women’s Medical University, Tokyo, Japan Critical Care 2014, 18(Suppl 1):P481 (doi: 10.1186/cc13671) P480 Work activities after 3-year follow-up in ICU patients with traumatic brain injury We used validated scales including the Glasgow Outcome Scale (GOS) and the Project for the Epidemiological Analysis of Critical Care Patients Quality of Life questionnaire. S174 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 2 (abstract P479). Discrimination. Results We studied 531 patients, age 40.35 ± 19.75 years, APACHE II 17 94±6 97points GlasgowComaScaleatadmission7 53±3 83points Figure 1 (abstract P479). Calibration. Figure 2 (abstract P479). Discrimination. (16.2% missing). Regarding work activities, after 1 year, 28.5% of 326 patients evaluated have no diffi culties with work, 4.6% have diffi culties but work as before, 10.1% work only part-time or have changed to a job requiring minimum eff ort and 56.7% of patients do not work. After 3 years, 41.2% of 238 patients evaluated have no diffi culties with work, 4.6% have diffi culties but work as before, 12.6% work only part-time or have changed to a job requiring minimum eff ort and 41.6% of patients do not work. Evolution between 1 and 3 years by the McNemar test was statistically signifi cant (P <0.001). A total of 173 patients were in similar situation, only one had deteriorated and in 62 (26.05%) patients the evaluation of work activity had improved. y Conclusion After 1 year of admission from the ICU with TBI, more than 50% of patients have diffi culties with work. After 3 years the number of patients who work has increased although approximately 40% of the surviving patients do not work. Enteral administration of antiepileptic agents could have effi cacy for prevention of post-traumatic seizures in severe traumatic brain injury K Mocjiduki, H Suzuki, A Kawasaki, M Hotta, M Namiki, T Harada, M Takeda, A Yaguchi Introduction Antiseizure prophylaxis is recommended for preventing only early post-traumatic seizures (PTS) in the guidelines for the management of severe traumatic brain injury (TBI) by the Brain Trauma Foundation. Phenytoin is recommended to reduce the incidence of early PTS prophylaxis. Early enteral nutrition has recently shown theoretical advantages for prevention of bacterial translocation to maintain normal turnover of gut mucosa and is commonly used for TBI patients. Our hypothesis is that the enteral administration of antiepileptic agents is also useful for early PTS. Figure 1 (abstract P479). Calibration. Figure 1 (abstract P479). Calibration. Results We studied 531 patients, age 40.35 ± 19.75 years, APACHE II 17.94 ± 6.97 points, Glasgow Coma Scale at admission 7.53 ± 3.83 points. Cranial tomography at admission was: diff use injury type I (10.4%), diff use injury type II (28.1%), diff use injury type III (24.5%), diff use injury type IV (8.3%), evacuated mass lesion (22.6%), non-evacuated mass lesion (6.2%). Hospital mortality was 28.6%, 171 (32.2%) patients died after 1 year (6.6% missing) and 181 (34.1%) died after 3 years p p g y Methods This retrospective observational study included all adult patients admitted to our tertiary academic medico-surgical ICU due to TBI from September 2011 to August 2012. Patients who have epilepsy as a past history were excluded. Clinical data were collected from electrical medical archives. The baseline characteristics collected were age, gender, diagnosis, antiepileptic agents, timing of start and adverse eff ects of those agents, and methods of administration. S175 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Of 65 patients with TBI, 25 patients (18 men, seven women; mean age 56.7 ± 20.1) who were administered antiepileptic agents for PTS prophylaxis were studied. Fifteen cerebral contusions, 10 acute subdural hematomas, nine traumatic subarachnoid hemorrhages, two cerebral infarctions, two pneumocephalus and one traumatic intracerebral hemorrhage were shown in 25 patients. All patients were alive 28 days after the injury. Fourteen patients (56%) were intravenously administered (13 phenytoin and one phenobarbital), while 11 patients (44%) were administered with enteral feeding (four valproates, four carbamazepine and three zonisamides) as PTS prophylaxis. Simulation-based education for cardiopulmonary resuscitation and airway management protocols: a brief report of a systematic review and meta-analysis Simulation-based education for cardiopulmonary resuscitation and airway management protocols: a brief report of a systematic review and meta-analysis A Cortegiani, V Russotto, A Naro, G Sanzo, G Grutta, SM Raineri, A Giarratano University of Palermo, Italy Critical Care 2014, 18(Suppl 1):P482 (doi: 10.1186/cc13672) y A Cortegiani, V Russotto, A Naro, G Sanzo, G Grutta, SM Raineri, A Giarratano University of Palermo, Italy Critical Care 2014, 18(Suppl 1):P482 (doi: 10.1186/cc13672) y A Cortegiani, V Russotto, A Naro, G Sanzo, G Grutta, SM Raineri, A Giarratano University of Palermo, Italy Critical Care 2014, 18(Suppl 1):P482 (doi: 10.1186/cc13672) f Methods All ambulances of the Hiroshima City Fire Department are equipped with a specially designed transmission device (RVT-SD200; Sony) that transmits high-resolution visual images and patient vital data using video cameras and a bio-monitor. We analyzed video data of OHCA patients transported by ambulance from November 2012 through December 2012, and evaluated the performance of CPR during transportation in accordance with the 2010 guidelines. The hands-off time was calculated as the time without chest compressions divided by the total CPR time. Introduction We aimed to summarize the effi cacy of simulation-based education in cardiopulmonary resuscitation and airway management [1]. Methods We searched the MEDLINE, Scopus and EMBASE databases for all peer-reviewed articles enrolling physicians/medical students in a simulation of either cardiopulmonary resuscitation or airway management protocols compared with no intervention or traditional teaching methods. We categorized the outcomes of the studies into four groups: task success, process skill, time skill, knowledge. Task success was defi ned as evaluation of successful completion of the task, process skill as evaluation of the procedure, time skill as the time required to complete the task, and knowledge as the objective assessment of conceptual understanding. When studies investigated more than one outcome, we considered the primary outcome, the overall measure or the most clinically relevant outcome. Results Thirty-two resuscitation episodes during transportation by ambulance were analyzed. Median CPR time per episode was 846 seconds (range 126 to 1,833 seconds). In total, the fraction of time without chest compression was 19.5 ± 7.6% (mean ± SD). Enteral administration of antiepileptic agents could have effi cacy for prevention of post-traumatic seizures in severe traumatic brain injury The average start day of PTS prophylaxis was 2.6 days after the injury by intravenous administration, and 2.2 days by enteral administration, respectively. Two patients with phenytoin showed hepatic dysfunction as an adverse eff ect and no patient showed early PTS both by intravenous and by enteral administrations. favor of simulation. Pooled eff ect size for process skill was 0.48 (0.11 to 0.84), for time skill was 0.29 (0.13 to 0.73) and for knowledge was 0.41 (0 to 0.84). Conclusion Simulation is an eff ective educational method to improve performance of physicians/medical students in the application of protocols for cardiopulmonary resuscitation and airway management. Reference 1. McGaghie WC, et al.: A critical review of simulation-based medical education research: 2003–2009. Med Educ 2010, 44:50-63. P483 Video analysis of cardiopulmonary resuscitation performance of ambulance crews during transportation g p H Giga1, T Otani1, T Sadamori1, K Une1, Y Kida1, K Ota1, J Itai1, S Yamaga1, S Kusunoki2, S Ohshimo1, Y Iwasaki1, N Hirohashi1, K Tanigawa1 1Hiroshima University, Hiroshima, Japan; 2Critical Care Medical Center, Hiroshima Prefectural Hospital, Hiroshima, Japan Critical Care 2014, 18(Suppl 1):P483 (doi: 10.1186/cc13673) y y Conclusion The present study has some limitations because it is a single-center retrospective analysis. However, enteral administration of antiepileptic agents could be useful for PTS prophylaxis. Considering cost, adverse eff ects and serum monitoring, there is a possibility of enteral administration of antiepileptic agents as an alternative to intravenous phenytoin. Introduction High quality of chest compressions during cardio- pulmonary resuscitation (CPR) is a critical determinant of outcome from out-of-hospital cardiac arrest (OHCA). Unfortunately, however, victims often do not receive adequate chest compression for various reasons, particularly during transportation. Recent studies have demonstrated the interruption time of chest compression using transthoracic impedance analysis, but more information is needed to evaluate the performance of CPR provided by ambulance crews and reveal reasons for hands off chest during CPR. Simulation-based education for cardiopulmonary resuscitation and airway management protocols: a brief report of a systematic review and meta-analysis Reasons for interruption and its fraction of time in total hands-off time were as follows: 36% accounted for rhythm analysis/pulse check, 31% for ventilation, 11% for setting up automated chest compression devices, 8% for tracheal intubation/placement of supraglottic airway devices, 4% for intravenous line placement/administration of adrenaline, 3% for rescuer change, and 7% for adjustment of patient position/correction of rescuer posture and others. y Results From 8,528 articles, we selected 24 studies (13 randomized controlled studies, eight pre–post studies, three case–control studies) involving 1,149 participants. Compared with no intervention or traditional teaching methods, simulation was associated with a signifi cant improvement from mild to moderate of all outcomes (Figure 1). Log of odds ratio for task success was 2.03 (0.46 to 3.59) in Conclusion The fraction of time without chest compression observed in this study was comparative with those found in other studies in spite of the diffi cult situations, such as during transportation. Most frequent reasons for hands-off time were rhythm analysis and ventilation even though the ambulance crews strictly adhered to the guidelines. Figure 1 (abstract P482). S176 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P484 Implementation of the PulsePoint smartphone application for crowd-sourcing bystander resuscitation SC Brooks1, H Worthington1, T Gonedalles2, B Bobrow3, LJ Morrison4 1Queen’s University, Kingston, Canada; 2St Michael’s, Toronto, Canada; 3University of Arizona Medical College, Phoenix, AZ, USA; 4University of Toronto, Canada Critical Care 2014, 18(Suppl 1):P484 (doi: 10.1186/cc13674) P484 with 100% survival and in-hospital ICU transfer. The cardiac arrest (CA) group depended on cause of CA: cardiac arrhythmia (n = 12), survival 50%; surgical etiology (nontraumatic), survival 50%, mean age 70; myocardial infarction (n = 12), survival 83%, mean age 61; sepsis (n = 6), survival 67%, mean age 48; drugs overdose (n = 2), survival 100%; traumatic cardiac arrest (n = 15), 89% mortality, mean age 41; and CA with unknown etiology, survival 38%, mean age 68. Conclusion Emergency room cardiac arrest arrivals are not correlated with ER-CPA total activity. Good survival rates are probably related to a special quick response protocol and trained teams. Outcomes research to detect potential improvement in nurse care is needed, and should be evidence guided. Worst results in survival rates must be detected as the main topics for nursing care development. Introduction Only a minority of patients suff ering out-of-hospital cardiac arrest receive any bystander cardiopulmonary resuscitation (CPR). What is the role of head computed tomography in post-resuscitation care? What is the role of head computed tomography in post-resuscitation care? Y Valzani1, A Marudi2, S Baroni2, G De Grandis2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P486 (doi: 10.1186/cc13676) p Y Valzani1, A Marudi2, S Baroni2, G De Grandis2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P486 (doi: 10.1186/cc13676) Y Valzani1, A Marudi2, S Baroni2, G De Grandis2, R Stacca2, E Bertellini2 1University of Modena and Reggio Emilia, Modena, Italy; 2Nuovo Ospedale Civile Sant’Agostino Estense, Modena, Italy Critical Care 2014, 18(Suppl 1):P486 (doi: 10.1186/cc13676) Methods We conducted a structured survey delivered via Survey Monkey optimized for mobile devices. All alerted PulsePoint users between 28 June 2012 and 1 November 2013 were sent an invitation to participate in the survey. Survey responses associated with test activations and emergency medical services (EMS) professionals on active duty were excluded from the analysis. Introduction Guidelines recommend to perform head computed tomography (CT) in nontraumatic cardiac arrest only if the patient remains unconscious after return of spontaneous circulation [1]. Nevertheless, in cardiac arrest due to neurological causes, head CT would dramatically change the therapeutic strategy in these patients [2]. The aim of this study is to present the experience of our ICU in nontraumatic out-of-hospital cardiac arrest (OHCA) patients and to demonstrate that neurological abnormalities can be the primary cause of cardiac arrest. y y Results Of 4,827 survey requests sent, 995 responded and completed our survey (response rate 21%). Respondents identifi ed themselves as fi refi ghters (30%), paramedics (19%), EMTs (16%), nurses (4.4%), MDs (0.83%) and other professions (50%). Of those who received a PulsePoint alert, 23% (157/690) responded by making their way towards the location of the emergency. Of those who responded, 70% (110/157) arrived on scene. Most of those who did not arrive said they saw professional rescuers already on scene and turned back (52%, 24/47). Of those who arrived on scene, only 34% (37/110) found a person unconscious and not breathing normally. The majority found a person on scene who was not suff ering cardiac arrest. Of those who arrived on scene prior to EMS and also found a cardiac arrest victim on scene who required resuscitation, 80% (8/10) performed bystander CPR. Simulation-based education for cardiopulmonary resuscitation and airway management protocols: a brief report of a systematic review and meta-analysis Bystander CPR is associated with improved odds for survival. The PulsePoint smartphone application alerts users in the vicinity of a cardiac arrest to facilitate immediate citizen bystander resuscitation. Addressing implementation barriers may provide an opportunity to increase eff ectiveness of the application. PulsePoint is currently active in over 400 communities in the United States. Our objective was to identify modifi able barriers to optimal implementation of the PulsePoint smartphone application. P485 Emergency room advanced life support after cardiac arrest: outcomes and survival, nursing care and team response A Villamor, A Tugas, Y Masguret, M Bayon, P Ceregido Barcelona Clinic Hospital, Barcelona, Spain Critical Care 2014, 18(Suppl 1):P485 (doi: 10.1186/cc13675) What is the role of head computed tomography in post-resuscitation care? Methods We collected data for all patients with nontraumatic OHCA admitted to the ICU from 1 January 2012 to 31 December 2012. We recorded mean age, male-to-female ratio, cause of cardiac arrest and mortality in the ICU. Results Thirty-four survive cardiac arrest patients were admitted to the ICU. Mean age was 66 years. The male-to-female ratio was 2:1. Sixteen patients died in the ICU. Head CT scans obtained in the immediate post- arrest period found signifi cant abnormalities in four patients (three subarachnoid hemorrhage and one ischemic ictus) with immediate modifi cation of treatment. Conclusion We observed a very high proportion of bystander CPR (80%) for victims of out-of-hospital cardiac arrest when PulsePoint users arrived before EMS. This suggests that optimized PulsePoint implementation may increase community bystander CPR rates. Alert specifi city for cardiac arrest may be too low (the cry wolf scenario) with current default activation criteria. Also, the current activation radius (0.5 mile) may be too large because EMS frequently arrive before the PulsePoint responder. i Conclusion Head CT abnormalities can be found in cardiac arrest patients. Further investigations are necessary to evaluate the impact of cardiac arrest from neurological causes and to establish the role of neuroimaging in these patients. f References References 1. Cocchi et al.: Intern Emerg Med 2010, 5:533-538. 2. Ball et al.: Can J Anesth 2005, 52:892-893. 1. Cocchi et al.: Intern Emerg Med 2010, 5:533-538. 1. Cocchi et al.: Intern Emerg Med 2010, 5:533-538. 2. Ball et al.: Can J Anesth 2005, 52:892-893. P487 To see or not to see: does video CPR perform better than telephone CPR? A Ghuysen1, A Delfosse1, S Stipulante2, A Donneau3, V D’Orio1 1CHU – Ulg Liège, Belgium; 2Federal Public Health Services, Liege, Belgium; 3Liège University, Liege, Belgium Critical Care 2014, 18(Suppl 1):P487 (doi: 10.1186/cc13677) Initial anticoagulation strategy for extracorporeal cardiopulmonary resuscitation patients p Critical Care 2014, 18(Suppl 1):P489 (doi: 10.1186/cc136 Introduction Out-of-hospital cardiac arrest (OOHCA) causes 60,000 UK and 300,000 US deaths each year. Survival to hospital discharge in the developed world has historically been 7 to 10% with obvious cognitive impairment in 10% of survivors. Primary percutaneous coronary intervention (PPCI) and targeted temperature management (TTM) (or at least hyperthermia avoidance) have been shown to improve survival in comatose patients post OOHCA. There is no reliable method to predict poor outcome on presentation. We aimed to identify factors associated with poor outcome in our single-centre regional referral OOHCA population. Y Iwashita, M Matsuduki, M Yukimitsu, K Yokoyama, T Nakata, A Yamamoto, K Ishikura, H Imai Mie University Hospital, Tsu, Mie, Japan Critical Care 2014, 18(Suppl 1):P488 (doi: 10.1186/cc13678) Introduction Extracorporeal cardiopulmonary resuscitation (ECPR) is increasingly being used in emergency and critical care medicine in Japan. Although a major complication of this procedure is bleeding, the optimal heparin dose and activated coagulation time (ACT) remain unknown. p p Methods We performed a pragmatic single-centre retrospective review over 18 months commencing 1 January 2011 of all patients admitted to our regional OOHCA centre ICU following successful resuscitation from OOHCA. In keeping with guidelines, all patients were assessed for suitability for PPCI and TTM. A good outcome was defi ned by a Pittsburgh Cognitive Performance Category (CPC) score of 1 to 2 (independence, mild impairment) on hospital discharge. A poor outcome was defi ned as death or CPC 3 to 4 (moderate to severe impairment, coma) on hospital discharge. CPC scoring was determined Methods We retrospectively evaluated the initial heparin doses, ACT values, and complications of patients who received ECPR between February 2011 and November 2013 at the Emergency and Critical Care Center, Mie University Hospital, Japan. Results ECPR was performed in 45 patients, and the ACT was evaluated in 32 patients. All patients were administered 3,000 U unfractionated heparin at the time of priming the circuit. Patients for whom cannulation took a longer time received an additional 2,000 to 3,000 U unfractionated heparin. The average heparin dose administered Table 1 (abstract P489). Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 was 53.6 U/kg body weight. The average ACT was 231.3 seconds. In 17 of the 32 patients, the ACT exceeded 200 seconds. Three patients experienced fatal bleeding in the chest wall, which could not be stabilized by conservative treatment. One patient developed a cerebral infarction. There were no signifi cant diff erences between the patients with fatal bleeding and those without fatal bleeding with regard to the heparin dose, ACT, and duration of CPR. every 2 minutes. A total of 120 students without prior CPR training were recruited from upper secondary school, during regular class hours, and randomly assigned to the t-CPR group (n = 60) versus the v-CPR group (n = 60). The Resusci®Anne SkillReporter™ manikin was used to evaluate CPR performance. Data were transferred from the manikin into a computerized database using the Laerdal SkillReporting System V.2.2.1 software. Further analysis was based on audio-recordings and video-recordings. Primary outcome measures were the results of the Cardiff evaluation test; the secondary measures were global scoring of a complete 7-minute period of CPR.i Conclusion According to the Extracorporeal Life Support Organization guidelines, the target ACT should be around 1.5 times the normal ACT. However, it is diffi cult to obtain the normal ACT in emergency situations. Many of our patients’ ACTs exceeded 200 seconds, and three patients experienced fatal bleeding that was possibly due to chest compression. Post-cardiac arrest patients often experience coagulopathy due to either cardiac arrest or hypothermia therapy. Therefore, an anticoagulation protocol other than the pulmonary extracorporeal membrane oxygenation protocol is required for ECPR patients. We evaluated our anticoagulation protocol for ECPR and observed that our patients’ ACTs frequently exceeded the target value and some experienced fatal bleeding. The anticoagulation protocol for post-CPR patients may need to be reconsidered. Results The mean chest compression rate increased signifi cantly in the v-CPR group as compared with t-CPR (110 ± 16 vs. 86 ± 28; P <0.0001), while depth remained constant (48 ± 13 mm vs. 47 ± 16 mm, P = NS). Hand positioning was correct in 91.7% of cases with v-CPR, but only in 68% with t-CPR (P = 0.001). The hands-off period was almost nonexistent in the v-CPR group (0 vs. 7 seconds; P = 0.0016), but the median no-fl ow time was signifi cantly greater in the v-CPR group (146 vs. 122 seconds, P < 0.0001). P488 pl Hammersmith Hospital, London, UK Critical Care 2014, 18(Suppl 1):P489 (doi: 10.1186/cc13679) P489 Predictors of poor outcome in out-of-hospital cardiac arrest JE Petrie, S Brett, R Stümpfl e Hammersmith Hospital, London, UK Critical Care 2014, 18(Suppl 1):P489 (doi: 10.1186/cc13679) P489 Predictors of poor outcome in out-of-hospital cardiac arrest JE Petrie, S Brett, R Stümpfl e Hammersmith Hospital, London, UK Critical Care 2014, 18(Suppl 1):P489 (doi: 10.1186/cc13679) Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 As a consequence, global evaluation of CPR performance revealed a signifi cant improvement in v-CPR group score as compared with the t-CPR group (6 vs. 5, P < 0.001). g Conclusion Video-assisted CPR using this original algorithm allows bystanders to reach compression rates and depths close to international guidelines and to reduce hands-off events during CPR. To see or not to see: does video CPR perform better than telephone CPR? Introduction An organized team response and trained nursing staff in the emergency room critical patient area (ER-CPA) are the main factors to determine cardiopulmonary resuscitation (CPR) success rates. The objective was to evaluate outcomes in the emergency room after advanced life support (ALS) by reanimation teams, to improve nursing care, quality of resuscitation and survival. Introduction The ALERT algorithm, a simple and eff ective com- pression-only telephone CPR (t-CPR) protocol, has been previously demonstrated to help bystanders initiate CPR. According to their worldwide availability, mobile phone communications may play an increasing role in emergency calls. Preliminary studies suggest that they might improve dispatcher’s understanding of the rescuer’s situation. However, there is currently no validated protocol for videoconference-assisted CPR (v-CPR). We initiated the present study to validate an original protocol of v-CPR based on the ALERT algorithm and to evaluate the potential benefi t of this assistance in comparison with classical t-CPR. Introduction The ALERT algorithm, a simple and eff ective com- pression-only telephone CPR (t-CPR) protocol, has been previously demonstrated to help bystanders initiate CPR. According to their worldwide availability, mobile phone communications may play an increasing role in emergency calls. Preliminary studies suggest that they might improve dispatcher’s understanding of the rescuer’s situation. However, there is currently no validated protocol for videoconference-assisted CPR (v-CPR). We initiated the present study to validate an original protocol of v-CPR based on the ALERT algorithm and to evaluate the potential benefi t of this assistance in comparison with classical t-CPR. q y Methods We included all adult patients receiving ALS in the ER-CPA during 1 year. Cases not allowed CPR are excluded. A retrospective design, multivariate with ALS performed, gender, age, cause of arrest, precedence, outcome and hospital derivation, and 100% of cases studied. Results A total of 149 patients were attended in the ER-CPA with ALS maneuvers during the studied period (9.23% of a total 1,613). Thirty-four of them were nonheart-beating donors. The protocol was performed with 23 eff ective donors and 31 organs were obtained for transplant. Respiratory arrest (n = 31) was the best outcome group, Methods We developed a strictly worded algorithm for v-CPR, adapted from the ALERT t-CPR protocol, with additional re-evaluation loops, S177 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Post Arrest Consult Team: a knowledge translation strategy for post-cardiac arrest care Results Of the 51 patients, 21 were S. Mean age was 70 years (± 15) in the S, of which 10 (48%) were male, and in the nonsurvivors (NS) mean age was 70 years (± 17) (P = 0.916) with 23 (77%) male patients (P = 0.042). The initial rhythm was asystole in 11 S and in 20 NS (P =0.386), pulseless electrical activity in two S and NS (P = 1), and ventricular fi brillation in eight S and six NS (P = 0.207). The arrest was witnessed in 15 NS and 16 S (P = 0.083). Lay rescuer BLS was performed in 17 NS and nine S (P = 0.4). A signifi cant diff erence in time of EC and start of ALS was observed between S (12 minutes; 8 to 15) and NS (14 minutes; 12 to 17) (P = 0.03). Mean initial rSO2 was 34% (± 23) and 24% (± 12.5) in S and NS (P = 0.077). We observed a negative correlation between mean initial rSO2 and time between EC and ALS (correlation coeffi cient –0.243; P = 0.041). Critical Care 2014, 18(Suppl 1):P492 (doi: 10.1186/cc13682) Critical Care 2014, 18(Suppl 1):P492 (doi: 10.1186/cc13682 Introduction Lack of standardized care contributes to low survival in admitted out-of-hospital cardiac arrest (OHCA) patients. The objective of our study was to implement a Post Arrest Consult Team (PACT) and improve the quality of care for admitted OHCA patients. Introduction Lack of standardized care contributes to low survival in admitted out-of-hospital cardiac arrest (OHCA) patients. The objective of our study was to implement a Post Arrest Consult Team (PACT) and improve the quality of care for admitted OHCA patients. p q y p Methods We conducted a prospective cohort study with concurrent controls from February 2011 to February 2013 in a network of 29 Toronto-area hospitals. The PACT was implemented in two hospitals and functioned as a consult service with a nurse and physician on- call 24/7. Patients from other network hospitals acted as concurrent controls. The PACT focused on four key processes of care: targeted temperature management (TTM); coronary angiography; avoidance of premature withdrawal of life-sustaining therapy (WLST <72 hours) on the basis of neuroprognostication; and electrophysiology assessment. We included nontraumatic OHCA patients who were >18 years old, survived at least 6 hours, and were comatose. We excluded patients with do-not-resuscitate orders, intracranial or other severe bleeding. P490 Results There were 247 admissions to the ICU following OHCA from 2010 to 2012. Seven patients were excluded for missing/incomplete data. In total, 102 patients (42.5%) survived to discharge. PAR ranged from –2 to 18. Zero was the most frequent score. Only one of 15 admissions with PAR >5 (PAR 13) survived. A total of 101 patients with PAR ≤5 survived (45%). Acute myocardial infarction was identifi ed as the precipitating event in 111 patients (46%). Mean initial cerebral saturation and time to start advanced life support in out-of-hospital cardiac arrest: are they correlated? C Genbrugge, C De Deyne, I Meex, F Jans, W Boer, J Dens ZOL, Genk, Belgium Critical Care 2014, 18(Suppl 1):P490 (doi: 10.1186/cc13680) Introduction During advanced life support (ALS) it is still impossible to predict return of spontaneous circulation (ROSC) or outcome. Cerebral saturation (rSO2) measurements with near-infrared spectroscopy can be used in cardiac arrest circumstances and could play a role in predicting ROSC or neurologic outcome [1]. It is known that an initial rhythm of asystole and a long no/low-fl ow time has a worse outcome. We measured rSO2 during ALS in out-of hospital cardiac arrest (OHCA) patients and compared the mean rSO2 during the fi rst minute with the time between the emergency call (EC) and start of ALS. Conclusion Only one of 240 OHCA patients admitted to the ICU over a 3-year period with PAR >5 survived until discharge. PAR scoring has been shown to be useful in helping decide the appropriateness of ICU admission for combined IHCA/OHCA. Our data support its use in isolated OHCA. PAR ≤5 appears to be poorly predictive of survival. However, PAR >5, combined with clinical evaluation, could help identify OHCA nonsurvivors and avoid ICU admissions that do not benefi t the patient. References 1. Bernard SA: Emerg Med 1998, 10:25-29. y Methods With IRB approval, rSO2 was prospectively measured between December 2011 and November 2013 in 51 OHCA cases with presumed cardiac cause during ALS. One sensor of the EQUANOX Advance was applied to the right side of the patient’s forehead when the medical emergency team arrived. The measurement was continued until death of the patient or arrival at the ICU. Survivors (S) are defi ned as sustained ROSC longer than 20 minutes. CPR data were collected using the Utstein CPR data registration. Post Arrest Consult Team: a knowledge translation strategy for post-cardiac arrest care We used generalized linear mixed models to assess whether PACT implementation was associated with higher odds of achieving each of the four targeted processes of care while adjusting for secular trends unrelated to the intervention. Conclusion A tendency towards higher mean initial rSO2 in S compared with NS was observed together with a negative correlation between mean initial rSO2 and time between the EC and start of ALS. Also a signifi cant diff erence in time between the EC and start of ALS between S and NS is observed. Larger studies are needed to confi rm the possible function of rSO2 as a surrogate for time between the EC and start of ALS. Reference 1. Ito N et al.: Resuscitation 2012, 83:46-50. P490 The Mann–Whitney test and Pearson chi- square were utilized for comparison of S and nonsurvivors (NS) data. The Pearson test was used to examine correlation. 2. Ebell MH: J Fam Pract 1992, 34:551-558. 3. O’Keefe S, et al.: Resuscitation 1994, 28:21-25. 4. Porter R, et al.: Crit Care 2011, 15:P299. Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 has been fully validated for predicting survival following OHCA. The Prognosis After Resuscitation (PAR) score was developed from meta- analysis in 1992. A score >5 (from seven variables) predicts nonsurvival following in-hospital cardiac arrest (IHCA) [2,3]. Porter and colleagues demonstrated that PAR >5 is a useful predictor of nonsurvival for combined IHCA/OHCA ICU admissions [4]. We aim to evaluate PAR application to adult ICU admissions at University Hospitals Bristol (UHB), UK following OHCA. has been fully validated for predicting survival following OHCA. The Prognosis After Resuscitation (PAR) score was developed from meta- analysis in 1992. A score >5 (from seven variables) predicts nonsurvival following in-hospital cardiac arrest (IHCA) [2,3]. Porter and colleagues demonstrated that PAR >5 is a useful predictor of nonsurvival for combined IHCA/OHCA ICU admissions [4]. We aim to evaluate PAR application to adult ICU admissions at University Hospitals Bristol (UHB), UK following OHCA. from physiotherapy and occupational therapy assessments in the clinical notes. Continuous data were analysed using a two-tailed t test or Mann–Whitney U test, categorical data with a chi-squared test. from physiotherapy and occupational therapy assessments in the clinical notes. Continuous data were analysed using a two-tailed t test or Mann–Whitney U test, categorical data with a chi-squared test. Results Sixty-nine patients were admitted to our ICU following successful resuscitation from OOHCA in the 18-month period. Presenting features and outcomes are shown in Table 1. Conclusion Good outcome was associated with a shockable rhythm on presentation, cardiac cause, arrest occurring in daylight hours (08:00 to 20:00) and shorter time between collapse and ROSC. Poor outcome was associated with placement of an advanced airway device, lower pH, higher base defi cit and higher lactate. g Methods The Innovian (Dräger) electronic clinical information database was searched retrospectively for all OHCA ICU admissions from 2010 to 2012. Data were extracted using an electronic pro forma in Microsoft Excel. Missing/incomplete data were excluded. Survival was defi ned as survival to discharge from the acute hospital (UHB). Initial anticoagulation strategy for extracorporeal cardiopulmonary resuscitation patients Presenting features and outcome at hospital discharge of ICU OOHCA patients Good outcome (CPC 1 to 2) Poor outcome (dead or CPC 3 to 4) Total P value Age 63.3 ± 16.8 63.6 ± 14.4 63.5 ± 14.7 NS Male sex 21/26 (81%) 29/43 (67%) 50/69 (72%) NS Arrest occurring 08:00 to 20:00 14/19 (74%) 16/36 (44%) 30/55 (55%) <0.05 VF/VT as initial rhythm 25/26 (96%) 25/43 (58%) 50/69 (72%) <0.001 Bystander CPR 12/26 (46%) 24/43 (56%) 36/69 (52%) NS Time from collapse to ROSC (minutes) 18.3 ± 11 32.3 ± 26 27.1 ± 23 <0.05 Advanced airway management 14/24 (58%) 38/42 (90%) 52/66 (79%) <0.005 Cardiac cause 24/26 (92%) 30/43 (70%) 54/69 (78%) <0.05 Angiography 19/26 (73%) 23/43 (53%) 42/69 (61%) NS Therapeutic hypothermia 24/26 (92%) 41/43 (95%) 64/69 (93%) NS Presenting pH 7.21 ± 0.11 7.08 ± 0.18 7.13 ± 0.17 <0.01 Presenting base defi cit (mmol/l) –7.9 ± 6.2 –13.8 ± 5.9 –11.5 ± 6.7 <0.001 Presenting lactate (mmol/l) 4.3 ± 3.0 8.3 ± 4.5 6.8 ± 4.4 <0.0005 Table 1 (abstract P489). Presenting features and outcome at hospital discharge of ICU OOHCA patients Table 1 (abstract P489). Presenting features and outcome at hospital discharge of ICU OOHCA patients Good outcome (CPC 1 to 2) Poor outcome (dead or CPC S178 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 P492 P492 Post Arrest Consult Team: a knowledge translation strategy for post-cardiac arrest care SC Brooks1, D Scales2, K Dainty2, S Gray2, R Pinto3, E Racz2, M Gaudio2, A Amaral2, A Baker2, M Chapman2, E Crystal2, P Dorian2, N Fam2, R Fowler2, J Friedrich2, M Madan2, G Rubenfeld2, O Smith4, LJ Morrison2 1Queen’s University, Kingston, Canada; 2University of Toronto, Canada; 3Sunnybrook Health Sciences Centre, Toronto, Canada; 4St Michael’s, Toronto, Canada Critical Care 2014 18(Suppl 1):P492 (doi: 10 1186/cc13682) P493 One-year assessment of in-hospital cardiac arrest M Ahmed, A Kochhar, O Rose Univeristy Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P493 (doi: 10.1186/cc13683) P493 One-year assessment of in-hospital cardiac arrest M Ahmed, A Kochhar, O Rose Univeristy Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P493 (doi: 10.1186/cc13683) survivors of cardiac arrest (CA). Despite this, in our country there are still few centers that apply hypothermia post CA regularly. We describe a Chilean experience with endovascular hypothermia post CA, in three ICUs of the same university clinical center [1-3]. Introduction This retrospective audit evaluated adult patients who suff ered in-hospital cardiac arrest (IHCA) against the recent National Confi dential Enquiry into Patient Outcome and Death (NCEPOD) report [1]. It looked specifi cally at the recognition of the acutely unwell, the interventions made, the decisions taken from admission through to the post-arrest period and the outcomes following cardiopulmonary resuscitation (CPR). The audit aims to guide future improvements in preventing cardiac arrest and enhancing end-of-life care decision-making.f Methods A descriptive cohort study. All surviving comatose patients after CA were included, and underwent endovascular hypothermia management according to protocol. CoolGard™ internal cooling equipment was used. Variables: delay between CA and hypothermia (34°C), time in hypothermia, complications, ventilatory and hemo- dynamic management. Main outcome measures: mortality and neuro- logical follow-up to 6 months with Glasgow Outcome Score Extended (GOSE). Results Twenty-seven patients were managed in the ICU post CA (18 outpatients). Twenty-four were men (89%), mean age 33.5 ± 19 years (16 to 76). Delay was considered (time between CA and achieving target temperature of 34°C): median 10.5 ± 3.3 hours (3 to 18 hours). Hypothermic maintenance (from when it reaches 34°C until it returns to 36°C): 24 ± 18 hours (24 to 48 hours). Complications of hypothermia: fi ve hypokalemia (18.5%), three ventricular arrhythmias (11%), one vein thrombosis (3.7%). There were no deaths during hypothermia. Hospital mortality was two cases (7.4%). At 6 months it was three (11%). Neurological outcome at discharge and 6 months are shown in Figure 1. Good outcome (GOSE 5 to 8) occurred in 11 patients (40%) at discharge. Good outcome at 6 months occurred in 20 patients (74%). Methods Medical notes of adult patients suff ering IHCA, over a 1-year period, were identifi ed and data were collected using a validated NCEPOD audit tool. Knowledge and use of therapeutic hypothermia in cardiac arrest victims among healthcare staff in Greece Conclusion The results from this audit highlight persistent defi ciencies in the care pathway for the acutely unwell patient. Improvement will be focused on earlier consultant review and better recognition of warning signs with appropriate action taken. Furthermore, earlier routine and senior clinician-led discussions on appropriate end-of-life care are vital. References Knowledge and use of therapeutic hypothermia in cardiac arrest victims among healthcare staff in Greece P Manthou1, T Katsoulas1, A Korobeli1, L Kiriakou2, G Fildissis1 1Department of Nursing University, Athens, Greece; 2Gonk Agioi Anargiroi Hospital, Kifi ssia, Greece Critical Care 2014, 18(Suppl 1):P495 (doi: 10.1186/cc13685) P Manthou1, T Katsoulas1, A Korobeli1, L Kiriakou2, G Fildissis1 1Department of Nursing University, Athens, Greece; 2Gonk Agioi Anargiroi Hospital, Kifi ssia, Greece Critical Care 2014, 18(Suppl 1):P495 (doi: 10.1186/cc13685) P Manthou1, T Katsoulas1, A Korobeli1, L Kiriakou2, G Fildissis1 1Department of Nursing University, Athens, Greece; 2Gonk Agioi Anargiroi Hospital, Kifi ssia, Greece Critical Care 2014, 18(Suppl 1):P495 (doi: 10.1186/cc13685) 1. Findlay G, et al.: Time to Intervene? A Review of Patients who Underwent Cardiopulmonary Resuscitation as a Result of an In-hospital Cardiorespiratory Arrest. London: NCEPOD; 2012. [http://www.ncepod.org.uk/ 2012report1/ downloads/CAP_summary.pdf] 1. Findlay G, et al.: Time to Intervene? A Review of Patients who Underwent Cardiopulmonary Resuscitation as a Result of an In-hospital Cardiorespiratory Arrest. London: NCEPOD; 2012. [http://www.ncepod.org.uk/ 2012report1/ downloads/CAP_summary.pdf] Introduction Therapeutic hypothermia (TH) improves the neurologic outcome of patients who survive after cardiac arrest but suff er from severe secondary neurological damage [1]. In 2010, the use of TH after cardiac arrest was included in the ERC guidelines. The purpose of this study was to investigate the knowledge of the medical and nursing staff on the implementation of TH in patients after cardiac arrest [2]. 2. Meaney PA, et al.: Crit Care Med 2010, 38:101-108. 2. Meaney PA, et al.: Crit Care Med 2010, 38:101-108. Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score H Davies, A Loosely, S Dolling, R Eve University Hospitals Bristol NHS Foundation Trust, Bristol, UK Critical Care 2014, 18(Suppl 1):P491 (doi: 10.1186/cc13681) Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score H Davies, A Loosely, S Dolling, R Eve University Hospitals Bristol NHS Foundation Trust, Bristol, UK Critical Care 2014, 18(Suppl 1):P491 (doi: 10.1186/cc13681) Results The primary analysis included 162 patients from two intervention hospitals and 892 from 27 control hospitals. Thirty- two percent of the patients were female and the mean age was 65.3 ± 16.5 years. Almost one-half (46%) of patients had a shockable initial cardiac arrest rhythm, 41% had bystander CPR, and 5% had an AED applied. PACT did not improve use of TTM (ratio of ORs = 1.03, 95% CI = 0.89 to 1.20), angiography for patients without ST-elevation H Davies, A Loosely, S Dolling, R Eve H Davies, A Loosely, S Dolling, R Eve University Hospitals Bristol NHS Foundation Trust, Bristol, UK Critical Care 2014, 18(Suppl 1):P491 (doi: 10.1186/cc13681) Introduction Out-of-hospital cardiac arrest (OHCA) has a poor prognosis even after successful resuscitation [1]. No scoring system S179 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 myocardial infarction (ratio of ORs = 1.10, 95% CI = 0.87 to 1.40), or electrophysiology assessment (ratio of ORs = 1.06, 95% CI = 0.81 to 1.38) as compared with concurrent control hospitals. Patients in the intervention group were less likely to have life support withdrawn within 72 hours on the basis of neuroprognosis compared with patients in the concurrent control group (ratio of ORs = 0.62, 95% CI = 0.39 to 0.98). Conclusion PACT was associated with reduced WLST <72 on the basis of neuroprognostication but did not improve other important post-cardiac arrest processes of care. Further work is underway to identify factors that infl uenced implementation. This will guide future consideration of the PACT model in other settings. myocardial infarction (ratio of ORs = 1.10, 95% CI = 0.87 to 1.40), or electrophysiology assessment (ratio of ORs = 1.06, 95% CI = 0.81 to 1.38) as compared with concurrent control hospitals. Predicting survival in patients admitted to intensive care following out-of-hospital cardiac arrest using the Prognosis After Resuscitation score Patients in the intervention group were less likely to have life support withdrawn within 72 hours on the basis of neuroprognosis compared with patients in the concurrent control group (ratio of ORs = 0.62, 95% CI = 0.39 to 0.98). Conclusion PACT was associated with reduced WLST <72 on the basis of neuroprognostication but did not improve other important post-cardiac arrest processes of care. Further work is underway to identify factors that infl uenced implementation. This will guide future consideration of the PACT model in other settings. P493 One-year assessment of in-hospital cardiac arrest M Ahmed, A Kochhar, O Rose Univeristy Hospital Lewisham, London, UK Critical Care 2014, 18(Suppl 1):P493 (doi: 10.1186/cc13683) These data included patient demographics, initial clerking and consultant review, patient care during the 48 hours prior to cardiac arrest, the resuscitation status of the patient, the resuscitation attempt, the post-cardiac arrest care and survival to discharge rates. p p g Results Medical notes were available for assessment for 69 out of the 82 patients that were identifi ed as having IHCA between 1 October 2011 and 30 September 2012. The frequency of IHCA showed no correlation to day of the week or month. Initial clerking was incomplete in history- taking (16% vs. 14% in NCEPOD) and in examination (46% vs. 24% in NCEPOD). The majority of patients were appropriately escalated in a timely fashion (94% vs. 82% in NCEPOD), but fi rst consultant review was delayed beyond 12 hours in 49% of cases (48% in NCEPOD). A total 81% of patients suff ered cardiac arrest 24 hours after admission (68% in NCEPOD). Warning signs for cardiac arrest were considered present in 59% of cases (75% in NCEPOD), with a signifi cant proportion going unrecognised (27%) despite multiple medical reviews. Out-of- hours CPR attempts (68% vs. 59% in NCEPOD) seemed be associated with poorer survival. The survival to discharge rate after in-hospital cardiac arrest was 10.1%. This compares with 14.6% in the NCEPOD data and 20% in larger studies [2]. Ninety per cent of patients had no documentation of resuscitation status (78% in NCEPOD). Conclusion Our series shows a low mortality and a very good neurological outcome. There was no mortality or severe complications associated with endovascular hypothermia. It is a safe and feasible technique implemented in Latin American critical care units. Even the delay in achieving the objective of hypothermia is very long. R f 1. Kory et al.: Resuscitation 2011, 82:15-20. 2. Seder et al.: Crit Care Med 2009, 37:S211-S222. 3. Holzer et al.: N Engl J Med 2010, 363:1256-1264. 1. Kory et al.: Resuscitation 2011, 82:15-20. 1. Kory et al.: Resuscitation 2011, 82:15-20. 2. Seder et al.: Crit Care Med 2009, 37:S211-S222. 3. Holzer et al.: N Engl J Med 2010, 363:1256-1264. Figure 1 (abstract P494). Figure 1 (abstract P494). P494 d f p p Methods Data were collected by an anonymous questionnaire designed for the purpose of research, addressed to medical and nursing staff of Greek hospitals. The questionnaire consisted of questions about knowledge and behavior related to TH induction, target temperature and duration of cooling. Information about the potential barriers to implementation of TH was also collected. Endovascular hypothermia after cardiac arrest in a Chilean ICU M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P494 (doi: 10.1186/cc13684) Figure 1 (abstract P494). Figure 1 (abstract P494). Endovascular hypothermia after cardiac arrest in a Chilean ICU M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P494 (doi: 10.1186/cc13684) Introduction For more than a decade, mild hypothermia has been a standard for enhancing the neurological prognosis in comatose Results We obtained 344 questionnaires from 16 hospitals. The population of the study was registered nurses (RN) (63.8%) and doctors S180 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 24 hours of targeting body temperature at 33°C or 36°C. These results suggest that hypothermia does not alter host immune response. Reference 24 hours of targeting body temperature at 33°C or 36°C. These results suggest that hypothermia does not alter host immune response. Reference 1. Nielsen N, et al.: Targeted temperature management at 33°C versus 36°C after cardiac arrest. N Engl J Med 2013, 369:2197-2206. 24 hours of targeting body temperature at 33°C or 36°C. These results suggest that hypothermia does not alter host immune response. Reference (36.2%). The majority of health staff (81.5%) had never implemented TH. A total 45.8% of respondents stated that the main reasons for not using TH were the lack of information and training about the method, the lack of nursing staff , the lack of available cooling methods and the required time. The most common methods of application were cold packs and intravenous fl uids. Only 30.2% of the doctors and 5.5% of the nurses (P <0.001) actually had the knowledge to implement TH, and this was demonstrated by correct answers. Of the respondents who answered that they did know the method, only 23.9% answered correctly; about the target temperature, the maintenance and rewarming phase. A total 59.1% of doctors, despite having attended the Advanced Cardiac Life Support seminar, were not able to answer correctly the knowledge questions. Continuous education of health professionals and the existence of a protocol were proposed by 65% of participants as the best way of increasing knowledge and adherence with ERC guidelines about TH. 1. Nielsen N, et al.: Targeted temperature management at 33°C versus 36°C after cardiac arrest. N Engl J Med 2013, 369:2197-2206. Endovascular hypothermia after cardiac arrest in a Chilean ICU M Canitrot, S Ugarte INDISA Clinic, Santiago, Chile Critical Care 2014, 18(Suppl 1):P494 (doi: 10.1186/cc13684) P497 P497 Derived electromyography is an accurate measure of shivering burden during targeted temperature management T May, DS Seder, GF Fraser, BM McCrum, CF Hoover, RR Riker Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P497 (doi: 10.1186/cc13687) Introduction Shivering complicates targeted temperature manage- ment (TTM) by increasing metabolism, oxygen consumption, rest ing energy expenditure, and carbon dioxide production and is associated with lower brain tissue oxygen levels; all of these may limit the eff ectiveness of TTM. However, the recognition and measurement of shivering are subjective and ill-defi ned. The Bedside Shivering Assessment Scale (BSAS) is the only validated tool to describe the intensity of shivering. We hypothesized that the derived electromyography (dEMG) value measured by the bispectral index monitor (BIS) would agree with energy expenditure due to shivering, compared with the BSAS. Conclusion Therapeutic hypothermia is rarely used in Greek hospitals. The level of knowledge is mainly related to the lack of education and the lack of information about new techniques. Programs for continuing education are necessary for the use of new therapeutic techniques in the fi eld of health. i References 1. Erb JL, Hravnak M, Rittenberger JC: Therapeutic hypothermia after cardiac arrest. Am J Nurs 2012, 112:38-44. 1. Erb JL, Hravnak M, Rittenberger JC: Therapeutic hypothermia after cardiac arrest. Am J Nurs 2012, 112:38-44. 2. Metzger JC, Eastman AL, Pepe PE: Year in review 2010: critical care – cardiac arrest and cardiopulmonary resuscitation. Crit Care 2011, 15:239. 2. Metzger JC, Eastman AL, Pepe PE: Year in review 2010: critical care – cardiac arrest and cardiopulmonary resuscitation. Crit Care 2011, 15:239. 2. Metzger JC, Eastman AL, Pepe PE: Year in review 2010: critical care – cardiac arrest and cardiopulmonary resuscitation. Crit Care 2011, 15:239. Methods We measured continuous indirect calorimetry during a 2 to 5 hour time span during targeted temperature management in 12 patients being treated for hypoxic ischemic encephalopathy after cardiac arrest. Patients were excluded if seizing, requiring >FiO2 0.5, exhibiting early spasticity, or not shivering. The BSAS was measured every 15 minutes by a blinded observer and shivering was treated for a BSAS ≥1, as per institutional protocol. The association of dEMG and BSAS as a predictor of resting energy exposure (REE) was measured using linear regression and Pearson’s correlation. P498 P498 Shivering during targeted temperature management after cardiac arrest: a physiologic description T May, DS Seder, GF Fraser, CF Hoover, RR Riker, BM McCrum Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P498 (doi: 10.1186/cc13688) P496 g g Results The study population included 12 patients. Average age was 54 years, nine patients were male, eight patients had a CPC of 1 to 3 on hospital discharge. There were a total of 182 measurements of BSAS, dEMG, and REE. There is improved correlation between REE and dEMG compared with REE and BSAS (0.24 (CI = 0.10 to 0.37) vs. 0.10 (CI = –0.04 to 0.24); P <0.001 vs. 0.14). Each increase in dEMG resulted in an increase of 14 kcal of energy expenditure (P = 0.003). Introduction Induced hypothermia is applied in the ICU and in the operating theater to reduce ischemia–reperfusion injury. It is thought that induced hypothermia may hamper the immune response and therefore carry the risk of acquiring or aggravating an infection. We investigated the eff ect of hypothermia on host response by comparing survivors of cardiac arrest in which body temperature was kept at either 33°C or 36°C. Conclusion Continuous dEMG power measured by Covidien BIS monitors is a more accurate and less subjective measure of shivering burden compared with the intermittent BSAS. Introducing dEMG into clinical practice may improve recognition of shivering, allow quantifi cation of the metabolic cost of shivering, and serve as a more reliable research tool for diagnostic and treatment strategies of shivering. Methods As a substudy to the Target Temperature Management trial [1] in which cardiac arrest patients admitted to the ICU were randomized to maintaining either 33°C (n = 11) or 36°C (n = 9) for 24 hours, blood was drawn at the start and end of the target temperature phase as well as after reaching normotemperature. Host response was measured via monocyte human leukocyte antigen-DR (HLA-DR) expression and via whole blood stimulation with TLR ligands lipopolysaccharide (LPS) and lipoteicoic acid (LTA) for 24 hours. Plasma levels of interleukin (IL)-1β, IL-1RA, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNFα), macrophage infl ammatory proteins (MIP)-1, monocyte chemotactic protein (MCP)- 1 and soluble CD40 ligand levels were determined with ELISA or Luminex. Statistics were by unpaired Mann–Whitney U tests. Shivering during targeted temperature management after cardiac arrest: a physiologic description arrest: a physiologic description T May, DS Seder, GF Fraser, CF Hoover, RR Riker, BM McCrum Maine Medical Center, Portland, ME, USA Critical Care 2014, 18(Suppl 1):P498 (doi: 10.1186/cc13688) y p y Results HLA-DR expression was decreased compared with healthy controls, without diff erences between 33°C and 36°C. Following whole blood stimulation with LPS, TNFα and IL-6 production was lower after cardiac arrest compared with healthy controls. The 33°C and 36°C groups diff ered at baseline in TNFα levels after LPS whole blood stimulation. After 24 hours of temperature management, there was no diff erence in both TNFα and IL-6 production between the groups following TLR ligand stimulation. After cessation of temperature management only TNFα levels increased after LTA stimulation. Plasma levels of IL-1RA, IL-8 and IL-10 were decreased after cardiac arrest compared with healthy controls. In plasma levels of IL-1β, MIP-1 and soluble CD40 ligand there were no diff erences between the 33°C and 36°C groups at all time points. Apart from baseline diff erences, there were no diff erences between 33°C and 36°C in plasma levels of IL-1RA, IL-8, IL-10 and MCP-1.l Introduction Targeted temperature management (TTM) is used to treat hypoxic ischemic encephalopathy (HIE), elevated intracranial pressure, status epilepticus, and other brain injuries. Shivering complicates TTM, and the associated energy burden and its response to treatment are poorly understood. We describe the pattern of shivering and response to neuromuscular blockade in a series of patients undergoing TTM after cardiac arrest using continuous indirect calorimetry. Methods With IRB approval, we studied 16 patients undergoing TTM for HIE with continuous calorimetry during their treatment. The calorimeter measures inspired and expired oxygen and carbon dioxide, which are used in the Weir equation to calculate resting energy expenditure (REE). We excluded patients known to be actively seizing, requiring FiO2 >0.5, with early spasticity, or who did not shiver. All patients received counterwarming, moderate analgosedation, and bolused vecuronium in response to visible shivering, measured hourly by nurses using the Bedside Shivering Assessment Scale. Conclusion Cardiac arrest induces a decrease in proinfl ammatory response. There were no diff erences in immune host response after S181 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Results Sixteen patients of average age 57 included 12 men. Twelve patients had CPC of 1 to 3 on hospital discharge. References References References 1. N Engl J Med 2002, 346:549-555. 2. Crit Care Med 2011, 39:1113-1125. 1. N Engl J Med 2002, 346:549-555. Serum phosphate concentration during the rewarming period after deep hypothermic circulatory arrest H Hayami1, O Yamaguchi2, S Ishida3, Y Koide4, T Gotoh2 1Yokohama Municipal Citizen’s Hospital, Yokohama, Japan; 2Yokohama City University Hospital, Yokohama, Japan; 3Yokohama City University Medical Center, Yokohama, Japan; 4Hayama Heart Center, Hayama, Japan Critical Care 2014, 18(Suppl 1):P501 (doi: 10.1186/cc13691) p Results Forty-three patients were admitted in Group 1, 28 in Group 2. Of these, 42% in both groups were following out-of-hospital (OOH) VF arrests. Cooling was attempted in 88% and 82% of OOH VF patients respectively. For patients with either in-hospital or non-VF/ VT cardiac arrests, the numbers cooled were 16% and 12.5%. Cooling initiation within 1 hour increased from 27 to 50%. Achievement of a target temperature of 32 to 34°C within 4 hours of ROSC was 55% and 50% respectively. Target maintenance for 12 to 24 hours after ROSC increased 79% to 100%. Avoidance of hypothermia <31°C for 48 hours after ROSC improved 95% to 100%. Slow rewarming at 0.25 to 0.5°C/ hour to 37°C was achieved in 76% and 90%. Avoidance of temperature >37.2°C for 48 hours after ROSC increased 84 to 100%. Of the patients cooled, survival with good neurological outcome was achieved in 52% in Group 1 and 88% in Group 2. Factors involved in prolonged eff ect of neuromuscular blockade in therapeutic hypothermia Factors involved in prolonged eff ect of neuromuscular blockade in therapeutic hypothermia p yp H Tanaka, T Mochizuki, S Ode, S Ishimatsu St Lukes International Hospital, Akashi-Chou Chuo-Ku, Japan Critical Care 2014, 18(Suppl 1):P500 (doi: 10.1186/cc13690) p yp H Tanaka, T Mochizuki, S Ode, S Ishimatsu St Lukes International Hospital, Akashi-Chou Chuo-Ku, Japan Critical Care 2014, 18(Suppl 1):P500 (doi: 10.1186/cc13690) Introduction Therapeutic hypothermia (TH) following out-of-hospital cardiac arrest (OHCA) improves neurological outcomes in such patients. During TH, neuromuscular blockade is used to control shivering in the patient. In our hospital, we use vecuronium as a neuromuscular blocker. However, occasionally, prolonged eff ects of vecuronium delay accurate evaluation of patients’ neurological function or extubation. Unfortunately, the factors involved in the prolonged eff ect of vecuronium in TH remain unclear. Conclusion The energy burden of shivering is underestimated by standard nutritional formulas in patients undergoing TTM after cardiac arrest. Subclinical shivering is associated with increased energy expenditure. Clinical recognition occurs long after the increase in metabolic activity, and persists for a signifi cant period of time after treatment. These fi ndings should infl uence how shivering is monitored and treated during TTM. Methods We conducted a retrospective cohort study of patients managed with TH following OHCA at our institution from April 2010 to September 2013. We defi ned full-muscle reaction to train-of-four stimulation (TOF) as the end of eff ects of vecuronium. In this study, the time from the end of vecuronium administration to full-muscle reaction to TOF was evaluated as the outcome. We calculated the adjusted hazard ratio (HR) for the outcome using Cox regression analysis after adjustment for age, gender, albumin levels, estimated glomerular fi ltration rate, temperature at the end of vecuronium administration, and total amount of vecuronium per kilogram of body weight. Temperature management following cardiac arrest: introducing a protocol improves compliance with targets P Creber, G Talling, M Oram Cheltenham General Hospital, Cheltenham, UK Critical Care 2014, 18(Suppl 1):P499 (doi: 10.1186/cc13689) Results In total, 52 patients were evaluated in this study (86.5% male; average age, 55.2 years). The median time from the end of vecuronium administration to full-muscle reaction to TOF was 660 minutes (maximum: 10,363 minutes; minimum: 72 minutes). Blood albumin levels on admission decreased the time (adjusted HR: 2.31, 95% CI: 1.073 to 5.004; P <0.05). However, other factors had no signifi cant diff erence on the time. The amount of vecuronium per kilogram of weight had no signifi cant diff erences (adjusted HR: 1.073, 95% CI: 0.869 to 1.325; P = 0.511). Introduction We audited the achievement of therapeutic hypothermia (TH) before and after the introduction of a cooling protocol. Instituting TH is recommended following the return of spontaneous circulation (ROSC) for many patients who survive a cardiac arrest [1,2]. The key intervention may be the avoidance of hyperthermia rather than cooling [3]. Conclusion The time from the end of vecuronium administration to full-muscle reaction to TOF decreased in relation to blood albumin levels on admission. Other factors had no signifi cant diff erence on the outcome time. Taken together, the outcome time may be determined by albumin levels and not by the administered amount of vecuronium per kilogram of body weight. Based on this result, we suggest that blood albumin levels on admission should be taken into consideration for appropriate use of vecuronium in TH. g Methods We conducted a chart review of all patients admitted to the Department of Critical Care (DCC) at our hospital following cardiac arrest over 2 years in 2010 to 2012 (Group 1). We recorded compliance with key recommendations produced by the Royal College of Anaesthetists [4] although we defi ned post-ROSC hyperthermia as >37.2°C rather than >38°C. A TH protocol was designed and personnel in the emergency department and DCC educated as to its use. Recommended practice was the infusion of cold i.v. normal saline (1 to 2 l) followed by the use of an intravascular cooling device (Alsius CoolGard™). Data collection was then undertaken after introduction of the protocol for all patients admitted to the DCC following cardiac arrest in November 2012 to 2013 (Group 2). Shivering during targeted temperature management after cardiac arrest: a physiologic description Sixteen shivering events were monitored with calorimetry among the 12 patients. The average rate of energy expenditure (without shivering) leading up to and following paralytic treatment was 1,425 kcal/hour (± 489 kcal/ hour) and 1,386 kcal/hour (± 235 kcal/hour). This was signifi cantly greater than the predicted basal energy expenditure (1,089 ± 222 kcal; P = 0.007 and P <0.001). Time from a change in baseline energy expenditure to recognition and treatment of clinical shivering was 57 (± 64) minutes, and from treatment with neuromuscular blockade to baseline energy expenditure was 30 (± 20) minutes. This accounts for a total diff erence of 15,223 kcal (± 10,997 kcal) before treatment and 7,113 kcal (± 3,706 kcal) after treatment for each shivering episode compared with baseline (P = 0.01 and P = 0.003). P502l Infl uence of baseline magnesium concentrations on shivering in therapeutic temperature modulation KL Johnson HCMC, Minneapolis, MN, USA Critical Care 2014, 18(Suppl 1):P502 (doi: 10.1186/cc13692) g Results Twenty patients receiving TTM for TBI were evaluated (March to October 2013). One-half of the patients maintained targeted BSAS scores <1 for the full duration of TTM (n = 10 of 20). Serum Mg levels at the initiation of TTM were observed to negatively correlate with the level of shivering, as indicated by the BSAS scoring system (P = 0.02). See Figure 1. g Results Twenty patients receiving TTM for TBI were evaluated (March to October 2013). One-half of the patients maintained targeted BSAS scores <1 for the full duration of TTM (n = 10 of 20). Serum Mg levels at the initiation of TTM were observed to negatively correlate with the level of shivering, as indicated by the BSAS scoring system (P = 0.02). See Figure 1. Introduction Therapeutic temperature modulation (TTM) is widely used in the care setting to improve outcomes of patients with traumatic brain injury (TBI). Through fever prevention, both oxygen utilization and caloric expenditure are reduced, so metabolic effi ciency can be maximized [1]. However, patient cooling is not without consequences and shivering is experienced by more than 70% of patients achieving TTM. Because shivering triggers an increase in metabolic demand, causing additional oxygen consumption and the promotion of catabolism, its prevention is ideal [2]. We set out to review the data surrounding the anti-shivering component of a normothermia protocol in the surgical ICU (SICU) of one Minnesota hospital. g Conclusion The literature suggests the positive impact of TTM on patient outcomes can be maximized with shivering prevention [2]. Current SICU practices provide a similar Mg loading dose for all patients, regardless of baseline Mg levels. In our observed patients, achieving a baseline serum Mg level >2 was associated with lower shivering scores throughout the TTM course. This supports the hypothesis that serum Mg concentrations prior to TTM are important predictors of shivering reduction, and suggests that loading doses of Mg should be tailored to the individual patient to achieve such levels. f Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Figure 1 (abstract P502). Occurrence of SND was defi ned as neurological disarrangement such as irritability, confusion, or delirium within 48 hours after operation. Occurrence of SND was defi ned as neurological disarrangement such as irritability, confusion, or delirium within 48 hours after operation. as irritability, confusion, or delirium within 48 hours after operation. Results Durations of CPB, aortic clamp, and circulatory arrest were 194 ± 51, 136 ± 53, and 70 ± 17 minutes respectively. Nine patients underwent ACP (89 ± 26 minutes) and 11 patients underwent RCP (56 ± 25 minutes). Concentration of ALac, ScvO2, and P changed at 2, 4, 6, 10 hours after CPB as follows: ALac 4.0 ± 1.9, 5.1 ± 2.6, 5.3 ± 3.3, 4.0 ± 2.8 mmol/l, ScvO2 71 ± 6.7, 67 ± 10, 67 ± 9.7, 65 ± 11%, and P 2.9 ± 0.9, 2.4 ± 0.7, 2.2 ± 1.0, 1.8 ± 0.8 mg/dl, respectively. Serum P recovered to 2.6 ± 1.4 at 18 hours after CPB. The incidence of hypophosphatemia (<2.6 mg/dl) and SND in our series were 18/20 (90%) and 14/20(70%) respectively. There was a correlation between minimum P and time to confi rm M6 on the GCS (P = 0.508, Fisher r-to-z transformation), but no signifi cant correlation between SND. The mortality rate during the fi rst 28 days was 5% (1/20). y Conclusion Serum phosphate decreased dramatically during the rewarming period after deep hypothermic circulatory arrest. There was a moderate correlation between minimum phosphate concentration and time to confi rm M6 on the GCS. Monitoring phosphate concentration might predict neurological recovery after deep hypothermic circulatory arrest. Figure 1 (abstract P502). attention paid to the anti-shivering portion of the protocol. Serum magnesium (Mg) levels were assessed prior to initiation of TTM and Bedside Shivering Assessment Scale (BSAS) scores were collected. attention paid to the anti-shivering portion of the protocol. Serum magnesium (Mg) levels were assessed prior to initiation of TTM and Bedside Shivering Assessment Scale (BSAS) scores were collected. Results Twenty patients receiving TTM for TBI were evaluated (March to October 2013). One-half of the patients maintained targeted BSAS scores <1 for the full duration of TTM (n = 10 of 20). Serum Mg levels at the initiation of TTM were observed to negatively correlate with the level of shivering, as indicated by the BSAS scoring system (P = 0.02). See Figure 1. Serum phosphate concentration during the rewarming period after deep hypothermic circulatory arrest H Hayami1, O Yamaguchi2, S Ishida3, Y Koide4, T Gotoh2 1Yokohama Municipal Citizen’s Hospital, Yokohama, Japan; 2Yokohama City University Hospital, Yokohama, Japan; 3Yokohama City University Medical Center, Yokohama, Japan; 4Hayama Heart Center, Hayama, Japan Critical Care 2014, 18(Suppl 1):P501 (doi: 10.1186/cc13691) H Hayami1, O Yamaguchi2, S Ishida3, Y Koide4, T Gotoh2 1Yokohama Municipal Citizen’s Hospital, Yokohama, Japan; 2Yokohama City University Hospital, Yokohama, Japan; 3Yokohama City University Medical Center, Yokohama, Japan; 4Hayama Heart Center, Hayama, Japan Critical Care 2014, 18(Suppl 1):P501 (doi: 10.1186/cc13691) Introduction Short-term neuropsychologic dysfunction (SND) is often observed during the postoperative period of open total arch repair (TAR) under deep hypothermic circulatory arrest (DHCA). We supposed that there might be a large consumption of high-energy phosphates during the rewarming period, and this might be associated with awakening and SND. Due to diffi culty in measuring such high-energy phosphates at the bedside, we measured the serum phosphate concentration (P) as a substitute, and assessed its infl uence on awakening and postoperative SND. Conclusion The institution of a temperature management protocol improved compliance with recommended goals, both in achieving hypothermia and in the avoidance of hyperthermia. References Methods Twenty patients with a mean age of 68 ± 11 years who underwent open TAR under DHCA applied at a temperature of 20°C, with antegrade cerebral perfusion (ACP) or retrograde cerebral perfusion (RCP), were enrolled. Arterial blood gas lactate concentration (ALac), ScvO2, and P were measured at 2, 4, 6, and 10 hours after weaning from cardiopulmonary bypass (CPB). We also measured time to confi rm M6 on the Glasgow Coma Scale (GCS), and incidence of SND. 1. Nolan JP, et al.: ERC Guidelines 2010. Resuscitation 2010, 81:1219-1276. 2. HACA Study Group: N Engl J Med 2002, 346:549-556. 3. Nielsen N, et al.: N Engl J Med 2013, 369:2197-2206. 4. Nolan J: Implementation of therapeutic hypothermia. In Raising the Standard: A Compendium of Audit Recipes. 3rd edition. Edited by Colvin J, Peden C. Royal College of Anaesthetists; 2012:200-201. 1. Nolan JP, et al.: ERC Guidelines 2010. Resuscitation 2010, 81:1219-1276. 2. HACA Study Group: N Engl J Med 2002, 346:549-556. 3. Nielsen N, et al.: N Engl J Med 2013, 369:2197-2206. 4. Nolan J: Implementation of therapeutic hypothermia. In Raising the Standard: A Compendium of Audit Recipes. 3rd edition. Edited by Colvin J, Peden C. Royal College of Anaesthetists; 2012:200-201. Serum phosphate concentration during the rewarming period after deep hypothermic circulatory arrest S182 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 Critical Care 2014, Volume 18 Suppl 1 http://ccforum.com/supplements/18/S1 References Methods A retrospective review was conducted looking at SICU patients managed with a normothermia protocol, with particular 1. Badjatia N: Crit Care Med 2009, 37:S250-S255. 2. Badjatia N, et al.: Crit Care Med 2009, 37:1893-1896.
https://openalex.org/W2612537002	http://repository.uwl.ac.uk/id/eprint/3313/1/Thabet-etal-2015-PTSD-Depression-and-Anxiety-among-Palestinian.pdf	English	null	PTSD, Depression, and Anxiety among Palestinian Women Victims of Domestic Violence in the Gaza Strip	British journal of education, society & behavioural science	2,015	cc-by	8,512	. Thabet1, Ahmad Abu Tawahina2, Tischler Victoria3 and Panos Vostanis4 1Department of Child and Adolescent Psychiatry, Al-Quds University, School of Public Health, Child Institute-Gaza, Palestine. 2Gaza Community Mental Health Program, Gaza, El Rasheed Street, Palestine. 3Department of Behavioural Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, UK. 4Department of Child Psychiatry, School of Medicine- Greenwood Institute of Child Health, School of Psychology Westcotes House Westcotes Drive Leicester LE3 OQU UK 1Department of Child and Adolescent Psychiatry, Al-Quds University, School of Public Health, Child Institute-Gaza, Palestine. 2 , 2Gaza Community Mental Health Program, Gaza, El Rasheed Street, Palestine. 3Department of Behavioural Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, UK. 3Department of Behavioural Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, UK. 4Department of Child Psychiatry, School of Medicine- Greenwood Institute of Child Health, School of Psychology Westcotes House, Westcotes Drive, Leicester LE3 OQU, UK. Authors’ contributions This work was carried out in collaboration between all authors. Authors A. A. Thabet and AAT designed the study, wrote the protocol. Authors A. A. Thabet, AAT, TV and PV wrote the first draft of the manuscript. Authors A. A. Thabet, AAT, TV and PV managed the literature searches. Authors A. A. Thabet, TV and PV analyses of the study performed the spectroscopy analysis and wrote the final draft. All authors read and approved the final manuscript. British Journal of Education, Society & Behavioural Science 11(2): 1-13, 2015, Article no.BJESBS.18960 ISSN: 2278-0998 SCIENCEDOMAIN international www.sciencedomain.org PTSD, Depression, and Anxiety among Palestinian Women Victims of Domestic Violence in the Gaza Strip A. A. Thabet1, Ahmad Abu Tawahina2, Tischler Victoria3 and Panos Vostanis4 1Department of Child and Adolescent Psychiatry, Al-Quds University, School of Public Health, Child Institute-Gaza, Palestine. 2Gaza Community Mental Health Program, Gaza, El Rasheed Street, Palestine. 3Department of Behavioural Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, UK. 4Department of Child Psychiatry, School of Medicine- Greenwood Institute of Child Health, School of Psychology Westcotes House, Westcotes Drive, Leicester LE3 OQU, UK. Authors’ contributions This work was carried out in collaboration between all authors. Authors A. A. Thabet and AAT designed the study, wrote the protocol. Authors A. A. Thabet, AAT, TV and PV wrote the first draft of the manuscript. Authors A. A. Thabet, AAT, TV and PV managed the literature searches. Authors A. A. Thabet, TV and PV analyses of the study performed the spectroscopy analysis and wrote the final draft. All authors read and approved the final manuscript. Article Information DOI: 10.9734/BJESBS/2015/18960 Editor(s): (1) Tompson Makahamadze, School for Conflict Analysis and Resolution, George Mason University, USA. Reviewers: (1) Anonymous, University of Sri Jaywardenepura, Sri Lanka. (2) Alfredo Rodríguez-Sedano, Department of Education, University of Navarre, Pamplona, Spain. (3) Anonymous, Cayetano Heredia University, Peru. (4) Anonymous, India. Complete Peer review History: http://sciencedomain.org/review-history/10284 Received 19th May 2015 Accepted 2nd July 2015 Published 30th July 2015 ABSTRACT Background: Domestic violence is a universal phenomenon which affects all family members and specially children and women. Common reactions to domestic violence are post-traumatic stress di d d i d i t Original Research Article British Journal of Education, Society & Behavioural Science 11(2): 1-13, 2015, Article no.BJESBS.18960 ISSN: 2278-0998 Article Information DOI: 10.9734/BJESBS/2015/18960 Editor(s): (1) Tompson Makahamadze, School for Conflict Analysis and Resolution, George Mason University, USA. Reviewers: (1) Anonymous, University of Sri Jaywardenepura, Sri Lanka. (2) Alfredo Rodríguez-Sedano, Department of Education, University of Navarre, Pamplona, Spain. (3) Anonymous, Cayetano Heredia University, Peru. (4) Anonymous, India. Complete Peer review History: http://sciencedomain.org/review-history/10284 Received 19th May 2015 Accepted 2nd July 2015 Published 30th July 2015 Original Research Article ABSTRACT Background: Domestic violence is a universal phenomenon which affects all family members and specially children and women. Common reactions to domestic violence are post-traumatic stress disorder, depression, and anxiety. *Corresponding author: E-mail: abdelazizt@hotmail.com; Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 Aims: The aim of this study is to find type and severity of domestic violence against Palestinian women in the Gaza Strip, and to investigate whether it is associated with mental health problems such as post-traumatic stress disorder, depression, and anxiety. Aims: The aim of this study is to find type and severity of domestic violence against Palestinian women in the Gaza Strip, and to investigate whether it is associated with mental health problems such as post-traumatic stress disorder, depression, and anxiety. p p y Methods: The study sample included 622 Palestinian women randomly selected from the five areas of the Gaza Strip aged from 18 to 50 years (mean age = 31.5 years). They were interviewed using questionnaires including Sociodemogrophic variables, Conflicts Tactics Scale, post-traumatic stress disorder scale, Beck Depression Inventory, Taylor manifestation Anxiety Scale. p y y y ults: The study showed that psychological assault was 56.91%, physical assault 37.3% sical injury 12.06%, and sexual assault was 7.14%. The study showed that domestic violence was significantly higher in women living in villages than in cities or camps. Also, women living in villages experienced more psychological abuse than women living in cities or camps. g p The study showed that 71 women (11.4%) had been diagnosed as post-traumatic stress disor 5% had moderate to severe depression, and 29.9% had very severe anxiety. The study showed that psychological assault toward women was positively correlated to depression, anxiety, and posttraumatic stress disorder. Also, there were significant positive relationships between physical assault and depression, anxiety, and posttraumatic stress symptoms. Physical injury and sexual assault were significantly positively related to post traumatic stress disorder, depression, and anxiety. p y Conclusion: The study showed that one third Palestinian women exposed to physical violence and half of them exposed to psychological violence which lead to post-traumatic stress disorder depression, and anxiety. So, a great need for more programs for women victims of domestic violence in Palestinian society are needed with well trained professionals in the field of psychological support and therapy. More specific programs should be established in Gaza to enable women of using new coping strategies with difficulties. ABSTRACT Also, training programs including primary health care professional such physicians, nurses, social service experts, and midwives should be provide to enable them of early detection of victims of violence and provide social support to these women. Keywords: Anxiety; depression; domestic violence; Palestinian women; PTSD. 1. INTRODUCTION Physical 2 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 violence from their partners was reported in 23– 49% of those women, and sexual violence was reported in 10–50% of them [4]. found that 23.3% suffered physical abuse from their husbands, 61.7% psychological abuse, and 10.9% sexual abuse at least once during the year 2005. There were higher rates of all forms of abuse in the West Bank compared to the Gaza Strip. For single, never-married women the rates were found to be 25% for physical abuse and 52.7% for psychological abuse experienced at least once during the year 2005. Similar to their ever-married counterparts, the West Bank had higher rates of abuse than the Gaza Strip. Other studies of domestic violence in non- Western contexts suggest that traditional norms may still exert an influence on the status of women. For example, a study of Sri Lankan medical students [5] and Jordanian men [6] revealed that strong patriarchal beliefs were used to justify violence against women. A previous study involving Palestinian women found that 13- 69% strongly agreed that wife beating was justified under certain circumstances, for example, when a wife was sexually unfaithful [7]. In Saudi Arabia, it was revealed that 30% of male respondents had been violent toward a female family member, with female misbehavior cited as the main reason for the abuse, in addition to disapproval over dress and conduct for unmarried women. The most likely targets of such violence were, in order of likelihood, sisters, mothers, sisters-in-law, aunts, and wives [13]. In a study of Qatari female students, 80% of whom were single never-married women, it was found that 39% were abused by their brothers, 38% by their fathers, and 22% reported abuse from their mothers [14]. Sociodemographic variables such as younger age and limited decision-making power are significant predictors of both physical and psychological abuse of single, never-married Palestinian women; religion was found to be significant for psychological abuse alone [8]. A number of previous studies have examined the incidence of domestic violence and attitudes towards male to female spousal abuse in Arabic countries and Islamic cultures. In a reported the results of two national surveys of married Palestinian woman living in the West Bank and Gaza strip. 1. INTRODUCTION Living in rural areas and camps, poverty, unemployment, being a Muslim, husbands has a low level of education and women having a higher educational level than their husbands were factors predicted domestic violence in women [6]. In Syria, 26% of married women reported at least three instances of abuse during the year, while weekly battering occurred among 3.3% of married women [9]. Similarly, Bedouin Arab women in Israel have a 48% lifetime exposure rate to violence in their families [10]. Another, family public health survey from Iraq documented that 83.1% of women reporting at least one form of marital control. Overall, younger married women were the most likely to report restrictions; 74.5% of those aged 15 to 24 years reported having to ask permission to seek health care, compared to 60.3% of those aged 40 to 49 years. As for emotional or psychological violence, 33.4% of women reported at least one form of violence and 21.2% of women experienced physical violence [11]. National research on violence against women in Turkey revealed 39% of women were physically and 15% sexually abused by their partner, whereas 18% were physically and 3% were sexually abused by a non-partner. The same research noted that the most common perpetrators in non-partner abuse were fathers (42%), mothers (32%), and brothers (16%) [15]. The data from the Spanish Women’s Institute report that 10% of women above the age of 18 have been exposed to intimate-partner violence [16]. In study were conducted with 96 women recruited from community health clinics serving low-income women in an urban U.S. city showed that all women had experienced some type of physical violence in the past year based on the eligibility criteria, most women experienced severe physical or sexual IPV in the past year (88.5%). In the past 6 months, 22% of women experienced no IPV or psychological IPV only, while 16% experienced minor physical or sexual intimate-partner violence and 62.5% experienced severe physical or sexual IPV. Fifty two percent of women were experiencing intimate-partner violence from their current partner, while 48% were experiencing IPV from a former partner [17]. In 2006, the Palestinian Central Bureau of Statistics (PCBS) conducted a Domestic Violence Survey examining instances of abuse among ever-married women; single, never- married women over 18 years; children; and the elderly [12]. 1. INTRODUCTION Palestine refugee camps, which have one of the highest population densities in the world. That camps contain two thirds of the population of Gaza Strip who had been uprooted from their lands in 1948. Gaza Strip is composed of five provinces: North Gaza, Gaza city, Middle area, Khan Younis and Rafah [1]. Palestine refugee camps, which have one of the highest population densities in the world. That camps contain two thirds of the population of Gaza Strip who had been uprooted from their lands in 1948. Gaza Strip is composed of five provinces: North Gaza, Gaza city, Middle area, Khan Younis and Rafah [1]. Gaza Strip is a narrow piece of land lying on the coast of the Mediterranean sea. Its position on the crossroads from Africa to Asia made it a target for occupiers and conquerors over the centuries. Gaza Strip is very crowded place with area 365 sq. Km and constitute 6.1% of total area of Palestinian territory land. The Gaza Strip is home to a population of more than 1.76 million people, including 1.26 million Palestine refugees. For the last decade, the socioeconomic situation in Gaza has been in steady decline. Years of conflict and closure have left 80 per cent of the population dependent on international assistance. The tightened blockade, imposed following the Hamas takeover of Gaza in June 2007, has decimated lives and livelihoods, resulting in the impoverishment and de- development of a highly skilled and well- educated society. Despite adjustments made to the blockade by the Government of Israel in June 2010, restrictions on imports and exports continue to severely hamper recovery and reconstruction. Over half a million Palestine refugees in Gaza live in the eight recognized The World Health Organization (WHO) defines violence as “the intentional use of physical force or power, threatened or actual, against oneself, another person, or against a group or community, that either results in or has a high likelihood of resulting in injury, death, psychological harm, maldevelopment, or deprivation” [2]. Others, refers domestic violence to a pattern of physical, sexual, and/or psychological behaviors perpetrated by a current or former intimate partner [3]. The WHO study called “Women's Health and Domestic Violence against Women” surveyed 10 countries and 24,000 women and showed prevalence of domestic violence. 2.2.2 Revised conflict tactics scale [34] Studies have demonstrated that living with a violent intimate male partner is harmful to the psychological conditions of the victimized women. Mental health consequences of exposure to domestic violence against women commonly include depression, posttraumatic stress disorder (PTSD), anxiety, suicidal behavior, abuse of alcohol and/or other substances, and personality disorders [26-34]. Conflict Tactics Scale included subscales measuring physical, sexual, and psychological, and injury due to domestic violence. A previous version of the CTS was used for a study on domestic violence in the Palestinian territories [6]. This self-report instrument measures the frequency of physical abuse, verbal violence, sexual violence, reasoning tactics, and injury within an intimate relationship. Participants were asked to report the frequency with which they performed the various behavioral conflict resolution tactics in the previous year. Specifically, 12 items from the Physical Assault subscale, 8 items from the Psychological Aggression subscale, 3 items from the Sexual Coercion subscale, and 6 items from the Injury subscale were used to measure intimate partner physical assault, psychological abuse, and sexual coercion. Participants were asked to report on the frequency of each of the abusive acts occurring in a partner conflict situation over The aim of this study is to find type and severity of domestic violence against Palestinian women in the Gaza Strip, and to investigate whether it is associated with mental health problems such as post-traumatic stress disorder, depression, and anxiety. 2.2.1 Socio demographic scale Experience of domestic violence is associated with numerous deleterious effects on mental, social and emotional wellbeing. For example, it is associated with posttraumatic stress disorder (PTSD) [20,21], anxiety, gynaecological and digestive disorders [22], and depression [23], low self-esteem [24]. The experience of domestic violence is associated with a number of demographic factors such as poverty [25]. Sociodemographic data were collected on the women’s socio-demographic characteristics, including age, number of children, place of residence, education, marital status, occupational status, and, if married, and husband’s occupation. 1. INTRODUCTION For ever-married women, it was In another study of community sample of 4,114 in Bangladesh women of age 15 to 49 years having 3 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 at least one child, more than half of the women (53.9%) were found to be exposed to IPV [18]. the Gaza Strip aged from 18 to 50 years. We selected the sample according to the population density of the Gaza Strip. We gave the data collectors the total number of women from each area (Gaza Strip is divided in to 5 areas). Each Data collector had the exact number of women to be interviewed. From Each area one street was chosen and every other home was entered and the women were informed about the purpose of the study and signed a consent form and were told that the information will kept confidential with the researcher co do the interview. Data collection was done by a will trained group of 10 community health workers. Data was collected in July to September 2009. There is overwhelming evidence that intimate partner violence (IPV) is a major contributor to the ill-health of its women survivors. For example, an extensive review of the literature found that IPV posed a significant risk to women’s physical health, including increased mortality, injury, disability, chronic pain. In a Taiwanese study of 109 abused women found that the medium- and high-risk groups for life- threatening situations accounted for 82.6% of all subjects, and 93.6% of all subjects were in a high score Yahia group of post-traumatic responses Among the 10 symptom dimensions of psycho- physiological symptoms, anxiety had the highest standardized mean score, followed by obsession, depression, and somatization. The life- threatening situations had significant positive correlation with the overall post-traumatic responses, the responses of intrusion, and the general severity index (GSI). Overall post- traumatic responses, intrusion, and avoidance were all positively associated with GSI [19]. These workers were based in the (Women Empowerment Project’-WEP). Women Empowerment Project had three centers one in Gaza City, Middle area, and South of Gaza- It is a part of (Gaza Community Mental Health- CMHP). 2.3 Statistical Analysis Data analyses were performed using SPSS Version 18 (SPSS, Inc. Chicago, United States). The frequencies of categorical data are presented. Established cut-off scores were used to provide rates of likely PTSD, depression, and anxiety. The relationship between domestic violence, demographic variables, PTSD, depression, and anxiety was investigated using the Spearman correlation test, as the continuous scores were not normally distributed. Differences in mean scores for violence exposure, mental health problems and sociodemographic variables were tested using one way ANOVA. Multivariate linear regression analyses were performed to determine predictors of total anxiety, PTSD, and depression scores by types of violence experienced. 2.2.5 Beck depression inventory [38] the past year using a 7-point scale, with the following response options: 0 = never, 1 = once, 2 = twice, 3 = 3 to 5 times, 4 = 6 to 10 times, 5 = 11 to 20 times, and 6 = more than 20 times. The total scores for each subscale were calculated by adding up the midpoints of the response categories for each item (e.g., the midpoint for the 6 to 10 times response was 8). In this study, the Cronbach’s alpha coefficient was .92 for the Physical Assault subscale, .88 for the Psychological Aggression subscale, .89 for the Sexual Coercion subscale, and .88 for the Injury subscale. the past year using a 7-point scale, with the following response options: 0 = never, 1 = once, 2 = twice, 3 = 3 to 5 times, 4 = 6 to 10 times, 5 = 11 to 20 times, and 6 = more than 20 times. The total scores for each subscale were calculated by adding up the midpoints of the response categories for each item (e.g., the midpoint for the 6 to 10 times response was 8). In this study, the Cronbach’s alpha coefficient was .92 for the Physical Assault subscale, .88 for the Psychological Aggression subscale, .89 for the Sexual Coercion subscale, and .88 for the Injury subscale. The long form of the BDI is composed of 21 questions or items, each with four possible responses. Each response is assigned a score ranging from zero to three (0, 1, 2, 3), indicating the severity of the symptom. Individual questions of the BDI -2 assess mood, pessimism, sense of failure, self-dissatisfaction, guilt, punishment, self-dislike, self-accusation, suicidal ideas, crying, irritability, social withdrawal, body image, work difficulties, insomnia, fatigue, appetite, weight loss, bodily preoccupation, and loss of libido. Items 1 to 13 assess symptoms that are psychological in nature, while items 14 to 21 assess more physical symptoms. The scores of the BDI-2 were, where a score ‹ 20 = no depression, 21- 31= mild depression, 32-41 = moderate depression, and 42 and above = severe depression. The Arabic version of the scale was used in the current study [39]. We calculated the reliability of the Beck Depression Inventory by using alpha Chronbach = 0.77. 2.1 Participants The study sample included 622 Palestinian women randomly selected from the five areas of 4 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 2.2.3 Posttraumatic stress disorder checklist [35] This checklist contains 17 items adapted from the DSM-IV PTSD symptom criteria. The 17 PTSD symptoms are rated by the participant for the previous month on a scale indicating the degree to which the respondent was bothered by a particular symptom from 1 (not at all) to 5 (extremely). Items can be categorized as follows: items 1-4, 17 are for criteria B (intrusive re- experiencing); items 5-11 are for criteria C) avoidance and numbness); and items 12-16 are for criteria D (hyperarousal). Respondents are asked to rate on a 5-point Likert scale (1 = not at all to 5 = extremely) the extent to which symptoms troubled them in the previous month. Using the recommended Posttraumatic Stress Disorder Checklist cutoff score of 50, Blanchard found cut of point of 44 [35]. Previous research has suggested using as a minimum sum either a score of 3 or 4 on a symptom for it to count as positive towards the diagnosis. This scale was used in previous studies and showed high reliability and validity [36]. In this study, the Cronbach’s alpha coefficient was high and acceptable .91. 2.2.4 The taylor manifestation anxiety scale (TMAS) [37] Taylor developed one of the first measures of chronic, manifest anxiety, Taylor’s Manifest Anxiety Scale (MAS). Taylor’s scale consisted of items selected from the Minnesota Multiphase Personality Inventory. We used the Arabic version with 50 items and answer is "Yes= 1" or "No= 0. The score ranged from (0- 16 no anxiety), (17- 20 Mild anxiety), (21- 26 moderate anxiety), and (27- 29 severe anxiety), and (30-50 very severe anxiety). 30 In this study, the Cronbach’s alpha coefficient was high and acceptable .79. 3.1 Sociodemographic Characteristics The selected sample included 622 Palestinian women. Mean age was 30.61 years (SD = 8.9). Most of women were married (64.1%). 22% were single, 8.8% were divorced and 5% were widowed. Almost 40% of women had finished secondary education, 10.8% had completed a diploma, and 20.7% had university degrees. The 5 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 3.2 Means, Standard Deviation, and Percentage of Domestic Violence majority (60.6%) lived in cities, 24.4% lived in refugee camps, and 15% lived in villages. For those women who were married, 68% of their husbands had finished secondary education. 11.8% had completed a high diploma, and 19.4% had university degrees. Eighty-eight percent of women were housewives, 2.4% were simple workers, and 9.3% were employees. Nearly 45% of women had 8 or more children, 33. 9% had 5-7 children, and 21.1% had 4 and less children. Almost two-thirds of the women were living on an average monthly family income of less than 270 US, 22.8% on less than $450 US and 11.6% were earning $451 or more (see Table 1). The study showed that psychological assault was 56.91%, physical assault 37.3%, physical injury was 12.06%, and sexual assault was 7.14% (see Table 2). 3.2.1 Sociodemographic variables and domestic violence In order to investigate the differences in violence and socioeconomic variables such as age, type of residence, education, place of residence, marital status, number of children, and monthly family income, a One way ANOVA test was performed in which each of the demographic variable was entered as independent variables and means of violence including subscales as dependent variable. In order to investigate the differences in violence and socioeconomic variables such as age, type of residence, education, place of residence, marital status, number of children, and monthly family income, a One way ANOVA test was performed in which each of the demographic variable was entered as independent variables and means of violence including subscales as dependent variable. Table 1. Sociodemographic characteristics of the study sample (N = 662) Table 1. Sociodemographic characteristics of the study sample (N = 662) Mean age of women in years Mean age =30.61, SD = 8.9 Marital status No % Single 137 22.0 Married 399 64.1 Divorced 55 8.8 Widowed 31 5.0 Type of residence City 377 60.6 Camp 152 24.4 Village 93 15.0 Women education Illiterate 15 2.4 Elementary 51 8.2 Primary 123 19.8 Secondary 233 37.5 Diploma 67 10.8 University 129 20.7 High diploma 3 .5 High education 1 .2 Place of residence North Gaza 134 21.5 Gaza 199 32.0 Middle area 95 15.3 Khan Younis 118 19.0 Rafah 76 12.2 Women job House wife 549 88.3 Simple worker 15 2.4 Employee 58 9.3 No of children 4 children and less 131 21.1 5-7 children 211 33.9 8 and more children 280 45.0 Family monthly income Less than US$ 270 408 65.6 US$ 271- 450 142 22.8 US$ 451-700 53 8.5 More than US$ 701 19 3.1 Age was recorded in three categories (18-29 years, 30-39 years, and 40-50 years). There were no statistically significant differences in exposure to all subscales of domestic violence between these age groups, or according to monthly family income, educational level of participants and occupational status. According to place of residence (village, city, and camp), post hoc tests revealed that total domestic violence was significantly higher in women living in villages than in cities or camps that women living in villages experienced more psychological abuse than women living in cities or camps (F= 4.56, p = 0.01) Table 2. 3.5 Depression in Women Depressive symptoms reported ranged from 0, which equated to no symptoms, to 57 symptoms, with a mean depression score of 18.50 (SD = 11.57). Using the established cutoff score on the BDI-II (Gareeb, 2000) where a score ‹ 20 = no depression, 21-31= mild depression, 32-41 = moderate depression, and 42 and 3.2.1 Sociodemographic variables and domestic violence In order to investigate differences in post- traumatic stress disorder scores according to socioeconomic variables such as age, type of residence, education, place of residence, marital status, number of children, and monthly family income, a One way ANOVA test was performed. Each of the demographic variables was entered as the independent variable and the PCL mean scores were entered as dependent variables. Women who were single, divorced, or widowed had higher post-traumatic stress disorder scores than women who were married (F=6.9, p = 0.001). Women living in cities had higher PTSD scores than those living in camps or villages (F= 5.66, p= 0.004). There were no significant differences in levels of post-traumatic stress disorder between groups according to monthly family income or educational level of women. A One way ANOVA test was performed in which each of the demographic variables were entered as independent variables with the mean scores for anxiety as dependent variables. Women who were single were more anxious than women who were married (F= 5.4, p = 0.001). No differences in reported anxiety were established between type of residence, educational level and monthly income. Linear regression analyses were performed to determine predictors of total anxiety scores by types of violence experienced. The following subscale items were found to predict anxiety, I felt physical pain that still hurt the next day because of the fight we had (B = 0.19, p = 0.001), he did something to spite me (B = 0.15, p = 0.004), he accused me of being a lousy lover (B = 0.12, p = 0.01), I stomped out of the room or yard (B = .09, p = 0.04). Linear regression analyses were performed to determine predictors of total post-traumatic stress disorder scores by types of violence experienced. The following subscale items were found to predict post-traumatic stress disorder: He insulted me (B = 0.15, p = 0.002), I passed out from being hit on the head by him (B = 0.15, p = 0.002), I stomped out of the room or yard (B = 0.15, p = 0.001), he pushed or shoved me (B = 0.14, p = 0.001), he did something to spite me (B = 0.14, p = 0.001), he burned or scalded me(B = 0.11, p = 0.01), I needed to go to the doctor because of the fight, but he did not (B = 0.11, p = 0.01). 3.2.1 Sociodemographic variables and domestic violence Means, standard deviation, and percentage of domestic violence Items Mean SD % Psychological assault 16.34 14.90 56.91 Physical assault 15.18 19.74 37.3 Physical injury 5.91 9.60 12.06 Sexual assault 1.5 4.31 7.14 3.3 Post Traumatic Stress Symptoms in Women Our results showed mean score for the post- traumatic stress disorder was 34.33 (SD= 12.62), with subscale mean scores of 9.96 (SD = 4.4) for intrusion, 14.10 (SD = 5.52 for avoidance, and 10.26 (SD = 4.41) for hyperarousal. Using the established cutoff point of 50 on the PCL, 71 women (11.4%) met the criteria for a diagnosis of post-traumatic stress disorder. Table 2. Means, standard deviation, and percentage of domestic violence Table 2. Means, standard deviation, and percentage of domestic violence Items Mean SD % Psychological assault 16.34 14.90 56.91 Physical assault 15.18 19.74 37.3 Physical injury 5.91 9.60 12.06 Sexual assault 1.5 4.31 7.14 3.3 Post Traumatic Stress Symptoms in Women 3.3 Post Traumatic Stress Symptoms in Women Our results showed mean score for the post- traumatic stress disorder was 34.33 (SD= 12.62), with subscale mean scores of 9.96 (SD = 4.4) for intrusion, 14.10 (SD = 5.52 for avoidance, and 10.26 (SD = 4.41) for hyperarousal. Using the established cutoff point of 50 on the PCL, 71 women (11.4%) met the criteria for a diagnosis of post-traumatic stress disorder. 6 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 scores: 15.6% had no anxiety, 11.3 had mild anxiety, 25.9% had moderate anxiety, 17.4% had severe anxiety, and 29.9% had very severe anxiety. In order to investigate differences in post- traumatic stress disorder scores according to socioeconomic variables such as age, type of residence, education, place of residence, marital status, number of children, and monthly family income, a One way ANOVA test was performed. Each of the demographic variables was entered as the independent variable and the PCL mean scores were entered as dependent variables. Women who were single, divorced, or widowed had higher post-traumatic stress disorder scores than women who were married (F=6.9, p = 0.001). Women living in cities had higher PTSD scores than those living in camps or villages (F= 5.66, p= 0.004). There were no significant differences in levels of post-traumatic stress disorder between groups according to monthly family income or educational level of women. R2 = 0.31, Std. error of the estimate = 10.49 3.4 Anxiety in Women The mean score for anxiety was 25.11 (SD = 9.57). Using the established anxiety cut off e 3. Linear regression analysis of total post-traumatic stress disorder and each of the violence items Table 3. Linear regression analysis of total post-traumatic stress disorder and each of the violence items Unstandardized coefficients Standardized coefficients t p 95.0% confidence interval for B B Std. error Beta Lower bound Upper bound He insulted me .69 .22 .15 3.21 .001 .27 1.12 I passed out from being hit on the head by him 1.01 .30 .15 3.41 .001 .43 1.59 I stomped out of the room or yard. .73 .20 .15 3.57 .001 .33 1.13 He pushed or shoved me. .73 .22 .14 3.26 .001 .29 1.17 He did something to spite me. .64 .21 .14 3.07 .001 .23 1.05 He burned or scalded me. -.85 .31 .11 -2.72 .01 -1.47 -.24 I needed to go to the doctor because of the fight, but he did not .74 .28 .11 2.61 .01 .18 1.30 R2 = 0 31 Std error of the estimate = 10 49 7 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 above = severe depression, 57.5% of participants reported no depression, 27.5% had mild, 12.9% had moderate, and 2.1% reported severe depression. items entered as independent variables. The following subscale violence items predicted depressive symptoms in women: Minor psychological aggression: ‘he insulted or swore at me’ (B= 0.15, p = 0.001), ‘he did something to spite me’ (B= 0.19, p = 0.001), I felt physical pain that still hurt the next day because of the fight we had’ (B= 0.16, p = 0.001), someone threatened to have sexual activity against my will (B= 0.11, p = 0.01) and he accused me of being a lousy lover (B= 0.12, p = 0.01). One way ANOVA was performed in which depression total scores were entered as the dependent variable and sociodemographic variables were entered as independent variables. Women who were single reported more depression than those who were divorced, widowed, or married (F=8.5, p = 0.001). No other significant differences were found in depression scores and other sociodemographic variables. R2 = 0.15, Std. error of the estimate= 4.81 3.6 Relationships of Domestic Violence, Women’s Symptoms of Posttraumatic Stress, Anxiety, and Depression In order to investigate which types of violence may predict depression scores, a linear regression analysis was performed. Total depression (BDI-II) scores were entered as the dependent variable and each of the violence Our results showed the direct relationships of domestic violence, women’s symptoms of posttraumatic stress, anxiety, and depression. Table 4. Linear regression analysis of total anxiety and each of the violence items Table 4. Linear regression analysis of total anxiety and each of the violence items Unstandardized coefficients Standardized coefficients t p 95.0% confidence interval for B B Std. error Beta Lower bound Upper bound (Constant) 21.41 0.48 44.40 0.001 20.47 22.36 I felt physical pain that still hurt the next day because of the fight we had 0.68 0.15 0.19 4.41 0.001 0.38 0.98 He did something to spite me. 0.47 0.14 0.15 3.26 0.001 0.18 0.75 He accused me of being a lousy lover. 0.45 0.17 0.12 2.70 0.01 0.12 0.78 I stomped out of the room or yard. 0.31 0.15 0.09 2.10 0.04 0.02 0.60 R2 = 0.19, Std. error of the estimate= 7.81 Table 5. Linear regression analysis of total depression and each of the violence items Unstandardized coefficients Standardized coefficients t p 95.0% confidence interval for B B Std. error Beta Lower bound Upper bound (Constant) 13.15 0.62 21.35 0.001 11.94 14.36 He insulted me 0.61 0.21 0.15 2.94 0.001 0.20 1.02 He did something to spite me. 0.81 0.20 0.19 4.10 0.001 0.42 1.20 I felt physical pain that still hurt the next day because of the fight we had 0.77 0.21 0.16 3.61 0.001 0.35 1.18 Did someone threatened to have sexual activity without your will 0.74 0.28 0.11 -2.70 0.01 1.28 0.20 He accused me of being a lousy lover. 0.60 0.23 0.12 2.66 0.01 0.16 1.05 R2 = 0.15, Std. error of the estimate= 4.81 Table 5. Linear regression analysis of total depression and each of the violence items 8 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 The study showed that psychological assault toward women was positively correlated to and depression (r= .42, p <.01) anxiety (r= .39, p <.01) and posttraumatic stress (r = .52, p <.01) symptoms. Also, there were significant positive relationships between physical assault and depression (r= .30, p <.01), anxiety (r= .31, p <.01), and posttraumatic stress (r = .41, p <.01) symptoms. 3.6 Relationships of Domestic Violence, Women’s Symptoms of Posttraumatic Stress, Anxiety, and Depression Also, there were significant positive relationships between physical injury and depression (r= .28, p <.01), anxiety (r= .33, p <.01) and posttraumatic stress (r = .39, p <.01) symptoms. Sexual assault was positively related to anxiety (r= .20, p <.01) and posttraumatic stress (r = .21, p <.01) symptoms. (53.9%) were exposed to IPV [18]. This study rate of domestic violence is more than found in study of WHO study which showed that 23–49% of women reported physical violence 10–50% reported sexual violence [4]. So, the effect of IPV on society is now viewed not only as a human right issue but also as a public health issue. Similarly, in study of Palestinian women living in refugee camps in Jordon showed that 78% experienced one form or more of intimate- partner violence. Nearly half of the women were victims to either two (24.3%) or three (22.7%) different types of intimate- partner violence. The most-reported type of intimate- partner violence to which the participants were victims was control by the partner (73.7%), followed by economic intimate- partner violence, reported by 53.3%, and emotional intimate-partner violence, reported by 50.3% of the women [40]. 4. DISCUSSION Means, standard deviations and intercorrelations of study variables 9 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 domestic violence survivors. This systems approach should take their needs into account, offer high-quality, timely, and sensitive physical and mental health care, and provide effective referrals to other formal support systems. Until these needs are incorporated into the health care system itself, survivors will continue to suffer the health consequences of violence, despite their universal access to care. Similarly the relationship between the domestic violence and mental health status of women previously reported in the literature, domestic violence has a significant impact on women’s mental health [26,27,28,31,32,42]. Also, in another study researcher found that among a cohort of primarily African American women, those who were both HIV-positive and experienced any IPV over their lifetime were 7.0 times more likely to report problems with depression, 4.9 times more likely to report problems with anxiety, and 12.55 times more likely to have attempted to commit suicide compared to women who did not experience IPV or were HIV-negative [43]. Some investigators found that women who had experienced IPV were over three times more likely than women without a trauma history to experience depression [44]. Furthermore, in their comparison study showed that the severity of symptoms was higher in the IPV depressive and the IPV- depressive/PTSD groups than in both the non- abused and the IPV -no-symptoms groups. Also, results showed that the prevalence of emotional disorders, anxiety, and somatoform disorders was 22.8%, 24.8%, and 16.9%, respectively [45]. Recently, other showed that the majority of women exhibited depressive symptoms (73%), findings have also shown, more severe physical and sexual IPV was found to be associated with depression in this sample of low-income abused women, which is consistent with prior research examining women experiencing IPV [17]. Moreover, in study of 50 women survivors of IPV showed that over half of women were experiencing either PTSD, clinical depression, or both at the time they were interviewed. Thirty six percent had active PTSD, 48% had depression, and 30% had both [46]. This study results were consistent with study of 308 Chinese women survivors of IPV recruited at community setting and at domestic violence shelters showed that psychological abuse was associated with mental health problems (depressive and PTSD symptoms) and also physical health problems (chronic pain) [47]. COMPETING INTERESTS Authors have declared that no competing interests exist. CONSENT All authors hereby declare that study had been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. REFERENCES 1. The United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA). Facts and figures. Available:http://www.unrwa.org/where-we- work/gaza-strip (Accessed 7 July 2015) 1. The United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA). Facts and figures. Available:http://www.unrwa.org/where-we- work/gaza-strip (Accessed 7 July 2015) 2. Etienne GK. World report on violence and health. World Health Organization. Geneva: Swizerland; 2002;1. Available:http://www.who.in/violence- injury-prevention/pdf (Accessed 7 July 2015) 4. DISCUSSION Other programs should be designed to include components aimed at reducing women’s use of avoidance coping. In addition we should focus on interventions that promote problem-focused coping and social support coping enhance self- efficacy and lead to more positive adjustment. Also, interventions should be designed as well to promote use of these more active approaches to coping with relationship conflict. Findings suggest that future research should examine women’s experience other types of violence such as political and community and their resilience and coping with such problem. It would be useful to examine subtypes of using violence and being victimized (i.e., physical, sexual, and psychological) because investigations at this more discrete level may reveal differential patterns of relationships among variables depending on the type of violence. 4. DISCUSSION The present study aimed to assess prevalence of domestic violence and relationship to mental health problems of women including PTSD, anxiety, and depression. As shown in our results Palestinian women in the Gaza Strip exposed to domestic violence which include psychological, physical assault, physical injury, and sexual assault. The study showed that 56.91% reported psychological and 37.3% reported physical assault, 12.06% reported physical injury, and 7.14% reported sexual assault. Our results consistent with previous study which found that 23.3% suffered physical abuse from their husbands, 61.7% psychological abuse, and 10.9% sexual abuse at least once during the year 2005 [12]. Similarly in study of Qatari female students, found that 39% were abused by their brothers, 38% by their fathers, and 22% reported abuse from their mothers [14]. Our findings were consistent with another study of Bangladesh women which more than half of the women This study showed that 11.4% met the criteria for a diagnosis of post-traumatic stress disorder, 47.1% had severe to very severe anxiety, and 15% of women reported moderate to severe depression. The study showed significant and positive relationships between of psychological, physical assault, injury, and sexual abuse and all women's mental health problems including post- traumatic stress disorder, depression, and anxiety. Similarly, others found moderate to severe depression to be a problem among battered women that was predicted by physical abuse coupled with daily stress [22]. Moreover, other study found that prevalence of major depressive disorder (MDD) among women who had endured intimate partner violence was 68 percent for lifetime MDD and 18 percent for current major depressive disorder [41]. Table 6. Means, standard deviations and intercorrelations of study variables M SD 1 2 3 4 5 6 7 8 9 10 1. Psychological assault 16.34 14.90 1 2. Physical assault 15.18 19.75 .69 1.00 3. Physical injury 5.91 9.60 .58 .76 1.00 4. Sexual assault 1.50 4.31 .37 .52 .59 1.00 5. Depression 18.50 11.57 .42 .30 .28 .08 1.00 6. Anxiety 25.14 8.65 .39 .31 .33 .20 .56 1.00 7. PTSD 34.32 12.62 .52 .41 .39 .21 .69 .60 1.00 8. Intrusion subscale 9.96 4.44 .50 .41 .39 .23 .55 .50 .86 1.00 9. Avoidance subscale 14.10 5.52 .43 .32 .31 .15 .64 .52 .90 .65 1.00 10. Arousal subscale 10.26 4.41 .45 .35 .33 .17 .61 .56 .87 .64 .68 1.00 Table 6. 5. CONCLUSION Our study highlights implications for mental health service management of women victims of domestic violence in the Palestinian society. To reduce the risk of adverse family and community violence, care plans should be in place to manage risk and specify action if a crisis does occur. Our findings suggest that a unifying approach overcoming the health issues of work/gaza-strip (Accessed 7 July 2015) 2. Etienne GK. World report on violence and health. World Health Organization. Geneva: Swizerland; 2002;1. Available:http://www.who.in/violence- injury-prevention/pdf (Accessed 7 July 2015) g p ( y ) 2. Etienne GK. World report on violence and health. World Health Organization. Geneva: Swizerland; 2002;1. A il bl h // h i / i l Available:http://www.who.in/violence- injury-prevention/pdf (Accessed 7 July 2015) Available:http://www.who.in/violence- injury-prevention/pdf (Accessed 7 July 2015) 10 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 3. Rodriguez MA, Bauer HM, McLough-lin E, Grumbach K. Screening and intervention for intimate partner abuse. JAMA. 1999; 282:468-474. 7 July 2015) 16. Ministerio DS, Política Social e Igualdad. IV informe anual del observatorio estatal de violencia contra la mujer. IV Annual Report by the National Observatory on Violence against Women; 2011. Available:http://www.msssi.gob.es/ssi/ violencia Genero/ publicaciones/ colecciones/PDFS_COLECCION/libro_14_ IV_ InformeAnual_Ingles. pdf 4. Hussain R, Khan A. Women's perceptions and experiences of sexual violence in marital relationships and its effect on reproductive health. Health Care for Women International. 2008;29:468-483. 5. Haj-Yahia M, de Zoysa R. Rates and psychological effects of exposure to family violence among Sri Lankan university students. Child Abuse & Neglect. 2008; 32(10):994-1002. _ _ g p 17. Illangasekare SL, Burke JG, McDonnell KA, Gielen AC. The impact of intimate partner violence, substance use, and HIV on depressive symptoms among abused low-income urban women. Journal of Interpersonal Violence. 2013;28(14):2831- 2848. ( ) 6. Haj-Yahia M. Wife abuse and battering in the sociocultural context of Arab society Family Process. 2000;39:237-255. 18. Hossain A, Sumi NS, Haque A, Bari B. Consequences of intimate partner violence against women on under-five child mortality in Bangladesh. Journal of Interpersonal Violence. 2014;29(8):1402-1417. 7. Haj-Yahia M. Perceptions of abusive and violent husbands by engaged Arab men in Israel. The Journal of Social Psychology. 1998;138(6):772-786. ( ) 8. Assaf S, Chaban S. Domestic violence against single, never-married women in the occupied Palestinian territory. Violence Against Women. 2013;19(3 ):422-441. 19. ( ) 19. Hou W, Wang H, Chungb H. Domestic violence against women in Taiwan: Their life-threatening situations, post-traumatic responses, and psycho-physiological symptoms. 5. CONCLUSION An interview study. International Journal of Nursing Studies. 2005;42:629- 636. 9. Maziak W, Asfar T. Physical abuse in low- income women in Aleppo, Syria. Health Care for Women International. 2003;24: 313-326. 20. Campbell JC. Health consequences of intimate partner violence. The Lancet. 2002;359(9314):1331-1336. 10. Cwikel J, Lev-Wiesel R, Al-Krenawi A. The physical and psychosocial health of Bedouin Arab women in the Negev region of Israel. Violence against Women. 2003; 9:240-257. ( ) 21. Woods SJ. Prevalence and patterns of post-traumatic stress in abused and post- abused women. Issues in Mental Health Nursing. 2000;21:309-324. World Health Organization. Switzerland. 11. Ministry of Health. Central Organization for Statistics & Information Technology, Ministry of Health Kurdistan, Kurdistan Regional Statistics Office, & WHO Iraq. Iraq family health survey report (IFHS, 2006/7); 2007. Iraq: World Health Organization. 22. Campbell JC, Lewandowski LA. Mental and physical health effects of intimate partner violence on women and children Psychiatric Clinics of North America. 1994; 20(2):353-374. g 12. Palestinian Central Bureau of Statistics. Domestic violence survey main findings: Summary of findings; 2006. Available:www.pcbs.gov.ps/ Portals/_PCBS/ Downloads/ (Accessed 7 July 2015) 23. Zlotnick C, Johnson DM, Kohn R. Intimate partner violence and long-term psychosocial functioning in a national sample of American women. Journal of Interpersonal Violence. 2006; 21:262-275. 13. Almosaed N. Violence against women: A cross-cultural perspective Journal of Muslim minority affairs. 2004;24(1):67-88. 24. Duxbury F. Recognising domestic violence in clinical practice using the diagnoses of posttraumatic stress disorder, depression and low self-esteem. British Journal of General Practice. 2006;294-300. y ( ) 14. Al-Ghanim KA. Violence against women in Qatari society. Journal of Middle East Women’s Studies. 2009;5(1):80-93. 25. Hampton RL, Carrillo R, Kim J. Domestic violence at the margins: Readings on race, class, gender, and culture. New Brunswick, NJ: Rutgers University Press. Domestic violence in African American communities. 15. National research on domestic violence against women in Turkey; 2009. Available:www.hips.hacettepe.edu.tr/eng/... /2008-TDVAW_Main_Report.pd (Accessed 11 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 In B. E. Richie, N. J. Sokoloff, C. Pratt (Eds.). 2005;127-141. partner violence. Journal of Family Violence. 2008;23:577-588. 34. Straus MA, Hamby SL, Boney-McCoy S, Sugarman DB. The revised conflict tactics scales (CTS-2). Journal of Family Issues. 1996;17(3 ):283-316. 26. Beck JG, McNiff J, Clapp JD, Olsen SA, Avery ML, Hagewood JH. Exploring negative emotion in women experiencing intimate partner violence. Shame, guilt, and PTSD behavior therapy. 2011;42:740-750. DOI: 710.1016/j. beth.2011.1004.1001. 35. 5. CONCLUSION Blanchard EB, Alexandr JJ, Buckley TC, Forneris CA. Psychometric properties of the PTSD checklist (PCL). Behaviour Research and Therapy. 1996;34(8):669- 673. 27. Devries K, Watts C, Yoshihama M, Kiss L, Schraiber LB, Deyessa N, WHO. Multi- country study team. Violence against women is strongly associated with suicide attempts: Evidence from the WHO multi- country study on women’s health and domestic violence against women. Social Science and Medicine. 2001;73:79-86. DOI: 10.1016/ j.socscimed. 36. Thabet AA, Abu Tawahina, A, El Sarraj E, Vostanis P. Exposure to war trauma and PTSD among parents and children in the Gaza Strip. European Child & Adolescent Psychiatry. 2008;17:191-199. 37. Taylor JA. Personality scale of manifest anxiety. Journal of Abnormal and Social Psychology.1953;48(2):285-290. 28. Ellsberg M, Jansen H, Heise L, Watts CH, Garcia-Moreno C. Intimate partner violence and women’s physical and mental health in the WHO multi country study on women’s health and domestic violence: An observational study. The Lancet. 2008; 371:1165-1172. 38. Beck AT, Steer RA, Garbin MG. Psychometric properties of the beck depression inventory twenty-five years of evaluation. Clinical Psychology Review. 1988;8:77-100. 39. Abdel-Khalek AM. Internal consistency of an Arabic adaptation of the beck depression inventory in four Arab countries. Psychological Reports. 1998;82:264-266. 29. Golier JA, Yehuda R, Bierer LM, Mitropoulou V, New AS, Schmeidler J. The relationship of borderline personality disorder to posttraumatic stress disorder and traumatic events. American Journal of Psychiatry. 2003;160:2018-2024. 40. Al-Modallal H1, Abuidhail J, Sowan A, Al- Rawashdeh A. Determinants of depressive symptoms in Jordanian working women. J Psychiatr Ment Health Nurs. 2010;17(7): 569-576. DOI: 10.1111/j.1365-2850.2010.01562.x. 30. Heru AM, Stuart GL, Rainey S, Eyre J, Recupero PR. Prevalence and severity of intimate partner violence and associations with family functioning and alcohol abuse in psychiatric inpatients with suicidal intent. Journal of Clinical Psychiatry. 2006;67:23- 29. 41. Stein MB, Kennedy C . Major depressive and post-traumatic stress disorder comorbidity in female victims of intimate partner violence. Journal of Affective Disorders. 2001;66:133-138. 31. La Flair LN, Bradshaw CP, Campbell JC. Intimate partner violence/ abuse and depressive symptoms among female health care workers: Longitudinal findings Women’s Health Issues. 2012;22:53-59. 42. Stuart GL, Moore TM, Gordon KP, Ramsey SE, Kahler CW. Psychopathology in women arrested for domestic violence Journal of Interpersonal Violence. 2006; 21:376-389. 32. Pico-Alfonso MA, Garcia-Linares MI, Celda-Navarro N, Blasco-Ros C, Echeburúa E, Martinez M. _________________________________________________________________________________ © 2015 Thabet et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peer-review history: The peer review history for this paper can be accessed here: http://sciencedomain.org/review-history/10284 5. CONCLUSION The impact of physical, psychological, and sexual intimate male partner violence on women’s mental health: Depressive symptoms, posttraumatic stress disorder, state anxiety, and suicide. Journal of Women’s Health Issues. 2006;15:599-611. DOI: 510.1089/jwh. 43. Gielen A, McDonnell KA, O’Campo PJ, Burke JG. Suicide risk and mental health indicators: Do they differ by abuse and HIV status? Women’s Health Issues. 2005; 15(2):89-95. 44. 44. Bonomi AE, Anderson ML, Reid RJ, Rivara FP, Carrell D, Thompson RS. Medical and psychosocial diagnoses in women with a history of intimate partner violence. Arch Intern Med. 2009;169(18):1692-1697. DOI: 10.1001/archinternmed. 33. Pico-Alfonso MA, Echeburúa E, Martinez M. Personality disorder symptoms in women as a result of chronic intimate male 12 Thabet et al.; BJESBS, 11(2): 1-13, 2015; Article no.BJESBS.18960 45. Savas N, Agridag G. The relationship between women’s mental health and domestic violence in semirural areas: A study in Turkey. Asia-Pacific Journal of Public Health. 2001;23(3):399 -407. intimate partner violence. Social Work in Mental Health. 2013;11:34-54. ( ) 46. Edmond T, Bowland S, Mansoo O. Use of mental health services by survivors of 13
https://openalex.org/W2155112172	https://europepmc.org/articles/pmc3072993?pdf=render	English	null	Asthma Prevalence Associated with Geographical Latitude and Regional Insolation in the United States of America and Australia	PloS one	2,011	cc-by	7,753	Asthma Prevalence Associated with Geographical Latitude and Regional Insolation in the United States of America and Australia Goran Krstic´* Fraser Health, Environmental Health Services, New Westminster, Canada Abstract Competing Interests: The author has declared that no competing interests exist. Competing Interests: The author has declared that no competing interests exist. * E-mail: Goran.Krstic@fraserhealth.ca * E-mail: Goran.Krstic@fraserhealth.ca PLoS ONE \| www.plosone.org Abstract Background: It has been proposed that vitamin D deficiency may be responsible for an increase in the prevalence of allergic diseases and asthma worldwide. Human ability to generate physiologically required quantities of vitamin D through sun exposure is decreasing with increasing geographical latitude. Objectives: Considering that vitamin D deficiency is usually due to lack of outdoor sun exposure, this study is designed to test the hypothesis that a higher prevalence of asthma should be expected at high relative to low geographical latitudes. Methods: Linear regression analyses are performed on asthma prevalence in the U.S. adult population vs. geographical latitude, insolation, air temperature, and air pollution (PM2.5) for 97 major metropolitan/micropolitan statistical areas of the continental United States of America and on general population asthma prevalence vs. geographical latitude in eight metropolitan areas of Australia. Results: A 10u change in geographical latitude from southern to northern regions of the Eastern Seaboard is associated with a 2% increase in adult asthma prevalence (p,0.001). Total insolation in winter months is almost as strong as latitude in its ability to explain the observed spatial variation in the prevalence of asthma (r2 = 0.43; p,0.001). Similar results are obtained using the Australian data (r2 = 0.73; p,0.01), suggesting a consistent association between the latitude/insolation and asthma prevalence worldwide. Conclusions: The results of this study suggest that, as a known modulator of the immune response closely linked with the geographical latitude and erythemal UV irradiation, vitamin D may play an important role in the development/exacerbation of asthma. Citation: Krstic´ G (2011) Asthma Prevalence Associated with Geographical Latitude and Regional Insolation in the United States of A ONE 6(4): e18492. doi:10.1371/journal.pone.0018492 Asthma Prevalence Associated with Geographical Latitude and Regional Insolation in the United States of America and Australia. PLoS 1371/journal.pone.0018492 Editor: Raymond J. Pickles, University of North Carolina at Chapel Hill, United States of America Editor: Raymond J. Pickles, University of North Carolina at Chapel Hill, United States of America Received September 28, 2010; Accepted March 9, 2011; Published April 8, 2011 Copyright: 2011 Goran Krstic´. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The author has no support or funding to report. Competing Interests: The author has declared that no competing interests exist. Asthma – U.S. data The data on asthma prevalence in the U.S. adult population (i.e., age 18 and over) is obtained from the U.S. Department of Health and Human Services (DHHS), Centers for Disease Control and Prevention (CDC) Behavioral Risk Factor Surveillance System [16]. The mean annual asthma prevalence in U.S. ‘‘adults who have been told they currently have asthma’’ for the period from 2006 to 2008 by Metropolitan/Micropolitan Statistical Area (MMSA) are included in this study. The obtained data relates to simple prevalence by MMSA and contains no patient-specific informa- tion. Therefore, there was no need to request an ethics committee approval or a written consent from the patients. All asthma prevalence data used in this study are freely available to download from online resources. The citation recommended by the CDC is used to reference the source of asthma prevalence information for this research paper. Asthma – Australian data The Australian general population asthma prevalence data by Local Government Area (LGA) for the period from 2004 to 2005 are obtained from the publically available online resource of the Australian Public Health Information Development Unit [18]. The studied metropolitan areas of Australia include: Sydney – New South Wales (NSW), Melbourne – Victoria (Vic), Brisbane – Queensland (Qld), Adelaide – South Australia (SA), Perth – Western Australia (WA), Hobart – Tasmania (Tas), Darwin – Northern Territory (NT), and Canberra – Australian Capital Territory (ACT). The prevalence of asthma in the U.S. adult population, as presented in Table 1 and Figure 1, is associated with geographical latitude (r2 = 0.22; p,0.001), annual mean insolation on horizontal surface (r2 = 0.15; p,0.001), and annual regional air temperature (r2 = 0.17; p,0.001). The association of asthma prevalence with the annual mean air pollution as PM2.5 is very weak and not statistically significant (r2 = 0.002; p = 0.66). Although the annual air temperature appears to be a marginally better predictor of asthma prevalence than the annual mean insolation in the studied population, both insolation (r2 = 0.48) and air temperature (r2 = 0.84) are correlated with the geographical latitude (Table 1). Statistical analysis Descriptive statistics and scatter plot analysis revealed no evidence of non-normality in the distribution of asthma preva- lence, geographical coordinates, insolation, air temperature, and air pollution data, allowing the use of linear regression analysis models to evaluate the relationships between the studied variables. Linear regression and correlation analyses are implemented to evaluate the strength, direction, and statistical significance of regression/correlation coefficients. The selected independent variables (i.e., regional horizontal surface insolation, latitude, air temperature, and air pollution) are compared in terms of their ability to explain the observed variation in the dependent/ response variable, the prevalence of asthma in the U.S. adult population, by calculating the coefficients of determination (r2). Linear regression equations are developed and tested for their ability to predict the prevalence of asthma in 97 major metropolitan/micropolitan areas of the continental U.S. and a subset of 39 areas from the Eastern Seaboard in response to meteorology and air pollution at different latitudes. The same approach is applied to test the correlation between the general population asthma prevalence and the geographical latitude in eight metropolitan areas of Australia. Descriptive statistics and scatter plot analysis revealed no evidence of non-normality in the distribution of asthma preva- lence, geographical coordinates, insolation, air temperature, and air pollution data, allowing the use of linear regression analysis models to evaluate the relationships between the studied variables. A continuity of geographical latitude and longitude is considered as important for reducing the influence of statistical outliers and implementing a meaningful regression analysis. Hence, the studied U.S. geographical area includes all continental states with the exception of Alaska. Asthma prevalence data are matched with the data on Air Quality Trends by Core Based Statistical Areas (CBSA), obtained from the U.S. Environmental Protection Agency (EPA) [17]. The resulting 97 matched and collated U.S. wide MMSAs/CBSAs are used in the statistical analyses presented in this paper. Air pollution and meteorological data The air pollution data for fine airborne particulate matter, aerodynamic diameter of less than 2.5 mm (i.e., PM2.5), are obtained from the U.S. EPA Air Trends online information resource [17]. Mean annual PM2.5 concentrations expressed in mg/m3 for the period from 1999 to 2008 by matched CBSAs/ MMSAs are included in this study. The mean annual insolation on horizontal surface and air temperature, expressed in kWh/m2/day and uC, respectively, for the MMSAs/CBSAs in the period from 1983 to 2005 are obtained from the National Aeronautics and Space Administration (NASA) Atmospheric Science Data Center [21]. Central latitudes and longitudes of cities/statistical areas, as presented by the USGS, are used to obtain the data on annual mean insolation and air temperature at specific geographical coordinates and regional elevation. Asthma Associated with Latitude and Insolation sunscreen, and the skin pigment melanin [12]. Higher prevalence of vitamin D deficiency has been found among inner-city African American youth with asthma when compared to non-asthmatic control subjects [13]. Geological Survey (USGS). The geographical coordinates for Australian metropolitan areas are obtained from the Guide to Australia at Charles Sturt University online resource, which is based on the 1996 data from the Australian Bureau of Statistics [20]. An intense annual insolation in the lower latitudes of the southern U.S. regions provides greater potential for the cutaneous vitamin D production in exposed individuals. It is estimated that over 95% of the variability in average daily UV dosages can be explained by the latitude and altitude, where the effect of the latitude on the UV irradiation on the Earth’s surface is much more significant than the altitude. The longitude is not statistically significant in terms of its ability to predict the intensity of the UV irradiance [14]. Considering that vitamin D is suspected to have a role in asthma development [8,15] and that vitamin D deficiency is usually due to lack of outdoor sun exposure [11,12], it is hypothesized in this study that a higher prevalence of asthma should be expected at high relative to low geographical latitudes. Introduction proposed that vitamin D deficiency may be responsible for an increase in the prevalence of allergic diseases and asthma worldwide, as more time is spent indoors with less exposure to sunlight, leading to a decreased cutaneous vitamin D production [8]. Exposure to solar ultraviolet radiation within a wavelength band of 290–315 nm (UV-B) and production of vitamin D in the skin is the primary source of vitamin D for many people [9], particularly for those who do not receive adequate vitamin D doses through diet. Both positive [1,2] and negative associations [3,4] between the prevalence of asthma/allergies and geographical latitude have been reported in the published literature. This is an ecological study designed to test the proposed hypothesis that the prevalence of asthma increases with increasing latitude due to a decreasing intensity of solar irradiation which effectively reduces the individual’s cutaneous generation of vitamin D. Maternal vitamin D deficiency/insufficiency is associated with an increased probability of developing asthma and allergy-related symptoms in early life of their offspring. Camargo, Devereux and colleagues concluded that specifically in the northeastern United States of America, an increased maternal intake of vitamin D from diet or supplements during pregnancy may decrease the risk of wheeze symptoms in early childhood [5,6]. Low levels of serum vitamin D in adults have been associated with impaired lung function, increased airway hyperresponsiveness, and reduced glucocorticoid response in asthma [7]. Litonjua and Weiss Higher rates of emergency department visits for acute allergic reactions have been observed in northeastern when compared to southern regions of the United States [10], suggesting an association with latitudinal difference in insolation. Kimlin and colleagues observed that the available levels of erythemal or vitamin D producing UV-B irradiation decreases dramatically as the latitude increases [11], particularly during the four cooler months (i.e., November to February). The efficiency of vitamin D production in the exposed skin depends on the dose of solar UV-B radiation, which can be curtailed by clothing, excess body fat, PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e18492 1 April 2011 \| Volume 6 \| Issue 4 \| e18492 Asthma Associated with Latitude and Insolation Geographical coordinates data Geographical latitudes and longitudes for the main cities in the MMSAs are obtained using Microsoft Research (MSR) Maps [19]. The geographical information and maps made available online by the MSR are supplied through their partnership with the U.S. The best predictor of asthma prevalence among the studied variables is the geographical latitude with an ability to explain up to 22% of the variation in the prevalence of asthma in the continental U.S. adult population. Based on the linear regression PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e18492 April 2011 \| Volume 6 \| Issue 4 \| e18492 2 Asthma Associated with Latitude and Insolation Table 1. Linear regression estimates of asthma prevalence in US adult population associated with latitude, annual mean insolation, air temperature and air pollution (PM2.5). Table 1. Linear regression estimates of asthma prevalence in US adult population associated with latitude, annual mean insolation, air temperature and air pollution (PM2.5). Table 1. Linear regression estimates of asthma prevalence in US adult population associated with latitude, annual mean insolation, air temperature and air pollution (PM2.5). Linear Regression Coefficient/Variable{ Model 1 - Asthma vs. Latitude Model 2 - Asthma vs. Insolation Model 3 - Asthma vs. Temperature Model 4 - Asthma vs. PM2.5 Model 5 - Insolation vs. Latitude Model 6 - Temperature vs. Latitude Y-axis Intercept 3.7460.90* 12.2260.96* 9.7760.34* 8.1360.52* 7.0860.31* 45.7461.50* Latitude (u) 0.1260.02* — — — 20.0760.01* 20.8760.04* Annual Mean Insolation (kWh/m2/day) — 20.9260.23* — — — — Annual Mean Air Temperature (uC) — — 20.1160.03* — — — Air Pollution (PM2.5) (mg/m3) — — — 0.0260.04 — — Number of Areas 97 97 97 97 97 97 Coefficient of Determination (r2) 0.22 0.15 0.17 0.002 0.48 0.84 p,0.001. {Plus-minus values are linear regression coefficients and standard errors (i.e., 6SE). doi:10.1371/journal.pone.0018492.t001 p,0.001. {Plus-minus values are linear regression coefficients and standard errors (i.e., 6SE). doi:10.1371/journal.pone.0018492.t001 p,0.001. {Plus-minus values are linear regression coefficients and standard errors (i.e., 6SE). doi:10.1371/journal.pone.0018492.t001 asthma is observed in Detroit, MI at 10.97% (latitude: 42.35u North) and the lowest in Miami, FL at 5.03% (latitude: 25.81u North). analysis, a 10u change in the geographical latitude from southern to northern U.S. regions is associated with a 1.2% increase in the prevalence of adult asthma. The highest prevalence of adult Figure 1. Asthma prevalence vs. Asthma Associated with Latitude and Insolation Bothe the regional insolation on horizontal surface (r2 = 0.21; p,0.001) and air temperature (r2 = 0.16; p,0.001) in winter months (i.e., November to February) are statistically significant predictors of asthma prevalence (Table 2, Figure 1). However, regional insolation during winter appears to be almost as effective as latitude in terms of its ability to predict the prevalence of asthma in the U.S. adult population. When compared to the results of regression analyses using annual mean values, a stronger association between latitude and insolation (r2 = 0.80; p,0.001) is observed for winter months, which is in agreement with the findings of Kimlin and colleagues [11]. metropolitan regions is positive, statistically significant, and in agreement with the results obtained using the U.S. adult asthma prevalence data (Figure 3). The results show that up to 73% of the variation in asthma prevalence in the Australian general population could be explained by the variation in the geographical latitude (r2 = 0.73; p,0.01). A 10u change in the geographical latitude from the North to the South is associated with approximately the same increase in asthma prevalence as presented in Figure 2 for the U.S. Eastern Seaboard region from the South to the North (i.e., ,2%). It is important to observe that latitudes in the southern hemisphere have negative values and that, although the relationship between the absolute latitude and asthma is positive, the correlation coefficient is mathematically negative. The correlation matrix in Table 3 shows that asthma prevalence is best explained by the variation in geographical latitude (r = 0.47) and winter insolation (r = 20.46), closely followed by annual air temperature (r = 20.42). Both the geographical longitude (r = 0.15) and air pollution (r = 0.04) showed weak and not statistically significant correlation with the prevalence of asthma in U.S. adult population. Latitude is best correlated with annual air temperature (r = 20.92), closely followed by winter insolation (r = 20.90). Discussion There are pros as well as cons associated with study designs in epidemiological research. An ecological study design, as presented in this and similar papers, has some known limitations in terms of its ability to provide reliable inferences about the population characteristics at individual level. As discussed by Robinson in 1950, ecological correlations cannot be validly used as substitutes for individual correlations [22]. Using the same principle one could argue that the opposite also applies, where one cannot make valid inferences about large populations and their possible interactions by focusing only on the individuals from those populations. Robinson indicated that the purpose of his paper is to prevent ‘‘the future computation of meaningless correlations’’. However, despite lacking the ability to deal with health risk factors at a small- scale individual level, well designed ecological studies provide meaningful correlations and inferences that are useful when dealing with national and international public health issues and health risk factors at a large-scale population level. In their revisiting of Robinson’s paper, Subramanian et al. [23] concluded that ‘‘… perils are posed by not only ecological fallacy but also individualistic fallacy’’. The regression analysis of a subset of data covering 39 major metropolitan/micropolitan areas in the U.S. regions of the Eastern Seaboard (i.e., New England, Middle Atlantic, and South Atlantic) shows a very strong association between asthma prevalence and winter insolation (r2 = 0.43; p,0.001). A 10u change in the geographical latitude from southern to northern regions of the Eastern Seaboard is associated with a 2% change in adult asthma prevalence (Figure 2). Considering that asthma prevalence estimates are more reliable for the U.S. states and regions with larger population size, having narrower 95% confidence intervals [16], and that the Eastern Seaboard is home to most highly populated U.S. cities, a regression analysis based on the data from this region is expected to be more reliable when compared to the data for all continental U.S. regions. Multiple regression analyses are performed on asthma preva- lence vs. insolation, air temperature, and air pollution in an attempt to identify independent effects of the studied predictors (Table 4). Geographical coordinates data latitude, air pollution (PM2.5), winter insolation, and winter temperature in adult population of 97 major metropolitan/micropolitan areas of continental U.S. doi:10.1371/journal.pone.0018492.g001 Figure 1. Asthma prevalence vs. latitude, air pollution (PM2.5), winter insolation, and winter temperature in adult population of 97 major metropolitan/micropolitan areas of continental U.S. doi:10.1371/journal.pone.0018492.g001 April 2011 \| Volume 6 \| Issue 4 \| e18492 PLoS ONE \| www.plosone.org 3 Asthma Associated with Latitude and Insolation Discussion Air temperature and insolation are the only statistically significant predictors of asthma in the continental U.S., where the air temperature appears to be the best predictor based on total annual data (annual mean air temperature: p,0.01; annual mean insolation: p = 0.17) and the insolation when only winter data is applied (winter mean insolation: p,0.05; winter mean air temperature: p = 0.31). Air pollution is not statistically significant predictor of asthma in any of the models. Although a similar study design was applied by Staples and colleagues [3], this is the first comprehensive study that includes the populations of the continental United States and Australia, covering over 100 metropolitan areas within a substantial latitudinal range from both the northern and the southern hemispheres. It is interesting that Staples and colleagues observed a negative correlation between the prevalence of asthma and latitude, which is in contrast to the results of the study on the basis of more recent data for both the U.S. and Australia as presented in The observed correlation between asthma prevalence and absolute values of geographical latitude for eight Australian Table 2. Linear regression estimates of asthma prevalence in US adult population associated with insolation and air temperature in winter months (November to February). Table 2. Linear regression estimates of asthma prevalence in US adult population associated with insolation and air temperature in winter months (November to February) Table 2. Linear regression estimates of asthma prevalence in US adult population associated with insolation and air temperature in winter months (November to February). Table 2. Linear regression estimates of asthma prevalence in US adult population associated with insolation and air temperature in winter months (November to February). Linear Regression Coefficient/ Variable{ Model 7 - Asthma vs. Insolation Model 8 - Asthma vs. Temperature Model 9 - Insolation vs. Latitude Model 10 - Temperature vs. Latitude Y-axis Intercept 10.7260.48 8.6060.13* 6.6460.21* 48.6862.61* Latitude (u) — — 20.1160.01* 21.1960.07* Winter Mean Insolation (kWh/m2/day) 20.9760.19* — — — Winter Mean Air Temperature (uC) — 20.0860.02* — — Number of Areas 97 97 97 97 Coefficient of Determination (r2) 0.21 0.16 0.80 0.76 *p,0.001. {Plus-minus values are linear regression coefficients and standard errors (i.e., 6SE). doi:10.1371/journal.pone.0018492.t002 April 2011 \| Volume 6 \| Issue 4 \| e18492 PLoS ONE \| www.plosone.org Asthma Associated with Latitude and Insolation Table 3. Discussion Correlation matrix for asthma prevalence in US adult population, latitude, longitude, air pollution (PM2.5), annual insolation, winter insolation, annual air temperature, and winter air temperature. Latitude (6) Longitude (6) Air Pollution (PM2.5) (mg/m3) Annual Insolation (kWh/m2/day) Winter Insolation (kWh/m2/day) Annual Tempera- ture (6C) Winter Tempera- ture (6C) Asthma (%) Latitude (6) 1.00 Longitude (6) 20.04 1.00 Air Pollution (PM2.5) (mg/m3) 20.15 0.43 1.00 Annual Insolation (kWh/m2/day) 20.69 20.54 20.29 1.00 Winter Insolation (kWh/m2/day) 20.90 20.24 20.13 0.91 1.00 Annual Temperature (6C) 20.92 0.13 0.29 0.51 0.75 1.00 Winter Temperature (6C) 20.87 0.04 0.24 0.51 0.73 0.97 1.00 Asthma (%) 0.47 0.15 0.04 20.38 20.46 20.42 20.40 1.00 doi:10.1371/journal.pone.0018492.t003 Table 3. Correlation matrix for asthma prevalence in US adult population, latitude, longitude, air pollution (PM2.5), annual insolation, winter insolation, annual air temperature, and winter air temperature. Table 3. Correlation matrix for asthma prevalence in US adult population, latitude, longitude, air pollution (PM2.5), annual insolation, winter insolation, annual air temperature, and winter air temperature. Table 3. Correlation matrix for asthma prevalence in US adult population, latitude, longitude, air pollution (PM2.5), annual insolation, winter insolation, annual air temperature, and winter air temperature. territories and across all age groups. In addition, asthma prevalence data were based on state/territory while geographical latitudes represented smaller areas of corresponding capital this paper. It should be taken into consideration that Staples et al. (2003) study was based on the 1995 Australian National Health Survey of approximately 54,000 people from all states and this paper. It should be taken into consideration that Staples et al. (2003) study was based on the 1995 Australian National Health Survey of approximately 54,000 people from all states and Figure 2. Asthma prevalence vs. latitude, air pollution (PM2.5), winter insolation, and winter temperature in adult population of 39 major metropolitan/micropolitan areas of the eastern seaboard. doi:10.1371/journal.pone.0018492.g002 Figure 2. Asthma prevalence vs. latitude, air pollution (PM2.5), winter insolation, and winter temperature in adult population of 39 major metropolitan/micropolitan areas of the eastern seaboard. doi:10.1371/journal.pone.0018492.g002 April 2011 \| Volume 6 \| Issue 4 \| e18492 PLoS ONE \| www.plosone.org 5 Asthma Associated with Latitude and Insolation Table 4. Multiple regression estimates of asthma prevalence in US adult population associated with annual and winter mean insolation, annual and winter mean air temperature and air pollution (PM2.5). Multiple Regression Coefficient/Variable{ Model 11 - Asthma vs. Annual Mean Insolation, Temperature, and PM2.5 Model 12 - Asthma vs. Discussion Winter Mean Insolation, Temperature, and PM2.5 Collinearity Statistics Collinearity Statistics Tolerance VIF# Tolerance VIF# Y-axis Intercept 10.8661.38* 10.0161.06* Annual Mean Insolation (kWh/m2/day) 20.4260.30 0.53 1.89 — Annual Mean Air Temperature (uC) 20.1060.03 0.53 1.89 — Winter Mean Insolation (kWh/m2/day) — 20.7260.32* 0.36 2.75 Winter Mean Air Temperature (uC) — 20.0360.03 0.35 2.87 Air Pollution (PM2.5) (mg/m3) 0.0460.05 0.65 1.53 0.0260.04 0.74 1.36 Number of Areas 97 97 Coefficient of Determination (R2) 0.22 0.22 Adjusted R2 0.19 0.20 *p,0.001; p,0.01; *p,0.05. {Plus-minus values are multiple regression coefficients and standard errors (i.e., 6SE). #VIF – Variance Inflation Factor. doi:10.1371/journal.pone.0018492.t004 Table 4. Multiple regression estimates of asthma prevalence in US adult population associated with annual and winter mean insolation, annual and winter mean air temperature and air pollution (PM2.5). Table 4. Multiple regression estimates of asthma prevalence in US adult population associated with annual and winter mean insolation, annual and winter mean air temperature and air pollution (PM2.5). Table 4. Multiple regression estimates of asthma prevalence in US adult population associated with annual and winter mean insolation, annual and winter mean air temperature and air pollution (PM2.5). regions. The authors indicated that the latitude ranges for some states/territories spanned over 10u (up to 19u for Queensland). Yet a single latitude coordinate was applied for each of the states/ territories, not necessarily representing the areas from which the cases of asthma were identified and clearly affecting the accuracy of the observed relationship between the asthma prevalence and geographical latitude in Australia. and found that the prevalence of eczema symptoms was positively associated with latitude. The authors observed a similar associa- tion in both 6–7 and 13–14 years age-groups in Europe and also among 6–7 years old children worldwide, suggesting that ‘‘… latitude and temperature affect the prevalence of eczema only indirectly, due to changes in behaviour and differences in sun exposure’’ (emphasis added). They concluded that ‘‘climate may affect the prevalence of asthma and atopic eczema in children’’. Franco et al. [1] showed a statistically significant positive correlation between geographical latitude and active asthma prevalence in the eight International Study of Asthma and Allergies in Childhood (ISAAC) centres in North-East Brazil. The authors found no relation between the tropical weather and high prevalence of childhood asthma in the studied population. April 2011 \| Volume 6 \| Issue 4 \| e18492 Asthma Associated with Latitude and Insolation Asthma Associated with Latitude and Insolation annual/winter solar irradiation [29,30]. This may result in a potentially severe deficiency if sufficient doses of vitamin D are not obtained through diet and/or supplementation. and asthma prevalence is air temperature and not insolation, one would expect cold weather in winter months to yield a stronger correlation of asthma prevalence with air temperature than insolation. A contrary is observed in this study, where winter insolation is almost as good as geographical latitude in terms of its ability to predict the prevalence of asthma. In addition, the results of multiple regression analyses presented in Table 4 confirm that, based on the data for winter insolation, winter air temperature, and air pollution, insolation is the best predictor of asthma. Different immunologic, genetic and environmental mechanisms are considered in the etiology of asthma and it is proposed that geographical variation in asthma prevalence could be due to gene- by-environment interactions [31]. Although cold air has been associated with worsened respiratory symptoms and exacerbation of asthma, ambient air temperature is expected to be a symptom trigger rather than a causal factor initiating respiratory diseases [32]. Asthma attacks can be triggered by cold air, irritating fumes, or fine airborne particulate matter [33]. Winter insolation is consistently a stronger predictor of asthma prevalence than winter air temperature in both the continental U.S. population and a subset of data for the Eastern Seaboard states. It appears that populations living in northern regions may have wider swings in vitamin D levels due to a significant decrease in vitamin D production during the winter months. When compared to those living in tropical areas, populations in the northern regions have much lower production of vitamin D in summer months which may lead to a weak physiological baseline at the beginning of winter. This condition could then progress into a vitamin D insufficiency or a severe deficiency in winter months, when regional insolation is at its annual minimum, potentially affecting the immune system and increasing the probability of respiratory infection and developing/exacerbating asthma. Vitamin D has been recognized as an important immuno- modulating factor with dendritic cells as its primary targets [34]. Calcitriol (1,25-dihydroxycholecalciferol), the main vitamin D metabolite, inhibits dendritic cell maturation and T-helper1 (Th1) cell differentiation, which has been described as a key mechanism of allergy development [35]. Asthma Associated with Latitude and Insolation The inhibition of Th1 cell differentiation leads to a predominance of Th2 cells, which has been implicated in asthma pathogenesis [36,37]. Wjst and Dold (1999) proposed that nutritional intake of vitamin D for rickets prophylaxis could be responsible for the increase in the prevalence of asthma in developed countries over the last three decades, suggesting that if protective antigen-reactive Th1 memory cells fail to develop the subsequent predominance of Th2 cells can trigger allergic reactions [38]. The results of statistical analyses presented in this paper indicate that the probability for the observed association between latitude/ insolation and asthma prevalence to be simply due to a chance is very small (i.e., p,0.01). In addition, consistent results are obtained using the data from the U.S. and Australia further reducing the probability that this may be just a property of the data distribution and not a biological or a physiological response. Camargo et al. (2011) studied the relationship between cord- blood levels of an active metabolite of vitamin D, 25-hydro- xyvitamin D (25[OH]D), and the risk of respiratory infection, wheezing, and asthma [48]. The authors concluded that cord- blood levels of 25[OH]D are inversely associated with the risk of respiratory infection and childhood wheezing but no association with incident asthma is observed. These results suggest that active metabolites of vitamin D could have an effect on reducing the frequency of respiratory infections, which may lead to a reduction in exacerbation of symptoms in those suffering from asthma. The results of statistical analyses presented in this paper indicate that the probability for the observed association between latitude/ insolation and asthma prevalence to be simply due to a chance is very small (i.e., p,0.01). In addition, consistent results are obtained using the data from the U.S. and Australia further reducing the probability that this may be just a property of the data distribution and not a biological or a physiological response. However, the mechanism of action of vitamin D and its active metabolites is quite complex and not yet fully understood. One should take into consideration that, in addition to the inhibition of dendritic cell maturation, activated vitamin D modulates the immune response through inhibition or enhancement on multiple levels of cell function, such as production of both pro- and anti- inflammatory cytokines, and inhibition of B cell differentiation, proliferation, and antibody secretion [34]. Asthma Associated with Latitude and Insolation Activated vitamin D enhances the development of interleukin-IL-10- and reduces the number of IL-6- and IL-17-secreting cells [39]. High levels of pro- inflammatory IL-6 and IL-17 cytokines [40,41,42] and low levels of an anti-inflammatory cytokine IL-10 have been observed in asthmatic patients [43,44], suggesting that a functional vitamin D insufficiency/deficiency may be responsible for an increased probability of developing asthma. Allergies and asthma could be the result of unbalanced metabolic transformation or inadequate vitamin D receptor (VDR) binding, leading to plasma accumula- tion of active/inactive vitamin D metabolites, predominance of pro-inflammatory cytokines and an impaired immuno-modula- tion. Camargo et al. (2011) studied the relationship between cord- blood levels of an active metabolite of vitamin D, 25-hydro- xyvitamin D (25[OH]D), and the risk of respiratory infection, wheezing, and asthma [48]. The authors concluded that cord- blood levels of 25[OH]D are inversely associated with the risk of respiratory infection and childhood wheezing but no association with incident asthma is observed. These results suggest that active metabolites of vitamin D could have an effect on reducing the frequency of respiratory infections, which may lead to a reduction in exacerbation of symptoms in those suffering from asthma. Allan et al. (2010) conducted a case-control study of vitamin D status and asthma in 160 adults aged between 15 and 80 years, 80 with physician-confirmed mild/moderate asthma and 80 age and gender-matched controls who had a smoking history of ,10 pack- years [49]. The majority of controls (i.e., 70%) were recruited from local surgery units and the remainder through the advertising in local press. The study showed no significant difference in the serum 25-hydroxyvitamin D3 concentrations between cases and controls, and no association between 25-hydroxyvitamin D3 levels and asthma severity or lung function. The authors concluded that this study does not find evidence to support the use of vitamin D as an adjunct to conventional therapy in asthma in adults. However, this study is based on a rather small group of individuals who were confined to a small geographical area with only a snapshot in time for a vitamin D status. Although high doses of vitamin D may not be useful in the treatment of adult asthma as an existing condition, the study by Allan et al. PLoS ONE \| www.plosone.org Discussion In an ecological analysis of geo-climatic variations in the prevalence of current asthma, allergic rhinitis and chronic cough, and phlegm in Italy, Zanolin et al. [4] found a negative correlation between asthma-like symptoms and geographical latitude. Rather than to focus specifically on the diagnosed asthma cases country- wide, the study is based on a random sample of 18,873 subjects with a response rate of 72.7% from different climatic regions, effectively representing less than 0.03% of the Italian total general population. The authors suggested that ‘‘variations in the prevalence of respiratory symptoms according to geo-climatic factors could provide important clues to the knowledge of the aetiology of asthma’’. Weiland et al. [2] studied the association between climate and atopic diseases using the data from 146 ISAAC centres worldwide Figure 3. Asthma prevalence vs. latitude in the population of 8 major metropolitan areas of Australia. doi:10.1371/journal.pone.0018492.g003 A report from the US CDC on The State of Childhood Asthma, United States, 1980–2005 [24] indicates that current asthma prevalence rates among children 0–17 years of age, by state, annual average for the period 2001–2005 are generally higher in the northeast region of the United States. These findings are in agreement with the results and observations for U.S. adult population, by metropolitan/micropolitan statistical area, present- ed in this paper. Ethnic groups with darker skin have higher prevalence of asthma when compared to those with lighter skin pigmentation [13,25,26,27]. Darker skin pigmentation is a form of evolutionary adaptation, providing protection against potentially harmful effects of excessive UV irradiation doses, developed in populations inhabiting tropical regions [28]. Therefore, when compared to natives of higher latitudes with lighter skin pigmentation, natives of tropical regions migrating from lower to higher latitudes may become disadvantaged in terms of their ability to synthesize physiologically required quantities of vitamin D under low levels of Figure 3. Asthma prevalence vs. latitude in the population of 8 major metropolitan areas of Australia. doi:10.1371/journal.pone.0018492.g003 PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e18492 6 References 16. U.S. Department of Health and Human Services (DHHS) - Centers for Disease Control (CDC) and Prevention (2008) Behavioral Risk Factor Surveillance System Survey: Prevalence and Trends Data by selected Metropolitan/ Micropolitan Statistical Areas (MMSA). Atlanta, Georgia (Accessed in May 2010 at http://apps.nccd.cdc.gov/brfss-smart/SelMMSAPrevData.asp). 1. Franco JM, Gurgel R, Sole D, Lu´cia Franc¸a V, Brabin B, et al. (2009) Socio- environmental conditions and geographical variability of asthma prevalence in Northeast Brazil. Allergol Immunopathol (Madr) 37(3): 116–21. 2. Weiland SK, Husing A, Strachan DP, Rzehak P, Pearce N, et al. (2004) Climate and the prevalence of symptoms of asthma, allergic rhinitis, and atopic eczema in children. Occup Environ Med 61: 609–615. 17. U.S. Environmental Protection Agency (2009) Air Trends: Air Quality Statistics by City and County (Accessed in May 2010 at http://epa.gov/air/airtrends/ factbook.html). 3. Staples JA, Ponsonby AL, Lim LLY, McMichael AJ (2003) Ecologic Analysis of Some Immune-Related Disorders, Including Type 1 Diabetes, in Australia: Latitude, Regional Ultraviolet Radiation, and Disease Prevalence. Environ Health Perspect 111: 518–523. 18. Public Health Information Development Unit (2010) A Social Health Atlas of Australia – Local Government Area (LGA) Data (Accessed in June 2010 at http:// www.publichealth.gov.au/data/a-social-health-atlas-of-australia_-2010.html). 4. Zanolin ME, Pattaro C, Corsico A, Bugiani M, Carrozzi L, et al. (2004) The role of climate on the geographic variability of asthma, allergic rhinitis and respiratory symptoms: results from the Italian study of asthma in young adults. Allergy 59(3): 306–14. 19. Microsoft Research (MSR) Maps (Sponsored by U.S. Geological Survey (USGS). Microsoft Bay Area Research Center (Accessed in May 2010 at http://msrmaps.com/advfind.aspx). 20. Charles Sturt University, Guide to Australia – Latitude and Longitude Search, 1996 Census Data from the Australian Bureau of Statistics (Accessed in June 2010 at http://www.csu.edu.au/australia/latlong/index.html). 5. Camargo CA Jr., Rifas-Shiman SL, Litonjua AA, Rich-Edwards JW, Weiss ST, et al. (2007) Maternal intake of vitamin D during pregnancy and risk of recurrent wheeze in children at 3 y of age. Am J Clin Nutr 85: 788–95. 21. National Aeronautics and Space Administration (NASA), Atmospheric Science Data Center, Surface Meteorology and Solar Energy: Interannual Variability (Accessed in May 2010 at http://eosweb.larc.nasa.gov/sse/). 6. Devereux G, Litonjua AA, Turner SW, Craig LCA, McNeill G, et al. (2007) Maternal vitamin D intake during pregnancy and early childhood wheezing. Am J Clin Nutr 85: 853–9. 22. Robinson WS (1950) Ecological correlations and the behavior of individuals. Am Sociol Rev 15(3): 351–357. 7. References Sutherland ER, Goleva E, Jackson LP, Stevens AD, Leung DY (2010) Vitamin D levels, lung function, and steroid response in adult asthma. Am J Respir Crit Care Med 181(7): 699–704. 23. Subramanian SV, Jones K, Kaddour A, Krieger N (2009) Revisiting Robinson: The perils of individualistic and ecologic fallacy. Int J Epidemiol 38: 342–360. 8. Litonjua AA, Weiss ST (2007) Is vitamin D deficiency to blame for the asthma epidemic? J Allergy Clin Immunol 120(5): 1031–5. 24. Akinbami LJ (2006) The State of Childhood Asthma, United States, 1980–2005. Advance data from vital and health statistics No. 381. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Center for Health Statistics (http://www.cdc.gov/nchs/data/ad/ ad381.pdf). 9. Grant WB (2007) Roles of solar UV radiation and vitamin D in human health and how to obtain vitamin D. Expert Rev Dermatol 2(5): 563–577. 10. Rudders SA, Espinola JA, Camargo CA, Jr. (2010) North-south differences in US emergency department visits for acute allergic reactions. Ann Allergy Asthma Immunol 104(5): 413–6. 25. Ginde AA, Espinola JA, Camargo CA Jr. (2008) Improved overall trends but persistent racial disparities in emergency department visits for acute asthma, 1993–2005. J Allergy Clin Immunol 122(2): 313–8. 11. Kimlin MG, Olds WJ, Moore MR (2007) Location and vitamin D synthesis: is the hypothesis validated by geophysical data? J Photochem Photobiol B 86(3): 234–9. 26. Meng YY, Babey SH, Hastert TA, Brown ER (2007) California’s racial and ethnic minorities more adversely affected by asthma. Policy Brief UCLA Cent Health Policy Res PB2007-3: 1–7. 12. Mead MN (2008) Benefits of Sunlight: A Bright Spot for Human Health. Environ Health Perspect 116(4): A161–A167. 27. Grant EN, Lyttle CS, Weiss KB (2000) The relation of socioeconomic factors and racial/ethnic differences in US asthma mortality. Am J Public Health 90(12): 1923–5. 13. Freishtat RJ, Iqbal SF, Pillai DK, Klein CJ, Ryan LM, et al. (2010) High prevalence of vitamin D deficiency among inner-city African American youth with asthma in Washington, DC. J Pediatr 156(6): 948–52. 28. Jablonski NG, Chaplin G (2000) The evolution of human skin coloration. J Hum Evol 39: 57–106. 14. Wang X, Gao W, Davis J, Becky O, George J, et al. (2007) Dependence of erythemally weighted UV radiation on geographical parameters in the United States. Proc SPIE 6679: 667903. 29. Conclusions At 21% for the continental U.S. and up to 43% for the Eastern Seaboard regions, total insolation in winter months is almost as strong as latitude in its ability to explain the observed spatial variation in the prevalence of asthma in the U.S. adult population. Acknowledgments I would like to thank the Anonymous reviewers for their constructive comments and suggested changes, and Dusˇica Krstic´ for the encourage- ment and enthusiastic support. The author had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Author Contributions Analyzed the data: GK. Wrote the paper: GK. Analyzed the data: GK. Wrote the paper: GK. Asthma Associated with Latitude and Insolation Asthma Associated with Latitude and Insolation some unknown effects of insolation and UV radiation cannot be eliminated based on the results of this study. However, in conjunction with other published studies suggesting a possible link between vitamin D and asthma, a significant decrease of vitamin D producing erythemal UV irradiation with an increase in latitude provides a plausible explanation for the observed geographical distribution of asthma prevalence in both the northern and the southern hemispheres. As a modulator of the immune response, vitamin D could have an influence on the frequency and severity of respiratory infections which may lead to exacerbation of symptoms from preexisting asthma. A U.S.-wide comprehensive study on the relationship between plasma vitamin D status and the prevalence of asthma in different age groups (e.g., children age 0–17 years), ethnic groups and geographical regions would provide a better understanding of the role that vitamin D may play in the frequency of respiratory infections, and the development/exacerbation of allergies and asthma. Although such a study may confirm or rule out vitamin D, in absence of other plausible leads in the published literature, it may be difficult to design a specific study to determine if there are other possible effects of insolation and UV radiation that could be associated with asthma. Similar results are obtained using the Australian data, suggesting a consistent association between the latitude/insolation and asthma prevalence worldwide. Taking into consideration confounding factors and possible limitations of an ecological study design, the results presented in this paper suggest that, as a known modulator of the immune response closely linked with geographical latitude and erythemal UV irradiation, vitamin D may play an important role in the development/exacerbation of asthma. Vitamin D is essential for the functional immune system and should be maintained at adequate levels not only in those suffering from asthma but also in the general population. Hence, community-level educational programs on asthma may benefit from including recommenda- tions on adequate diet and vitamin D supplementation to prevent severe deficiencies, particularly among ethnic groups with darker skin pigmentation inhabiting high geographical latitudes. Asthma Associated with Latitude and Insolation does not address possible effects of a long- term vitamin D deficiency/insufficiency, an impaired immuno- modulation, and how these parameters may affect the prevalence of asthma in large populations and over large geographical areas with significantly different levels of annual UV-B insolation. The observed correlation between geographical longitude and annual insolation (r = 20.54) presented in Table 3 is in agreement with the estimates of monthly mean erythemal UV irradiation values for the U.S. and Canada [45], showing an increase of solar irradiation in the direction from northeastern to southwestern regions of North America. Considering that total insolation is almost perfectly correlated with the UV component of the total solar irradiation on the Earth’s surface (r.0.96, [46,47]), the correlation between latitude and total insolation presented in this paper could be used to predict the effect of erythemal UV irradiation on the skin production of vitamin D in the studied population. The results of linear regression analyses performed in this study provide evidence that the geographical latitude can be used to predict the prevalence of asthma in the U.S. adult population. It is interesting that in winter months the association of asthma prevalence with air temperature is weaker than the association with insolation. If the factor associated with geographical latitude Air temperature and confounding factors that could be associated with the geographical latitude, such as socio-economic status, regional diet, demographic structure and ethnic origin, or April 2011 \| Volume 6 \| Issue 4 \| e18492 April 2011 \| Volume 6 \| Issue 4 \| e18492 7 Asthma Associated with Latitude and Insolation 30. Genuis SJ, Schwalfenberg GK, Hiltz MN, Vaselenak SA (2009) Vitamin D status of clinical practice populations at higher latitudes: Analysis and applications. Int J Environ Res Public Health 6: 151–173. 42. Neveu WA, Allard JL, Raymond DM, Bourassa LM, Burns SM, et al. (2010) Elevation of IL-6 in the allergic asthmatic airway is independent of inflammation but associates with loss of central airway function. Respir Res 11: 28. g y p but associates with loss of central airway function. Respir Res 11: 28. 31. Subbarao P, Mandhane PJ, Sears MR (2009) Asthma: epidemiology, etiology and risk factors. CMAJ 181(9): E181–E190. 43. Matsumoto K, Inoue H, Fukuyama S, Tsuda M, Ikegami T, et al. (2004) Decrease of interleukin-10-producing T cells in the peripheral blood of severe unstable atopic asthmatics. Int Arch Allergy Immunol 134(4): 295–302. J ( ) 32. Koskela HO (2007) Cold-air provoked respiratory symptoms: the mechanisms and management. Int J Circumpolar Health 66(2): 91–100. 44. Tomita K, Lim S, Hanazawa T, Usmani O, Stirling R, et al. (2002) Attenuated production of intracellular IL-10 and IL-12 in monocytes from patients with severe asthma. Clin Immunol 102(3): 258–66. 33. George RB, Owens MW (1991) Bronchial asthma. Dis Mon 37(3): 137–96. 34. Cutolo M (2009) Vitamin D and autoimmune rheumatic diseases. Rheumatol- ogy 48: 210–212. 45. Fioletov VE, Kimlin MG, Krotkov N, McArthur LJB, Kerr JB, et al. (2004) UV index climatology over the United States and Canada from ground-based and satellite estimates. J Geophys Res 109: D22308. 35. Wjst M (2006) The vitamin D slant on allergy. Pediatr Allergy Immunol 17(7): 477–83. 36. Bharadwaj AS, Bewtra AK, Agrawal DK (2007) Dendritic cells in allergic airway inflammation. Can J Physiol Pharmacol 85(7): 686–99. 46. Trabea AA, Salem I (2001) Empirical Relationship for Ultraviolet Solar Radiation Over Egypt. Egypt J Sol 24(1): 123–132. 37. Aiba S (2007) Dendritic cells: importance in allergy. Allergol Int 56(3): 201–8. 47. Murillo W, Can˜adab J, Pedro´s G (2003) Correlation between global ultraviolet (290–385 nm) and global irradiation in Valencia and Cordoba (Spain). Renew Energ 28(3): 409–418. 38. Wjst M, Dold S (1999) Genes, factor X, and allergens: what causes allergic diseases? Allergy 54: 757–759. 39. Correale J, Ysrraelit MC, Gaita´n MI (2009) Immunomodulatory effects of vitamin D in multiple sclerosis. Brain 132: 1146–1160. 48. Camargo CA Jr., Ingham T, Wickens K, Thadhani R, Silvers KM, et al. References Hintzpeter B, Scheidt-Nave C, Mu¨ller MJ, Schenk L, Mensink GB (2008) Higher prevalence of vitamin D deficiency is associated with immigrant background among children and adolescents in Germany. J Nutr 138: 1482–1490. 15. Schauber J, Gallo RL (2008) Vitamin D deficiency and asthma: Not a strong link – yet. J Allergy Clin Immunol 121(3): 782–784. PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e18492 April 2011 \| Volume 6 \| Issue 4 \| e18492 8 PLoS ONE \| www.plosone.org Asthma Associated with Latitude and Insolation (2011) Cord-blood 25-Hydroxyvitamin D levels and risk of respiratory infection, wheezing, and asthma. Pediatrics 127(1): e180–e187. 40. Wong CK, Ho CY, Ko FWS, Chan CHS, Ho ASS, et al. (2001) Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin Exp Immunol 125(2): 177–83. 49. Allan K, Devereux G, McNeill G, Wilson A, Avenell A, et al. (2010) A case- control study of vitamin D status and asthma in adults. Proceedings of the Nutrition Society 69(OCE6): E475. ( ) 41. Molet S, Hamid Q, Davoine F, Nutku E, Taha R, et al. (2001) IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. J Allergy Clin Immunol 108(3): 430–8. PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e18492 9
W3186789171.txt	https://link.springer.com/content/pdf/10.1365/s35764-021-00347-6.pdf	de		Best Practices für Marketing-Automation-Einsteiger	Wirtschaftsinformatik & Management	2,021	cc-by	3,111	Schwerpunkt Best Practices für MarketingAutomation-Einsteiger Automatisierte Marketing-Kampagnen zur Leadgenerierung sind schon längst keine Zukunftsmusik mehr, sondern Realität. Über 8000 Softwareprodukte machen die Auswahl eines geeigneten Marketing-Automation-Software-Stacks aber alles andere als leicht. Welches System passt am besten zum eigenen Unternehmen? Welche Funktionen und Integrationen braucht man wirklich? Neueinsteiger, aber auch Unternehmen, die bereits ein System haben und umsteigen möchten, fühlen sich sehr leicht von den zahllosen Möglichkeiten überwältigt. Wie der Start mit Marketing-Automation für B2B trotzdem gelingt, zeigen unsere praxiserprobten Best Practices. Alex Schoepf Wirtschaftsinformatik & Management 2021 • 13 (4): 280–289 https://doi.org/10.1365/s35764-021-00347-6 Angenommen: 11. Juni 2021 Online publiziert: 20. Juli 2021 © Der/die Autor(en) 2021 280 Wirtschaftsinformatik & Management 4 \| 2021 Schwerpunkt Erfolgsentscheidende Vorarbeiten sorgfältig umsetzen Mit einem Marketing-Automation-System sind automatisierte Omnichannel-Kampagnen auch für KMU einfach und effizient umsetzbar. Leider sind Missverständnisse gerade bei Einsteigern aber an der Tagesordnung. Denn allein das Automatisieren von E‑Mails führt längst nicht zu mehr Erfolg. Marketing-Automation ist ein Weg, der sich auch für Inbound Marketing bewährt hat. Dieses lebt davon, dass potenzielle Kunden das Unternehmen durch eigene Recherche finden. Den Ausschlag für Erfolg mit MarketingAutomation geben in aller Regel die Vorarbeiten, und zwar noch vor dem Start der allerersten Kampagne. Natürlich spielen auch technische Fragen eine Rolle beim Einführen eines Marketing-Automation-Systems, wesentlich erfolgsentscheidender ist es jedoch, vorab strategische Fragen zu klären. Unternehmen, die sehr früh wissen, für welche konkreten Aufgaben/Prozesse sie Marketing-Automation einsetzen wollen, können strategische Entscheidungen wohlbegründet treffen. Üblich ist eine Einrichtungsphase von zwei Wochen, bis das Marketing-Automation-System für die erste Kampagne startklar ist. Unsere Best Practices eignen sich sowohl für Einsteiger als auch für erfahrene Nutzer, die mehr aus ihrem System herausholen möchten. Kundenbedürfnisse präzise erfassen Leadgenerierung, mehr Traffic oder einfach zusätzlich Brand Awareness? Die Ziele, die mit Onlinemarketing erreicht werden können, sind ganz verschieden. Genauso muss sich auch automatisiertes Marketing präzise an Kundenbedürfnissen orientieren und passgenau auf die Empfänger zugeschnitten sein. Das traditionelle Konzept der über demografische Daten definierten Zielgruppe ist jedoch vorbei. Heute arbeiten wir mit der Buyer Persona, die wir besser kennen und verstehen, da sie von uns befragt wurde. Dieser idealtypische Wunschkunde repräsentiert die Herausforderungen, Ziele und Träume, beinhaltet aber auch handfeste psychografische Verhaltensdaten. Alex Schoepf () (Ing. ETH/BWI) ist Gründer und CEO der 4results AG und Schweizer Pionier im Bereich Marketing-Automation. Seit vielen Jahren ist Marketing-Automation seine Leidenschaft und er ist ein gefragter Referent zu diesem Thema. Er hat mehr als 60 Marketing-Automation-Systeme evaluiert und mit mehr als 12 selbst gearbeitet. Mit seiner langjährigen Erfahrung im Produktmanagement und in der B2B-Marketingleitung auf Kundenseite versteht er die Praxisherausforderungen. Er ist Autor und doziert MarketingAutomation an verschiedenen Schweizer Fachhochschulen. Mehr Informationen unter: https://www.marketingautomation. tech/ueber. alex@marketingautomation.tech HWZ Hochschule für Wirtschaft Zürich, Zürich, Schweiz 1 Customer Journey definieren Ebenfalls noch vor der ersten Automation feststehen sollte die Customer Journey, also die Kundenreise, der Zielgruppe. Welche Kanäle und ContentFormate nutzen die Zielkunden am liebsten? Welche Kontaktpunkte spielen bei der Kundenreise eine Rolle? Wo können wir sie vereinfachen und Innovationen einfließen lassen? Diese und weitere Fragen zur Reise des Kunden vom ersten Kontakt bis zum glücklichen Abschluss hält das CustomerJourney-Modell fest. Da sich die Interessenten jedoch nicht stereotypisch verhalten und den einen oder anderen Kontaktpunkt überspringen, sind die Lebenszyklusstadien zu definieren. Lässt sich genau bestimmen, in welchem Stadium sich ein Lead befindet, bedeutet dies einen wertvollen Hinweis auf seine Verkaufsreife und die nächstmöglichen optimalen Aktionen. Mit Funnel und Lifecycle Stages arbeiten Noch mehr Präzision erhalten Customer Journey und die zugehörigen Funnels, wenn Unternehmen zusätzlich die Phasen der Kundenreise definieren. Wirtschaftsinformatik & Management 4 \| 2021 281 Schwerpunkt Dazu werden einzelne Abschnitte definiert, die signifikant für die Abschlussreife eines Leads sind. Dies hilft Unternehmen dabei, die Kaufbereitschaft genau abzupassen und nicht zu früh in den direkten Kontakt zu gehen. Welche Phasen bzw. Lifecycle Stages die Personas eines Unternehmens aufweisen, hängt stark von der Leistung bzw. dem Produkt ab. Branchenabhängig kann ein Kontakt beispielsweise als Newsletterabonnent starten, dann zum Lead werden und sich folgend zum qualifizierten Lead für Marketing oder den Vertrieb weiterentwickeln. Erster Sales-Kontakt ist dann erst ab der darauf folgenden Opportunityphase sinnvoll. Aktiver Kunde ist der Lead erst nach dem erfolgreichen Abschluss (Abb. 1). Mit individuellem Marketing bzw. Sales Funnel entwerfen Unternehmen ein Modell, das potenzielle Kunden von der Leadgenerierung bis zum Abschluss leitet. Entlang des Funnel ist reichlich Platz und Gelegenheit für Automationen, welche den Lead in jeder Phase begleiten. Gemeinsam mit den Buyer Personas bildet die individuelle Customer Journey die Basis für eine kundenzentrierte Content-Entwicklung, aber auch Automationen. Evergreen-Inhalte vorbereiten Wer bereits Buyer Personas und zugehörige Customer Journeys erstellt hat, besitzt ein gutes Gefühl für Themen und Fragen, die bei den Zielkunden hoch im Kurs stehen. Auch für den Einsatz mit Marketing-Automation sind gute Inhalte, die reichlich Mehrwert bieten und zur Lifecycle Stage passen, unverzichtbar. Evergreen Content sollte zudem dem Verhalten des Leads entgegenkommen, um möglichst einfach konsumierbar zu bleiben. Mit Marketing-Automation sind viele Formate zur Bespielung der unterschiedlichsten Kanäle möglich, da es sich um ein Omnichannel-Tool handelt: Landingpages, E‑Mails, Fachartikel, Webinare, E‑Books und viele weitere ziehen potenzielle Kunden an. Bereits vor der Implementierung über einen Grundstock an Evergreen-Inhalten zu verfügen, ist vorteilhaft. So kann gleich nach der Installation mit dem Aufbau erster Automationen begonnen werden. Abb. 1 Marketing und Sales Funnel. (Quelle: marketingautomation.tech) MARKETING Funnel SALES Funnel GENERIEREN TRAFFIC ERFASSEN A 282 KOMMUNIZIEREN ANGEBOT B FOLLOW-UP KREIEREN ABSCHLUSS C LIEFERUNG D Wirtschaftsinformatik & Management 4 \| 2021 Schwerpunkt Individualisiertes Lead Scoring nutzen Noch mehr machen Unternehmen aus ihren Leads mit Lead Scoring. Dabei handelt es sich um ein individualisiertes Punktesystem, welches das Verhalten eines jeden Leads misst. Jeder Lead, der aufgrund seiner Merkmale das Potenzial besitzt, Kunde zu werden, gilt als Marketing qualifizierter Lead (MQL). Verhaltensbasiert bekommt der Lead eine zuvor festgelegte Punktzahl für das Ausführen der verschiedensten Aktionen zugewiesen. Über den Besuch bestimmter Webseiten, das Öffnen von E‑Mails, Whitepaper-Downloads und weitere Aktionen sammelt der Lead Punkte bis zu einer festgelegten Schwelle, welche die Abschlussreife markiert. Leads, welche diesen Wert erreichen, werden dann an den Verkauf weitergegeben, der die Qualifizierung überprüft und feststellt, ob es sich um einen Sales qualifizierten Lead (SQL) handelt. Ist dies der Fall, wird der Lead zu einem Sales Accepted Lead (SAL), einem vom Verkauf akzeptierten Lead. Sowohl die Punktzahl für Aktionen und Verhalten als auch die Schwelle zur Abschlussreife hängen stark vom Unternehmen und dem vertriebenen Produkt ab. Am besten startet man deswegen mit einem einfachen Basismodell, das individuell angepasst wird. Mit weiteren Optimierungen und dem Einsatz von künstlicher Intelligenz und Maschinenlernen (KI/ML) entsteht mit der Zeit ein ausgereiftes Lead Scoring, das Unternehmen zuverlässig „heiße“ Leads anzeigen kann. Zusammenfassung • Marketing-Automation unterstützt kleine und mittlere Unternehmen (KMU) beim Umsetzen automatisierter Omnichannel-Kampagnen. • Optimal eingestellte Marketing-Automation sichert die Wettbewerbsfähigkeit des Unternehmens und beschleunigt die digitale Transformation. • Eine individuelle personalisierte Ansprache bei automatisierten Kampagnen erhöht nicht nur die Konversionsrate, sondern auch die Kundenzentrierung – und das rund um die Uhr. Künstliche Intelligenz (KI) und Maschinenlernen (ML) für mehr Präzision Künstliche Intelligenz und Maschinenlernen sind heute schon fester Bestandteil von Marketing-Automation-Plattformen. Diese Unterstützungssysteme versetzen die Marketing-Automation-Software in die Lage, selbst Probleme zu lösen, die für uns Menschen schwierig sind. Im Verarbeiten der Analytikdaten, aber auch bei der Beurteilung der Leadqualität, dem intelligenten Segmentieren, Lead Scoring, Abschlussvorhersagen, Bestimmung des optimalen Versandzeitpunkts, dem Empfehlen von Produkten und passenden Inhalten sind KI-Anwendungen für Marketing-Automation heute nicht mehr wegzudenken. Segmentierung Leads können sehr unterschiedliche Bedürfnisse haben. Damit jeder Lead möglichst die für ihn am besten geeigneten Informationen bekommt und eine treffende Personalisierung möglich ist, gilt Segmentierung als wichtiger Bestandteil der Kampagnenvorbereitung. Zielgenau werden Segmentierungen, wenn maximal sechs Kriterien definiert werden, beispielsweise Persona, Branche, aber natürlich auch Produktinteresse, Position im Sales Funnel und weitere. Welche Merkmale dabei wichtig sind, hängt nicht zuletzt vom Unternehmen und den Produkten oder Dienstleistungen ab. Marketing-Automation kann Segmentierungen auch selbst erledigen und Leads auf Basis der identifizierten Merkmale in die entsprechend geeigneten Kampagnen überführen. Nutzersegmente lassen sich bereits vor der Implementierung festlegen und dann gleich bei der Installation hinzufügen. Wirtschaftsinformatik & Management 4 \| 2021 283 Schwerpunkt Tiefe Integration ausgewählter Systeme Kernthese 1 Marketing-AutomationSysteme eignen sich wegen der einfachen Bedienung und geringen Kosten auch für KMU. Das Einführen von Marketing-Automation hat eher weniger den Charakter eines großen IT-Projekts, da der Großteil der Produkte als SaaS-Lösung genutzt wird. Das Anschaffen neuer Hardware oder großer Server bleibt Unternehmen daher erspart. Essenziell ist in der Praxis jedoch die reibungslose Kommunikation der Marketing-Automation-Lösung mit allen anderen beteiligten Softwaresystemen des Unternehmens, wie beispielsweise dem CRM/ERP oder mit der Webinarsoftware und der Terminbuchung. Eine umfangreiche eigene Schnittstellenprogrammierung ist aber auch hier bei vielen Produkten wiederum nicht notwendig, da sie über eigene App-Marktplätze Schnittstellen als Plug-ins zur Verfügung stellen. Diese werden nach dem Plug-Play-Prinzip installiert und erfordern keinen Programmieraufwand. Der Testaufwand bleibt aber. Es mag natürlich reizen, möglichst viele Systeme zu integrieren, um eine maßgeschneiderte Lösung zu erhalten. In der Praxis hat es sich jedoch gezeigt, dass es besser ist, sich Zeit zu lassen und auf wenige, sorgfältig ausgewählte Systeme zu setzen, die dann tief integriert werden und zuverlässig funktionieren. Die Komplexität erhöht sich mit jeder weiteren Integration und macht dadurch das Gesamtsystem fehleranfälliger. Die obige Grafik zeigt, welche Unterschiede in der Tiefe der Integration bestehen können, vom Synchronisieren einfacher Listen bis zum Starten von Automationen aus einer anderen Software wie z. B. dem CRM. Tiefe Integrationen bedeuten oft, dass ein Teil eines Fensters von einer anderen Software ist (Abb. 2). Einfach starten und langsam aufbauen Das Spektrum an Einsatzbereichen für Marketing-Automation ist sehr breit [1] und einfache erste Schritte sind ideal. Die verschiedenen Punkte der Marketing-Automation-Best-Practice veranschaulicht die obige Grafik und ein agiles Vorgehen ist empfehlenswert. Diejenigen Elemente, die funktionieren, werden beibehalten, alles, was noch Nachbesserung benötigt, erhält Abb. 2 284 Integration von Leadgenerierungssoftware. (Quelle: marketingautomation.tech) TAGS, CUSTOM FIELDS LISTS, TAGS, CUSTOM FIELDS ALLE 3 CUSTOM FIELDS, LIST, TAGS, OR FORM # INTEGRATIONSNIVEAU SUBSCRIBE TO A LIST 1 Schwach √ 2 Minimal v v 3 Guter Standard v v v 4 Besser v v v v 5 Am besten v v v v ADD TO AUTOMATION/ DEAL v Wirtschaftsinformatik & Management 4 \| 2021 Schwerpunkt Schritt für Schritt weitere Ausarbeitung. Die reine Implementierung ist ein Sprint, das Nachoptimieren bis zur bestmöglichen Performance eher ein Marathon. Dafür sind regelmäßige Gesundheitschecks mit einem Experten zu empfehlen (Abb. 3). Welches ist die ideale erste Automation? Die wenigsten Leads sind sofort kaufbereit. Auch wenn dies stark von Branche und Produkt bzw. Dienstleistung abhängt, ist in der Mehrzahl der Fälle Vorarbeit notwendig, bis der Kaufentscheid ansteht. Unternehmen, die sich einen möglichst schnellen Erfolg mit Marketing-Automation wünschen, sollten dies in Betracht ziehen, und möglichst weit oben in der Customer Journey mit der ersten Automatisierung beginnen. Leads, die bereits kurz vor der Entscheidung stehen, lassen sich schneller gewinnen. Beginnt ein Unternehmen mit ersten Automatisierungen in der Awarenessphase, in der sich potenzielle Kunden erst ihrer Probleme und möglicher Lösungswege bewusst werden, verliert es viel Zeit und letzten Endes auch Leads. Wie geht es nach der ersten Automation weiter? Egal für welchen Einstieg sich ein Unternehmen für seine Reise mit Marketing-Automation entscheidet – Optimierungen gehören stets dazu. Erreicht Abb. 3 Stufenweises Aufbauen von personalisierter Marketing-Automation. (Quelle: marketingautomation.tech in Anlehnung an smartinsights) Kontrolliert Beginner Definiert Beginn Automa sierung gezielte E-Mails Reguläre Reports für Öffnungsrate und Klicks Mehr als einfaches Klicktracking von Marketingaktivitäten SegmentTracking Quan fiziert Beginn Integra on Op miert Integriertes Lebenszyklus Targe ng Wertberichterstattung Hindernisrate und Aktivitätsengagement Levels. Antwortdaten > CRMSystem/ Datenbank E-Mail-Funktionen & Tracking Einfaches Antworten-Tracking Qualitätsliste Nicht steuerbar Optionen für Listenerstellung Einhaltung des Datenschutzgesetzes Verbesserte Listenqualität Reaktivierungs- & Entfernungsmöglichkeiten Präferenzzentrum Targetierung (Relevanz) Keine: Massenmailing Demografisch Einfache TriggerMöglichkeiten Komplette LifecycleSegmentierung Lead Scoring & Bewertung Zusätzliche Sequenzen hinzufügen Angebote & Kommunikationsstrategie Newsletter und E-Mail-Kampagne Marketing-Automation (z.B. Willkommens-E-Mail) Mehr Automationen (z.B. automatisierte Kundenpflege) Komplette LifecycleAutomation (z.B. E-MailReaktivierung) Verbesserte Automationen und Frequentierung Inhalte, Formate & Vorlagen Einfache Headers Verschiedene Templates Dynamische ContentAnpassung Video-Vorschau Mobile Optimierungen Fortgeschrittener EchtzeitContent (z.B. Wetter, Ort) Zustellung Nicht überprüfbar Report Report mit ESP Feedbackschlaufen und Zustellservice Kontinuierliche Überwachung A/B-Tests mit verschiedenen Betreffzeilen/ Angeboten Strukturiertes Angebot/ Betreffzeilen Testing Layout-Vorschau A/B-Testing Multivariates Testing Einsatz von künstlicher Intelligenz und maschinellem Lernen Optimierungen Keine Wirtschaftsinformatik & Management 4 \| 2021 285 Schwerpunkt die erste Kampagne ihr Ziel nicht sofort, ist dies jedoch kein Grund, sie zu stoppen oder gleich die nächste, komplett neu konzipierte Kampagne aufzusetzen. Erfolg versprechender ist es, bestehende Kampagnen anhand von Analytics-Daten weiter zu optimieren und nicht jedes Mal das Rad neu zu erfinden. Auch hier sind gute Vorarbeiten wichtig. Für jede Buyer Persona sollte ein Szenario in Form eines stringenten Prozesses entworfen werden, der dann anhand der gewonnenen Daten immer weiter optimiert werden kann. So lassen sich die Prozesse für Leadgenerierung, aber auch das komplette Leadmanagement und die dahinterstehenden Funnels gezielt optimieren. Eine geringere Anzahl an Einzelkampagnen, die dafür stets nachoptimiert werden, ist wesentlich Erfolg versprechender, als unüberschaubar viele Kampagnen nebeneinander herlaufen zu lassen. Experimente für mehr Conversions Kernthese 2 Strategieplanung und Vorbereitung sind wichtige Faktoren für eine erfolgreiche Automation. Testen sollten Unternehmen bereits in der Evaluationsphase der MarketingAutomation-Software mit dem Demozugang. Dazu gehören Vorlagen für die verschiedenen E‑Mail-Programme und Variantentests mit verschiedenen Anreden, Betreffzeilen und grafischen Elementen. Einsteiger treffen mit einfachen ersten Automationen eine gute Entscheidung. In solchen ersten Kampagnen können alle beteiligten Mitarbeiter Erfahrungen mit der Software und ihrer Funktionsweise sammeln. Mit Experimenten bessere Conversions zu erzielen, ist mit Marketing-Automation gut möglich. Hier ist eine positive Kultur notwendig, welche die Teams im Ausprobieren bestärkt und auf diese Weise innovative Wege entwickelt, denn Marketing-Automation-Software vereinfacht das Austesten der verschiedensten Varianten beträchtlich. Gut dokumentieren Damit der Überblick in der Fülle an Möglichkeiten und Tests nicht verloren geht, ist sauberes Dokumentieren der verschiedenen Aspekte (Mitteilungen/Journey/Zeit/Engagement/Systeme) wichtig. Gebündelt in einer einzigen Dokumentation bleiben Änderungen und Einstellungen auch über längere Zeit nachvollziehbar. Auch für neu hinzukommende Teammitglieder ist das Einarbeiten dann wesentlich einfacher. Nutzt man einheitliche Namenskonventionen für die Arbeit mit der Automatisierung, bleibt die Orientierung in den Kampagnen auch langfristig erhalten. Umgang mit Daten – ein sensibles Thema Marketing-Automation ist in besonders hohem Maß eine datenbasierte Marketingmethode, bei der die Nutzerdaten quasi der „Treibstoff “ sind. Diese Softwaresysteme sind dazu in der Lage, selbst Daten zu sammeln und diese dann wiederum als Kampagnenauslöser und zur Personalisierung zu nutzen. Umso wichtiger ist eine exzellente und aktuelle Datenqualität für den Erfolg automatisierter Kampagnen. Ohne eine konkrete Datenmanagementstrategie ist dies kaum im erforderlichen Ausmaß erreichbar. 286 Wirtschaftsinformatik & Management 4 \| 2021 Schwerpunkt Reportings und Analytik Der Erfolg einer Werbemaßnahme, ganz gleich ob automatisiert oder nicht, wird über Analytikdaten ermittelt. Weil Daten und deren Analyse das Herz erfolgreicher Marketing-Automation ausmachen, kommen die meisten Systeme bereits mit einer eigenen Analytikfunktion. Weitere Analysetools stellen jedoch unter Umständen eine wichtige Ergänzung dar und liefern in der Kombination miteinander ein noch besseres Bild der Performance. Ideal ist ein Dashboard, welches die Werte aus mehreren Datenquellen anzeigt. Dies schafft einen wirklich ganzheitlichen Überblick über alle Marketingmaßnahmen und erlaubt noch mehr Kundenzentriertheit. Da Analytiksoftware aber sehr viele Werte ausliefern kann, stehen Unternehmen vor der Entscheidung, die wichtigsten Merkmale zu bestimmen. Zahlreiche Systeme arbeiten heute bereits mit KI und Maschinenlernen, um die gewonnenen Daten aufzubereiten. Der Übersichtlichkeit halber sollten zum regelmäßigen Monitoring aber trotzdem nur die wichtigsten Werte herangezogen werden. Auf individualisierten Dashboards liefern sie dann den idealen Überblick über Kampagnenperformance und weitere Vorgänge. Auch hier ist viel Vorarbeit im Unternehmen möglich, beispielsweise in Form einer Liste, welche die wichtigsten zu analysierenden Werte zusammenstellt. Diese bilden dann eine gute Grundlage, um Optimierungspotenziale von Kampagnen zu identifizieren und entsprechende Optimierungsmaßnahmen zu schaffen, denn ohne Daten sind keine zielgerichteten Optimierungen möglich. Kernthese 3 Best Practices geben erste Orientierung für den Einstieg. Know-how beschaffen Startet ein Unternehmen zum ersten Mal mit Marketing-Automation sind möglicherweise auch grundlegende Funktionen nicht bekannt. Qualifizierte Fachkräfte aus dem Bereich Marketing-Automation einzustellen, wäre ideal, dies ist aber nicht immer möglich. Deswegen sind die Trainingsprogramme des Herstellers ein Faktor, der auch bei der Systemwahl eine Rolle spielen sollte. Sind die Trainings eher als einmaliges Onboarding gedacht oder gibt es eine umfassende Reihe an Webinaren in einer Akademie mit einem größeren User-Forum? Die gute Erreichbarkeit und Schnelligkeit des Supports zu üblichen Geschäftszeiten gilt ebenfalls als wichtiger Faktor. Gerade in der Startphase treten unvorhergesehene Fragen auf, die eine schnelle Beantwortung verlangen. Schlecht erreichbarer Support hat dann das Potenzial, den gesamten Zeitplan durcheinanderzubringen. Einsteiger profitieren bei der Vorbereitung und Durchführung der ersten Kampagne in aller Regel von externen Experten. Sie helfen beim Verstehen und Einbinden der Software in die komplette Unternehmensinfrastruktur, kennen Funktionen und die Auswirkungen sehr spezifischer Einstellungen. Zudem können sie Mitarbeiter exakt auf das eingesetzte Produkt schulen und unterstützen. Erste Schritte mit einer Investition in die Zukunft Ganz ohne Frage sind die ersten Schritte mit Marketing-Automation herausfordernd und selten erleben Einsteiger sofort nach dem Start der ersten Wirtschaftsinformatik & Management 4 \| 2021 287 Schwerpunkt Handlungsempfehlung • Frühzeitige Strategie- und Content- planung macht das Tool schnell nutzbar. • Umfangreiche Tests und lückenlose Dokumentationen helfen, Marketing-Automation schnell zu verstehen und die Leistung zu steigern. • Externe Unterstützung hilft nicht nur bei der Systemwahl und der ersten Kampagne, sondern auch bei späteren Healthchecks. Kampagne eine Welle des Erfolgs. Die hier dargestellten Best Practices geben jedoch den Weg vor, der notwendig ist, um längerfristig mit dieser Marketingmethode erfolgreich zu sein. Auch wenn die ersten Gehversuche an viel Vorbereitung gekoppelt sind, rechtfertigen mittel- bis langfristig die exzellenten Resultate diesen Einsatz durchaus. Marketing-Automation ist immer für den Kunden da – rund um die Uhr an jedem Wochentag. Zu bedenken ist dabei auch, dass Marketing-Automation-Software nicht nur für automatisiertes Inbound-Marketing genutzt werden kann. Die meisten Systeme bringen alle Funktionen mit, um interne Workflows zu optimieren und auf diese Weise eine weitere Effizienzsteigerung zu erreichen. Hier sind die Unternehmen selbst gefordert, die Bereitschaft zum Wandel mitzubringen und diese Investition in den zukünftigen Erfolg anzupacken. Funding. Open access funding provided by University of Applied Sciences in Business Administration Zurich HWZ. Open Access. Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenz veröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/deed.de. Literatur [1] Schoepf, A. (2020). Mehr Erfolg mit Marketing Automation. Wie man automatisiert Neukunden generiert und bis zu 30 Prozent mehr verkauft (4. Aufl.). Norderstedt: BoD – Books on Demand. Mehr zum Thema finden Sie online www.springerprofessional.de/wum 288 Wirtschaftsinformatik & Management 4 \| 2021 Hier steht eine Anzeige. 123
https://openalex.org/W4210469687	https://www.frontiersin.org/articles/10.3389/fitd.2021.771030/pdf	English	null	Achievements, Gaps, and Emerging Challenges in Controlling Malaria in Ethiopia	Frontiers in tropical diseases	2,022	cc-by	4,795	Achievements, Gaps, and Emerging Challenges in Controlling Malaria in Ethiopia Amsalu Woldie Yalew 1,2,3* 1 Department of Environmental Sciences, Informatics and Statistics, Ca’ Foscari University of Venice, Venice, Italy, 2 Economic Analysis of Climate Impacts and Policy Division, Euro-Mediterranean Center on Climate Change, Venice, Italy, 3 RFF-CMCC European Institute on Economics and the Environment, Venice, Italy Controlling malaria is one of the top health sector priorities in Ethiopia. The concrete prevention, control, and treatment interventions undertaken in the past two decades have substantially reduced the morbidity and mortality attributable to malaria. Emboldened by these past achievements, Ethiopia envisages to eliminate malaria by 2030. Realizing this ambition, however, needs to further strengthen the ﬁnancial, technical, and institutional capacities to address the current as well as emerging challenges. It particularly needs to step up measures pertaining to diagnosis, domestic resource mobilization, vector surveillance, and seasonal weather forecasting. HIGHLIGHTS • Ethiopia has made a remarkable progress in terms of controlling malaria, especially, since 2004. • It is further campaigning towards a “malaria free Ethiopia”. Correspondence: Amsalu Woldie Yalew amsaluwoldie.yalew@unive.it p g g p • This requires building strong cross-sectoral and cross-border coordination capacity. p g g p • This requires building strong cross-sectoral and cross-border coordination capacity. • It also needs to scale up research and surveillance on emerging malaria vectors. • The implications of irrigation and hydropower dams on malaria transmission should not be undermined. • The implications of irrigation and hydropower dams on malaria transmission should not be undermined. Specialty section: This article was submitted to Disease Prevention and Control Policy, a section of the journal Frontiers in Tropical Diseases Received: 05 September 2021 Accepted: 09 December 2021 Published: 25 January 2022 Citation: Yalew AW (2022) Achievements, Gaps, and Emerging Challenges in Controlling Malaria in Ethiopia. Front. Trop. Dis. 2:771030. doi: 10.3389/ﬁtd.2021.771030 Specialty section: This article was submitted to Disease Prevention and Control Policy, a section of the journal Frontiers in Tropical Diseases Received: 05 September 2021 Accepted: 09 December 2021 Published: 25 January 2022 Specialty section: This article was submitted to Disease Prevention and Control Policy, a section of the journal Frontiers in Tropical Diseases Received: 05 September 2021 Accepted: 09 December 2021 Published: 25 January 2022 Citation: Yalew AW (2022) Achievements, Gaps, and Emerging Challenges in Controlling Malaria in Ethiopia. Front. Trop. Dis. 2:771030. doi: 10.3389/ﬁtd.2021.771030 Specialty section: This article was submitted to Disease Prevention and Control Policy, a section of the journal Frontiers in Tropical Diseases Edited by: Edited by: Son H. Nghiem, Grifﬁth Health, Grifﬁth University, Australia Keywords: malaria risk, malaria control, malaria funding, health policy, Ethiopia Reviewed by: Megha Raj Banjara, Tribhuvan University, Nepal Jun Feng, Shanghai Municipal Center for Disease Control and Prevention (SCDC), China Reviewed by: Megha Raj Banjara, Tribhuvan University, Nepal Jun Feng, Shanghai Municipal Center for Disease Control and Prevention (SCDC), China Correspondence: Amsalu Woldie Yalew amsaluwoldie.yalew@unive.it INTRODUCTION Malaria is one of the major infectious tropical diseases with substantial socio-economic repercussions in the sub-Saharan Africa region. In 2019, WHO’s African Region accounted for about 94% of malaria cases while only six African countries (Nigeria, Democratic Republic of the Congo, Tanzania, Mozambique, Niger, and Burkina Faso) accounted for about 51% of all malaria deaths globally (1). The number of estimated malaria cases in Africa in 2019 were 215 million (1). POLICY BRIEF published: 25 January 2022 doi: 10.3389/ﬁtd.2021.771030 Keywords: malaria risk, malaria control, malaria funding, health policy, Ethiopia POLICY RESPONSES TO MALARIA Section 2 gives a brief overview of the malaria risk in Ethiopia. Section 3 succinctly presents the malaria prevention and control measures followed by the past achievements, the current gaps, and emerging issues related to malaria control measures in Section 4. This is followed by some recommendations in Section 5, and conclusions in Section 6. Ethiopia started scaling up prevention and control measures in 2004 (5). Notable large-scale interventions include distribution of long-lasting insecticidal nets (LLINs), and indoor residual spraying (IRS), and introduction of additional diagnosis and treatment mechanisms such as artemisinin-based combination therapy (ACT), artemether lumefantrine (AL), and rapid diagnostic test (RDT) (5, 7, 13). Furthermore, in2010 itstarted implementing test- treat policy, i.e., administration of antimalarial drugs based on test results (5). Figure 2, which is adapted from (5), depicts the timeline for major interventions between 1995 and 2016. Citation: Malaria-related morbidity and mortality entail substantial private costs (e.g., direct costs due to clinical treatments, and indirect costs due to reduced labor productivity) and societal costs (e.g., increasing public health expenditure, and effects on labor, investment, and tourism ﬂows) in many tropical countries (2). January 2022 \| Volume 2 \| Article 771030 Frontiers in Tropical Diseases \| www.frontiersin.org Yalew Malaria Control in Ethiopia Yalew There are ﬁve distinct malaria risk strata classiﬁed based on annual parasite incidence (API) per 1,000 population, elevation, and expert opinions (7). According to the latest malaria risk classiﬁcation, about 18% of the population lives in high (API ≥ 50)andmoderate(10≤API<50)riskstrata.Suchclassiﬁcationsare important as they facilitate the design and implementation of most appropriate interventions per strata (5, 7, 14). It worth noting here that the latest stratiﬁcation (7) and percentage distribution of population living under each risk stratum is different from the previous stratiﬁcations and percentage distributions such as, for example,theonein2014(14).Figure1collatesthetwostratiﬁcation maps together. See also that the latest stratiﬁcation adds one more stratum which is very low risk. Currently, more than 50% of the population in Ethiopia is exposed to the risk of malaria infection (3–5). Despite the range of prevention measures undertaken in the last two decades, malaria remainstobeoneofthetoptencausesofmorbidityandmortalityin Ethiopia (6) with substantial repercussions for the macroeconomy (7,8).The effectsofmalariapropelinto themacroeconomythrough two main channels. The ﬁrst is through agricultural labor productivity changes as the malaria transmission seasons (September to December, and April to May) coincide with the main agriculture harvest seasons (5, 7, 8). Agriculture is the main sourceofemployment(≈75%)andmerchandiseexportearnings(≈ 80%) in Ethiopia (9). The second way is through government budget allocation and ﬁscal balance as government is the main health service provider (4). Seen against the forgoing conditions in Ethiopia, even a marginal increase in the risk of malaria has profound socio-economic implications making malaria disease public health as well as economic problem (8). Frontiers in Tropical Diseases \| www.frontiersin.org POLICY RESPONSES TO MALARIA Ethiopia has been ﬁghting malaria through formal institutions for more than ﬁve decades (5, 13). Today, malaria is one of its top national health and economic development priorities (7). In line with this, especially since 2004, the Government of Ethiopia together with its international partners has implemented a series of malaria prevention, control, and elimination programs (7) including preparing malaria guidelines (15, 16) and strategies (7, 17–19), and conducting surveys (14, 20, 21). The guidelines present detailed procedures on implementing and reporting various malaria vector control, diagnosis and treatment, and surveillance and response activities (15, 16). The periodic surveys produce evidence needed to formulate as well as evaluate different policy measures. All in all, policy wise, Ethiopia is stepping up its efforts to move from controlling (17) to eliminating (7) malaria. It is therefore important to assess the status of malaria risk, and tocontinuouslyevaluatethepreventionandcontrolmeasuresinthe country. It is equally important to identify the existing policy and implementation gaps, and emerging challenges that may undermine (or even reverse) the progress made so far. This paper aims to contribute its part in this regard. It brieﬂy discusses the status of malaria risk, the past achievements, gaps, and emerging challenges in ﬁghting malaria in Ethiopia. The study is a narrative overview that aims to brieﬂy synthesize the existing knowledge, and todraw implications for future research and policy makers (10–12). It is therefore important to assess the status of malaria risk, and tocontinuouslyevaluatethepreventionandcontrolmeasuresinthe country. It is equally important to identify the existing policy and implementation gaps, and emerging challenges that may undermine (or even reverse) the progress made so far. This paper aims to contribute its part in this regard. It brieﬂy discusses the status of malaria risk, the past achievements, gaps, and emerging challenges in ﬁghting malaria in Ethiopia. The study is a narrative overview that aims to brieﬂy synthesize the existing knowledge, and todraw implications for future research and policy makers (10–12). The remainder of the paper is structured as follows. Section 2 gives a brief overview of the malaria risk in Ethiopia. Section 3 succinctly presents the malaria prevention and control measures followed by the past achievements, the current gaps, and emerging issues related to malaria control measures in Section 4. This is followed by some recommendations in Section 5, and conclusions in Section 6. The remainder of the paper is structured as follows. OVERVIEW OF MALARIA RISK A B FIGURE 1 \| Malaria risk stratiﬁcation in Ethiopia, 2014 (A), and 2020 (B). A B FIGURE 1 \| Malaria risk stratiﬁcation in Ethiopia, 2014 (A), and 2020 (B). A FIGURE 1 \| Malaria risk stratiﬁcation in Ethiopia, 2014 (A), and 2020 (B). B B A A FIGURE 1 \| Malaria risk stratiﬁcation in Ethiopia, 2014 (A), and 2020 (B). FIGURE 2 \| Timeline of major malaria control, diagnosis, and treatment interventions in Ethiopia, 1995-2016. FIGURE 2 \| Timeline of major malaria control, diagnosis, and treatment interventions in Ethiopia, 1995-2016. country offers malaria diagnosis or treatment (4). Table 1 presents the key trends in public health services, in malaria control measures, and in malaria disease in the past two decades. number of deaths due to malaria declined by 54% between 2000 and 2016 (31) while the age-standardized mortality rate of malaria has declined by 96.5% between 1990 and 2015 (32). Ethiopia is also on a good track in terms of meeting its Global Technical Strategy for Malaria (2016-2030) (1, 7). ACHIEVEMENTS, GAPS, AND EMERGING ISSUES These past achievements encouraged the country to set more ambitious future goals. It envisages to reduce malaria morbidity and mortality, relative to 2020, by 50% in 2025 and to eliminate malaria by 2030 (7). More speciﬁcally, it aims to reduce malaria deaths per 100,000 population at risk (from 0.36 to 0.1), and the number of reported cases (from 1.7 million to 0.7 million) between 2019/20 and 2025/26 (7). Seen against these past OVERVIEW OF MALARIA RISK Malaria transmission in Ethiopia is seasonal, unstable, and often characterized by highly focal and large-scale cyclic epidemics (1, 7). Areas lying at altitudes between 1600 and 2000 meters above sea level (masl) are in general epidemic prone hypo-endemic zones of malaria (4) although some studies could also detected malaria in areas higher than 2000 masl (5, 7). Altitude, climate, environmental changes (e.g., due to dams, roads construction, agricultural projects), and housing conditions are important determinants of malaria risk and transmission in Ethiopia (3, 7). Overall, more than 50% of the total population in Ethiopia is exposed to the risk of malaria infection (3–5). Every year, four to ﬁve million people are affected by malaria (3, 13) while a major epidemic occurs every ﬁve to eight years (7). Remarkable progress has been made afterwards. The share of households in malarious areas (≤2000 masl) that possess at least one LLINs and received IRS have, respectively, reached 85% and 93% in 2019 (22). The introduction of RDTs in 2004 was a signiﬁcant step forward in terms of case detection and management (3) since RDTs are easy to use and to deploy in rural areas (23) where 80% of Ethiopians live. These mass scale deployment of malaria speciﬁc inputs were also accompanied by the deployment of health extension workers (HEW) in rural areas which conduct home-to-home outreach activities, and provide basic curative, promotive, and preventive services at health posts (24). The reported malaria cases remain higher than one million cases per annum. On an average, about 1.6 million malaria cases (more than 60% is related to the P. falciparum species) are reported between 2010 and 2019 (1, 5). The rate of the P. falciparum species is especially higher in the lowland regions (7). The malaria-speciﬁc interventions are also complemented with the overall increase in the number of health infrastructure (e.g., public health facilities and professionals) which profoundly improved malaria case management (13, 24). Notwithstanding the limitations, currently, about 68% of the facilities in the January 2022 \| Volume 2 \| Article 771030 2 Malaria Control in Ethiopia Yalew Yalew country offers malaria diagnosis or treatment (4) Table 1 number of deaths due to malaria declined by 54% between 2000 A B FIGURE 1 \| Malaria risk stratiﬁcation in Ethiopia, 2014 (A), and 2020 (B). FIGURE 2 \| Timeline of major malaria control, diagnosis, and treatment interventions in Ethiopia, 1995-2016. Achievements The range of malaria prevention and control measures discussed in the preceding section are paying off. Mortality and morbidity attributable to malaria have signiﬁcantly declined (7, 30). The January 2022 \| Volume 2 \| Article 771030 Frontiers in Tropical Diseases \| www.frontiersin.org 3 Malaria Control in Ethiopia Yalew TABLE 1 \| Public health services, malaria interventions, and malaria disease in Ethiopia, 2000-2019. Indicator Unit 2000/01 2004/05 2009/10 2014/15 2018/19 Source Public Health Services Health facilities Number 3,502 6,162 16,450 20,183 21,154 (22, 25–28) Health professionals Number 19,529 19,823 39,558 71,529 87,800 (22, 25–28) Health extension workers Number 0 2,737 34,396 42,336 42,336 (22, 25–28) Malaria interventions Households in the malarious areas with IRS conducted during the year Percent 10.5 20 57.2 90 93 (20, 22, 25, 26, 29) Households in malarious area that have at least one LLINs Percent 5.7 68.9 55.2 63.6 85 (4, 20–22, 25, 29) Malaria disease Incidence of malaria (per 1,000 population at risk) Number 157 190 126 55 32 (30) Malaria deaths (per 100,000 people) Number 29.6 29.9 3.7 2.4 3.2 (30) Health facilities in 2000/01 and 2004/05 include non-public facilities. Health professionals include physicians, health ofﬁcer, nurses, pharmacist, laboratory technicians, and environmental and sanitation experts. Deploying health extension workers started in 2004. The percentages for the IRS in 2000/01 and 2004/05 are relative to total households in the country while the percentage for the LLINs in 2000/01 refer to any type of mosquito nets. TABLE 1 \| Public health services, malaria interventions, and malaria disease in Ethiopia, 2000-2019. Health facilities in 2000/01 and 2004/05 include non-public facilities. Health professionals include physicians, health ofﬁcer, nurses, pharmacist, laboratory technicians, and environmental and sanitation experts. Deploying health extension workers started in 2004. The percentages for the IRS in 2000/01 and 2004/05 are relative to total households in the country while the percentage for the LLINs in 2000/01 refer to any type of mosquito nets. found in (23). The readiness for malaria diagnostic capacity is 55% in private health facilities compared to 80% in government health facilities (4). achievements and the ongoing efforts, Ethiopia’s aim to eliminate malaria by 2030 seems ambitious but attainable. (13). There are however outstanding gaps and emerging challenges that should be addressed to keep this momentum of ﬁghting malaria. Some of them are highlighted below. Achievements On the other hand, sufﬁcient and sustained amount offunding is required to keep the momentum of ﬁghting malaria (24). The generous funding from external sources has been one of the major reasons behind the past success (5, 13). Development assistance, for instance, contributes about 57% of an estimated US$ 81.2 million total spending on malaria in 2016 (34). See also Figure 3 which is based on (34). The Global Fund and the US Presidents’ Malaria Initiative (PMI) are the two main sources of external funds (7). Ethiopia received close to US$ 0.5 billion between 2008 and 2021 from the PMI funds (35). The biggest share of the external funds is spent on ﬁxed costs and commodities such as LLINs, ACTs, and RDTs (7, 35). As such, it is fair to argue that the prospects of malaria elimination goals partly hinges on the ﬁnancial commitments by the international donors. The amount of external funds are however expected to decline, and thus domestic sources should ﬁll the gap. For example, the Government of Ethiopia anticipates ﬁnancing about 56% of the total spending required to implement the malaria elimination plan by 2025/26 (7). In spite of this increased budgetary Gaps Diagnosis is one of the key factors in controlling and eliminating malaria. In Ethiopia, 63% of the health facilities provide malaria diagnosis testing (4). Of the alternative methods of malaria diagnosis (33), a microscopic diagnosis allows for the identiﬁcation of parasitemia percentage, parasitic morphology, and speciation (23). The microscopic diagnosis in Ethiopia is limited. Health facilities that offered malaria diagnosis by microscopy (17%) was much lower than health facilities that offered diagnosis by clinical symptoms (42%) and RDT (54%) (4). Diagnosis by microscopy is available only in 7% of the rural health facilities (4). This represents a major diagnosis capacity gap seen against the fact that about 80% of the population lives in rural areas (7) with poor housing conditions, and thus at higher risk of infection (3). A recent review on the relative advantages and limitations of different malaria diagnostic methods can be FIGURE 3 \| Malaria spending in Ethiopia by source, 2016. January 2022 \| Volume 2 \| Article 771030 Frontiers in Tropical Diseases \| www.frontiersin.org Malaria Control in Ethiopia Yalew CONCLUSIONS Ethiopia has been undertaking a wide range of policy measures to control malaria, especially, after 2004. Consequently, mortality and morbidity attributable to malaria have declined signiﬁcantly. To keep the momentum of ﬁghting malaria, however, Ethiopia needs to strengthen its institutional capacity pertaining to domestic resource mobilization, diagnosis by microscopy, vector surveillance, and climate information processing and seasonal weather forecasting. These actions need, among others, to layout and enhance cross-sectoral coordination (e.g., with irrigation, hydropower, and climate change), and cross- border cooperation (e.g., for better surveillance of vectors) mechanisms. Future public budget allocation to ﬁght malaria should factor in these and other emerging challenges. Emerging Challenges • Consider dam reservoir management as one of malaria vector control tools (48). • Consider dam reservoir management as one of malaria vector control tools (48). g g g The past progress in ﬁghting malaria is threatened by a set of emerging challenges due to such as mosquito resistance to insecticides, the emergence of new vectors, the potential side effects of irrigation and hydropower reservoirs, and climate change and variability (7). Recent evidence shows that local vectors are generally resistant to dichloro-diphenyl-trichloroethane (DDT) and pyrethroids (35), and the LLINs (36). The use of DDT for IRS was of course discontinued in 2007 (5). The emergence of new vectors such as the A.stephensi, which were not previously widely known, poses yet another challenge (1, 7). Anopheles stephensi mosquitoes breed predominately in urban settings preferably in man-made water containers and poses risk for increased transmission of P. falciparum and P.vivax (37). Currently, the A. stephensi vector is widely distributed and established in the eastern parts of Ethiopia (38). On the other hand, Ethiopia is expanding irrigation and hydropower dams that were found intensifying malaria transmission (39). For instance, malaria incidence was about 32% among households in villages with irrigation micro-dams compared to 19% in villages with no micro-dams in northern Ethiopia (40). The side effects are much more pronounced in the lowland and midland ecological settings (41) where the country is recently eyeing to expand its large-scale irrigated agriculture. On top of this, temperature suitability for malaria is climbing into the highlands of Ethiopia (42, 43) because of which the prevalence of malaria is projected to increase (44). A case study in northern Ethiopia shows that climate change may increase area suitable for malaria transmission by 94 to 114% by 2050 (45). Overall, countrywide, up to 130 million people may be at risk of malaria by 2070 (46) that could induce substantial economic costs (8, 42). • Scale up research (38), and surveillance (37) capacity particularly with regard to the emerging vectors. • Pursue regional cooperation to control cross-border malaria transmission through migration (24), and to surveille the emerging vectors (38). ACTIONABLE RECOMMENDATIONS commitment from the government, however, implementing the current Ethiopia Malaria Elimination Strategic Plan (2021/22- 2025/26) will still face about US$ 167.9 million ﬁnancial gap (7). Diversifying the sources of funds is particularly important in light of unforeseen global and domestic challenges such as the COVID- 19 pandemics that may affect the priority areas of the government as well as international partners (7). Therefore, in order to maintain the momentum of ﬁghting malaria, it needs to scale up measures related to funding, climate services, and vector control. More speciﬁcally, it needs to: • Raise the domestic resource mobilization capacity (7). • Enhance climate information processing capacity (47). AUTHOR CONTRIBUTIONS The author conﬁrms being the sole contributor of this work and has approved it for publication. 8. Yalew AW. Revisiting Economic Burdens of Malaria in the Face of Climate Change: A Conceptual Analysis for Ethiopia. Int J Climate Change Strategies Manage (2021) 13(1):1–18. doi: 10.1108/IJCCSM-05-2020-0045 8. Yalew AW. Revisiting Economic Burdens of Malaria in the Face of Climate Change: A Conceptual Analysis for Ethiopia. Int J Climate Change Strategies Manage (2021) 13(1):1–18. doi: 10.1108/IJCCSM-05-2020-0045 14. Ethiopian Public Health Institute. Ethiopian National Malaria Indicator Survey 2015. Addis Ababa (2016). y 15. Ministry of Health. National Malaria Guidelines. 3rd Edition. Addis Ababa (2012). 13. Bugssa G, Tedla K. Feasibility of Malaria Elimination in Ethiopia. Ethiopian J Health Sci (2020) 30(4):607–14. doi: 10.4314/ejhs.v30i4.16 REFERENCES Addis Ababa (2006). 38. Balkew M, Mumba P, Dengela D, Yohannes G, Getachew D, Yared S, et al. Geographical Distribution of Anopheles Stephensi in Eastern Ethiopia. Parasit Vectors (2020) 13:135. doi: 10.1186/s13071-020-3904-y 18. Ministry of Health. National Strategic Plan for Malaria Prevention, Control and Elimination in Ethiopia: 2011-2015. Addis Ababa (2010). 39. Yewhalaw D, Legesse W, Van Bortel W, Gebre-Selassie S, Kloos H, Duchateau L, et al. Malaria and Water Resource Development: The Case of Gilgel-Gibe Hydroelectric Dam in Ethiopia. Malaria J (2009) 8:21. doi: 10.1186/1475- 2875-8-21 Ministry of Health. National Malaria Elimination Roadmap. Addis 20. Ministry of Health. Ethiopian National Malaria Indicator Survey 2007. Addis Ababa (2008). 21. Ethiopian Health and Nutrition Research Institute. Ethiopia National Malaria Indicator Survey 2011. Addis Ababa (2012). 40. Ersado L. Small-Scale Irrigation Dams, Agricultural Production, and Health: Theory and Evidence From Ethiopia. In: World Bank Policy Research Working Paper, World Bank, Washington, DC. (2005). p. 3494. 22. Ministry of Health. Health and Health Related Indicators, 2018/19. Addis Ababa (2019). 41. Kibret S, Wilson GG, Ryder D, Tekie H, Petros B. Malaria Impact of Large Dams at Different Eco-Epidemiological Settings in Ethiopia. Trop Med Health (2017) 45:4. doi: 10.1186/s41182-017-0044-y 23. Mbanefo A, Kumar N. Evaluation of Malaria Diagnostic Methods as a Key for Successful Control and Elimination Programs. Trop Med Infect Dis (2020) 5:102. doi: 10.3390/tropicalmed5020102 42. Ministry of Health. National Health Adaptation Plan to Climate Change: 2018-2020. Addis Ababa (2018). p 24. PATH. Ethiopia: Charting the Path to Malaria Elimination. Addis Ababa (2019). 43. Lyon B, Dinku T, Raman A, Thomson MC. Temperature Suitability for Malaria Climbing the Ethiopian Highlands. Environ Res Lett (2017) 12:064015. doi: 10.1088/1748-9326/aa64e6 25. Ministry of Health. Health and Health Related Indicators, 2014/15. Addis Ababa (2015). 26. Ministry of Health. Health and Health Related Indicators, 2009/10. Addis Ababa (2010). 44. Taye G, Kaba M, Woyessa A, Deressa W, Simane B, Kumie A, et al. Modeling Effect of Climate Variability on Malaria in Ethiopia. Ethiopian J Health Dev (2015) 29(3):183–96. 27. Ministry of Health. Health and Health Related Indicators, 2004/05. Addis Ababa (2005). 28. Ministry of Health. Health and Health Related Indicators, 2000/01. Addis Ababa (2001). 45. Abrha H,Hagos H,BrhaneE,Hadgu M, Mamo G.Spatio-TemporalDynamics of Malaria Expansion Under Climate Change in Semi-Arid Areas of Ethiopia. Environ Hazards (2019) 18(5):400–13. doi: 10.1080/17477891.2019.1609405 29. Central Statistical Agency and ORC Macro. Ethiopia Demographic and Health Survey 2005. Addis Ababa (2006). 46. World Health Organization. REFERENCES Climate and Health Country Proﬁle: Ethiopia (2015). Available at: https://apps.who.int/iris/handle/10665/208861 (Accessed July 10, 2021). 30. Roser M, Ritchie H. Malaria (2019). Available at: https://ourworldindata.org/ malaria (Accessed November 17, 2021). 31. Girum T, Shumbej T, Shewangizaw M. Burden of Malaria in Ethiopia, 2000- 2016: Findings From the Global Health Estimates 2016. Trop Dis Travel Med Vaccines (2019) 5:11. doi: 10.1186/s40794-019-0090-z 47. Ghebreyesus TA, Tadesse Z, Jima D, Bekele E, Mihretie A, Yihdego YY, et al. Using Climate Information in the Health Sector. Field Actions Sci Rep (2009) 2 (2):63–7. doi: 10.5194/facts-2-63-2009 32. Deribew A, Dejene T, Kebede B, Tessema GA, Melaku YA, Misganaw A, et al. Incidence, Prevalence and Mortality Rates of Malaria in Ethiopia From 1990 to 2015: Analysis of the Global Burden of Diseases 2015. Malaria J (2017) 16:271. doi: 10.1186/s12936-017-1919-4 48. Kibret S, Wilson GG, Ryder D, Tekie H, Petros B. Environmental and Meteorological Factors Linked to Malaria Transmission Around Large Dams at Three Ecological Settings in Ethiopia. Malaria J (2019) 18:54. doi: 10.1186/s12936-019-2689-y 33. Center for Disease Control and Prevention. Malaria Diagnosis (United States) (2021). Available at: https://www.cdc.gov/malaria/diagnosis_treatment/ diagnosis.html (Accessed November 15, 2021). Conﬂict of Interest: The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 34. Haakenstad A, Harle AC, Tsakalos G, Micah AE, Dieleman JL. Tracking Spending on Malaria by Source in 106 Countries, 2000-16: An Economic Modelling Study. Lancet Infect Dis (2019) 19(7):703–16. doi: 10.1016/S1473- 3099(19)30165-3 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 35. The President’s Malaria Initiative. Ethiopia Malaria Operational Plans (2021). Available at: https://www.pmi.gov/where-we-work/ethiopia/ (Accessed July 19, 2021). 36. Kendie FA, W/Kiros TH, Semegn EN, Ferede MW. Prevalence of Malaria Among Adults in Ethiopia: A Systematic Review and Meta-Analysis. J Trop Med (2021) 8863002:9. doi: 10.1155/2021/8863002 Copyright © 2022 Yalew. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). REFERENCES 1. World Health Organization. World Malaria Report 2020. Geneva (2020). 1. World Health Organization. World Malaria Report 2020. Geneva (2020). g 9. National Bank of Ethiopia. Annual Economic Report-2019/20. Addis Ababa (2021). 2. Gallup JL, Sachs JD. The Economic Burden of Malaria. Am J Trop Med Hyg (2001) 64(1):85–96. doi: 10.4269/ajtmh.2001.64.85 10. Pare G, Trudel M, Jaana M, Kitsiou S. Synthesizing Information Systems Knowledge: A Typology of Literature Reviews. Inf Manage (2015) 52:183–99. doi: 10.1016/j.im.2014.08.008 3. Ayele DG, Zewotir TT, Mwambi HG. Prevalence and Risk Factors of Malaria in Ethiopia. Malaria J (2012) 11:195. doi: 10.1186/1475-2875-11-195 11. Cronin P, Ryan F, Coughlan M. Undertaking a Literature Review: A Step-by-Step Approach. Br J Nurs (2008) 17(1):38–43. doi: 10.12968/bjon.2008.17.1.28059 4. Ethiopian Public Health Institute. Services Availability and Readiness Assessment. Addis Ababa (2018). 5. Taffese HW, Hemming-Schroeder E, Koepﬂi C, Tesfaye G, Lee M, Kazura J, et al. Malaria Epidemiology and Interventions in Ethiopia From 2001 to 2016. Infect Dis Poverty (2018) 7:103. doi: 10.1186/s40249-018-0487-3 12. Green BN, Johnson CD, Adams A. Writing Narrative Literature Reviews for Peer-Reviewed Journals: Secrets of the Trade. J Chiropr Med (2006) 5(3):101– 17. doi: 10.1016/S0899-3467(07)60142-6 12. Green BN, Johnson CD, Adams A. Writing Narrative Literature Reviews for Peer-Reviewed Journals: Secrets of the Trade. J Chiropr Med (2006) 5(3):101– 17. doi: 10.1016/S0899-3467(07)60142-6 13. Bugssa G, Tedla K. Feasibility of Malaria Elimination in Ethiopia. Ethiopian J Health Sci (2020) 30(4):607–14. doi: 10.4314/ejhs.v30i4.16 6. Misganaw A, Melaku YA, Tessema GA, Deribew A, Deribe K, Abera S, et al. National Disability-Adjusted Life Years (DALYs) for 257 Diseases and Injuries in Ethiopia, 1990–2015: Findings From the Global Burden of Disease Study 2015. Popul Health Metrics (2017) 15:28. doi: 10.1186/s12963-017-0146-0 14. Ethiopian Public Health Institute. Ethiopian National Malaria Indicator Survey 2015. Addis Ababa (2016). g Popul Health Metrics (2017) 15:28. doi: 10.1186/s12963-017-0146-0 y 15. Ministry of Health. National Malaria Guidelines. 3rd Edition. Addis Ababa (2012). 7. Ministry of Health. National Malaria Elimination Strategic Plan: 2021-2025. Addis Ababa (2020). 7. Ministry of Health. National Malaria Elimination Strategic Plan: 2021-2025. Addis Ababa (2020). 16. Ministry of Health. National Malaria Guidelines. 4th Edition. Addis Ababa (2018). 16. Ministry of Health. National Malaria Guidelines. 4th Edition. Addis Ababa (2018). January 2022 \| Volume 2 \| Article 771030 Frontiers in Tropical Diseases \| www.frontiersin.org 5 Malaria Control in Ethiopia Yalew Yalew 17. Ministry of Health. National Five-Year Strategic Plan for Malaria Prevention and Control in Ethiopia: 2006-2010. January 2022 \| Volume 2 \| Article 771030 REFERENCES The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 37. Tadesse FG, Ashine T, Teka H, Esayas E, Messenger LA, Chali W, et al. Anopheles Stephensi Mosquitoes as Vectors of Plasmodium Vivax and P. Falciparum, Horn of Africa, 2019. Emerg Infect Dis (2021) 27(2):603–8. doi: 10.3201/eid2702.200019 January 2022 \| Volume 2 \| Article 771030 Frontiers in Tropical Diseases \| www.frontiersin.org
https://openalex.org/W2807297020	https://www.hal.inserm.fr/inserm-01810195/file/41598_2018_Article_26584_2018_Eq9.pdf	English	null	Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system	Scientific reports	2,018	cc-by	8,402	To cite this version: Delphine Garnier, Ruoya Li, Frédéric Delbos, Angélique Fourrier, Camille Collet, et al.. Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system. Scientific Reports, 2018, 8 (1), pp.8222 - 8222. 10.1038/s41598-018-26584-1. inserm- 01810195 Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system Delphine Garnier, Ruoya Li, Frédéric Delbos, Angélique Fourrier, Camille Collet, Christiane Guguen-Guillouzo, Christophe Chesné, Tuan Huy Nguyen Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system Fortunately, to maintain its homeostasis the liver does possess the impressive capacity to regenerate thanks to the proliferation of mature hepatocytes in the healthy tissue1. However in some cases of extreme severe injury, resident stem cells may take over to reconstitute liver tissue if the unique proliferative capacity of hepatocytes is not sufficient enough to compensate for the lost2,3. A lot of works have addressed the question of the nature of these liver stem cells, also referred to as liver progenitor cells or oval cells, however their location and characteristics are still not well understood4,5. In terms of therapy, whether the damage is induced by a virus, an oncogene, a drug or even surgical removal, the best way to treat hepatic disease when liver regeneration is inadequate remains the transplantation of the whole organ or of some fraction of the liver6. However the dramatic shortage of liver organs available for trans- plantation makes it difficult to satisfy the medical needs. To compensate for the lack of liver donors some other alternatives have to be found, including transplantation of hepatocytes7,8. Despite several decades of investigation and optimization of different cell culture systems, the in vitro amplification of hepatocytes on long-term remains still very challenging, mainly due to a lost of their differentiation features and a very poor proliferation potential in culture9. Tissue engineering has received a lot of attention lately, as recent progress has opened new perspectives, for example to generate patient-specific human hepatocytes by the differentiation of pluripotent stem cells into hepatocytes10, either from induced pluripotent stem cells (iPS)11–13 or embryonic stem cells (ES)14–16. These new stem cells sources to generate hepatocytes-like cells are very promising but at this time there are still limitations, 1INSERM, Université de Nantes, Centre de Recherche en Transplantation et Immunologie UMR 1064, Nantes, France. 2Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France. 3CRCINA INSERM U1232, Institut de Recherche en Santé de l’Université de Nantes, 8 quai Moncousu, Nantes, France. 4Biopredic International, Saint-Grégoire, France. Correspondence and requests for materials should be addressed to D.G. (email: delphine. garnier@univ-nantes.fr) The liver is an essential organ that guarantees detoxification and many metabolic functions, including the syn- thesis of some plasma proteins, production of hormones and bile, regulation of cholesterol and glucose storage. Any liver injury can therefore have dramatic consequences, possibly leading to hepatic pathologies such as hep- atitis, liver fibrosis, cirrhosis or hepatocarcinoma. HAL Id: inserm-01810195 https://inserm.hal.science/inserm-01810195v1 Submitted on 7 Jun 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. www.nature.com/scientificreports www.nature.com/scientificreports ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system Received: 12 February 2018 Accepted: 3 May 2018 Published: xx xx xxxx Delphine Garnier1,2,3, Ruoya Li4, Frédéric Delbos1,2, Angélique Fourrier1,2, Camille Collet1,2, Christiane Guguen-Guillouzo4, Christophe Chesné4 & Tuan Huy Nguyen1,2 Delphine Garnier1,2,3, Ruoya Li4, Frédéric Delbos1,2, Angélique Fourrier1,2, Camille Collet1,2, Christiane Guguen-Guillouzo4, Christophe Chesné4 & Tuan Huy Nguyen1,2 Despite decades of investigation on the proliferation of adult human primary hepatocytes, their expansion in vitro still remains challenging. To later be able to consider hepatocytes as a cell therapy alternative or bridge to liver transplantation, dramatically impeded by a shortage in liver donors, the first step is having an almost unlimited source of these cells. The banking of transplantable hepatocytes also implies a protocol for their expansion that can be compatible with large-scale production. We show that adult human primary hepatocytes when grown in 3D organoids are easily amplified, providing a substantial source of functional hepatocytes ready for transplantation. Following their plating, differentiated human hepatocytes are amplified during a transient and reversible step as liver progenitors, and can subsequently be converted back to mature differentiated hepatocytes. The protocol we propose is not only compatible with automated and high-throughput cell culture systems, thanks to the expansion of hepatocytes in suspension, but also guarantees the generation of a high number of functional cells from the same patient sample, with a relatively easy set up. 1 ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 show that adult human primary hepatocytes when grown in 3D organoids are easily amplified, providing a substantial source of functional hepatocytes ready for transplantation. Following their plating, differentiated human hepatocytes are amplified during a transient and reversible step as liver progenitors, and can subsequently be converted back to mature differentiated hepatocytes. The protocol we propose is not only compatible with automated and high-throughput cell culture systems, thanks to the expansion of hepatocytes in suspension, but also guarantees the generation of a high number of functional cells from the same patient sample, with a relatively easy set up. The liver is an essential organ that guarantees detoxification and many metabolic functions, including the syn- thesis of some plasma proteins, production of hormones and bile, regulation of cholesterol and glucose storage. Any liver injury can therefore have dramatic consequences, possibly leading to hepatic pathologies such as hep- atitis, liver fibrosis, cirrhosis or hepatocarcinoma. www.nature.com/scientificreports/ including the cost and time needed to maintain and differentiate cells into hepatocytes, as well as the potential genetic instability associated with the reprogramming strategy, compromising pitfall that will have to be consid- ered with regard to clinical applications. g pp To reconstitute more faithfully the in vivo hepatic environment, 3D culture systems have also generated an increasing interest over the last decades. Few years ago Takebe et al. were able to generate in vitro some vascu- larized 3D liver buds resembling the adult liver tissue, by combining three cell types in the same 3D structure, that is endodermal cells derived from induced pluripotent stem (iPS), human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs)17. The functionality of their system was validated by the rescue of drug-induced liver failure in mice after transplantation of human liver buds. However, when speaking about replacing liver transplantation by hepatocytes transplantation in human, that implies an almost unlimited source of mature human hepatocytes to be able to meet the needs. Despite their capacity to mimic in vitro liver tissues, it is difficult to consider liver buds model for the high scale production of human mature hepatocytes, as it rather favors the functionality/differentiation of liver cells than their proliferation, and requires a fastidious protocol. yf p q p On the other hand, along this line of 3D cell culture, more recently Huch et al. and other teams showed the long-term expansion of adult human liver cells as 3D organoids culture18–20. By allowing the restoration of a 3D configuration and the contact with the extracellular matrix (ECM), combined with the proper cocktail of growth factors and small molecules (EM expansion media), they managed to maintain Epcam (marker of liver progen- itors) positive liver cells and induce their proliferation on long term, starting from liver tissue. A population of liver progenitors with high proliferative capacity emerged from this 3D culture, that once transferred to differen- tiation media (DM) became fully functional hepatocytes capable of engraftment in different mouse models18. This work does represent a striking progress for the generation of human adult hepatocytes, however the model may suffer of some limitations when it comes to the transfer to clinical or industrial applications21. Results Human hepatocytes from different cell batches grow differently in 3D organoids. Starting from the protocol of Huch et al.18 to grow liver cells in 3D organoids we first tested this culture system on different batches of liver cells, except that contrary to this study we did not sort Epcam positive cells but used cryopre- served mature human hepatocytes. These cells were isolated from the healthy tissue of three different patients, each suffering from a different pathology (Fig. 1A). When analyzing the number of organoids in the three cell batches separately, it appears that there is a lot of variability between patient samples (Fig. 1B, upper panel). In parallel the cell count at different time points showed that the kinetic of proliferation differs from batch to batch, with a peak in proliferation around 30 days after plating and a decline in growth thereafter (Fig. 1B, middle panel). Of note, despite the drop in the growth rate overtime, cells did not all died by 50 days but a cell fraction still continued to proliferate for more than 2 months. In parallel the FACS analysis of the three different batches of hepatocytes showed that they are all negative for Epcam marker (Fig. 1B lower panel, Supplementary Fig. 1A and Supplementary Methods). This was also confirmed by immunofluorescence experiment on hepatocytes 24 h after plating (Supplementary Fig. 1B). p g pp y g To further investigate the properties of hepatocytes growing in 3D organoids, we decided to focus on the batch HEP187269, that showed a better progression of proliferation between week 1 and 2, a higher number of cells per well and a better proliferation rate on long term (Fig. 1B). This batch was used for all the subsequent experiments. Human hepatocytes plated as 3D organoids acquire features of liver progenitors. We then com- pared the expression of different mature/progenitor liver cell markers, in short term adherent hepatocyte culture 24, 48 and 72 h after plating, and in long term 3D organoid culture around 40 days after plating and close to 2 months after plating (when cell number dramatically decreases, see Fig. 1B). www.nature.com/scientificreports/ In particular, the 3D organoid culture consists in growing cells inside a polymerized Matrigel drop, that by being very fragile and easily breakable poses a problem on the practical side when thinking of large-scale production.h Therefore we did investigate a new protocol to improve long term culture of human hepatocytes, and enable a transfer to industrial production thanks to a compatible process of culture. With this paper we propose a new technic to amplify human hepatocytes as 3D organoids in suspension, without the need of sorting Epcam positive cells. Besides the advantage of suspension culture, suitable for large-scale production in bioreactors, our work is a clear improvement of the previous protocol because of its increased efficiency at maturation towards functional hepatocytes, it also suggests that this cell culture system could be more stable over time. Expansion of human primary hepatocytes in vitro through their amplification as liver progenitors in a 3D organoid system Fortunately, to maintain its homeostasis the liver does possess the impressive capacity to regenerate thanks to the proliferation of mature hepatocytes in the healthy tissue1. However in some cases of extreme severe injury, resident stem cells may take over to reconstitute liver tissue if the unique proliferative capacity of hepatocytes is not sufficient enough to compensate for the lost2,3. A lot of works have addressed the question of the nature of these liver stem cells, also referred to as liver progenitor cells or oval cells, however their location and characteristics are still not well understood4,5. In terms of therapy, whether the damage is induced by a virus, an oncogene, a drug or even surgical removal, the best way to treat hepatic disease when liver regeneration is inadequate remains the transplantation of the whole organ or of some fraction of the liver6. However the dramatic shortage of liver organs available for trans- plantation makes it difficult to satisfy the medical needs. To compensate for the lack of liver donors some other alternatives have to be found, including transplantation of hepatocytes7,8. Despite several decades of investigation and optimization of different cell culture systems, the in vitro amplification of hepatocytes on long-term remains still very challenging, mainly due to a lost of their differentiation features and a very poor proliferation potential in culture9. Tissue engineering has received a lot of attention lately, as recent progress has opened new perspectives, for example to generate patient-specific human hepatocytes by the differentiation of pluripotent stem cells into hepatocytes10, either from induced pluripotent stem cells (iPS)11–13 or embryonic stem cells (ES)14–16. These new stem cells sources to generate hepatocytes-like cells are very promising but at this time there are still limitations, 1INSERM, Université de Nantes, Centre de Recherche en Transplantation et Immunologie UMR 1064, Nantes, France. 2Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France. 3CRCINA INSERM U1232, Institut de Recherche en Santé de l’Université de Nantes, 8 quai Moncousu, Nantes, France. 4Biopredic International, Saint-Grégoire, France. Correspondence and requests for materials should be addressed to D.G. (email: delphine. garnier@univ-nantes.fr) ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 1 www.nature.com/scientificreports/ Results t p g ( y , g ) When plated as adherent on collagen I substrate, human hepatocytes display a high level of hepatic functions, however the proliferation rate is very low and the survival limited: as early as 72 h hepatocytes start to activate apoptosis pathways and cells die by 2 weeks9. In parallel when liver cells are cultured as 3D organoids, the survival can be maintained on long term and an active proliferation can be obtained18. We did compare those two different cell culture systems, and indeed hepatocytes cultured as adherent express a high level of differentiation markers such that Albumin, CYP3A4 or CYP3A7 (Fig. 2A-Adh). On the contrary hepatocyte organoids maintained on long term do not express those markers or at a very low level (Figs 2A–3D). Interestingly that coincides with the induction of markers of hepatic progenitors, such as a 500 fold induction of Cytokeratin 19 (CK19) compared to adherent hepatocytes, 25 fold induction of Epcam or 15 fold induction of Sox9 (Fig. 2B). According to the Ki67 mRNA level, the proliferation rate is also dramatically increased in 3D organoids, with a 150 fold induction (Fig. 2C-ORGt1). Of note is that the second time point used in 3D culture shows a decline in proliferation as well as in the expression of progenitor markers (Fig. 2BC-ORGt2). Coherently with the measurement of cell number over time (Fig. 1B), there is clearly an exhaustion of organoids cell culture when getting close to 2 months cell culture. ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 2 www.nature.com/scientificreports/ Figure 1. Variability in kinetic of growth of human hepatocytes when cultured as 3D organoids, depending o the cell batch. (A) Characteristics of human hepatocytes batches used. (B) Top panel shows organoids countin per well, 1 and 2 weeks after plating. Middle panel indicates the cell number production per well of a 24-wells plate over time. Cells were dissociated and counted once a week, and plated back in the same conditions. And the respective percentage of Epcam positive cells in each batch, measured by FACS, is shown in lower panel. Figure 1. Variability in kinetic of growth of human hepatocytes when cultured as 3D organoids, depending on the cell batch. (A) Characteristics of human hepatocytes batches used. (B) Top panel shows organoids counting per well, 1 and 2 weeks after plating. Results (A) The expression of mature hepatocytes markers was analyzed by qPCR in adherent hepatocytes at 24, 48 and 72 h after plating (Adh), or in hepatocyte organoids (3D) after 2–3 passages (t1; around 40 days after plating) and 2 weeks later (t2). (B) The expression of markers of liver progenitors was analyzed in the same conditions, as well as Ki67 proliferation marker (C). The expression was normalized compared to the expression in adherent human hepatocytes 24 h post-plating. (N = 3). after few weeks. When looking at 3D systems, following Huch protocol18 hepatocytes formed organoids that could be maintained on long term (Fig. 3A-third picture), but interestingly similar structures could also be obtained when culturing cells in suspension in media containing some Matrigel (Fig. 3A-fourth picture). Cell culture conditions were the same in both systems, except that in the classic 3D organoids culture cells were stuck inside a drop of polymerized Matrigel, whereas in our suspension system organoids were floating in media containing 10% Matrigel (see schematic representation Fig. 3B). The measurement of organoids count and average size 3 weeks after plating did not show a significant difference between organoids grown in Matrigel drop or in Matrigel suspension (Fig. 3C). The viability inside the organoids formed was also comparable (Fig. 3D). Analysis of pro- liferation suggested that the growth rate could be slightly higher in Matrigel suspension compared to Matrigel drop, as indicated by the immunofluorescence experiment on the proliferation marker Ki67 (Fig. 3E). The mRNA expression of Ki67 was also increased in Matrigel suspension (Fig. 3F), however the difference was not significant. Therefore data suggest a proliferation benefit for the 3D Matrigel suspension system, further experiments will be needed to confirm it. Cells amplified in 3D Matrigel suspension organoids are also liver progenitors, exhibiting a low expression of differentiation markers. To study whether plating mature hepatocytes in 3D organoid culture would lead to the amplification of the same cell type, in Matrigel drops or in Matrigel suspension, we then analysed their mRNA profiles by qPCR. As shown earlier (Fig. 2), when differentiated hepatocytes are cultured in 3D organoids in Huch & Clevers conditions (Matrigel drops)18, cells exhibit a high level of markers of liver pre- cursors and lose their differentiation markers, evidencing an enrichment of a liver progenitors population. Results Middle panel indicates the cell number production per well of a 24-wells plate over time. Cells were dissociated and counted once a week, and plated back in the same conditions. And the respective percentage of Epcam positive cells in each batch, measured by FACS, is shown in lower panel. While short term adherent culture goes with highly differentiated hepatocytes, long term 3D organoid culture is rather associated with highly proliferating progenitor cells. Plating mature human hepatocytes as 3D culture therefore leads to an enrichment in hepatic progenitors. While short term adherent culture goes with highly differentiated hepatocytes, long term 3D organoid culture is rather associated with highly proliferating progenitor cells. Plating mature human hepatocytes as 3D culture therefore leads to an enrichment in hepatic progenitors. Liver organoids can also be amplified in Matrigel suspension instead of Matrigel embedding. In an attempt to develop a similar 3D culture system that would not necessitate inclusion of the organoids inside polymerized Matrigel, different culture settings have been tried. Adherent culture is a well-known culture system for hepatocytes (Fig. 3A-first picture), but survival is limited to few days and proliferation is very low9. In our hands growing hepatocytes in suspension initially gave rise to clusters of cells that looked healthy and growing (Fig. 3A-second picture), however we were not able to dissociate the spheroids formed, and cells eventually died ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 3 www.nature.com/scientificreports/ Figure 2. Culture of human hepatocytes as 3D organoids leads to long term survival, loss of mature hepatocyte markers, and enrichment in liver progenitors markers. (A) The expression of mature hepatocytes markers was analyzed by qPCR in adherent hepatocytes at 24, 48 and 72 h after plating (Adh), or in hepatocyte organoids (3D) after 2–3 passages (t1; around 40 days after plating) and 2 weeks later (t2). (B) The expression of markers of liver progenitors was analyzed in the same conditions, as well as Ki67 proliferation marker (C). The expression was normalized compared to the expression in adherent human hepatocytes 24 h post-plating. (N = 3). Figure 2. Culture of human hepatocytes as 3D organoids leads to long term survival, loss of mature hepatocyte markers, and enrichment in liver progenitors markers. Results When comparing the level of expression of those markers with the one measured in 3D Matrigel suspension culture, we observed that the mRNA expression of progenitors markers is very similar (Fig. 4A-lower panel). This was confirmed by immunofluorescence experiment, that showed similar levels of staining for Epcam and CK19 in both 3D conditions (Fig. 4B). On the other hand, organoids seem to exhibit a higher level of expression of differ- entiation markers when cultured as 3D in Matrigel suspension (Fig. 4A-upper panel). Indeed Albumin expression was close to 12 fold higher in organoids maintained in Matrigel suspension compared to Matrigel drops, whereas HNF4 expression was increased by a factor 5. Other markers such that AFP, CYP3A4 and CYP3A7 were not significantly increased, even though they tend to show higher levels of expression in Matrigel suspension. Overall ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 4 www.nature.com/scientificreports/ Figure 3. 3D organoid culture of mature human hepatocytes in Matrigel drops or in Matrigel suspension display comparable levels of viability and growth on short-term. (A) Observation by phase contrast microscopy of human hepatocytes cultured as adherent cells, in suspension, or maintained in 3D inside Matrigel drop or in Matrigel suspension (bar graph = 50 μm). (B) Schematic representation of the protocol settings. (C) Average organoid counts 3 weeks after plating as 3D, in Matrigel drops (3D-D - black bars) or in Matrigel suspension (3D-S - white bars) (number of organoids per well of a 24-wells plate) (N = 2). (D) Analysis of cell viability with Live (green)/Dead (red) staining kit (bar graph = 50 μm). Proliferation was analyzed by Ki67 l Figure 3. 3D organoid culture of mature human hepatocytes in Matrigel drops or in Matrigel suspension display comparable levels of viability and growth on short-term. (A) Observation by phase contrast microscopy of human hepatocytes cultured as adherent cells, in suspension, or maintained in 3D inside Matrigel drop or in Matrigel suspension (bar graph = 50 μm). (B) Schematic representation of the protocol settings. (C) Average organoid counts 3 weeks after plating as 3D, in Matrigel drops (3D-D - black bars) or in Matrigel suspension (3D-S - white bars) (number of organoids per well of a 24-wells plate) (N = 2). (D) Analysis of cell viability with Live (green)/Dead (red) staining kit (bar graph = 50 μm). Results Proliferation was analyzed by Ki67 immunofluorescence (E) (bar graph = 100 μm) and qPCR (F) (N = 3). ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 5 www.nature.com/scientificreports/ Figure 4. 3D organoids cultured in Matrigel suspension also allows amplification of liver progenitors, while maintaining a higher level of differentiated functions. (A) The expression of different liver progenitors/ mature hepatocyte/proliferation markers was analyzed at the mRNA level by qPCR, in 3D culture of human hepatocytes inside Matrigel drop (3D-D - black bars) or in suspension (3D-S - white bars), after 2–3 passages (t1; close to 40 days after plating) and 2 weeks later (t2). Data were normalized to the expression in Matrigel drop organoids. (N = 3) (B) The expression of some of those markers was validated at the protein level by immunofluorescence (bar graph = 100 μm). Figure 4. 3D organoids cultured in Matrigel suspension also allows amplification of liver progenitors, while maintaining a higher level of differentiated functions. (A) The expression of different liver progenitors/ mature hepatocyte/proliferation markers was analyzed at the mRNA level by qPCR, in 3D culture of human hepatocytes inside Matrigel drop (3D-D - black bars) or in suspension (3D-S - white bars), after 2–3 passages (t1; close to 40 days after plating) and 2 weeks later (t2). Data were normalized to the expression in Matrigel drop organoids. (N = 3) (B) The expression of some of those markers was validated at the protein level by immunofluorescence (bar graph = 100 μm). Figure 4. 3D organoids cultured in Matrigel suspension also allows amplification of liver progenitors, while maintaining a higher level of differentiated functions. (A) The expression of different liver progenitors/ mature hepatocyte/proliferation markers was analyzed at the mRNA level by qPCR, in 3D culture of human hepatocytes inside Matrigel drop (3D-D - black bars) or in suspension (3D-S - white bars), after 2–3 passages (t1; close to 40 days after plating) and 2 weeks later (t2). Data were normalized to the expression in Matrigel drop organoids. (N = 3) (B) The expression of some of those markers was validated at the protein level by immunofluorescence (bar graph = 100 μm). those data therefore demonstrate that whether it be in Matrigel drops or in Matrigel suspension, 3D organoids cultured in those conditions exhibit features of liver progenitors, with similar levels of expression. Results However, the decrease in features of mature hepatocytes observed in parallel is less pronounced in Matrigel suspension culture, that exhibit a significantly higher level of expression of differentiation markers such that Albumin and HNF4. This observation therefore suggests that if inducing these progenitors grown as 3D organoids to differentiate, to give back mature hepatocytes, Matrigel suspension culture could present an advantage. Organoids transferred to differentiation conditions exhibit a more mature profile when cul- tured in Matrigel suspension compared to the Matrigel drop system. In light of the previous observation of a higher level of expression of Albumin and HNF4 in Matrigel suspension organoids, we then interrogated whether this could be correlated to a better inclination to differentiate when transferred to the DM differentiation media Indeed after 2 weeks in differentiation media in Matrigel suspension cells did exhibit a The expression of CYP3A4 and CYP3A7 seemed to be higher in Matrigel suspension too, however the difference was not significant. Surprisingly this differentiation was associated with a slight increase in the expression of the progenitors markers Epcam and Sox9 (Fig. 5-lower panel). expression compared to culture in 3D drops (Fig. 5-upper panel). The expression of CYP3A4 and CYP3A7 seemed to be higher in Matrigel suspension too, however the difference was not significant. Surprisingly this differentiation was associated with a slight increase in the expression of the progenitors markers Epcam and Sox9 (Fig. 5-lower panel). ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 Organoids transferred to differentiation conditions exhibit a more mature profile when cul- tured in Matrigel suspension compared to the Matrigel drop system. In light of the previous observation of a higher level of expression of Albumin and HNF4 in Matrigel suspension organoids, we then interrogated whether this could be correlated to a better inclination to differentiate when transferred to the DM differentiation media Indeed after 2 weeks in differentiation media in Matrigel suspension cells did exhibit a Organoids transferred to differentiation conditions exhibit a more mature profile when cul- tured in Matrigel suspension compared to the Matrigel drop system. In light of the previous observation of a higher level of expression of Albumin and HNF4 in Matrigel suspension organoids, we then interrogated whether this could be correlated to a better inclination to differentiate when transferred to the DM differentiation media. Indeed after 2 weeks in differentiation media, in Matrigel suspension cells did exhibit a more than 800 times higher level of Albumin, 150 times higher level of AFP, and close to 20 times more HNF4 g p p g p y g observation of a higher level of expression of Albumin and HNF4 in Matrigel suspension organoids, we then interrogated whether this could be correlated to a better inclination to differentiate when transferred to the DM differentiation media. Indeed after 2 weeks in differentiation media, in Matrigel suspension cells did exhibit a more than 800 times higher level of Albumin, 150 times higher level of AFP, and close to 20 times more HNF4 ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 6 www.nature.com/scientificreports/ Figure 5. Induction of differentiation of 3D organoids into mature hepatocytes is more efficient if cultured in Matrigel suspension. The mRNA expression level of different liver progenitor/mature hepatocyte/proliferation markers was analyzed by qPCR, in 3D culture of human hepatocytes inside Matrigel drops (3D-D – black bars) or in Matrigel suspension (3D-S – white bars), in expansion (EM) or differentiation media (DM). (N = 3). Figure 5. Induction of differentiation of 3D organoids into mature hepatocytes is more efficient if cultured in Matrigel suspension. The mRNA expression level of different liver progenitor/mature hepatocyte/proliferation markers was analyzed by qPCR, in 3D culture of human hepatocytes inside Matrigel drops (3D-D – black bars) or in Matrigel suspension (3D-S – white bars), in expansion (EM) or differentiation media (DM). (N = 3). Figure 5. Induction of differentiation of 3D organoids into mature hepatocytes is more efficient if cultured in Matrigel suspension. The mRNA expression level of different liver progenitor/mature hepatocyte/proliferation markers was analyzed by qPCR, in 3D culture of human hepatocytes inside Matrigel drops (3D-D – black bars) or in Matrigel suspension (3D-S – white bars), in expansion (EM) or differentiation media (DM). (N = 3). expression compared to culture in 3D drops (Fig. 5-upper panel). Discussion T In fact a recent work demonstrated that rat terminally differentiated hepatocytes can be converted into bipotent liver progenitors, also called chemically induced liver progenitors (CLiPs), with some of the chemicals included in the EM media27. When looking at the number of cells amplified, that would on the other hand mean that the dedifferentiation process would concern only a fraction of the initial plated population.hif f p y p p p The question whether the amplified cells were directly converted from differentiated hepatocytes or from some underlying precursors cells will be further investigated, it still remains from our observations that starting from purified human hepatocytes we can generate a progenitor population with an elevated proliferation poten- tial, while keeping the capacity to differentiate into mature hepatocytes. This is of considerable interest when considering the relatively easy procedure, and the great benefit of having the capacity of producing a big quantity of hepatocytes from short supply. To add on to the breakthrough made by Huch et al.18, which was to introduce 3D organoid expansion of liver progenitors, our results therefore suggest that an even more accessible source of hepatic cells would be frozen purified hepatocytes, commercially available and without the need of FACS-sorting EpCAM positive cells or using freshly isolated cells.i p p g y Our major finding is the demonstration that, when culturing 3D organoids in a suspension media that con- tains diluted Matrigel instead of embedded inside a pure Matrigel drop, culture conditions can be really improved to obtain 1- a more efficient differentiation, 2- a process compatible with production in bioreactors, and 3- poten- tially a higher rate of proliferation and a better stability on long term.f y g p y g First using Huch et al. differentiation media we report that cells maintained as 3D organoids in Matrigel sus- pension, while showing features of progenitors, preserve a higher level of expression of differentiation markers. That results in a more efficient conversion into mature hepatocytes when shifted to DM media, compared to 3D organoid in Matrigel drops (Fig. 5). This constitutes a remarkable advantage, as this new procedure not only could save time and money on hepatocytes production, but those observations also suggest that in the context of transplantation cells would most probably be more prone to finish their maturation in vivo, leading to optimized functionality of transplanted cells and rapidity of response in case of acute liver injury. Discussion T To overcome the serious shortage of donor livers the optimization of liver cell therapy is becoming essential, therefore different technologies are now developed for the generation of liver cells7. This includes differentiation of hepatocytes-like cells from iPS or ES cells, however some major issues remain especially with regard to their functionality, as the maturity displayed by those cells is questionable, and their genetic stability is uncertain yet22. Th The direct expansion of adult hepatocytes from healthy tissue would therefore be the method of choice, none- theless the culture of human mature hepatocytes in vitro is limited in time and is subject to a quick loss of func- tionality. Thanks to the 3D organoid protocol set up by Huch et al. in18, differentiated human hepatocytes can be produced in large quantity starting from the same batch of cells, avoiding variability from batch to batch and allowing a standardization of their phenotype. g p yp Based on this model, we propose a new 3D organoid system to produce in vitro large quantities of human mature hepatocytes starting from a low number of cryopreserved cells, with a protocol of culture in suspension allowing an easy cell passaging and large-scale amplification.i i Of note is the fact that whereas Huch et al. started from EpCAM positive cells, we did first start from human hepatocytes as fully differentiated hepatocytes. While they grew in 3D in expansion medium, the cells started to exhibit a phenotype of liver progenitors with a high proliferation capacity (Fig. 2). This was paralleled by a decrease in hepatocyte-specific functions, which is reversible as cells reacquire a differentiated phenotype when they are switched to the DM maturation media (Fig. 5). Surprisingly in contrast to Huch et al. who claimed that the Epcam negative hepatocyte fraction can not generate organoids, our results show that purified hepatocytes are able to develop into organoids with features of liver progenitors (Fig. 2). Their expression of SOX9, CK19 and 7 www.nature.com/scientificreports/ EpCAM however attest of their ductal origin, asking the question of the origin of these cells. Several hypotheses could explain this discrepancy. One cannot exclude the presence of some very rare EpCAM positive ductal con- taminant cells that took over the prevalent hepatocyte population. The uncovered plasticity of mature hepatocytes also allows us to consider some dedifferentiation into liver progenitors, as revealed by several previous works23–26. Discussion T y p p y p j y Secondly, on a practical aspect we designed an adaptation of the 3D liver organoids culture that is suitable on a large-scale, thanks to the suspension culture. Indeed this attribute makes the process adaptable for the culture inside bioreactors in large volumes of media. Importantly, while the 10% Matrigel suspension culture gave rise to organoids with a similar morphology and phenotype compared to Matrigel drop culture, this was not the case for classical suspension culture. By comparison the same hepatocytes incubated in suspension in the EM media, devoid of Matrigel, never formed organoids that could be maintained in culture, and rather ended up in aggre- gates (Fig. 3A- second picture) that eventually died. This difference may be explained by the crucial role played by the contact with the ECM, provided by the presence of Matrigel in the media28. p y p g Finally, when looking at the proliferation activity in cell culture, over time 3D organoids experience a decrease in proliferation (Fig. 1). They can be maintained in culture for several months, however their growth rate declines overtime. Similarly Huch et al. also observed a prolongation of the cell doubling time over time. Our preliminary data will have to be confirmed, especially on long term culture, but they suggest that the proliferation index would be maintained at a higher level over time when organoids are cultured in Matrigel suspension compared to Matrigel drop 3D culture (Fig. 3), offering a potential further optimization of the system by delaying this cellular exhaustion observed in culture.ii In summary, we hope these findings will be beneficial for the research on liver failure treatment, by providing a new way to generate large quantities of mature hepatocytes, after an intermediary step of expansion as bipotent progenitors growing in suspension. This protocol presents the advantage of using some easily accessible starting material, that is cryopreserved hepatocytes without FACS sorting. Finally, converting a system previously estab- lished as 3D solid polymer into a suspension culture makes it suitable for the production of hepatocytes in large containers and cell culture automation. As a perspective for a clinical use, this in vitro matrigel-expanded hepatocytes could be used in the context of external bioartificial liver devices with encapsulation, as some bioartificial liver devices with hepatic cells have been safely used for treating patients with end-stage liver diseases29–32. Methods ll l Cell culture. Cryoplateable primary human hepatocytes. Cryopreserved primary human hepatocytes were provided by Biopredic, and thawed according to the manufacturer instructions. Briefly, human primary hepat- ocytes were isolated from tumor-free margin of the resected liver tissue by a two-step collagenase perfusion technique. After washing steps, the obtained hepatocytes were purified by Percoll density gradient and then cry- opreserved using an optimized freezing process. After thawing, cells were plated on Collagen I-coated plates, and maintained in William’s E GlutaMAXTM medium (Life Technologies) supplemented with 4 μg/mL Bovine Insulin (Sigma) and 50 μM Hydrocortisone (Sigma). Details of hepatocytes batches are indicated in Fig. 1. Culture as 3D organoids in Matrigel drops (3D-D). Primary hepatocytes were cultured as 3D organoids accord- ing to the protocol set up by Huch and Clevers18. Briefly, after centrifugation primary hepatocytes were mixed with growth factor reduced Matrigel (Corning), and a 50 μL drop of this solution was plated in the middle of each well of a 24-well plate. For the first set of experiments, comparing organoïds formation in three differ- ent batches of human hepatocytes (Fig. 1), 30,000 cells per well have been plated. For the other experiments, focusing on HEP187269, 300,000 cells per well were used. After solidification of Matrigel the EM expansion media was added, composed of AdDMEM/F12 (Life Technologies) supplemented with 1% N2 and 1% B27 (Life ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 8 www.nature.com/scientificreports/ technologies), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma), 10 mM Nicotinamide (Sigma), 5 μM A83.01 (Tocris), 10 μM FSK (Tocris), and the growth factors: 50 ng/mL EGF (R&D Systems), 500 ng/mL Rspo1 (Peprotech), 100 ng/mL FGF10 (Miltenyi Biotec), 25 ng/mL HGF (Miltenyi Biotec). During the 3 first days after plating, the media was supplemented with 25 ng/mL Noggin (Peprotech), 50 ng/mL Wnt3a (R&D Systems), and 10 μM Y27632 (Stem Cell Technologies). technologies), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma), 10 mM Nicotinamide (Sigma), 5 μM A83.01 (Tocris), 10 μM FSK (Tocris), and the growth factors: 50 ng/mL EGF (R&D Systems), 500 ng/mL Rspo1 (Peprotech), 100 ng/mL FGF10 (Miltenyi Biotec), 25 ng/mL HGF (Miltenyi Biotec). During the 3 first days after plating, the media was supplemented with 25 ng/mL Noggin (Peprotech), 50 ng/mL Wnt3a (R&D Systems), and 10 μM Y27632 (Stem Cell Technologies). Culture as 3D organoids in Matrigel suspension (3D-S). Methods ll l Statistical analysis was performed using two-tailed unpaired t-test with GraphPad Prism software (GraphPad, USA). Statistical analysis. All results are presented as number of replicates (N) and mean value of replicates ± SD. Statistical analysis was performed using two-tailed unpaired t-test with GraphPad Prism software (GraphPad, USA). References 1. Michalopoulos, G. K. Hepatostat: Liver regeneration and normal liver tissue maintenance. Hepatology 65, 1384–1392 (2017). 2 Mi h l l G K & Kh Z Li St C ll E i t l Fi di d I li ti f H Li Di 2. Michalopoulos, G. K. & Khan, Z. Liver Stem Cells: Experimental Findings and Implications for Human Liver Disease. Gastroenterology 149, 876–882 (2015). gy ( ) 3. Fausto, N. & Campbell, J. S. The role of hepatocytes and oval cells in liver regeneration and repopulation. Mechanisms of Developmen 120, 117–130 (2003). 3. Fausto, N. & Campbell, J. S. The role of hepatocytes and oval cells in liver regeneration and repopulation. Mechanisms of Development 120, 117–130 (2003). 4 Kopp J L Grompe M & Sander M Stem cells versus plasticity in liver and pancreas regeneration Nat Cell Biol 18 238–245 h 120, 117–130 (2003). 4. Kopp, J. L., Grompe, M. & Sander, M. Stem cells versus plasticity in liver and pancreas regeneration. Nat. Cell Biol. 18, 238–245 ( ) , ( ) 4. Kopp, J. L., Grompe, M. & Sander, M. Stem cells versus plasticity in liver and pancreas regeneration. Nat. Cell Biol. 18, 238–245 (2016). ( ) 5. Miyajima, A., Tanaka, M. & Itoh, T. Stem/progenitor cells in liver development, homeostasis, regeneration, and reprogramming. Cell Stem Cell 14, 561–574 (2014). 6. Dhawan, A. Clinical human hepatocyte transplantation: Curre 6. Dhawan, A. Clinical human hepatocyte transplantation: Current status and challenges. Liver Transplant. 21, S39–S44 (2015). 7. Forbes, S. J., Gupta, S. & Dhawan, A. Cell therapy for liver disease: From liver transplantation to cell factory. Journal of Hepatolog 62, S157–S169 (2015). 8. Bhatia, S. N., Underhill, G. H., Zaret, K. S. & Fox, I. J. Cell and t 8. Bhatia, S. N., Underhill, G. H., Zaret, K. S. & Fox, I. J. Cell and tissue engineering for liver disease. Sci. Transl. Med. 6, 245sr2 (2014). 9. Guguen-Guillouzo, C. & Guillouzo, A. General review on in vitro hepatocyte models and their applications. Methods in molecular biology (Clifton, N.J.) 640, 1–40 (2010). gy ft 0. Schwartz, R. E., Fleming, H. E., Khetani, S. R. & Bhatia, S. N. Pluripotent stem cell-derived hepatocyte-like cells. Biotechnology Advances 32, 504–513 (2014).fi 11. Si-Tayeb, K. et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297–305 (2010).h 2. Hannoun, Z., Steichen, C., Dianat, N., Weber, A. Methods ll l Alternatively primary hepatocytes were cultured as 3D organoids in 10% Matrigel suspension, in similar conditions of culture except organoids were floating in Huch and Clevers EM media18 containing 10% of growth factors-reduced Matrigel, instead of embedded in a Matrigel drop. After dissociation of organoids the cell suspension was plated on Ultra-Low Attachment plates (Corning). Differentiation of organoids into hepatocytes. Differentiation of organoids into hepatocytes was induced as pre- viously described18. Briefly, after amplification organoids were first cultured in EM media supplemented by BMP7 (25 ng/ml) for 7–10 days, then split and plated back in the same media for 2–4 days. Organoids were subsequently switched to the DM differentiation media, composed as follows: AdDMEM/F12 medium (Life Technologies), 1% N2 and 1% B27 (Life Technologies), 50 ng/mL EGF (R&D Systems), 10 nM gastrin (Sigma), 25 ng/mL HGF (Miltenyi Biotec), 100 ng/mL FGF19 (R&D Systems), 500 nM A83.01 (Tocris), 10 μM DAPT (Sigma), 25 ng/mL BMP7 (Peprotech), and 30 μM Dexamethasone (Sigma). RNA isolation and Real-time RT-PCR. Total RNA was isolated and purified from cells using the RNeasy Mini Kit (Qiagen). Real-time reverse-transcription was performed starting from 5 ng RNA, with a one-step RT-PCR kit using Taqman technology (AgPath-ID™ One-Step RT-PCR, Life Technologies) and the spe- cific following probes: Albumin Hs00910225_m1; AFP Hs00173490_m1; HNF4A Hs00230853_m1; CYP3A4 Hs00604506_m1; CYP3A7 Hs00426361_m1; Cytokeratin 19 Hs00761767_s1; Epcam Hs00901885_m1; SOX9 Hs00165814_m1; Ki67 Hs00267195_m1)(Life Technologies) and using the Applied Biosystems ViiA 7 Real-Time PCR System. GAPDH was used as a housekeeping gene. Results were normalized as specified in figures legends. Immunocytochemistry. Cells were fixed in 4% PFA. After permeabilization in 0.5% Triton X-100, cells were blocked with 1% BSA and incubated overnight at 4 °C with the following primary antibodies: goat anti-Epcam antibody (R&D Systems), mouse anti-CK19 (Dako) or rabbit anti-Ki67 (Abcam). Secondary antibodies were then incubated for 1 h at room temperature (Life Technologies), and Prolong Gold mounting media with DAPI (Life Technologies) was used for mounting on a glass slide. Live/Dead staining kit. Viability on cells was assessed by using the Live/Dead Viability/Cytotoxicity Kit for mammalian cells (ThermoFisher Scientific), according to the manufacturer instructions. Briefly, a mix of 2 µM calcein AM and 4 µM EthD-1 was prepared and incubated with cells for 45 min at room temperature, the prepa- ration was then observed under a Zeiss fluorescent microscope. Statistical analysis. All results are presented as number of replicates (N) and mean value of replicates ± SD. Acknowledgementsh g This work was supported by ANR LabCom -14-LAB3-0001 - HULIVER, ANR-14-CE16-0026 pStemHepTher and by the IHU-Cesti project (French government financial support via the investment of the future program ANR-10-IBHU-005 and supports from Nantes Metropole and the Pays de la Loire Region). Author Contributions D.G., R.L., C.C., C.G.G. and T.H.N. conceived the experiment(s), D.G., F.D., A.F. and C.C. conducted the experiment(s), D.G. analysed the results and wrote the manuscript, R.L., C.G.G., C.C. and T.H.N. corrected the manuscript. All authors reviewed the manuscript. D.G., R.L., C.C., C.G.G. and T.H.N. conceived the experiment(s), D.G., F.D., A.F. and C.C. conducted the experiment(s), D.G. analysed the results and wrote the manuscript, R.L., C.G.G., C.C. and T.H.N. corrected the manuscript. All authors reviewed the manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 19. Broutier, L. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and genetic manipulation. Nat. Protoc. 11, 1724–43 (2016).l g p ( ) 20. Rennert, K. et al. A microfluidically perfused three dimensional human liver model. Biomaterials 71, 119–131 (2015).h l 21. Schneeberger, K. et al. Converging biofabrication and organoid technologies: The next frontier in hepatic and intestinal t engineering? Biofabrication 9 (2017). g g f 2. Rezvani, M., Grimm, A. A. & Willenbring, H. Assessing the therapeutic potential of lab-made hepatocytes. Hepatology 64, 287–294 (2016). 3. Krause, P., Unthan-Fechner, K., Probst, I. & Koenig, S. Cultured hepatocytes adopt progenitor characteristics and display bipoten capacity to repopulate the liver. Cell Transplant. 23, 805–817 (2014). p y p p p 4. Chen, Y., Wong, P. P., Sjeklocha, L., Steer, C. J. & Sahin, M. B. Mature hepatocytes exhibit unexpected plasticity by direc dedifferentiation into liver progenitor cells in culture. Hepatology 55, 563–574 (2012). f 25. Tarlow, B. D. et al. Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes. Cell Stem Cell 15, 605–618 (2014).l ( ) 26. Yimlamai, D. et al. Hippo pathway activity influences liver cell fate. Cell 157, 1324–1338 (2014). 27. Katsuda, T. et al. Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity. Cell Stem Cell 20, 41–55 (2017).ff 8. Kourouklis, A. P., Kaylan, K. B. & Underhill, G. H. Substrate stiffness and matrix composition coordinately control the differentiation of liver progenitor cells. Biomaterials 99, 82–94 (2016).ii p g 9. No, D. Y., Jeong, G. S. & Lee, S. H. Immune-protected xenogeneic bioartificial livers with liver-specific microarchitecture and hydrogel-encapsulated cells. Biomaterials 35, 8983–8991 (2014).i 29. No, D. Y., Jeong, G. S. & Lee, S. H. Immune-protected hydrogel-encapsulated cells. Biomaterials 35, 8983–8991 (2014 y g p 30. Lu, J. et al. A New Fluidized Bed Bioreactor Based on Diversion-Type Microcapsule Suspension for Bioartificial Liver Systems. PLoS One 11, e0147376 (2016). F S l O h fl d d b d b l b fi l l I J A f O ( ) ( ) 1. Figaro, S. et al. Optimizing the fluidized bed bioreactor as an external bioartificial liver. Int. J. Artif. Organs 40, 196–203 (2017).h 32. Thompson, J. et al. Extracorporeal cellular therapy (ELAD) in severe alcoholic hepatitis: A multinational, prospective, controlled, randomized trial. Liver Transplant. 24, 380–393 (2018). References & Dubart-Kupperschmitt, A. The potential of induced pluripotent stem cell derived hepatocytes. Journal of Hepatology 65, 182–199 (2016). p y f p gy 3. Du, C., Narayanan, K., Leong, M. F. & Wan, A. C. A. Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering. Biomaterials 35, 6006–6014 (2014).h i 14. Tolosa, L. et al. Transplantation of hESC-derived hepatocytes protects mice from liver injury. Stem Cell Res. Ther. 6, 246 (2015) C l ffi d ff f h f h b ll h b k l i 14. Tolosa, L. et al. Transplantation of hESC-derived hepatocytes p h 5. Hay, D. C. et al. Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating live development in vivo. Stem Cells 26, 894–902 (2008).ff 16. Tasnim, F., Phan, D., Toh, Y.-C. & Yu, H. Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Biomaterials 70, 115–125 (2015). g ( ) 17. Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013). 18. Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312 (2015). 17. Takebe, T. et al. Vascularized and functional human liver from an iPSC derived organ bud transplant. Nature 499, 481 484 (2013). 18. Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312 (2015). ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 9 ScIentIfIc REPOrTS \| (2018) 8:8222 \| DOI:10.1038/s41598-018-26584-1 © The Author(s) 2018 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26584-1. Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018- Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26584-1. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. ublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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W4200178190.txt	https://www.frontiersin.org/articles/10.3389/fpls.2021.674510/pdf	en		Table_3.docx	null	2,021	cc-by	6,061	ORIGINAL RESEARCH published: 10 December 2021 doi: 10.3389/fpls.2021.674510 Evaluation and Pre-selection of New Grapevine Genotypes Resistant to Downy and Powdery Mildew, Obtained by Cross-Breeding Programs in Spain Leonor Ruiz-García 1 , Pilar Gago 2 , Celia Martínez-Mora 1 , José Luis Santiago 2 , Diego J. Fernádez-López 1 , María del Carmen Martínez 2 and Susana Boso 2* 1 Department of Biotechnology, Genomics and Plant Breeding, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, Murcia, Spain, 2 Department of Viticulture and Forestry, Misión Biológica de Galicia (Consejo Superior de Investigaciones Científicas, CSIC), Salcedo, Spain Edited by: Omer Frenkel, Agricultural Research Organization (ARO), Israel Reviewed by: Eva Maria Zyprian, Institut für Rebenzüchtung, Julius Kühn-Institut, Germany Marisa Maia, University of Lisbon, Portugal Correspondence: Susana Boso susanab@mbg.csic.es Specialty section: This article was submitted to Plant Pathogen Interactions, a section of the journal Frontiers in Plant Science Received: 01 March 2021 Accepted: 22 November 2021 Published: 10 December 2021 Citation: Ruiz-García L, Gago P, Martínez-Mora C, Santiago JL, Fernádez-López DJ, Martínez MC and Boso S (2021) Evaluation and Pre-selection of New Grapevine Genotypes Resistant to Downy and Powdery Mildew, Obtained by Cross-Breeding Programs in Spain. Front. Plant Sci. 12:674510. doi: 10.3389/fpls.2021.674510 The need to develop an environmentally friendly, sustainable viticulture model has led to numerous grapevine improvement programmes aiming to increase resistance to downy and powdery mildew. The success of such programmes relies on the availability of protocols that can quantify the resistance/susceptibility of new genotypes, and on the existence of molecular markers of resistance loci that can aid in the selection process. The present work assesses the degree of phenotypic resistance/susceptibility to downy and powdery mildew of 28 new genotypes obtained from crosses between “Monastrell” and “Regent.” Three genotypes showed strong combined resistance, making them good candidates for future crosses with other sources of resistance to these diseases (pyramiding). In general, laboratory and glasshouse assessments of resistance at the phenotype level agreed with the resistance expected from the presence of resistanceassociated alleles of simple sequence repeat (SSR) markers for the loci Rpv3 and Ren3 (inherited from “Regent”), confirming their usefulness as indicators of likely resistance to downy and powdery mildew, respectively, particularly so for downy mildew. Keywords: downy mildew, powdery mildew, resistance, grapevine, marker-assisted breeding, Vitis INTRODUCTION Downy and powdery mildew (caused by Plasmopara viticola and Erysiphe necator, respectively) are some of the most important diseases of grapevines worldwide. Both reduce crop yield and quality leading to economic losses. Vitis vinifera is highly susceptible to both, while American and Asian vine species are much more resistant, perhaps due to their co-evolution with the causal pathogens (Armijo et al., 2016). The most efficient way to deal with them is chemical control, but this can have a negative biological and ecological impact; sustainable and environmentally friendly viticulture requires such treatment be reduced. Since the discovery of sources of resistance to these pathogens, many grapevine genetic improvement programmes around the world have tried to produce quality grapevine hybrids carrying the resistance genes of wild American vine species - but this has not been easy (Töpfer et al., 2011; Villano and Aversano, 2020). We still lack knowledge on the molecular Frontiers in Plant Science \| www.frontiersin.org 1 December 2021 \| Volume 12 \| Article 674510 Ruiz-García et al. Grapevine Genotypes Resistant to Mildews of nine SSR markers registered in the NCBI database2 (Supplementary Table 1). bases of such resistance, and several backcrosses are often required before a hybrid of sufficient winemaking quality and lasting disease resistance is obtained. Such programmes have involved the use of American vine species, e.g., Vitis riparia, Vitis rupestris, Muscadinia rotundifolia, Vitis cinerea, etc., or species from the Far East, such as Vitis piasezkii, Vitis amurensis, Vitis romanetii, or Vitis vinifera Kishmish vatkana (Merdinoglu et al., 2018; Maul and Töpfer, 2019). These show either partial resistance, or, in the case of M. rotundifolia and V. piasezkii, complete resistance (Wan et al., 2007; Bellin et al., 2009; Casagrande et al., 2011; Gessler et al., 2011). Many studies have also tried to compare the degree of resistance to P. viticola and E. necator of the hybrids produced in different improvement programmes (Hoffmann et al., 2008; Kozma et al., 2009; Vezzulli et al., 2017, 2019; Bove et al., 2020; Possamai et al., 2020). To date, 31 grapevine genomic regions have been associated with resistance to downy mildew (Rpv loci) and 13 with resistance to powdery mildew (Run loci and Ren loci) according to the table of loci for traits in grapevine relevant for breeding and genetics (update April 30, 20211 ). The availability of markers that reveal the presence of these loci could allow for the marker-assisted selection (MAS) of likely resistant genotypes (Eibach et al., 2007; Kozma et al., 2009; Vezzulli et al., 2019; Zini et al., 2019). The resistance actually shown by a genotype can then be tested in the field and under controlled laboratory/glasshouse conditions following established protocols (Brown et al., 1999; Boso et al., 2006, 2010, 2014; Prajongjai et al., 2014). New material resistant to both diseases coming out of improvement programmes includes the variety “Regent.” Obtained at the Julius-Kühn Institute in Germany, its pedigree includes American vines carrying Ren3, Ren9, Rpv3, Rpv4, and Rpv11 (Fischer et al., 2004; Welter et al., 2007; Zendler et al., 2017). The aim of the present work was to assess the degree of phenotypic resistance/susceptibility to downy and powdery mildew of 28 “Monastrell” × “Regent” hybrids selected for their carriage of resistance-associated alleles of simple sequence repeat (SSR) markers for the loci Rpv3 and Ren3, and thus being likely resistant to these diseases. The results allow for the selection of genotypes that could be used in future crosses with other sources of resistance to downy and powdery mildew (pyramiding). Amplification of Resistance-Associated Molecular Markers of Rpv3 and Ren3 The resistance-associated molecular markers of Rpv3 and Ren3 used in this work and their sequences are shown in Supplementary Table 2. PCR analyses to detect the presence of the SSR alleles-associated to resistance were performed according to Bayo-Canha et al. (2019). “Monastrell” and “Regent” were used as negative and positive controls, respectively. Resistance to Downy and Powdery Mildew Resistance to downy mildew was examined using the leaf disc test (laboratory conditions) (Rumbolz et al., 2002). Susceptibility to powdery mildew was examined using the (modified) method of Wang et al. (1995) (glasshouse conditions). Resistance to both diseases was also recorded via the use of official OIV descriptors (Organisation Internationale de la Vigne et du Vin [OIV], 2009), with small modifications. All tests were performed in triplicate. “Monastrell” and “Regent” were used as controls for the resistance assays. Plant Material Used At least 60 cuttings with 2–3 buds were taken in January from each of the field-grown genotypes. These were disinfected, dipped in paraffin wax and preserved in a cold chamber for at least 8 weeks to encourage later sprouting in a glasshouse under controlled conditions of temperature and humidity (25◦ C, RH > 95%, 16 h white light at 400–700 nm and 8 h darkness). Thirty plants of each genotype were used to examine resistance to either disease. Pathogen Material Plasmopara viticola and Erysiphe necator were obtained from plants naturally infected in the experimental vineyards of the Misión Biológica de Galicia (MBG-CSIC). For P. viticola, sporangia for inocula were propagated following the method of Rumbolz et al. (2002). For E. necator, no propagation was needed since plenty of conidia were already available on infected ‘Castañal’ host plants. To prepare the inoculum, fresh conidia were collected using a small paintbrush, placed in centrifuge tubes, and diluted with 50 ml of distilled water with 0.05% Tween 20, thus obtaining a final concentration of 50,000 conidia ml−1 . MATERIALS AND METHODS Plant Material The study material included 28 new genotypes obtained by crosses in 2012 (Ruiz-García et al., 2014) between “Monastrell” (susceptible to downy mildew and with medium resistance to powdery mildew) and “Regent” which carries the loci Rpv3 and Ren3 (Fischer et al., 2004; Welter et al., 2007). The identity of the parentals and of the 28 genotypes produced from them was performed by PCR amplification 1 Resistance to Downy Mildew (Laboratory Leaf Disc Tests) The 5th or 6th unfurled leaf on a green shoot of each of the 30 plants per genotype grown in the glasshouse (see above) was detached. Leaf discs were prepared and incubated according to Rumbolz et al. (2002). Disease incidence and disease severity were visually analysed as independent variables at 5 days postinoculation (dpi) according to the method of Boso et al. (2014). 2 https://www.vivc.de/ Frontiers in Plant Science \| www.frontiersin.org 2 https://www.ncbi.nlm.nih.gov/ December 2021 \| Volume 12 \| Article 674510 Ruiz-García et al. Grapevine Genotypes Resistant to Mildews and low severity (8%) scores. With respect to descriptor code OIV 452-1, “Monastrell” showed a score of 1, and “Regent” a score of 5 (Table 1). Among the 27 new genotypes tested, disease incidence ranged between 9 and 100%, and disease severity between 5 and 67%. With respect to the same descriptor code, and taking incidence and severity into account, six genotypes showed a resistance score of 9, six a score of 7, four a score of 5, one a score of 3, and 10 a score of 1. It should be noted that 12 genotypes (3_032, 3_082, 4_032, 4_063, 4_082, 5_022, 5_033, 5_060, 5_078, 5_107, 6_046, and 6_080) were transgressive with respect to the resistance donor “Regent” (OIV = 5), and showed greater resistance (OIV = 9 and 7). “Monastrell” and genotypes 3_094, 4_005, 4_037, 4_011, 4_136, 3_070, 3_073, 4_001, and 3_052 were the least resistant of all; they showed significantly higher incidence and severity scores (Table 1 and Supplementary Figure 3). Genotype 6_080 was significantly more resistant than the rest, with low severity and incidence scores (Figure 1a). Genotypes 5_078, 4_082, 4_063, 5_022, and 5_107 showed somewhat less resistance, with slightly higher incidence values but similar severity values. The remaining genotypes showed medium resistance, with intermediate disease incidence and severity scores. Resistance to Powdery Mildew (Glasshouse Conditions) Ten plants per genotype were challenged on their adaxial leaf surfaces by spraying with the prepared conidial suspension. They were then incubated for 5–6 days at 24◦ C, at an RH of <55%, and under long day conditions (white light at 400–700 nm, 16 h light and 8 h dark). At 5–6 dpi, disease incidence was calculated as the number of leaves with sporulating lesions per total number of leaves per plant, and disease severity as the percentage of leaf area showing symptoms of sporulation. Disease Assessment Using Organisation Internationale de la Vigne et du Vin Descriptors Descriptors recommended by the OIV [OIV452-1 for downy mildew (leaf discs inoculated with P. viticola sporangia) and OIV455-1 for powdery mildew (leaf in glasshouse inoculated with E. necator)] (Organisation Internationale de la Vigne et du Vin [OIV], 2009). Different scores for resistance to downy and powdery mildew are available in Supplementary Figures 1, 2. Statistical Analysis The differences between the studied variables were analysed by ANOVA using the fixed effects model (p < 0.001). Following ANOVA, significant F values were subjected to comparison using Fisher’s protected least significant difference (LSD) test (p < 0.05). The association between phenotypic resistance and that expected from the possession of resistance-associated alleles of the SSR markers was determined via the Chi squared test. All calculations were made using SAS V8.1 software (SAS Institute, Cary, NC; 2000). Powdery Mildew “Monastrell” showed high incidence (50%) and severity (65%) scores, while “Regent” showed scores of only 25 and 8%, respectively (p < 0.01). With respect to descriptor code OIV 455-1, “Monastrell” had a score of 5, while “Regent” scored 9. For the 28 new genotypes tested, disease incidence ranged between 0 and 53%, while severity ranged from 0 to 65%. With respect to the same descriptor code, and taking incidence and severity into account, 22 genotypes showed a resistance score of 9, two a score of 7, three a score of 5, and one a score of 3 (Table 1). With respect to descriptor code OIV 455-1, genotype 5_060 showed transgressive segregation and less strong resistance (OIV = 3) than “Monastrell” (OIV = 5). Genotype 5_060 and “Monastrell” showed significantly higher incidence and severity scores compared to the rest (Table 1; Figure 1b; Supplementary Figure 3). Genotypes 3_082, 4_063 and 4_124 showed medium resistance (lower severity score). “Regent” grouped with the genotypes showing the lowest incidence and severity values (Table 1 and Supplementary Figure 3); genotypes 3_058 and 5_107 showed no symptoms of disease at all (Table 1). RESULTS Presence of Resistance-Associated Simple Sequence Repeat Alleles Rpv3 Of the 28 genotypes examined, 18 were positive for the resistance-associated SSR allele UDV305_299 bp, 18 for UDV737_279 bp, 18 for UDV108_238 bp, and 18 for GF188_392 bp (Supplementary Table 3). The 18 genotypes that carried all four alleles were deemed likely resistant to downy mildew. Ren3 Of the 28 genotypes examined, 20 were positive for the resistanceassociated SSR allele GF15-42_199 bp, 19 for GF15-28_341 bp, 21 for GF15-30_446 bp, and 21 for VChr15CenGen06_283 bp (Supplementary Table 3). The 19 genotypes that carried all these alleles were deemed likely resistant to powdery mildew. Nine genotypes (genotypes 4_136, 5_022, 5_033, 5_107, 6_018, 6_025, 6_046, 6_080, and 6_125) carried the resistanceassociated SSR alleles for both Rpv3 and Ren3 (Supplementary Table 3), and were deemed likely resistant to both diseases. Combined Resistance to Both Diseases Genotypes 6_080, 5_107 and 4_082 showed the greatest combined resistance to both diseases, with incidence and severity values much lower than those shown even by “Regent” (Table 1). Phenotypic Resistance Association Between Phenotypic Resistance and Possession of Resistance-Associated Simple Sequence Repeat Alleles Downy Mildew Rpv3 “Monastrell” showed high disease incidence (100%) and severity scores (58%), while “Regent” showed medium incidence (54%) Supplementary Table 4 shows the association between possession of the SSR alleles UDV305_299 bp, UDV737_279 bp, Frontiers in Plant Science \| www.frontiersin.org 3 December 2021 \| Volume 12 \| Article 674510 Ruiz-García et al. Grapevine Genotypes Resistant to Mildews TABLE 1 \| Mean disease severity (DS), disease incidence (DI), OIV scores, and resistance genotype for downy mildew and powdery mildew. Phenotype post-inoculation with downy mildew Vine material DS (%) S.D. Monastrell 58.3a Regent 8.3cd 3_016 Phenotype post-inoculation with powdery mildew DI (%) S.D. OIV (452-1) DS (%) S.D. DI (%) S.D. OIV (455-1) Genotype 14.43 100.0a 0.00 1 65.0a 13.23 49.3ab 4.04 5 Susceptible 2.89 54.0bcd 2.00 5 8.3c 2.89 25.0de 0.00 9 DM_PM 5.0d 0.00 52.0bcde 0.00 5 0.0d 0.00 2.3ij 2.52 9 DM 3_025 not data not data not data not data not data 25.0b 0.00 13.6fgh 321% 9 DM 3_032 5.0d 0.00 36.3fghi 3.51 7 5.0cd 0.00 21.6ef 5.77 9 DM 3_052 46.6ab 20.21 100.0a 0.00 1 5.0cd 0.00 5.0hij 0.00 9 PM 3_058 30.0bc 5.00 100.0a 0.00 1 0.0d 0.00 0.0j 0.00 9 PM 3_070 50.0ab 25.00 100.0a 0.00 1 5.0cd 0.00 12.6fghi 2.52 9 PM 3_073 48.3ab 37.86 100.0a 0.00 1 5.0cd 0.00 1.6j 2.89 9 PM 3_082 5.0d 0.00 45.6cdef 2.08 7 25.0b 0.00 45.0ab 18.03 5 DM 3_094 63.3a 12.58 100.0a 0.00 1 5.0cd 0.00 0.0j 0.00 9 PM 4_001 48.3ab 37.86 100.0a 0.00 1 25.0b 0.00 33.3cd 14.43 7 PM 4_005 66.67a 14.43 100.0a 0.00 1 5.0cd 0.00 0.0j 0.00 9 PM 4_011 50.0ab 25.00 100.0a 0.00 1 5.0cd 0.00 1.6j 2.89 9 PM 4_032 11.6cd 11.55 40.0defg 0.00 7 5.0cd 0.00 28.0de 5.20 7 DM 4_037 55.0a 8.66 100.0a 0.00 1 5.0cd 0.00 9.3ghij 4.04 9 PM 4_063 5.0d 0.00 23.3ij 9.87 9 25.0b 0.00 40.0bc 15.00 5 DM DM 4_082 5.0d 0.00 20.0jk 0.00 9 5.0cd 0.00 6.6ghij 2.89 9 4_124 11.6cd 11.55 65.0b 17.32 3 25.0b 0.00 40.0bc 15.00 5 DM 4_136 50.0ab 25.00 100.0a 0.00 1 5.0cd 0.00 17.3efg 5.86 9 DM_PM 5_022 18.3cd 11.55 25.6hij 18.01 9 5.0cd 0.00 1.6j 2.89 9 DM_PM 5_033 5.0d 0.00 44.3cdef 12.01 7 5.0cd 0.00 23.3def 7.64 9 DM_PM 5_060 5.0d 0.00 38.9efgh 10.65 7 65.0a 13.23 53.3a* 5.77 3 DM 5_078 5.0d 0.00 20.0jk 4.00 9 5.0cd 0.00 10.0ghij 0.00 9 PM 5_107 5.0d 0.00 30.0ghij 0.00 9 1.6d 0.00 0.0j 0.00 9 DM_PM 6_018 5.0d 0.00 55.6bc 25.58 5 5.0cd 0.00 21.6ef 2.89 9 DM_PM 6_025 5.0d 0.00 55.3bc 21.57 5 5.0cd 0.00 7.5ghij 3.54 9 DM_PM 6_046 11.6cd 11.55 40.3defg 4.51 7 5.0cd 0.00 6.6ghij 2.89 9 DM_PM 6_080 5.0d 0.00 8.6k 4.16 9 5.0cd 0.00 3.0hij 1.73 9 DM_PM 6_125 11.0cd 12.17 51.3bcde 1.15 5 5.0cd 0.00 1.6j 2.89 9 DM_PM LSD (0.05) 24.49 14.15 5.71 2.95 a Means S.D., standard deviation. with the same letter are not significantly different (LSD test). *Result for resistance based on the molecular findings: PM, Powdery mildew resistant; DM, Downy mildew resistant; PM_DM, Powdery and downy midew resistant. Ren3 UDV108_238 bp, and GF18-8_392 bp and the actual phenotypic resistance shown to downy mildew. Of the 16 genotypes (3_016, 3_032, 3_082, 4_032, 4_063, 4_082, 5_022, 5_033, 5_060, 5_078, 5_107, 6_018, 6_025, 6_046, 6_080, and 6_125) with resistance equal to or greater than that shown by “Regent” (OIV 452-1 = 9, 7, and 5), 15 (all genotypes except for 5_078) possessed all four alleles (Supplementary Table 3). Genotype 5_078 gave a false negative result for resistance based on the molecular findings (Table 1). Of the 11 genotypes (3_052, 3_058, 3_070, 3_073, 3_082, 3_094, 4_001, 4_005, 4_011, 4_032, 4_037, 4_063, 4_082, 4_124, and 4_136) showing less resistance than “Regent” (OIV 452-1 = 1 and 3), nine (all genotypes except for 4_124 and 4_136) did not have all the above alleles (Supplementary Table 3). Genotypes 4_124 and 4_136 gave false positive results for resistance based on molecular findings (Table 1). The carriage of all four alleles was significantly associated (χ2 = 19.57, p ≤ 0.001) with actual phenotypic resistance. Frontiers in Plant Science \| www.frontiersin.org Supplementary Table 4 also shows the association between detection of the SSR alleles GF15-42_199 bp, GF15-28_341 bp, GF15-30_446 bp, and VChr15CenGen06_283 bp and actual phenotypic resistance shown to powdery mildew. Of the 24 genotypes (3_016, 3_025, 3_032, 3_052, 3_058, 3_070, 3_073, 3_094, 4_001, 4_005, 4_011, 4_032, 4_037, 4_082, 4_136, 5_022, 5_033, 5_078, 5_107, 6_018, 6_025, 6_046, 6_080, and 6_125) with resistance similar to “Regent” (OIV 4551 = 7 and 9), 19 (all genotypes except 3_016, 3_025, 3_032, 4_032 and 4_082) carried all four alleles (Supplementary Table 3). Genotypes 3_016; 3_025; 3_032; 4_032; 4_082 gave false negative results based on molecular findings (Table 1). The four genotypes (3_082, 4_063, 4_124 and 5_060) that showed actual phenotypic resistance well below that shown by “Regent” (OIV 455-1 = 1, 3 or 5) did not carry all four alleles (Supplementary Table 3). The carriage of all four alleles was 4 December 2021 \| Volume 12 \| Article 674510 Ruiz-García et al. Grapevine Genotypes Resistant to Mildews different degrees of resistance/susceptibility were seen among those genotypes generally classified as resistant or susceptible (Table 1). The greatest resistance to downy mildew was shown by genotypes 6_080 and 4_082, while 4_005 showed the greatest susceptibility. With respect to powdery mildew, genotypes 3_058 and 5_107 showed the greatest resistance, while 5_060 showed the greatest susceptibility. The present data support the idea that transgressive segregation is common in plant breeding populations, with a number of recombinants appearing as outliers with respect to the resistance shown by the parental phenotypes (Mackay et al., 2021). With regard to downy mildew descriptor code OIV 452-1, 12 genotypes were transgressive with respect to the resistance donor “Regent” (OIV = 5), showing greater resistance than that genotype (OIV = 7 or 9). Similar results were obtained by Vezzulli et al. (2019) in a segregating population for resistance to downy mildew. These extreme phenotypes suggest the presence of unidentified resistance factors that segregate in the breeding populations and result in minor but significant effects. The causes of transgressive segregation may be genetic (positive or negative complementation of additive alleles, epistatic interactions of unique parental attributes, the unmasking of recessive alleles from a heterozygous parent, or any combinations of these mechanisms) or environmental. Zanghelini et al. (2019) agree with the present hyopthesis that a more environmentally friendly way to control grapevine disease would be to select new genotypes with combinations of resistance loci. In the present work, genotypes 6_080, 5_107 and 4_082 showed the greatest resistance to both downy and powdery mildew, and indeed these genotypes have been selected as the best progenitors in an IMIDA breeding program with the aim of combining their characteristics with those conferred by other resistance loci to downy mildew (e.g., Rpv10) and powdery mildew (e.g., Ren1), and thus help maintain the durability of resistance (pyramiding) (Eibach et al., 2007). The possession of alleles of the SSRs used as markers of resistance was significantly associated with the actual phenotypic resistance to downy (especially) and powdery mildew, confirming that these markers can be used in plant improvement programmes designed to reduce the current use of pesticides. With respect to downy mildew, only two false positives were obtained (by genotypes 4_124 and 4_136, i.e., they were supposedly resistant but actually susceptible), and one false negative (by genotype 5_078, i.e., supposedly susceptible but actually showed resistance). With respect to powdery mildew, five false negative results were noted (provided by genotypes 3_016, 3_025, 3_032, 4_032, and 4_082. The additional fine mapping of the areas of the genome where different R-loci have been identified might provide more robust markers for use in marker-assisted selection (Zini et al., 2019; Zendler et al., 2021). Overall, these results confirm that MAS can be of great use in traditional improvement programmes, allowing for the selection of material with resistance to disease. However, MAS is not always as efficient as expected, possibly due to a relatively loose association between QTL alleles and the level of infection (Hospital, 2009). FIGURE 1 \| (a) Images of leaf discs from genotypes showing different degrees of resistance to downy mildew (6 dpi), as well as greater resistance than “Regent” (no sporulation) or greater susceptibility than “Monastrell” (white downy mildew sporulation on the abaxial surface of leaf discs). (A) “Regent”; (B) “Monastrell”; (C) genotype 6_080 (resistant); (D) genotype 4_001 (susceptible). (b) Images of plants, grown under glasshouse conditions, belonging to genotypes showing different degrees of resistance to powdery mildew (6 dpi). (A) “Regent”; (B) “Monastrell”; (C) genotype 5_107 (resistant); (D) 5_060 (susceptible). significantly associated (χ2 = 10.70, p ≤ 0.025) with actual phenotypic resistance. DISCUSSION The present leaf disc and plant inoculation results for resistance are reminiscent of those reported by other authors (Staudt and Kassemeyer, 1995; Prajongjai et al., 2014; Vezzulli et al., 2017) who indicate that non-vinifera hybrids are not fully resistant to downy and powdery mildew, and that the degree of resistance is a segregable trait. Indeed, in the present work, Frontiers in Plant Science \| www.frontiersin.org 5 December 2021 \| Volume 12 \| Article 674510 Ruiz-García et al. Grapevine Genotypes Resistant to Mildews The introduction of resistance genes from Vitis species into a V. vinifera variety is a long and costly process, and in any event resistance may be overcome by particularly virulent pathogens. This is why the durability of resistance is crucial - particularly in the case of a perennial species like the vine. Plants that combine several resistance factors might be expected to show greater durability of resistance, even if they display the same level of resistance as those bearing only one resistance factor. MAS can be used to identify those genotypes that combine desired resistance factors, thus helping in the generation of varieties of greater potential for resistance durability. However, it does not appear to be so useful in identifying small-effect loci that can enhance the protection conferred by major genes and thus improve their durability (Merdinoglu et al., 2018). Hence the importance of combining MAS with phenotypic characterisation; this should allow for the better determination of the degree of resistance. Breeding programs should be vigilant of any advances made in molecular biology and genomic selection that might help construct varieties with highly durable resistance (Meuwissen et al., 2001; Merdinoglu et al., 2018). In conclusion, cross-breeding programs generate great variation and allow for the selection of new genotypes that can promote a more sustainable and environmentally friendly form of viticulture - as long as the winemaking quality of their grapes is confirmed. This variation may include extreme phenotypes that show greater resistance than the actual donor. The combination of phenotypic characterisation and molecular selection is very useful, allowing the degree of resistance achieved in new genotypes, and the durability of that resistance, to be more accurately determined. The very resistant lines discussed in the present work provide valuable material for obtaining durably resistant genotypes, and should help characterise the molecular basis of resistance to downy and powdery mildew. contributed to the final version and read and approved the final manuscript. FUNDING This work was financed by the European Regional Development Fund (80%) through Projects PO07-037, FEDER1420-04, and FEDER1420-29, with the collaboration of the Region of Murcia (20%). ACKNOWLEDGMENTS The authors thank Iván González, Elena Zubiaurre, and Ana Fuentes-Denia for technical assistance and Sergio LucasMiñano, Adrián Yepes-Hita, and José Antonio MartínezJiménez for plant management in the field. PCR products were separated by capillary electrophoresis in an ABI Prism 3730 sequencer at the Unidad de Genómica-Universidad Complutense de Madrid/Parque Científico de Madrid, Spain. The English manuscript was prepared by Adrian Burton (www.physicalevidence.es). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. 674510/full#supplementary-material Supplementary Figure 1 \| Resistance to downy mildew as recorded by OIV 452-1 descriptors (leaf discs inoculated with P. viticola sporangia). The scores for resistance to downy mildew were: OIV-1, very low resistance (dense sporulation over large lesions, 100% of discs affected); OIV-3, low resistance (dense sporulation over medium-sized lesions, 65–99% of discs affected; OIV-5, medium resistance (little sporulation over small-mid size lesions, 47–64% of discs affected; OIV-7, strong resistance (scant sporulation over small lesions; 31–46% of discs affected); OIV-9, very strong resistance (scant sporulation over small lesions; <30% of discs affected). DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author. Supplementary Figure 2 \| Resistance to powdery mildew as recorded by descriptor OIV 455-1 (leaf in glasshouse inoculated with E. necator). The scores for resistance to powdery mildew were: 1, very low resistance [dense sporulation over the entire leaf surface (Adaxial) (estimating an average for all leaves)], >75 of plants affected); 3, low resistance (dense sporulation over 65–100%, 50–75% of plants affected); 5, medium resistance (sporulation over 25–65%, 36–50% of plants affected); 7, strong resistance (scant sporulation over 5–25, 25–35% of plants affected); 9, very strong resistance (sporulation over ≤5%, <25% plants affected). AUTHOR CONTRIBUTIONS LR-G, SB, and MM proposed the study, planned and directed it, set goals, undertook experimental work, analysed and interpreted the results, and wrote the draft of the manuscript. JS helped to wrote the draft of the manuscript. PG and CM-M undertook experimental work and helped to wrote the draft of the manuscript. DF-L undertook experimental work. All authors Supplementary Figure 3 \| Relationship between disease incidence and severity for downy (leaf disc test) and powdery (glasshouse observation) mildew. Alleles of DMR = SSR alleles associated with resistance to downy mildew (all four alleles present). Alleles of PMR = SSR alleles associated with resistance to downy mildew (all four alleles present). DM_PM resistant to downy and powdery mildew. REFERENCES strategies and host response scenarios. Front. Plant Sci. 7:382. doi: 10.3389/ fpls.2016.00382 Bayo-Canha, A., Costantini, L., Fernandez-Fernandez, J. I., Martinez-Cutillas, A., and Ruiz-Garcia, L. (2019). QTLs related to berry acidity identified in a wine Armijo, G., Schlechter, R., Agurto, M., Munoz, D., Nunez, C., and Arce-Johnson, P. (2016). Grapevine pathogenic microorganisms: understanding infection Frontiers in Plant Science \| www.frontiersin.org 6 December 2021 \| Volume 12 \| Article 674510 Ruiz-García et al. Grapevine Genotypes Resistant to Mildews grapevine population grown in warm weather. Plant Mol. Biol. Rep. 2019:11456. doi: 10.1007/s11105-019-01145-6 Bellin, D., Peressotti, E., Merdinoglu, D., Wiedemann-Merdinoglu, S., AdamBlondon, A. F., Cipriani, G., et al. (2009). Resistance to Plasmopara viticola in grapevine ‘Bianca’ is controlled by a major dominant gene causing localised necrosis at the infection site. Theor. Appl. Genet. 120, 163–176. doi: 10.1007/ s00122-009-1167-2 Boso, S., Martinez, M. C., Unger, S., and Kassemeyer, H.-H. (2006). Evaluation of foliar resistance to downy mildew in different cv. Albariño clones. Vitis 4, 23–27. Boso, S., Alonso-Villaverde, V., Santiago, J. L., Gago, P., Dürrenberg, M., Duggelin, M., et al. (2010). Macro- and microscopic leaf characteristics of six grapevine genotypes (Vitis spp.) with different susceptibilities to grapevine downy mildew. Vitis 49, 43–50. Boso, S., Alonso-Villaverde, V., Gago, P., Santiago, J. L., and Martínez, M. C. (2014). Susceptibility to downy mildew (Plasmopara viticola) of different Vitis varieties. Crop Prot. 63, 26–35. doi: 10.1016/j.cropro.2014.04.018 Bove, F., Savary, S., Willocquet, L., and Rossi, V. (2020). Designing a modelling structure for the grapevine downy mildew pathosystem. Eur. J. Plant Pathol. 157, 251–268. doi: 10.1007/s10658-020-01974-2 Brown, M. V., Moore, J. N., Fenn, P., and McNew, R. W. (1999). Comparison of leaf disk, greenhouse, and field screening procedures for evaluation of grape seedlings for downy mildew resistance. HortScience 34, 331–333. doi: 10.21273/ HORTSCI.34.2.331 Casagrande, K., Falginella, L., Castellarin, S. D., Testolin, R., and Di Gaspero, G. (2011). Defence responses in Rpv3-dependent resistance to grapevine downy mildew. Planta 234, 1097–1109. doi: 10.1007/s00425-011-1461-5 Di Gaspero, G., Cipriani, G., Marrazzo, M. T., Andreetta, D., Prado Castro, M. J., Peterlunger, E., et al. (2005). Isolation of (AC)n-microsatellites in Vitis vinifera L. and analysis of genetic background in grapevines under marker assisted selection. Mol. Breed. 15, 11–20. doi: 10.1007/s11032-004-1362-4 Di Gaspero, G., Copetti, D., Coleman, C., Castellarin, S. D., Eibach, R., Kozma, P., et al. (2012). Selective sweep at the Rpv3 locus during grapevine breeding for downy mildew resistance. Theor. Appl. Genet. 124, 277–286. doi: 10.1007/ s00122-011-1703-8 Eibach, R., Zyprian, E., Welter, L., and Töpfer, R. (2007). The use of molecular markers for pyramiding resistance genes in grapevine breeding. Vitis 46, 120– 124. Fischer, B. M., Salakhutdinov, I., Akkurt, M., Eibach, R., Edwards, K. J., Töpfer, R., et al. (2004). Quantitative trait locus analysis of fungal disease resistance factors on a molecular map of grapevine. Theor. Appl. Genet. 108, 501–515. doi: 10.1007/s00122-003-1445-3 Gessler, C., Pertot, I., and Perazzolli, M. (2011). Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathol. Mediterr. 50, 3–44. Hoffmann, S., Di Gaspero, G., Kovacs, L., Howard, S., Kiss, E., Galbacs, Z., et al. (2008). Resistance to Erysiphe necator in the grapevine ‘Kishmish vatkana’ is controlled by a single locus through restriction of hyphal growth. Theor. Appl. Genet. 116, 427–438. doi: 10.1007/s00122-0070680-4 Hospital, F. (2009). Challenges for effective marker-assisted selection in plants. Genetica 136, 303–310. doi: 10.1007/s10709-0089307-1 Kozma, P., Kiss, E., Hoffmann, S., Galbacs, Z., and Dula, T. (2009). Using the powdery mildew resistant Muscadinia rotundifolia and Vitis vinifera ‘Kishmish vatkana’ for breeding new cultivars. Acta Hortic. 827, 559–564. doi: 10.17660/ ActaHortic.2009.827.97 Mackay, I. J., Cockram, J., Howell, P., and Powell, W. (2021). Understanding the classics: the unifying concepts of transgressive segregation, inbreeding depression and heterosis and their central relevance for crop breeding. Plant Biotechnol. J. 19, 26–34. doi: 10.1111/pbi. 13481 Maul, E., and Töpfer, R. (2019). VIVC-Vitis International Variety Catalogue. Available online at: http://www.vivc.de/ (accessed on May 31, 2019) Merdinoglu, D., Schneider, C., Prado, E., Wiedemann-Merdinoglu, S., and Mestre, P. (2018). Breeding for durable resistance to downy and powdery mildew in grapevine. OENO One 52, 203–209. doi: 10.20870/oeno-one.2018.52.3. 2116 Frontiers in Plant Science \| www.frontiersin.org Meuwissen, T. H. E., Hayes, B. J., and Goddard, M. E. (2001). Prediction of total genetic value using genome-wide dense marker maps. Genetics 157, 1819–1829. doi: 10.1093/genetics/157.4.1819 Organisation Internationale de la Vigne et du Vin [OIV] (2009). Code des caractères descriptifs des variétés et espèces de Vitis. Deuxième édition. Paris: Dedon. 232. Possamai, T., Migliaro, D., Gardiman, M., Velasco, R., and De Nardi, B. (2020). Rpv mediated defense responses in grapevine offspring resistant to Plasmopara viticola. Plants 9:781. doi: 10.3390/plants906 0781 Prajongjai, T., Poolsawat, O., Pornbungkerd, P., Wongkaew, S., and Tantasawat, P. A. (2014). Evaluation of grapevines for resistance to downy mildew (Plasmopara viticola) under laboratory and field conditions S. Afr. J. Enol. Viticult. 35, 43–50. doi: 10.21548/35-1-983 Ruiz-García, L., Fuentes, A., García, B., Martínez, J. A., and MartínezCutillas, A. (2014). Selection of new fungal disease resistant grapevine varieties generated by crosses involving Monastrell. Proceedings of the 7th International Workshop on Grapevine Downy and Powdery Mildew. Vitoria. 155–156. Rumbolz, J., Wirtz, S., Kassemeyer, H.-H., Guggenheim, R., Schäfer, E., and Büche, C. (2002). Sporulation of Plasmopara viticola: differentiation and light regulation. Plant Biol. 4, 413–422. doi: 10.1055/s-200232342 Staudt, G., and Kassemeyer, H. (1995). Evaluation of downy mildew resistance in various accessions of wild Vitis species. Vitis 34, 225–228. Töpfer, R., Hausmann, L., and Eibach, R. (2011). “Molecular breeding,” in Genetics, genomics, and breeding of grapes, eds A.-F. Adam-Blondon, J.-M. MartinezZapater, and C. Kole (Boca Raton: CRC Press), 160–185. doi: 10.1201/b1 0948 Van Heerden, C. J., Phyllis, B., Abraham, V., and Renée, P. (2014). Detection of downy and powdery mildew resistance QTL in a ‘Regent’ x ‘RedGlobe’ population. Euphytica 200, 281–295. doi: 10.1007/s10681-014-1167-4 Vezzulli, S., Vecchione, A., Stefanini, M., and Zulini, L. (2017). Downy mildew resistance evaluation in 28 grapevine hybrids promising for breeding programs in Trentino region (Italy). Eur. J. Plant Pathol. 150, 485–495. doi: 10.1007/ s10658-017-1298-2 Vezzulli, S., Malacarne, G., Masuero, D., Vecchione, A., Dolzani, C., Goremykin, V., et al. (2019). The Rpv3-3 haplotype and stilbenoid induction mediate downy mildew resistance in a grapevine interspecific population. Front. Plant Sci. 10:234. doi: 10.3389/fpls.2019.00234 Villano, C., and Aversano, R. (2020). Towards grapevine (Vitis vinifera L.) mildews resistance: molecular defence mechanisms and new breeding technologies. Italus Hortus 27, 1–17. doi: 10.26353/j.itahort/2020.3.0117 Wan, Y., Schwaniniger, H., He, P., and Wang, Y. (2007). Comparison of resistance to powdery mildew and downy mildew in Chinese wild grapes. Vitis 2007, 132–136. Wang, Y., Li, Y., He, P., Chen, J., Lamikanra, O., and Lu, J. (1995). Evaluation of foliar resistance to Uncinula necator in Chinese wild Vitis species. Vitis 34, 159–164. Welter, L. J., Göktürk-Baydar, N., Akkurt, M., Maul, E., Eibach, R., Töpfer, R., et al. (2007). Genetic mapping and localization of quantitative trait loci affecting fungal disease resistance and leaf morphology in grapevine (Vitis vinifera L). Mol. Breed. 20, 359–374. doi: 10.1007/s11032-007-9097-7 Zanghelini, J. A., Bogo, A., Dal Vesco, L. L., Gomes, B. R., Mecabo, C. V., Herpich, C. H., et al. (2019). Response of PIWI grapevine cultivars to downy mildew in highland region of southern Brazil. Eur. J. Plant Pathol. 154, 1051–1058. doi: 10.1007/s10658-019-01 725-y Zendler, D., Schneider, P., Topfer, R., and Zyprian, E. (2017). Fine mapping of Ren3 reveals two loci mediating hypersensitive response against Erysiphe necator in grapevine. Euphytica 213:68. doi: 10.1007/s10681-0171857-9 Zendler, D., Topfer, R., and Zyprian, E. (2021). Confirmation and fine mapping of the resistance locus Ren9 from the grapevine cultivar ‘Regent’. Plants 2021:24. doi: 10.3390/plants10010024 Zini, E., Dolzani, C., Stefanini, M., Gratl, V., Bettinelli, P., Nicolini, D., et al. (2019). R-loci arrangement versus downy and powdery mildew resistance level: a Vitis hybrid survey. Int. J. Mol. Sci. 20:3526. doi: 10.3390/ijms2014 3526 7 December 2021 \| Volume 12 \| Article 674510 Ruiz-García et al. Grapevine Genotypes Resistant to Mildews Zyprian, E., Ochßner, I., Schwander, F., Šimon, S., Hausmann, L., Bonow-Rex, M., et al. (2016). Quantitative trait loci affecting pathogen resistance and ripening of grapevines. Mol. Genet. Genom. 291, 1573–1594. doi: 10.1007/s00438-0161200-5 the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2021 Ruiz-García, Gago, Martínez-Mora, Santiago, Fernádez-López, Martínez and Boso. 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https://openalex.org/W4283709413	https://www.researchsquare.com/article/rs-1729255/latest.pdf	English	null	Infants with Macrosomia and Infants of Diabetic Mothers Have Increased Carotid Artery Intima- Media Thickness in Childhood	Research Square (Research Square)	2,022	cc-by	7,252	Infants with Macrosomia and Infants of Diabetic Mothers Have Increased Carotid Artery Intima- Media Thickness in Childhood Hacer Yapicioglu (  hyapicioglu@cu.edu.tr Cukurova University Sultan Ceren Seckin Cukurova University Ahmet Yontem Cukurova University Dincer Yildizdas Cukurova University Hacer Yapicioglu (  hyapicioglu@cu.edu.tr ) Sultan Ceren Seckin Cukurova University Ahmet Yontem Cukurova University Dincer Yildizdas Cukurova University Abstract Purpose: Incidence of diabetes during pregnancy is increasing worldwide, and intrauterine hyperglycemia exposure may have long-term adverse effects on the cardiovascular health of children. We investigated risk of atherosclerosis and carotid intima-media thickness (CIMT) in infants born macrosomic and in infants of diabetic mothers (IDM) at the age of 8–9 years. Method: 49 infants of diabetic mothers (IDM group) and 13 macrosomic infants (Macrosomic group) were included in the study. They were compared with 26 age-matched healthy children with birth weight appropriate for gestational age born to non-diabetic mothers (Control group). Anthropometric measurements, atherosclerosis risk factors and CIMT measurements were performed. Results: There was no significant difference between the groups in terms of age, gender, actual anthropometric measurements, blood pressure measurements, laboratory parameters or atherosclerosis risk factors. Gestational age was lower in the IDM group (p<0.001), while birth weight was higher in the Macrosomic group (p<0.001). High-density lipoprotein cholesterol level was lower in the IDM group than the other groups. Duration of exclusive and total breastfeeding were lower in IDM group than in Control group (p<0.001 for both). Body mass index, skinfold thickness, waist-to-hip ratio and waist-to-height ratio were higher in those breastfed for less than 6 months in the IDM group. The CIMT values were statistically higher in IDM [0.43±.0.047 (0.34-0.60)] and Macrosomic [0.40±0.055 (0.33-0.50)] groups than Control group [0.34±0.047 (0.26-0.45)]. Conclusion, CIMT values were higher in IDM and Macrosomic groups. This indicates intrauterine exposure in both groups. Breastfeeding seems very important for IDMs. Research Article Keywords: Atherosclerosis risk factors, carotid artery intima-media thickness, infants of diabetic mothers, macrosomic infants, breastfeeding Posted Date: June 28th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1729255/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: June 28th, 2022 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/18 1. Introduction Incidence of gestational diabetes mellitus (GDM) is increasing worldwide and many babies are exposed to hyperglycemia inutero [1]. Comparison of siblings before and after the development of maternal DM show the role of epigenetic transmission on long-term negative effects on the cardiovascular health of the offspring [2]. Fetal 'poor metabolic memory' resulting from intrauterine hyperglycemia, hyperinsulinemia and leptin resistance cause offspring obesity, diabetes mellitus (DM) and metabolic disturbances, endothelial dysfunction and hypertension causing cardiovascular diseases (CVD) [2]. Alterations in insulin-like growth factor-1 and growth hormone axis are also risk factors for CVDs; both hypersecretion and hyposecretion of growth hormone increase the risk [3]. Hyperinsulinemia in the fetus caused acts as a growth hormone and results in increased fetal fat tissue, glycogen stores and body mass [4]. Both macrosomic infants and infants of diabetic mothers (IDM) have an increased risk of obesity in childhood and a high growth trajectory, resulting in an emphasis on such factors as antenatal counseling, breastfeeding and complementary feeding to reduce the effect of this adverse condition [5– 7] Incidence of gestational diabetes mellitus (GDM) is increasing worldwide and many babies are exposed to hyperglycemia inutero [1]. Comparison of siblings before and after the development of maternal DM show the role of epigenetic transmission on long-term negative effects on the cardiovascular health of the offspring [2]. Fetal 'poor metabolic memory' resulting from intrauterine hyperglycemia, hyperinsulinemia and leptin resistance cause offspring obesity, diabetes mellitus (DM) and metabolic disturbances, endothelial dysfunction and hypertension causing cardiovascular diseases (CVD) [2]. Alterations in insulin-like growth factor-1 and growth hormone axis are also risk factors for CVDs; both hypersecretion and hyposecretion of growth hormone increase the risk [3]. Hyperinsulinemia in the fetus caused acts as a growth hormone and results in increased fetal fat tissue, glycogen stores and body mass [4]. Both macrosomic infants and infants of diabetic mothers (IDM) have an increased risk of obesity in childhood and a high growth trajectory, resulting in an emphasis on such factors as antenatal counseling, breastfeeding and complementary feeding to reduce the effect of this adverse condition [5– 7] Page 2/18 Ultrasonographic measurement of carotid intima-media thickness (CIMT) is a non-invasive, safe, validated and reproducible technique for the assessment of the risk of subclinical vascular disease [8]. Increased CIMT is a strong predictor of future CVDs, including coronary heart disease, myocardial infarction, stroke, and peripheral artery disease. 2.2. Exclusion criteria for the Patient and Control groups 2.2. Exclusion criteria for the Patient and Control groups Children with a gestational age of < 34 weeks, birth weight of < 1500 g, with congenital anomalies, chromosomal anomalies and a history of birth asphyxia, children with any neurological disorder or mental retardation, children with orthopedic disorders that may affect measurements, children with known chronic kidney disease, congenital heart disease (except for hemodynamically insignificant ventricular septal defect, atrial septal defect, and patent ductus arteriosus), Kawasaki disease, childhood cancers or chronic inflammatory bowel disease, chronic infections (Hepatitis B, C, HIV etc.), previously diagnosed dyslipidemia, genetic and metabolic disorders, and children whose parents did not give their consent were not included in the study. Blood samples were gained after 8–12 hours of fasting and stored at -80°C until biochemical study. 2.1. Study Design The study included children of mothers with Type 1 DM, Type 2 DM and GDM who were born in Cukurova University Hospital in 2011 and 2012 (IDM group), and the children of healthy mothers with a birth weight of > 4000 g (Macrosomic group). The “Control group” consisted of children born in the same years to healthy, non-diabetic mothers, who were appropriate for gestational age and not macrosomic. The study was approved by Çukurova University Ethics Committee (Date: October 4, 2019, Decision No.: 30). 1. Introduction Dietary factors, lipid profile characteristics, obesity, diabetes and high blood pressure (BP) are among the risk factors for CVDs [9]. Macrosomic neonates of healthy and diabetic mothers were reported to have significantly higher abdominal aorta intima media thickness measurements compared to controls [10, 11]. A significant association was established between birth weight and CIMT values also in childhood [7]. To the best of our knowledge, there are few studies dealing with IDMs and macrosomic infants during childhood which evaluate multiple atherosclerosis risk factors and CIMT together [12–14). We hypothesized that CIMT may be higher in IDMs and macrosomic infants during childhood at 8–9 years of age compared to children born to healthy mothers and we examined dietary factors, physical activity, obesity, lipid profiles, insulin resistance, blood pressure (BP) and CIMT measurements in the study. Early CVD A family history of coronary artery stent, coronary bypass surgery or stroke in women before the age of 65, and in men before the age of 55 among first- and second-degree relatives was considered as "early cardiovascular disease". According to the pediatric blood pressure categories for children aged 1–13 years, blood pressure was defined as normal, elevated or hypertension [18]. Carotid intima-media thickness was assessed by a single experienced reader (AY) who was blinded to the patients and controls via high-resolution ultrasound (Mindray Resona7, Mindray Bio-Medical Electronics Co., Ltd., China) with an L14-6s linear 5.1–12.5-MHz probe. CIMT was measured three times at the posterior wall, 1 cm distal to the bifurcation of carotid artery with the child in the supine position [8]. The children were positioned with a small turn to the contralateral side with the neck in supine position and mean of the three measurements was noted. Abdominal obesity An age- and gender-specific waist circumference in the > 90th percentile or a waist circumference-to- height ratio of ≥ 0.5 was defined as abdominal obesity [16]. Dyslipidemia The presence of one or more of the followings- plasma triglycerides (TG), low density lipoprotein (LDL) cholesterol and total cholesterol (TC) levels higher than the reference values, or high density lipoprotein (HDL) cholesterol lower than the reference values- was considered as dyslipidemia [9]. Skinfold thickness was measured using the Skinfold method at the midpoint of the upper arm using a caliper. Overweight/Obesity Children with an age- and gender-specific body mass index (BMI) of 85–94% and > 95% were considered overweight and obese, respectively [17]. Insulin resistance in children Fasting plasma glucose (FPG) and simultaneous plasma insulin levels were measured. A HOMA-IR value of > 2.22 in girls and > 2.67 in boys was considered insulin resistance [15]. Page 3/18 Page 3/18 Page 3/18 2.4. Statistical Analysis IBM SPSS Statistics (Version 20.0. Armonk, NY: IBM Corp.) was used for statistical assessment. Categorical data were expressed as numbers and percentages, and continuous data as mean, deviation, median and interquartile range. Pearson’s Chi-square test was used for comparison of categorical variables; a Mann-Whitney U test was used to compare quantitative data between two independent groups; and a Kruskal-Wallis H test was used to compare three or more groups. Tamhane’s T2 test was used for poc-hoc analysis. Correlations between CIMT and age, lipid profile, BMI percentiles and BP levels were assessed with Pearson’s correlation and regression analyses. p < 0.05 was considered statistically significant. Page 4/18 3. Results According to inclusion criteria, there were 73 IDMs and 29 macrosomic infants born to healthy mothers at Cukurova University Hospital in 2011 and 2012. All parents were called, but the parents of 60 IDMs and 21 macrosomic infants could be reached and informed about the study. Parents of 49/60 (81.6%) IDM (IDM group) and 13/21 (61.9%) macrosomic infants (Macrosomic group) agreed to participate in the study. In the IDM group, five mothers had Type 1, 19 had Type 2 and 25 had gestational DM. Of these mothers, 36 (73.5%) were treated with insulin during pregnancy. Control Group included 26 healthy children who were born to healthy mothers. Characteristics of the infants in groups are shown in Table 1. There was a statistical difference in birth weight and gestational age between the groups (p < 0.001 for both), with birth weight being higher in the Macrosomic group and gestational age lower in the IDM group. Duration of exclusive breastfeeding was longer in Control group than in IDM group (p < 0.001), while there was no significant difference in exclusive breastfeeding between the Macrosomic and Control groups or between the Macrosomic and IDM groups (Table 1). Atherosclerosis risk factors and family history of early CVD of Control, IDM and Macrosomic groups were not different (Table 2). HDL cholesterol level was significantly lower in IDM group than in Control (p = 0.006) and Macrosomic groups (p = 0.024), Table 2. CIMT measurements of the children were statistically significantly higher in both IDM and Macrosomic groups than in Control group (p < 0.001 and p = 0.008, respectively), there was no difference between IDM and Macrosomic groups, Table 3. Page 5/18 Table 1 General Characteristics of the Control, Infants of Diabetic Mothers and Macrosomic Group Control (n = 26) IDM (n = 49) Macrosomic (n = 13) Mean ± SD Med. (min- max) Mean ± SD Med. (min- max) Mean ± SD Med. 3. Results (min- max) p Age (months) 98.9 ± 9.0 100 (85–113) 99.9 ± 7.5 98 (89–118) 97.8 ± 11.4 95 (83–120) 0.468 Body weight (kg) 30.4 ± 8.3 31 (18.7–57.1) 31.4 ± 9.6 31 (18.5–67.6) 33.5 ± 6.8 32 (24.5–48) 0.343 Gestational age (weeks) 39.2 ± 1.4 40 (34–40) 37.5 ± 1.7 38 (34–40) 39.6 ± 1 40 (38–41) < 0.001 Birth weight (g) 3138 ± 404 3000 (2400– 3900) 3439 ± 809 3460 (1640– 5250) 4298 ± 176 4280 (4040– 4550) < 0.001 Duration of exclusive breastfeeding (months) 5.7 ± 1.1 6 (0–6) 3 ± 3.1 2 (0–12) 4 ± 2.5 6 (0–6) < 0.001 n (%) n (%) n (%) p Duration of breastfeeding Never breastfed 0–6 months 6–12 months 12–24 month 0 1 (3.8) 4 (1.5) 21 (80) 3 (6.1) 14 (28.5) 13 (26.5) 19 (38.7) 1 (7.6) 1 (7.6) 4 (30.7) 7 (53.8) 0.019 IDM: Infants of diabetic mothers Page 6/18 Page 6/18 Page 6/18 Table 2 Atherosclerosis risk factors in the Control, Infant of Diabetic Mothers and Macrosomic groups Control (n = 26) IDM (n = 49) Macrosomic (n = 13) n (%) n (%) n (%) p Obesity 2 (8) 13 (27.7) 3 (23) 0.152 Abdominal obesity 14 (53.8) 22 (44.8) 8 (61.5) 0.508 Hypertension 0 3 (6) 0 * Dyslipidemia 12 (46.1) 16 (32.6) 6 (46.1) 0.434 Insulin resistance 2 (7.6) 8 (16.3) 0 0.099 Impaired fasting plasma glucose (100–125 mg/dL) 1 (3.8) 8 (16.3) 0 0.099 Family history of early CVD 11 (42.3) 23 (46.9) 7 (53.8) 0.783 Smoking exposure 13 (50) 27 (56.3) 6 (46.2) 0.763 Physical inactivity (< 1 hr/day activity) 20 (76.9) 36 (73.5) 8 (61.5) 0.587 Screen time ≥ 1 hr/day 21 (80.7) 38 (77.5) 11 (84.6) 0.840 mg/dL Mean ± SD Med. (min-max) Mean ± SD Med. (min-max) Mean ± SD Med. (min-max) p HDL cholesterol 60.3 ± 13.9 58 (29–91) 50.4 ± 8.5 50.5 (31–66) 62.3 ± 13.3 59 (43–96) 0.001 LDL cholesterol 81.4 ± 29 80.5 (42–182) 89.5 ± 29.5 84 (41.6–199) 97.5 ± 24.4 91.6 (66–140) 0.164 TC 162 ± 34.2 162 (108–260) 157.1 ± 33.1 153 (98–288) 175.8 ± 22 168 (146–208) 0.063 TG 103.3 ± 58.1 85.5 (44–306) 87.9 ± 45.3 76 (37–287) 78.6 ± 35.7 66 (38–152) 0.221 *Not evaluated due to the insufficient number of patients. 3. Results IDM: Infants of diabetic mothers, CVD: Cardiovascular disease, HDL: High density lipoprotein, LDL: Low density lipoprotein, TC: Total cholesterol, TG: Triglyceride Table 2 Atherosclerosis risk factors in the Control, Infant of Diabetic Mothers and DM: Infants of diabetic mothers, CVD: Cardiovascular disease, HDL: High density lipoprotein, LDL: Low density lipoprotein, TC: Total cholesterol, TG: Triglyceride Table 3 Carotid intima-media thickness measurements in Control, Infants of Diabetic Mothers and Macrosomic groups Control (n = 26) Mean ± SD Med. (min-max) IDM (n = 49) Mean ± SD Med. (min-max) Macrosomic (n = 13) Mean ± SD Med. (min-max) p CIMT (mm) 0.34 ± 0.047 0.34 (0.26–0.45) 0.43 ± 0.047 0.43 (0.34–0.60) 0.40 ± 0.055 0.41 (0.33–0.50) < 0.001 IDM: Infants of diabetic mothers, CIMT: Carotid artery intima media thickness Data were expressed as mean ± SEM and median (minimum-maximum) Table 3 Table 3 Table 3 Carotid intima-media thickness measurements in Control, Infants of Diabetic Mothers and Macrosomic groups 34 (38.6%) of children had a BMI of ≥ 85%. There was no difference in CIMT values of those with a BMI of ≥ 85 and < 85 (p = 0.698). The within-group comparisons also yielded no difference (Table 4). No significant correlation was found in Control and Macrosomic groups between children's CIMT and BMI, systolic BP, diastolic BP, HDL, LDL, TC and TG. In IDM group, there was a weak positive correlation between systolic BP and CIMT (r = 0.310, p = 0.030), and a moderate positive correlation between diastolic BP and CIMT (r = 0.486, p < 0.001) values. No significant correlation was found among the other parameters. Evaluating all children together revealed CIMT to be weakly positively correlated with diastolic BP (r = 0.213, p = 0.048), weakly positively correlated with systolic BP (r = 0.204, p = 0.059) and weakly negatively correlated with HDL (r=-0.371, p < 0.001). Page 8/18 Page 8/18 Table 4 Carotid intima-media thickness measurements of all children according to body mass index percentiles of < 85 and ≥ 85 CIMT measurement (mm) of children with BMI < 85% CIMT measurement (mm) of children with BMI ≥ 85% Mean ± SD Med. (min-max) Mean ± SD Med. 3. Results (min-max) Overall (n = 88) (n = 54) (n = 34) p 0.40 ± 0.06 0.40 (0.26–0.51) 0.41 ± 0.07 0.41 (0.28–0.60) 0.698 Control (n = 26) (n = 18) (n = 8) 0.35 ± 0.05 0.35 (0.26–0.45) 0.34 ± 0.04 0.34 (0.28–0.39) 0.849 IDM (n = 49) (n = 31) (n = 18) 0.43 ± 0.03 0.43 (0.37–0.51) 0.44 ± 0.06 0.44 (0.34–0.60) 0.551 Macrosomic (n = 13) (n = 5) (n = 8) 0.41 ± 0.03 0.41 (0.36–0.44) 0.41 ± 0.07 0.41 (0.33–0.50) 0.933 IDM: Infants of diabetic mothers, BMI: Body mass index, CIMT: Carotid artery intima media thickness Table 4 IDM: Infants of diabetic mothers, BMI: Body mass index, CIMT: Carotid artery intima media thickness Table 5 compares the CIMT values of infants born to mothers with pregestational DM (Type 1 DM and Type 2 DM) and GDM. Maternal HbA1C was found to be significantly higher in the pregestational DM group (6.9 ± 1.3 vs. 6.0 ± 0.7), (p = 0.012), while no significant difference was observed in the other parameters such as gestational age, birth weight, weight, BMI, skinfold thickness and abdominal obesity. In addition, no statistically significant difference was found in CIMT measurements, Table 5. Table 6 compares the IDM group in terms of total breastfeeding durations of < 6 months and ≥ 6 months. BMI, skinfold thickness, waist circumference, waist-to-hip ratio and waist-to-height ratio were found to be significantly higher in children who had been breastfed for < 6 months in total. Difference in CIMT measurements was borderline significant (p = 0.057). Page 9/18 Table 5 Carotid artery intima media thickness measurements of infants born to mothers with pregestational diabetes mellitus and gestational diabetes mellitus Carotid artery intima media thickness measurements of infants born to mothers with pregestational diabetes mellitus and gestational diabetes mellitus diabetes mellitus and gestational diabetes mellitus Pregestational DM (n = 24) Mean ± SD Med. (min-max) GDM (n = 25) Mean ± SD Med. (min-max) p CIMT (mm) 0.42 ± 0.03 0.42 (0.37–0.53) 0.44 ± 0.05 0.44 (0.34–0.60) 0.238 CIMT: Carotid artery intima media thickness, DM: diabetes mellitus, GDM: Gestational diabetes mellitus CIMT: Carotid artery intima media thickness, DM: diabetes mellitus, GDM: Gestational diabetes mellitus Table 6 Comparison of the Infants of Diabetic Mothers group according to total breastfeeding duration IDM, breastfeeding for < 6 months (n = 17) Mean ± SD Med. 4. Discussion In the present study we have reported higher CIMT values in macrosomic born infants and IDMs at 8–9 years old age supporting our hypothesis. The incidence of diabetes during pregnancy is increasing. According to International Diabetes Federation, it is estimated that 16.7% of live births in 2021 had some form of hyperglycemia during pregnancy, the vast majority (80.3%) of which were GDM [1]. In the present study we have reported higher CIMT values in macrosomic born infants and IDMs at 8–9 years old age supporting our hypothesis. The incidence of diabetes during pregnancy is increasing. According to International Diabetes Federation, it is estimated that 16.7% of live births in 2021 had some form of hyperglycemia during pregnancy, the vast majority (80.3%) of which were GDM [1]. Hyperglycemia during pregnancy is one of the leading causes of maternal and fetal morbidity and mortality, and is associated with short- and long-term outcomes. According to Barker's hypothesis of "fetal origins", fetal environment and endocrine status changes lead to adaptations that can permanently change physiology and metabolism, and these adaptations predispose individuals to CVDs and metabolic and endocrine disorders in adulthood [19]. Studies based on this hypothesis suggest that intrauterine exposure to hyperglycemia causes fetal programming that predisposes the infant to obesity, hypertension, CVDs, metabolic syndrome and Type 2 DM in the long term [2]. Hyperglycemia during pregnancy is one of the leading causes of maternal and fetal morbidity and mortality, and is associated with short- and long-term outcomes. According to Barker's hypothesis of "fetal origins", fetal environment and endocrine status changes lead to adaptations that can permanently change physiology and metabolism, and these adaptations predispose individuals to CVDs and metabolic and endocrine disorders in adulthood [19]. Studies based on this hypothesis suggest that intrauterine exposure to hyperglycemia causes fetal programming that predisposes the infant to obesity, hypertension, CVDs, metabolic syndrome and Type 2 DM in the long term [2]. The international, multiethnic, and observational Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study, which examined the association of maternal hyperglycemia with adverse perinatal outcomes found birth weight and cord C peptide levels of newborns- whose mothers had hyperglycemia but not DM- exhibited a strong and persistent association with maternal glucose levels [20]. 4. Discussion HAPO Follow- up Study, in turn, reported maternal glucose levels during pregnancy to be independently associated with childhood adiposity, including overweight, obesity, skinfold thickness, body fat percentage and waist circumference in pubertal children aged 10–14 years [12]. Similarly, studies of children aged 5–10 years reported a positive correlation between maternal glucose levels during pregnancy and obesity in children [13, 14]. In the present study comparing IDMs and macrosomic infants with control children at 8–9 years old age, gestational age was lower in the IDM group, while birth weight was higher in the Macrosomic group (p < 0.001 for both]. IDMs are at increased risk of macrosomia and being large for gestational age, and we attribute the similarity of the mean birth weight to that of the Control group, but lower than in the Macrosomic group to their lower gestational age at birth, as the gestational age of IDMs was two weeks less than the other two groups. IDM group had the lowest gestational age, which can be explained by increased risk of both indicated preterm labor and spontaneous preterm labor in diabetic women [21]. In the present study comparing IDMs and macrosomic infants with control children at 8–9 years old age, gestational age was lower in the IDM group, while birth weight was higher in the Macrosomic group (p < 0.001 for both]. IDMs are at increased risk of macrosomia and being large for gestational age, and we attribute the similarity of the mean birth weight to that of the Control group, but lower than in the Macrosomic group to their lower gestational age at birth, as the gestational age of IDMs was two weeks less than the other two groups. IDM group had the lowest gestational age, which can be explained by increased risk of both indicated preterm labor and spontaneous preterm labor in diabetic women [21]. Studies about the effects of maternal obesity and overnutrition on metabolic programming in children during prenatal, perinatal and postnatal periods show that each of the factors indicating excessive food intake, such as prepregnancy maternal obesity and weight gain during pregnancy [22], macrosomic birth [23] and intrauterine hyperglycemic environment [12, 20] are associated with childhood obesity and poor cardiovascular outcomes. Macrosomic infants with non-diabetic mothers were also shown to have higher plasma insulin levels [24] and a larger area of pancreatic β-cell hyperplasia than those born at normal weight to nondiabetic mothers [25]. 3. Results (min-max) IDM, breastfeeding for ≥ 6 months (n = 32) Mean ± SD Med. (min-max) p BMI (kg/m2) 20.7 ± 5.5 20 (14.2–35) 16.2 ± 2.3 16 (12.6–21) 0.001 Skinfold thickness (mm) 13.7 ± 6.5 12 (6–26) 8.6 ± 3 8 (4–18) 0.002 Waist circumference (cm) 74 ± 13.7 74 (55–101) 61.9 ± 7.3 60 (54–81) 0.002 Waist-to-hip ratio 0.93 ± 0.06 0.95 (0.83-1) 0.89 ± 0.05 0.89 (0.81–1.01) 0.036 Waist-to-height ratio 0.54 ± 0.09 0.52 (0.43–0.74) 0.47 ± 0.05 0.45 (0.40–0.61) 0.001 CIMT (mm) 0.45 ± 0.05 0.44 (0.35–0.60) 0.42 ± 0.03 0.42 (0.37–0.51) 0.057 IDM: Infants of diabetic mothers, BMI: body mass index, CIMT: Carotid artery intima media thickness Comparison of the Infants of Diabetic Mothers group according to tota IDM: Infants of diabetic mothers, BMI: body mass index, CIMT: Carotid artery intima media thickness 4. Discussion CIMT measurements are widely used to assess the risk of subclinical vascular disease in adulthood. Similar to adults, researches in children have found that hypertension [26], familial hypercholesterolemia [27], overweight and obesity [28], and Type 1 DM [29] lead to an increase in Page 11/18 Page 11/18 CIMT measurements when compared to the age-matched healthy population. The present study established similar rates of risk factors -family history of early CVD, secondhand smoke exposure, obesity, dyslipidemia and physical inactivity- for atherosclerosis within all groups, while CIMT was higher in the IDM and Macrosomic groups compared to the Control group. While it is known that atherosclerosis starts during the intrauterine period in IDMs [11, 30], our study found higher CIMT values in both the IDM and Macrosomic groups, suggesting that macrosomic birth may also have an effect on metabolic programming as in IDM [23–25, 31]. Similar to our study, Akcakus et al [11] showed that both macrosomic newborns of diabetic and nondiabetic mothers had higher abdominal intima media thickness than the control newborns and the lowest HDL cholesterol level was in the macrosomic infants of diabetic mothers. Diabetes mellitus is not a homogenous disease process. Type 1, 2 and GDM result dysglycaemia differently during fetal development. In the present study, we did not detect any difference in CIMT values of children whose mothers had preexisting DM or GDM although HbA1C levels of preexisting DM were higher. Child and adolescent obesity demonstrates a strong association with early atherosclerosis. Carotid IMT is increased in overweight and obese children [32]. Therefore, we examined CIMT measurements in overweight or obese children in our study. CIMT measurements were not different in overweight and obese children overall and within the individual groups. Based on these findings, it can be concluded that CIMT is affected due rather to intrauterine exposure than the extrauterine exposure. HAPO Follow-up Study found that exposure to hyperglycemia inutero was significantly associated with elevated plasma glucose levels and insulin resistance in children, independent of maternal and childhood BMIs and family history of diabetes [33]. In the present study, rate of children with increased HOMA-IR values and impaired FPG was 16.3% (8 children) in IDM group and 7.6% (2 children) in Control group. In Macrosomic group, there were no children with insulin resistance and impaired FPG. Although rate of insulin resistance and impaired FPG was higher in the IDM group, difference was statistically insignificant (Table 2). 4. Discussion International Diabetes Federation [34] recommends oral glucose testing (OGT] as the diagnosing procedure of choice for screening at- risk children for glucose homeostasis abnormalities. Unfortunately, no OGT test was administered in the present study, and so our results might not have fully reflected the abnormality in the glucose metabolism. HAPO Follow-up Study found that exposure to hyperglycemia inutero was significantly associated with elevated plasma glucose levels and insulin resistance in children, independent of maternal and childhood BMIs and family history of diabetes [33]. In the present study, rate of children with increased HOMA-IR values and impaired FPG was 16.3% (8 children) in IDM group and 7.6% (2 children) in Control group. In Macrosomic group, there were no children with insulin resistance and impaired FPG. Although rate of insulin resistance and impaired FPG was higher in the IDM group, difference was statistically In the present study, lipid profiles of the groups revealed a statistically significant difference in HDL levels and a borderline significant difference in TC levels. The lowest HDL levels were recorded in the IDM group. Akcakus et al [11] reported lower HDL and higher TC and LDL levels in macrosomic IDM compared to control group. We may conclude that exposure starting in the intrauterine period in IDMs continues into childhood. In the study by Tam et al. [35] GDM exposure was found to be associated with high systolic BP and diastolic BP, and low HDL levels in children at the age of 7–10 years, similar to our results. However, when the study re-evaluated children at an average age of 15 years, BP and lipid profiles of the groups were similar [36]. Although our study identified lower HDL levels in IDM aged 8–9 years, lipid Page 12/18 Page 12/18 profile of children should be assessed again in different ages to evaluate the long-term effect of maternal hyperglycemia. Various studies involving children have shown that newly diagnosed hypertension [26], familial hypercholesterolemia [27], overweight and obesity [28], and type 1 DM [29] increase CIMT. In this study, CIMT was weakly positively correlated with diastolic and systolic BP, and borderline weakly negatively correlated with HDL in all children. Similar to the present study, low HDL cholesterol [37] and high BP [38] were associated with an increase in CIMT in children. 4. Discussion We can only detect a correlation in IDM group; there was a weak positive correlation between systolic BP and a moderate positive correlation between diastolic BP and CIMT. In the present study, a pairwise comparison revealed a significantly shorter duration of exclusive breastfeeding in IDM group than in control group. Only 38.7% of the IDM group was breastfed for 12–24 months. We believe that this may be attributed to the more hospitalization rates of IDMs after birth, causing mother and baby separation and leading to decreased milk production. Another factor may be that parents use more formula due to concern that the baby may have hypoglycemia. Breastfeeding during infancy, however, is very important, and as shown in the present study it seems much more important for IDMs. In low-socioeconomic societies in particular, duration of breastfeeding should be longer, as the risk of being overweight is 45% lower in children with maternal GDM breastfed for more than 3 months, compared to those breastfed for less than 3 months at the age of 2–8 years [39]. In 6–13 years old aged children, waist circumference, BMI and visceral and subcutaneous fat were found to be higher in IDMs breastfed for less than 6 months while no difference was found in anthropometric measurements breastfed for more than 6 months, regardless of whether their mothers were diabetic or not [40]. In the present study, the duration of “exclusive breastfeeding” was very low, with a mean of 3 months in IDMs. Similar to previous studies [39, 40], significantly higher BMI, waist circumference, skinfold thickness, waist-to-hip and waist-to-height ratios were recorded in children breastfed for less than 6 months compared with children breastfed for more than 6 months in IDM group; also CIMT measurement was borderline significantly higher. These findings indicate the importance of breastfeeding in IDMs. Limitations of our study include the small sample size, especially in the Macrosomic group. Furthermore, the fact that the study was conducted in a single healthcare facility makes generalization difficult. Plasma lipid, insulin, and glucose parameters require at least 8 hours of fasting, and the results can be misleading if the patient is not properly fasted. Some children may not have complied with the fasting requirement. 5. Conclusion We found higher CIMT measurements in IDM group. This supports “Barker’s hypothesis”, which states that antenatal exposure to hyperglycemia leads to adaptations that permanently alter the fetal physiology and metabolism, and that these adaptations predispose such children to cardiovascular, Page 13/18 Page 13/18 metabolic and endocrine disorders in adulthood. The CIMT measurements, however, were also higher in the macrosomic children born to non-diabetic mothers than in the Control group, suggesting that factors other than maternal diabetes may also be involved. The shorter duration of breastfeeding in the IDM group and the higher BMI, skinfold thickness, waist circumference, waist circumference-to-hip ratio and waist circumference-to-height ratio in children breastfed for less than 6 months suggest that breastfeeding is very important in IDMs. Nutrition is very important both in intrauterine and extrauterine periods, especially in IDMs. There is a need for further studies examining similar variables, involving a larger sample. Abbreviations BMI body mass index BP blood pressure CIMT carotid intima-media thickness CVD cardiovascular diseases DM diabetes mellitus FPG fasting plasma glucose GDM gestational diabetes mellitus HAPO Hyperglycemia and Adverse Pregnancy Outcome HDL High density lipoprotein IDM infants of diabetic mothers LDL Low density lipoprotein TC Total cholesterol TG Triglyceride Declarations Code availability: N/A Authors' contributions: Conceptualization: H.Y., S.C.C.; Methodology: H.Y., D.Y.; Data curation: S.C.C., A.Y.; Formal analysis and investigation: A.Y., S.C.C.; Writing - original draft preparation: S.C.C., H.Y.; Writing - review and editing: H.Y., D.Y.; Resources: S.C.C., A.Y.; Supervision: H.Y., D.Y.. Funding: The study was funded Cukurova University 2021, TTU-13484 Conflicts of interest/Competing interests: N/A Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. The study was approved by Çukurova University Ethics Committee (Date: October 4, 2019, Decision No.: 30). Consent to participate: Informed consent was obtained from legal guardians to participate. Consent to participate: Informed consent was obtained from legal guardians to participate. Consent for publication: Informed consent was obtained from legal guardians for publication. Consent for publication: Informed consent was obtained from legal guardians for publication. Availability of data and material: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Availability of data and material: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Code availability: N/A Funding: The study was funded Cukurova University 2021, TTU-13484 Conflicts of interest/Competing interests: N/A Funding: The study was funded Cukurova University 2021, TTU-13484 Conflicts of interest/Competing interests: N/A Declarations Page 14/18 References 1. International Diabetes Federation. IDF Diabetes Atlas, 10th Edition. 2021, . International Diabetes Federation. IDF Diabetes Atlas, 10th Edition. 2021, . 2. Vrachnis N, Antonakopoulos N, Iliodromiti Z, Dafopoulos K, Siristatidis C, Pappa KI, et al. Impact of maternal diabetes on epigenetic modifications leading to diseases in the offspring. Exp Diabetes Res 2012; 2012: 538474. doi: 10.1155/2012/538474. 2. Vrachnis N, Antonakopoulos N, Iliodromiti Z, Dafopoulos K, Siristatidis C, Pappa KI, et al. Impact of maternal diabetes on epigenetic modifications leading to diseases in the offspring. Exp Diabetes Res 2012; 2012: 538474. doi: 10.1155/2012/538474. 3. Merzouk H, Madani S, Prost J, Loukidi B, Meghelli-Bouchenak M, Belleville J. Changes in serum lipid and lipoprotein concentrations and compositions at birth and after 1 month of life in macrosomic infants of insulin-dependent diabetic mothers. Eur J Pediatr 1999; 158: 750–6. doi: 10.1007/s004310051194. 3. Merzouk H, Madani S, Prost J, Loukidi B, Meghelli-Bouchenak M, Belleville J. Changes in serum lipid and lipoprotein concentrations and compositions at birth and after 1 month of life in macrosomic infants of insulin-dependent diabetic mothers. Eur J Pediatr 1999; 158: 750–6. doi: 10.1007/s004310051194. 4. Eidelman AI, Samueloff A. The pathophysiology of the fetus of the diabetic mother. Semin Perinatol 2002, 26: 232–6. doi: 10.1053/sper.2002.34215. 5. Hammoud NM, Visser GH, van Rossem L, Biesma DH, Wit JM, de Valk HW. Long-term BMI and growth profiles in offspring of women with gestational diabetes. Diabetologia 2018; 61:1037–45. doi: org/10.1007/s00125-018-4584-4. 6. Dugas C, Perron J, Kearney M, Mercier R, Tchernof A, Marc I, et al. Postnatal prevention of childhood obesity in offspring prenatally exposed to gestational diabetes mellitus: where are we now. Obes Facts 2017; 10: 396–406. doi: 10.1159/000477407. 7. Dratva J, Breton CV, Hodis HN, Mack WJ, Salam MT, Zemp E, et al. Birth weight and carotid artery intima-media thickness. J Pediatr 2013; 162: 906 – 11. e1- 2. doi: 10.1016/j.jpeds.2012.10.060. Page 15/18 Page 15/18 8. Stein J. American Society of Echocardiography Carotid Intima-Media Thickness Task Force. Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media Thickness Task Force. Endorsed by the Society for Vascular Medicine. J Am Soc Echocardiogr 2008; 21: 93–111. doi: 10.1016/j.echo.2007.11.011. 8. Stein J. American Society of Echocardiography Carotid Intima-Media Thickness Task Force. References Use of carotid ultrasound to identify subclinical vascular disease and evaluate cardiovascular disease risk: a consensus statement from the American Society of Echocardiography Carotid Intima-Media Thickness Task Force. Endorsed by the Society for Vascular Medicine. J Am Soc Echocardiogr 2008; 21: 93–111. doi: 10.1016/j.echo.2007.11.011. 9. Expert Panel on Integrated Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents. Expert panel on integrated guidelines for cardiovascular health and risk reduction in children and adolescents: summary report. Pediatrics 2011; 128: S213–56. doi: 10.1542/peds.2009- 2107C. 10. Koklu E, Akcakus M, Kurtoglu S, Koklu S, Yikilmaz A, Coskun A, et al. Aortic intima-media thickness and lipid profile in macrosomic newborns. Eur J Pediatr 2007; 166: 333–8. doi: 10.1007/s00431-006- 0243-8. 11. Akcakus M, Koklu E, Baykan A, Yikilmaz A, Coskun A, Gunes T, et al. Macrosomic newborns of diabetic mothers are associated with increased aortic intima-media thickness and lipid concentrations. Horm Res 2007;67:277–83. https://doi.org/10.1159/000098157. 12. Lowe WL, Lowe LP, Kuang A, Catalano PM, Nodzenski M, Talbot O, et al. Maternal glucose levels during pregnancy and childhood adiposity in the Hyperglycemia and Adverse Pregnancy Outcome Follow-up Study. Diabetologia 2019; 62:598–610. https://doi.org/10.1007/s00125-018-4809-6. 13. Tam WH, Ma RCW, Ozaki R, Li AM, Chan MHM, Yuen LY, et al. In utero exposure to maternal hyperglycemia increases childhood cardiometabolic risk in offspring. Diabetes Care 2017; 40: 679– 86. doi: 10.2337/dc16-2397. 14. Landon MB, Mele L, Varner MW, Casey BM, Reddy UM, Wapner RJ, et al. The relationship of maternal glycemia to childhood obesity and metabolic dysfunction. J Matern Fetal Neonatal Med 2020; 33: 33–41. doi: 10.1080/14767058.2018.1484094. 14. Landon MB, Mele L, Varner MW, Casey BM, Reddy UM, Wapner RJ, et al. The relationship of maternal glycemia to childhood obesity and metabolic dysfunction. J Matern Fetal Neonatal Med 2020; 33: 33–41. doi: 10.1080/14767058.2018.1484094. 15. Kurtoğlu S, Hatipoğlu N, Mazıcıoğlu M, Kendirici M, Keskin M, Kondolot M. Insulin resistance in obese children and adolescents: HOMA- IR cut- off levels in the prepubertal and pubertal periods. J Clin Res Pediatr Endocrinol 2010; 2: 100–6. doi: 10.4274/jcrpe.v2i3.100. 15. Kurtoğlu S, Hatipoğlu N, Mazıcıoğlu M, Kendirici M, Keskin M, Kondolot M. Insulin resistance in obese children and adolescents: HOMA- IR cut- off levels in the prepubertal and pubertal periods. J Clin Res Pediatr Endocrinol 2010; 2: 100–6. doi: 10.4274/jcrpe.v2i3.100. 16. Zimmet P, Alberti G, Kaufman F, Tajima N, Silink M, Arslanian S, et al. The metabolic syndrome in children and adolescents. References Lancet 2007; 369: 2059–61. doi: 10.1016/S0140-6736(07)60958-1. 16. Zimmet P, Alberti G, Kaufman F, Tajima N, Silink M, Arslanian S, et al. The metabolic syndrome in children and adolescents. Lancet 2007; 369: 2059–61. doi: 10.1016/S0140-6736(07)60958-1. 17. Neyzi O, Bundak R, Gökçay G, Günöz H, Furman A, Darendeliler F, et al. Reference values for weight, height, head circumference, and body mass index in Turkish children. J Clin Res Pediatr Endocrinol 2015; 7: 280–93. doi: 10.4274/jcrpe.2183. 18. Flynn JT, Kaelber DC, Baker-Smith CM, Blowey D, Carroll AE, Daniels SR, et al. Clinical practice guideline for screening and management of high blood pressure in children and adolescents. Pediatrics 2017; 140: e20171904. doi: 10.1542/peds.2017-1904. 18. Flynn JT, Kaelber DC, Baker-Smith CM, Blowey D, Carroll AE, Daniels SR, et al. Clinical practice guideline for screening and management of high blood pressure in children and adolescents. Pediatrics 2017; 140: e20171904. doi: 10.1542/peds.2017-1904. 19. Farnetani I, Fanos V. David Barker: the revolution that anticipates existence. JPNIM 2014; 3: e030111–e030111. doi: 10.7363/030111. Page 16/18 20. HAPO Study Cooperative Research Group, Metzger BE, Lowe LP, Dyer AR, Trimble ER, Chaovarindr U, et al. Hyperglycemia and adverse pregnancy outcomes. N Engl J Med 2008; 358: 1991–2002. doi: 10.1056/NEJMoa0707943. 21. E kbom P, Damm P, Feldt-Rasmussen B, Feldt-Rasmussen U, Jensen DM, Mathiesen ER. Elevated third-trimester haemoglobin A1c predicts preterm delivery in type 1 diabetes. J Diabetes Complications 2008; 22: 297–302. doi: 10.1016/j.jdiacomp.2007.03.008. 22. Fraser A, Tilling K, Macdonald-Wallis C, Sattar N, Brion M-J, Benfield L, et al. Association of maternal weight gain in pregnancy with offspring obesity and metabolic and vascular traits in childhood. Circulation 2010; 121: 2557–64. doi: 10.1161/CIRCULATIONAHA.109.906081. 23. Schaefer-Graf UM, Pawliczak J, Passow D, Hartmann R, Rossi R, Bührer C, et al. Birth weight and parental BMI predict overweight in children from mothers with gestational diabetes. Diabetes Care 2005; 28: 1745–50. doi: 10.2337/diacare.28.7.1745. 24. Hoegsberg B, Gruppuso PA, Coustan DR. Hyperinsulinemia in macrosomic infants of nondiabetic mothers. Diabetes Care 1993; 16: 32–6. https:// doi: 10.2337/diacare.16.1.32. 25. Pinar H, Pinar T, Singer DB. Beta-cell hyperplasia in macrosomic infants and fetuses of nondiabetic mothers. Pediatr Dev Pathol 2000; 3: 48–52. doi: 10.1007/s100240050006. 26. Sorof JM, Alexandrov AV, Cardwell G, Portman RJ. Carotid artery intimal-medial thickness and left ventricular hypertrophy in children with elevated blood pressure. Pediatrics 2003; 111: 61–6. doi: 10.1542/peds.111.1.61. 27. Virkola K, Pesonen E, Åkerblom H, Siimes MA. References The association between blood pressure and carotid intima-media thickness in children: a systematic review. Cardiol Young 2017; 27: 1295–305. doi: 10.1017/S1047951117000105. 39. Schaefer-Graf UM, Hartmann R, Pawliczak J, Passow D, Abou-Dakn M, Vetter K, et al. Association of breast-feeding and early childhood overweight in children from mothers with gestational diabetes mellitus. Diabetes Care 2006; 29: 1105–7. doi: 10.2337/diacare.2951105. 39. Schaefer-Graf UM, Hartmann R, Pawliczak J, Passow D, Abou-Dakn M, Vetter K, et al. Association o breast-feeding and early childhood overweight in children from mothers with gestational diabetes mellitus. Diabetes Care 2006; 29: 1105–7. doi: 10.2337/diacare.2951105. 40. Crume TL, Ogden L, Maligie M, Sheffield S, Bischoff KJ, McDuffie R, et al. Long-TermiImpact of neonatal breastfeeding on childhood adiposity and fat distribution among children exposed to diabetes in utero. Diabetes Care 2011; 34: 641–5. doi: 10.2337/dc10-1716. 40. Crume TL, Ogden L, Maligie M, Sheffield S, Bischoff KJ, McDuffie R, et al. Long-TermiImpact of neonatal breastfeeding on childhood adiposity and fat distribution among children exposed to diabetes in utero. Diabetes Care 2011; 34: 641–5. doi: 10.2337/dc10-1716. References Cholesterol and carotid artery wall in children and adolescents with familial hypercholesterolaemia: a controlled study by ultrasound. Acta Paediatr 1997; 86: 1203–7. doi: 10.1111/j.1651-2227.1997.tb14847.x. 28. Woo KS, Chook P, Yu CW, Sung RY, Qiao M, Leung SS, et al. Effects of diet and exercise on obesity- related vascular dysfunction in children. Circulation 2004; 109:1 981-6. doi: 10.1161/01.CIR.0000126599.47470.BE. 29. Atabek ME, Kurtoglu S, Pirgon O, Baykara M. Arterial wall thickening and stiffening in children and adolescents with type 1 diabetes. Diabetes Res Clin Pract 2006;74: 33–40. doi: 10.1016/j.diabres.2006.03.004. 30. Asmussen I. Ultrastructure of human umbilical arteries. Studies on arteries from newborn children delivered by nonsmoking, white group D, diabetic mothers. Circ Res 1980; 47: 620–6. doi: 10.1161/01.res.47.4.620. 31. Kapral N, Miller SE, Scharf RJ, Gurka MJ, DeBoer MD. Associations between birthweight and overweight and obesity in school-age children. Pediatr Obes 2018; 13: 333–41. doi: 10.1111/ijpo.12227. 32. Meyer AA, Kundt G, Steiner M, Schuff-Werner P, Kienast W. Impaired flow-mediated vasodilation, carotid artery intima-media thickening, and elevated endothelial plasma markers in obese children: the impact of cardiovascular risk factors. Pediatrics 2006; 117: 1560–7. doi: 10.1542/peds.2005- 2140. Page 17/18 Page 17/18 33. Lowe Jr WL, Scholtens DM, Kuang A, Linder B, Lawrence JM, Lebenthal Y, et al. Hyperglycemia and Adverse Pregnancy Outcome Follow-up Study (HAPO FUS): maternal gestational diabetes mellitus and childhood glucose metabolism. Diabetes Care 2019; 42: 372–80. doi: 10.2337/dc18-1646. 34. Global guideline for type 2 diabetes. International Diabetes Federation Guideline Development Group. Diabetes Res Clin Pract 2014; 104: 1–52. https:// doi: 10.1016/j.diabres.2012.10.001. 34. Global guideline for type 2 diabetes. International Diabetes Federation Guideline Development Group. Diabetes Res Clin Pract 2014; 104: 1–52. https:// doi: 10.1016/j.diabres.2012.10.001. 35. Tam WH, Ma RCW, Yang X, Ko GTC, Tong PCY, Cockram CS, et al. Glucose intolerance and cardiometabolic risk in children exposed to maternal gestational diabetes mellitus in utero. Pediatrics 2008; 122: 1229–34. doi: 10.1542/peds.2008-0158. 36. Tam WH, Ma RCW, Yang X, Li AM, Ko GTC, Kong APS, et al. Glucose intolerance and cardiometabolic risk in adolescents exposed to maternal gestational diabetes: a 15-year follow-up study. Diabetes Care 2010;33:1382–4. doi: 10.2337/dc09-2343. 37. Ayer JG, Harmer JA, Nakhla S, Xuan W, Ng MKC, Raitakari OT, et al. HDL-Cholesterol, blood pressure, and asymmetric dimethylarginine are significantly associated with arterial wall thickness in children. Arterioscler Thromb Vasc Biol 2009; 29: 943–9. doi: 10.1161/ATVBAHA.109.184184. 38. Day TG, Park M, Kinra S. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. GraphicalabstractEJP.docx Legendstographicalabstract.docx Page 18/18
https://openalex.org/W3215897669	https://vuir.vu.edu.au/46419/1/High-intensity%20training%20induces%20non-stoichiometric%20changes%20in%20the%20mitochondrial%20proteome%20of%20human%20skeletal%20muscle%20without%20re.pdf	English	null	High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content	Nature communications	2,021	cc-by	23,459	This is the Published version of the following publication This is the Published version of the following publication Granata, Cesare, Caruana, Nikeisha, Botella, Javier, Jamnick, Nicholas, Huynh, Kevin, Kuang, Jujiao, Janssen, Hans A, Reljic, Boris, Mellett, Natalie A, Laskowski, Adrienne, Stait, Tegan L, Frazier, Ann, Coughlan, Melinda T, Meikle, Peter, Thorburn, David, Stroud, David A and Bishop, David (2021) High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content. Nature Communications, 12 (1). pp. 1-18. ISSN 2041-1723 The publisher’s official version can be found at https://www.nature.com/articles/s41467-021-27153-3 Note that access to this version may require subscription. Downloaded from VU Research Repository https://vuir.vu.edu.au/46419/ ARTICLE Results and discussion Increases in mitochondrial content underlie changes in mar- kers of mitochondrial biogenesis and mitochondrial respira- tory function following training. The traditional view of mitochondrial biogenesis proposes that changes in individual mitochondrial proteins are stoichiometrically linked to changes in mitochondrial content. Although this has been questioned previously17, it remains an unresolved matter. To challenge this paradigm, we employed an experimental model in which 10 men were sequentially exposed to different volumes (normal- [NVT], high- [HVT] and reduced- [RVT] volume training) of high- intensity interval training (HIIT) with biopsies taken at baseline (BL) and post-NVT (PN), post-HVT (PH), and post-RVT (PR) (Fig. 1a). This design was chosen as we have previously demon- strated that changes (increases or decreases) in training volume lead to changes in mitochondrial content and markers of mito- chondrial biogenesis9,18–20. Weekly training volumes for the three phases are shown in Fig. 1b; changes in physiological and per- formance parameters are presented in Table 1. p g A key biological function of mitochondria is energy conversion via oxidative phosphorylation (OXPHOS), which is carried out by the four multi-protein complexes (complexes I to IV [CI-CIV]) of the electron transport chain (ETC) and F0F1-ATP synthase (or CV)13. Assembly of the OXPHOS complexes is an intricate process requiring the coordination of two genomes (nuclear and mitochondrial) and non-subunit proteins known as assembly factors14. Only two studies have investigated training-induced changes in OXPHOS complexes in humans15,16, reporting that exercise differentially modulates the assembly and composition of respiratory chain complexes - including their high molecular weight supercomplex (SC) assemblies. Furthermore, no study has investigated if training-induced changes in chaperones, assembly factors, and single OXPHOS subunits occur in conjunction with, or precede, changes in the complete, functional complexes. Many other processes support assembly and maintenance of the OXPHOS complexes, including mitochondrial protein import and assembly pathways, mitochondrial DNA transcription and translation, metabolite carriers, and pathways producing critical cofactors such as coenzyme Q and iron-sulphur proteins (Fe–S)14. In turn, these processes are reliant on pathways sup- porting mitochondrial biogenesis more broadly, such as lipid biogenesis and maintenance of mitochondrial morphology. Whether these processes and pathways are activated by exercise training and at which speciﬁc times remains unknown. p p We ﬁrst veriﬁed that changes in training volume induced changes in the yield of mitochondrial proteins isolated from muscle biopsies (Fig. 1c), and citrate synthase (CS) activity (Fig. High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content Cesare Granata 1,2,9,11✉, Nikeisha J. Caruana 1,3,11, Javier Botella 1, Nicholas A. Jamnick1,4, Kevin Huynh 5, Jujiao Kuang 1, Hans A. Janssen1, Boris Reljic 3,10, Natalie A. Mellett5, Adrienne Laskowski2, Tegan L. Stait6, Ann E. Frazier 6,7, Melinda T. Coughlan 2,5, Peter J. Meikle 5, David R. Thorburn 6,7,8, David A. Stroud 3,6,12✉& David J. Bishop 1,12✉ Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible, inexpensive therapeutic intervention that can improve mitochondrial bioener- getics and quality of life. By combining multiple omics techniques with biochemical and in silico normalisation, we removed the bias arising from the training-induced increase in mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the overall increase in mitochondrial content. Our ﬁndings suggest enhancing electron ﬂow to oxidative phos- phorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and do not support the hypothesis that training- induced supercomplex formation enhances mitochondrial bioenergetics. Our study provides an analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding, and calling for careful reinterpretation of previous ﬁndings. 1 Institute for Health and Sport (iHeS), Victoria University, Melbourne, VIC 3011, Australia. 2 Department of Diabetes, Central Clinical School, Monash University, Melbourne, VIC 3004, Australia. 3 Department of Biochemistry and Pharmacology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC 3010, Australia. 4 Metabolic Research Unit, School of Medicine and Institute for Mental and Physical Health and Clinical Translation (iMPACT), Deakin University, Geelong, VIC, Australia. 5 Baker Heart & Diabetes Institute, Melbourne, VIC 3004, Australia. 6 Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC 3052, Australia. 7 Department of Paediatrics, The University of Melbourne, Melbourne, VIC 3052, Australia. 8 Victorian Clinical Genetics Services, Royal Children’s Hospital, Melbourne, VIC 3052, Australia. 9Present address: Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research, Heinrich Heine University, 40225 Düsseldorf, Germany. 10Present address: Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, 3800 Melbourne, Australia. 11These authors contributed equally: Cesare Granata, Nikeisha J. Caruana. 12These authors jointly supervised this work: David A. Stroud, David J. Bishop. High-intensity training induces non-stoichiometric changes in the mitochondrial proteome of human skeletal muscle without reorganisation of respiratory chain content ✉email: cesare.granata@monash.edu; david.stroud@unimelb.edu.au; david.bishop@vu.edu.au 1 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 M M itochondria are the main site of energy conversion in the cell and have critical roles in other essential biolo- gical processes1. Owing to this, defects in mitochondria have been implicated in multiple diseases, medical conditions, and aging2–5. The development of interventions to improve the content and function of mitochondria is therefore important to enhance quality of life and to extend life expectancy. patterns of adaptation. In addition, we provide evidence that training-induced changes in the abundance and/or organisation of SCs did not contribute to improvements in mitochondrial bioenergetics. Our study provides an analytical approach that expands our fundamental understanding of training-induced mitochondrial biogenesis. These ﬁndings challenge our current knowledge and call for careful reinterpretation of previous results, while also supporting the development of revised hypotheses on how training alters mitochondrial protein content and function, and provide an important resource with implications for both health and disease. q y p y Exercise training is one of the most widely accessible inter- ventions or “therapies” to stimulate mitochondrial biogenesis6,7. As arguably the most extensive and also natural perturbation, exercise represents an excellent experimental model for under- standing both the complex nature of mitochondrial biogenesis and the plasticity of the skeletal muscle mitochondrial proteome in humans8. It is well accepted that exercise training induces an increase in mitochondrial content and respiratory function in human skeletal muscle9. However, our knowledge of mitochon- drial adaptations is limited to a small fraction of the mitochon- drial proteome10, and is complicated by large training-induced changes in mitochondrial content, which limit the ability to interpret adaptations in individual proteins independent of the overall change in mitochondrial content. To account for this, most studies to date have either performed analyses in mito- chondrial isolates obtained after differential centrifugation or have employed a crude normalisation strategy involving the use of markers of mitochondrial content, such as citrate synthase (CS) activity9,11. Nonetheless, due to limitations in both approaches11,12, there remain major gaps in our understanding of the magnitude, timing, and direction of change in individual mitochondrial proteins and related functional pathways, inde- pendent of the overall change in mitochondrial content. k b l l f f h d NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 EXTRACELLULAR MATRIX ORGANIZATION (GO:0030198) EXTRACELLULAR STRUCTURE ORGANIZATION (GO:0043062) EXTERNAL STRUCTURE ORGANIZATION (GO:0045229) REGULATION OF ANGIOGENESIS (GO:0045765) REGULATION OF CELL MIGRATION (GO:0030334) COMBINED SCORE 200 100 50 150 0 mtribo −1 −0.5 0 0.5 1 CI CII CIII CIV CV −1 −0.5 0 0.5 1 OXPHOS assembly TCA SLC25A MICOS PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL NVT HVT RVT 0 3 6 9 12 Weekly training volume (MJ) P < 0.001 P < 0.001 P < 0.001 0 200 400 600 800 CS activity nmol min-1 mg protein -1 P < 0.001 P = 0.009 P = 0.001 BL PN PH PR BL PN PH PR 0 1 2 3 μg of crude mitochondrial protein/mg of muscle P < 0.001 P < 0.001 P < 0.001 a b c d HVT 3 weeks 40 HIIT sessions RVT 1 week 6 HIIT sessions NVT 2 weeks 6 HIIT sessions BL PN PH PR g f e -1 0 0.5 1 -0.5 Log2FC PN vs. BL PR vs. PH PH vs. PN a HVT 3 weeks 40 HIIT sessions RVT 1 week 6 HIIT sessions NVT 2 weeks 6 HIIT sessions BL PN PH PR a HVT 3 weeks 40 HIIT sessions RVT 1 week 6 HIIT sessions PH PR NVT HVT RVT 0 3 6 9 12 Weekly training volume (MJ) P < 0.001 P < 0.001 P < 0.001 0 200 400 600 800 CS activity nmol min-1 mg protein -1 P < 0.001 P = 0.009 P = 0.001 BL PN PH PR BL PN PH PR 0 1 2 3 μg of crude mitochondrial protein/mg of muscle P < 0.001 P < 0.001 P < 0.001 b c d f 0 200 400 600 800 CS activity nmol min-1 mg protein -1 P < 0.001 P = 0.009 P = 0.001 BL PN PH PR d d b c f E g f e -1 0 0.5 1 -0.5 Log2FC PN vs. BL PR vs. PH PH vs. Results and discussion 1d) - a commonly used biomarker of mitochondrial content21, as previously reported6,9,20,22–24. We then conﬁrmed that classic markers of mitochondrial biogenesis, such as the protein content of OXPHOS subunits assessed in whole-muscle lysates (Supplementary Fig. 1a, upper panels), as well as measurements of mitochondrial respiratory function, such as mitochondrial respiration in permeabilised ﬁbres and ETC enzyme activity (Supplementary Fig. 1b, c, respectively, all upper panels) changed, for the most part, as a consequence of changes in training volume. RVT resulted in no change in any of these markers, suggesting that the training stimulus was sufﬁcient to prevent a decrease in these parameters. Subsequently, to investigate the underpinnings of training-induced changes in the above markers, we normalised these values by CS activity - a widely used normalisation strategy to account for differences in mitochondrial content9,20,21,25. This demonstrated that, for the most part, absolute changes in markers of mitochondrial biogenesis and respiratory function were stoichiometrically associated with the overall increase in mitochondrial content (Supplementary Fig. 1a, b, c, all lower panels). This is consistent with previous ﬁndings20 and with the notion that absolute changes in markers of mitochondrial respiratory function (i.e., mitochondrial respiration and mitochondrial enzyme activity) induced by manipulation of training volume are mainly driven by the overall change in mitochondrial content, rather than by altered mitochondrial efﬁciency per se9,20,26. Here, we employed a 3-phase training intervention, combined with multiple omics techniques (transcriptomics, proteomics, and lipidomics) and a normalisation strategy that removed the bias induced by large alterations in mitochondrial content, to inves- tigate mitochondrial adaptations in human skeletal muscle. Our ﬁndings revealed an intricate and timely remodelling of the mitochondrial transcriptome, proteome, and lipidome, indepen- dent of changes in overall mitochondrial content. Post-normal- isation, we identiﬁed 185 differentially expressed mitochondrial proteins, representing multiple protein functional classes, as well as bioenergetic and metabolic pathways, that followed distinct Disentangling changes in the mitochondrial proteome from the general increase in mitochondrial content observed post- training. The above ﬁndings seem to indicate that altering training volume results in a stoichiometric relationship between NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 This demonstrated the presence of a signiﬁcant proportion of co-isolating non-mitochondrial proteins, with enrichment analysis indicating these to be predominantly of myoﬁbrillar origin (Supplementary Fig. 2a), consistent with most mitochondria in skeletal muscle being tightly associated with myoﬁbrils23. Surprisingly, we still observed a relative increase in the abundance of mitochondrial proteins within mitochondrial isolates following training (Fig. 2a, upper panel, magenta proﬁles), an artefact that likely stems from differences in mitochondrial protein enrichment (MPE) - the contribution of mitochondrial protein intensities in biochemically isolated support these ﬁndings with quantitative proteomics. To negate the general increase in mitochondrial content following exercise training (Fig. 1d) and to avoid this inherent bias on our proteomics measurements, we performed label-free quantitative (LFQ) proteomics on equal amounts of mitochondria-enriched fractions (biochemical enrichment of mitochondrial proteins using differential centrifugation). We quantiﬁed 1411 proteins − 726 of which were annotated as mitochondrial based on either the Mitocarta2.028 or the Integrated Mitochondrial Protein Index (IMPI; Known and Predicted Mitochondrial)29 databases (Sup- plementary Data 3). Considering only the high-conﬁdence IMPI (Known Mitochondrial) dataset, mitochondrial proteins repre- sented 41% of the total number of proteins identiﬁed in our mitochondria-enriched fraction (584 annotated mitochondrial proteins), contributing to 32% of the overall protein abundance based on raw intensity data for each protein (Supplementary Data 3). This demonstrated the presence of a signiﬁcant proportion of co-isolating non-mitochondrial proteins, with enrichment analysis indicating these to be predominantly of myoﬁbrillar origin (Supplementary Fig. 2a), consistent with most mitochondria in skeletal muscle being tightly associated with myoﬁbrils23. Surprisingly, we still observed a relative increase in the abundance of mitochondrial proteins within mitochondrial isolates following training (Fig. 2a, upper panel, magenta proﬁles), an artefact that likely stems from differences in mitochondrial protein enrichment (MPE) - the contribution of mitochondrial protein intensities in biochemically isolated p g The above ﬁndings highlight that the standard approach of relying on biochemical enrichment to eliminate the bias arising from different MPEs across time points is insufﬁcient and that an additional strategy is needed. We therefore complemented our biochemical enrichment approach with a statistical correction strategy consisting of (i) removing proteins that were identiﬁed in less than 70% of samples, (ii) removing all except high-conﬁdence mitochondrial proteins (IMPI Known Mitochondrial), and (iii) undertaking variance stabilising normalisation (VSN) (see Methods section for more detail). This resulted in the retention of 498 mitochondrial proteins normalised across the dataset with high conﬁdence (Supplementary Data 4). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 a Study design; coloured boxes indicate a training phase: normal-volume training (NVT), high-volume training (HVT), and reduced-volume training (RVT), whereas open boxes indicate a testing session and resting skeletal muscle biopsy: baseline (BL), post-NVT (PN), post-HVT (PH), and post-RVT (PR). b Weekly training volume during the NVT, HVT, and RVT training phase; training volume was calculated by multiplying the absolute exercise intensity in Watts by the effective duration of exercise training in minutes (excluding the warm up and the rest periods between intervals) by the total number of training sessions in each phase. c Mitochondrial yield per mg of tissue achieved during mitochondria isolation of human vastus lateralis biopsy samples (biochemical enrichment of mitochondrial protein). d Citrate synthase (CS) enzyme activity assessed in whole-tissue (vastus lateralis) homogenates. For panel b–d source data are provided as a Source Data ﬁle and P values are indicated on the ﬁgure to three decimal places; for P values that were truncated, the corresponding accurate P values were: HVT vs. NVT: P = 3.9e−14; RVT vs. NVT: P = 3.8e−5; HVT vs. NVT: P = 1.1e−12 in panel b; PH vs. BL: P = 1.9e−6; PR vs. BL: P = 1.6e−5; PH vs. PN: P = 8.3e−4 in panel c; and PR vs. BL: P = 5.3e−4 in panel d. e Heatmap of differentially expressed transcripts between training phases determined with an adjusted P < 0.05 (Benjamini–Hochberg). Row clustering determined by unsupervised hierarchical cluster analysis. f Proﬁle plots showing relative scaled transcripts of subunits of the ﬁve oxidative phosphorylation (OXPHOS) complexes (CI to CV), mitochondrial ribosomes (mtribo), OXPHOS assembly, TCA cycle, SLC25As, and mitochondrial contact site and cristae organising system (MICOS) complex. Transcripts grouped according to known literature (see Methods section). g Biological process (BP) gene ontology of all differentially expressed transcripts as in e; the top ﬁve biological processes by adjusted p value, as determined by Enrichr (see Methods section), are displayed with their combined score (the full list is presented in Supplementary Data 2). HIIT high-intensity interval training, MJ megajoules, CI-V complex I–V, IB immunoblotting. Datasets b–d (n = 10) were analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05. Filled triangles, empty diamonds, and empty and ﬁlled circles represent individual values; the maxima of each bar represents the mean value. Datasets e–g, n = 5. Table 1 Participants’ physiological and endurance performance measurements. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Filled triangles, empty diamonds, and empty and ﬁlled circles represent individual values; the maxima of each bar represents the mean value. Datasets e–g, n = 5. Table 1 Participants’ physiological and endurance performance measurements. Measurement BL PN PH PR Age 22.3 ± 5.2 – – – Height 178.1 ± 10.7 – – – Body mass 78.8 ± 12.1 79.0 ± 12.9 78.0 ± 12.5 77.9 ± 11.8# BMI 24.8 ± 2.9 24.9 ± 3.2 24.6 ± 3.0 24.5 ± 2.9# V̇O2Peak 46.7 ± 8.2 47.7 ± 8.8 50.5 ± 6.0# 50.0 ± 6.0 Ẇ LT 174.1 ± 30.7 182.5 ± 33.0 222.2 ± 36.2# 219.6 ± 31.4# Ẇ peak 231.1 ± 43.2 233.2 ± 43.5 266.3 ± 42.4# 264.5 ± 40.4# 20k-TT time 2321.0 ± 168.1 2309.1 ± 257.4 2094.6 ± 130.8# 2030.1 ± 140.5# BL baseline, PN post normal-volume training, PH post high-volume training, PR post reduced-volume training, BMI body mass index [kg m−2], V̇O2Peak peak oxygen uptake [mL min−1 kg−1], Ẇ LT power at the lactate threshold [Watts], Ẇ peak peak power output [Watts], 20k-TT 20-km time trial [seconds]. Source data are provided as a Source Data ﬁle. P values reported in the table were: P = 0.040 for PR vs. PN in “Body mass”; P = 0.041 for PR vs. PN in “BMI”; P = 0.004 for PH vs. BL, P = 0.046 for PH vs. PN, and P = 0.014 for PR vs. BL in “V̇O2Peak”; P = 4.5e−10 for PH vs. BL, P = 2.5e−8 for PH vs. PN, P = 1.5e−9 for PR vs. BL, and P = 9.6e−8 for PR vs. PN in “Ẇ LT”; P = 1.1e−8 for PH vs. BL, P = 3.8e−8 for PH vs. PN, P = 3.3e−8 for PR vs. BL, and P = 1.1e−7 for PR vs. PN in “Ẇ peak”; P = 4.3e−6 for PH vs. BL, P = 1.0e−5 for PH vs. PN, P = 4.6e−8 for PR vs. BL, and P = 1.0e−7 for PR vs. PN in “20k-TT time”. All values are mean ± SD; n = 10 for all analyses; all datasets analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05 * vs. BL, # vs. PN. Fig. 1 A complex network of transcriptional responses underpins training-induced increases in mitochondrial content. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Measurement BL PN PH PR Age 22.3 ± 5.2 – – – Height 178.1 ± 10.7 – – – Body mass 78.8 ± 12.1 79.0 ± 12.9 78.0 ± 12.5 77.9 ± 11.8# BMI 24.8 ± 2.9 24.9 ± 3.2 24.6 ± 3.0 24.5 ± 2.9# V̇O2Peak 46.7 ± 8.2 47.7 ± 8.8 50.5 ± 6.0# 50.0 ± 6.0 Ẇ LT 174.1 ± 30.7 182.5 ± 33.0 222.2 ± 36.2# 219.6 ± 31.4# Ẇ peak 231.1 ± 43.2 233.2 ± 43.5 266.3 ± 42.4# 264.5 ± 40.4# 20k-TT time 2321.0 ± 168.1 2309.1 ± 257.4 2094.6 ± 130.8# 2030.1 ± 140.5# BL baseline, PN post normal-volume training, PH post high-volume training, PR post reduced-volume training, BMI body mass index [kg m−2], V̇O2Peak peak oxygen uptake [mL min−1 kg−1], Ẇ LT power at the lactate threshold [Watts], Ẇ peak peak power output [Watts], 20k-TT 20-km time trial [seconds]. Source data are provided as a Source Data ﬁle. P values reported in the table were: P = 0.040 for PR vs. PN in “Body mass”; P = 0.041 for PR vs. PN in “BMI”; P = 0.004 for PH vs. BL, P = 0.046 for PH vs. PN, and P = 0.014 for PR vs. BL in “V̇O2Peak”; P = 4.5e−10 for PH vs. BL, P = 2.5e−8 for PH vs. PN, P = 1.5e−9 for PR vs. BL, and P = 9.6e−8 for PR vs. PN in “Ẇ LT”; P = 1.1e−8 for PH vs. BL, P = 3.8e−8 for PH vs. PN, P = 3.3e−8 for PR vs. BL, and P = 1.1e−7 for PR vs. PN in “Ẇ peak”; P = 4.3e−6 for PH vs. BL, P = 1.0e−5 for PH vs. PN, P = 4.6e−8 for PR vs. BL, and P = 1.0e−7 for PR vs. PN in “20k-TT time”. All values are mean ± SD; n = 10 for all analyses; all datasets analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05 * vs. BL, # vs. PN. Table 1 Participants’ physiological and endurance performance measurements. BL baseline, PN post normal-volume training, PH post high-volume training, PR post reduced-volume training, BMI body mass index [kg m−2], V̇O2Peak peak oxygen uptake [mL min−1 kg−1], Ẇ LT power at the lactate threshold [Watts], Ẇ peak peak power output [Watts], 20k-TT 20-km time trial [seconds]. Source data are provided as a Source Data ﬁle. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 P values reported in the table were: P = 0.040 for PR vs. PN in “Body mass”; P = 0.041 for PR vs. PN in “BMI”; P = 0.004 for PH vs. BL, P = 0.046 for PH vs. PN, and P = 0.014 for PR vs. BL in “V̇O2Peak”; P = 4.5e−10 for PH vs. BL, P = 2.5e−8 for PH vs. PN, P = 1.5e−9 for PR vs. BL, and P = 9.6e−8 for PR vs. PN in “Ẇ LT”; P = 1.1e−8 for PH vs. BL, P = 3.8e−8 for PH vs. PN, P = 3.3e−8 for PR vs. BL, and P = 1.1e−7 for PR vs. PN in “Ẇ peak”; P = 4.3e−6 for PH vs. BL, P = 1.0e−5 for PH vs. PN, P = 4.6e−8 for PR vs. BL, and P = 1.0e−7 for PR vs. PN in “20k-TT time”. All values are mean ± SD; n = 10 for all analyses; all datasets analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05 * vs. BL, # vs. PN. mitochondria relative to all protein intensities - at different time points. Indeed, the MPE for the four different time points in the present study was signiﬁcantly different (Fig. 2a, middle panel) and corresponded to 24.5% at baseline (BL), 31.5% post-NVT (PN), 39.3% post-HVT (PH), and 34.5% post-RVT (PR) (Supplementary Data 3) - a pattern consistent with our assessment of mitochondrial content (compare Fig. 2a, middle panel with Fig. 1c, d). support these ﬁndings with quantitative proteomics. To negate the general increase in mitochondrial content following exercise training (Fig. 1d) and to avoid this inherent bias on our proteomics measurements, we performed label-free quantitative (LFQ) proteomics on equal amounts of mitochondria-enriched fractions (biochemical enrichment of mitochondrial proteins using differential centrifugation). We quantiﬁed 1411 proteins − 726 of which were annotated as mitochondrial based on either the Mitocarta2.028 or the Integrated Mitochondrial Protein Index (IMPI; Known and Predicted Mitochondrial)29 databases (Sup- plementary Data 3). Considering only the high-conﬁdence IMPI (Known Mitochondrial) dataset, mitochondrial proteins repre- sented 41% of the total number of proteins identiﬁed in our mitochondria-enriched fraction (584 annotated mitochondrial proteins), contributing to 32% of the overall protein abundance based on raw intensity data for each protein (Supplementary Data 3). ARTICLE ARTICLE BL baseline, PN post normal-volume training, PH post high-volume training, PR post reduced-volume training, BMI body mass index [kg m−2], V̇O2Peak peak oxygen uptake [mL min−1 kg−1], Ẇ LT power at the lactate threshold [Watts], Ẇ peak peak power output [Watts], 20k-TT 20-km time trial [seconds]. Source data are provided as a Source Data ﬁle. P values reported in the table were: P = 0.040 for PR vs. PN in “Body mass”; P = 0.041 for PR vs. PN in “BMI”; P = 0.004 for PH vs. BL, P = 0.046 for PH vs. PN, and P = 0.014 for PR vs. BL in “V̇O2Peak”; P = 4.5e−10 for PH vs. BL, P = 2.5e−8 for PH vs. PN, P = 1.5e−9 for PR vs. BL, and P = 9.6e−8 for PR vs. PN in “Ẇ LT”; P = 1.1e−8 for PH vs. BL, P = 3.8e−8 for PH vs. PN, P = 3.3e−8 for PR vs. BL, and P = 1.1e−7 for PR vs. PN in “Ẇ peak”; P = 4.3e−6 for PH vs. BL, P = 1.0e−5 for PH vs. PN, P = 4.6e−8 for PR vs. BL, and P = 1.0e−7 for PR vs. PN in “20k-TT time”. All values are mean ± SD; n = 10 for all analyses; all datasets analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05 * vs. BL, # vs. PN. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Fig. 1 A complex network of transcriptional responses underpins training-induced increases in mitochondrial content. a Study design; coloured boxes indicate a training phase: normal-volume training (NVT), high-volume training (HVT), and reduced-volume training (RVT), whereas open boxes indicate a testing session and resting skeletal muscle biopsy: baseline (BL), post-NVT (PN), post-HVT (PH), and post-RVT (PR). b Weekly training volume during the NVT, HVT, and RVT training phase; training volume was calculated by multiplying the absolute exercise intensity in Watts by the effective duration of exercise training in minutes (excluding the warm up and the rest periods between intervals) by the total number of training sessions in each phase. c Mitochondrial yield per mg of tissue achieved during mitochondria isolation of human vastus lateralis biopsy samples (biochemical enrichment of mitochondrial protein). d Citrate synthase (CS) enzyme activity assessed in whole-tissue (vastus lateralis) homogenates. For panel b–d source data are provided as a Source Data ﬁle and P values are indicated on the ﬁgure to three decimal places; for P values that were truncated, the corresponding accurate P values were: HVT vs. NVT: P = 3.9e−14; RVT vs. NVT: P = 3.8e−5; HVT vs. NVT: P = 1.1e−12 in panel b; PH vs. BL: P = 1.9e−6; PR vs. BL: P = 1.6e−5; PH vs. PN: P = 8.3e−4 in panel c; and PR vs. BL: P = 5.3e−4 in panel d. e Heatmap of differentially expressed transcripts between training phases determined with an adjusted P < 0.05 (Benjamini–Hochberg). Row clustering determined by unsupervised hierarchical cluster analysis. f Proﬁle plots showing relative scaled transcripts of subunits of the ﬁve oxidative phosphorylation (OXPHOS) complexes (CI to CV), mitochondrial ribosomes (mtribo), OXPHOS assembly, TCA cycle, SLC25As, and mitochondrial contact site and cristae organising system (MICOS) complex. Transcripts grouped according to known literature (see Methods section). g Biological process (BP) gene ontology of all differentially expressed transcripts as in e; the top ﬁve biological processes by adjusted p value, as determined by Enrichr (see Methods section), are displayed with their combined score (the full list is presented in Supplementary Data 2). HIIT high-intensity interval training, MJ megajoules, CI-V complex I–V, IB immunoblotting. Datasets b–d (n = 10) were analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 PN e mtribo −1 −0.5 0 0.5 1 CI CII CIII CIV CV −1 −0.5 0 0.5 1 OXPHOS assembly TCA SLC25A MICOS PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL f EXTRACELLULAR MATRIX ORGANIZATION (GO:0030198) EXTRACELLULAR STRUCTURE ORGANIZATION (GO:0043062) EXTERNAL STRUCTURE ORGANIZATION (GO:0045229) REGULATION OF ANGIOGENESIS (GO:0045765) REGULATION OF CELL MIGRATION (GO:0030334) COMBINED SCORE 200 100 50 150 0 g g g EXTRACELLULAR MATRIX ORGANIZATION (GO:0030198) EXTRACELLULAR STRUCTURE ORGANIZATION (GO:0043062) EXTERNAL STRUCTURE ORGANIZATION (GO:0045229) REGULATION OF ANGIOGENESIS (GO:0045765) REGULATION OF CELL MIGRATION (GO:0030334) COMBINED SCORE 200 100 50 150 0 g training-induced changes in mitochondrial content and markers of mitochondrial biogenesis and mitochondrial respiratory function9. However, whether there is also a ﬁxed stoichiometry between changes in mitochondrial content and individual mito- chondrial proteins, protein functional classes, and metabolic pathways, remains undetermined. To gain further insight into the effects of training on mitochondria we ﬁrst employed RNA sequencing (RNA-seq) based transcriptomics (Supplementary Data 1). We identiﬁed 1206 transcripts differentially expressed across the three training phases (Fig. 1e and Supplementary Data 2), and we observed the expected increase in gene tran- scripts encoding subunits of the OXPHOS complexes (Fig. 1f, upper panels, compare with Supplementary Fig. 1a, upper panels) and enzymes of the tricarboxylic acid (TCA) cycle (Fig. 1f, central lower panel), consistent with previous studies in humans27. However, transcripts for genes encoding other mitochondrial proteins did not all follow the same trend (Fig. 1f, lower panels). Moreover, gene ontology enrichment analysis of the differentially expressed transcripts did not identify signiﬁcant alterations of pathways involved in mitochondrial respiratory function (Fig. 1g and Supplementary Data 2), despite the increase in mitochondrial respiration and ETC enzyme activity reported above (Supple- mentary Fig. 1b, c, respectively). This reveals a greater complexity in the transcriptional responses to exercise than previously hypothesised; i.e., not all mitochondria-related gene transcripts changed in the same direction and with a similar magnitude following different training phases (Fig. 1f). As transcriptomics suggested there may not be a ﬁxed stoichiometry between training-induced changes in mitochon- drial content and individual mitochondrial proteins, we sought to TURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153- rease in mitochondrial proteins with different training umes had been compensated for, indicating that our To assess the power of our normalisation strategy, we compared results from non-normalised and BESt-normalised f 45% Mitochondrial translation 55% Translation 72% Respiratory electron transport, ATP synthesis 15% Cristae formation 13% Protein localisation 100% Metabolism of amino acids & derivatives 50% Cristae formation 50% Mitochondrial protein import Metabolism 51% TCA cycle & respiratory electron transport 25% 14% Mitochondrial protein import 10% Metabolism of amino acids & derivatives 45% Mitochondrial Fatty Acid β-Oxidation 30% TCA cycle 25% Metabolism of amino acids & derivatives c a BL PN PH PR 1.8 P P NON-NORMALIZED BL PN PH PR BESt-NORMALISED = 0.014 = 0.002 BL PN PH PR MPE (raw values x1012) 0 0.6 1.2 P = 0.033 b −4e+08 −2e+08 0e+00 2e+08 4e+08 −1e+09 0e+00 1e+09 2e+09 Dimension 1 Dimension 2 BL PN PH PR d Holloway 2009 Present study Egan 2011 Hostrup 2018 163 8 0 9 2 1 0 2 9 0 0 2 1 5 0 −2 −1 0 1 2 Log2FC CLUSTER-3 CLUSTER-4 CLUSTER-6 CLUSTER-5 CLUSTER-2 CLUSTER-1 PN vs. BL PR vs. PH PH vs. PN e g Unchanged Downregulated Upregulated PR vs. PN 472 14 12 PR vs. BL 393 62 43 PH vs. BL 366 71 61 PR vs. PH 498 PH vs. PN 464 23 11 PN vs. BL 396 61 41 CI CII CIII CIV CV mtribo PH PR PN BL BESt- NORMALIZED NON- NORMALIZED PH PR PN BL ase in mitochondrial proteins with different training mes had been compensated for, indicating that our hemical enrichment and statistical correction (BESt-normal- on) was successful (Fig. 2a, lower panel). In further support of normalisation strategy, multidimensional scaling analysis wed good segregation of samples corresponding to the rent time points (Fig 2b) To asses compared proteomics values revea and mitoch marked incr panels) res c a BL PN PH PR 1.8 P P NON-NORMALIZED BL PN PH PR BESt-NORMALISED = 0.014 = 0.002 BL PN PH PR MPE (raw values x1012) 0 0.6 1.2 P = 0.033 b −4e+08 −2e+08 0e+00 2e+08 4e+08 −1e+09 0e+00 1e+09 2e+09 Dimension 1 Dimension 2 BL PN PH PR d Holloway 2009 Present study Egan 2011 Hostrup 2018 163 8 0 9 2 1 0 2 9 0 0 2 1 5 0 g Unchanged Downregulated Upregulated PR vs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Unlike CS activity, which is a single value for each sample, our normalisation strategy utilises the trends of hundreds of proteins and reduces the confounding effect of potential outliers. This is extremely important, given that previous studies have shown a greater than 5-fold difference in training-induced synthesis rate between fast and slow synthesised mitochondrial proteins in human skeletal muscle11,30,31. Plotting the resulting data revealed that the general NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 PN 472 14 12 PR vs. BL 393 62 43 PH vs. BL 366 71 61 PR vs. PH 498 PH vs. PN 464 23 11 PN vs. BL 396 61 41 CI CII CIII CIV CV mtribo PH PR PN BL BESt- NORMALIZED NON- NORMALIZED PH PR PN BL f 45% Mitochondrial translation 55% Translation 72% Respiratory electron transport, ATP synthesis 15% Cristae formation 13% Protein localisation 100% Metabolism of amino acids & derivatives 50% Cristae formation 50% Mitochondrial protein import Metabolism 51% TCA cycle & respiratory electron transport 25% 14% Mitochondrial protein import 10% Metabolism of amino acids & derivatives 45% Mitochondrial Fatty Acid β-Oxidation 30% TCA cycle 25% Metabolism of amino acids & derivatives 11 trup 8 −2 −1 0 1 2 Log2FC CLUSTER-3 CLUSTER-4 CLUSTER-6 CLUSTER-5 CLUSTER-2 CLUSTER-1 PN vs. BL PR vs. PH PH vs. PN e f 45% Mitochondrial translation 55% Translation Metabolism 51% TCA cycle & respiratory electron transport 25% 14% Mitochondrial protein import 10% Metabolism of amino acids & derivatives 45% Mitochondrial Fatty Acid β-Oxidation 30% TCA cycle 25% Metabolism of amino acids & derivatives −2 −1 0 1 2 Log2FC CLUSTER-3 CLUSTER-2 CLUSTER-1 e c CI CII CIII CIV CV mtribo PH PR PN BL BESt- NORMALIZED NON- NORMALIZED PH PR PN BL a BL PN PH PR 1.8 P P NON-NORMALIZED BL PN PH PR BESt-NORMALISED = 0.014 = 0.002 BL PN PH PR MPE (raw values x1012) 0 0.6 1.2 P = 0.033 a c e CI 45% Mitochondrial translation 55% Translation 72% Respiratory electron transport, ATP synthesis 15% Cristae formation 13% Protein localisation 100% Metabolism of amino acids & derivatives 50% Cristae formation 50% Mitochondrial protein import Metabolism 51% TCA cycle & respiratory electron transport 25% 14% Mitochondrial protein import 10% Metabolism of amino acids & derivatives 45% Mitochondrial Fatty Acid β-Oxidation 30% TCA cycle 25% Metabolism of amino acids & derivatives CLUSTER-3 CLUSTER-4 CLUSTER-6 CLUSTER-5 CLUSTER-2 CLUSTER-1 PN vs. BL PR vs. PH PH vs. PN CLUSTER-1 CLUSTER-2 increase in mitochondrial proteins with different training volumes had been compensated for, indicating that our biochemical enrichment and statistical correction (BESt-normal- isation) was successful (Fig. 2a, lower panel). In further support of our normalisation strategy, multidimensional scaling analysis showed good segregation of samples corresponding to the different time points (Fig. 2b). To assess the power of our normalisation strategy, we compared results from non-normalised and BESt-normalised proteomics values. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Whereas analysis of non-normalised intensity values revealed a lack of change in the abundance of OXPHOS and mitochondrial ribosomal subunits post-NVT, followed by a marked increase post-HVT, and a decrease post-RVT (Fig. 2c, left panels), results from BESt-normalised values showcased a very 72% Respiratory electron transport, ATP synthesis 15% Cristae formation 13% Protein localisation 100% Metabolism of amino acids & derivatives 50% Cristae formation 50% Mitochondrial protein import Metabolism 51% TCA cycle & respiratory electron transport 25% 14% Mitochondrial protein import 10% Metabolism of amino acids & derivatives BL PN PH PR b −4e+08 −2e+08 0e+00 2e+08 4e+08 −1e+09 0e+00 1e+09 2e+09 Dimension 1 Dimension 2 BL PN PH PR d Holloway 2009 Present study Egan 2011 Hostrup 2018 163 8 0 9 2 1 0 2 9 0 0 2 1 5 0 CLUSTER-3 CLUSTER-4 CLUSTER-6 CLUSTER-5 PN vs. BL PR vs. PH PH vs. PN g Unchanged Downregulated Upregulated PR vs. PN 472 14 12 PR vs. BL 393 62 43 PH vs. BL 366 71 61 PR vs. PH 498 PH vs. PN 464 23 11 PN vs. BL 396 61 41 CV mtribo PH PR PN BL PH PR PN BL NATURE COMMUNICATIONS \| (2021)12 7056 \| htt //d i /10 1038/ 41467 021 27153 3 \| t / t i ti 5 CLUSTER-3 d Holloway 2009 Present study Egan 2011 Hostrup 2018 163 8 0 9 2 1 0 2 9 0 0 2 1 5 0 PR vs PH PH vs PN b −4e+08 −2e+08 0e+00 2e+08 4e+08 −1e+09 0e+00 1e+09 2e+09 Dimension 1 Dimension 2 BL PN PH PR b d Holloway 2009 Hostrup 2018 CLUSTER-4 Present study g PH vs. BL PN vs. BL 396 61 41 PR vs. BL PH vs. PN 464 23 11 g PR vs. PH PN vs. BL Unchanged PR vs. PN 472 14 12 PR vs. PH 498 CLUSTER-5 PH vs. BL PR vs. BL PR vs. PN 12 CLUSTER-6 Downregulated 393 62 43 Upregulated 366 71 61 14 Upregulated Unchanged Downregulated To assess the power of our normalisation strategy, we compared results from non-normalised and BESt-normalised proteomics values. Whereas analysis of non-normalised intensity values revealed a lack of change in the abundance of OXPHOS and mitochondrial ribosomal subunits post-NVT, followed by a marked increase post-HVT, and a decrease post-RVT (Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Differentially expressed proteins for each time point comparison were identiﬁed by linear modelling with multiple comparison adjustment made with a P value of <0.01 using the Benjamini–Hochberg method, as shown in Supplementary Data 7. BL baseline, PN post-NVT, PH post-HVT, PR post-RVT. n = 10 for all analyses. Fig. 2 Correcting for changes in mitochondrial content reveals differentially expressed protein clusters with divergent prioritisation in response to training. a Training-induced differences in the mitochondrial protein content of isolated mitochondrial (IM) fractions obtained from vastus lateralis muscle biopsies. Upper panel: proﬁle plot of all non-normalised intensity values displaying “known mitochondrial” (IMPI “Known Mitochondrial”) (magenta) and non-mitochondrial (salmon) proteins co-precipitating during mitochondrial isolation. Proteins identiﬁed in less than 70% of samples were removed. Middle panel: mitochondrial protein enrichment (MPE) obtained by adding the raw intensity values from LC-MS/MS analysis of all “known mitochondrial” proteins (IMPI “Known Mitochondrial”) at the four different time points. Filled triangles, empty diamonds, and empty and ﬁlled circles represent individual values; the maxima of each bar represents the mean value; analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05 have been identiﬁed by sampling muscle only at the beginning and end of this study). different picture, as the content of OXPHOS subunits was markedly decreased (deprioritised) post-NVT, and showed discreet and speciﬁc changes (mainly modest increases) thereafter (Fig. 2c, right panels; results discussed in more detail later), whereas mitochondrial ribosomal subunits increased to a greater extent than mitochondrial content (prioritised) (Fig. 2c, right panels). These ﬁndings demonstrate the power of our BESt- normalisation strategy compared to classical approaches, as it enables an unbiased comparison of the changes in individual mitochondrial proteins relative to the overall increase in mitochondrial content following training, providing clear evi- dence that these changes are not stoichiometrically linked. As a means of comparison, we also employed the classical approach of normalising the abundances of speciﬁc proteins derived from SDS-PAGE and immunoblot analysis on isolated mitochondrial fractions by CS activity; this demonstrates that while this widely used approach partially corrects for changes in MPE, it fails to recapitulate most of the ﬁndings observed with our BESt- normalisation strategy (Supplementary Fig. 2b). Unsupervised hierarchical clustering of all differentially expressed mitochondrial proteins revealed six clusters (Fig. 2e) with distinct patterns of change in response to training (Supplementary Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Fig. 2 Correcting for changes in mitochondrial content reveals differentially expressed protein clusters with divergent prioritisation in response to training. a Training-induced differences in the mitochondrial protein content of isolated mitochondrial (IM) fractions obtained from vastus lateralis muscle biopsies. Upper panel: proﬁle plot of all non-normalised intensity values displaying “known mitochondrial” (IMPI “Known Mitochondrial”) (magenta) and non-mitochondrial (salmon) proteins co-precipitating during mitochondrial isolation. Proteins identiﬁed in less than 70% of samples were removed. Middle panel: mitochondrial protein enrichment (MPE) obtained by adding the raw intensity values from LC-MS/MS analysis of all “known mitochondrial” proteins (IMPI “Known Mitochondrial”) at the four different time points. Filled triangles, empty diamonds, and empty and ﬁlled circles represent individual values; the maxima of each bar represents the mean value; analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing; P < 0.05 (n = 10 for all groups). Lower panel: same as upper panel but following removal of non-mitochondrial proteins and statistical correction (BESt-) normalisation. Source data are provided as a Source Data ﬁle. b Multidimensional scaling analysis showing segregation of samples obtained from IM fractions of BESt-normalised proteomics values. c Scaled proﬁle plots showing the relative abundance of subunits of OXPHOS complexes and mtribo of the non-normalised intensity values of “known mitochondrial” proteins (IMPI “Known Mitochondrial”) following valid value ﬁltering (left panels), and values obtained after BESt-normalisation (right panels). d Venn diagram representing the overlap in differentially expressed proteins between the present study and previous literature utilising young healthy humans23,32,33. The bubble size for each study is proportional to the number of differentially expressed proteins identiﬁed (this was not possible for the overlapping size). e Heatmap of BESt-normalised differentially expressed mitochondrial proteins (IMPI “Known Mitochondrial”), identiﬁed with an adjusted P < 0.01 (Benjamini–Hochberg) determined by linear model ﬁt using limma with empirical Bayes method. Row clustering determined by unsupervised hierarchical cluster analysis. f Venn diagram representation of the gene ontology of the six clusters identiﬁed in e obtained from enrichment analysis (using the ClueGO [v2.5.6] application in Cytoscape [v3.7.1]; see Methods section) of all proteins belonging to each cluster performed separately (Supplementary Data 5). g Venn diagram representations of the number of BESt-normalised differentially expressed mitochondrial proteins (IMPI “Known Mitochondrial”) between each of the time points. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 2c), which also conﬁrmed the absence of a ﬁxed stoichiometry between training-induced changes in mito- chondrial content and mitochondrial protein functional classes. Enrichment analysis of differentially expressed proteins within each of the six clusters revealed them to be enriched in, but not always exclusively contain, proteins involved in fatty acid β- oxidation (FAO), mitochondrial translation, the TCA cycle, OXPHOS, metabolism of amino acids, and cristae formation, respectively (Fig. 2f and Supplementary Data 5). Because not all proteins belonging to each individual Reactome pathway segregated within one cluster (Supplementary Fig. 2d), we compared our 185 differentially expressed proteins with proteins belonging to the six aforementioned functional classes (Reactome R-HSA) and other related pathways; matching proteins (Supple- mentary Data 6) were used to generate the individual pathway heatmaps presented in Fig. 3 and to discuss our ﬁndings. In addition, because Reactome, like other protein databases, only provides an extensive but not exhaustive list of proteins involved in each pathway, we complemented our heatmaps with other proteins identiﬁed through literature (see Methods section for more information on how pathways in Supplementary Data 6 and Fig. 3 were generated). From this point onwards, unless speciﬁed otherwise, post BESt- normalisation proteomics results will be presented, enabling the investigation of training-induced changes in mitochondrial proteins without the confounding inﬂuence of changes in mitochondrial content. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 2c, left panels), results from BESt-normalised values showcased a very To assess the power of our normalisation strategy, we compared results from non-normalised and BESt-normalised proteomics values. Whereas analysis of non-normalised intensity values revealed a lack of change in the abundance of OXPHOS and mitochondrial ribosomal subunits post-NVT, followed by a marked increase post-HVT, and a decrease post-RVT (Fig. 2c, left panels), results from BESt-normalised values showcased a very To assess the power of our normalisation strategy, we compared results from non-normalised and BESt-normalised proteomics values. Whereas analysis of non-normalised intensity values revealed a lack of change in the abundance of OXPHOS and mitochondrial ribosomal subunits post-NVT, followed by a marked increase post-HVT, and a decrease post-RVT (Fig. 2c, left panels), results from BESt-normalised values showcased a very increase in mitochondrial proteins with different training volumes had been compensated for, indicating that our biochemical enrichment and statistical correction (BESt-normal- isation) was successful (Fig. 2a, lower panel). In further support of our normalisation strategy, multidimensional scaling analysis showed good segregation of samples corresponding to the different time points (Fig. 2b). increase in mitochondrial proteins with different training volumes had been compensated for, indicating that our biochemical enrichment and statistical correction (BESt-normal- isation) was successful (Fig. 2a, lower panel). In further support of our normalisation strategy, multidimensional scaling analysis showed good segregation of samples corresponding to the different time points (Fig. 2b). TURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 5 Discovery of mitochondrial proteins and functional classes and metabolic pathways differentially affected by exercise training. Discovery of mitochondrial proteins and functional classes and metabolic pathways differentially affected by exercise training. We next interrogated our BESt-normalised mitochondrial pro- teome to gain new insights into non-stoichiometric changes in individual mitochondrial proteins following three training phases designed to induce different effects on mitochondrial biogenesis. Using linear modelling (comparable to a multiple samples ANOVA), we identiﬁed 185 mitochondrial proteins that were differentially expressed across the three training phases (Supple- mentary Data 5). This is ~10-fold greater than the number of proteins reported to change post-training in previous human skeletal muscle studies23,32–34 (Fig. 2d). This increased number of proteins was likely related to our normalisation strategy, and did not seem to depend on the large training volume utilised (120 proteins were differentially expressed after the modest training volume of the NVT phase alone), nor was the result of comparing multiple time points (118 differentially expressed proteins would To pinpoint the origin of training-induced changes in individual mitochondrial proteins, we performed unpaired t-tests between group pairs following permutation-based false discovery rate (FDR) correction. Using a conservative approach, we matched the differentially expressed proteins identiﬁed by each t-test comparison with the 185 differentially expressed proteins across the three training phases; this analysis, which includes all relevant comparisons, including those between each time point against baseline, can be found in Supplementary Data 7. The majority of changes occurred during the initial NVT phase (Fig. 2g and Supplementary Data 7), where 102 proteins were differentially expressed, suggesting that the greatest number of mitochondrial adaptations took place within the ﬁrst few training sessions and these became harder to obtain as the training progressed (Fig. 2e, note the strongest changes are found in the TURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 6 vs. BL” column). Our study also conﬁrms that the training ulus during the RVT phase was sufﬁcient to prevent a loss of tation (Fig. 2g and Supplementary Data 7). The lack of ge in mitochondrial markers in the current study is in present study (2.14 MJ/week for 1 week vs. 0.32 MJ/we 2 weeks, respectively). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 UQCR10 UQCRB UQCRC2 UQCRFS1 UQCRC1 SDHB SDHA MP68 ATP5J ATP5B ATP5H ATP5O ATP5A1 MT-CO3 MT−ND5 NDUFC2 NDUFA13 NDUFS4 NDUFB4 NDUFA10 NDUFS2 NDUFB11 NDUFS6 NDUFB7 Subunits OXPHOS Succinate SUCLG2 SUCLG1 SUCLA2 SDHB SDHA COQ7 COQ3 COQ9 COQ5 CPT1B PLIN5 ACSL1 ACSL3 ALDH3A2 CI CIV CII CV CIII Q UQCRC1 SDHA MP68 MT-CO3 MT−ND5 OXPHOS COQ7 CPT1B CI CIV CII CV CIII Q N vs. BL” column). Our study also conﬁrms that the training mulus during the RVT phase was sufﬁcient to prevent a loss of ptation (Fig 2g and Supplementary Data 7) The lack of present study (2.14 MJ/week for 1 week vs. 0.32 MJ/we 2 weeks, respectively). UQCR10 UQCRB UQCRC2 UQCRFS1 UQCRC1 SDHB SDHA MP68 ATP5J ATP5B ATP5H ATP5O ATP5A1 MT-CO3 NDUFB5 NDUFA2 MT−ND5 NDUFC2 NDUFA13 NDUFS4 NDUFB4 NDUFA10 NDUFS2 NDUFB11 NDUFS6 NDUFB7 Subunits OXPHOS BCS1L ATPAF1 COA3 TACO1 COX19 TIMMDC1 ACAD9 NDUFAF3 NUBPL ECSIT Assembly Factors CI CIV CII CV CIII Q 3 Overview of training-induced changes in mitochondrial protein functional classes and metabolic pathways. Protein functional classes abolic pathways (deﬁned in Supplementary Data 6) as determined in Supplementary Data 7. Row clustering determined by unsupervised hier er analysis. BL baseline, PN post-NVT, PH post-HVT, PR post-RVT, ROS reactive oxygen species, FAO fatty acid β-oxidation, CoQ Coenzyme rboxylic acid cycle, OXPHOS oxidative phosphorylation. UQCR10 UQCRB UQCRC2 UQCRFS1 UQCRC1 SDHB SDHA MP68 ATP5J ATP5B ATP5H ATP5O ATP5A1 MT-CO3 NDUFB5 NDUFA2 MT−ND5 NDUFC2 NDUFA13 NDUFS4 NDUFB4 NDUFA10 NDUFS2 NDUFB11 NDUFS6 NDUFB7 Subunits OXPHOS BCS1L ATPAF1 COA3 TACO1 COX19 TIMMDC1 ACAD9 NDUFAF3 NUBPL ECSIT Assembly Factors . 3 Overview of training-induced changes in mitochondrial protein functional classes and metabolic pathways. Protein functional classes and/or tabolic pathways (deﬁned in Supplementary Data 6) as determined in Supplementary Data 7. Row clustering determined by unsupervised hierarchica ster analysis. BL baseline, PN post-NVT, PH post-HVT, PR post-RVT, ROS reactive oxygen species, FAO fatty acid β-oxidation, CoQ Coenzyme Q, TCA arboxylic acid cycle, OXPHOS oxidative phosphorylation. Fig. 3 Overview of training-induced changes in mitochondrial protein functional classes and metabolic pathways. Protein functional classes and/or metabolic pathways (deﬁned in Supplementary Data 6) as determined in Supplementary Data 7. Row clustering determined by unsupervised hierarchical cluster analysis. BL baseline, PN post-NVT, PH post-HVT, PR post-RVT, ROS reactive oxygen species, FAO fatty acid β-oxidation, CoQ Coenzyme Q, TCA tricarboxylic acid cycle, OXPHOS oxidative phosphorylation. present study (2.14 MJ/week for 1 week vs. 0.32 MJ/week for 2 weeks, respectively). “PN vs. Discovery of mitochondrial proteins and functional classes and metabolic pathways differentially affected by exercise training. UQCR10 UQCRB UQCRC2 UQCRFS1 UQCRC1 SDHB SDHA MP68 ATP5J ATP5B ATP5H ATP5O ATP5A1 MT-CO3 NDUFB5 NDUFA2 MT−ND5 NDUFC2 NDUFA13 NDUFS4 NDUFB4 NDUFA10 NDUFS2 NDUFB11 NDUFS6 NDUFB7 Subunits OXPHOS BCS1L ATPAF1 COA3 TACO1 COX19 TIMMDC1 ACAD9 NDUFAF3 NUBPL ECSIT Assembly Factors TCA Cycle Isocitrate α - ketoglutarate Succinyl CoA Succinate Fumarate Malate Oxaloacetate Citrate MDH2 ACO2 FH CS SUCLG2 SUCLG1 SUCLA2 SDHB SDHA IDH2 IDH3B IDH3A IDH3G FAHD1 CoQ Biosynthesis COQ7 COQ3 COQ9 COQ5 Mitochondrial Translation MRPL18 MRPS5 MRPS22 MRPL4 MRPS27 GFM1 TUFM PTCD3 MTIF2 TSFM Protein Folding HSPA9 TRAP1 HSPD1 HSPE1 TIMM44 Response to ROS GPX1 GPX4 SOD2 PRDX5 TXN2 CYCS −2 −1 0 1 2 Log2FC PN vs. BL PR vs. PH PH vs. PN DLD FAO CPT1B ETFB ETFA PLIN5 ACSF2 ACOT9 ECHS1 DECR1 HADH MCEE ACAA2 MECR ACAT1 HSD17B8 HMGCL HSD17B10 HADHA ACSL1 ACSL3 MLYCD ALDH3A2 Cristae Formation CHCHD3 IMMT APOOL APOO MTX2 MTX1 OMA1 TMEM11 Pyruvate AcCoA ME2 DLD CI CIV CII CV CIII Q CoQ e - donors IVD DHODH L2HGDH ETFDH GPD2 DLD Overview of training-induced changes in mitochondrial protein functional classes and metabolic pathways. Protein functional classes a olic pathways (deﬁned in Supplementary Data 6) as determined in Supplementary Data 7. Row clustering determined by unsupervised hier r analysis. BL baseline, PN post-NVT, PH post-HVT, PR post-RVT, ROS reactive oxygen species, FAO fatty acid β-oxidation, CoQ Coenzyme boxylic acid cycle, OXPHOS oxidative phosphorylation. RE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 ARTI NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Mitochondrial Translation MRPL18 MRPS5 MRPS22 MRPL4 MRPS27 GFM1 TUFM PTCD3 MTIF2 TSFM Protein Folding HSPA9 TRAP1 HSPD1 HSPE1 TIMM44 Response to ROS GPX1 GPX4 SOD2 PRDX5 TXN2 CYCS −2 −1 0 1 2 Log2FC PN vs. BL PR vs. PH PH vs. PN Cristae Formation CHCHD3 IMMT APOOL APOO MTX2 MTX1 OMA1 TMEM11 Mitochondrial Translation MRPL18 MRPS5 MRPS22 MRPL4 MRPS27 GFM1 TUFM PTCD3 MTIF2 TSFM Response to ROS TCA Cycle Isocitrate α - ketoglutarate Succinyl CoA Succinate Fumarate Malate Oxaloacetate Citrate MDH2 ACO2 FH CS SUCLG2 SUCLG1 SUCLA2 SDHB SDHA IDH2 IDH3B IDH3A IDH3G FAHD1 CoQ Biosynthesis COQ7 COQ3 COQ9 COQ5 DLD FAO CPT1B ETFB ETFA PLIN5 ACSF2 ACOT9 ECHS1 DECR1 HADH MCEE ACAA2 MECR ACAT1 HSD17B8 HMGCL HSD17B10 HADHA ACSL1 ACSL3 MLYCD ALDH3A2 Pyruvate AcCoA ME2 DLD CoQ e - donors IVD DHODH L2HGDH ETFDH GPD2 DLD FAO CPT1B ETFB ETFA PLIN5 ACSF2 ACOT9 ECHS1 DECR1 HADH MCEE ACAA2 MECR ACAT1 HSD17B8 HMGCL HSD17B10 HADHA ACSL1 ACSL3 MLYCD ALDH3A2 BL” column). Our study also conﬁrms that the training present study (2.14 MJ/week for 1 week vs. 0.32 MJ/week UQCR10 UQCRB UQCRC2 UQCRFS1 UQCRC1 SDHB SDHA MP68 ATP5J ATP5B ATP5H ATP5O ATP5A1 MT-CO3 NDUFB5 NDUFA2 MT−ND5 NDUFC2 NDUFA13 NDUFS4 NDUFB4 NDUFA10 NDUFS2 NDUFB11 NDUFS6 NDUFB7 Subunits OXPHOS BCS1L ATPAF1 COA3 TACO1 COX19 TIMMDC1 ACAD9 NDUFAF3 NUBPL ECSIT Assembly Factors Isocitrate α - ketoglutarate Succinyl CoA Succinate Fumarate Malate Oxaloacetate Citrate MDH2 ACO2 FH SUCLG2 SUCLG1 SUCLA2 SDHB SDHA IDH2 IDH3B IDH3A IDH3G FAHD1 CoQ Biosynthesis COQ7 COQ3 COQ9 COQ5 DLD CPT1B ETFB ETFA PLIN5 ACSF2 ACOT9 ECHS1 DECR1 HADH MCEE ACAA2 MECR ACAT1 HSD17B8 HMGCL HSD17B10 HADHA ACSL1 ACSL3 MLYCD ALDH3A2 ME2 CI CIV CII CV CIII Q CoQ e - donors IVD DHODH L2HGDH ETFDH GPD2 DLD erview of training-induced changes in mitochondrial protein functional classes and metabolic pathways. Protein functional classes and/ c pathways (deﬁned in Supplementary Data 6) as determined in Supplementary Data 7. Row clustering determined by unsupervised hierarch alysis. BL baseline, PN post-NVT, PH post-HVT, PR post-RVT, ROS reactive oxygen species, FAO fatty acid β-oxidation, CoQ Coenzyme Q, T ylic acid cycle, OXPHOS oxidative phosphorylation. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Of note, SDHA and SDHB, subunits of CII - the only TCA cycle enzyme that also participates in the ETC - clustered with the majority of OXPHOS subunits (discussed below) and decreased during NVT. While training-induced changes in some TCA cycle proteins have been previously reported23,33, our data extends these ﬁndings by revealing training-induced increases in ACO2, SUCLG1, SUCLG2, SUCLA2, and FAHD1, noting that that these enzymes adapted mostly in a coordinated fashion. p y Proteins involved in FAO segregated mainly within cluster-1 and cluster-3 (Fig. 2f; Supplementary Fig. 2d and Supplementary Data 5). Our data demonstrated a large and concerted increase in these proteins post-NVT, with minimal (HVT) and no (RVT) changes thereafter (Fig. 3 and Supplementary Data 7). Proteins increased post-NVT included chain shortening enzymes involved in FAO (ACAA2, ACSF2, ECHS1, HADH, HSD17B10, MECR, MLYCD) and enzymes required to convert unsaturated fatty acids into intermediates of FAO (DECR1 and HSD17B8). Similar increases were also observed for ETFA and ETFB, which transfer electrons produced during FAO to the OXPHOS system, further highlighting the early increase in FAO. HMGCL and ACAT1, two enzymes involved in the ketogenesis pathway, were also increased, suggesting that excess acetyl-CoA generated by enhanced FAO may be fed into ketogenesis. Taken together, these ﬁndings indicate that the synthesis of proteins involved in both the TCA cycle and FAO pathways is prioritised in the early stages of an endurance training intervention; these increases may have contributed, at least in part, to the training-induced increase in mitochondrial respiration (Supplementary Fig. 1b, upper panels). Of relevance to the increase in FAO reported above, we observed a post-NVT decrease in TGs (Fig. 4d), a class of lipids used as an important source of energy production during exercise40; however, likely due to the high number of multiple comparisons in our bioinformatic analysis (n = 779), no signiﬁcant changes in single TG species were detected (Supple- mentary Fig. 3). The decrease in TGs in our study is consistent with an increase in fatty acid turnover, and is corroborated by the post-NVT increase in FAO enzymes (Fig. 3). Finally, changes in TGs (Fig. 4d), which are not found in mitochondria but rather in lipid droplets41, mirrored changes in PLIN5 (Fig. 3). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Post-normalisation proﬁle plots of CL species indicated that, despite changes in individual CL species, the general increase in mitochondrial content with different training volumes had been compensated for by our normalisation strategy (Fig. 4a; right panel), and the lipid proﬁles could be used to readily seg- regate samples across different time points (Fig. 4b). From this point on we will present post-normalisation lipidomics results, unless speciﬁed otherwise. We identiﬁed 182 differentially expressed lipid species (Fig. 4c and Supplementary Data 9). Similar to what we observed in our proteomics dataset, and consistent with previous research in humans36,37, our study highlighted the divergent response to training of different lipid classes and species relative to overall changes in mitochondrial content (Fig. 4d). However, in contrast to proteomics, where the greatest number of changes were observed early (during NVT) and no changes were observed during RVT, our lipidomics assessment showed the most signiﬁcant changes to occur during the latter two stages (Supplementary Fig. 3). This suggests a delayed response to changes in lipid content compared to proteins, and may reﬂect overall mitochondrial biogenesis as training-induced changes in lipid species were mainly related to mitochondrial membrane- based lipids (CLs, phosphatidylethanolamines and phosphatidyl- cholines) and the triglycerides (TGs) (Supplementary Fig. 3). Contrary to previous training studies in obese humans36–38, we did not observe reductions in muscle ceramides [with the exception of Cer(d18:1/18:0) during HVT] or diacylglycerol content in our healthy, young men following training. While this could indicate a different response of lipid species in mitochon- dria isolates (our study) compared to whole-muscle lysates36–38, as previously suggested39, it could also highlight adaptation differences between obese and healthy individuals. A complete dataset detailing the differentially expressed lipids between each of the four time points (including comparisons of all time points vs. baseline) can be found in Supplementary Data 9. Mitochondria prioritise the TCA cycle and FAO in response to NVT. Proteins involved in the TCA cycle pathway grouped mainly within cluster-3 and, to a lesser extent, cluster-1 (Fig. 2f; Supplementary Fig. 2d and Supplementary Data 5). This pathway was characterised by a large and concerted increase post-NVT (12 of 16 enzymes were signiﬁcantly increased), with many of these proteins remaining upregulated compared to BL both post-HVT and post-RVT (Fig. 3 and Supplementary Data 7). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 BL” column). Our study also conﬁrms that the training stimulus during the RVT phase was sufﬁcient to prevent a loss of adaptation (Fig. 2g and Supplementary Data 7). The lack of change in mitochondrial markers in the current study is in contrast with the signiﬁcant reductions reported in a previous study20; this is likely to stem from the greater training volume and/or the shorter duration of the RVT phase employed in the Proteins important for the translation of mitochondrial pro- teins are sensitive to training volume. We next examined the six over-represented pathways highlighted by our enrichment TURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 7 7 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 analysis, beginning with the pathway related to mitochondrial protein translation (Fig. 3), which mostly segregated within cluster-2 (Fig. 2f; Supplementary Fig. 2d; and Supplementary Data 5). Speciﬁcally, we identiﬁed proteins from both the small 28S (MRPS5, MRPS22, MRPS27) and large 39S (MRPL4, MRPL18) subunits of the mitochondrial ribosome, as well as proteins involved in translation initiation (MTIF2) and elonga- tion (GFM1, PTCD3, TSFM, TUSM; Fig. 3). Whereas only four proteins were upregulated post-NVT, eight of the 10 aforemen- tioned proteins were upregulated post-HVT, with six remaining elevated post-RVT (Supplementary Data 7). These ﬁndings demonstrate that training-induced changes occur in these or related mitochondrial translation proteins in young, healthy humans, in line with observations previously made in older (65–80 years) individuals34. Although the inﬂuence of training duration cannot be completely ruled out, our ﬁndings also indi- cate that changes in proteins involved in mitochondrial protein translation may be sensitive to training volume, consistent with a study observing a positive association between these proteins and physical activity levels in healthy individuals (20–87 years)35. (Supplementary Data 8) were quantiﬁed. Indeed, changes in the mitochondria-speciﬁc cardiolipins (CLs), the biomarker most strongly associated with mitochondrial content in human skeletal muscle21, mirrored changes in training volume and in non- normalised mitochondrial proteins (compare Fig. 4a, left panel with Fig. 2a, upper panel; Supplementary Data 8). Therefore, to ensure that our lipidomics data were corrected for changes in MPE in our fractions, we applied a similar approach to the one used for proteomics where lipidomic results were normalised according to the trend observed for the CL class (see Methods section). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 d c a PN vs. BL PR vs. PH PH vs. PN −2 −1 0 1 2 Log2FC Neutral/Other Sphingolipids Cardiolipin Glycerophospholipids Lipids −5e+04 0e+00 5e+04 10e+04 −10e+04 −5e+04 0e+00 5e+04 10e+04 15e+04 Dimension 1 Dimension 2 BL PN PH PR b Scaled z-scored Mean −1.0 −0.5 0.0 0.5 1.0 NORMALISED TO CL −1.0 −0.5 0.0 0.5 1.0 NON-NORMALISED PH PR PN BL PH PR PN BL Scaled Concentration Mean FA PC PE LPC Cer(d) LPC(O) LPE(P) PE(O) PC(O) CE TG TG(O) COH PC(P) LPI LPC(P) Sph Ubiquinone PE(P) PG PI PA GM3 Hex2Cer DG AcylCarnitine LPE PS −0.5 0.0 0.5 1.0 Sphingolipids Glycerophospholipids Neutral Lipids/Other −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 SM −0.5 0.0 0.5 1.0 PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL a b −5e+04 0e+00 5e+04 10e+04 −10e+04 −5e+04 0e+00 5e+04 10e+04 15e+04 Dimension 2 BL PN PH PR b −1.0 −0.5 0.0 0.5 1.0 NORMALISED TO CL PH PR PN BL a NORMALISED TO CL Scaled z-scored Mean −1.0 −0.5 0.0 0.5 1.0 NON-NORMALISED PH PR PN BL c A training-induced increase in OXPHOS subunits is not a priority to enhance mitochondrial respiration. We next exam- ined the OXPHOS pathway, which segregated almost entirely in cluster-4 (Fig. 2f; Supplementary Fig. 2d and Supplementary Data 5). Our analysis provided excellent coverage of OXPHOS proteins and identiﬁed 78 OXPHOS subunits. We report training-induced changes in 26 OXPHOS subunits (Fig. 3), equivalent to 33% of all subunits identiﬁed. Differentially expressed OXPHOS proteins included subunits from all six of the modules required for the assembly of CI, both catalytic subunits of CII, as well as subunits from CIII, one of the three core sub- units of CIV, and proteins found in both functional domains of CV. Speciﬁcally, we observed a large and coordinated (23 sub- units) decrease post-NVT, with no changes during HVT and/or d c PN vs. BL PR vs. PH PH vs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 PN −2 −1 0 1 2 Log2FC Neutral/Other Sphingolipids Cardiolipin Glycerophospholipids Lipids −10e+04 −5e+04 0e+00 5e+04 10e+04 15e+04 Dimension 1 Scaled Concentration Mean FA PC PE LPC Cer(d) LPC(O) LPE(P) PE(O) PC(O) CE TG TG(O) COH PC(P) LPI LPC(P) Sph Ubiquinone PE(P) PG PI PA GM3 Hex2Cer DG AcylCarnitine LPE PS −0.5 0.0 0.5 1.0 Sphingolipids Glycerophospholipids Neutral Lipids/Other −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 SM −0.5 0.0 0.5 1.0 PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 9 c PN vs. BL PR vs. PH PH vs. PN −2 −1 0 1 2 Log2FC Neutral/Other Sphingolipids Cardiolipin Glycerophospholipids Lipids XPHOS subunits is not a espiration. We next exam- egregated almost entirely in ig. 2d and Supplementary llent coverage of OXPHOS HOS subunits. We report XPHOS subunits (Fig. 3), equivalent to 33% of all subunits identiﬁed. Differentially expressed OXPHOS proteins included subunits from all six of the modules required for the assembly of CI, both catalytic subunits of CII, as well as subunits from CIII, one of the three core sub- units of CIV, and proteins found in both functional domains of CV. Speciﬁcally, we observed a large and coordinated (23 sub- units) decrease post-NVT, with no changes during HVT and/or d R vs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Since PLIN5 is a protein that tethers mitochondria to lipid droplets and regulates the release of lipids for FAO42,43, we hypothesise that training-induced changes in TGs may reﬂect increased associa- tion of lipid droplets with mitochondria. While few studies have evaluated the effects of different training interventions on skeletal muscle lipid species, our results add to the growing evidence that changes with training are speciﬁc to the exercise prescription and that training-induced changes in individual lipid species and lipid classes are not stoichiometrically linked. Lipidomics highlights a divergent response to training of dif- ferent lipid classes. To highlight speciﬁc changes in fatty acid composition, LC-MS/MS based comparative lipidomics was performed using the same mitochondrial isolates used for pro- teomics. A total of 779 species representing 30 lipid classes NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 8 8 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 In contrast to the only previous report showing a post-training redistribution of CIII and CIV (but not CI) into SCs in elderly individuals15, and recent research in mouse skeletal muscle showing redistribution of CIV into SCs54, our analysis revealed no signiﬁcant changes through- out the intervention (Fig. 5b, lower panels). This demonstrates that in young healthy men endurance training does not alter the ETC complex distribution within major and minor SC assem- blies. While the contrasting ﬁndings may relate to either species differences (i.e., mice vs. humans) or differences in the participants’ average age (66 vs. 22 y) and baseline V̇O2max (1.9 vs. 3.6 L min−1), as well as the training interventions (48 sessions of moderate-intensity training in 16 weeks vs. 52 sessions of HIIT in 9 weeks), our results are consistent with the concept that SC assemblies are a structural feature of the ETC rather than a phenomenon conferring enhanced bioenergetics53. In line with this, our data indicated that the greatest changes in maximal mitochondrial respiration ([ETF + CI + II]P) occurred in parallel with the greatest changes in SC abundance (compare Fig. 5b with 5c, both upper panels). However, following normalisation by MPE, both of these adaptations were no longer signiﬁcant (compare Fig. 5b, middle panels with Fig. 5c, lower panel). Taken together, our ﬁndings do not support the hypothesis that training-induced changes in the abundance or organisation of SCs contribute to improvements in mitochondrial respiration. Conversely, our data indicate that training-induced changes in both mitochondrial respiration and SC formation could largely be attributed to increases in mitochondrial content. The coordinated assembly of OXPHOS complexes involves various assembly factors, chaperones, and protein translocation components, and has been suggested to be in the order of days48. Whereas increases in OXPHOS assembly factors occurred mostly post-HVT (Fig. 3 and Supplementary Data 7), changes in subunits of the mitochondrial ribosome (Fig. 2c, lower right panel), which synthesise the 13 OXPHOS subunits encoded by mitochondrial DNA, and in several chaperone proteins regulating import and folding of proteins destined for the mitochondrial matrix, took place earlier (post-NVT; Fig. 3 and Supplementary Data 7). Chaperone proteins upregulated post-NVT included HSPA9 (mtHsp70), HSPD1 (mtHsp60), HSPE1 (mtHsp10), and TRAP1 (mtHsp75), whereby TIMM44 was increased during HVT (Fig. 3 and Supplementary Data 7). All ﬁve proteins remained elevated compared to BL both post-HVT and post-RVT. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Changes in SLC25A4 (ANT1), a protein catalysing the exchange of cytosolic ADP and mitochondrial ATP across the mitochondrial inner membrane44, mirrored these changes (Supplementary Data 7). The post-NVT decrease in OXPHOS subunits indicates that during times of increased mitochondrial biogenesis, synthesis of proteins involved in OXPHOS pathways is deprioritised. Moreover, our results demonstrated a large post-NVT increase in TCA cycle and FAO related enzymes - the major providers of reducing equivalents (NADH and FADH2) to the OXPHOS system45; this suggests that enhancement of these two metabolic pathways may be more important to improve mitochondrial respiration following exer- cise training (Supplementary Fig. 1b, upper panels) than an increase in OXPHOS subunits. Training-induced changes in mitochondrial content, but not changes in the abundance and/or organisation of SCs, mediate improvements in mitochondrial respiration. A previous study reported that training increases the formation of ETC SCs - high molecular weight assemblies comprised of CI, CIII and CIV51 - and suggested this to contribute to improvements in mitochon- drial respiration15. Although SCs were originally proposed to support enhanced ETC function52, their role has become con- troversial, with multiple groups reporting that they confer no bioenergetic advantage53. To provide further insight, we per- formed BN-PAGE analysis of ETC SCs (Fig. 5a). We observed increases in SC abundance with increasing training volumes, achieving signiﬁcance post-HVT (Fig. 5b, upper panels); however, these increases were no longer signiﬁcant upon normalisation by MPE (Fig. 5b, middle panels). An increase in reducing equivalents coupled with the deprioritisation of the OXPHOS machinery could lead to increased reactive oxygen species (ROS) generation. Here we report an immediate post-NVT increase in the abundance of enzymes involved in protection from oxidative stress, such as PRDX5 (the most increased protein), TXN2, and GPX146 (Fig. 3 and Supplementary Data 7). Moreover, both SOD2, the mitochondrial ROS scavenger46, and PRDX5 were increased post-HVT and post-RVT. These ﬁndings suggest that training- induced synthesis of proteins involved in the protection from oxidative stress was emphasised early during the training intervention and maintained throughout, consistent with the notion that exercise training provides protection against oxidative stress46,47. We next assessed whether exercise training affects the distribution of ETC complexes into SCs. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Fig. 4 Lipidomics highlights a divergent response to training of different lipid classes. a All-lipids proﬁle plot of non-normalised (left panel) and cardiolipin (CL) class-normalised (right panel) intensity values from mitochondrial isolates, displaying all individual CL species (gold) and all other lipid species (grey). b Multidimensional scaling analysis showing segregation of samples obtained from IM fractions of lipidomics values normalised by the entire CL class. c Heatmap of differentially expressed lipids after normalisation by the entire CL class between training phases determined by linear model ﬁt using limma with empirical Bayes method with an adjusted P < 0.05 (Benjamini–Hochberg) to account for multiple comparisons. Row clustering determined by unsupervised hierarchical cluster analysis. d Proﬁle plots of the scaled concentration mean of different lipid classes. For each proﬁle plot, all the individual lipid species identiﬁed within the speciﬁc class are represented in colour over the entire lipidome (grey) at each time point. The intensity values for all lipid species were normalised by the entire CL class to adjust for changes in mitochondrial content. BL baseline, PN post-NVT, PH post-HVT, PR post-RVT. n = 10 for all analyses. SM sphingomyelins, Cer(d) ceramides, GM3 monosialodihexosylganglioside, Hex2Cer dihexosylceramide, Sph sphingosine, LPC lysophosphatidylcholines, LPC(O) lysoalkylphosphatidylcholines, LPC(P) lysoalkenylphosphatidylcholines, PG phosphatidylglycerols, LPE lysophosphatidylethanolamines, LPE(P) lysoalkenylphosphatidylethanolamines, LPI lysophosphatidylinositols, PA phosphatidic acid, PE phosphatidylethanolamines, PE(O) alkylphosphatidylethanolamines, PE(P) alkenylphosphatidylethanolamines, PI phosphatidylinositols, PC phosphatidylcholines, PC(O) alkylphosphatidylcholines, PC(P) alkenylphosphatidylcholines, PS phosphatidylserines, CE cholesterol ester, COH cholesterol, DG diacylglycerols, FA fatty acids, TG triacylglycerols, TG(O) alkyl triacylglycerols. RVT (Fig. 3 and Supplementary Data 7). Changes in SLC25A4 (ANT1), a protein catalysing the exchange of cytosolic ADP and mitochondrial ATP across the mitochondrial inner membrane44, mirrored these changes (Supplementary Data 7). The post-NVT decrease in OXPHOS subunits indicates that during times of increased mitochondrial biogenesis, synthesis of proteins involved in OXPHOS pathways is deprioritised. Moreover, our results demonstrated a large post-NVT increase in TCA cycle and FAO related enzymes - the major providers of reducing equivalents (NADH and FADH2) to the OXPHOS system45; this suggests that enhancement of these two metabolic pathways may be more important to improve mitochondrial respiration following exer- cise training (Supplementary Fig. 1b, upper panels) than an increase in OXPHOS subunits. TIM, respectively; Supplementary Data 3), which are required for importing nuclear-encoded subunits50, most did not change signiﬁcantly with training, suggesting that their levels remain stable relative to changes in mitochondrial content. RVT (Fig. 3 and Supplementary Data 7). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 PH −2 −1 0 1 2 Log2FC −10e+04 −5e+04 0e+00 5e+04 10e+04 15e+04 Dimension 1 Scaled Concentration Mean FA PC PE LPC Cer(d) LPC(O) LPE(P) PE(O) PC(O) CE TG TG(O) COH PC(P) LPI LPC(P) Sph Ubiquinone PE(P) PG PI PA GM3 Hex2Cer DG AcylCarnitine LPE PS −0.5 0.0 0.5 1.0 Sphingolipids Glycerophospholipids Neutral Lipids/Other −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 SM −0.5 0.0 0.5 1.0 PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL tps://doi org/10 1038/s41467-021-27153-3 \| www nature com/naturecommunications 9 Dimension 1 d Scaled Concentration Mean FA PC PE LPC Cer(d) LPC(O) LPE(P) PE(O) PC(O) CE TG TG(O) COH PC(P) LPI LPC(P) Sph Ubiquinone PE(P) PG PI PA GM3 Hex2Cer DG AcylCarnitine LPE PS −0.5 0.0 0.5 1.0 Sphingolipids Glycerophospholipids Neutral Lipids/Other −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 SM −0.5 0.0 0.5 1.0 PH PR PN BL PH PR PN BL PH PR PN BL PH PR PN BL d Scaled Concentration Mean PC PE LPC Cer(d) LPC(O) LPE(P) PE(O) PC(O) PC(P) LPI LPC(P) Sph PE(P) PG PI PA GM3 Hex2Cer LPE PS Sphingolipids Glycerophospholipids 0 0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 SM −0.5 0.0 0.5 1.0 d Sphingolipids Neutral Lipids/Other A training-induced increase in OXPHOS subunits is not a priority to enhance mitochondrial respiration. We next exam- ined the OXPHOS pathway, which segregated almost entirely in cluster-4 (Fig. 2f; Supplementary Fig. 2d and Supplementary Data 5). Our analysis provided excellent coverage of OXPHOS proteins and identiﬁed 78 OXPHOS subunits. We report training-induced changes in 26 OXPHOS subunits (Fig. 3), equivalent to 33% of all subunits identiﬁed. Differentially expressed OXPHOS proteins included subunits from all six of the modules required for the assembly of CI, both catalytic subunits of CII, as well as subunits from CIII, one of the three core sub- units of CIV, and proteins found in both functional domains of CV. Speciﬁcally, we observed a large and coordinated (23 sub- units) decrease post-NVT, with no changes during HVT and/or 9 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 1 1 1 2 2 4 5 6 3 CI-NDUFA9 CIII-UQCRC2 CIV-COX IV 2 - CIII2+CIV 660 450 132 66 kDa CL(18:2_18:1_18:1_16:1) CL(18:2_18:1_18:1_18:1) CL(18:2_18:1_18:2_20:1) CL(18:2_18:1_18:2_20:2) CL(18:2_18:1_18:2_22:6) CL(18:2_18:2_18:1_16:1) CL(18:2_18:2_18:1_18:1) CL(18:2_18:2_18:1_20:2) CL(18:2_18:2_18:1_20:3) CL(18:2_18:2_18:1_22:6) CL(18:2_18:2_18:2_18:3) CL(34:1_34:1) 0 9e+04 1.8e+04 2.7e+04 CLs average concentration CL(18:2_18:1_18:2_18:1) CL(18:2_18:2_18:2_18:1) CL(18:2_18:2_18:2_18:2) 8e+05 6e+05 4e+05 2e+05 0 d BL PN PH PR OMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 A ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Except for HSPE123, none of the above chaperones have previously been reported to respond to exercise. Owing to the notion that all of these chaperones are known to interact49, our data indicate a coordinated increase in the protein quality control system in response to exercise training. While we detected many subunits of the translocases of the outer and inner membrane (TOM and NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 10 he importance of CLs for the assembly and stability of rial membrane protein complexes and ETC SCs55,56, interrogated our normalised mitochondrial lipidome. ysis demonstrated an extensive remodelling of CL on (Fig. 5d); notably, following NVT and HVT, we a decrease in the abundance of tetra-linoleoyl CL skeletal muscle57. Accompanying this were concomita in CLs containing oleic acid (18:1) and to a l palmitoleic acid (16:1) acyl chains. This may reﬂe relative availability of dietary linoleic acid due t mitochondrial biogenesis, since the nature of CL catalysed by TAZ, which was unaltered in our datase c b a [ETF+CI+II]P 0 40 80 120 160 200 240 P < 0.001 P < 0.001 P = 0.008 BL PN PH PR Mass-specific MR pmol O2 s-1 mg wet weight-1 [ETF+CI+II]P/MPE 0 1 2 3 4 5 Mt-specific MR [pmol O2 s-1 mg wet weight-1 MPE-1]x1010 BL PN PH PR 0.0 0.5 1.0 OXPHOS complexes distribution from IM by BN-PAGE (digitonin) 0 4 8 12 OXPHOS SCs/MPE x1012 from IM by BN-PAGE (digitonin) 0 1 2 3 4 5 OXPHOS SCs from IM by BN-PAGE (digitonin) 0 1 2 3 4 5 6 7 0 1 2 3 4 0.0 0.5 1.0 0.0 0.5 1.0 0 2 4 6 0 4 8 12 P = 0.003 P = 0.045 P = 0.020 P = 0.038 P = 0.003 P = 0.043 BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR IB: CI-NDUFA9 IB: CIV-COXIV IB: CIII-UQCRC2 CI-NDUFA9 CIV-COXIV CIII-UQCRC2 SCs CIII2 CIII2+ CIV CIV CIV2 1 - SCs 4 - CIII2 5 - CIV2 6 - CIV 3 - LMW CI int. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 c b a [ETF+CI+II]P 0 40 80 120 160 200 240 P < 0.001 P < 0.001 P = 0.008 BL PN PH PR Mass-specific MR pmol O2 s-1 mg wet weight-1 [ETF+CI+II]P/MPE 0 1 2 3 4 5 Mt-specific MR [pmol O2 s-1 mg wet weight-1 MPE-1]x1010 BL PN PH PR 0.0 0.5 1.0 OXPHOS complexes distribution from IM by BN-PAGE (digitonin) 0 4 8 12 OXPHOS SCs/MPE x1012 from IM by BN-PAGE (digitonin) 0 1 2 3 4 5 OXPHOS SCs from IM by BN-PAGE (digitonin) 0 1 2 3 4 5 6 7 0 1 2 3 4 0.0 0.5 1.0 0.0 0.5 1.0 0 2 4 6 0 4 8 12 P = 0.003 P = 0.045 P = 0.020 P = 0.038 P = 0.003 P = 0.043 BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR BL PN PH PR IB: CI-NDUFA9 IB: CIV-COXIV IB: CIII-UQCRC2 CI-NDUFA9 CIV-COXIV CIII-UQCRC2 SCs CIII2 CIII2+ CIV CIV CIV2 1 - SCs 4 - CIII2 5 - CIV2 6 - CIV 3 - LMW CI int. 1 1 1 2 2 4 5 6 3 CI-NDUFA9 CIII-UQCRC2 CIV-COX IV 2 - CIII2+CIV 660 450 132 66 kDa b 0 1 2 3 4 5 OXPHOS SCs from IM by BN-PAGE (digitonin) P = 0.003 P = 0.045 BL PN PH PR IB: CI-NDUFA9 t. NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications ARTICLE For panels b–d ﬁlled triangles, empty diamonds, and empty and ﬁlled circles represent individual values; the maxima of each bar represents the mean value; n = 10 for all analyses; datasets b and c were analysed by repeated measures one-way ANOVA followed by Tukey’s post hoc testing, except for the lower right panel of b, which was analysed by Friedman test followed by Dunn’s post hoc testing, as not normally distributed; P < 0.05. Fig. 5 Changes in mitochondrial content without reorganisation of SCs occur alongside improvements in mitochondrial respiration. a Representative BN-PAGE blots of isolated mitochondria (IM) fractions from human vastus lateralis muscles (images from baseline [BL] IM fractions). Band 1 (SCs): mature supercomplexes (SCs) consisting of complex I + IIIn + IVn; band 2 (CIII2 + IV): a SC consisting of CIII and CIV; band 3 (LMW CI int): low molecular weight intermediate of CI (band not present in all samples); band 4 (CIII2): CIII dimer; band 5 (CIV2): CIV dimer; band 6 (CIV): CIV monomer. b Top panels: protein content of SCs of the electron transport chain (ETC) by BN-PAGE in IM fractions from human vastus lateralis muscle biopsies (antibodies probed on separate membranes); middle panels: values from top panels normalised by mitochondrial protein enrichment (MPE); lower panels: distribution of ETC complexes into SCs from images obtained in b at each time point. Source data are provided as a Source Data ﬁle. All samples analysed were derived The mitochondrial contact site and cristae organising system (MICOS) is pivotal for the formation of cristae junctions, providing the extended membrane surface hosting OXPHOS complexes65. The MICOS associates with the outer membrane sorting and assembly machinery (SAM) to yield the mitochon- drial intermembrane space bridging complex (MIB), thus linking the mitochondrial inner and outer membranes65. Eight proteins involved in cristae formation were differentially expressed in our study: APOO (MIC26), APOOL (MIC27), CHCHD3 (MIC19) and MIC60 from the MICOS complex; MTX1 and MTX2 from the SAM complex; TMEM11, a protein associating with multiple MICOS subunits for cristae biogenesis66; and OMA1, a mitochondrial protease regulating OPA167 - a key protein implicated in cristae remodelling68 (Fig. 3 and Supplementary Data 6). Seven of these proteins, which almost entirely grouped within cluster-6 (Fig. 2f; Supplementary Fig. 2d and Supplemen- tary Data 5), were signiﬁcantly decreased post-HVT, with four being decreased post-RVT (Supplementary Data 7), suggesting a training-induced deprioritisation of these proteins. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 Fig. 5 Changes in mitochondrial content without reorganisation of SCs occur alongside improvements in mitochondrial respiration. a Representative BN-PAGE blots of isolated mitochondria (IM) fractions from human vastus lateralis muscles (images from baseline [BL] IM fractions). Band 1 (SCs): mature supercomplexes (SCs) consisting of complex I + IIIn + IVn; band 2 (CIII2 + IV): a SC consisting of CIII and CIV; band 3 (LMW CI int): low molecular weight intermediate of CI (band not present in all samples); band 4 (CIII2): CIII dimer; band 5 (CIV2): CIV dimer; band 6 (CIV): CIV monomer. b Top panels: protein content of SCs of the electron transport chain (ETC) by BN-PAGE in IM fractions from human vastus lateralis muscle biopsies (antibodies probed on separate membranes); middle panels: values from top panels normalised by mitochondrial protein enrichment (MPE); lower panels: distribution of ETC complexes into SCs from images obtained in b at each time point. Source data are provided as a Source Data ﬁle. All samples analysed were derived from the same experiment and blots were processed in parallel. c Top panel: maximal mass-speciﬁc mitochondrial respiration (MR) in permeabilised human vastus lateralis muscle ﬁbres with convergent electron input through ETF + CI + CII ([ETF + CI + II]P); lower panel: values of mitochondrial- (mt-) speciﬁc MR, obtained after normalising values from the top panel by MPE ([ETF + CI + II]P/MPE). Results for the entire substrate-uncoupler-inhibitor- titration (SUIT) protocol are shown in Supplementary Fig. 1b. P values are indicated on the ﬁgure to three decimal places; for P values that were truncated, the corresponding accurate P values were: PH vs. BL: P = 5.2e−5; PR vs. BL: P = 4.5e−4. Source data are provided as a Source Data ﬁle. d Training-induced changes in all individual cardiolipin (CL) species that were identiﬁed as differentially expressed (as determined in Fig. 4c, by linear model ﬁt using limma with empirical Bayes method with an adjusted P < 0.05 [Benjamini–Hochberg]). Source data are provided as a Source Data ﬁle. PN post-NVT, PH post- HVT, PR post-RVT, IB immunoblotting. ARTICLE This is consistent with the minimal changes observed in cristae density following short-term (<3 months) exercise training in obese individuals69. Thus, our results support the notion that cristae remodelling is not a priority during short-term training and that changes in other mitochondrial proteins are more important to adapt to the higher metabolic demand of exercise. Despite MICOS-MIB subunits being known to interact with CLs70, we report no correlation between the levels of MICOS-MIB subunits and changes in CLs content following different training volumes. compensation in CL content is in line with our observation that increased training volume does not lead to changes in ETC SC assembly (Fig. 5b middle and lower panels). Coenzyme Q biosynthesis and cristae formation are not prioritised during high-volume endurance training. Despite enrichment analysis suggesting cluster-5 to be enriched in metabolism of amino acids and derivatives, upon further inspection we identiﬁed this cluster to predominantly contain proteins involved in the biosynthesis and function of coenzyme Q (CoQ or ubiquinone; Fig. 2f; Supplementary Fig. 2d and Sup- plementary Data 5) - an enzyme with an essential role as an electron transfer lipid in OXPHOS processes59, which has been shown to be altered following exercise training in mouse skeletal muscle54. We identiﬁed changes in four constituents (COQ3, COQ5, COQ7, COQ9) of the CoQ synthome (Fig. 3 and Sup- plementary Data 6), a multi-subunit complex necessary for the biosynthesis of CoQ60. We report no changes post-NVT, con- sistent with previous research investigating the effects of short- duration (<3 weeks) exercise training on CoQ content in human skeletal muscle61. However, post-HVT we report a concerted reduction in three of these proteins, which also persisted post- RVT (Supplementary Data 7). This indicates that CoQ bio- synthesis is not prioritised in response to high-volume endurance training, suggesting that enhancing the CoQ pool may not be necessary to support an increase in mitochondrial respiration in human skeletal muscle of young healthy individuals. y g y CoQ is an electron carrier shuttling electrons derived from the TCA cycle (via CI and CII), pyrimidine biosynthesis (via DHODH), glycolysis (via GPD2), and FAO (via EFTDH) to Complex III45. Moreover, other enzymes located near or on the inner mitochondrial membrane feeding electrons to CoQ, such as IVD62, DLD63 and L2HGDH64, were also identiﬁed in our study (Supplementary Data 6). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 0 1 2 3 4 5 6 7 0 1 2 3 4 P = 0.020 P = 0.038 P = 0.003 P = 0.043 BL PN PH PR BL PN PH PR IB: CIV-COXIV IB: CIII-UQCRC2 b 0 4 8 12 OXPHOS SCs/MPE x1012 from IM by BN-PAGE (digitonin) 0 2 4 6 0 4 8 12 BL PN PH PR BL PN PH PR BL PN PH PR 0.0 0.5 1.0 OXPHOS complexes distribution from IM by BN-PAGE (digitonin) 0.0 0.5 1.0 0.0 0.5 1.0 BL PN PH PR BL PN PH PR BL PN PH PR CI-NDUFA9 CIV-COXIV CIII-UQCRC2 SCs CIII2 CIII2+ CIV CIV CIV2 2 2 2 CL(18:2_18:1_18:1_16:1) CL(18:2_18:1_18:1_18:1) CL(18:2_18:1_18:2_20:1) CL(18:2_18:1_18:2_20:2) CL(18:2_18:1_18:2_22:6) CL(18:2_18:2_18:1_16:1) CL(18:2_18:2_18:1_18:1) CL(18:2_18:2_18:1_20:2) CL(18:2_18:2_18:1_20:3) CL(18:2_18:2_18:1_22:6) CL(18:2_18:2_18:2_18:3) CL(34:1_34:1) 0 9e+04 1.8e+04 2.7e+04 CLs average concentration CL(18:2_18:1_18:2_18:1) CL(18:2_18:2_18:2_18:1) CL(18:2_18:2_18:2_18:2) 8e+05 6e+05 4e+05 2e+05 0 d BL PN PH PR d Given the importance of CLs for the assembly and stability of mitochondrial membrane protein complexes and ETC SCs55,56, we again interrogated our normalised mitochondrial lipidome. Our analysis demonstrated an extensive remodelling of CL composition (Fig. 5d); notably, following NVT and HVT, we observed a decrease in the abundance of tetra-linoleoyl CL (18:2_18:2_18:2_18:2), the dominant form of CLs found in skeletal muscle57. Accompanying this were concomitant increases in CLs containing oleic acid (18:1) and to a lesser extent palmitoleic acid (16:1) acyl chains. This may reﬂect a lack in relative availability of dietary linoleic acid due to increased mitochondrial biogenesis, since the nature of CL remodelling catalysed by TAZ, which was unaltered in our dataset, appears to be controlled predominantly by lipid availability58. This 11 ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 prioritisation extended to all three levels investigated (i.e., the transcriptome, proteome, and lipidome) and between levels, as demonstrated by the delayed adaptive response of lipids com- pared to proteins. Testing procedures. Participants were required to avoid any vigorous exercise for the 48 h preceding each performance test (72 h for the skeletal muscle biopsy), from alcohol and any exercise for 24 h before testing, and from food and caffeine consumption for the 2 h preceding each test. Similar tests were performed at the same time of the day throughout the study to avoid variations caused by changes in circadian rhythm. p p A striking and unexpected ﬁnding was the early (post-NVT) deprioritisation of OXPHOS subunit formation, which indicates that the overall training-induced increase in biogenesis and the proliferation of mitochondria is greater than the increase in speciﬁc components of the OXPHOS machinery. This deprioritisation of OXPHOS subunit biogenesis becomes even more striking when considering the early increase in mitochondrial respiration, as well as proteins involved in the TCA cycle and FAO processes - two of the main suppliers of reducing equivalents to the OXPHOS system. This increase could not be explained by changes in the abundance and/or organisation of SCs, as previously hypothesised52, since we observed no change in these parameters. Our ﬁndings therefore add to the growing evidence that SC formation does not confer enhancements in mitochondrial bioenergetics53. The deprioritisation of OXPHOS subunit biogenesis is also unlikely to be related to the time required to assemble large multi-protein complexes48, as the relative amount of OXPHOS subunits remained reduced throughout the intervention (lasting several weeks), whereas mitochondrial respiration, as well as TCA cycle and FAO process, remained upregulated throughout. Instead, the increase in mitochondrial respiration was likely supported by the inherent reserve capacity of mitochondria (i.e., their ability to respond to sudden increases in energy requirements)71, which suggests that enhancing electron ﬂow to OXPHOS is more important to increase ATP production than an increase in the components of the OXPHOS machinery. The relative decrease in proteins important for both CoQ biosynthesis and cristae formation further substantiates these ﬁndings. GXT. ARTICLE A graded exercise test was performed on an electronically braked cycle ergometer (Lode Excalibur v2.0, Groningen, The Netherlands) to determine peak oxygen uptake (V̇O2Peak), peak power output (Ẇ Peak), the power attained at the lactate threshold (Ẇ LT) using the modiﬁed DMax method74, and the training intensity for each training phase. The test consisted of consecutive 4-min stages at constant power output; the test starting intensity (range: 45–77 W) and the intensity increase of each stage (range: 17–28 W) were chosen so as to obtain at least 8 time points for the determination of the Ẇ LT75 and were based on parti- cipants’ ﬁtness levels. An identical protocol was used at all four time points for each participant. Prior to the test, and in the last 30 s of each stage, venous blood samples were taken for measurement of blood lactate concentration ([La−]). Participants were instructed to keep a cadence > 60 rpm and were only allowed access to cadence. The test was stopped when a participant reached volitional exhaustion or cadence dropped below 60 rpm for over 10 s. The Ẇ Peak was determined as the power of the last completed stage plus an additional 25% of the stage increase wattage for every additional minute completed. At the end of each GXT, after a 5-min recovery, a veriﬁcation exhaustive bout was performed at an intensity equivalent to V̇O2Peak to conﬁrm the highest measured V̇O2Peak75. 20k-TT. Cycling time trials were performed on an electronically braked cycle ergometer (Velotron, RacerMate, Seattle, WA, USA) after a 6-min cycling warm-up (4 min at 66% of Ẇ LT followed by 2 min at Ẇ LT), and 2 min of rest. Participants were only allowed access to cadence and completed distance. Gas Analysis during the GXT. During the GXT, expired air was continuously analysed for O2 and CO2 concentrations via a gas analyser (Moxus 2010, AEI Technologies, Pittsburgh, PA, USA), which was calibrated immediately before each test. V̇O2 values were recorded every 15 s and the average of the two highest consecutive 15-s values was recorded as a participant’s V̇O2Peak. p p Venous blood sampling: Venous blood samples (~1 mL) were collected during the GXT from a cannula inserted in the antecubital vain for the determination of venous blood [La-] using a blood-lactate analyser (2300 STAT Plus; YSI, Yellow Spring, OH, USA). ARTICLE Although the inﬂuence of training duration cannot be completely ruled out, the above ﬁndings were the result of changes in only one of the several exercise prescription programming variables (i.e., training volume). Future research should investigate the effects of manipulat- ing other programming variables (e.g., exercise intensity, frequency and recovery between sessions), the type of exercise (e.g., running, swimming and resistance training), and different populations (e.g., differences in age, sex and health status) utilising a similar approach. These interventions are likely to induce adaptations that are speciﬁc and presumably different from the one presented here. Because of the well-documented therapeutic beneﬁts of exercise training72,73, the knowledge generated by our ﬁndings, which are readily available in supplemental tables, together with the results from future research, could be mined by exercise and health professionals to design focused and personalised training interventions. Muscle biopsies. A biopsy needle with suction under local anaesthesia (1% xylo- caine) was used to obtain vastus lateralis muscle biopsies at rest at the following four time points: BL, PN, PH and PR. After being cleaned of excess blood, con- nective and fat tissue muscle biopsies were divided as follows: ~10 mg was immediately immersed in ~2 mL of ice-cold BIOPS for measurements of mito- chondrial respiration, whereas the remainder was promptly frozen in liquid nitrogen and stored at −80 °C for follow-up analyses. Training intervention. All training sessions were performed on an electronically braked cycle ergometer (Velotron, RacerMate, USA), following an 8-min warm up (see 20k-TT) and consisted of HIIT (2:1 work-to-rest ratio). Training intensity was set relative to Ẇ LT (rather than Ẇ Peak) so as to induce similar metabolic and cardiac stresses amongst participants of differing ﬁtness levels76. Exercise intensity was maintained between Ẇ LT and Ẇ Peak throughout the entire study so that training volume was the only manipulated variable between the three phases. NVT phase. This consisted of 6 HIIT sessions within 2 weeks of 5–7 4-min cycling intervals interspersed with a 2-min recovery at 60 W. Exercise intensities were deﬁned as [Ẇ LT + x(Ẇ Peak-Ẇ LT)], with x increasing from 0.5 to 0.7 throughout the phase. ARTICLE Similar to the decrease reported above in CI and CII subunits post-NVT, we report decreases in four of these enzymes during HVT, with three of them remaining downregulated both post-HVT and post-RVT; conversely, IVD was increased during NVT and DHODH was elevated post-HVT (Fig. 3 and Supplementary Data 7). The lack of a clear correlation between the content of the above proteins and that of CoQ may be ascribed to changing preferences for energy utilisation over the course of the training intervention, as also indicated by the differential prioritisation of the various metabolic pathways described above. Concluding remarks. The present study utilised the power of multiple omics techniques, and an in silico normalisation strategy eliminating the bias introduced by changes in mitochondrial content, to unearth an intricate and previously undemonstrated network of differentially prioritised mitochondrial adaptations to exercise training in men. We identiﬁed 185 differentially expressed mitochondrial proteins, an ~10-fold greater number than previous studies23,32,33. This increased depth of analysis enabled us to identify the time-dependent and complex remo- delling of the mitochondrial proteome following different phases of endurance exercise training. We demonstrate that training- induced changes in individual proteins, protein functional classes, and metabolic pathways follow markedly different patterns of adaptation, which are not stoichiometrically linked to the overall training-induced increase in mitochondrial content. Moreover, we show that the lack of stoichiometry and the differential NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 12 Muscle analyses This analysis was conducted on n = 5 par- ticipants (20 samples in total); samples were sequenced (100 base pair, single reads) on the Illumina NovaSeq 6000 platform at the Australian Genome Research Facility (AGRF). Transcriptome assembly was completed at AGRF with reads screened for presence of any Illumina adaptor/over-represented sequences and cross-species contamination. Per base sequence quality for all samples was >96% bases above Q30. Cleaned sequence reads were aligned against the Homo sapiens genome (Build version HG38). The STAR aligner (v2.5.3a) was used to map reads to the genomic sequences. Counts of reads mapping to each known gene were summarised to provide the matrix used for further analysis. Mitochondrial isolation for SDS- and BN-PAGE assessment of ETC subunits, complexes and SCs. Frozen skeletal muscle samples (~30 mg) were homogenised with 2 × 20 strokes in a Potter-Elvehjem tissue grinder attached to a rotating drill (~1000 rpm) in 5 mL solution A (1 mM EDTA, 220 mM mannitol, 20 mM HEPES- KOH [pH = 7.6], 70 mM sucrose, 2 mg/mL BSA, 0.5 mM PMSF) and spun at 800 × g for 5 min at 4 °C. The supernatant was collected, whereas the pellet was re- homogenised as above in 5 mL of solution A to maximise extraction. The two supernatants were mixed and further spun at 800 g for 5 min at 4 °C. The ensuing supernatant was then spun at 10,000 × g for 20 min at 4 °C, and the pellet was resuspended in 200 µL of sucrose buffer (0.5 M sucrose, 10 mM HEPES-KOH [pH = 7.6], 0.5 mM PMSF). Protein concentration was determined by the bicinchoninic acid method according to the manufacturer’s instructions (BCA Protein Assay Kit, Pierce-Thermo Fisher Scientiﬁc, Melbourne, Australia). This measurement was used to generate Fig. 1c. Bioinformatic analysis of RNA-seq data. For downstream RNA-seq analysis the R package limma80 was used to conduct the differential expression analysis from count data. Count data was normalised using calcNormFactors in the edgeR package in R. Linear modelling was performed in limma to identify transcripts that were differentially expressed across the three pairwise comparisons (i.e., PN vs. BL, PH vs. PN, and PR vs. PH). The resulting differential expression values were ﬁltered for an adjusted P value < 0.05 using the Benjamini–Hochberg method. Differentially expressed transcripts were visualised in the heatmap in Fig. 1e using hierarchical clustering with the “average” method. Muscle analyses Enzymatic activity. Enzyme activities were determined spectrophotometrically in post-600g supernatants of skeletal muscle homogenates according to the method described for respiratory chain complexes I–IV and citrate synthase77. Brieﬂy, ~20 mg of skeletal muscle sample was homogenised in sucrose/mannitol con- taining buffer using a glass/glass homogeniser and spun for 10 min at 600 g and 4 °C. The supernatant was then subjected to two freeze/thaw cycles and stored at −80 °C until measurement of respiratory chain enzymes. CI was assayed as rotenone-sensitive NADH:CoQ1 oxidoreductase by monitoring the decrease in absorbance due to NADH oxidation at 340 nm. For CII, activity was measured as succinate:CoQ1 oxidoreductase by measuring CoQ1 reduction at 280 nm. CIII was assayed as decylbenzylquinol; cytochrome c oxidoreductase by following the increase in absorbance resulting from cytochrome c reduction at 550 nm. CIV was measured as cytochrome c oxidase by following the decrease in absorbance resulting from cytochrome c oxidation at 550 nm. To assay the CS catalysed production of coenzyme A (CoA.SH) from oxaloacetate, the generation of free sulfhydryl groups was monitored using the thiol reagent 5,5’-dithio-bis-(2-nito- benzoic acid) (DTNB), which reacts spontaneously with the sulfhydryl groups to produce 5-thio-2-nitrobenzoate anions. CS speciﬁc activity was measured by fol- lowing the increase in absorbance resulting from the formation of 5-thio-2 nitrobenzoate anions at 412 nm. Following the enzyme measurements, the amount of protein in each sample was determined using a bicinchoninic acid assay and activity calculated as initial rates (complexes I, II and citrate synthase) or as ﬁrst- order rate constants (complexes III and IV). Mitochondrial respiration protocol. Muscle analyses The substrate-uncoupler-inhibitor titration (SUIT) protocol13 used was as follows: 0.2 mM octanoylcarnitine and 2 mM malate ([ETF]L: leak respiration state [L] in the absence of adenylates and limitation of ﬂux by electron input through electron transfer ﬂavoprotein [ETF]); 3 mM MgCl2 and 5 mM ADP ([ETF]P: maximal OXPHOS capacity [P] with saturating levels of ADP and limitation of ﬂux by electron input through ETF); 5 mM pyruvate ([ETF + CI]P: P with saturating levels of ADP and limitation of ﬂux by convergent electron input through ETF + CI); 10 mM succinate ([ETF + CI + II]P: P with saturating levels of ADP and limitation of ﬂux by convergent electron input through ETF + CI + CII); 10 μM cytochrome c (outer mitochondrial membrane integrity test); 0.75-1.5 μM carbonyl cyanide 4-(triﬂuoromethoxy) phenylhy- drazone (FCCP) via stepwise titration ([ETF + C + II]E, maximal electron trans- port chain capacity [E] with saturating levels of ADP and limitation of ﬂux by convergent electron input through ETF + CI + CII); 0.5 μM rotenone ([CII]E: E with saturating levels of ADP and limitation of ﬂux by electron input through CII); and 5 μM antimycin A (residual non-mitochondrial oxygen consumption [ROX]). Data are presented as mass-speciﬁc mitochondrial respiration [pmol O2 s−1 mg−1 wet weight] and as mitochondrial-speciﬁc respiration [pmol O2 s−1 mg−1 wet weight/CS activity]. RNA-seq analysis. Approximately 10–15 mg of frozen muscle was used to isolate RNA using the RNeasy Mini Kit (Qiagen, Canada) according to the manufacturer’s instructions. Samples were homogenised using the TissueLyser II (Qiagen, Canada). RNA concentration and purity were determined spectrophotometrically (NanoDrop 2000, Thermo Fisher Scientiﬁc, Wilmington, DE, USA) at 260 and 280 nm. RNA integrity was assessed using an Agilent Bioanalyser according to manufacturer’s instructions. The RNA was stored at −80 °C. Preparation of whole-muscle lysates for SDS-PAGE assessment of ETC subunits. Frozen skeletal muscle samples (~10 mg) were homogenised in a TissueLyser for 2 × 2 min at maximum speed in ice-cold lysis buffer (1:20 w/v) containing 50 mM Tris- HCl, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and a phosphatase/protease inhibitor (5872, Cell Signaling Technology, Danvers, MA, USA). Homogenates were rotated end- over-end at 4 °C for 1 h and protein concentration was determined in triplicate using a commercial colorimetric assay (Bio-Rad Protein Assay kit-II, Australia). Sequencing and assembly of RNA-seq. Muscle analyses Categorical columns for the generation of Fig. 1f were determined by identifying the transcripts to known descriptions in the literature; MICOS81, TCA cycle82, SLC25A44 and Assembly Factors83. An enrichment analysis using Enrichr [https://maayanlab.cloud/ Enrichr/84,85] was then performed on the same list of differentially altered tran- scripts with the results shown in Fig. 1g and Supplementary Data 2. SDS-PAGE. Both whole-muscle lysates (7.5 μg) and mitochondrial isolates (5 μg) were separated by electrophoresis using 12 or 15% SDS-PAGE gels, as previously described78, and blotted with a total OXPHOS (ab110411, Abcam, Cambridge, MA, USA) or with a single CI (ab110242, Abcam, Cambridge, MA, USA) antibody where separation with CIV was not optimal using the total OXPHOS antibody. All antibodies were used at the ﬁnal concentration suggested by the manufacturer (7.2 μg/mL [ab110411] and 0.5 μg/mL [ab110242]). BN-PAGE. Mitochondrial isolates (6–15 μg) were separated by electrophoresis using 3–12% NativePAGE gels (Life Technologies Australia, Mulgrave, Australia) as previously described79. A 4 g/g digitonin/protein ratio was used for assessment of SCs. The following primary antibodies were used: NADH:ubiquinone oxidor- eductase subunit A9 (NDUFA9; ab14713), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2; ab14745) and cytochrome c oxidase subunit IV (COX IV; ab14744) (all Abcam, Cambridge, MA, USA). All antibodies were used at the ﬁnal concentration suggested by the manufacturer (1 μg/mL for all three antibodies used). Mitochondrial isolation for proteomics and lipidomics assessment. Frozen skeletal muscle samples (~30 mg) were homogenised with 2 × 20 strokes in a Potter- Elvehjem tissue grinder attached to a rotating drill (~1000 rpm) in 3 mL solution B (1 mM EDTA, 220 mM mannitol, 20 mM HEPES-KOH [pH = 7.6], 70 mM sucrose, 0.5 mM PMSF) and spun at 1000 × g for 5 min at 4 °C. The supernatant was further spun at 12,000 × g for 10 min at 4 °C, and the ensuing pellet was resuspended in 200 µL of sucrose buffer (0.5 M sucrose, 10 mM HEPES-KOH [pH = 7.6]). Protein concentration was determined by the bicinchoninic acid method according to the manufacturer’s instructions (BCA Protein Assay Kit, Pierce-Thermo Fisher Scientiﬁc, Melbourne, Australia). Two IM fractions per sample (50 µg each) were spun at 12,000 × g for 10 min at 4 °C; after removal of the supernatant, the pellets were stored frozen at −80 °C for subsequent proteomics and lipidomics analysis. ARTICLE To control for dietary effects on muscle metabolism, participants were provided with a standardised dinner ( k k 1 b d ( ) d b h d ( ) k 1 p p p (55 kJ kg−1 body mass (BM), providing 2.1 g carbohydrate (CHO) kg−1 BM, 0.3 g fat kg−1 BM, and 0.6 g protein kg−1 BM) and breakfast (41 kJ kg−1 BM, providing 1.8 g CHO kg−1 BM, 0.2 g fat kg−1 BM, and 0.3 g protein kg−1 BM), to be con- sumed 15 and 3 h prior to the muscle biopsy, respectively. Methods P ti i t Participants and ethics approval. Ten healthy men volunteered to take part in this study (physiological and performance parameters are presented in Table 1). Potential participants were deemed suitable if aged 18–35 y, were moderately- trained (i.e., <4 h per week of unstructured aerobic activity for half a year prior to the study), not regularly engaged in cycling-based sports, and were non-smokers and medication free prior to and during the study. Participants underwent a medical screening to exclude conditions that may have precluded their participa- tion (e.g., cardiovascular, musculoskeletal and/or metabolic problems), and were informed of the study requirements, risks, and beneﬁts, before giving written informed consent. Approval for the study’s procedures, which conformed to the standards set by the latest revision of the Declaration of Helsinki, was granted by the Victoria University Human Research Ethics Committee (HRE15-126). HVT phase. Participants performed HIIT twice a day for 20 consecutive days; training sessions consisted of either 7–10 4-min intervals interspersed with a 2-min recovery at 60 W at intensities ranging from [Ẇ LT + 0.5(Ẇ Peak-Ẇ LT)] to [Ẇ LT + 0.8(Ẇ Peak-Ẇ LT)], or 15–20 2-min intervals at intensities ranging from [Ẇ LT + 0.5(Ẇ Peak-Ẇ LT)] to [Ẇ LT + 0.95(Ẇ Peak-Ẇ LT)], interspersed with a 1-min recovery at 60 W. Single session duration increased from ~45 min to 60 min. RVT phase. The RVT phase consisted of 6 HIIT sessions in 6 days; participants performed 10, 9, 8, 7, 6, and 4, 4-min intervals interspersed with a 2-min recovery at 60 W, at an intensity of [Ẇ LT + x(Ẇ Peak- Ẇ LT)], with x increasing from 0.5 to 0.7 throughout the phase. Study design. The study consisted of three consecutive training phases: the nor- mal- (NVT), high- (HVT) and reduced- (RVT) training volume phase (Fig. 1a). Each training phase was preceded (and followed) by performance testing, which included a 20-km cycling time trial (20k-TT), a graded exercise test (GXT) (par- ticipants were previously familiarised with both tests), and a resting muscle biopsy. Overall study duration was ~9 weeks. Physical activity and nutritional control. Physical activity and dietary patterns were maintained throughout the study and were monitored with the use of food and physical activity recall diaries. The last three meals prior to each performance test undertaken during baseline testing were recorded by each participant and were 13 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunicatio ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 replicated thereafter before the same type of test. To control for dietary effects on muscle metabolism, participants were provided with a standardised dinner (55 kJ kg−1 body mass (BM), providing 2.1 g carbohydrate (CHO) kg−1 BM, 0.3 g fat kg−1 BM, and 0.6 g protein kg−1 BM) and breakfast (41 kJ kg−1 BM, providing 1.8 g CHO kg−1 BM, 0.2 g fat kg−1 BM, and 0.3 g protein kg−1 BM), to be con- sumed 15 and 3 h prior to the muscle biopsy, respectively. replicated thereafter before the same type of test. To control for dietary effects on muscle metabolism, participants were provided with a standardised dinner (55 kJ kg−1 body mass (BM), providing 2.1 g carbohydrate (CHO) kg−1 BM, 0.3 g fat kg−1 BM, and 0.6 g protein kg−1 BM) and breakfast (41 kJ kg−1 BM, providing 1.8 g CHO kg−1 BM, 0.2 g fat kg−1 BM, and 0.3 g protein kg−1 BM), to be con- sumed 15 and 3 h prior to the muscle biopsy, respectively. Fibre preparation for high-resolution respirometry. Fresh muscle ﬁbres were mechanically separated in ice-cold BIOPS (in mM: 2.77 CaK2EGTA, 7.23 K2EGTA, 5.77 Na2ATP, 6.56 MgCl2, 20 taurine, 50 MES, 15 Na2phosphocreatine, 20 imi- dazole and 0.5 dithiothreitol adjusted to pH 7.113, followed by permeabilisation by gentle agitation for 30 min at 4 °C in BIOPS containing 50 μg/mL of saponin, and three 5-min washes in MiR05 (in mM, unless speciﬁed: 0.5 EGTA, 3 MgCl2, 60 K- lactobionate, 20 taurine, 10 KH2PO4, 20 HEPES, 110 sucrose and 1 g/L BSA essentially fatty acid-free, pH 7.1)13. Mitochondrial respiration was measured in duplicate (from 2 to 3 mg wet weight of muscle ﬁbres) in MiR05 at 37 °C using the high-resolution Oxygraph-2k (Oroboros, Innsbruck, Austria). To avoid potential oxygen diffusion limitation, oxygen concentration was maintained between 270 and 480 nmol mL−1 by re-oxygenation via direct syringe injection of O2. replicated thereafter before the same type of test. ARTICLE Supernatants were transferred into a 96-well plated, dried down and reconstituted in 50 µL H2O saturated butanol and sonicated for 10 min. After the addition of 50 µL of methanol with 10 mM ammonium formate, samples were centrifuged at 4000 rpm on a plate centrifuge and transferred into glass vials with inserts for mass spectrometry analysis. Raw ﬁles were analysed using the MaxQuant platform88 version 1.6.1.0, searching against the Uniprot human database containing reviewed, canonical variants in FASTA format (June 2018) and a database containing common contaminants by the Andromeda search engine89. Default search parameters for a label-free quantiﬁcation (LFQ) experiment were used with modiﬁcations. In brief, “Label-free quantiﬁcation” was set to “LFQ” using a minimum ratio count of 2. Cysteine carbamidomethylation was used as a ﬁxed modiﬁcation, and N-terminal acetylation and methionine oxidation were used as variable modiﬁcations. False discovery rates of 1% for proteins and peptides were applied by searching a reverse database, and ‘match from and to’, ‘match between runs’ options were enabled with a match time window of 0.7 min. Unique and razor peptides with a minimum ratio count of 2 were used for quantiﬁcation. Targeted lipidomics analysis. LC-MS/MS was performed according to previously published methods, with slight modiﬁcation for tissue samples98. Sample extracts were analysed using either (i) an AB Sciex Qtrap 4000 mass spectrometer coupled to an Agilent 1200 HPLC system for CL assessment, as described preciously99 or (ii) an Agilent 6490 QQQ mass spectrometer coupled with an Agilent 1290 series HPLC system for assessment of all other lipid species98. Lipids run on the Agilent 6490 were measured using scheduled multiple reaction monitoring with the fol- lowing conditions: isolation widths for Q1 and Q3 were set to “unit” resolution (0.7 amu), gas temperature 150 °C, nebuliser 20 psi, sheath gas temperature 200 °C, gas ﬂow rate 17 L/min, capillary voltage 3500 V and sheath gas ﬂow 10 L/min. The list of multiple reaction monitoring (MRM) used and the chromatographic con- ditions were described previously98. Chromatographic peaks were integrated using Mass Hunter (B.09.00, Agilent) and assigned to lipid species using MRMs and retention time, with quantiﬁcation derived from the ratio of each peak with its corresponding internal standard. Bioinformatic analysis of proteomics data. ARTICLE The next day, samples were acidiﬁed with triﬂuoroacetic acid (1% [v/v] ﬁnal concentration) and centrifuged for 5 min at 20,100 × g at RT. The supernatants were desalted on pre-activated (100% acet- onitrile [ACN]) and pre-equilibrated (0.1% TFA, 2% ACN) Empore™SPE Disks (matrix active group polystyrene-divinylbenzene [SDB-XC] Merck, Bayswater, VIC, Australia) stage tips87 made in-house, before being washed (0.1% TFA, 2% ACN) and eluted in 0.1% TFA, 80% ACN. Samples were concentrated under vacuum and reconstituted in 0.1% TFA, 2% ACN. After 15 min sonication and subsequent vortexing samples were centrifuged at 20,100 × g at RT before esti- mation of peptide concentration (Direct Detect, Merck). Approximately 600–800 ng of peptides were analysed on a Thermo Q Exactive™Plus mass spec- trometer coupled to an Ultimate 3000 HPLC (both Thermo Fisher Scientiﬁc, Mulgrave, VIC, Australia). Peptides were ﬁrst loaded onto a trap column (Dionex- C18, 100 Å, 75 µm x 2 cm; Thermo Fisher Scientiﬁc) at an isocratic ﬂow of 5 µL min−1 of 2% (v/v) ACN containing 0.1 % (v/v) formic acid (FA) for 5 min before switching the trap-column in line with the analytical column (Dionex-C18, 100 Å, 75 µm x 50 cm; Thermo Fisher Scientiﬁc). The separation of peptides was per- formed at 300 nL min−1 using a nonlinear ACN gradient of buffer A (2% ACN, 0.1% FA) and buffer B (80% ACN, 0.1% FA) over 125 min including void and equilibration. Data were collected in positive mode using Data Dependent Acquisition using m/z 375–1400 as MS scan range, HCD for MS/MS of the 15 most intense ions with z ≥2. Other instrument parameters were: MS1 scan at 70,000 resolution (at 200 m/z), MS maximum injection time 50 ms, AGC target 3e6, stepped normalised collision energy of 27, 30, 32, isolation window of 1.6 m/z, MS/ MS resolution 17,500, MS/MS AGC target of 5e4, MS/MS maximum injection time 50 ms, minimum intensity was set at 2e3 and dynamic exclusion was set to 30 s. Lipid extraction for lipidomics. Mitochondrial isolates were extracted using a modiﬁed single-phase chloroform/methanol extraction as described previously97. In brief, 20 volumes of chloroform:methanol (2:1) were added to the sample along with a series of internal standards. Samples were vortexed and centrifuged on a rotary mixer for 10 min. Following sonication on a sonicator bath for 30 min, samples were rested for 20 min prior to centrifugation at 13,000×g for 10 min. ARTICLE The R package limma80 was used to conduct the differential expression analysis of MaxQuant LFQ intensities (extracted from proteinGroups.txt) after ﬁrst performing normalisation using variance stabilising normalisation (VSN) as found in the limma package. Identi- ﬁcations labelled by MaxQuant as ‘only identiﬁed by site’, ‘reverse’ and ‘potential contaminant’ were removed. Proteins having <70% valid values were removed and remaining missing data was imputed using QRILC method from the imputeLCMD package in R90. Differential expression analysis was performed between BL and then each subsequent time point to show comparative changes between each of the training volumes, though comparisons across all three training phases were accounted for within the analysis. Linear modelling was determined using eBayes in the limma package. Proteins that were differentially expressed across all three training phases were identiﬁed by ﬁltering for an adjusted P value < 0.01 using the Benjamini–Hochberg method. For the mitochondrial normalisation, all “Known Mitochondrial” proteins identiﬁed using the Integrated Mitochondrial Protein Index (IMPI)29 were subset from the rest of the dataset, and then followed the normalisation and statistical validation as described above. Heatmaps were pro- duced using hierarchical clustering using the “complete” method. Gene ontology of the clusters was determined by taking the proteins identiﬁed in the cluster and performing an enrichment analysis using the ClueGO (v2.5.6) application in Cytoscape (v3.7.1) using default settings except for a GO tree interval of 3 to 5, with only the Biological Processes Ontology switched on (Fig. 2f and Supplementary Fig. 2a). Full tables of identiﬁed proteins and post-normalisation differentially expressed proteins in their respective clusters and their annotations can be found in Supplementary Data 3–7. Bioinformatic analysis of lipidomics data. For lipidomics we identiﬁed that the increase in CLs in the un-normalised data was due to a training-induced increase in MPE in our mitochondrial isolates. To eliminate this bias, all lipid species were normalised by total CL amount (Supplementary Data 8). Lipid species were log transformed before undergoing differential expression analysis in limma80, with linear models using the eBayes function. The resulting differential expression values were ﬁltered for an adjusted P value < 0.05 using Benjamini–Hochberg method (Supplementary Data 9). Heatmaps were visualised using hierarchical clustering using the “complete” method. Proﬁle plots were determined by taking the mean of the z-score for all participants’ samples for each lipid species at each time point (Fig. 4d). Forest plots are depicted using LogFC results from limma analysis (Supplementary Fig. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 HSD17B1093, and PLIN542 were added based on their involvement with FAO and lipid metabolism; GPD245,94, GPX1 [R-HSA-3299685 and46], and GPX446 where not presented within this pathway as their primary function suggested their inclusion in different pathways of Fig. 3; SAR1B was removed because its main function is the regulation of vesicle budding95. “OXPHOS” was based on Reactome pathway R-HSA:1632000 (Respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins); ETFA92, ETFB92, ETFDH45, TRAP149, and CYCS (R-HSA-3299685) were not presented within this pathway as their primary function and/or training-induced changes suggested their inclusion in different pathways of Fig. 3 was more appropriate; based on the main function of its constituent proteins the “OXPHOS” pathway was then subdivided in 2 subgroups: “OXPHOS - subunits”, to which MP6896 was added, and “OXPHOS - Assembly factors” to which ATPAF114, BCS1L14, and COA314 were added. “Response to ROS” was based on Reactome pathway: R-HSA- 3299685 (Detoxiﬁcation of Reactive Oxygen Species); GPX4 was added based on its ability to reduce H2O246. “Protein Folding” was based on literature searches: TRAP1, HSPA9, HSPD1, HSPE1, and TIMM44 were all added based on their involvement in protein folding and quality control mechanisms49. “CoQ biosynthesis” was based on Reactome pathway R-HSA-2142789 (Ubiquinol biosynthesis). “CoQ e- donors” was based on literature searches: DHODH45, ETFDH45, GPD245, IVD62, DLD63 and L2HGDH64, were all added based on their function to feed electrons into the respiratory chain via CoQ. “Cristae formation” was based on Reactome pathway R-HSA-8949613 (Cristae formation); OMA167,68 was added based on its involvement in cristae formation, whereas ATP5A1, ATP5J, ATP5B, ATP5H, and ATP5O (all based on R-HSA:163200), as well as HSPA949 were not presented within this pathway as their primary function suggested their inclusion in different pathways of Fig. 3. Proteomics. IM fractions were prepared for proteomics analysis as previously described86 with minor modiﬁcations. Brieﬂy, 50 μg of frozen mitochondrial iso- lates were solubilised in 20 µL of 8 M urea, 40 mM chloroacetamide, 10 mM tris(2- carboxyethyl)phosphine, 100 mM Tris, pH 8.1; this was followed by 15 min of sonication in a water bath sonicator and 30 min of shaking (1500 rpm, at 37 °C). The urea concentration was reduced to 2 M with H2O prior to protein digestion with trypsin (Promega, Alexandria, NSW, Australia) at a 1:60 trypsin:protein ratio and subsequent overnight digestion at 37 °C. Muscle analyses For both SDS- and BN-PAGE, protein bands were visualised using a Bio-Rad ChemiDoc imaging system and bands were quantiﬁed using Bio-Rad Image Lab 5.0 software (Bio-Rad laboratories, Gladesville, NSW, Australia). An internal standard (made of a mixture of all samples) was loaded in each SDS- and BN- PAGE gel, and each lane was normalised to this value, to reduce gel-to-gel variability. 14 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunicatio References 1. Spinelli, J. B. & Haigis, M. C. The multifaceted contributions of mitochondria to cellular metabolism. Nat. Cell Biol. 20, 745–754 (2018). 30. Scalzo, R. L. et al. Greater muscle protein synthesis and mitochondrial biogenesis in males compared with females during sprint interval training. FASEB J. 28, 2705–2714 (2014). 2. Gorman, G. S. et al. Mitochondrial diseases. Nat. Rev. Dis. Prim. 2, 1–22 (2016). 3. Lin, M. T. & Beal, M. F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 (2006). 31. Shankaran, M. et al. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics. J. Clin. Investig. 126, 288–302 (2016). g 4. López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M. & Kroemer, G. The hallmarks of aging. Cell 153, 1194–1217 (2013). 32. Holloway, K. V. et al. Proteomic investigation of changes in human vastus lateralis muscle in response to interval‐exercise training. Proteomics 9, 5155–5174 (2009). 5. Nunnari, J. & Suomalainen, A. Mitochondria: in sickness and in health. Cell 148, 1145–1159 (2012). 33. Hostrup, M., Onslev, J., Jacobson, G. A., Wilson, R. & Bangsbo, J. Chronic β2‐ adrenoceptor agonist treatment alters muscle proteome and functional adaptations induced by high intensity training in young men. J. Physiol. 596, 231–252 (2018). 6. Holloszy, J. O. Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J. Biol. Chem. 242, 2278–2282 (1967). 7. Hoppeler, H., Lüthi, P., Claassen, H., Weibel, E. R. & Howald, H. The ultrastructure of the normal human skeletal muscle. Pﬂügers Arch. 344, 217–232 (1973). 34. Robinson, M. M. et al. Enhanced protein translation underlies improved metabolic and physical adaptations to different exercise training modes in young and old humans. Cell Metab. 25, 581–592 (2017). 8. Neufer, P. D. et al. Understanding the cellular and molecular mechanisms of physical activity-induced health beneﬁts. Cell Metab. 22, 4–11 (2015). 35. Ubaida-Mohien, C. et al. Physical activity associated proteomics of skeletal muscle: being physically active in daily life may protect skeletal muscle from aging. Front. Physiol. 10, 312 (2019). 9. Granata, C., Jamnick, N. A. & Bishop, D. J. Training-induced changes in mitochondrial content and respiratory function in human skeletal muscle. Sports Med. 48, 1809–1828 (2018). 36. Dube, J. et al. Effects of weight loss and exercise on insulin resistance, and intramyocellular triacylglycerol, diacylglycerol and ceramide. Diabetologia 54, 1147–1156 (2011). 10. Granata, C., Jamnick, N. A. & Bishop, D. J. Data availability 21. Larsen, S. et al. Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects. J. Physiol. 590, 3349–3360 (2012). Source data to interpret, verify and extend this research is provided with this paper. The mass spectrometry proteomics data has been deposited in the ProteomeXchange Consortium via the PRIDE partner repository under accession code PXD026219. The transcriptomic data has been deposited in the NCBI and can be found under the BioProject PRJNA732106. The lipidomics data has been deposited in the NIH Common Fund’s National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, under Project ID ST001907 [https://doi.org/10.21228/M8G69Q]. Source data are provided with this paper. Source data to interpret, verify and extend this research is provided with this paper. The mass spectrometry proteomics data has been deposited in the ProteomeXchange 22. Barnard, R. J., Edgerton, V. R. & Peter, J. Effect of exercise on skeletal muscle. I. Biochemical and histochemical properties. J. Appl. Physiol. 28, 762–766 (1970). 23. Egan, B. et al. 2‐D DIGE analysis of the mitochondrial proteome from human skeletal muscle reveals time course‐dependent remodelling in response to 14 consecutive days of endurance exercise training. Proteomics 11, 1413–1428 (2011). 24. Morgan, T., Cobb, L., Short, F., Ross, R. & Gunn, D. in Muscle Metabolism During Exercise p. 87–95 (Springer, 1971). ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 17. Ørtenblad, N. Mitochondrial increase in volume density with exercise training: More, larger or better? Acta Physiol. 222, e12976 (2018). level of statistical signiﬁcance was set a priori at P < 0.05. GraphPad Prism (v. 8.4.2) was used for all statistical non-omics analyses. Differential expression for all omics was completed in R (v.3.6.3), using the packages, limma and edgeR to determine signiﬁcantly enriched results between the groups. Signiﬁcance was determined by an adjusted p value < 0.05 for the transcriptomics and lipidomics and 0.01 for the 18. Bishop, D. J. et al. High-intensity exercise and mitochondrial biogenesis: current controversies and future research directions. Physiology 34, 56–70 (2019). j p p p proteomics. Statistical details can be found in the associated ﬁgure legends. All ﬁgures were prepared using Adobe Illustrator (CC2018.22.1). 19. Bishop, D. J., Granata, C. & Eynon, N. Can we optimise the exercise training prescription to maximise improvements in mitochondria function and content? Biochim. Biophys. Acta 1840, 1266–1275 (2014). 20. Granata, C., Oliveira, R. S. F., Little, J. P., Renner, K. & Bishop, D. J. Mitochondrial adaptations to high-volume exercise training are rapidly reversed after a reduction in training volume in human skeletal muscle. FASEB J. 30, 3413–3423 (2016). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Code availability 25. Meinild Lundby, A. K. et al. Exercise training increases skeletal muscle mitochondrial volume density by enlargement of existing mitochondria and not de novo biogenesis. Acta Physiol. 222, e12905 (2018). The R scripts used for all omics analyses described above are deposited on GitHub and available through https://doi.org/10.5281/zenodo.5576974. There are no restrictions placed on accessibility of this code. 26. Bishop, D. J., Botella, J. & Granata, C. CrossTalk opposing view: exercise training volume is more important than training intensity to promote increases in mitochondrial content. J. Physiol. 597, 4115–4118 (2019). Received: 19 April 2021; Accepted: 26 October 2021; Received: 19 April 2021; Accepted: 26 October 2021; 27. Pillon, N. J. et al. Transcriptomic proﬁling of skeletal muscle adaptations to exercise and inactivity. Nat. Commun. 11, 1–15 (2020). 28. Calvo, S. E., Clauser, K. R. & Mootha, V. K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2016). 29. Smith, A. C. & Robinson, A. J. MitoMiner v4. 0: an updated database of mitochondrial localization evidence, phenotypes and diseases. Nucleic Acids Res. 47, D1225–D1228 (2019). ARTICLE 3). Higher classes were determined through descriptions of the literature as assigned by the Lipid Metabolites and Pathways Strategy (Lipid MAPS)100. The heatmaps presented in Fig. 3 and discussed in the Results section were generated according to Reactome pathways and/or literature searches (Supplementary Data 6). Proteins involved in two or more pathways were either presented in both pathways, or were assigned to the pathway involving the protein’s primary function and/or most closely matching their training-induced changes. Speciﬁcally, “mitochondrial translation” was based on Reactome pathway R-HSA:5368287 (Mitochondrial translation). “TCA cycle” was based on Reactome pathway “R-HSA:71403 (Citric acid cycle [TCA cycle]); NNT was removed because its main physiological function is the generation of NADPH91. “FAO” was based on Reactome pathway R-HSA:556833 (Metabolism of Lipids); ETFA92, ETFB92, Statistical analysis. All values are reported as means ± SD, unless otherwise speci- ﬁed. For non-omics analyses: outliers were ﬁrst removed using the ROUT method set at Q = 1%101. Normally distributed datasets (Shapiro–Wilk test P > 0.05) were analysed by a repeated measures one-way ANOVA followed by Tukey’s correction post hoc testing. Non-normally distributed datasets (Shapiro–Wilk test P < 0.05) were transformed using, in order, log(Y), 1/Y, √Y, until normality was met, before being analysed as above; datasets that remained non-normal following three independent transformation attempts were analysed using the non-parametric Friedman test on the raw data followed by Dunn’s correction post hoc testing. The 15 TURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications ARTICLE 43, e47–e47 (2015). and mammalian mitochondria. EMBO J. 19, 1777–1783 (200 52. Lapuente-Brun, E. et al. Supercomplex assembly determines electron ﬂux in the mitochondrial electron transport chain. Science 340, 1567–1570 (2013). 81. Eramo, M. J., Lisnyak, V., Formosa, L. E. & Ryan, M. T. The ‘mitochondrial contact site and cristae organising system’(MICOS) in health and human disease. J. Biochem. 167, 243–255 (2020). 53. Hirst, J. Open questions: respiratory chain supercomplexes—why are they there and what do they do? BMC Biol. 16, 111 (2018). 82. Martínez-Reyes, I. & Chandel, N. S. Mitochondrial TCA cycle metabolites control physiology and disease. Nat. Commun. 11, 1–11 (2020). 54. Gonzalez-Franquesa, A. et al. Mass-spectrometry-based proteomics reveals mitochondrial supercomplexome plasticity. Cell Rep. 35, 109180 (2021). 83. Hock, D. H., Robinson, D. R. & Stroud, D. A. Blackout in the powerhouse: clinical phenotypes associated with defects in the assembly of OXPHOS complexes and the mitoribosome. Biochem. J. 477, 4085–4132 (2020). 55. Gebert, N. et al. Mitochondrial cardiolipin involved in outer-membrane protein biogenesis: implications for Barth syndrome. Curr. Biol. 19, 2133–2139 (2009). 84. Chen, E. Y. et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinform. 14, 128 (2013). 56. Pfeiffer, K. et al. Cardiolipin stabilizes respiratory chain supercomplexes. J. Biol. Chem. 278, 52873–52880 (2003). 85. Kuleshov, M. V. et al. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res. 44, W90–W97 (2016). 57. Ikon, N. & Ryan, R. O. Cardiolipin and mitochondrial cristae organization. Biochim. Biophys. Acta Biomembr. 1859, 1156–1163 (2017). 86. Stroud, D. A. et al. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature 538, 123 (2016). 58. Malhotra, A., Xu, Y., Ren, M. & Schlame, M. Formation of molecular species of mitochondrial cardiolipin. 1. A novel transacylation mechanism to shuttle fatty acids between sn-1 and sn-2 positions of multiple phospholipid species. Biochim. Biophys. Acta 1791, 314–320 (2009). 87. Kulak, N. A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319 (2014). 88. Cox, J. & Mann, M. MaxQuant enables high peptide identiﬁcation rates, individualized ppb-range mass accuracies and proteome-wide protein quantiﬁcation. Nat. Biotechnol. 26, 1367–1372 (2008). 59. Wang, Y. & Hekimi, S. The complexity of making ubiquinone. Trends Endocrinol. Metab. 30, 929–943 (2019). 60. Stefely, J. A. & Pagliarini, D. J. ARTICLE Regulation of OPA1 processing and mitochondrial fusion by AAA i d OMA1 J C ll Bi l 187 1023 1036 (2009) 67. Ehses, S. et al. Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1. J. Cell Biol. 187, 1023–1036 (2009). 98. Huynh, K. et al. High-throughput plasma lipidomics: detailed mapping of the associations with cardiometabolic risk factors. Cell Chem. Biol. 26, 71–84. e74 (2019). m-AAA protease isoenzymes and OMA1. J. Cell Biol. 187, 1023–1036 (2009). 68. Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional 68. Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured ﬁlroblasts. Nat. Protoc. 2, 287 (2007). p g mitochondria from mouse liver, muscle and cultured ﬁlroblasts. Nat. Protoc. 2, 287 (2007). 99. Tan, S. M. et al. Complement C5a induces renal injury in diabetic kidney disease by disrupting mitochondrial metabolic agility. Diabetes 69, 83–98 (2020). 69. Nielsen, J. et al. Plasticity in mitochondrial cristae density allows metabolic capacity modulation in human skeletal muscle. J. Physiol. 595, 2839–2847 (2017). 100. Fahy, E., Cotter, D., Sud, M. & Subramaniam, S. Lipid classiﬁcation, structures and tools. Biochim. Biophys. Acta 1811, 637–647 (2011). 70. Weber, T. A. et al. APOOL is a cardiolipin-binding constituent of the Mitoﬁlin/MINOS protein complex determining cristae morphology in mammalian mitochondria. PLoS ONE 8, e63683 (2013). 101. Motulsky, H. J. & Brown, R. E. Detecting outliers when ﬁtting data with nonlinear regression–a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinform. 7, 123 (2006). 71. Desler, C. et al. Is there a link between mitochondrial reserve respiratory capacity and aging? J. Aging Res. 2012, 192503 (2012). 72. Booth, F. W., Gordon, S. E., Carlson, C. J. & Hamilton, M. T. Waging war on modern chronic diseases: primary prevention through exercise biology. J. Appl. Physiol. 88, 774–787 (2000). ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-021-27153-3 75. Jamnick, N. A., Botella, J., Pyne, D. B. & Bishop, D. J. Manipulating graded exercise test variables affects the validity of the lactate threshold and V˙ O2peak. PLoS ONE 13, e0199794 (2018). 45. Wang, Y. & Hekimi, S. Understanding ubiquinone. Trends Cell Biol. 26, 367–378 (2016). 46. Powers, S. K. et al. Exercise-induced oxidative stress: friend or foe? Journal of sport and health science. J Sport Health Sci. 9, 415–425 (2020). p 76. Baldwin, J., Snow, R. J. & Febbraio, M. A. Effect of training status and relative exercise intensity on physiological responses in men. Med. Sci. Sports Exerc. 32, 1648–1654 (2000). p p 47. LaHue, S. C., Comella, C. L. & Tanner, C. M. The best medicine? The inﬂuence of physical activity and inactivity on Parkinson’s disease. Mov. Disord. 31, 1444–1454 (2016). p p 47. LaHue, S. C., Comella, C. L. & Tanner, C. M. The best medicine? The inﬂuence of physical activity and inactivity on Parkinson’s disease. Mov. inﬂuence of physical activity and inactivity on Parkinson’s disease. Mov. Disord. 31, 1444–1454 (2016). 77. Frazier, A. E., Vincent, A. E., Turnbull, D. M., Thorburn, D. R. & Taylor, R. W. Assessment of mitochondrial respiratory chain enzymes in cells and tissues. Methods Cell Biol. 155, 121–156 (2020). 48. McKenzie, M., Lazarou, M., Thorburn, D. R. & Ryan, M. T. Analysis of mitochondrial subunit assembly into respiratory chain complexes using Blue Native polyacrylamide gel electrophoresis. Anal. Biochem. 364, 128–137 (2007). 78. Granata, C., Oliveira, R. S. F., Little, J. P., Renner, K. & Bishop, D. J. Training intensity modulates changes in PGC-1α and p53 protein content and mitochondrial respiration, but not markers of mitochondrial content in human skeletal muscle. FASEB J. 30, 959–970 (2016). 49. Voos, W. Chaperone–protease networks in mitochondrial protein homeostasis. Biochim. Biophys. Acta 1833, 388–399 (2013). p y 50. Wiedemann, N. & Pfanner, N. Mitochondrial machineries for protein import and assembly. Annu. Rev. Biochem. 86, 685–714 (2017). 79. Jha, P., Wang, X. & Auwerx, J. Analysis of mitochondrial respiratory chain supercomplexes using blue native polyacrylamide gel electrophoresis (BN‐ PAGE). Curr. Protoc. Mouse Biol. 6, 1–14 (2016). y ( ) 51. Schägger, H. & Pfeiffer, K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19, 1777–1783 (2000). 80. Ritchie, M. E. et al. limma powers differential expression analyses for RNA- sequencing and microarray studies. Nucleic Acids Res. ARTICLE Biochemistry of mitochondrial coenzyme Q biosynthesis. Trends Biochem. Sci. 42, 824–843 (2017). quantiﬁcation. Nat. Biotechnol. 26, 1367–1372 (2008). 89. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011). y 61. Svensson, M. et al. Effect of Q10 supplementation on tissue Q10 levels and adenine nucleotide catabolism during high-intensity exercise. Int. J. Sport Nutr. Exerc. Metab. 9, 166–180 (1999). 90. Lazar, C. ImputeLCMD: A Collection Of Methods For Left-censored Missing Data Imputation. R package, version 2.0. 62. Finocchiaro, G., Ito, M. & Tanaka, K. Puriﬁcation and properties of short chain acyl-CoA, medium chain acyl-CoA, and isovaleryl-CoA dehydrogenases from human liver. J. Biol. Chem. 262, 7982–7989 (1987). p p g 91. Kampjut, D. & Sazanov, L. A. Structure and mechanism of mitochondrial proton-translocating transhydrogenase. Nature 573, 291–295 (2019). 92. Watmough, N. J. & Frerman, F. E. The electron transfer ﬂavoprotein: ubiquinone oxidoreductases. Biochim. Biophys. Acta 1797, 1910–1916 (2010). 63. Xia, L., Björnstedt, M., Nordman, T., Eriksson, L. C. & Olsson, J. M. Reduction of ubiquinone by lipoamide dehydrogenase: an antioxidant regenerating pathway. Eur. J. Biochem. 268, 1486–1490 (2001). 93. Schulz, H. Beta oxidation of fatty acids. Biochim. Biophys. Acta 1081, 109–120 (1991). 94. Mráček, T., Drahota, Z. & Houštěk, J. The function and the role of the mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissues. Biochim. Biophys. Acta 1827, 401–410 (2013). y 64. Oldham, W. M., Clish, C. B., Yang, Y. & Loscalzo, J. Hypoxia-mediated increases in L-2-hydroxyglutarate coordinate the metabolic response to reductive stress. Cell Metab. 22, 291–303 (2015). 65. Huynen, M. A., Mühlmeister, M., Gotthardt, K., Guerrero-Castillo, S. & Brandt, U. Evolution and structural organization of the mitochondrial contact site (MICOS) complex and the mitochondrial intermembrane space bridging (MIB) complex. Biochim. Biophys. Acta 1863, 91–101 (2016). 95. Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 81, 153–208 (2001). 96. Fujikawa, M., Ohsakaya, S., Sugawara, K. & Yoshida, M. Population of ATP synthase molecules in mitochondria is limited by available 6.8‐kDa proteolipid protein (MLQ). Genes Cells 19, 153–160 (2014). 66. Guarani, V. et al. QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology. Elife 4, e06265 (2015). 97. Weir, J. M. et al. Plasma lipid proﬁling in a large population-based cohort. J. Lipid Res. 54, 2898–2908 (2013). p y p gy f 67. Ehses, S. et al. References Principles of exercise prescription, and how they inﬂuence exercise-induced changes of transcription factors and other regulators of mitochondrial biogenesis. Sports Med. 48, 1541–1559 (2018). 37. Shepherd, S. O. et al. Lipid droplet remodelling and reduced muscle ceramides following sprint interval and moderate-intensity continuous exercise training in obese males. Int. J. Obes. 41, 1745–1754 (2017). 11. Groennebaek, T. et al. Utilization of biomarkers as predictors of skeletal muscle mitochondrial content after physiological intervention and in clinical settings. Am. J. Physiol. Endocrinol. Metab. 318, E886–E889 (2020). 38. Bruce, C. R. et al. Endurance training in obese humans improves glucose tolerance and mitochondrial fatty acid oxidation and alters muscle lipid content. Am. J. Physiol. Endocrinol. Metab. 291, E99–E107 (2006). 12. Franko, A. et al. Efﬁcient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti- TOM22 magnetic beads. PLoS ONE 8, e82392 (2013). 39. Perreault, L. et al. Intracellular localization of diacylglycerols and sphingolipids inﬂuences insulin sensitivity and mitochondrial function in human skeletal muscle. JCI insight 3, e96805 (2018). g 13. Pesta, D. & Gnaiger, E. in Mitochondrial Bioenergetics: Methods and Protocols 810 (eds Carlos M. Palmeira & Antonio J. Moreno) 25–58 (Springer Science +Business Media, 2012). 40. Goodpaster, B. H. & Kelley, D. E. Role of muscle in triglyceride metabolism. Curr. Opin. Lipidol. 9, 231–236 (1998). 41. Walther, T. C. & Farese, R. V. Jr Lipid droplets and cellular lipid metabolism. Annu. Rev. Biochem. 81, 687–714 (2012). 14. Signes, A. & Fernandez-Vizarra, E. Assembly of mammalian oxidative phosphorylation complexes I–V and supercomplexes. Essays Biochem. 62, 255–270 (2018). 42. Benador, I. Y., Veliova, M., Liesa, M. & Shirihai, O. S. Mitochondria bound to lipid droplets: where mitochondrial dynamics regulate lipid storage and utilization. Cell Metab. 29, 827–835 (2019). 15. Greggio, C. et al. Enhanced respiratory chain supercomplex formation in response to exercise in human skeletal muscle. Cell Metab. 25, 301–311 (2017). 43. Wang, H. et al. Perilipin 5, a lipid droplet-associated protein, provides physical and metabolic linkage to mitochondria. J. Lipid Res. 52, 2159–2168 (2011). 16. Wyckelsma, V. L. et al. Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, ﬁbre type and high‐ intensity exercise training. J. Physiol. 595, 3345–3359 (2017). 44. Palmieri, F. The mitochondrial transporter family SLC25: identiﬁcation, properties and physiopathology. Mol. Asp. Med. 34, 465–484 (2013). 16 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications Additional information Research Facility (AGRF). This study was funded by grants from the ANZ-MASON Foundation (to D.J.B), the Australian Research Council (Discovery Project DP140104165 to D.J.B), the Australian National Health and Medical Research Council (NHMRC Project Grant 1140906 to D.A.S.; NHMRC Fellowships 1140851 to D.A.S. and 1155244 to D.R.T.), JDRF Australia (JDRF Career Development to M.T.C.), and the Australian Research Council Special Research Initiative in Type 1 Juvenile Diabetes (to M.T.C.). We acknowledge the support of the Mito Foundation for the provision of instrumentation and the Victorian Government’s Operational Infrastructure Support Program. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-27153-3. Correspondence and requests for materials should be addressed to Cesare Granata, David A. Stroud or David J. Bishop. Peer review information Nature Communications thanks Atul Deshmukh, Martin Picard and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprints C.G., D.A.S. and D.J.B. conceptualised the study. C.G., D.A.S. and D.J.B. devised the study methodology. C.G., N.A.J. and H.A.J. performed study validation. C.G., N.A.J. and H.A.J. delivered the training and performed sample collection. C.G., J.B., J.K., B.R., A.L., T.L.S., and A.E.F. performed biochemical analyses. C.G., B.R., and D.A.S., performed proteomic analysis. K.H. and N.A.M. performed lipidomic analysis. RNA-seq analysis was performed at the Australian Genome Research Facility (AGRF). C.G., N.J.C. and D.A.S. performed statistical and bioinformatic analysis. C.G., N.J.C. and D.A.S. delivered the visualisation. C.G., D.A.S. and D.J.B. wrote the manuscript. M.T.C., P.J.M., D.R.T., D.A.S. and D.J.B. provided super- vision. D.R.T., M.T.C., D.A.S. and D.J.B. funded the research. C.G., D.A.S. and D.J.B. have primary responsibility for ﬁnal content. Data collection took place at Victoria University. Muscle analysis took place at Victoria University, Monash University, Murdoch Children’s Research Institute, the Baker Heart & Diabetes Institute, AGRF, and the Bio21 Molecular Science & Biotechnology Institute (The University of Melbourne). All persons designated as authors qualify for authorship, and all those qualifying for authorship are listed. All authors have read and approved the ﬁnal manuscript. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Acknowledgements We thank the participants for their time and commitment to this study. The authors would like to acknowledge all members of the Stroud and Bishop labs for input into interpretation and presentation of data. We thank the Bio21 Mass Spectrometry and Proteomics Facility (MMSPF) for provision of instrumentation, training and technical support. We acknowledge the use of the services and facilities of the Australian Genome 73. Pedersen, B. K. & Saltin, B. Evidence for prescribing exercise as therapy in chronic disease. Scand. J. Med. Sci. Sports 16, 3–63 (2006). 74. Bishop, D. J., Jenkins, D. G., McEniery, M. & Carey, M. F. Relationship between plasma lactate parameters and muscle characteristics in female cyclists. Med. Sci. Sports Exerc. 32, 1088–1093 (2000). 17 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications Competing interests © The Author(s) 2021 The authors declare no competing interests. NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunications 18 NATURE COMMUNICATIONS \| (2021) 12:7056 \| https://doi.org/10.1038/s41467-021-27153-3 \| www.nature.com/naturecommunicatio
https://openalex.org/W4248838274	https://dash.harvard.edu/bitstream/1/37068128/1/5877366.pdf	English	null	Erratum: Sleep and survival among women with breast cancer: 30 years of follow-up within the Nurses’ Health Study	British journal of cancer	2,018	cc-by	439	Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:37068128 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citation Trudel-Fitzgerald, Claudia, Eric S Zhou, Elizabeth M Poole, Xuehong Zhang, Karin B Michels, A Heather Eliassen, Wendy Y Chen, Michelle D Holmes, Shelley S Tworoger, and Eva S Schernhammer. 2018. “Sleep and survival among women with breast cancer: 30 years of follow-up within the Nurses’ Health Study.” British Journal of Cancer 118 (6): e6. doi:10.1038/ bjc.2017.437. http://dx.doi.org/10.1038/bjc.2017.437. Published Version doi:10.1038/bjc.2017.437 This work is published under the standard license to publish agree- ment. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution- NonCommercial-Share Alike 4.0 Unported License. Correction to: British Journal of Cancer (2017) 116, 1239–1246. doi:10.1038/bjc.2017.85; published online 30 March 2017 Since the publication of this manuscript, the authors realised affiliation 7 was listed incorrectly. The correct address is now listed above. British Journal of Cancer (2018) 118, e6 \| doi: 10.1038/bjc.2017.437 Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Correction to: British Journal of Cancer (2017) 116, 1239–1246. doi:10.1038/bjc.2017.85; published online 30 March 2017 Published online 9 January 2018 r 2018 Cancer Research UK. All rights reserved 0007 – 0920/18 Sleep and survival among women with breast cancer: 30 years of follow-up within the Nurses’ Health Study Claudia Trudel-Fitzgerald1, Eric S Zhou2,3, Elizabeth M Poole4, Xuehong Zhang4,5, Karin B Michels6, A Heather Eliassen4,5, Wendy Y Chen2,3,4, Michelle D Holmes4,5, Shelley S Tworoger4,5 and Eva S Schernhammer4,5,7 1Department of Social and Behavioral Sciences, Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA; 2Department of Medicine, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA; 3Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; 4Department of Medicine, Channing Division of Network Medicine, Brigham and Women’s Hospital, 181 Longwood Avenue, Boston, MA 02115, USA; 5Department of Epidemiology, Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA; 6Department of Epidemiology, UCLA Fielding School of Public Health, 650 Charles E Young Dr S, Los Angeles, CA 90095, USA and 7Department of Epidemiology, Medical University of Vienna, Kinderspitalgasse 15, Vienna 1090, Austria j p Since the publication of this manuscript, the authors realised affiliation 7 was listed incorrectly. The correct address is now listed above. Published online 9 January 2018 r 2018 Cancer Research UK. All rights reserved 0007 – 0920/18 www.bjcancer.com \| DOI:10.1038/bjc.2017.437 1
https://openalex.org/W4241305518	https://curis.ku.dk/ws/files/241816539/3886_Article_Text_15358_1_10_20200522.pdf	English	null	Situating Boundary Work: Chronic Disease Prevention in Danish Hospitals	Professions and professionalism	2,020	cc-by	8,665	Citation for published version (APA): Pedersen, I. K. (2020). Situating Boundary Work: Chronic Disease Prevention in Danish Hospitals. Professions and Professionalism, 10(1), e3362. https://doi.org/10.7577/pp.3886 Download date: 24. Oct. 2024 university of copenhagen university of copenhagen https://doi.org/10.7577/pp.3362 ISSN: 1893-1049 Situating Boundary Work Chronic Disease Prevention in Danish Hospitals Pedersen, Inge Kryger Published in: Professions and Professionalism DOI: 10.7577/pp.3886 Publication date: 2020 Document version Publisher's PDF, also known as Version of record Document version Publisher's PDF, also known as Version of record Download date: 24. Oct. 2024 Abstract This paper investigates how health professions compete and cooperate in addressing emerging local work tasks defined in relation to new globalized health challenges, such as type 2 diabetes. It identifies which professional groups have claimed responsibility for the tasks and by means of which kinds of interactions and infighting. The materials entail workplace-related artefacts and documents; in-depth interviews and extended conversations with health professionals about goals, dilemmas, and practices linked to prevention of lifestyle-related diseases; and site visits at Danish hospitals. Grounding Abbott’s framework of jurisdictions and his meso-level vocabulary in a situated account of professional boundary work, the analysis follows the ways that nurses in particular create, and sometimes stabilize or standardize, techniques for a disease prevention programme less than a decade old. The paper argues that processual theory of boundary work would benefit from grounding in a situated account of forms of professional boundaries within emerging jurisdictional tasks. Situating Boundary Work: Chronic Disease Prevention in Danish Hospitals Inge Kryger Pedersen University of Copenhagen Inge Kryger Pedersen University of Copenhagen Contact: Inge Kryger Pedersen, University of Copenhagen, Denmark, ikp@soc.ku.dk Keywords Health promotion, lifestyle modifications, health professions, work practices, boundary objects, workplace artefacts Introduction Reducing the global burden of noncommunicable diseases (NCDs), such as type 2 diabetes, is an overriding priority in the work of the World Health Organization (WHO). NCDs are the leading cause of death globally (WHO, 2014, p. xi) and health professionals are mobilized to identify individuals at risk. However, it is not always evident which professions are responsible for this work. From a global perspective, some crucial and increasing risks for NCDs are highlighted by the Global Study of Disease Burden 2015: obesity, high fasting plasma glucose, and alcohol use (Murray et al., 2016). It is well known that such health risk factors are prevalent among patients at hospitals and might aggravate their pathway and clinical outcome (Oppedal et al., 2011). Interventions aimed at helping patients in healthcare settings to quit daily smoking, control alcohol use and nutrition, and attain a healthy level of physical activity—so-called clinical health promotion—have been indicated to improve treatment results, and also have proved cost-effective (Tønnesen, Svane, Groene & Chiou, 2016, p. 13). However, we have few insights into situated work practices for handling the new health challenges. In Denmark and elsewhere, health professionals in hospitals are mobilized to identify individuals at risk as a way of reducing the probability of their developing, or worsening, the most common and deadly NCDs. In the case of so-called lifestyle-related disease prevention, the transnational authority of the WHO has laid the global epistemic foundation for raising the professional and political stakes of this challenge. The WHO has estimated that four lifestyle factors combined—weight, exercise, diet, and smoking—are associated with an 80 per cent reduction in the risk of common and deadly chronic diseases (Mathers & Loncar, 2005), and lifestyle-related disease prevention figures prominently as a policy idea in numerous public health reports, including in Denmark. Within the last decade, all Danish public hospitals have implemented a screening programme among their inpatients to evaluate their habits of smoking, nutrition, alcohol intake, and physical activity, thereby deciding whether interventions should be recommended to prevent or avoid worsening chronic diseases. Yet, which professions or professional groups are supposed to handle such tasks at hospitals—and how? Drawing on empirical material gathered from Danish hospitals in 2017 and 2018, this paper investigates how health professions compete and cooperate in addressing emerging local work tasks defined in relation to the new globalized health challenges. © 2020 the authors. License: https://creativecommons.org/licenses/by/4.0/ (CC-BY 4.0) www.professionsandprofessionalism.com © 2020 the authors. License: https://creativecommons.org/licenses/by/4.0/ (CC-BY 4.0) www.professionsandprofessionalism.com © 2020 the authors. License: https://creativecommons.org/licenses/by/4.0/ (CC-BY 4.0) www.professionsandprofessionalism.com 1 Situating Boundary Work: Chronic Disease Prevention Introduction It will be demonstrated how nurses account mostly for those who compete, participate, and collaborate in this new area of possible inter-professionality, as well as assuming responsibility for its coordination. In the analysis, professional boundary work is a key concept that covers the varieties of situated work in which professions and professional actors engage in order to forge jurisdictional claims, including symbolic boundaries within and between professions (Gieryn, 1999; Liu, 2015). Such dynamics are demonstrated by considering the lifestyle-related disease prevention programme as a boundary object, first 2 2 Situating Boundary Work: Chronic Disease Prevention and foremost by situating the boundary work addressing this specific programme. It is argued that the empirical material of local professional work indicates how nurses re- establish professional boundaries in a new territory. The concluding discussion addresses how processual theory of boundary work can benefit from grounding in a situated account of forms of professional boundaries within emerging jurisdictional tasks. In the following section, the theoretical framework and contribution, along with the concepts informing this paper, are outlined, inspired as they are by the American sociologist Andrew Abbott’s sociology of professions, as well as by sociological literature on professional boundary work. Theoretical frame: Professional boundary work on the g Exploring how new health promotion initiatives alter the foundations for professional jurisdiction, this paper traces the emergence of prevention of lifestyle-related diseases as a trans-local professional proto-jurisdiction by concentrating on profession-driven interventions and initiatives in Denmark. The term “trans-local” refers here to a focus on how professional groups compete and cooperate in creating local change in relation to a border-transcending challenge (Blok, Lindstrøm, Meilvang & Pedersen, 2018). The term “proto-jurisdiction” captures the way professions renegotiate established boundaries under conditions of institutional change (Abbott, 1995). In this case, prevention of lifestyle-related diseases has emerged in the form of novel professional work tasks over the past 10-15 years, thereby still constituting a relatively elastic and ambiguous arena where, in particular, medical doctors and nurses lay claim to certain degrees of control. In order to contribute new insights into the intra- and inter-professional responses to an emerging work task arena—or proto-jurisdiction of health professions, related to global health problems—this paper employs a jurisdictional (Abbott, 1988, 2005) research approach. According to Abbott (1988, p. 20), professional relations are organized via jurisdictions, defined as “problem-spaces” that link professional groups to particular work tasks over which they claim expert authority. However, how health professionals impact upon, and are themselves influenced by, changing local as well as global contexts for their work is a question yet to be systematically explored within sociological research on global transformations and changes in professionals’ work. Addressing lifestyle-related disease prevention is frequently articulated as a border-transcending global challenge that requires new transnational forms of professional expertise and political regulation (Faulconbridge & Muzio, 2011; Kuhlmann & Saks, 2008). Rather than take such “global” claims for granted, this paper will demonstrate how prevention of lifestyle-related diseases as a new trans-local professional proto-jurisdiction is enacted in a workplace arena. In line with this approach, the analysis will ground Abbott’s framework (1988, 2005) in a more situated account of professional boundary work and follow the way health professionals create and sometimes stabilize or standardize techniques for lifestyle-related disease prevention at Danish hospitals. 3 3 Situating Boundary Work: Chronic Disease Prevention Considering professional boundary work as “situated” involves in this case a focus on both conflict and cooperation at the workplace level between health professionals and professions, or professional segments (Bucher & Strauss, 1961), not to mention occupational strategies that emphasize specific knowledge, training, and skills (Apesoa- Varano, 2013). Theoretical frame: Professional boundary work on the Given this paper’s aim to forge an analytic vocabulary capable both empirically and conceptually of keeping its focus on dynamic professional interactions within workplace boundaries, I join related attempts at furthering an interactionist approach to inter-professional change (e.g., Liu, 2018). Situating boundary work hints at Liu’s very important work on lawyers (e.g., Liu, 2015) and professional change, which places at the centre of attention the situated interaction of professionals and professional groups over, within, and across boundaries. Yet, in defining “boundary maintenance”, he does not include situated workplace-based professional interactions in his three-fold distinction, which also encompasses “boundary making” and “boundary blurring” (Liu, 2018, pp. 48-49; see also Blok, Lindstrøm, Meilvang & Pedersen 2019, p. 595). Likewise, “situating boundary work” critically hints at Abbott’s more abstract work on “linked ecologies” (2005) and his general approach to the workplace arena in The System of Professions (1988). As argued elsewhere (Blok et al., 2019), Abbott is pointing to outcomes rather than the means and tactics of boundary work (see for example Abbott’s listing of settlement types, 1988, pp. 69- 79). In this paper, interactionist concepts are chosen as a way of exploring situated tactics and workplace processes involved in the making and maintaining of inter-professional boundaries, herein the concept of boundary object (Star & Griesemer, 1989). By viewing a lifestyle-related disease prevention programme as a boundary object, this paper will show how health professionals articulate their prevention work tasks in terms that are close to what Abbott has called the “jurisdictional dispute” (1988, pp. 69-79). Considering a boundary object implies an analysis of how it functions as a coordinating, but also a contested, object between professional groups. To use Star’s characterization of such objects, they are “a sort of arrangement that allow different groups to work together without consensus”, and what matters for boundary objects is “how practices structure, and language emerges, for doing things together” (Star, 2010, p. 602). The forms this work may take overall are not arbitrary, Star emphasizes. Such forms have arisen owing to “information and work requirements” (Star, 2010, p. 602), and the usefulness of the “boundary object” is underlined at the organizational level in particular (Star, 2010, p. 612). The analytical scope in this paper is concretized to the hospital as the workplace level in which health professionals are developing and using a disease prevention programme—or escaping it, or experiencing limited access to it. Theoretical frame: Professional boundary work on the Thus, for example, “workplace artifact”, as defined by Bechky (2003), will illustrate workplace relations and how task boundaries are created, maintained, or challenged. By viewing a lifestyle-related disease prevention programme as a boundary object, this paper will show how health professionals articulate their prevention work tasks in terms that are close to what Abbott has called the “jurisdictional dispute” (1988, pp. 69-79). Whereas social boundaries often refer to lines of demarcation (Lamont & Molnár, 2002), Liu characterizes boundary work as a social process and defines the boundary for a profession 4 Situating Boundary Work: Chronic Disease Prevention as “a site of conflict and cooperation between two or more professional or non-professional actors seeking to establish jurisdictions over similar work” (Liu, 2018, p. 46). As Liu also has suggested (2015, 2018), the varieties of situated boundary work in which professions and professional actors engage in order to forge jurisdictional claims, niches, and linkages have yet to be further explored. Within the goal of tracing “prevention of lifestyle-related diseases” as a new set of professional tasks and identities, I will draw on Liu’s notion of boundary and typology of boundary work. In situating boundary work, the discussion will, as mentioned above, deviate from Liu’s proposed typology (Liu, 2015, 2018), since Liu does not retain the definitions of all the forms (i.e., boundary maintenance) at a situated workplace level. By these means, I aim to capture the significant ways that Danish nurses seek to navigate emerging task arenas on the ground as they encounter new health challenges. Context, methods, and analytical strategy The Danish hospital sector is mainly publicly owned and regulated through political- administrative decisions. Serving a population of about 5.8 million, the health system is relatively small and the actual number of public hospitals about 50 (see e.g., Kirkpatrick, Dent, & Jespersen, 2011, pp. 494-495, for more information about the Danish health system). Most hospitals are specialized in the treatment of diseases, and yet in Danish hospitals, as well as those in many other countries, health professionals are mobilized to identify individuals at risk in order to reduce the development of the most common and deadly so-called lifestyle-related chronic diseases. In Denmark, there have been inter- and intra-professional discussions about this issue. Who should handle the prevention of such diseases and how? Indeed, the concepts and practices of health promotion and disease prevention are not new to hospitals. However, concepts, along with practices, have changed over recent decades. The “KRAM screening & intervention” is a less than a decade-old implementation at Danish hospitals, implying that in principle all inpatients should be asked about their so-called lifestyle habits. KRAM (in English: SNAP) is an acronym for smoking, nutrition, alcohol intake, and physical activity—at Danish hospitals, related to lifestyle disease prevention. Managing prevention of such diseases may be presumed to be carried out within an inter-professional area of expertise. In considering “KRAM screening & intervention” as a professional boundary object, I was able in undertaking my research to capitalize on current health policy developments and to “study the ways in which these changes in occupational frontiers were being managed by staff in the workplace” (Allen, 2000, p. 335). As argued elsewhere (Blok et al., 2019), under such conditions of institutional change, and without external specifications about how such tasks should be addressed, inter-professional boundary work takes on a specific importance in that it pertains simultaneously to a renegotiation of established workplace routines. 5 Methods This paper draws on research I carried out in four hospitals, mainly in the northern part of Denmark, between May 2017 and June 2018. The study included site visits, field observations, conversations, and in-depth interviews with health professionals and managers about goals, dilemmas, and practices related to “KRAM screening & intervention” (see Table 1 below). Observations were carried out on one of the wards through attendance at “KRAM screening” of new inpatients and “KRAM conversation” about recommended interventions with inpatients close to being discharged from the hospital. Elsewhere in the organization, I observed among other activities patient education about “KRAM risk factors”. On the ward, I mainly accompanied some of the nurses, but also one of the healthcare assistants. I gained insight into informal collaboration on the ward and how personnel communicated among themselves both directly and by means of aids such as laminated pocket sheets, flow charts of working processes, noticeboards, lists of inpatients with messages about the KRAM screening, discharge sheets, etc. Managers at the hospitals’ divisions for health promotion information all agreed to participate and gave access to the hospitals in which observations, conversations, and interviews were performed. One of them acted as a gatekeeper and granted access to the ward studied here, as well as to patient education meetings about lifestyle risk factors. I generated data through field notes, as far as possible recorded contemporaneously in a notebook; through tape-recorded in-depth semi-structured interviews with ward nurses and other nurses, doctors, dietitians, and clinical and HR managers; as well as through spontaneous or planned extended, sometimes repeat conversations (not tape-recorded, but collected thorough notes), with healthcare assistants, nurses, physiotherapists, and a non- smoking consultant. In all, I engaged in a total of 25 interviews and extended conversations. Table 1. Methods Overview of data sources Site visits Interviews Participants Documents Lifestyle disease prevention Danish hospitals (4), one repeat four times (1-4 days per visit); workplace artefacts (23 items) In-depth interviews (11), extended conversations (14); transcribed material, in sum 578 pages Nurses (12), dietitians (4), physiotherapists (4), doctors (2), others (e.g., healthcare assistants) (3) Prevention policy (7) and practical intervention materials (32), educational programmes (9) In Denmark, there are no institutional boards for the approval of social science studies, but the study was carried out in accordance with the ethical guidelines for the social sciences as 6 6 Situating Boundary Work: Chronic Disease Prevention specified by The Norwegian National Research Ethics Committees (NESH, 2016). Consent from the individuals or their representatives was obtained and confidentiality promised. Thus, all identifying information has been removed from the material used in this paper. The interviews and not least the extended conversations proved to be an important source of data. The perspectives from different professional groups have afforded an enhanced understanding of my observations. I have compared my own empirical data with materials from different sources in order to make more thorough judgements as to how I should interpret the data or material. This material included documents (e.g., standard procedures and registration forms), reports (for example, by the WHO and the Danish National Health Authorities), workplace artefacts, and educational programmes that have developed or revised their curricula to include disease prevention (see Table 1 above). Analytical strategy This paper’s qualitative approach follows Abbott’s contextual sociology (2001) in stressing the concrete relations, settings, and situations in which professional power is shaped and exercised. The empirical work therefore also includes site visits and observations. The methodology of interviewing provides access to agendas, understandings, and opinions of health professionals based on their daily work and experiences with health promotion and prevention of chronic diseases. The study was directed at eliciting the health professionals’ own terms for and ideas about their practices and experiences related to “KRAM screening & intervention”. The practices, strategies, and rhetorical devices they employed in exercising occupational demarcation will be treated here as examples of professional boundary work (Gieryn, 1999; see also Allen, 2000, p. 327). The Danish hospital context is an arena where disease prevention and health promotion are not clearly demarcated as the domain of one specific profession, and so in this analysis it will be demonstrated how different forms of professional boundary work are under way. A situated analysis of boundary work in this instance makes for the first step to discussing professional and wider institutional change. Drawing on Clarke, Friese, & Washburn’s Situational analysis in practice, it is assumed that “the conditions of the situation are in the situation” (2015, p. 98, their italics for emphasis). This means that there is not a “context” for such a notion of situation: Instead, “the conditional elements of the situation need to be specified in the analysis of the situation itself as they are constitutive of it” (Clarke et al., 2015, p. 98, their italics for emphasis). This notion of situation—I will not dwell here on philosophical or other disciplines where this notion is seminal (see, for example, Dewey, 1949[1938])—derives its heuristic value when Clarke et al. (2015, p. 99) suggest making different kinds of mapping: situational maps; social worlds/arenas maps; positional maps. The mapping has been useful to analyse across different types of material and elements, and the initial situational mapping suggests much inter- and intra-professional repositioning of tasks. The following analysis draws on maps of the kind that lay out “the major human, 7 7 Situating Boundary Work: Chronic Disease Prevention nonhuman, discursive, historical, symbolic, cultural, political, and other elements in the research situation of concern” (Clarke et al., 2015, p. 100) while also indicating relations among selected elements such as political issues or initiatives and how professionals handle such issues. Situated boundary work y In this section, the findings will encapsulate the ways professionals on the ground—in this case, nurses in particular—seek to navigate emerging task arenas that are not always well defined. These findings are structured into three subsections concentrating in particular on nurses’ efforts to extend, defend, or refashion established work boundaries when handling “KRAM screening & intervention” at Danish hospitals. Analytical strategy The overall question underlying the situational mapping was: Who or what matters to “KRAM screening & intervention”? Worth mentioning here is the notion of “workplace artifacts” (Bechky, 2003), which below includes many elements ranging from flow charts to laminated pocket sheets to the manner in which health professionals use the procedure of “KRAM screening & intervention”. Preventive work emerges as a possible arena for nurses in particular, wherein workplace artefacts are developed and demonstrate important professional coordination work at stake. Effecting control over the process of screening and intervention When a nurse engaged in developing a flow chart for the process of “KRAM screening & intervention” mentioned that healthcare assistants were interested in becoming involved in the screening task, she emphasized the importance of knowing about specific diagnoses. In particular, she stressed the importance of knowing about instances of comorbidity in order to administer the programme, not least the intervention part, without risks: … when you recognize the criteria for a metabolic syndrome, you have to intervene yourself, based on: What are the criteria? And then you say: “Okay, then, in fact I can see my patient really has the precursors to metabolic syndrome. We need to start some kind of KRAM prevention!” This means we’re confronted with certain issues that demand the development of healthcare assistants’ skills to be able to handle the task like the nurses can. (…) This means when we produce guidelines and test them in clinical settings, and we see there are different levels of skill, then … we see there are differences in how to read guidelines. These are our experiences. And there’s no consensus on in how much detail the tasks should be described. It’s clear that because something’s obvious to a nurse, it doesn’t mean it’s obvious to a healthcare assistant. And I think this may be the same for a nurse and a doctor. What appear as arguments over safety—namely the importance of having insights into the medical implications of comorbidity, thereby triggering an ethical dilemma if healthcare assistants cross perceived occupational boundaries—might be in fact disguised turf wars. 8 Situating Boundary Work: Chronic Disease Prevention Arguing for the importance of having achieved knowledge of pathology to handle the screening and intervention programme in a safe and proper manner may indeed be seen as a marker of an occupational identity, thus marking the legitimate boundaries between the nurses’ and healthcare assistants’ training. The flow chart of the screening and intervention process documented lifestyle risk factors, as well as different medical conditions, with respect to occupational knowledge and authority. However, this nurse’s account also allows for the possibility of knowledge transfer that Abbott calls “workplace assimilation” (1988, pp. 65-66). In particular, when there is too much work to do, which is the case in many hospital wards, subordinate professionals or nonprofessionals (have to) learn from a given profession’s knowledge system. Effecting control over the process of screening and intervention When I asked a nurse if doctors could complete the prevention talk with patients, she replied that this was an option and sometimes it happened, but not in a systematic way: Maybe they tell the patient not to smoke—and sometimes they ask about alcohol use, but they don’t complete a KRAM screening. Maybe they tell the patient not to smoke—and sometimes they ask about alcohol use, but they don’t complete a KRAM screening. She and more nurses from this ward also emphasized that the discharge sheet for following up on the “KRAM screening & intervention” was a nurses’ tool. The head nurse related how one of the doctors wanted to add something to the discharge sheet: Yet he hasn’t received it [the authority to modify the sheet]. This [his suggestion] can’t currently be included. Here, on this sheet, we’re focusing on some other aspects. The current political claim of prevention of lifestyle-related diseases seems to have created among certain segments of the nurses an ambition to establish a domain of professional practice that is relatively removed from doctors’ control. As for the doctors’ concern, attempts to control the “KRAM screening & intervention” can be interpreted as a strategy for maintaining their dominance, whereas for nurses, as illustrated above, this programme can be understood as an opportunity to extend their task area by means of workplace artefacts, for example, flow charts and discharge sheets used as jurisdictional tools. Effecting control over the process of screening and intervention Yet, as we will see below, other nurses emphasized that theoretical training equivalent to a nurse’s education should be required. A nurse who worked as a coordinator of health promotion at another hospital emphasized as well that all aspects of the KRAM screening should be carried out by qualified staff who had finished a Professional Bachelor’s degree programme: I’ve argued that KRAM advisers [appointed in each ward] as a minimum should hold a Professional BA degree. It means they should be physiotherapists or occupational therapists in their department and midwives in the maternity department, and dietitians and nurses. (…) We had some healthcare assistants who wanted to work with it. (…) However, we maintain that although they’re allowed to talk to the patients about health promotion—this isn’t an issue— those who should serve as the KRAM advisers and have the responsibility in the ward, and who can be asked about concerns regarding organizational issues— “What’s meaningful in your area? What do you prefer in this case?” and so on— this [the nurse’s emphasis] should come under the KRAM advisers’ area, for those who hold a Professional BA degree. Nurses’ professional rhetoric about healthcare assistants can be seen as occupational identity work that may be considered a variant of boundary work. Nonetheless, how the labour was divided varied from ward to ward. In ward A, there was too much work for nurses to do all the KRAM screening and so healthcare assistants did it as well. However, the nurses in ward A distinguished between identifying lifestyle habits and motivating patients. A head nurse said that healthcare assistants, like the nurses themselves, can and in fact do complete KRAM screenings to identify lifestyle habits: Within some areas, the healthcare assistants have a much greater focus on lifestyle than us [the nurses]—in their training as well. 9 9 Situating Boundary Work: Chronic Disease Prevention Yet when it came to disease prevention as a follow-up to the KRAM screening and thereby the task of communicating with patients before they were discharged, the same head nurse stated: But they [healthcare assistants] are unable to undertake the talk with patients about prevention. But they [healthcare assistants] are unable to undertake the talk with patients about prevention. Only nurses completed the “KRAM conversation” to motivate patients close to being discharged. Instruments to maintain authority How established work boundaries were defended, in order to be maintained, was evident from the way the interviewees linked the task area to training necessary to handle the programme of “KRAM screening & intervention”. One of the interviewees, a nurse with an MA degree and a supervisor for health professionals involved in prevention of lifestyle- related diseases, said: I’ve argued that as a professional encountering a patient with lifestyle-related problems, you need to have achieved a certain background knowledge. The reason is that first off, you need knowledge and, second, the situation implies the art of communication. And you definitely have this if you’ve graduated with a Professional BA degree. You’ve been trained, then, in some psychology and 10 Situating Boundary Work: Chronic Disease Prevention communication, also pedagogy. Moreover, you’ve succeeded in passing lots of exams where you certainly can delve more deeply into some issues. And you’ve practised how to convey [these ideas] to other people—and this is the deepest you can go. You must feel 100 per cent confident in what you want to convey to others. Those who hold a Professional BA degree have tested this ability. Thus, she emphasized that the “KRAM screening & intervention” was made possible through securing the appropriate patient communication derived through training and its grounds for developing qualifications. Although “health promoter” is one of seven so-called “roles for doctors” included in the specialty training of Danish medical doctors, this area is not much evident or prestigious among doctors at Danish hospitals. As a medical doctor said: I don’t have the full overview of what’s known, but of course many people have studied different … what should we call them? Pedagogical methods? However, it’s not the case that I have one [the doctor’s emphasis] way of doing things. It’s a bit like common sense. And I have thought about this in connection with helping our nurses … with what I’ll call “nursing professionalism”—the ability to do this [lifestyle disease prevention and health promotion]. And where … do they position their professionalism? Is it only a matter of common sense or do they simply have a method they use? And this … right here, it’s not really my strength. And I don’t think it’s our nurses’ either. I don’t think so. Instruments to maintain authority This example of downgrading the prevention tasks, and thereby the work of subordinate professionals, sheds some light on the status of lifestyle disease prevention and health- promoting work tasks and how this may affect where the occupational boundaries are drawn. Not least, as demonstrated in this subsection, the doctor’s account lends a fresh perspective on how established work boundaries are defended when handling the programme of “KRAM screening & intervention”. As Allen has noted (2000, p. 332), two of nursing’s key occupational boundaries are those at the interface with medicine and with support staff, respectively. The accounts above illustrate how the downgrading of these tasks undertaken by the subordinate profession ensures these professionals retain a lower status. This applies also when new work tasks have to be handled. In order to undertake those tasks not covered in basic training, some of the nurses within this area of prevention and health promotion—in particular those with managerial responsibilities—had acquired extended training certificates such as an MA degree within a relevant area of education, for example, an MA in Public Health. The nurses serving as consultants or managers had reasoned the additional degree would prove advantageous for them. It has been suggested that doctors in this instance, as in some others, have dumped more low-prestige work on occupational groups lower in the implicit hierarchy. Yet changes 11 Situating Boundary Work: Chronic Disease Prevention in Danish university-based doctor training programmes (SST, 2013) suggest as well that, far from giving up prevention as a task, this profession is also slowly annexing the agenda. With the public hospital system in constant flux and a general shortage of doctors and not least of nurses, as well as a steadily increasing focus on efficiency, it seems—as a third scenario— that occupational groups such as healthcare assistants could assume an enlarged role in the increased efforts to prioritize work tasks related to disease prevention and health promotion. In what follows, I will demonstrate, however, how stable boundaries are kept alive by refashioning occupational boundaries with a variety of workplace artefacts developed by nurses. Boundary re-establishment in new territory A nurse coordinating the “KRAM screening & intervention” programme at a local hospital stressed the importance of developing the necessary tools and procedures for this specific programme. She talked about the frustration experienced by nurses in particular when they felt that they had transformed patients previously considered “normal” into “problems”; such shifts resulted not only from the classification of patients into “high risk” categories within the screening system, but also from the placement of so-called lifestyle habits onto an agenda of patient communication in the hospital setting: They [the health professionals doing the screening] tell me: “Whatever I try to say to the patient and however I say it—then I seem to end up communicating in one way or another, ‘You’re wrong about this!’” The nurse showed how she herself had developed laminated pocket sheets with helpful knowledge, models, and advice used in particular by nurses in the patient-professional encounter. She explained that the staff themselves had enquired about tools they could use to handle situations with patients when required to ask them about their lifestyle habits, and that they experienced patients who went quiet when it was recommended they stop smoking: If you’re too quick to present your agenda: “You need to stop smoking because of this and that …”, then the patient probably won’t say a word. You’re better off asking: “What do you know about smoking? How is it related to your prostate?” This approach will make your message more appealing. So, we’ve created some exercises and (…) the staff asked me: “Can’t you make a framework [produce in writing a form of guidelines they can draw upon in these situations]?” She summed up: When do we know we have important knowledge to contribute to work practice? When the staff ask to get it laminated! 12 Situating Boundary Work: Chronic Disease Prevention This is an example of how the staff needed some tools and received a variety of workplace artefacts, in particular laminated sheets as pedagogical tools, for refashioning their skills to motivate the patient in a non-blaming way. Moreover, this example illustrates how the training in communicative skills was used to justify responsibility for such refashioning. Indeed, it turned out that how nurses defined their work boundaries was central to the interactional accomplishment of the division of labour among nursing, medical, and assistant staff when handling tasks of lifestyle-related disease prevention and health promotion in the hospital setting. This observation can be seen in light of specific aspects of the Danish healthcare management reform of 2007, not to mention how, since the mid- 1980s, both the nursing and other medical professions in Denmark actively have sought to lay claim to the jurisdiction of hospital management (Kirkpatrick et al., 2011). Moreover, since the late 2000s, who does what in the hospitals’ KRAM screening division of labour has not been explicitly addressed either at the national or counties level. Responsibility for agreeing to the division of labour is left to local determination, that is, certain doctors at the respective hospitals produce individual plans for staff involved in the screening programme. However, in the workplace settings observed, it appeared to be the nurses’ domain to produce plans for staff involved in the actual KRAM screening. At the level of everyday work practices, including new tasks and established routines, nurses are still sorting out the jurisdictional boundaries, with much inter- and intra-professional repositioning of tasks. National health policy, as well as regional regulations and reforms, is a conditional element, among others, of work task situations in Danish hospitals. From the accounts of health professionals were evident a spectrum of responses to their work environment—from feeling used to continually experiencing changing conditions and pragmatically reshuffling features of how they are accustomed to doing their work, to accommodating themselves by taking control of or suggesting new initiatives and using them for professional purposes (see also Allen, 2000, p. 339; Dent, 2008). Control of education and training also is vital to retaining professional jurisdiction (Abbott, 1988; see also Allen, 2000, p. 341). Concluding discussion: Situating boundary work g g y In this section, I will discuss and sum up the ways processual theory of boundary work can benefit—or not—from grounding theoretical frameworks in the workplace arena in order to analyse conflicts or other dynamics involved in professional proto-jurisdiction. The focus, then, is on situated boundary work when a jurisdiction is not resolved. In sum, this paper’s findings indicate that the onus for defining the boundaries of nursing within the arena of prevention and health promotion at Danish hospitals currently is left sometimes to the local hospital. Second, the findings hypothesize that this trans-local professional proto- jurisdiction is likely to emerge as the next site of well-known inter-professional struggles between doctors and nurses. Indeed, medical doctors have included “health promoter” as one of seven important roles in their specialty training programme. However, at some hospitals’ health promotion units, which often are run by nurses as managers, doctors in training draw upon services from the unit. The findings have demonstrated how nurses justify their role and obtain qualifications, hereby distinguishing themselves within this area, which is what Liu (2015, p. 3) has referred to as boundary making. Considering “KRAM screening & intervention” as a boundary object, this paper has drawn on Star and Griesemer’s (1989) understanding of such objects as artefacts fulfilling specific functions in bridging intersecting practices, as well as on Star’s (2010) perception of such objects as certain arrangements that allow different professional groups to work together. This paper’s findings have demonstrated such dynamics of “KRAM screening & intervention”, first and foremost by situating the boundary work addressing this specific boundary object. In using this situating approach, which includes investigation of work practices and workplace artefacts, it has been possible to indicate only slightly other important components in jurisdictional work, such as political agendas, changes in organizations and education, as well as the linking of health professionals to transnational activities (e.g., WHO). The findings are based mainly on one kind of mapping out of the three available from Clarke et al.’s (2015) SA method, namely situational and relational mapping and not (yet) social world/arena and positional mapping. This focus hinders the “linked ecology” approach (Abbott, 2005) from being fully implemented. She summed up: In the field of disease prevention and health promotion, not only nurses, but also nutritionists, for example, are changing their jurisdictional claims; educational programmes are revising or have developed their curricula to include specialization within this field. The curricula for a Danish Professional BA degree in Nutrition and Health have changed in recent years (cf. curriculum 2010 compared with curriculum 2016) to include a specialty in health promotion and disease prevention. Thus, at the educational level, different professional areas indeed are involved in developing the field of disease prevention and health promotion. As such, health professionals’ boundary work is linked not only to the workplace arena, but also to universities as well as to political institutions (cf. Abbott, 2005, about “linked ecologies”). However, the fact that this field is still developing implies that the practical tasks of handling disease prevention and health 13 Situating Boundary Work: Chronic Disease Prevention promotion at Danish hospitals form a substantial part of what makes up the (proto-)jurisdictional boundaries. Concluding discussion: Situating boundary work However, analysing professional boundary work through the lifestyle-related disease prevention programme considered as a boundary object prevents losing sight of the workplace arena and should be seen as an important step towards grounding Abbott’s meso-level vocabulary in situated interactions among professionals. The findings have illustrated how nurses in particular not only are (re-)constructing their own boundaries, but also the boundaries of other occupational groups. Most work tasks in a hospital fall within certain areas over which a specific profession has established 14 Situating Boundary Work: Chronic Disease Prevention jurisdiction. However, the everyday tasks involving disease prevention and health- promoting work at Danish hospitals are not externally coordinated in their details. As the findings have demonstrated, this work is in many cases negotiable. Sometimes the jurisdictional relations in the workplace are blurred (see also Abbott, 1988, p. 66; Liu, 2015, p. 3). As Christiansen, Taasen, Hagstrøm, Kjellaug, & Norenberg (2017, p. 1), among others, have noted, borderlines between professions can also be “areas of contact that link social worlds and open opportunities for collaboration, learning, and development”, although the delineation between professions “has traditionally been used to construct boundaries around tasks and fields of knowledge and to exclude others”. In the case of creating and implementing the programme of “KRAM screening & intervention”, there are several tasks and situations requiring cooperation and coordination to solve problems and meet challenges, for example, when working under time pressure. As Abbott has noted, in the workplace, boundaries between professional jurisdictions can disappear or at least become very blurred, “particularly in overworked worksites” (1988, p. 65). However, although Danish hospital wards in general are lacking nurses, in the study sites, it was nurses in particular, who had developed specific tools for doing the “KRAM screening & intervention” work. This paper has mainly focused on demarcation work and less on collaboration when analysing professional boundary work. A jurisdictional approach will often produce certain results and the findings therefore should reflect this perspective. In this study, nurses emphasized that they had the advantage of certain ideas about the most appropriate sites for prevention and knowledge linked to the dangers of health risks, along with an understanding of the importance of distinctly classifying the “normal” and the “pathological”; and they stressed, too, that they knew “how to communicate with patients”. Such statements from nurses could be considered conflictual as well as collaborative. Concluding discussion: Situating boundary work Nurse managers also were establishing alliances with other professional groups with a Professional BA degree, such as physiotherapists, dietitians, midwives, and occupational therapists, while at the same time resisting coming under the control of the medical doctors. The establishing of inter-professional alliances, as well as demarcations, was an important element in the examined boundary work. In future research, collaborative learning will need more attention as well (see also Christiansen et al., 2017). Medical doctors at Danish hospitals are in many cases formally responsible for areas related to and projects centred on prevention and health promotion. However, in practice, disease preventive work is emerging as a possible arena in which nurses might contest the hierarchized division of labour and management competencies (see also Kirkpatrick et al., 2011)—in part because the commitment of doctors to the prevention agenda seems to fluctuate considerably. Here, WHO recommendations and standards may well come to serve as a resource for nurses to reformulate their projects in ways that simultaneously enjoy professional and political legitimacy. At the same time, the nursing contribution to this new task could entail nurses’ “rejection of the old hierarchy of prestige which elevated technical 15 Situating Boundary Work: Chronic Disease Prevention (medical) tasks over bedside (nursing) care” (Allen, 2000, p. 334). Future—and transnational—studies are needed to follow up on this issue. Grounded in empirical material, this paper’s findings have distinguished nurses’ efforts to extend, or defend, or refashion established work boundaries when handling “KRAM screening & intervention” at Danish hospitals. Nurses, along with managers who were trained as nurses, have developed and initiated a number of workplace artefacts to show their competency in practising and managing a process of boundary work. These artefacts include, for example, flow charts and self-instruction packages, and specific discharge sheets for KRAM health risk intervention, but also measures supplementing training and education. The focus on such activities, which considers the programme of “KRAM screening & intervention” as a boundary object, perceived as a set of work arrangements allowing different professional groups to work collaborate, has situated the boundary work. The findings have gained analytical purchase for documenting relations that emerge when task boundaries are challenged. It has been illustrated how the development of workplace artefacts, sometimes distributed as laminated pocket paper sheets used in the patient- professional encounter, as well as flow charts outlining procedures for screening and intervention processes, can serve as jurisdictional tools. Concluding discussion: Situating boundary work In these cases, this is especially true for nurses engaged in hospitals’ disease prevention work. Thus, by situating processes of boundary making, or maintenance, or blurring (cf. Liu, 2015), this paper can set the situational foundations for moving to more mapping—aimed at tracing the wider local, national, and transnational professional, university, and political linkages at work in the emergence of new jurisdictional tasks (see also Blok et al., 2019). “Prevention of lifestyle-related diseases” considered as a trans-local professional proto- jurisdiction is likely to emerge as the next site of a well-known inter-professional struggle. In particular, the struggle will take place between medical doctors and nurses—but also among other health professionals involved—and this issue can differ transnationally. Thus, the definition and meaning of task areas are likely to become the subject of intense conflict. However, with the current shortage of health professionals in Denmark and elsewhere, nurses do not so much need to reduce the role of possible competitors as to use the new prevention tasks to strengthen their claims for a more elevated status. Indeed, the demonstrations of boundary work presented in this paper indicate this conclusion. More research is needed to affirm whether doctors, nurses, healthcare assistants, or other professions or occupational groups, perhaps in particular segments within a profession (Bucher & Strauss, 1961), in fact have most to gain from leveraging ties both to local and global health problems. Overall, the interactive effect of the developments within Danish hospitals has created some jurisdictional ambiguity at the work boundaries between the professions of medicine and nursing in particular, and maybe also between nurses and healthcare assistants. This ambiguity raises many questions about the future jurisdiction of 16 Situating Boundary Work: Chronic Disease Prevention prevention of lifestyle-related diseases in other countries as well, since NCDs represent a global problem. This paper’s grounding of Abbott’s framework of linked ecologies and his meso-level vocabulary in a more situated account of professional boundary work hopefully has opened the way to exploring how health professionals create, and sometimes stabilize or standardize, techniques for prevention of lifestyle-related diseases at hospitals, not merely in Denmark but elsewhere. The paper has situated all the different forms of boundary work in Liu’s typology (2015)—not only boundary making and blurring, as Liu himself has undertaken, but also boundary maintenance—by drawing on Bechky’s (2003) notion of “workplace artifacts”. Concluding discussion: Situating boundary work Using this methodology, we have seen how nurses specifically are creating, but also stabilizing and maintaining jurisdictional claims, niches, and linkages. Yet, the ways nurses in particular seek to navigate emerging task arenas, which are not well defined, have been captured only at a situated workplace level, meaning the material does not allow me to draw conclusions about general jurisdictional claims. This type of qualitative study, developed to collect varied material, has been more concerned with obtaining reasonable grounds for the relevance of essential issues to the sociology of professions in order to discuss how prevention of lifestyle-related diseases, addressed as a proto- jurisdictional task, can contribute insights into the dynamics of boundary work on the ground. Building on empirical observations from ongoing research, I have taken some steps towards fleshing out the trajectories of a novel trans-local professional proto-jurisdiction by sketching an analysis of situated work practices that address emerging local work tasks related to a globalized health challenge. This is only a starting point for further empirical enquiry, for example, elucidating the role of international networks such as Health Promoting Hospitals (e.g., Tønnesen et al., 2016), standards, and professional practices in framing the jurisdictional boundaries of global and local health problems. Further enquiry, I hope, will indicate a number of interesting directions for research more generally, both empirically and in terms of theory construction—thus addressing a research agenda resulting from a more comprehensive assessment of trans-local professional projects, including how their linked ecologies, such as those of politics and the university, are transnationalized. Acknowledgements This work was supported by the Danish Council for Independent Research—Social Sciences, grant number DFF—6109-00063. I would like to thank the anonymous reviewers for their fruitful comments, and my colleagues as well, Anders Blok, Maria Duclos Lindstrøm, and Marie Leth Meilvang, on earlier versions of this paper. 17 Situating Boundary Work: Chronic Disease Prevention Article history Received: 26 Apr 2019 Accepted: 30 Aug 2020 Published: 22 May 2020 References Abbott, A. (1988). The system of professions: An essay on the division of expert labor. Chicago: University of Chicago Press. https://doi.org/10.7208/chicago/9780226189666.001.0001 Abbott, A. (1995). Boundaries of social work or social work of boundaries? The social service review lecture. Social Science Review, 69(4), 545-562. https://doi.org/10.1086/604148 Abbott, A. (2001). Time matters: On theory and method. Chicago: University of Chicago Press. Abbott, A. (2005). Linked ecologies: States and universities as environments for professions. Sociological Theory, 23(3), 245-274. https://doi.org/10.1111/j.0735-2751.2005.00253.x Allen, D. (2000). Doing occupational demarcation: The ‘boundary-work’ of nurse managers in a district general hospital. Journal of Contemporary Ethnography, 29(3), 326-356. https://doi.org/10.1177/089124100129023936 Apesoa-Varano, E. C. (2013). Interprofessional conflict and repair: A study of boundary work in the hospital. Sociological Perspectives, 56(3), 327-349. https://doi.org/10.1525/sop.2013.56.3.327 Bechky, B. A. (2003). Object lessons: Workplace artifacts as representations of occupational jurisdiction. American Journal of Sociology, 109(3), 720-752. https://doi.org/10.1086/379527 Blok, A., Lindstrøm M. D., Meilvang, M. L., & Pedersen, I. K. (2018). Trans-local professional projects: Re-scaling the linked ecology of expert jurisdictions. Journal of Professions and Organization, 5(2), 106-122. https://doi.org/10.1093/jpo/joy003 Blok, A., Lindstrøm M. D., Meilvang, M. L., & Pedersen, I. K. (2019). Ecologies of boundaries: modes of boundary work in professional proto-jurisdictions. Symbolic Interaction, 42(4), 588-617. https://doi.org/10.1002/symb.428 Bucher, R., & Strauss, A. (1961): Professions in process. The American Journal of Sociology, 66(4), 325-334. https://doi.org/10.1086/222898 Christiansen, B., Taasen, I., Hagstrøm, N., Kjellaug, K. H., & Norenberg, D. L. (2017). Collaborative learning at the boundaries: Hallmarks within a rehabilitation context. Professions & Professionalism, 7(3), 1-15. https://doi.org/10.7577/pp.2121 18 Situating Boundary Work: Chronic Disease Prevention Situating Boundary Work: Chronic Disease Prevention Clarke, A. E., Friese, C., & Washburn, R. (Eds.) (2015). Situational analysis in practice. Mapping research with grounded theory. London: Routledge. https://doi.org/10.4324/9781315420134 Clarke, A. E., Friese, C., & Washburn, R. (Eds.) (2015). Situational analysis in practice. Mapping research with grounded theory. London: Routledge. https://doi.org/10.4324/9781315420134 Dent, M. (2008). Medicine, nursing and changing professional jurisdictions in the UK. In D. Muzio, S. Ackroyd, & J. F. Chanlat (Eds.), Redirections in the study of expert labour (pp. 101-117). London: Palgrave Macmillan. https://doi.org/10.1057/9780230592827_5 Dewey, J. (1949[1938]). Logic. The theory of inquiry. New York: Henry Holt and Company. Faulconbridge, J., & Muzio, D. (2011). Professions in a globalizing world: Towards a transnational sociology of the professions. International Sociology, 27(1), 136-152. https://doi.org/10.1177/0268580911423059 Gieryn, T. F. (1999). Cultural boundaries of science. Credibility on the line. Chicago: University of Chicago Press. Kirkpatrick, I., Dent, M., & Jespersen, P. K. (2011). The contested terrain of hospital management: Professional projects and healthcare reform in Denmark. Current Sociology, 59(4), 489-506. https://doi.org/10.1177/0011392111402718 Kuhlmann, E., & Saks, M. (Eds.) (2008). Rethinking governance: International directions in healthcare. Bristol: Policy Press. https://doi.org/10.1332/policypress/9781861349569.001.0001 Lamont, M., & Molnár, V. (2002). The study of boundaries in the social sciences. Annual Review of Sociology, 28, 167-195. https://doi.org/10.1146/annurev.soc.28.110601.141107 Liu, S. (2015). Boundary work and exchange: The formation of a professional service market. Symbolic Interaction, 38(1), 1-21. https://doi.org/10.1002/symb.137 Liu, S. (2018). Boundaries and professions: Towards a processual theory of action. Journal of Professions and Organization, 5(1). 45-57. https://doi.org/10.1093/jpo/jox012 Liu, S. (2018). Boundaries and professions: Towards a processual theory of action. Journal of Professions and Organization, 5(1). 45-57. https://doi.org/10.1093/jpo/jox012 Mathers, C. D., & Loncar, D. (2005). Updated projections of global mortality and burden of disease, 2002-2030: Data sources, methods and results. Geneve: World Health Organization. https://doi.org/10.1371/journal.pmed.0030442 Mathers, C. D., & Loncar, D. (2005). Updated projections of global mortality and burden of disease, 2002-2030: Data sources, methods and results. Geneve: World Health Organization. https://doi.org/10.1371/journal.pmed.0030442 Murray, C. J. L. et al. (2016) Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: A systematic analysis for the Global Burden of Disease Study 2015. Lancet, 388(10053), 1659-1724. https://doi.org/10.1016/S0140-6736(16)31679-8 NESH (2016). Guidelines for research ethics in the social sciences, humanities, law and theology. Oslo: The National Committee for Research Ethics in the Social Sciences and the Humanities. 19 Situating Boundary Work: Chronic Disease Prevention Situating Boundary Work: Chronic Disease Prevention Oppedal, K., Nesvåg, S., Pedersen, B., Skjøtskift, S., Aarstad, A. K., Ullaland, S., . . . Tønnesen, H. (2011). Health and the need for health promotion in hospital patients. European Journal of Public Health, 21(6). 744-749. https://doi.org/10.1093/eurpub/ckq148 Star, S. L. (2010). This is not a boundary object: Reflections on the origins of a concept. Science, Technologies & Human Values, 35(5), 601-617. https://doi.org/10.1177/0162243910377624 Star, S. L., & Griesemer, J. R. (1989). Institutional ecology, ‘translations' and boundary objects: Amateurs and professionals in Berkeley's Museum of Vertebrate Zoology, 1907-39. Social Studies of Science, 19(3), 387-420. https://doi.org/10.1177/030631289019003001 STS (2013). De syv lægeroller. Copenhagen: Sundhedsstyrelsen. TS (2013). De syv lægeroller. Copenhagen: Sundhedsstyrelsen. Tønnesen, H., Svane, J. K., Groene, O., & Chiou, S.-T. (2016). The WHO-HPH recognition project: Fast-track implementation of clinical health promotion—a protocol for multi-center RCT. Clinical Health Promotion, 6(1), 13-20. https://doi.org/10.29102/clinhp.16003 WHO (2014). Global Status Report on Noncommunicable Diseases. Geneva: World Health Organization. 20 20
https://openalex.org/W4292649926	https://zenodo.org/records/7018005/files/PK_article_82821.pdf	English	null	Osodendron gen. nov. (Leguminosae, Caesalpinioideae), a new genus of mimosoid legumes of tropical Africa	PhytoKeys	2,022	cc-by	9,095	PhytoKeys 205: 453–470 (2022) doi: 10.3897/phytokeys.205.82821 https://phytokeys.pensoft.net PhytoKeys 205: 453–470 (2022) doi: 10.3897/phytokeys.205.82821 https://phytokeys.pensoft.net PhytoKeys 205: 453–470 (2022) doi: 10.3897/phytokeys.205.82821 https://phytokeys.pensoft.net RESEARCH ARTICLE emic editor: Colin E. Hughes \| Received 27 February 2022 \| Accepted 25 July 2022 \| Published 22 August 202 Citation: Koenen EJM (2022) Osodendron gen. nov. (Leguminosae, Caesalpinioideae), a new genus of mimosoid legumes of tropical Africa. In: Hughes CE, de Queiroz LP, Lewis GP (Eds) Advances in Legume Systematics 14. Classification of Caesalpinioideae Part 1: New generic delimitations. PhytoKeys 205: 453–470. https://doi.org/10.3897/ phytokeys.205.82821 Abstracth The genus Osodendron is here newly described to accommodate three species and one subspecies of African mimosoid legumes. These taxa have previously been included by several authors in Albizia, Cathormion and/or Samanea, but they have been shown to be phylogenetically unrelated to any of these, being in stead the sister-group of the recently described Neotropical genus Robrichia, which is similar in vegetative morphology and especially its very similar indumentum, but is decidedly different in pod morphology. A taxonomic treatment of the three species in the genus is presented, with species descriptions, photo graphs, distribution maps and an identification key. The type species Osodendron altissimum (Hook. f.) E.J.M. Koenen occurs in swamp and riverine rainforest and gallery forests, with the typical subsp. altissi mum widespread across tropical Africa, while Osodendron altissimum subsp. busiraensis (G.C.C. Gilbert) E.J.M. Koenen is only known from the Busira river catchment in the western part of the Democratic Republic of Congo. Of the other two species, Osodendron dinklagei (Harms) E.J.M. Koenen is a com mon tree of rainforest and the forest-savannah transition including semi-deciduous and secondary forest as well as gallery forest and is restricted to Upper Guinea and the similar, but vegetatively more variable Osodendron leptophyllum (Harms) E.J.M. Koenen occupies comparable vegetation types in Lower Guinea and extends marginally into the Sudanian and Zambezian savannahs in gallery forest. Copyright Erik J. M. Koenen. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Albizia, Cathormion, ingoid clade, Fabaceae, Samanea, taxonomy Osodendron gen. nov. (Leguminosae, Caesalpinioideae), a new genus of mimosoid legumes of tropical Africa Erik J. M. Koenen1 1 Evolutionary Biology & Ecology, Université Libre de Bruxelles, Faculté des Sciences, Campus du Solbosch - CP 160/12, Avenue F.D. Roosevelt, 50, 1050 Brussels, Belgium Corresponding author: Erik J. M. Koenen (erikk_botany@gmx.com) Introduction The taxonomy and classification of the mimosoid legumes has seen significant changes over the last 35 years, including the disintegration of Acacia Mill. (Pedley 1986; Luckow et al. 2003; Miller and Seigler 2012), multiple contentious reclassifications of the ingoid mimosoids by several authors (e.g. Nielsen 1981; Barneby and Grimes 1996, 1997; Lewis and Rico-Arce 2005; reviewed by Brown 2008), as well as the clade not being recognised as a subfamily (LPWG 2017) anymore, being instead subsumed into a recircumscribed Caesalpinioideae. Following phylogenetic (Luckow et al. 2003; Bouchenak-Khelladi et al. 2010) and phylogenomic (Koenen et al. 2020; Ringelberg et al. 2022) studies, several further changes have recently been proposed (e.g. Souza et al. 2013; Soares et al. 2021). g p p g In all this turmoil, a number of African species that have variously been placed in Al bizia Durazz., Cathormion Hassk. and/or Samanea Merr., have been somewhat neglected by most authors. While Barneby and Grimes (1996) mentioned the similarities of some of these species to South-American taxa and Nielsen (1992) excluded them from Cathormion, they have not been formally dealt with and, therefore, Lewis and Rico-Arce (2005) referred them to the ‘dustbin’ genus Albizia (see Koenen et al. 2020) pending further studies on that genus. This was clearly unsatisfying and botanists working on the African flora have con tinued treating these species in Cathormion and Samanea (e.g. Hawthorne and Jongkind 2006: see note on p. 860), but this is an equally poor solution. The phylogenomic studies of Koenen et al. (2020) and Ringelberg et al. (2022) have elucidated the evolutionary relation ships of these species for the first time. First, the type species of Cathormion was shown to be nested in Albizia and the generic name has been synonymised with that genus by Koenen et al. (2020), while the African Cathormion obliquifoliolatum (De Wild.) G.C.C. Gilbert & Boutique and Cathormion rhombifolium (Benth.) Keay were shown to be closely related to South American Hydrochorea Barneby & J.W. Grimes and are transferred to that genus by Soares et al. (2022). This leaves us with three species, Cathormion altissimum (Hook. f.) Hutch. & Dandy, Samanea dinklagei (Harms) Keay and Samanea leptophylla (Harms) Brenan & Brummit that were placed in the Inga clade by Koenen et al. (2020), but which cannot be placed decisively in any existing mimosoid genus. These three species have since been resolved by Ringelberg et al. Keywords Albizia, Cathormion, ingoid clade, Fabaceae, Samanea, taxonomy Copyright Erik J. M. Koenen. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) 454 Introduction (2022) as the sister-group to the recently established South and Central American genus Robrichia (Barneby & J.W. Grimes) A.R.M. Luz & E.R. Souza (syn. Enterolobium sect. Robrichia Barneby & J.W. Grimes; Souza et al. 2022). y y While these African species are clearly similar to Robrichia in sharing the same ferrugi nous pubescence on young parts, as well as having very similar inflorescence morphology, the African species display wider variation in leaf size, leaflet number and shape, but differ most notably in pod morphology. The species of Robrichia are characterised by contorted (“ear-shaped”) indehiscent pods (Fig. 1G, H; Souza et al. 2022: Fig. 2D, E, J) which led them to be previously classified in Enterolobium Mart., while the related African species have twisted to coiled articulated lomentiform pods (C. altissimum, Figs 1A, 2G, H), straight to slightly curved woody pods (S. dinklagei, Figs 1C, 3F) or intermediate straight to slightly curved articulated pods (S. leptophylla/C. eriorhachis, Figs 1D, 3K). The woody pods were likened to Samanea and the articulated pods are superficially similar to Cathor mion s.s.; hence, these species have been placed in either or both these genera in the past. 455 Taxonomy of Osodendron gen. nov. Given the similarities and sister-group relationship with Robrichia, one could ar gue that these species might be accommodated in that genus, perhaps as a separate sec tion. However, the generic description of Robrichia would then have to be considerably expanded to include the greater variation in leaf morphology and especially to encom pass the different pod types. Given that the pods of Robrichia are highly characteristic, adding these African taxa to that genus would be undesirable and these two groups are equally conveniently accommodated in two separate genera. Therefore, I establish the new genus Osodendron E.J.M. Koenen to accommodate these three African species. g p Much confusion about relationships and generic classification in mimosoids (espe cially ingoids) has been caused by homoplasious fruit characters (Ringelberg et al. 2022) and the lomentiform pod type is one of the most intriguing because it has apparently evolved multiple times associated with repeated adaptations to periodically inundated areas and/or riparian habitats to facilitate hydrochorous seed dispersal (Aviles Peraza et al. 2022; Ringelberg et al. 2022; Soares et al. 2022). Indeed, when comparing the pods of Osodendron spp. to those of some Albizia species (Fig. 1B) or Hydrochorea (Fig. Introduction 1F), one can understand why these have all been previously included in a broadly circumscribed Cathormion and why a close relationship between Osodendron and Robrichia was not suspected (Fig. 1G, H), even though Barneby and Grimes (1996: p. 247) noted that Ro brichia schomburgkii (Benth.) A.R.M. Luz & E.R. Souza and Samanea dinklagei are very similar and essentially only differ in their fruits. To aid understanding of the differences in fruit morphology amongst the genera that are either closely related to Osodendron or with which these species were thought to share affinities, the critical pod characters of these groups are summarised globally in Table 1. It is clear that Osodendron can be sepa rated from the continental African species of Albizia and Hydrochorea by at least some of the features of the fruit, especially dehiscence, but it becomes more complicated when taking Madagascan, Asian, Australian and Pacific Albizia into account. Further studies Table 1. Fruit characters of Osodendron and morphologically similar genera. fruit shape pod valve texture septate between seeds dehiscence Osodendron – Africa straight, slightly curved, coiled and/or twisted woody, papery to thinly ligneous or aerenchymous yes indehiscent or tardily breaking up into articles Robrichia – Neotropics contorted/ “ear-shaped” woody yes indehiscent Hydrochorea – African continent (sensu Soares et al. (2022)) straight to falcate or slightly curved papery yes loment, seeds dispersed in 1-seeded articles Hydrochorea – Neotropics (sensu Soares et al. (2022)) straight to falcate or slightly curved papery or ligneous to woody yes loment, cryptoloment, follicular or indehiscent Albizia – African continent straight papery no dehiscent Albizia – Madagascar straight or twisted papery, thinly ligneous or woody variable dehiscent or indehiscent Albizia – Asia, Australia and Oceania (incl. Cathormion s.s.) straight, slightly curved or coiled/contorted papery or woody variable dehiscent or tardily breaking up into articles Albizia sect. Arthrosamanea (= Pseudalbizzia) – Neotropics straight papery, coriaceous or ligneous variable dehiscent, indehiscent, loment or cryptoloment Samanea – Neotropics straight to slightly curved thick and ligneous and/or fleshy yes indehiscent Table 1. Fruit characters of Osodendron and morphologically similar genera. Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) 456 are required to understand the morphological differences between Albizia and its allies and segregates, but it is clear that the genus Albizia, under its current circumscription, with the inclusion of Cathormion s.s. Introduction and exclusion of the New World species (these be ing transferred to Pseudalbizzia Britton & Rose by Aviles Peraza et al. 2022) is monophy letic (Koenen et al. 2020; Ringelberg et al. 2022; Koenen et al. unpubl. data). Regardless of this wider global context, Osodendron can be readily separated from all other ingoid genera on the African continent, for which I include an identification key below. Identification key to the continental African genera of ingoid mimosoids Identification key to the continental African genera of ingoid mimosoid (i.e. the ingoid clade sensu Koenen et al. (2020), which, apart from Senegalia Raf. and Faidherbia A. Chev., equates to all mimosoid genera native to the African continent with stamens fused into a tube): 1 Plants with prickles, stamens usually free or sometimes connate at base......... ...................................................................................................... Senegalia – Plants usually unarmed, rarely with spinescent shoots or stipular spines, sta mens fused into a tube or at least connate at base ........................................2 2 Shrubs with elastically dehiscent pods............................................................ ..................................................Afrocalliandra E.R. Souza & L.P. Queiroz – Trees or rarely shrubs with indehiscent, non-elastically dehiscent or lomenti form pods....................................................................................................3 3 Trees with stipular spines, inflorescence spicate............................Faidherbia – Usually unarmed trees or with spinescent shoots, but never stipular spines, inflorescences capitate..................................................................................4 4 Fruit non-septate, with papery valves, usually dehiscent..................... Albizia – Fruit septate, with thin fibrous or thick woody valves, indehiscent or lomen tiform..........................................................................................................5 5 Peripheral flowers of capitulum on pedicels at least 1 mm long, leaves with (1–)2–3 pairs of pinnae, fruit lomentaceous with seeds dispersed as 1-seeded articles......................................................................................Hydrochorea – Peripheral flowers on pedicels up to 0.5 mm long, leaves with (3–)5–30(–35) pairs of pinnae, fruit indehiscent or, if lomentiform, only tardily breaking up into articles................................................................................ Osodendron Osodendron E.J.M. Koenen, gen. nov. urn:lsid:ipni.org:names:77303836-1 Type. Osodendron altissimum (Hook. f.) E.J.M. Koenen. Osodendron E.J.M. Koenen, gen. nov. urn:lsid:ipni.org:names:77303836-1 457 Taxonomy of Osodendron gen. nov. Diagnosis. Osodendron differs from Robrichia by pods being either straight to twisted or coiled, but not contorted and ear-shaped. Diagnosis. Osodendron differs from Robrichia by pods being either straight to twisted or coiled, but not contorted and ear-shaped. Description. Trees, or rarely shrubs, unarmed or sometimes with spine-like pro jections on twigs or spine-like outgrowths on adventitious roots, resting buds peru late with scales distinctly different from stipules. Indumentum of usually dense fer ruginous pubescence on twigs, petioles, rachis and pinna rachises, stipules, bud scales and peduncles. Leaves with (3–)5–30(–34) pairs of pinnae, opposite or sometimes (the lowermost pairs) sub-opposite, with a single petiolar nectary usually present, as well as nectaries between at least some of the pinna pairs on abaxial surface of the rachis and often also between the upper 1–many leaflet pairs on the pinna-rachis es, the lowermost pair of pinnae usually distinctly shorter than others, pinnae with (7–)13–40(–48) leaflet pairs, one of the two leaflets of the lowermost pair usually reduced to a small paraphyllidium or lacking. Identification key to the continental African genera of ingoid mimosoids Inflorescences sub-globose capitula, dimorphic, borne on peduncles arising from axillary fascicles, sometimes arranged in short compound pseudoracemes with the leaves suppressed (not fully develop ing) and caducous as apparent from the presence of leaf scars in the pseudoracemes, these pseudoracemes developing below the foliage. Flowers sessile or shortly stipi tate, 4- or 5-merous, with fused calyx and corolla, androecium consisting of 10–25 stamens that are fused in the lower part to form a staminal tube, pollen compound in 32-celled polyads, central flowers more robust with a broader nectariferous base and longer staminal tube exserted beyond corolla tube. Fruits septate, indehiscent or tardily breaking up into articles, either lomentiform, twisted and strongly curved to coiled or weakly to not articulate and slightly curved to straight, seeds with a hard testa and open or closed pleurogram. p p g Distribution and habitat. Three species in tropical Africa, from Senegal in the west to the Democratic Republic of Congo in the east and Zambia and Angola in the south. Typically occurring in rainforest and extending into the savannah zone in gallery forest. Etymology. The genus is named after “Oso”, a food that is prepared in West Africa (Ghana and Nigeria) by fermenting the seeds of the type species O. altissimum and grinding them into a protein-rich paste that is subsequently cooked and eaten as either a main food, a delicacy or as a condiment to flavour soups and stews (Popoola et al. 2004; Jolaoso et al. 2014). Distribution and habitat. Three species in tropical Africa, from Senegal in the west to the Democratic Republic of Congo in the east and Zambia and Angola in the south. Typically occurring in rainforest and extending into the savannah zone in gallery forest.h Distribution and habitat. Three species in tropical Africa, from Senegal in the west to the Democratic Republic of Congo in the east and Zambia and Angola in the south. Typically occurring in rainforest and extending into the savannah zone in gallery forest. Etymology. The genus is named after “Oso”, a food that is prepared in West Africa (Gh d Ni i ) b f i h d f h i O l i i d Etymology. The genus is named after “Oso”, a food that is prepared in West Africa (Ghana and Nigeria) by fermenting the seeds of the type species O. Identification key to the continental African genera of ingoid mimosoids altissimum and grinding them into a protein-rich paste that is subsequently cooked and eaten as either a main food, a delicacy or as a condiment to flavour soups and stews (Popoola et al. 2004; Jolaoso et al. 2014). Notes. Osodendron is closely related to the Central and South American Robrichia, which was originally described as a section of Enterolobium by Barneby and Grimes (1996) and later segregated as a new genus by Souza et al. (2022). In the phylogeny of Ringelberg et al. (2022), the sampled species of both genera form sister-lineages. In habit, leaves and flowers, the two genera are very similar; however, the numbers of pinnae per leaf and leaflets per pinna and leaflet dimensions show greater variation in Osodendron with especially relatively large leaflets in the type species compared to Robrichia. The clearest difference between the two genera is to be found in the fruits, where those of Robrichia are contorted (or “ear-shaped”) and indehiscent, while those of Osodondron are either straight to falcate and indehiscent or twisted to spirally-coiled and lomentiform, only tardily breaking up into articles. 458 Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) Species of Osodendron can be easily distinguished from African Albizia species by the sub-opposite lower few pinna-pairs of which the lowermost is usually distinctly shorter (half or two thirds the length of the next few pairs of pinnae), as well as the strong reduction or lack of one of the two leaflets of the lowermost leaflet pair on each pinna (in Albizia, the lowermost pair of pinnae is only slightly shorter at most and the lowermost leaflet pair is not reduced or lacking one leaflet). The fruits are also notably different, with the inertly dehiscent pods of continental African Albizia spp. never woody nor articulate and always flat and papery (although in several Asian, Australian and Madagascan Albizia spp., indehiscent woody or tardily dehiscent articulate pods occur, for example, Fig. 1B; Table 1). Identification key to the species of Osodendron 1 Leaves with fewer than 10 pairs of pinnae; fruits coiled or twisted, rarely only curved, flowers with 20–25 stamens.................... 1. Osodendron altissimum – Leaves with more than 10 pairs of pinnae; fruit straight or somewhat curved, but not coiled nor twisted, flowers with 10–14 stamens...............................2 2 Leaves of mature individuals (not those on coppice shoots) lacking nectar ies between upper leaflet pairs, fruit a thick woody indehiscent pod, with uniform flat valves, not distinctly swollen over the seeds and rectangular in cross-section – West Africa (Upper Guinea)............................................... ...........................................................................2. Osodendron dinklagei – Leaves usually with nectaries between the upper 1–3 leaflet pairs, fruit usu ally a flat sub-lomentiform indehiscent pod or sometimes a thicker woody pod, articulately swollen around the seeds, elliptic to circular in cross-section – Central and East Africa (Lower Guinea, Congolia and gallery forests in adjacent savannahs).........................................3. Osodendron leptophyllum 1. Osodendron altissimum (Hook. f.) E.J.M. Koenen, comb. nov. urn:lsid:ipni.org:names:77303837-1 Figs 1A, 2 Pithecellobium altissimum (Hook. f.) Oliv., Fl. Trop. Afr. [Oliver et al.] 2: 364. 1871. Feuilleea altissima (Hook. f.) Kuntze, Revis. Gen. Pl. 1: 187. 1891.l Pithecellobium stuhlmannii Taub., Pflanzenw. Ost-Afrikas C 193. 1895. Type mate rial: Democratic Republic of Congo, Bataibo bei Duki, 850 m, Stuhlmann 2773 (holotype: B†), neotype here designated: Democratic Republic of Congo, Irumu, Bequaert 4887 (BR!). Albizia passargei Harms, Bot. Jahrb. 26: 253. 1899. Type material: Cameroon, Ngaoundéré, Passarge 164 (holotype: B†), neotype here designated: Cameroon, Bordure du Meng près des lacs de Boubala, Letouzey 2562 (P [P03502143] digital image!). 459 Taxonomy of Osodendron gen. nov. Figure 1. Confusing pods of Cathormion s.l. A pods of Osodendron altissimum that are very sim B pods of the Australian species Albizia moniliformis (DC.) F. Muell. (syn. Cathormion umbellatum moniliforme (DC.) Brummit) C pods of Osodendron dinklagei and D pods of Osodendron leptop that were likened to those of E Samanea (S. saman (Jacq.) Merr. is pictured here) and/or Cath F pods of Hydrochorea obliquifoliolata (De Wild.) E.J.M. Koenen (syn. Cathormion obliquifolio that are superficially similar to those of O. leptophyllum in particular; hence, this species having als classified in a broadly circumscribed Cathormion in the past, but note that the pods become disarti while still on the tree rather than tardily as in Osodendron and Albizia moniliformis G and H pods closest relative of Osodendron, the Neotropical genus Robrichia (R. schomburgkii is pictured here). I A Jos Stevens, voucher unknown B Russel Cumming, voucher unknown C J. Alves Pereira 2664 D R. Gutzwiller 1865 (P) E J.F. Maxwell 91-300 (L) F B. Descoings 8278 (MPU) G Cristoph M voucher unknown H José Benito Quezada Bonilla J B Quezada Bonilla 623 (HULE MO) A Figure 1. Confusing pods of Cathormion s.l. A pods of Osodendron altissimum that are very similar to B pods of the Australian species Albizia moniliformis (DC.) F. Muell. (syn. Cathormion umbellatum subsp. moniliforme (DC.) Brummit) C pods of Osodendron dinklagei and D pods of Osodendron leptophyllum that were likened to those of E Samanea (S. saman (Jacq.) Merr. is pictured here) and/or Cathormion F pods of Hydrochorea obliquifoliolata (De Wild.) E.J.M. Koenen (syn. Cathormion obliquifoliolatum) that are superficially similar to those of O. Figure 1. Confusing pods of Cathormion s.l. A pods of Osodendron altissimum that are very similar to B pods of the Australian species Albizia moniliformis (DC.) F. Muell. (syn. Cathormion umbellatum subsp. moniliforme (DC.) Brummit) C pods of Osodendron dinklagei and D pods of Osodendron leptophyllum that were likened to those of E Samanea (S. saman (Jacq.) Merr. is pictured here) and/or Cathormion F pods of Hydrochorea obliquifoliolata (De Wild.) E.J.M. Koenen (syn. Cathormion obliquifoliolatum) that are superficially similar to those of O. leptophyllum in particular; hence, this species having also been classified in a broadly circumscribed Cathormion in the past, but note that the pods become disarticulated while still on the tree rather than tardily as in Osodendron and Albizia moniliformis G and H pods of the closest relative of Osodendron, the Neotropical genus Robrichia (R. schomburgkii is pictured here). Images: A Jos Stevens, voucher unknown B Russel Cumming, voucher unknown C J. Alves Pereira 2664 (MA) D R. Gutzwiller 1865 (P) E J.F. Maxwell 91-300 (L) F B. Descoings 8278 (MPU) G Cristoph Moning, voucher unknown H José Benito Quezada Bonilla, J.B. Quezada Bonilla 623 (HULE, MO). A from www.africanplants.senckenberg.de B–H from www.gbif.org B–G distributed under a Creative Commons BY-NC-SA 4.0 Licence H distributed under a Creative Commons BY-NC-SA 3.0 Licence. 1. Osodendron altissimum (Hook. f.) E.J.M. Koenen, comb. nov. urn:lsid:ipni.org:names:77303837-1 Figs 1A, 2 leptophyllum in particular; hence, this species having also been classified in a broadly circumscribed Cathormion in the past, but note that the pods become disarticulated while still on the tree rather than tardily as in Osodendron and Albizia moniliformis G and H pods of the closest relative of Osodendron, the Neotropical genus Robrichia (R. schomburgkii is pictured here). Images: A Jos Stevens, voucher unknown B Russel Cumming, voucher unknown C J. Alves Pereira 2664 (MA) D R. Gutzwiller 1865 (P) E J.F. Maxwell 91-300 (L) F B. Descoings 8278 (MPU) G Cristoph Moning, voucher unknown H José Benito Quezada Bonilla, J.B. Quezada Bonilla 623 (HULE, MO). A from www.africanplants.senckenberg.de B–H from www.gbif.org B–G distributed under a Creative Commons BY-NC-SA 4.0 Licence H distributed under a Creative Commons BY-NC-SA 3.0 Licence. 460 Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) Cathormion altissimum (Hook. f.) Hutch. & Dandy, Fl. W. Trop. Afr. [Hutchinson & Dalziel] 1: 364. 1928; Hutchinson & Dandy in Kew Bull. 1928 (10): 401. 1928. Arthrosamanea altissima (Hook. f.) G.C.C. Gilbert & Boutique, Bull. Jard. Bot. État Bruxelles 22: 182. 1952. Inga altissima (Hook.f.) Roberty, Bull. Inst. Franç. Afrique Noire, A. 16: 343. 1954. Cathormion altissimum (Hook. f.) Hutch. & Dandy, Fl. W. Trop. Afr. [Hutchinson & Dalziel] 1: 364. 1928; Hutchinson & Dandy in Kew Bull. 1928 (10): 401. 1928. Arthrosamanea altissima (Hook. f.) G.C.C. Gilbert & Boutique, Bull. Jard. Bot. État Bruxelles 22: 182. 1952. Inga altissima (Hook.f.) Roberty, Bull. Inst. Franç. Afrique Noire, A. 16: 343. 1954. ( ) y, p [ Dalziel] 1: 364. 1928; Hutchinson & Dandy in Kew Bull. 1928 (10): 401. 1928. Arthrosamanea altissima (Hook. f.) G.C.C. Gilbert & Boutique, Bull. Jard. Bot. État B ll 22 182 1952 Bruxelles 22: 182. 1952. Bruxelles 22: 182. 1952. Inga altissima (Hook.f.) Roberty, Bull. Inst. Franç. Afrique Noire, A. 16: 343. 1954. Inga altissima (Hook.f.) Roberty, Bull. Inst. Franç. Afrique Noire, A. 16: 343. 1954. Basionym. Albizia altissima Hook. f. Niger Fl. [W. J. Hooker]. 332. 1849. Type material. Ghana, Cape Coast, 7/41, T. Vogel 18 (lectotype here designated from amongst the syntypes: K!); Nigeria, Aboh, 8/41, T. Vogel 28 (syntype: K!). g y yp g g y yp Description. Tree or rarely shrub, unarmed or sometimes with blunt spine-like projections on young twigs (W. Hawthorne, personal communication), 5–35 m tall, stem to 40 cm in diameter. 1. Osodendron altissimum (Hook. f.) E.J.M. Koenen, comb. nov. urn:lsid:ipni.org:names:77303837-1 Figs 1A, 2 Branches covered with many minute round lenticels 0.1–0.2 mm, twigs, stipules, bud scales, all leaf axes and peduncles brown pubescent, often densely so. Foliage consisting of bipinnate leaves with opposite to sub-opposite pinnae with closely-spaced, distinctly discolourous leaflets, slightly wider in the mid dle with the apical pair distinctly distally projected, in the basal pair often one or both leaflets reduced to paraphyllidia. The inflorescences consisting of sub-spherical capitula emerging in axillary fascicles, often aggregated into pseudoracemes emerging from the perulate resting buds, with the leaves not fully developing and caducous. Stipules deltoid to linear, 2–8 ×1.0–1.8 mm, caducous, perulae deltoid, 2–4 × 1.8– 3 mm. Leaves with (3–)5–7(–8) pairs of pinnae, apical pairs usually slightly longer and the basal pair sometimes much shorter with fewer leaflets, petiole 1.3–2.5 cm, pulvinate, adaxially shallowly canaliculate and often laterally and abaxially grooved, with a circular to elliptic sessile cupular nectary ca. 0.8–2.1 mm in diameter locat ed ca. mid-petiole, rachis (1.8–)4.5–11(–14.5) cm, adaxially shallowly canaliculate and often laterally and abaxially grooved, with 2–5 circular to elliptic sessile cupular nectaries between or just below the distal pairs of pinnae ca. 0.5 mm in diameter, pinna-rachises (2.4–)3.5–8(–10.2) cm, proximally pulvinate, laterally compressed, with elliptic to circular sessile cupular nectaries of 0.2–0.8 × 0.2–0.5 mm between the upper 1–4(–15) leaflet pairs, pinnae with (7–)13–20(–25) leaflet pairs, very often the abaxial leaflet of the lowermost pair reduced to a paraphyllidium, leaflets ses sile, pulvinate, inequilaterally hastate or weakly sigmoid, with acute or sometimes obtuse apex or (in subsp. busiraensis) weakly rhombic with oblique base and mostly obtuse apex or sometimes apiculate, (4–)7–14(–18) × (1.5–)3–4 mm, asymmetrically palmately-pinnate secondary venation with 7–10 veins starting from leaflet base next to the mid-rib and (7–)15–22 major and intercalary secondary veins laterally from the mid-rib, brochidodromous to craspedodromous, distinctly prominent on both surfaces when dry (or obscure on adaxial surface in subsp. busiraensis), tertiary vena tion finely reticulate, hardly visible on adaxial surface. Capitula ca. 35–50-flowered, on peduncles 1.8–3.2 cm, dimorphic with 3–9 central flowers with broader base and longer staminal tubes, bracts lanceolate to lanceolate-spatulate, ca. 0.9–1.2 × 0.1– 0.2 mm, ciliate in upper half, caducous. Peripheral flowers slenderly campanulate Taxonomy of Osodendron gen. nov. 461 with pale green or greenish-white calyx and corolla, subsessile to shortly pedicellate, pedicel to ca. 0.5 mm, sparsely pubescent. Calyx 5-merous, 2–3.2 mm long, the deltoid lobes ca. 1. Osodendron altissimum (Hook. f.) E.J.M. Koenen, comb. nov. urn:lsid:ipni.org:names:77303837-1 Figs 1A, 2 0.5 × 0.5 mm, ciliate at the apex and often also a few scattered short hairs on the outer surface of calyx tube and lobes, otherwise glabrous, corolla 5-mer ous, 5–7.5 mm long, glabrous, androecium of ca. 20–25 stamens, filaments white, ca. 12–17 mm long, fused into a tube at the base for ca. 2–3 mm, anthers yellow, basifixed, pollen aggregated into 32-celled flattened polyads. Pistil ca. 13–18 mm long, the ovary ca. 3 mm with a few scattered hairs on distal half and the funnel- shaped stigma extending ca. 1 mm beyond the anthers. Central flowers similar, but sessile, broadly campanulate and staminal tube ca. 5.5–8.5 mm long (i.e. the stamens are approximately the same length, but the fused tube is longer than in peripheral flowers), the tube exserted beyond the corolla. Fruit a spirally-curved, twisted or coiled lomentiform pod, usually articulate between the seeds, breaking up tardily after abscission in water or on the forest floor, often with arenchymous swollen meso carp for flotation, (10–)18–29(–30) × (0.8–)1.1–1.7 × 0.2–0.6 cm, (3–)9–20(–23) seeded. Seeds plano-compressed, nearly round or slightly elliptic, ca. 5–7.5 × 4–6 × 1.5–2 mm, chestnut brown with a small 2.1–3.2 × 1–1.5 mm elliptic to obovate or oblong open pleurogram. Notes. A specimen seen at Kew, of Espírito Santo 1858, collected in Guinée- Bissau, is doubtfully placed in this species. This specimen is highly unusual in the number of pairs of pinnae (13) and leaflet pairs (14–35) and the leaflets appear relatively small and elongate (even though they appear not fully expanded), while having the typical asymmetrical hastate shape. Furthermore, the fruit is not clearly articulate and also differs in being only somewhat curved rather than twisted or coiled. Brenan and Brummitt (1965) also cited this material as being unusual and speculated that it may be a distinct taxon. I do not share this opinion as I do not think it is sufficiently morphologically distinct. It is perhaps more likely of hybrid origin with O. dinklagei, which occurs sympatrically, as the other parent. However, this unusual specimen resembles O. altissimum much more closely than O. din klagei and the number of pinnae and leaflets and dimensions of the leaflets clearly distinguish it from that species. The holotype of Pithecellobium stuhlmannii was destroyed at B and no surviving isotypes are known. 1. Osodendron altissimum (Hook. f.) E.J.M. Koenen, comb. nov. urn:lsid:ipni.org:names:77303837-1 Figs 1A, 2 According to the Flora of East Tropical Africa (Brenan 1959), the original material was collected in the Democratic Republic of Congo, west of Lake Al bert. The specimen of Bequaert 4887 from BR is from the same region, includes leaves, flowers and immature fruit and matches the description in the protologue and is here selected as the neotype of P. stuhlmannii. The holotype of Albizia passargei was also destroyed at B, but Villiers (1989) placed the name in synonymy with C. altissimum, based on the description in the protologue. A specimen of Letouzey 2562 from P, comprising leaves and flowers, is here chosen as the neotype as it was collected in N Cameroon (ca. 150 km SW of the type locality) and matches the description in the protologue. Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) 462 Key to subspecies of O. altissimum: 1 Leaflets typically asymmetrically hastate and weakly sigmoid, apex acute, usu ally > 3× as long as wide; fruits spirally-curved, coiled or twisted, 1-seeded ar ticles (11–)13–17 mm wide and > (3–)4 mm thick when ripe – widespread in Lower and Upper Guinea......1.1. Osodendron altissimum subsp. altissimum – Leaflets weakly rhombic with oblique base, apex obtuse, ≤ 3× as long as wide; fruits spirally-coiled, 1-seeded articles 8–11 mm wide and 2–3 mm thick when ripe – Local in west Democratic Republic of Congo............................. ..........................................1.2. Osodendron altissimum subsp. busiraensis 1.1 Osodendron altissimum subsp. altissimum Distribution. Widespread across Upper and Lower Guinea and Congolia, from Guin ea-Bissau in the west to the Democratic Republic of Congo in the east and Angola in the south (Fig. 2A), also reported from Uganda and Zambia (Brenan 1959). g p g Ecology. Fresh-water swamp forest and along rivers in primary and secondary, evergreen or semi-deciduous rainforest. Representative specimens studied. Guinée-Bissau: Espírito Santo 3810 & 3832 (frts., BR); Côte d’Ivoire: Oldeman 968 (fls, BR); Cameroon: A.J.M. Leeuwen berg 8868 (fr., BR), B. Sonké 5499 (fr., BR); Gabon: N. Hallé 2119 (fls, frts, BR); Democratic Republic of Congo: F. Seret 461 (fls, BR); 1.2. Osodendron altissimum subsp. busiraensis (G.C.C. Gilbert & Boutique) E.J.M. Koenen, stat. nov. et comb. nov. urn:lsid:ipni.org:names:77303839-1 Basionym. Arthrosamanea altissima var. busiraensis G.C.C. Gilbert & Boutique, Fl. Congo Belge 3: 183. 1952. Type material. Democratic Republic of Congo, Eala surroundings, Wangata- Watsiko, October 1943, Germain 1604 (holotype: BR! [BR0000015846686], isotype: P [P00418269] digital image!). Type material. Democratic Republic of Congo, Eala surroundings, Wangata- Watsiko, October 1943, Germain 1604 (holotype: BR! [BR0000015846686], isotype: P [P00418269] digital image!). Note. This subspecies was originally described as an infraspecific variety. I have considered treating it as a distinct species, but after studying material held at BR, I concluded it cannot be reliably separated from the typical subspecies across its entire range. The material stands out in its combination of slightly different leaf shape and smaller pods, but material outside the Busira river catchment in the Democratic Re public of Congo, as well as in other parts of Africa, is rather variable in leaf shape and pod size. Given that both these differences are consistently present in most of the mate rial from that region, i.e. the variation is geographically structured within this variable and widespread species, subspecies is more appropriate than varietal rank and I treat it here as such. Note. This subspecies was originally described as an infraspecific variety. I have considered treating it as a distinct species, but after studying material held at BR, I concluded it cannot be reliably separated from the typical subspecies across its entire range. The material stands out in its combination of slightly different leaf shape and smaller pods, but material outside the Busira river catchment in the Democratic Re public of Congo, as well as in other parts of Africa, is rather variable in leaf shape and pod size. 1.1 Osodendron altissimum subsp. altissimum Given that both these differences are consistently present in most of the mate rial from that region, i.e. the variation is geographically structured within this variable and widespread species, subspecies is more appropriate than varietal rank and I treat it here as such. Taxonomy of Osodendron gen. nov. 463 Figure 2. Osodendron altissimum A distribution of the two subspecies as per the legend, based on data from Gbif.org with the addition of occurrence records for O. altissimum subsp. busiraensis from specimens at BR B trunk on riverbank C bipinnate leaf D close-up of leaflets E close-up of rachis apex showing nectary F inflorescences G unripe pod H mature pod. Images: B, F, H William Hawthorne (FHO) C–E, G David Harris (E), from www.africanplants.senckenberg.de. Taxonomy of Osodendron gen. nov. 4 Taxonomy of Osodendron gen. nov. 463 Taxonomy of Osodendron gen. nov. 463 Figure 2. Osodendron altissimum A distribution of the two subspecies as per the legend, based on data from Gbif.org with the addition of occurrence records for O. altissimum subsp. busiraensis from specimens at BR B trunk on riverbank C bipinnate leaf D close-up of leaflets E close-up of rachis apex showing nectary F inflorescences G unripe pod H mature pod. Images: B, F, H William Hawthorne (FHO) Figure 2. Osodendron altissimum A distribution of the two subspecies as per the legend, based on data from Gbif.org with the addition of occurrence records for O. altissimum subsp. busiraensis from specimens at BR B trunk on riverbank C bipinnate leaf D close-up of leaflets E close-up of rachis apex showing nectary F inflorescences G unripe pod H mature pod. Images: B, F, H William Hawthorne (FHO) C E G David Harris (E) from www africanplants senckenberg de Figure 2. Osodendron altissimum A distribution of the two subspecies as per the legend, based on data from Gbif.org with the addition of occurrence records for O. altissimum subsp. busiraensis from specimens at BR B trunk on riverbank C bipinnate leaf D close-up of leaflets E close-up of rachis apex showing nectary F inflorescences G unripe pod H mature pod. Images: B, F, H William Hawthorne (FHO) C–E, G David Harris (E), from www.africanplants.senckenberg.de. Distribution. Local to the Busira river catchment in the western Democratic Republic of Congo (Fig. 2A). Distribution. Local to the Busira river catchment in the western Democratic Republic of Congo (Fig. 2A). 2. Osodendron dinklagei (Harms) E.J.M. Koenen, comb. nov. Albizia dinklagei (Harms) Harms, Bot. Jahrb. Syst. 53(3–5): 455, in obs. 1915. Pithecellobium dinklagei (Harms) Harms, Notizbl. Bot. Gart. Berlin-Dahlem 8: 145. 1922. Cathormion dinklagei (Harms) Hutch. & Dandy, Fl. W. Trop. Afr. [Hutchinson & Dalziel] 1: 364. 1928; Hutchinson & Dandy in Kew Bull. 401. 1928. Samanea dinklagei (Harms) Keay, Kew Bull. 8(4): 488. 1954. Basionym. Mimosa dinklagei Harms, Bot. Jahrb. Syst. 26(3–4): 253. 1899. Type material. Liberia, Grand Bassa, 21 May 1897, Dinklage 1827 (holotype: B†, isotype: K! [K000044068]). Basionym. Mimosa dinklagei Harms, Bot. Jahrb. Syst. 26(3–4): 253. 1899. Description. Tree, up to 30 m tall, twigs with rows of lenticels and becoming shal lowly grooved with age. Twigs, stipules, perulate resting buds, all leaf-axes and peduncles covered in a densely villose ferruginous indumentum. Foliage consisting of very finely divided bipinnately compound leaves, the tiny leaflets ciliate and somewhat discolour ous. The inflorescences consisting of sub-spherical capitula emerging from axillary fasci cles of coeval leaves, sometimes in compound ramiflorous pseudoracemes with the leaves not fully developing and caducous. Stipules narrowly deltoid or lanceolate to linear 3.5–4.3(–9) × ca. 1 mm, caducous leaving conspicuous glabrous scars, perulae deltoid, ca. 2 × 1.5 mm. Leaves with 10–34 pairs of pinnae, often with one or a few much reduced pinnae at the base, petiole 5–11 mm, pulvinate and adaxially flattened at the base, usually with a sessile or shortly stipitate cupular nectary 0.5–0.7 mm in diameter just below the lowermost pinna pair, rachis grooved and adaxially canaliculate although obscured by dense indumentum, with a 1–1.5 mm long deciduous mucro, 6.5–16(–21) cm, with 3–8(–11) interpinnal subsessile cupular nectaries apically, these sometimes button-shaped and on a 0.7 mm long stipe, pinnae obscurely pulvinate, with an adaxial ridge and a tiny 0.3–0.4 mm long mucro at the apex, 1.0–2.5(–4.5) cm, usually without nectaries, except on larger leaves (of juveniles or coppice shoots) with cupular nectaries between the upper 1–3 leaflet pairs, pinnae with (9–)27–40(–48) leaflet pairs, the abaxial leaflet of the lowermost pair usually reduced to a paraphyllidium, leaflets inequilaterally linear, sessile and pulvinate, with strongly oblique base and acute apex, 0.8–3 × 0.2–0.8 mm, venation obscure on the adaxial surface, palmately-pinnate brochidodromous, with three secondary veins emerging from the base next to the clearly stronger mid-rib on the adaxial side, 2–3 further secondary veins on each side and sparse reticulate tertiary vena tion. Capitula 1–3 per leaf axil, ca. 1.1 Osodendron altissimum subsp. altissimum E l A h i l b i p g g Ecology. As the typical subspecies. g g Ecology. As the typical subspecies. Representative specimens studied. Democratic Republic of Congo: Boyekoli Ebale Congo Expedition 2010 104 (fls, BR); J. Louis 2052 (BR) & 13196 (BR); Robyns 653 (BR); Ghesquiere 2787 (BR); G. Hulstaert 607 (BR). Representative specimens studied. Democratic Republic of Congo: Boyekoli Ebale Congo Expedition 2010 104 (fls, BR); J. Louis 2052 (BR) & 13196 (BR); Robyns 653 (BR); Ghesquiere 2787 (BR); G. Hulstaert 607 (BR). Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) 464 2. Osodendron dinklagei (Harms) E.J.M. Koenen, comb. nov. 40–60-flowered, on peduncles 1.8–3.5(–4.1) cm, dimorphic with 1(–3) central flower(s) that are more robust and with a longer, exserted staminal tube. Peripheral flowers ca. 0.7 mm broad at the base, the basal ones on a pedi cel of ca. 0.4–0.6 mm, the others sub-sessile to sessile closer to the apex. Calyx slenderly campanulate, green, 5-merous, 1.6–3.1 mm long, densely puberulous on outer surface, the deltoid lobes ca. 0.4 × 0.4 mm, corolla green, 4- or 5-merous, 3–5.5 mm long, pubescent, the lobes 1.8–2 mm long, stamens 10–14, filaments white, 14 mm long, Taxonomy of Osodendron gen. nov. 465 Figure 3. A–F Osodendron dinklagei: A trunk of adult specimen in terra firme forest B foliage C detail of pinnae D detail of resting bud and petiolar nectary E inflorescence F pods G distribution map of O. dinklagei and O. leptophyllum as per the legend based on data from Gbif.org H–K O. leptophyllum: H trunk I bipin nate leaf J close-up of leaflets K twig and pods. Images: A–D, F William Hawthorne (FHO) E Xander van der Burgt (K) H–J David Harris (E) K Paul Latham H–K from www.africanplants.senckenberg.de. Figure 3. A–F Osodendron dinklagei: A trunk of adult specimen in terra firme forest B foliage C detail of pinnae D detail of resting bud and petiolar nectary E inflorescence F pods G distribution map of O. dinklagei and O. leptophyllum as per the legend based on data from Gbif.org H–K O. leptophyllum: H trunk I bipin nate leaf J close-up of leaflets K twig and pods. Images: A–D, F William Hawthorne (FHO) E Xander van der Burgt (K) H–J David Harris (E) K Paul Latham H–K from www.africanplants.senckenberg.de. Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) 466 basally fused into a tube ca. 3 mm long, anthers light yellow, basifixed, pollen released in 32-celled polyads, pistil ca. 18 mm long with a sessile ovary ca. 2.5 mm long, puberulent on upper half with white hairs, stigma shallowly funnel-shaped. Central flower(s) similar to peripheral flowers, but ca. 1.8 mm broad at the base, calyx 2.0–2.4 mm long, corolla ca. 4.5 mm long, staminal tube ca. 6 mm long (i.e. the stamens are approximately the same length, but fused for a greater part than in peripheral flowers). 2. Osodendron dinklagei (Harms) E.J.M. Koenen, comb. nov. Fruit an indehiscent woody pod up to 13–18 × 2–2.2(–2.5) × 0.3–0.6 cm, straight or slightly curved and with uniformly thick valves, except for the distinctly thickened margins, not swollen over the seeds, 16–24 seeded when well-fertilised, seeds rounded elliptical, only slightly laterally compressed, greenish light brown, 6–7.5 × 4–5 × 3–3.5 mm when dry, with a hard testa and an elongate closed pleurogram 5–6 × 2–2.5 mm. Distribution. Upper Guinea, from Guinea-Bissau to Ghana (Fig. 3G). Ecology. Rainforest, gallery forest, wooded grassland, edges of mangrove s Representative specimens examined. Guinée-Bissau: Espírito Santo 1747 (K) & 1864 (K) & 1871 (K) & 2697 (K). Sierra Leone: X. van der Burgt 1994 (K). 3. Osodendron leptophyllum (Harms) E.J.M. Koenen, comb. nov. urn:lsid:ipni.org:names:77303841-1 Fig. 3H–K Albizia eriorhachis Harms syn. nov., Bot. Jahrb. Syst. 53(3–5): 456. 1915. Type ma terial: Chevalier 7777 (lectotype here designated from amongst the syntypes: P [P00418361] digital image!, isolectotypes: P [P00418360] digital image!, P [P00418362] digital image!, L [L.1992190] digital image!).l Albizia flamignii De Wild., Pl. Bequaert. 3: 49. 1925. Type material: Democratic Republic of Congo, Kitobola, 12 Sept 1910, Flamigni 265 (lectotype designated here: BR! [BR0000016145290], isolectotypes: BR! [BR0000016145283], BR! [BR0000016145306], P [P00418365], digital image!). g g Cathormion eriorhachis (Harms) Dandy syn. nov., in F. W. Andrews, Fl. Pl. Anglo- Egypt. Sudan ii. 155. 1952. Type: Based on Albizia eriorhachis Harms. Arthrosamanea leptophylla var. guineensis G.C.C. Gilbert & Boutique, Bull. Jard. Bot. État Bruxelles 22: 182. 1952. Type material: Democratic Republic of Congo, Yangambi, réserve flore Isalowe, févr. 1940, Louis 16368 (BR!). l Samanea guineensis (G.C.C. Gilbert & Boutique) Brenan & Brummitt, Bol. Soc. Brot. sér. 2, 39: 202. 1965. Type: Based on Arthrosamanea leptophylla var. guineensis G.C.C. Gilbert & Boutique. Basionym. Albizia leptophylla Harms, Bot. Jahrb. Syst. 53(3–5): 455. 1915. Type material. Democratic Republic of Congo, Kimuenza, 17 km S of Leo poldville, August 1910, Mildbread 3520 (lectotype (designated by Villiers (1989) as holotype, here corrected): HBG [HBG519160] digital image!, isolectotypes: HBG [HBG519161] digital image!, P [P00418364] digital image!). Taxonomy of Osodendron gen. nov. 467 Description. Tree or more rarely a shrub, unarmed or with spine-like outgrowths near the base of the bole perhaps associated with adventitious roots (W. Hawthorne, personal communication), to 30 m tall and to 1.25 m in stem diameter, twigs dark with densely scattered small pustular lenticels or sometimes with lighter coloured corky bark. 2. Osodendron dinklagei (Harms) E.J.M. Koenen, comb. nov. Twigs, stipules, perulate resting buds, all leaf-axes and peduncles covered in dense ferruginous villose indumentum. Foliage consisting of finely divided bipinnate leaves, the leaflets variably ciliate, sometimes with only very few hairs on the margins, the lamina usually glabrous, but sometimes also with a few scattered hairs along the mid- rib on lower surface or rarely pilose or with appressed long hairs on both surfaces es pecially in lower half, usually distinctly discolourous. The inflorescences consisting of sub-spherical capitula emerging from axillary fascicles of coeval leaves, usually a few below the leaves in the axils of caducous leaves on the same shoots or sometimes also with a few short ramiflorous pseudoracemes, with all leaves caducous, lower down on the branch. Stipules elliptically oblong to asymetrically oblanceolate or falcate, 5–8(–9) × 2–3 mm, more scarcely pubescent or nearly glabrous on adaxial surface, caducous leaving conspicuous scars, perulae deltoid to ovoid, ca. 1.5–5 × 1.5–3.5 mm. Leaves with (10–)12–30(–35) pairs of pinnae, the lower 1–2 pairs of pinnae often shorter with fewer leaflets, petiole pulvinate and flattened on adaxial side at the base, (0.5– )0.8–2.2(–3) cm long, petiolar nectary usually present, cupular and circular, sessile or shortly stipitate and 0.6–1 mm in diameter, in variable position ranging from just above the pulvinule to just below the basal pair of pinnae, rachis canaliculate although often obscured due to dense pubescence, 8–15(–22) cm long, with 1–4(–6) cupular nectaries between the upper pairs of pinnae, these 0.3–0.6 mm in diameter, pinnae pulvinate and with an adaxial ridge, (0.5–)2.5–5(–7.5) cm long, nearly always the abaxial leaflet of the lowermost pair reduced to a paraphyllidium and the adaxial one somewhat smaller than the other leaflets, with elliptical nectaries ca. 0.1–0.25 mm in diameter between the upper 1–3 leaflet pairs, pinnae with (17–)24–35(–42) leaflet pairs, leaflets sessile, pulvi nate, asymmetrically oblong to oblanceolate, base oblique to sometimes hastate, apex rounded to acute, (2–)3–6(–9) × (0.3–)0.5–1.2(–2) mm, venation palmately-pinnate brochidodromous, with 2–4 basal veins adaxially next to the mid-rib and 5–8 further secondary veins on each side and reticulate tertiary venation, apart from the mid-rib, all venation often obscure in smaller leaves, otherwise the secondary venation prominent, sometimes only on the lower surface. Capitula 2–4 arranged in fascicles, on pedun cles ca. 2.7–4.5 cm, dimorphic with ca. 50–70 peripheral flowers and a single elon gated central flower. Bracteoles spatulate, ca. 0.5 mm long, pubescent on outer surface. 2. Osodendron dinklagei (Harms) E.J.M. Koenen, comb. nov. Flowers white, the peripheral ones on pedicels 0.75–2.5 mm. Calyx slenderly campanu late, green, 5-merous, 2–2.5 mm long, densely puberulous on outer surface, the deltoid lobes ca. 0.25 mm long, corolla 4–4.5 mm long, pubescent on outer surface, the lobes 1.8–2 mm long, stamens 10–14, the filaments white, 11–14 mm long, basally fused into a tube 1.5–2.5 mm long, anthers basifixed, pollen released in 32-celled polyads, pistil on a ca. 0.25–0.75(–1.25) mm long stipe, ovary ca. 1–1.5 mm long, pubescent, style 12–16 mm long, stigma shallowly funnel-shaped. Central flower(s) similar to periph eral flowers, but sessile and longer, calyx ca. 2.5 mm long, corolla ca. 6–6.5 mm long, Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) 468 staminal tube 4–6 mm long, exserted beyond corolla tube. Fruit a dark brown to black indehiscent pod that may tardily disintegrate into 1–multiple-seeded parts, the slightly thickened margins usually straight or also often articulate especially around aborted seeds, the valves papery and thin or with somewhat thickened mesocarp, but not really ligneous, when ripe, swollen over the seeds, when well-fertilised, 12–28(–32)-seeded, (4.5–)10–16(–20) × 0.9–1.3 × 0.4–0.7 cm. Seeds yellowish-brown, 5.5–7.5 × 3–4.5 × 2–3 mm with hard testa and a darker closed pleurogram of 4.5–5.5 × 1.2–1.5 mm. Distribution. Lower Guinea and Congolia in Cameroon, Central African Repub lic, Gabon, Republic of the Congo, Democratic Republic of Congo, Angola (Fig. 3G). Ecology. Forest edges and gallery forest. Representative specimens examined. Cameroon: J. Mildbread 8584 (K); Demo cratic Republic of Congo: Luidi, Thysville, Lusolo, 16 October 1959, P. Compere 610 (BR), Isangi, 21 February 1950, Callens 2363 (BR), Petite vallées d’affluente de la Belan zovi, près son confluent avec la Lubimbe, 26 January 1949, A. Michelson 877 (BR, K). pl y Notes. Osodendron leptophylla is highly variable in number of pinnae per leaf, num ber and size of leaflets and density and length of the indumentum. No consistent corre lations between any of these variations have been found suggesting that the material re ferred to two heterotypic synonyms is best included under O. leptophyllum. The first of these is Albizia eriorhachis, described by Harms in the same publication as A. leptophylla. Specimens from Cameroon, Central African Republic and Gabon, identified as Cathor mion eriorhachis, appear to be similar to typical O. leptophyllum, but with relatively large leaflets and denser indumentum. 2. Osodendron dinklagei (Harms) E.J.M. Koenen, comb. nov. However, the material is not distinct enough to merit recognition as a separate species, given the large variation in leaf dimensions and indu mentum in O. leptophyllum across its range and is here placed in its synonymy.h The second is Samanea guineensis, which was originally described as a variety of Arthrosamanea leptophylla, based on more numerous pairs of pinnae and leaflets per leaf, but Brenan and Brummitt (1965) found it to be distinctive enough to recognise it at species rank. Apart from the difference in foliage, they noted differences in the indumentum of the calyx and corolla (glabrous to subglabrous, instead of densely pu berulent) and a longer exserted staminal tube. I have observed material that fits their observations (e.g. Jean Louis 10051, BR), but in other collections with the leaf type of “guineensis”, I have observed dense pubescence on the calyx (e.g. Michelson 877, BR) and vice versa specimens with ca. 15 pairs of pinnae and a subglabrous calyx (Letouzey 9816). Villiers (1989) also judged S. guineensis to be conspecific with S. lepto phylla, having also seen numerous specimens with intermediate foliage. I follow Villiers (1989) and place this name in the synonymy of O. leptophyllum. References Aviles Peraza G, Koenen EJM, Riina R, Hughes CE, Ringelberg JJ, Carnevali Fernández-Con cha G, Ramírez Morillo IM, Can Itza LL, Tamayo-Cen I, Ramírez Prado JH, Cornejo X, Mattapha S, Duno de Stefano R (2022) Re-establishment of the genus Pseudalbizzia (Le guminosae, Caesalpinioideae: mimosoid clade): the New World species formerly placed in Albizia. In: Hughes CE, de Queiroz LP, Lewis GP (Eds) Advances in Legume Systematics 14. Classification of Caesalpinioideae Part 1: New generic delimitations. PhytoKeys 205: 371–400. https://doi.org/10.3897/phytokeys.205.76821 Barneby RC, Grimes J (1996) Silk tree, guanacaste, monkey’s earring: A generic system of the synandrous Mimosaceae of the Americas. Part I. Abarema, Albizia, and allies. Memoirs of the New York Botanical Garden 74(1). Barneby RC, Grimes J (1997) Silk tree, guanacaste, monkey’s earring: A generic system of the synandrous Mimosaceae of the Americas. Part II. Pithecellobium, Cojoba, and Zygia. Mem oirs of the New York Botanical Garden 74(2). Bouchenak-Khelladi Y, Maurin O, Hurter J, van der Bank M (2010) The evolutionary his tory and biogeography of Mimosoideae (Leguminosae): An emphasis on African aca cias. Molecular Phylogenetics and Evolution 57(2): 495–508. https://doi.org/10.1016/j. ympev.2010.07.019 Brenan JP (1959) Flora of tropical East Africa. Leguminosae: subfamily Mimosoideae. Royal Botanic Gardens, Kew. Brenan JPM, Brummitt RK (1965) New and little-known species from the Flora Zambesiaca area: 19. Leguminosae-Mimosoideae. Boletim da Sociedade Broteriana, serie 2, 39: 189–205. Brown GK (2008) Systematics of the tribe Ingeae (Leguminosae-Mimosoideae) over past 25 years. Muelleria 26(1): 27–42. https://doi.org/10.5962/p.292491 Hawthorne WD, Jongkind CC (2006) Woody plants of Western African forests, A guide to the forest trees, shrubs and lianes from Senegal to Ghana. Royal Botanic Gardens, Kew, London. Hawthorne WD, Jongkind CC (2006) Woody plants of Western African forests, A guide to the forest trees, shrubs and lianes from Senegal to Ghana. Royal Botanic Gardens, Kew, London. Jolaoso AA, Ajayi JO, Ogunmuyiwa SIO, Albert OM (2014) Functional, proximate and mineral iti f l k f t d l (f t d d f C th i lti i Jolaoso AA, Ajayi JO, Ogunmuyiwa SIO, Albert OM (2014) Functional, proximate and mineral composition of a lesser known fermented legume (fermented seeds of Cathormion altissimum, Oso). Journal of Emerging Trends in Engineering and Applied Sciences 5(2): 129–134. Acknowledgements I thank Gwilym Lewis for acting as my SYNTHESYS+ host which made for an excellent visit to the legume collections at K. I also thank Jan Wieringa, William Hawthorne and Colin Hughes for useful comments that greatly improved the manuscript. My research Taxonomy of Osodendron gen. nov. 469 was supported by the Swiss National Science Foundation (Early.Postdoc.Mobility fel lowship P2ZHP3_199693 to EJMK) and by the SYNTHESYS+ project http://www. synthesys.info/ financed by European Community Research Infrastructure Action under the H2020 Integrating Activities Programme, Project number 823827. References Koenen EJM, Kidner CA, de Souza ÉR, Simon MF, Iganci JRV, Nicholls JA, Brown GK, de Queiroz LP, Luckow MA, Lewis GP, Pennington RT, Hughes CE (2020) Hybrid capture of 964 nuclear genes resolves evolutionary relationships in the mimosoid legumes and re veals the polytomous origins of a large pantropical radiation. American Journal of Botany 107(12): 1710–1735. https://doi.org/10.1002/ajb2.1568 Lewis GP, Rico Arce ML (2005) Tribe Ingeae. In: Lewis G, Schrire B, Mackinder B, Lock M (Eds) Legumes of the World. Kew: Royal Botanic Gardens, 193–213. Erik J. M. Koenen / PhytoKeys 205: 453–470 (2022) 470 LPWG (2017) A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny – The Legume Phylogeny Working Group (LPWG). Taxon 66(1): 44–77. https://doi.org/10.12705/661.3 Luckow M, Miller JT, Murphy DJ, Livshultz T (2003) A phylogenetic analysis of the Mi mosoideae (Leguminosae) based on chloroplast DNA sequence data. In: Klitgaard B, Bruneau A (Eds) Advances in Legume Systematics Part 10, Royal Botanic Gardens, Kew, London, 197–220. Miller JT, Seigler DS (2012) Evolutionary and taxonomic relationships of Acacia s.l. (Legu minosae: Mimosoideae). Australian Systematic Botany 25(3): 217–224. https://doi. org/10.1071/SB11042 Nielsen IC (1981) Ingeae. In: Polhill RM, Raven PH (Eds) Advances in Legume Systematics, Part. 2. Royal Botanic Gardens, Kew, London, 173–190. Nielsen IC (1992) Mimosaceae (Leguminosae-Mimosoideae). Flora Malesiana-Series 1. Sper matophyta 11(1): 1–226. Pedley L (1986) Derivation and dispersal of Acacia (Leguminosae), with particular reference to Australia, and the recognition of Senegalia and Racosperma. Botanical Journal of the Lin nean Society 92(3): 219–254. https://doi.org/10.1111/j.1095-8339.1986.tb01429.x Popoola TOS, Jolaoso AA, Afolabi RO (2004) Microbiology of the production of Oso, a condi ment made by fermenting seeds of Cathormion altissimum. Tropical Science 44(4): 187– 189. https://doi.org/10.1002/ts.166 Ringelberg JJ, Koenen EJM, Iganci JR, de Queiroz LP, Murphy DJ, Gaudeul M, Bruneau A, Luckow M, Lewis GP, Hughes CE (2022) Phylogenomic analysis of 997 nuclear genes reveals the need for extensive generic re-delimitation in Caesalpinioideae (Leguminosae). In: Hughes CE, de Queiroz LP, Lewis GP (Eds) Advances in Legume Systematics 14. Clas sification of Caesalpinioideae Part 1: New generic delimitations. PhytoKeys 205: 3–58. https://doi.org/10.3897/phytokeys.205.85866 Soares MVB, Guerra E, Morim MP, Iganci JRV (2021) Reinstatement and recircumscription of Jupunba and Punjuba (Fabaceae) based on phylogenetic evidence. Botanical Journal of the Linnean Society 196(4): 456–479. https://doi.org/10.1093/botlinnean/boab007 Soares MVB, Koenen EJM, Iganci JRV, Morim MP (2022) A new generic circumscription of Hydrochorea (Leguminosae, Caesalpinioideae, mimosoid clade) with an amphi-Atlantic distribution. References In: Hughes CE, de Queiroz LP, Lewis GP (Eds) Advances in Legume System atics 14. Classification of Caesalpinioideae Part 1: New generic delimitations. PhytoKeys 205: 401–438. https://doi.org/10.3897/phytokeys.205.82775 Souza ER, Lewis GP, Forest F, Schnadelbach AS, Van Der Berg C, Queiroz LP (2013) Phylogeny of Calliandra (Leguminosae-Mimosoideae) based on nuclear and plastid molecular mark ers. Taxon 62(6): 1201–1220. https://doi.org/10.12705/626.2 Souza ER, Monteiro Luz AR, Rocha L, Lewis GP (2022) Molecular and morphological analysis supports the separation of Robrichia as a genus distinct from Enterolobium (Leguminosae: Caesalpinioideae: Mimosoid clade). Systematic Botany 47(1): 268–277. https://doi.org/1 0.1600/036364422X16442669847067 Villiers JF (1989) Leguminosae-Mimosoideae. Flore du Gabon (31): 3–174.

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